Energy and Technology Review

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poggio, A.J.; Mayall, B.H.

1989-04-01

The Lawrence Livermore National Laboratory (LLNL) is an acknowledged world center for analytical cytology. This leadership was recognized by the Regents of the University of California (UC), who in 1982 established and funded the Program for Analytical Cytology to facilitate the transfer of this technology from scientists at LLNL to their University colleagues, primarily through innovative collaborative research. This issue of Energy and Technology Review describes three of the forty projects that have been funded in this way; chosen to illustrate the potential medical application of the research. Analytical cytology is a relatively new field of biomedical research that ismore » increasingly being applied in clinical medicine. It has been particularly important in unraveling the complexities of the human immune system and in quantifying the pathobiology of malignancy. Defined as the characterization and measurement of cells and cellular constituents for biological and medical purposes, analytical cytology bridges the gap between the quantitative discipline of molecular biology and the more qualitative disciplines of anatomy and pathology. It is itself multidisciplinary in nature. Two major approaches to analytical cytology are flow cytometry and image cytometry. In each of these research techniques, cells are measured one at a time in an automated device. In flow instruments, the cells are dispersed in fluid suspension and pass in single file through a beam of laser light to generate optical signals that are measured. In image cytometry, cells are dispersed on a slide and are imaged through a microscope onto an electronic imaging and analysis system that processes the cell image to extract measurements of interest.« less

Two-Photon Flow Cytometry

NASA Technical Reports Server (NTRS)

Zhog, Cheng Frank; Ye, Jing Yong; Norris, Theodore B.; Myc, Andrzej; Cao, Zhengyl; Bielinska, Anna; Thomas, Thommey; Baker, James R., Jr.

2004-01-01

Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantitation is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two- channel detection and two-photon excitation flow cytometry (T(sup 3)FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo. Real-time detection of fluorescent microbeads in the vasculature of mouse ear demonstrates the ability to do flow cytometry in vivo. The conditions required to perform quantitative in vivo cytometry on labeled cells will be presented.

A novel image-based quantitative method for the characterization of NETosis

PubMed Central

Zhao, Wenpu; Fogg, Darin K.; Kaplan, Mariana J.

2015-01-01

NETosis is a newly recognized mechanism of programmed neutrophil death. It is characterized by a stepwise progression of chromatin decondensation, membrane rupture, and release of bactericidal DNA-based structures called neutrophil extracellular traps (NETs). Conventional ‘suicidal’ NETosis has been described in pathogenic models of systemic autoimmune disorders. Recent in vivo studies suggest that a process of ‘vital’ NETosis also exists, in which chromatin is condensed and membrane integrity is preserved. Techniques to assess ‘suicidal’ or ‘vital’ NET formation in a specific, quantitative, rapid and semiautomated way have been lacking, hindering the characterization of this process. Here we have developed a new method to simultaneously assess both ‘suicidal’ and ‘vital’ NETosis, using high-speed multi-spectral imaging coupled to morphometric image analysis, to quantify spontaneous NET formation observed ex-vivo or stimulus-induced NET formation triggered in vitro. Use of imaging flow cytometry allows automated, quantitative and rapid analysis of subcellular morphology and texture, and introduces the potential for further investigation using NETosis as a biomarker in pre-clinical and clinical studies. PMID:26003624

Label-free high-throughput imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mahjoubfar, A.; Chen, C.; Niazi, K. R.; Rabizadeh, S.; Jalali, B.

2014-03-01

Flow cytometry is an optical method for studying cells based on their individual physical and chemical characteristics. It is widely used in clinical diagnosis, medical research, and biotechnology for analysis of blood cells and other cells in suspension. Conventional flow cytometers aim a laser beam at a stream of cells and measure the elastic scattering of light at forward and side angles. They also perform single-point measurements of fluorescent emissions from labeled cells. However, many reagents used in cell labeling reduce cellular viability or change the behavior of the target cells through the activation of undesired cellular processes or inhibition of normal cellular activity. Therefore, labeled cells are not completely representative of their unaltered form nor are they fully reliable for downstream studies. To remove the requirement of cell labeling in flow cytometry, while still meeting the classification sensitivity and specificity goals, measurement of additional biophysical parameters is essential. Here, we introduce an interferometric imaging flow cytometer based on the world's fastest continuous-time camera. Our system simultaneously measures cellular size, scattering, and protein concentration as supplementary biophysical parameters for label-free cell classification. It exploits the wide bandwidth of ultrafast laser pulses to perform blur-free quantitative phase and intensity imaging at flow speeds as high as 10 meters per second and achieves nanometer-scale optical path length resolution for precise measurements of cellular protein concentration.

Imaging flow cytometry for phytoplankton analysis.

PubMed

Dashkova, Veronika; Malashenkov, Dmitry; Poulton, Nicole; Vorobjev, Ivan; Barteneva, Natasha S

2017-01-01

This review highlights the concepts and instrumentation of imaging flow cytometry technology and in particular its use for phytoplankton analysis. Imaging flow cytometry, a hybrid technology combining speed and statistical capabilities of flow cytometry with imaging features of microscopy, is rapidly advancing as a cell imaging platform that overcomes many of the limitations of current techniques and contributed significantly to the advancement of phytoplankton analysis in recent years. This review presents the various instrumentation relevant to the field and currently used for assessment of complex phytoplankton communities' composition and abundance, size structure determination, biovolume estimation, detection of harmful algal bloom species, evaluation of viability and metabolic activity and other applications. Also we present our data on viability and metabolic assessment of Aphanizomenon sp. cyanobacteria using Imagestream X Mark II imaging cytometer. Herein, we highlight the immense potential of imaging flow cytometry for microalgal research, but also discuss limitations and future developments. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantitative assessment of cancer cell morphology and motility using telecentric digital holographic microscopy and machine learning.

PubMed

Lam, Van K; Nguyen, Thanh C; Chung, Byung M; Nehmetallah, George; Raub, Christopher B

2018-03-01

The noninvasive, fast acquisition of quantitative phase maps using digital holographic microscopy (DHM) allows tracking of rapid cellular motility on transparent substrates. On two-dimensional surfaces in vitro, MDA-MB-231 cancer cells assume several morphologies related to the mode of migration and substrate stiffness, relevant to mechanisms of cancer invasiveness in vivo. The quantitative phase information from DHM may accurately classify adhesive cancer cell subpopulations with clinical relevance. To test this, cells from the invasive breast cancer MDA-MB-231 cell line were cultured on glass, tissue-culture treated polystyrene, and collagen hydrogels, and imaged with DHM followed by epifluorescence microscopy after staining F-actin and nuclei. Trends in cell phase parameters were tracked on the different substrates, during cell division, and during matrix adhesion, relating them to F-actin features. Support vector machine learning algorithms were trained and tested using parameters from holographic phase reconstructions and cell geometric features from conventional phase images, and used to distinguish between elongated and rounded cell morphologies. DHM was able to distinguish between elongated and rounded morphologies of MDA-MB-231 cells with 94% accuracy, compared to 83% accuracy using cell geometric features from conventional brightfield microscopy. This finding indicates the potential of DHM to detect and monitor cancer cell morphologies relevant to cell cycle phase status, substrate adhesion, and motility. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

NASA Astrophysics Data System (ADS)

Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

2016-09-01

Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

Fluorine-19 nuclear magnetic resonance of chimeric antigen receptor T cell biodistribution in murine cancer model.

PubMed

Chapelin, Fanny; Gao, Shang; Okada, Hideho; Weber, Thomas G; Messer, Karen; Ahrens, Eric T

2017-12-18

Discovery of effective cell therapies against cancer can be accelerated by the adaptation of tools to rapidly quantitate cell biodistribution and survival after delivery. Here, we describe the use of nuclear magnetic resonance (NMR) 'cytometry' to quantify the biodistribution of immunotherapeutic T cells in intact tissue samples. In this study, chimeric antigen receptor (CAR) T cells expressing EGFRvIII targeting transgene were labeled with a perfluorocarbon (PFC) emulsion ex vivo and infused into immunocompromised mice bearing subcutaneous human U87 glioblastomas expressing EGFRvIII and luciferase. Intact organs were harvested at day 2, 7 and 14 for whole-sample fluorine-19 ( 19 F) NMR to quantitatively measure the presence of PFC-labeled CAR T cells, followed by histological validation. NMR measurements showed greater CAR T cell homing and persistence in the tumors and spleen compared to untransduced T cells. Tumor growth was monitored with bioluminescence imaging, showing that CAR T cell treatment resulted in significant tumor regression compared to untransduced T cells. Overall, 19 F NMR cytometry is a rapid and quantitative method to evaluate cell biodistribution, tumor homing, and fate in preclinical studies.

Quantitative image cytometry measurements of lipids, DNA, CD45 and cytokeratin for circulating tumor cell identification in a model system

NASA Astrophysics Data System (ADS)

Futia, Gregory L.; Qamar, Lubna; Behbakht, Kian; Gibson, Emily A.

2016-04-01

Circulating tumor cell (CTC) identification has applications in both early detection and monitoring of solid cancers. The rarity of CTCs, expected at ~1-50 CTCs per million nucleated blood cells (WBCs), requires identifying methods based on biomarkers with high sensitivity and specificity for accurate identification. Discovery of biomarkers with ever higher sensitivity and specificity to CTCs is always desirable to potentially find more CTCs in cancer patients thus increasing their clinical utility. Here, we investigate quantitative image cytometry measurements of lipids with the biomarker panel of DNA, Cytokeratin (CK), and CD45 commonly used to identify CTCs. We engineered a device for labeling suspended cell samples with fluorescent antibodies and dyes. We used it to prepare samples for 4 channel confocal laser scanning microscopy. The total data acquired at high resolution from one sample is ~ 1.3 GB. We developed software to perform the automated segmentation of these images into regions of interest (ROIs) containing individual cells. We quantified image features of total signal, spatial second moment, spatial frequency second moment, and their product for each ROI. We performed measurements on pure WBCs, cancer cell line MCF7 and mixed samples. Multivariable regressions and feature selection were used to determine combination features that are more sensitive and specific than any individual feature separately. We also demonstrate that computation of spatial characteristics provides higher sensitivity and specificity than intensity alone. Statistical models allowed quantification of the required sensitivity and specificity for detecting small levels of CTCs in a human blood sample.

Functional characterization of neotropical snakes peripheral blood leukocytes subsets: Linking flow cytometry cell features, microscopy images and serum corticosterone levels.

PubMed

de Carvalho, Marcelo Pires Nogueira; Queiroz-Hazarbassanov, Nicolle Gilda Teixeira; de Oliveira Massoco, Cristina; Sant'Anna, Sávio Stefanini; Lourenço, Mariana Mathias; Levin, Gabriel; Sogayar, Mari Cleide; Grego, Kathleen Fernandes; Catão-Dias, José Luiz

2017-09-01

Reptiles are the unique ectothermic amniotes, providing the key link between ectothermic anamniotes fish and amphibians, and endothermic birds and mammals; becoming an important group to study with the aim of providing significant knowledge into the evolutionary history of vertebrate immunity. Classification systems for reptiles' leukocytes have been described by their appearance rather than function, being still inconsistent. With the advent of modern techniques and the establishment of analytical protocols for snakes' blood by flow cytometry, we bring a qualitative and quantitative assessment of innate activities presented by snakes' peripheral blood leukocytes, thereby linking flow cytometric features with fluorescent and light microscopy images. Moreover, since corticosterone is an important immunomodulator in reptiles, hormone levels of all blood samples were measured. We provide novel and additional information which should contribute to better understanding of the development of the immune system of reptiles and vertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vivo flow cytometry and time-resolved near-IR angiography and lymphography

NASA Astrophysics Data System (ADS)

Galanzha, Ekaterina I.; Tuchin, Valery V.; Brock, Robert W.; Zharov, Vladimir P.

2007-05-01

Integration of photoacoustic and photothermal techniques with high-speed, high-resolution transmission and fluorescence microscopy shows great potential for in vivo flow cytometry and indocyanine green (ICG) near-infrared (IR) angiography of blood and lymph microvessels. In particular, the capabilities of in vivo flow cytometry using rat mesentery and nude mouse ear models are demonstrated for real-time quantitative detection of circulating and migrating individual blood and cancer cells in skin, mesentery, lymph nodes, liver, kidney; studying vascular dynamics with a focus on lymphatics; monitoring cell traffic between blood and lymph systems; high-speed imaging of cell deformability in flow; and label-free real-time monitoring of single cell extravasation from blood vessel lumen into tissue. As presented, the advantages of ICG IR-angiography include estimation of time resolved dye dynamics (appearance and clearance) in blood and lymph microvessels using fluorescent and photoacoustic modules of the integrated technique. These new approaches are important for monitoring and quantifying metastatic and apoptotic cells; comparative measurements of plasma and cell velocities; analysis of immune responses; monitoring of circulating macromolecules, chylomicrons, bacteria, viruses and nanoparticles; molecular imaging. In the future, we believe that the integrated technique presented will have great potential for translation to early disease diagnoses (e.g. cancer) or assessment of innovative therapeutic interventions in humans.

Systems biology and clinical cytomics: The 10th Leipziger Workshop and the 3rd International Workshop on Slide-Based Cytometry, Leipzig, Germany, April 2005.

PubMed

Tárnok, Attila; Valet, Günther K; Emmrich, Frank

2006-01-01

Despite very significant technical and software improvements in flow cytometry (FCM) since the 1980's, the demand for a cytometric technology combining both quantitative cell analysis and morphological documentation in Cytomics became evident. Improvements in microtechnology and computing permit nowadays similar quantitative and stoichiometric single cell-based high-throughput analyses by microscopic instruments, like Slide-Based Cytometry (SBC). SBC and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures during early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance not only as a cytomics technology platform but also because of low quantities of required reagents and biological material. The exact knowledge of the location of each cell on the slide permits repetitive restaining and reanalysis of specimens. Various separate measurements of the same specimen can be ultimately fused to one database increasing the information obtained per cell. Relocation and optical evaluation of cells as typical SBC feature, can be of integral importance for cytometric analysis, since artifacts can be excluded and morphology of measured cells can be documented. Progress in cell analytic: In the SBC, new horizons can be opened by the new techniques of structural and functional analysis with the high resolution from intracellular and membrane (confocal microscopy, nanoscopy, total internal fluorescence microscopy (TIRFM), and tissue level (tissomics), to organ and organism level (in vivo cytometry, optical whole body imaging). Predictive medicine aims at the detection of changes in patient's state prior to the manifestation of the disease or the complication. Such instances concern immune consequences of surgeries or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high risk patients in cancer cytostatic therapy. Preventive anti-infectious or anti-shock therapy as well as curative chemotherapy in combination with stem cell transplantation may provide better survival chances for patient at concomitant cost containment. Predictive medicine-guided optimization of therapy could lead to individualized medicine that gives significant therapeutic effect and may lower or abrogate potential therapeutic side effects. The 10th Leipziger Workshop combined with the 3rd International Workshop on SBC aimed to offer new methods in Image- and Slide-Based Cytometry for solutions in clinical research. It moved towards practical applications in the clinics and the clinical laboratory. This development will be continued in 2006 at the upcoming Leipziger Workshop and the International Workshop on Slide-Based Cytometry.

The role of RhD agglutination for the detection of weak D red cells by anti-D flow cytometry.

PubMed

Grey, D E; Davies, J I; Connolly, M; Fong, E A; Erber, W N

2005-04-01

Anti-D flow cytometry is an accurate method for quantifying feto-maternal haemorrhage (FMH). However, weak D red cells with <1000 RhD sites are not detectable using this methodology but are immunogenic. As quantitation of RhD sites is not practical, an alternative approach is required to identify those weak D fetal red cells where anti-D flow cytometry is inappropriate. We describe a simple algorithm based on RhD agglutination and flow cytometry peak separation. All weak D (n = 34) gave weak agglutination with RUM-1 on immediate spin (grading

A general method for bead-enhanced quantitation by flow cytometry

PubMed Central

Montes, Martin; Jaensson, Elin A.; Orozco, Aaron F.; Lewis, Dorothy E.; Corry, David B.

2009-01-01

Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry. PMID:17067632

Changes in cell morphology due to plasma membrane wounding by acoustic cavitation

PubMed Central

Schlicher, Robyn K.; Hutcheson, Joshua D.; Radhakrishna, Harish; Apkarian, Robert P.; Prausnitz, Mark R.

2010-01-01

Acoustic cavitation-mediated wounding (i.e., sonoporation) has great potential to improve medical and laboratory applications requiring intracellular uptake of exogenous molecules; however, the field lacks detailed understanding of cavitation-induced morphological changes in cells and their relative importance. Here, we present an in-depth study of the effects of acoustic cavitation on cells using electron and confocal microscopy coupled with quantitative flow cytometry. High resolution images of treated cells show that morphologically different types of blebs can occur after wounding conditions caused by ultrasound exposure as well as by mechanical shear and strong laser ablation. In addition, these treatments caused wound-induced non-lytic necrotic death resulting in cell bodies we call wound-derived perikarya (WD-P). However, only cells exposed to acoustic cavitation experienced ejection of intact nuclei and nearly instant lytic necrosis. Quantitative analysis by flow cytometry indicates that wound-derived perikarya are the dominant morphology of nonviable cells, except at the strongest wounding conditions, where nuclear ejection accounts for a significant portion of cell death after ultrasound exposure. PMID:20350691

The evolution of phase holographic imaging from a research idea to publicly traded company

NASA Astrophysics Data System (ADS)

Egelberg, Peter

2018-02-01

Recognizing the value and unmet need for label-free kinetic cell analysis, Phase Holograhic Imaging defines its market segment as automated, easy to use and affordable time-lapse cytometry. The process of developing new technology, meeting customer expectations, sources of corporate funding and R&D adjustments prompted by field experience will be reviewed. Additionally, it is discussed how relevant biological information can be extracted from a sequence of quantitative phase images, with negligible user assistance and parameter tweaking, to simultaneously provide cell culture characteristics such as cell growth rate, viability, division rate, mitosis duration, phagocytosis rate, migration, motility and cell-cell adherence without requiring any artificial cell manipulation.

Development of CD3 cell quantitation algorithms for renal allograft biopsy rejection assessment utilizing open source image analysis software.

PubMed

Moon, Andres; Smith, Geoffrey H; Kong, Jun; Rogers, Thomas E; Ellis, Carla L; Farris, Alton B Brad

2018-02-01

Renal allograft rejection diagnosis depends on assessment of parameters such as interstitial inflammation; however, studies have shown interobserver variability regarding interstitial inflammation assessment. Since automated image analysis quantitation can be reproducible, we devised customized analysis methods for CD3+ T-cell staining density as a measure of rejection severity and compared them with established commercial methods along with visual assessment. Renal biopsy CD3 immunohistochemistry slides (n = 45), including renal allografts with various degrees of acute cellular rejection (ACR) were scanned for whole slide images (WSIs). Inflammation was quantitated in the WSIs using pathologist visual assessment, commercial algorithms (Aperio nuclear algorithm for CD3+ cells/mm 2 and Aperio positive pixel count algorithm), and customized open source algorithms developed in ImageJ with thresholding/positive pixel counting (custom CD3+%) and identification of pixels fulfilling "maxima" criteria for CD3 expression (custom CD3+ cells/mm 2 ). Based on visual inspections of "markup" images, CD3 quantitation algorithms produced adequate accuracy. Additionally, CD3 quantitation algorithms correlated between each other and also with visual assessment in a statistically significant manner (r = 0.44 to 0.94, p = 0.003 to < 0.0001). Methods for assessing inflammation suggested a progression through the tubulointerstitial ACR grades, with statistically different results in borderline versus other ACR types, in all but the custom methods. Assessment of CD3-stained slides using various open source image analysis algorithms presents salient correlations with established methods of CD3 quantitation. These analysis techniques are promising and highly customizable, providing a form of on-slide "flow cytometry" that can facilitate additional diagnostic accuracy in tissue-based assessments.

Fluorescence-based detection and quantification of features of cellular senescence.

PubMed

Cho, Sohee; Hwang, Eun Seong

2011-01-01

Cellular senescence is a spontaneous organismal defense mechanism against tumor progression which is raised upon the activation of oncoproteins or other cellular environmental stresses that must be circumvented for tumorigenesis to occur. It involves growth-arrest state of normal cells after a number of active divisions. There are multiple experimental routes that can drive cells into a state of senescence. Normal somatic cells and cancer cells enter a state of senescence upon overexpression of oncogenic Ras or Raf protein or by imposing certain kinds of stress such as cellular tumor suppressor function. Both flow cytometry and confocal imaging analysis techniques are very useful in quantitative analysis of cellular senescence phenomenon. They allow quantitative estimates of multiple different phenotypes expressed in multiple cell populations simultaneously. Here we review the various types of fluorescence methodologies including confocal imaging and flow cytometry that are frequently utilized to study a variety of senescence. First, we discuss key cell biological changes occurring during senescence and review the current understanding on the mechanisms of these changes with the goal of improving existing protocols and further developing new ones. Next, we list specific senescence phenotypes associated with each cellular trait along with the principles of their assay methods and the significance of the assay outcomes. We conclude by selecting appropriate references that demonstrate a typical example of each method. Copyright © 2011 Elsevier Inc. All rights reserved.

Automated analysis of individual sperm cells using stain-free interferometric phase microscopy and machine learning.

PubMed

Mirsky, Simcha K; Barnea, Itay; Levi, Mattan; Greenspan, Hayit; Shaked, Natan T

2017-09-01

Currently, the delicate process of selecting sperm cells to be used for in vitro fertilization (IVF) is still based on the subjective, qualitative analysis of experienced clinicians using non-quantitative optical microscopy techniques. In this work, a method was developed for the automated analysis of sperm cells based on the quantitative phase maps acquired through use of interferometric phase microscopy (IPM). Over 1,400 human sperm cells from 8 donors were imaged using IPM, and an algorithm was designed to digitally isolate sperm cell heads from the quantitative phase maps while taking into consideration both the cell 3D morphology and contents, as well as acquire features describing sperm head morphology. A subset of these features was used to train a support vector machine (SVM) classifier to automatically classify sperm of good and bad morphology. The SVM achieves an area under the receiver operating characteristic curve of 88.59% and an area under the precision-recall curve of 88.67%, as well as precisions of 90% or higher. We believe that our automatic analysis can become the basis for objective and automatic sperm cell selection in IVF. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

CytometryML and other data formats

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2006-02-01

Cytology automation and research will be enhanced by the creation of a common data format. This data format would provide the pathology and research communities with a uniform way for annotating and exchanging images, flow cytometry, and associated data. This specification and/or standard will include descriptions of the acquisition device, staining, the binary representations of the image and list-mode data, the measurements derived from the image and/or the list-mode data, and descriptors for clinical/pathology and research. An international, vendor-supported, non-proprietary specification will allow pathologists, researchers, and companies to develop and use image capture/analysis software, as well as list-mode analysis software, without worrying about incompatibilities between proprietary vendor formats. Presently, efforts to create specifications and/or descriptions of these formats include the Laboratory Digital Imaging Project (LDIP) Data Exchange Specification; extensions to the Digital Imaging and Communications in Medicine (DICOM); Open Microscopy Environment (OME); Flowcyt, an extension to the present Flow Cytometry Standard (FCS); and CytometryML. The feasibility of creating a common data specification for digital microscopy and flow cytometry in a manner consistent with its use for medical devices and interoperability with both hospital information and picture archiving systems has been demonstrated by the creation of the CytometryML schemas. The feasibility of creating a software system for digital microscopy has been demonstrated by the OME. CytometryML consists of schemas that describe instruments and their measurements. These instruments include digital microscopes and flow cytometers. Optical components including the instruments' excitation and emission parts are described. The description of the measurements made by these instruments includes the tagged molecule, data acquisition subsystem, and the format of the list-mode and/or image data. Many of the CytometryML data-types are based on the Digital Imaging and Communications in Medicine (DICOM). Binary files for images and list-mode data have been created and read.

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry.

PubMed

Pugsley, Haley R

2017-07-21

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

PubMed Central

Pugsley, Haley R.

2017-01-01

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner. PMID:28784946

Visualization of Pulmonary Clearance Mechanisms via Noninvasive Optical Imaging Validated by Near-Infrared Flow Cytometry

PubMed Central

Zhou, Haiying; Gunsten, Sean P.; Zhegalova, Natalia G.; Bloch, Sharon; Achilefu, Samuel; Holley, J. Christopher; Schweppe, Daniel; Akers, Walter; Brody, Steven L.; Eades, William; Berezin, Mikhail Y.

2016-01-01

In vivo optical imaging with near-infrared (NIR) probes is an established method of diagnostics in preclinical and clinical studies. However, the specificities of these probes are difficult to validate ex vivo due to the lack of NIR flow cytometry. To address this limitation, we modified a flow cytometer to include an additional NIR channel using a 752 nm laser line. The flow cytometry system was tested using NIR microspheres and cell lines labeled with a combination of visible range and NIR fluorescent dyes. The approach was verified in vivo in mice evaluated for immune response in lungs after intratracheal delivery of the NIR contrast agent. Flow cytometry of cells obtained from the lung bronchoalveolar lavage demonstrated that the NIR dye was taken up by pulmonary macrophages as early as four-hours post-injection. This combination of optical imaging with NIR flow cytometry extends the capability of imaging and enables complementation of in vivo imaging with cell-specific studies. PMID:25808737

Assessing FRET using spectral techniques.

PubMed

Leavesley, Silas J; Britain, Andrea L; Cichon, Lauren K; Nikolaev, Viacheslav O; Rich, Thomas C

2013-10-01

Förster resonance energy transfer (FRET) techniques have proven invaluable for probing the complex nature of protein-protein interactions, protein folding, and intracellular signaling events. These techniques have traditionally been implemented with the use of one or more fluorescence band-pass filters, either as fluorescence microscopy filter cubes, or as dichroic mirrors and band-pass filters in flow cytometry. In addition, new approaches for measuring FRET, such as fluorescence lifetime and acceptor photobleaching, have been developed. Hyperspectral techniques for imaging and flow cytometry have also shown to be promising for performing FRET measurements. In this study, we have compared traditional (filter-based) FRET approaches to three spectral-based approaches: the ratio of acceptor-to-donor peak emission, linear spectral unmixing, and linear spectral unmixing with a correction for direct acceptor excitation. All methods are estimates of FRET efficiency, except for one-filter set and three-filter set FRET indices, which are included for consistency with prior literature. In the first part of this study, spectrofluorimetric data were collected from a CFP-Epac-YFP FRET probe that has been used for intracellular cAMP measurements. All comparisons were performed using the same spectrofluorimetric datasets as input data, to provide a relevant comparison. Linear spectral unmixing resulted in measurements with the lowest coefficient of variation (0.10) as well as accurate fits using the Hill equation. FRET efficiency methods produced coefficients of variation of less than 0.20, while FRET indices produced coefficients of variation greater than 8.00. These results demonstrate that spectral FRET measurements provide improved response over standard, filter-based measurements. Using spectral approaches, single-cell measurements were conducted through hyperspectral confocal microscopy, linear unmixing, and cell segmentation with quantitative image analysis. Results from these studies confirmed that spectral imaging is effective for measuring subcellular, time-dependent FRET dynamics and that additional fluorescent signals can be readily separated from FRET signals, enabling multilabel studies of molecular interactions. © 2013 International Society for Advancement of Cytometry. Copyright © 2013 International Society for Advancement of Cytometry.

Synthetic aperture tomographic phase microscopy for 3D imaging of live cells in translational motion

PubMed Central

Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.

2009-01-01

We present a technique for 3D imaging of live cells in translational motion without need of axial scanning of objective lens. A set of transmitted electric field images of cells at successive points of transverse translation is taken with a focused beam illumination. Based on Hyugens’ principle, angular plane waves are synthesized from E-field images of a focused beam. For a set of synthesized angular plane waves, we apply a filtered back-projection algorithm and obtain 3D maps of refractive index of live cells. This technique, which we refer to as synthetic aperture tomographic phase microscopy, can potentially be combined with flow cytometry or microfluidic devices, and will enable high throughput acquisition of quantitative refractive index data from large numbers of cells. PMID:18825263

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

NASA Astrophysics Data System (ADS)

Regmi, Raju; Mohan, Kavya; Mondal, Partha Pratim

2014-09-01

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

PubMed

Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

2017-06-28

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

A quartz nanopillar hemocytometer for high-yield separation and counting of CD4+ T lymphocytes

NASA Astrophysics Data System (ADS)

Kim, Dong-Joo; Seol, Jin-Kyeong; Wu, Yu; Ji, Seungmuk; Kim, Gil-Sung; Hyung, Jung-Hwan; Lee, Seung-Yong; Lim, Hyuneui; Fan, Rong; Lee, Sang-Kwon

2012-03-01

We report the development of a novel quartz nanopillar (QNP) array cell separation system capable of selectively capturing and isolating a single cell population including primary CD4+ T lymphocytes from the whole pool of splenocytes. Integrated with a photolithographically patterned hemocytometer structure, the streptavidin (STR)-functionalized-QNP (STR-QNP) arrays allow for direct quantitation of captured cells using high content imaging. This technology exhibits an excellent separation yield (efficiency) of ~95.3 +/- 1.1% for the CD4+ T lymphocytes from the mouse splenocyte suspensions and good linear response for quantitating captured CD4+ T-lymphoblasts, which is comparable to flow cytometry and outperforms any non-nanostructured surface capture techniques, i.e. cell panning. This nanopillar hemocytometer represents a simple, yet efficient cell capture and counting technology and may find immediate applications for diagnosis and immune monitoring in the point-of-care setting.We report the development of a novel quartz nanopillar (QNP) array cell separation system capable of selectively capturing and isolating a single cell population including primary CD4+ T lymphocytes from the whole pool of splenocytes. Integrated with a photolithographically patterned hemocytometer structure, the streptavidin (STR)-functionalized-QNP (STR-QNP) arrays allow for direct quantitation of captured cells using high content imaging. This technology exhibits an excellent separation yield (efficiency) of ~95.3 +/- 1.1% for the CD4+ T lymphocytes from the mouse splenocyte suspensions and good linear response for quantitating captured CD4+ T-lymphoblasts, which is comparable to flow cytometry and outperforms any non-nanostructured surface capture techniques, i.e. cell panning. This nanopillar hemocytometer represents a simple, yet efficient cell capture and counting technology and may find immediate applications for diagnosis and immune monitoring in the point-of-care setting. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr11338d

Novel image cytometric method for detection of physiological and metabolic changes in Saccharomyces cerevisiae.

PubMed

Chan, Leo L; Kury, Alexandria; Wilkinson, Alisha; Berkes, Charlotte; Pirani, Alnoor

2012-11-01

The studying and monitoring of physiological and metabolic changes in Saccharomyces cerevisiae (S. cerevisiae) has been a key research area for the brewing, baking, and biofuels industries, which rely on these economically important yeasts to produce their products. Specifically for breweries, physiological and metabolic parameters such as viability, vitality, glycogen, neutral lipid, and trehalose content can be measured to better understand the status of S. cerevisiae during fermentation. Traditionally, these physiological and metabolic changes can be qualitatively observed using fluorescence microscopy or flow cytometry for quantitative fluorescence analysis of fluorescently labeled cellular components associated with each parameter. However, both methods pose known challenges to the end-users. Specifically, conventional fluorescent microscopes lack automation and fluorescence analysis capabilities to quantitatively analyze large numbers of cells. Although flow cytometry is suitable for quantitative analysis of tens of thousands of fluorescently labeled cells, the instruments require a considerable amount of maintenance, highly trained technicians, and the system is relatively expensive to both purchase and maintain. In this work, we demonstrate the first use of Cellometer Vision for the kinetic detection and analysis of vitality, glycogen, neutral lipid, and trehalose content of S. cerevisiae. This method provides an important research tool for large and small breweries to study and monitor these physiological behaviors during production, which can improve fermentation conditions to produce consistent and higher-quality products.

Noninvasive imaging of multiple myeloma using near infrared fluorescent molecular probe

NASA Astrophysics Data System (ADS)

Hathi, Deep; Zhou, Haiying; Bollerman-Nowlis, Alex; Shokeen, Monica; Akers, Walter J.

2016-03-01

Multiple myeloma is a plasma cell malignancy characterized by monoclonal gammopathy and osteolytic bone lesions. Multiple myeloma is most commonly diagnosed in late disease stages, presenting with pathologic fracture. Early diagnosis and monitoring of disease status may improve quality of life and long-term survival for multiple myeloma patients from what is now a devastating and fatal disease. We have developed a near-infrared targeted fluorescent molecular probe with high affinity to the α4β1 integrin receptor (VLA-4)overexpressed by a majority of multiple myeloma cells as a non-radioactive analog to PET/CT tracer currently being developed for human diagnostics. A near-infrared dye that emits about 700 nm was conjugated to a high affinity peptidomimmetic. Binding affinity and specificity for multiple myeloma cells was investigated in vitro by tissue staining and flow cytometry. After demonstration of sensitivity and specificity, preclinical optical imaging studies were performed to evaluate tumor specificity in murine subcutaneous and metastatic multiple myeloma models. The VLA-4-targeted molecular probe showed high affinity for subcutaneous MM tumor xenografts. Importantly, tumor cells specific accumulation in the bone marrow of metastatic multiple myeloma correlated with GFP signal from transfected cells. Ex vivo flow cytometry of tumor tissue and bone marrow further corroborated in vivo imaging data, demonstrating the specificity of the novel agent and potential for quantitative imaging of multiple myeloma burden in these models.

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry.

PubMed

Wang, Meiyao; Misakian, Martin; He, Hua-Jun; Bajcsy, Peter; Abbasi, Fatima; Davis, Jeffrey M; Cole, Kenneth D; Turko, Illarion V; Wang, Lili

2014-01-01

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells. Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry. The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10(5) and (0.85 ± 0.11) × 10(5), respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC. Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Annexin-V/quantum dot probes for multimodal apoptosis monitoring in living cells: improving bioanalysis using electrochemistry

NASA Astrophysics Data System (ADS)

Montón, Helena; Parolo, Claudio; Aranda-Ramos, Antonio; Merkoçi, Arben; Nogués, Carme

2015-02-01

There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry.There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry. Electronic supplementary information (ESI) available: Optical microscopy images of apoptotic induced cell cultures at different times and negative control of flow cytometry. See DOI: 10.1039/c4nr07191c

ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing.

PubMed

Chen, Xingqi; Shen, Ying; Draper, Will; Buenrostro, Jason D; Litzenburger, Ulrike; Cho, Seung Woo; Satpathy, Ansuman T; Carter, Ava C; Ghosh, Rajarshi P; East-Seletsky, Alexandra; Doudna, Jennifer A; Greenleaf, William J; Liphardt, Jan T; Chang, Howard Y

2016-12-01

Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.

Mass cytometry: a highly multiplexed single-cell technology for advancing drug development.

PubMed

Atkuri, Kondala R; Stevens, Jeffrey C; Neubert, Hendrik

2015-02-01

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srivastava, Nimisha; Singh, Anup K

Microfluidic devices and methods for flow cytometry are described. In described examples, various sample handling and preparation steps may be carried out within a same microfluidic device as flow cytometry steps. A combination of imaging and flow cytometry is described. In some examples, spiral microchannels serve as incubation chambers. Examples of automated sample handling and flow cytometry are described.

qFlow Cytometry-Based Receptoromic Screening: A High-Throughput Quantification Approach Informing Biomarker Selection and Nanosensor Development.

PubMed

Chen, Si; Weddell, Jared; Gupta, Pavan; Conard, Grace; Parkin, James; Imoukhuede, Princess I

2017-01-01

Nanosensor-based detection of biomarkers can improve medical diagnosis; however, a critical factor in nanosensor development is deciding which biomarker to target, as most diseases present several biomarkers. Biomarker-targeting decisions can be informed via an understanding of biomarker expression. Currently, immunohistochemistry (IHC) is the accepted standard for profiling biomarker expression. While IHC provides a relative mapping of biomarker expression, it does not provide cell-by-cell readouts of biomarker expression or absolute biomarker quantification. Flow cytometry overcomes both these IHC challenges by offering biomarker expression on a cell-by-cell basis, and when combined with calibration standards, providing quantitation of biomarker concentrations: this is known as qFlow cytometry. Here, we outline the key components for applying qFlow cytometry to detect biomarkers within the angiogenic vascular endothelial growth factor receptor family. The key aspects of the qFlow cytometry methodology include: antibody specificity testing, immunofluorescent cell labeling, saturation analysis, fluorescent microsphere calibration, and quantitative analysis of both ensemble and cell-by-cell data. Together, these methods enable high-throughput quantification of biomarker expression.

Fluorescent-Antibody Measurement Of Cancer-Cell Urokinase

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.

1993-01-01

Combination of laboratory techniques provides measurements of amounts of urokinase in and between normal and cancer cells. Includes use of fluorescent antibodies specific against different forms of urokinase-type plasminogen activator, (uPA), fluorescence microscopy, quantitative analysis of images of sections of tumor tissue, and flow cytometry of different uPA's and deoxyribonucleic acid (DNA) found in suspended-tumor-cell preparations. Measurements provide statistical method for indicating or predicting metastatic potentials of some invasive tumors. Assessments of metastatic potentials based on such measurements used in determining appropriate follow-up procedures after surgical removal of tumors.

Identification and characterization of neutrophil extracellular trap shapes in flow cytometry

NASA Astrophysics Data System (ADS)

Ginley, Brandon; Emmons, Tiffany; Sasankan, Prabhu; Urban, Constantin; Segal, Brahm H.; Sarder, Pinaki

2017-03-01

Neutrophil extracellular trap (NET) formation is an alternate immunologic weapon used mainly by neutrophils. Chromatin backbones fused with proteins derived from granules are shot like projectiles onto foreign invaders. It is thought that this mechanism is highly anti-microbial, aids in preventing bacterial dissemination, is used to break down structures several sizes larger than neutrophils themselves, and may have several more uses yet unknown. NETs have been implied to be involved in a wide array of systemic host immune defenses, including sepsis, autoimmune diseases, and cancer. Existing methods used to visually quantify NETotic versus non-NETotic shapes are extremely time-consuming and subject to user bias. These limitations are obstacles to developing NETs as prognostic biomarkers and therapeutic targets. We propose an automated pipeline for quantitatively detecting neutrophil and NET shapes captured using a flow cytometry-imaging system. Our method uses contrast limited adaptive histogram equalization to improve signal intensity in dimly illuminated NETs. From the contrast improved image, fixed value thresholding is applied to convert the image to binary. Feature extraction is performed on the resulting binary image, by calculating region properties of the resulting foreground structures. Classification of the resulting features is performed using Support Vector Machine. Our method classifies NETs from neutrophils without traps at 0.97/0.96 sensitivity/specificity on n = 387 images, and is 1500X faster than manual classification, per sample. Our method can be extended to rapidly analyze whole-slide immunofluorescence tissue images for NET classification, and has potential to streamline the quantification of NETs for patients with diseases associated with cancer and autoimmunity.

Hessian-based quantitative image analysis of host-pathogen confrontation assays.

PubMed

Cseresnyes, Zoltan; Kraibooj, Kaswara; Figge, Marc Thilo

2018-03-01

Host-fungus interactions have gained a lot of interest in the past few decades, mainly due to an increasing number of fungal infections that are often associated with a high mortality rate in the absence of effective therapies. These interactions can be studied at the genetic level or at the functional level via imaging. Here, we introduce a new image processing method that quantifies the interaction between host cells and fungal invaders, for example, alveolar macrophages and the conidia of Aspergillus fumigatus. The new technique relies on the information content of transmitted light bright field microscopy images, utilizing the Hessian matrix eigenvalues to distinguish between unstained macrophages and the background, as well as between macrophages and fungal conidia. The performance of the new algorithm was measured by comparing the results of our method with that of an alternative approach that was based on fluorescence images from the same dataset. The comparison shows that the new algorithm performs very similarly to the fluorescence-based version. Consequently, the new algorithm is able to segment and characterize unlabeled cells, thus reducing the time and expense that would be spent on the fluorescent labeling in preparation for phagocytosis assays. By extending the proposed method to the label-free segmentation of fungal conidia, we will be able to reduce the need for fluorescence-based imaging even further. Our approach should thus help to minimize the possible side effects of fluorescence labeling on biological functions. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Semi-automatized segmentation method using image-based flow cytometry to study sperm physiology: the case of capacitation-induced tyrosine phosphorylation.

PubMed

Matamoros-Volante, Arturo; Moreno-Irusta, Ayelen; Torres-Rodriguez, Paulina; Giojalas, Laura; Gervasi, María G; Visconti, Pablo E; Treviño, Claudia L

2018-02-01

Is image-based flow cytometry a useful tool to study intracellular events in human sperm such as protein tyrosine phosphorylation or signaling processes? Image-based flow cytometry is a powerful tool to study intracellular events in a relevant number of sperm cells, which enables a robust statistical analysis providing spatial resolution in terms of the specific subcellular localization of the labeling. Sperm capacitation is required for fertilization. During this process, spermatozoa undergo numerous physiological changes, via activation of different signaling pathways, which are not completely understood. Classical approaches for studying sperm physiology include conventional microscopy, flow cytometry and Western blotting. These techniques present disadvantages for obtaining detailed subcellular information of signaling pathways in a relevant number of cells. This work describes a new semi-automatized analysis using image-based flow cytometry which enables the study, at the subcellular and population levels, of different sperm parameters associated with signaling. The increase in protein tyrosine phosphorylation during capacitation is presented as an example. Sperm cells were isolated from seminal plasma by the swim-up technique. We evaluated the intensity and distribution of protein tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h under different experimental conditions. We used an antibody against FER kinase and pharmacological inhibitors in an attempt to identify the kinases involved in protein tyrosine phosphorylation during human sperm capacitation. Semen samples from normospermic donors were obtained by masturbation after 2-3 days of sexual abstinence. We used the innovative technique image-based flow cytometry and image analysis tools to segment individual images of spermatozoa. We evaluated and quantified the regions of sperm where protein tyrosine phosphorylation takes place at the subcellular level in a large number of cells. We also used immunocytochemistry and Western blot analysis. Independent experiments were performed with semen samples from seven different donors. Using image analysis tools, we developed a completely novel semi-automatic strategy useful for segmenting thousands of individual cell images obtained using image-based flow cytometry. Contrary to immunofluorescence which relies on the analysis of a limited sperm population and also on the observer, image-based flow cytometry allows for unbiased quantification and simultaneous localization of post-translational changes in an extended sperm population. Interestingly, important data can be independently analyzed by looking to the frame of interest. As an example, we evaluated the capacitation-associated increase in tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h. As previously reported, protein tyrosine phosphorylation increases in a time-depending manner, but our method revealed that this increase occurs differentially among distinct sperm segments. FER kinase is reported to be the enzyme responsible for the increase in protein tyrosine phosphorylation in mouse sperm. Our Western blot analysis revealed for the first time the presence of this enzyme in human sperm. Using our segmentation strategy, we aimed to quantify the effect of pharmacological inhibition of FER kinase and found a marked reduction of protein tyrosine phosphorylation only in the flagellum, which corresponded to the physical localization of FER in human sperm. Our method provides an alternative strategy to study signaling markers associated with capacitation, such as protein tyrosine phosphorylation, in a fast and quantitative manner. None. This is an in vitro study performed under controlled conditions. Chemical inhibitors are not completely specific for the intended target; the possibility of side effects cannot be discarded. Our results demonstrate that the use of image-based flow cytometry is a very powerful tool to study sperm physiology. A large number of cells can be easily analyzed and information at the subcellular level can be obtained. As the segmentation process works with bright-field images, it can be extended to study expression of other proteins of interest using different antibodies or it can be used in living sperm to study intracellular parameters that can be followed using fluorescent dyes sensitive to the parameter of interest (e.g. pH, Ca2+). Therefore, this a versatile method that can be exploited to study several aspects of sperm physiology. This work was supported DGAPA (IN203116 to C. Treviño), Fronteras-CONACyT No. 71 and Eunice Kennedy Shriver National Institute of Child Health and Human Development NIH (RO1 HD38082) to P.E. Visconti and by a Lalor Foundation fellowship to M.G. Gervasi. A. Matamoros is a student of the Maestría en Ciencias Bioquímicas-UNAM program supported by CONACyT (416400) and DGAPA-UNAM. A. Moreno obtained a scholarship from Red MacroUniversidades and L. Giojalas obtained a schloarhip from CONICET and Universidad Nacional de Cordoba. The authors declare there are not conflicts of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email:journals.permissions@oup.com

Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds

NASA Astrophysics Data System (ADS)

Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H. G.; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

2016-06-01

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ~150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ~150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01908k

In Vivo Myeloperoxidase Imaging and Flow Cytometry Analysis of Intestinal Myeloid Cells.

PubMed

Hülsdünker, Jan; Zeiser, Robert

2016-01-01

Myeloperoxidase (MPO) imaging is a non-invasive method to detect cells that produce the enzyme MPO that is most abundant in neutrophils, macrophages, and inflammatory monocytes. While lacking specificity for any of these three cell types, MPO imaging can provide guidance for further flow cytometry-based analysis of tissues where these cell types reside. Isolation of leukocytes from the intestinal tract is an error-prone procedure. Here, we describe a protocol for intestinal leukocyte isolation that works reliable in our hands and allows for flow cytometry-based analysis, in particular of neutrophils.

Flow cytometry microscopy and hyperspectral imaging of microcystis, cyanobacteria and algae

EPA Science Inventory

The detection of algae and cyanobacteria is an important step in assessing water quality. Studies were initiated using microscopy, flow cytometry and hyperspectral imaging with two fresh water species that could be grown in the laboratory: Microcystis Aeruginosa (cyanobacteria),...

Inhibition of Breast Cancer-Induced Angiogenesis by a Diverged Homeobox Gene

DTIC Science & Technology

2006-05-01

and then harvested for flow cytometry using appropriate antibodies. Ad.Gax blocked the expression of VCAM-1, E-selectin, and ICAM-1. DOD Idea Award...solution. Bands were visualized by chemiluminescence using the ECL-Plus reagent (Amersham, Piscataway, NJ). Flow Cytometry Cells were harvested after...33), all of whose down-regulation we have confirmed using real time quantitative RT-PCR, Western blot, and flow cytometry (Fig. 5). Moreover, Gax

Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy.

PubMed

Dardikman, Gili; Nygate, Yoav N; Barnea, Itay; Turko, Nir A; Singh, Gyanendra; Javidi, Barham; Shaked, Natan T

2018-03-01

We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new technique on cancer cells, obtaining nucleus refractive index values that are lower than those of the cytoplasm, coinciding with recent findings. We believe that the proposed technique has the potential to be used for flow cytometry, where full 3-D refractive index tomography is too slow to be implemented during flow.

Application of image flow cytometry for the characterization of red blood cell morphology

NASA Astrophysics Data System (ADS)

Pinto, Ruben N.; Sebastian, Joseph A.; Parsons, Michael; Chang, Tim C.; Acker, Jason P.; Kolios, Michael C.

2017-02-01

Red blood cells (RBCs) stored in hypothermic environments for the purpose of transfusion have been documented to undergo structural and functional changes over time. One sign of the so-called RBC storage lesion is irreversible damage to the cell membrane. Consequently, RBCs undergo a morphological transformation from regular, deformable biconcave discocytes to rigid spheroechinocytes. The spherically shaped RBCs lack the deformability to efficiently enter microvasculature, thereby reducing the capacity of RBCs to oxygenate tissue. Blood banks currently rely on microscope techniques that include fixing, staining and cell counting in order to morphologically characterize RBC samples; these methods are labor intensive and highly subjective. This study presents a novel, high-throughput RBC morphology characterization technique using image flow cytometry (IFC). An image segmentation template was developed to process 100,000 images acquired from the IFC system and output the relative spheroechinocyte percentage. The technique was applied on samples extracted from two blood bags to monitor the morphological changes of the RBCs during in vitro hypothermic storage. The study found that, for a given sample of RBCs, the IFC method was twice as fast in data acquisition, and analyzed 250-350 times more RBCs than the conventional method. Over the lifespan of the blood bags, the mean spheroechinocyte population increased by 37%. Future work will focus on expanding the template to segregate RBC images into more subpopulations for the validation of the IFC method against conventional techniques; the expanded template will aid in establishing quantitative links between spheroechinocyte increase and other RBC storage lesion characteristics.

Flow Cytometry, Microscopy and Hyperspectral Imaging of microcystis, Cyanobacteria, and Algae- SETAC

EPA Science Inventory

The detection of cyanobacteria algae, and picoplankton, in water is an important step in assessing water quality. Studies were initiated using fluorescence microscopy, flow cytometry and hyperspectral imaging with two fresh water species that were cultured in the laboratory:Micr...

CytometryML: a markup language for analytical cytology

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Stephanie H.; Leif, Suzanne B.

2003-06-01

Cytometry Markup Language, CytometryML, is a proposed new analytical cytology data standard. CytometryML is a set of XML schemas for encoding both flow cytometry and digital microscopy text based data types. CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. These schemas provide representations for the keywords in FCS 3.0 and will soon include DICOM microscopic image data. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. A preliminary version of a list mode binary data type, which does not presently exist in DICOM, has been designed. This binary type is required to enhance the storage and transmission of flow cytometry and digital microscopy data. Index files based on Waveform indices will be used to rapidly locate the cells present in individual subsets. DICOM has the advantage of employing standard file types, TIF and JPEG, for Digital Microscopy. Using an XML schema based representation means that standard commercial software packages such as Excel and MathCad can be used to analyze, display, and store analytical cytometry data. Furthermore, by providing one standard for both DICOM data and analytical cytology data, it eliminates the need to create and maintain special purpose interfaces for analytical cytology data thereby integrating the data into the larger DICOM and other clinical communities. A draft version of CytometryML is available at www.newportinstruments.com.

Biomarkers of Selenium Chemoprevention of Prostate Cancer

DTIC Science & Technology

2005-01-01

than Se-Met in inhibiting Flow Kit from BD Pharmigen (San Diego, CA). Stained cells were then quantified by flow cytometry , and the data were analyzed...decrease in Quantitation of Apoptosis by Flow Cytometry . PC-3 cells were plated at cell number accumulation by MSA was related to cell cycle arrest, we... flow exposed to either 5 or 10Mm MSA for 48 or 72 h. Adherent cells harvested by mild cytometry of ethanol-permeabilized cells stained with Pl. Synchro

Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds.

PubMed

Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H G; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

2016-06-02

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

Measurement of Lipid Accumulation in Chlorella vulgaris via Flow Cytometry and Liquid-State ¹H NMR Spectroscopy for Development of an NMR-Traceable Flow Cytometry Protocol

PubMed Central

Bono Jr., Michael S.; Garcia, Ravi D.; Sri-Jayantha, Dylan V.; Ahner, Beth A.; Kirby, Brian J.

2015-01-01

In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r 2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols. PMID:26267664

Measurement of lipid accumulation in Chlorella vulgaris via flow cytometry and liquid-state ¹H NMR spectroscopy for development of an NMR-traceable flow cytometry protocol.

PubMed

Bono, Michael S; Garcia, Ravi D; Sri-Jayantha, Dylan V; Ahner, Beth A; Kirby, Brian J

2015-01-01

In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.

BRAF and MEK inhibitor therapy eliminates nestin expressing melanoma cells in human tumors.

PubMed

Doxie, Deon B; Greenplate, Allison R; Gandelman, Jocelyn S; Diggins, Kirsten E; Roe, Caroline E; Dahlman, Kimberly B; Sosman, Jeffrey A; Kelley, Mark C; Irish, Jonathan M

2018-05-19

Little is known about the in vivo impacts of targeted therapy on melanoma cell abundance and protein expression. Here, 21 antibodies were added to an established melanoma mass cytometry panel to measure 32 cellular features, distinguish malignant cells, and characterize dabrafenib and trametinib responses in BRAF V 600mut melanoma. Tumor cells were biopsied before neoadjuvant therapy and compared to cells surgically resected from the same site after 4 weeks of therapy. Approximately 50,000 cells per tumor were characterized by mass cytometry and computational tools t-SNE/viSNE, FlowSOM, and MEM. The resulting single cell view of melanoma treatment response revealed initially heterogeneous melanoma tumors were consistently cleared of Nestin expressing melanoma cells. Melanoma cells subsets that persisted to week 4 were heterogeneous but expressed SOX2 or SOX10 proteins and specifically lacked surface expression of MHC I proteins by MEM analysis. Traditional histology imaging of tissue microarrays from the same tumors confirmed mass cytometry results, including persistence of NES- SOX10+ S100β+ melanoma cells. This quantitative single cell view of melanoma treatment response revealed protein features of malignant cells that are not eliminated by targeted therapy. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

2017-05-01

The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO 2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen-sufficient and nitrogen-deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae-based biofuel production. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Masks in Imaging Flow Cytometry

PubMed Central

Dominical, Venina; Samsel, Leigh; McCoy, J. Philip

2016-01-01

Data analysis in imaging flow cytometry incorporates elements of flow cytometry together with other aspects of morphological analysis of images. A crucial early step in this analysis is the creation of a mask to distinguish the portion of the image upon which further examination of specified features can be performed. Default masks are provided by the manufacturer of the imaging flow cytometer but additional custom masks can be created by the individual user for specific applications. Flawed or inaccurate masks can have a substantial negative impact on the overall analysis of a sample, thus great care must be taken to ensure the accuracy of masks. Here we discuss various types of masks and cite examples of their use. Furthermore we provide our insight for how to approach selecting and assessing the optimal mask for a specific analysis. PMID:27461256

Cell-free measurements of brightness of fluorescently labeled antibodies

PubMed Central

Zhou, Haiying; Tourkakis, George; Shi, Dennis; Kim, David M.; Zhang, Hairong; Du, Tommy; Eades, William C.; Berezin, Mikhail Y.

2017-01-01

Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures. PMID:28150730

Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

PubMed Central

Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

2008-01-01

Background Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Methods Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). Results We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. Conclusion The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes. PMID:18627634

Extended Field Laser Confocal Microscopy (EFLCM): combining automated Gigapixel image capture with in silico virtual microscopy.

PubMed

Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

2008-07-16

Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes.

B-ALL minimal residual disease flow cytometry: an application of a novel method for optimization of a single-tube model.

PubMed

Shaver, Aaron C; Greig, Bruce W; Mosse, Claudio A; Seegmiller, Adam C

2015-05-01

Optimizing a clinical flow cytometry panel can be a subjective process dependent on experience. We develop a quantitative method to make this process more rigorous and apply it to B lymphoblastic leukemia/lymphoma (B-ALL) minimal residual disease (MRD) testing. We retrospectively analyzed our existing three-tube, seven-color B-ALL MRD panel and used our novel method to develop an optimized one-tube, eight-color panel, which was tested prospectively. The optimized one-tube, eight-color panel resulted in greater efficiency of time and resources with no loss in diagnostic power. Constructing a flow cytometry panel using a rigorous, objective, quantitative method permits optimization and avoids problems of interdependence and redundancy in a large, multiantigen panel. Copyright© by the American Society for Clinical Pathology.

CytometryML: a data standard which has been designed to interface with other standards

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2007-02-01

Because of the differences in the requirements, needs, and past histories including existing standards of the creating organizations, a single encompassing cytology-pathology standard will not, in the near future, replace the multiple existing or under development standards. Except for DICOM and FCS, these standardization efforts are all based on XML. CytometryML is a collection of XML schemas, which are based on the Digital Imaging and Communications in Medicine (DICOM) and Flow Cytometry Standard (FCS) datatypes. The CytometryML schemas contain attributes that link them to the DICOM standard and FCS. Interoperability with DICOM has been facilitated by, wherever reasonable, limiting the difference between CytometryML and the previous standards to syntax. In order to permit the Resource Description Framework, RDF, to reference the CytometryML datatypes, id attributes have been added to many CytometryML elements. The Laboratory Digital Imaging Project (LDIP) Data Exchange Specification and the Flowcyt standards development effort employ RDF syntax. Documentation from DICOM has been reused in CytometryML. The unity of analytical cytology was demonstrated by deriving a microscope type and a flow cytometer type from a generic cytometry instrument type. The feasibility of incorporating the Flowcyt gating schemas into CytometryML has been demonstrated. CytometryML is being extended to include many of the new DICOM Working Group 26 datatypes, which describe patients, specimens, and analytes. In situations where multiple standards are being created, interoperability can be facilitated by employing datatypes based on a common set of semantics and building in links to standards that employ different syntax.

Cytological study of DNA content and nuclear morphometric analysis for aid in the diagnosis of high-grade dysplasia within oral leukoplakia.

PubMed

Yang, Xi; Xiao, Xuan; Wu, Wenyan; Shen, Xuemin; Zhou, Zengtong; Liu, Wei; Shi, Linjun

2017-09-01

To quantitatively examine the DNA content and nuclear morphometric status of oral leukoplakia (OL) and investigate its association with the degree of dysplasia in a cytologic study. Oral cytobrush biopsy was carried out to obtain exfoliative epithelial cells from lesions before scalpel biopsy at the same location in a blinded series of 70 patients with OL. Analysis of nuclear morphometry and DNA content status using image cytometry was performed with oral smears stained with the Feulgen-thionin method. Nuclear morphometric analysis revealed significant differences in DNA content amount, DNA index, nuclear area, nuclear radius, nuclear intensity, sphericity, entropy, and fractal dimension (all P < .01) between low-grade and high-grade dysplasia. DNA content analysis identified 34 patients with OL (48.6%) with DNA content abnormality. Nonhomogeneous lesion (P = .018) and high-grade dysplasia (P = .008) were significantly associated with abnormal DNA content. Importantly, the positive correlation between the degree of oral dysplasia and DNA content status was significant (P = .004, correlation coefficient = 0.342). Cytology analysis of DNA content and nuclear morphometric status using image cytometry may support their use as a screening and monitoring tool for OL progression. Copyright © 2017 Elsevier Inc. All rights reserved.

An automated quantitative DNA image cytometry system detects abnormal cells in cervical cytology with high sensitivity.

PubMed

Wong, O G; Ho, M W; Tsun, O K; Ng, A K; Tsui, E Y; Chow, J N; Ip, P P; Cheung, A N

2018-03-26

To evaluate the performance of an automated DNA-image-cytometry system as a tool to detect cervical carcinoma. Of 384 liquid-based cervical cytology samples with available biopsy follow-up were analyzed by both the Imager System and a high-risk HPV test (Cobas). The sensitivity and specificity of Imager System for detecting biopsy proven high-grade squamous intraepithelial lesion (HSIL, cervical intraepithelial neoplasia [CIN]2-3) and carcinoma were 89.58% and 56.25%, respectively, compared to 97.22% and 23.33% of HPV test but additional HPV 16/18 genotyping increased the specificity to 69.58%. The sensitivity and specificity of the Imager System for predicting HSIL+ (CIN2-3+) lesions among atypical squamous cells of undetermined significance samples were 80.00% and 70.53%, respectively, compared to 100% and 11.58% of HPV test whilst the HPV 16/18 genotyping increased the specificity to 77.89%. Among atypical squamous cells-cannot exclude HSIL, the sensitivity and specificity of Imager System for predicting HSIL+ (CIN2-3+) lesions upon follow up were 82.86% and 33.33%%, respectively, compared to 97.14% and 4.76% of HPV test and the HPV 16/18 genotyping increased the specificity to 19.05%. Among low-grade squamous intraepithelial lesion cases, the sensitivity and specificity of the Imager System for predicting HSIL+ (CIN2-3+) lesions were 66.67% and 35.71%%, respectively, compared to 66.67% and 29.76% of HPV test while HPV 16/18 genotyping increased the specificity to 79.76%. The overall results of imager and high-risk HPV test agreed in 69.43% (268) of all samples. The automated imager system and HPV 16/18 genotyping can enhance the specificity of detecting HSIL+ (CIN2-3+) lesions. © 2018 John Wiley & Sons Ltd.

Updating the maize karyotype by chromosome DNA sizing.

PubMed

Silva, Jéssica Coutinho; Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo

2018-01-01

The karyotype is a basic concept regarding the genome, fundamentally described by the number and morphological features of all chromosomes. Chromosome class, centromeric index, intra- and interchromosomal asymmetry index, and constriction localization are important in clinical, systematic and evolutionary approaches. In spite of the advances in karyotype characterization made over the last years, new data about the chromosomes can be generated from quantitative methods, such as image cytometry. Therefore, using Zea mays L., this study aimed to update the species' karyotype by supplementing information on chromosome DNA sizing. After adjustment of the procedures, chromosome morphometry and class as well as knob localization enabled describing the Z. mays karyotype. In addition, applying image cytometry, DNA sizing was unprecedentedly measured for the arms and satellite of all chromosomes. This way, unambiguous identification of the chromosome pairs, and hence the assembly of 51 karyograms, were only possible after the DNA sizing of each chromosome, their arms and satellite portions. These accurate, quantitative and reproducible data also enabled determining the distribution and variation of DNA content in each chromosome. From this, a correlation between DNA amount and total chromosome length evidenced that the mean DNA content of chromosome 9 was higher than that of chromosome 8. The chromosomal DNA sizing updated the Z. mays karyotype, providing insights into its dynamic genome with regards to the organization of the ten chromosomes and their respective portions. Considering the results and the relevance of cytogenetics in the current scenario of comparative sequencing and genomics, chromosomal DNA sizing should be incorporated as an additional parameter for karyotype definition. Based on this study, it can be affirmed that cytogenetic approaches go beyond the simple morphological description of chromosomes.

Updating the maize karyotype by chromosome DNA sizing

PubMed Central

2018-01-01

The karyotype is a basic concept regarding the genome, fundamentally described by the number and morphological features of all chromosomes. Chromosome class, centromeric index, intra- and interchromosomal asymmetry index, and constriction localization are important in clinical, systematic and evolutionary approaches. In spite of the advances in karyotype characterization made over the last years, new data about the chromosomes can be generated from quantitative methods, such as image cytometry. Therefore, using Zea mays L., this study aimed to update the species’ karyotype by supplementing information on chromosome DNA sizing. After adjustment of the procedures, chromosome morphometry and class as well as knob localization enabled describing the Z. mays karyotype. In addition, applying image cytometry, DNA sizing was unprecedentedly measured for the arms and satellite of all chromosomes. This way, unambiguous identification of the chromosome pairs, and hence the assembly of 51 karyograms, were only possible after the DNA sizing of each chromosome, their arms and satellite portions. These accurate, quantitative and reproducible data also enabled determining the distribution and variation of DNA content in each chromosome. From this, a correlation between DNA amount and total chromosome length evidenced that the mean DNA content of chromosome 9 was higher than that of chromosome 8. The chromosomal DNA sizing updated the Z. mays karyotype, providing insights into its dynamic genome with regards to the organization of the ten chromosomes and their respective portions. Considering the results and the relevance of cytogenetics in the current scenario of comparative sequencing and genomics, chromosomal DNA sizing should be incorporated as an additional parameter for karyotype definition. Based on this study, it can be affirmed that cytogenetic approaches go beyond the simple morphological description of chromosomes. PMID:29293613

Imaging cytometry in a plastic ultra-mobile system

NASA Astrophysics Data System (ADS)

Martínez Vázquez, R.; Trotta, G.; Paturzo, M.; Volpe, A.; Bernava, G.; Basile, V.; Ancona, A.; Ferraro, P.; Fassi, I.; Osellame, R.

2017-03-01

We present a cost-effective and highly-portable plastic prototype that can be interfaced with a cell phone to implement an optofluidic imaging cytometry platform. It is based on a PMMA microfluidic chip that fits inside an opto-mechanical platform fabricated by a 3D printer. The fluorescence excitation and imaging is performed using the LED and the CMOS from the cell phone increasing the compactness of the system. A custom developed application is used to analyze the images and provide a value of particle concentration.

Meta-analysis of two computer-assisted screening methods for diagnosing oral precancer and cancer.

PubMed

Ye, Xiaojing; Zhang, Jing; Tan, Yaqin; Chen, Guanying; Zhou, Gang

2015-11-01

The early diagnosis of oral precancer and cancer is crucial and could have the highest impact on improving survival rates. A meta-analysis was conducted to compare the accuracy between the OralCDx brush biopsy and DNA-image cytometry in diagnosing both conditions. Bibliographic databases were systematically searched for original relevant studies on the early diagnosis of oral precancer and oral cancer. Study characteristics were evaluated to determine the accuracy of the two screening strategies. Thirteen studies (eight of OralCDx brush biopsy and five of DNA-image cytometry) were identified as having reported on 1981 oral mucosa lesions. The meta-analysis found that the area under the summary receiver operating characteristic curves of the OralCDx brush biopsy and DNA-image cytometry were 0.8879 and 0.9885, respectively. The pooled sensitivity, specificity, and diagnostic odds ratio of the OralCDx brush biopsy were 86% (95% CI 81-90), 81% (95% CI 78-85), and 20.36 (95% CI 2.72-152.67), respectively, while these modalities of DNA-image cytometry were 89% (95% CI 83-94), 99% (95% CI 97-100), and 446.08 (95% CI 73.36-2712.43), respectively. Results of a pairwise comparison between each modality demonstrated that specificity, area under the curve (AUC), and Q(∗) index of DNA-image cytometry was significantly higher than that of the OralCDx brush biopsy (Z=2.821, p<0.05; Z=1.711, p<0.05; Z=1.727, p<0.05), but no significant difference in sensitivity was found (Z=1.520, p>0.05). In conclusion, the meta-analysis of the published studies indicated that DNA-image cytometry is more accurate than the OralCDx brush biopsy in diagnosing oral precancer and oral cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

CytometryML binary data standards

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2005-03-01

CytometryML is a proposed new Analytical Cytology (Cytomics) data standard, which is based on a common set of XML schemas for encoding flow cytometry and digital microscopy text based data types (metadata). CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. The separation of the large binary data objects (list mode and image data) from the XML description of the metadata permits the metadata to be directly displayed, analyzed, and reported with standard commercial software packages; the direct use of XML languages; and direct interfacing with clinical information systems. The separation of the binary data into its own files simplifies parsing because all extraneous header data has been eliminated. The storage of images as two-dimensional arrays without any extraneous data, such as in the Adobe Photoshop RAW format, facilitates the development by scientists of their own analysis and visualization software. Adobe Photoshop provided the display infrastructure and the translation facility to interconvert between the image data from commercial formats and RAW format. Similarly, the storage and parsing of list mode binary data type with a group of parameters that are specified at compilation time is straight forward. However when the user is permitted at run-time to select a subset of the parameters and/or specify results of mathematical manipulations, the development of special software was required. The use of CytometryML will permit investigators to be able to create their own interoperable data analysis software and to employ commercially available software to disseminate their data.

rFRET: A comprehensive, Matlab-based program for analyzing intensity-based ratiometric microscopic FRET experiments.

PubMed

Nagy, Peter; Szabó, Ágnes; Váradi, Tímea; Kovács, Tamás; Batta, Gyula; Szöllősi, János

2016-04-01

Fluorescence or Förster resonance energy transfer (FRET) remains one of the most widely used methods for assessing protein clustering and conformation. Although it is a method with solid physical foundations, many applications of FRET fall short of providing quantitative results due to inappropriate calibration and controls. This shortcoming is especially valid for microscopy where currently available tools have limited or no capability at all to display parameter distributions or to perform gating. Since users of multiparameter flow cytometry usually apply these tools, the absence of these features in applications developed for microscopic FRET analysis is a significant limitation. Therefore, we developed a graphical user interface-controlled Matlab application for the evaluation of ratiometric, intensity-based microscopic FRET measurements. The program can calculate all the necessary overspill and spectroscopic correction factors and the FRET efficiency and it displays the results on histograms and dot plots. Gating on plots and mask images can be used to limit the calculation to certain parts of the image. It is an important feature of the program that the calculated parameters can be determined by regression methods, maximum likelihood estimation (MLE) and from summed intensities in addition to pixel-by-pixel evaluation. The confidence interval of calculated parameters can be estimated using parameter simulations if the approximate average number of detected photons is known. The program is not only user-friendly, but it provides rich output, it gives the user freedom to choose from different calculation modes and it gives insight into the reliability and distribution of the calculated parameters. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Time-Domain Microfluidic Fluorescence Lifetime Flow Cytometry for High-Throughput Förster Resonance Energy Transfer Screening

PubMed Central

Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

2015-01-01

Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving fluorescence lifetime measurements, thereby providing statistically significant quantitative data for analysis of large cell populations. © 2014 International Society for Advancement of Cytometry PMID:25523156

Rise of the micromachines: microfluidics and the future of cytometry.

PubMed

Wlodkowic, Donald; Darzynkiewicz, Zbigniew

2011-01-01

The past decade has brought many innovations to the field of flow and image-based cytometry. These advancements can be seen in the current miniaturization trends and simplification of analytical components found in the conventional flow cytometers. On the other hand, the maturation of multispectral imaging cytometry in flow imaging and the slide-based laser scanning cytometers offers great hopes for improved data quality and throughput while proving new vistas for the multiparameter, real-time analysis of cells and tissues. Importantly, however, cytometry remains a viable and very dynamic field of modern engineering. Technological milestones and innovations made over the last couple of years are bringing the next generation of cytometers out of centralized core facilities while making it much more affordable and user friendly. In this context, the development of microfluidic, lab-on-a-chip (LOC) technologies is one of the most innovative and cost-effective approaches toward the advancement of cytometry. LOC devices promise new functionalities that can overcome current limitations while at the same time promise greatly reduced costs, increased sensitivity, and ultra high throughputs. We can expect that the current pace in the development of novel microfabricated cytometric systems will open up groundbreaking vistas for the field of cytometry, lead to the renaissance of cytometric techniques and most importantly greatly support the wider availability of these enabling bioanalytical technologies. Copyright © 2011 Elsevier Inc. All rights reserved.

Rise of the Micromachines: Microfluidics and the Future of Cytometry

PubMed Central

Wlodkowic, Donald; Darzynkiewicz, Zbigniew

2011-01-01

The past decade has brought many innovations to the field of flow and image-based cytometry. These advancements can be seen in the current miniaturization trends and simplification of analytical components found in the conventional flow cytometers. On the other hand, the maturation of multispectral imaging cytometry in flow imaging and the slide-based laser scanning cytometers offers great hopes for improved data quality and throughput while proving new vistas for the multiparameter, real-time analysis of cells and tissues. Importantly, however, cytometry remains a viable and very dynamic field of modern engineering. Technological milestones and innovations made over the last couple of years are bringing the next generation of cytometers out of centralized core facilities while making it much more affordable and user friendly. In this context, the development of microfluidic, lab-on-a-chip (LOC) technologies is one of the most innovative and cost-effective approaches toward the advancement of cytometry. LOC devices promise new functionalities that can overcome current limitations while at the same time promise greatly reduced costs, increased sensitivity, and ultra high throughputs. We can expect that the current pace in the development of novel microfabricated cytometric systems will open up groundbreaking vistas for the field of cytometry, lead to the renaissance of cytometric techniques and most importantly greatly support the wider availability of these enabling bioanalytical technologies. PMID:21704837

Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging.

PubMed

Zeng, Yan; Xu, Jin; Li, Dong; Li, Li; Wen, Zilong; Qu, Jianan Y

2012-07-01

We demonstrate a label-free in vivo flow cytometry in zebrafish blood vessels based on two-photon excited autofluorescence imaging. The major discovery in this work is the strong autofluorescence emission from the plasma in zebrafish blood. The plasma autofluorescence provides excellent contrast for visualizing blood vessels and counting blood cells. In addition, the cellular nicotinamide adenine dinucleotide autofluorescence enables in vivo imaging and counting of white blood cells (neutrophils).

Correlation of Cell Surface Biomarker Expression Levels with Adhesion Contact Angle Measured by Lateral Microscopy.

PubMed

Walz, Jenna A; Mace, Charles R

2018-06-05

Immunophenotyping is typically achieved using flow cytometry, but any influence a biomarker may have on adhesion or surface recognition cannot be determined concurrently. In this manuscript, we demonstrate the utility of lateral microscopy for correlating cell surface biomarker expression levels with quantitative descriptions of cell morphology. With our imaging system, we observed single cells from two T cell lines and two B cell lines adhere to antibody-coated substrates and quantified this adhesion using contact angle measurements. We found that SUP-T1 and CEM CD4+ cells, both of which express similar levels of CD4, experienced average changes in contact angle that were not statistically different from one another on surfaces coated in anti-CD4. However, MAVER-1 and BJAB K20 cells, both of which express different levels of CD20, underwent average changes in contact angle that were significantly different from one another on surfaces coated in anti-CD20. Our results indicate that changes in cell contact angles on antibody-coated substrates reflect the expression levels of corresponding antigens on the surfaces of cells as determined by flow cytometry. Our lateral microscopy approach offers a more reproducible and quantitative alternative to evaluate adhesion compared to commonly used wash assays and can be extended to many additional immunophenotyping applications to identify cells of interest within heterogeneous populations.

Objective detection of apoptosis in rat renal tissue sections using light microscopy and free image analysis software with subsequent machine learning: Detection of apoptosis in renal tissue.

PubMed

Macedo, Nayana Damiani; Buzin, Aline Rodrigues; de Araujo, Isabela Bastos Binotti Abreu; Nogueira, Breno Valentim; de Andrade, Tadeu Uggere; Endringer, Denise Coutinho; Lenz, Dominik

2017-02-01

The current study proposes an automated machine learning approach for the quantification of cells in cell death pathways according to DNA fragmentation. A total of 17 images of kidney histological slide samples from male Wistar rats were used. The slides were photographed using an Axio Zeiss Vert.A1 microscope with a 40x objective lens coupled with an Axio Cam MRC Zeiss camera and Zen 2012 software. The images were analyzed using CellProfiler (version 2.1.1) and CellProfiler Analyst open-source software. Out of the 10,378 objects, 4970 (47,9%) were identified as TUNEL positive, and 5408 (52,1%) were identified as TUNEL negative. On average, the sensitivity and specificity values of the machine learning approach were 0.80 and 0.77, respectively. Image cytometry provides a quantitative analytical alternative to the more traditional qualitative methods more commonly used in studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantification of green fluorescent protein by in vivo imaging, PCR, and flow cytometry: comparison of transgenic strains and relevance for fetal cell microchimerism.

PubMed

Fujiki, Yutaka; Tao, Kai; Bianchi, Diana W; Giel-Moloney, Maryann; Leiter, Andrew B; Johnson, Kirby L

2008-02-01

Animal models are increasingly being used for the assessment of fetal cell microchimerism in maternal tissue. We wished to determine the optimal transgenic mouse strain and analytic technique to facilitate the detection of rare transgenic microchimeric fetal cells amongst a large number of maternal wild-type cells. We evaluated two strains of mice transgenic for the enhanced green fluorescent protein (EGFP): a commercially available, commonly used strain (C57BL/6-Tg(ACTB-EGFP)10sb/J) (CAG) and a newly created strain (ROSA26-EGFP) using three different techniques: in vivo and ex vivo fluorescent imaging (for whole body and dissected organs, respectively), PCR amplification of gfp, and flow cytometry (FCM). By fluorescent imaging, organs from CAG mice were 10-fold brighter than organs from ROSA26-EGFP mice (P < 0.0001). By PCR, more transgene from CAG mice was detected compared to ROSA26-EGFP mice (P = 0.04). By FCM, ROSA26-EGFP cell fluorescence was more uniform than CAG cells. A greater proportion of cells from ROSA26-EGFP organs were positive for EGFP than cells from CAG organs, but CAG mice had a greater proportion of cells with the brightest fluorescent intensity. Each transgenic strain possesses characteristics that make it useful under specific experimental circumstances. The CAG mouse model is preferable when experiments require brighter cells, whereas ROSA26-EGFP is more appropriate when uniform or ubiquitous expression is more important than brightness. Investigators must carefully select the transgenic strain most suited to the experimental design to obtain the most consistent and reproducible data. In vivo imaging allows for phenotypic evaluation of whole animals and intact organs; however, we did not evaluate its utility for the detection of rare, fetal microchimeric cells in the maternal organs. Finally, while PCR amplification of a paternally inherited transgene does allow for the quantitative determination of rare microchimeric cells, FCM allows for both quantitative and qualitative evaluations of fetal cells at very high sensitivity in a plethora of maternal organs. (c) 2008 International Society for Analytical Cytology

The use of flow cytometry to monitor chitin synthesis in regenerating protoplasts of Candida albicans.

PubMed

Hector, R F; Braun, P C; Hart, J T; Kamarck, M E

1990-01-01

Flow cytometry was used to monitor chitin synthesis in regenerating protoplasts of the yeast Candida albicans. Comparisons of cells stained with Calcofluor White, a fluorochrome with known affinity for chitin, and cells incubated in the presence of N-[3H]-acetylglucosamine, the precursor substrate for chitin, showed a linear relationship between fluorescence and incorporation of label over time. Changes in both the fluorescence and light scatter of regenerating protoplasts treated with inhibitors of fungal chitin synthase were also quantitated by flow cytometry.

Characterization of neutrophils and macrophages from ex vivo cultured murine bone marrow for morphologic maturation and functional responses by imaging flow cytometry

PubMed Central

Pelletier, Margery G. H.; Szymczak, Klaudia; Barbeau, Anna M.; Prata, Gianna N.; O’Fallon, Kevin S.; Gaines, Peter

2016-01-01

Neutrophils and macrophages differentiate from common myeloid progenitors in the bone marrow, where they undergo nuclear morphologic changes during maturation. During this process, both cell types acquire critical innate immune functions that include phagocytosis of pathogens, and for neutrophils the release of nuclear material called nuclear extracellular traps (NETs). Primary cells used to study these functions are typically purified from mature mouse tissues, but bone marrow-derived ex vivo cultures provide more abundant numbers of progenitors and functionally mature cells. Routine analyses of these cells use conventional microscopy and flow cytometry, which present limitations; microscopy is laborious and subjective, whereas flow cytometry lacks spatial resolution. Here we describe methods to generate enriched populations of neutrophils or macrophages from cryopreserved mouse bone marrow cultured ex vivo, and to use imaging flow cytometry that combines the resolution of microscopy with flow cytometry to analyze cells for morphologic features, phagocytosis, and NETosis. PMID:27663441

Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

PubMed Central

Tak For Yu, Zeta; Guan, Huijiao; Ki Cheung, Mei; McHugh, Walker M.; Cornell, Timothy T.; Shanley, Thomas P.; Kurabayashi, Katsuo; Fu, Jianping

2015-01-01

Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology (‘AlphaLISA’) in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45 min with a limit of detection down to 10 pg mL−1. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications. PMID:26074253

Mathematic modeling the relationship of bacteria number in a dairy product and the color difference measured by a CCD image sensor

NASA Astrophysics Data System (ADS)

Zhou, Zhen; Zhao, Zhigang; Chen, Dongkui; Liu, Yuping

2005-01-01

Although many methods, such as bacteria plate count, flow cytometry and impedance method have been broadly used in the dairy industry to quantitate bacteria numbers around the world, none of them is a quick, low cost and easy one. In this study, we proposed to apply the color difference theory in this field to establish a mathematic model to quantitate bacteria number in fresh milk. Preliminary testing results not only indicate that the application of the color difference theory to the new system is practical, but also confirm the theoretical relationship between the numbers of bacteria, incubation time and color difference. The proof of the principal study in this article further suggests that the novel method has the potential to replace the traditional methods to determine bacteria numbers for the food industry.

Dynamic measurements of flowing cells labeled by gold nanoparticles using full-field photothermal interferometric imaging

NASA Astrophysics Data System (ADS)

Turko, Nir A.; Roitshtain, Darina; Blum, Omry; Kemper, Björn; Shaked, Natan T.

2017-06-01

We present highly dynamic photothermal interferometric phase microscopy for quantitative, selective contrast imaging of live cells during flow. Gold nanoparticles can be biofunctionalized to bind to specific cells, and stimulated for local temperature increase due to plasmon resonance, causing a rapid change of the optical phase. These phase changes can be recorded by interferometric phase microscopy and analyzed to form an image of the binding sites of the nanoparticles in the cells, gaining molecular specificity. Since the nanoparticle excitation frequency might overlap with the sample dynamics frequencies, photothermal phase imaging was performed on stationary or slowly dynamic samples. Furthermore, the computational analysis of the photothermal signals is time consuming. This makes photothermal imaging unsuitable for applications requiring dynamic imaging or real-time analysis, such as analyzing and sorting cells during fast flow. To overcome these drawbacks, we utilized an external interferometric module and developed new algorithms, based on discrete Fourier transform variants, enabling fast analysis of photothermal signals in highly dynamic live cells. Due to the self-interference module, the cells are imaged with and without excitation in video-rate, effectively increasing signal-to-noise ratio. Our approach holds potential for using photothermal cell imaging and depletion in flow cytometry.

Cytometry metadata in XML

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Stephanie H.

2016-04-01

Introduction: The International Society for Advancement of Cytometry (ISAC) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). CytometryML will serve as a common metadata standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). The datatypes are primarily based on the Flow Cytometry and the Digital Imaging and Communication (DICOM) standards. A small section of the code was formatted with standard HTML formatting elements (p, h1, h2, etc.). Results:1) The part of MIFlowCyt that describes the Experimental Overview including the specimen and substantial parts of several other major elements has been implemented as CytometryML XML schemas (www.cytometryml.org). 2) The feasibility of using MIFlowCyt to provide the combination of an overview, table of contents, and/or an index of a scientific paper or a report has been demonstrated. Previously, a sample electronic publication, EPUB, was created that could contain both MIFlowCyt metadata as well as the binary data. Conclusions: The use of CytometryML technology together with XHTML5 and CSS permits the metadata to be directly formatted and together with the binary data to be stored in an EPUB container. This will facilitate: formatting, data- mining, presentation, data verification, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate a publication's adherence to the MIFlowCyt standard, promote interoperability and should also result in the textual and numeric data being published using web technology without any change in composition.

Multi-channel imaging cytometry with a single detector

NASA Astrophysics Data System (ADS)

Locknar, Sarah; Barton, John; Entwistle, Mark; Carver, Gary; Johnson, Robert

2018-02-01

Multi-channel microscopy and multi-channel flow cytometry generate high bit data streams. Multiple channels (both spectral and spatial) are important in diagnosing diseased tissue and identifying individual cells. Omega Optical has developed techniques for mapping multiple channels into the time domain for detection by a single high gain, high bandwidth detector. This approach is based on pulsed laser excitation and a serial array of optical fibers coated with spectral reflectors such that up to 15 wavelength bins are sequentially detected by a single-element detector within 2.5 μs. Our multichannel microscopy system uses firmware running on dedicated DSP and FPGA chips to synchronize the laser, scanning mirrors, and sampling clock. The signals are digitized by an NI board into 14 bits at 60MHz - allowing for 232 by 174 pixel fields in up to 15 channels with 10x over sampling. Our multi-channel imaging cytometry design adds channels for forward scattering and back scattering to the fluorescence spectral channels. All channels are detected within the 2.5 μs - which is compatible with fast cytometry. Going forward, we plan to digitize at 16 bits with an A-toD chip attached to a custom board. Processing these digital signals in custom firmware would allow an on-board graphics processing unit to display imaging flow cytometry data over configurable scanning line lengths. The scatter channels can be used to trigger data buffering when a cell is present in the beam. This approach enables a low cost mechanically robust imaging cytometer.

PET Imaging of Tumor-Associated Macrophages with 89Zr-Labeled High-Density Lipoprotein Nanoparticles

PubMed Central

Pérez-Medina, Carlos; Tang, Jun; Abdel-Atti, Dalya; Hogstad, Brandon; Merad, Miriam; Fisher, Edward A.; Fayad, Zahi A.; Lewis, Jason S.; Mulder, Willem J.M.; Reiner, Thomas

2015-01-01

Tumor-associated macrophages (TAMs) are increasingly investigated in cancer immunology and are considered a promising target for better and tailored treatment of malignant growth. Although TAMs also have high diagnostic and prognostic value, TAM imaging still remains largely unexplored. Here, we describe the development of reconstituted high-density lipoprotein (rHDL)–facilitated TAM PET imaging in a breast cancer model. Methods Radiolabeled rHDL nanoparticles incorporating the long-lived positron-emitting nuclide 89Zr were developed using 2 different approaches. The nanoparticles were composed of phospholipids and apolipoprotein A-I (apoA-I) in a 2.5:1 weight ratio. 89Zr was complexed with deferoxamine (also known as desferrioxamine B, desferoxamine B), conjugated either to a phospholipid or to apoA-I to generate 89Zr-PL-HDL and 89Zr-AI-HDL, respectively. In vivo evaluation was performed in an orthotopic mouse model of breast cancer and included pharmacokinetic analysis, biodistribution studies, and PET imaging. Ex vivo histologic analysis of tumor tissues to assess regional distribution of 89Zr radioactivity was also performed. Fluorescent analogs of the radiolabeled agents were used to determine cell-targeting specificity using flow cytometry. Results The phospholipid- and apoA-I–labeled rHDL were produced at 79% ± 13% (n = 6) and 94% ± 6% (n = 6) radiochemical yield, respectively, with excellent radiochemical purity (>99%). Intravenous administration of both probes resulted in high tumor radioactivity accumulation (16.5 ± 2.8 and 8.6 ± 1.3 percentage injected dose per gram for apoA-I– and phospholipid-labeled rHDL, respectively) at 24 h after injection. Histologic analysis showed good colocalization of radioactivity with TAM-rich areas in tumor sections. Flow cytometry revealed high specificity of rHDL for TAMs, which had the highest uptake per cell (6.8-fold higher than tumor cells for both DiO@Zr-PL-HDL and DiO@Zr-AI-HDL) and accounted for 40.7% and 39.5% of the total cellular DiO@Zr-PL-HDL and DiO@Zr-AI-HDL in tumors, respectively. Conclusion We have developed 89Zr-labeled TAM imaging agents based on the natural nanoparticle rHDL. In an orthotopic mouse model of breast cancer, we have demonstrated their specificity for macrophages, a result that was corroborated by flow cytometry. Quantitative macrophage PET imaging with our 89Zr-rHDL imaging agents could be valuable for noninvasive monitoring of TAM immunology and targeted treatment. PMID:26112022

Technical Advance: Live-imaging analysis of human dendritic cell migrating behavior under the influence of immune-stimulating reagents in an organotypic model of lung

PubMed Central

Nguyen Hoang, Anh Thu; Chen, Puran; Björnfot, Sofia; Högstrand, Kari; Lock, John G.; Grandien, Alf; Coles, Mark; Svensson, Mattias

2014-01-01

This manuscript describes technical advances allowing manipulation and quantitative analyses of human DC migratory behavior in lung epithelial tissue. DCs are hematopoietic cells essential for the maintenance of tissue homeostasis and the induction of tissue-specific immune responses. Important functions include cytokine production and migration in response to infection for the induction of proper immune responses. To design appropriate strategies to exploit human DC functional properties in lung tissue for the purpose of clinical evaluation, e.g., candidate vaccination and immunotherapy strategies, we have developed a live-imaging assay based on our previously described organotypic model of the human lung. This assay allows provocations and subsequent quantitative investigations of DC functional properties under conditions mimicking morphological and functional features of the in vivo parental tissue. We present protocols to set up and prepare tissue models for 4D (x, y, z, time) fluorescence-imaging analysis that allow spatial and temporal studies of human DCs in live epithelial tissue, followed by flow cytometry analysis of DCs retrieved from digested tissue models. This model system can be useful for elucidating incompletely defined pathways controlling DC functional responses to infection and inflammation in lung epithelial tissue, as well as the efficacy of locally administered candidate interventions. PMID:24899587

Label-free counting of circulating cells by in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Zhou, Quanyu; Yang, Ping; Wang, Qiyan; Pang, Kai; Zhou, Hui; He, Hao; Wei, Xunbin

2018-02-01

Melanoma, developing from melanocytes, is the most serious type of skin cancer. Circulating melanoma cells, the prognosis marker for metastasis, are present in the circulation at the early stage. Thus, quantitative detection of rare circulating melanoma cells is essential for monitoring tumor metastasis and prognosis evaluation. Compared with in vitro assays, in vivo flow cytometry is able to identify circulating tumor cells without drawing blood. Here, we built in vivo photoacoustic flow cytometry based on the high absorption coefficient of melanoma cells, which is applied to labelfree counting of circulating melanoma cells in tumor-bearing mice.

Image-based quantification and mathematical modeling of spatial heterogeneity in ESC colonies.

PubMed

Herberg, Maria; Zerjatke, Thomas; de Back, Walter; Glauche, Ingmar; Roeder, Ingo

2015-06-01

Pluripotent embryonic stem cells (ESCs) have the potential to differentiate into cells of all three germ layers. This unique property has been extensively studied on the intracellular, transcriptional level. However, ESCs typically form clusters of cells with distinct size and shape, and establish spatial structures that are vital for the maintenance of pluripotency. Even though it is recognized that the cells' arrangement and local interactions play a role in fate decision processes, the relations between transcriptional and spatial patterns have not yet been studied. We present a systems biology approach which combines live-cell imaging, quantitative image analysis, and multiscale, mathematical modeling of ESC growth. In particular, we develop quantitative measures of the morphology and of the spatial clustering of ESCs with different expression levels and apply them to images of both in vitro and in silico cultures. Using the same measures, we are able to compare model scenarios with different assumptions on cell-cell adhesions and intercellular feedback mechanisms directly with experimental data. Applying our methodology to microscopy images of cultured ESCs, we demonstrate that the emerging colonies are highly variable regarding both morphological and spatial fluorescence patterns. Moreover, we can show that most ESC colonies contain only one cluster of cells with high self-renewing capacity. These cells are preferentially located in the interior of a colony structure. The integrated approach combining image analysis with mathematical modeling allows us to reveal potential transcription factor related cellular and intercellular mechanisms behind the emergence of observed patterns that cannot be derived from images directly. © 2015 International Society for Advancement of Cytometry.

Relationship between DNA damage response, initiated by camptothecin or oxidative stress, and DNA replication, analyzed by quantitative 3D image analysis.

PubMed

Berniak, K; Rybak, P; Bernas, T; Zarębski, M; Biela, E; Zhao, H; Darzynkiewicz, Z; Dobrucki, J W

2013-10-01

A method of quantitative analysis of spatial (3D) relationship between discrete nuclear events detected by confocal microscopy is described and applied in analysis of a dependence between sites of DNA damage signaling (γH2AX foci) and DNA replication (EdU incorporation) in cells subjected to treatments with camptothecin (Cpt) or hydrogen peroxide (H2O2). Cpt induces γH2AX foci, likely reporting formation of DNA double-strand breaks (DSBs), almost exclusively at sites of DNA replication. This finding is consistent with the known mechanism of induction of DSBs by DNA topoisomerase I (topo1) inhibitors at the sites of collisions of the moving replication forks with topo1-DNA "cleavable complexes" stabilized by Cpt. Whereas an increased level of H2AX histone phosphorylation is seen in S-phase of cells subjected to H2O2, only a minor proportion of γH2AX foci coincide with DNA replication sites. Thus, the increased level of H2AX phosphorylation induced by H2O2 is not a direct consequence of formation of DNA lesions at the sites of moving DNA replication forks. These data suggest that oxidative stress induced by H2O2 and formation of the primary H2O2-induced lesions (8-oxo-7,8-dihydroguanosine) inhibits replication globally and triggers formation of γH2AX at various distances from replication forks. Quantitative analysis of a frequency of DNA replication sites and γH2AX foci suggests also that stalling of replicating forks by Cpt leads to activation of new DNA replication origins. © 2013 International Society for Advancement of Cytometry. Copyright © 2013 International Society for Advancement of Cytometry.

DNA Content Differences Between Male and Female Chicken (Gallus gallus domesticus) Nuclei and Z and W Chromosomes Resolved by Image Cytometry

PubMed Central

Mendonça, Maria Andréia Corrêa; Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo

2010-01-01

Chicken red blood cells (CRBCs) are widely used as standards for DNA content determination. Cytogenetic data have shown that the Z sex chromosome is approximately twice as large as the W, so that the DNA content differs to some extent between male (ZZ) and female (ZW) chickens. Despite this fact, male and female CRBCs have been indiscriminately used in absolute genome size determination. Our work was conducted to verify whether the DNA content differences between male and female Gallus gallus domesticus “Leghorn” nuclei and ZZ/ZW chromosomes can be resolved by image cytometry (ICM). Air-dried smears stained by Feulgen reaction were used for nuclei analysis. Chicken metaphase spreads upon Feulgen staining were analyzed for obtaining quantitative information on the Z and W chromosomes. Before each capture session, we conducted quality control of the ICM instrumentation. Our results from nuclear measurements showed that the 2C value is 0.09 pg higher in males than in females. In chromosomes, we found that the Z chromosome shows 200% more DNA content than does the W chromosome. ICM demonstrated resolution power to discriminate low DNA content differences in genomes. We suggest prudence in the general use of CRBC 2C values as standards in comparative cytometric analysis. (J Histochem Cytochem 58:229–235, 2010) PMID:19875846

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

PubMed Central

Zhu, Hongying; Ozcan, Aydogan

2013-01-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 μm over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water. PMID:23603893

Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2013-04-11

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. - 10 μm over a very large field-of-view of - 81 mm(2). This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

Imaging Flow Cytometry Analysis to Identify Differences of Survival Motor Neuron Protein Expression in Patients With Spinal Muscular Atrophy.

PubMed

Arakawa, Reiko; Arakawa, Masayuki; Kaneko, Kaori; Otsuki, Noriko; Aoki, Ryoko; Saito, Kayoko

2016-08-01

Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.

Statistical performance of image cytometry for DNA, lipids, cytokeratin, & CD45 in a model system for circulation tumor cell detection.

PubMed

Futia, Gregory L; Schlaepfer, Isabel R; Qamar, Lubna; Behbakht, Kian; Gibson, Emily A

2017-07-01

Detection of circulating tumor cells (CTCs) in a blood sample is limited by the sensitivity and specificity of the biomarker panel used to identify CTCs over other blood cells. In this work, we present Bayesian theory that shows how test sensitivity and specificity set the rarity of cell that a test can detect. We perform our calculation of sensitivity and specificity on our image cytometry biomarker panel by testing on pure disease positive (D + ) populations (MCF7 cells) and pure disease negative populations (D - ) (leukocytes). In this system, we performed multi-channel confocal fluorescence microscopy to image biomarkers of DNA, lipids, CD45, and Cytokeratin. Using custom software, we segmented our confocal images into regions of interest consisting of individual cells and computed the image metrics of total signal, second spatial moment, spatial frequency second moment, and the product of the spatial-spatial frequency moments. We present our analysis of these 16 features. The best performing of the 16 features produced an average separation of three standard deviations between D + and D - and an average detectable rarity of ∼1 in 200. We performed multivariable regression and feature selection to combine multiple features for increased performance and showed an average separation of seven standard deviations between the D + and D - populations making our average detectable rarity of ∼1 in 480. Histograms and receiver operating characteristics (ROC) curves for these features and regressions are presented. We conclude that simple regression analysis holds promise to further improve the separation of rare cells in cytometry applications. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Multiplexed and Microparticle-based Analyses: Quantitative Tools for the Large-Scale Analysis of Biological Systems

PubMed Central

Nolan, John P.; Mandy, Francis

2008-01-01

While the term flow cytometry refers to the measurement of cells, the approach of making sensitive multiparameter optical measurements in a flowing sample stream is a very general analytical approach. The past few years have seen an explosion in the application of flow cytometry technology for molecular analysis and measurements using micro-particles as solid supports. While microsphere-based molecular analyses using flow cytometry date back three decades, the need for highly parallel quantitative molecular measurements that has arisen from various genomic and proteomic advances has driven the development in particle encoding technology to enable highly multiplexed assays. Multiplexed particle-based immunoassays are now common place, and new assays to study genes, protein function, and molecular assembly. Numerous efforts are underway to extend the multiplexing capabilities of microparticle-based assays through new approaches to particle encoding and analyte reporting. The impact of these developments will be seen in the basic research and clinical laboratories, as well as in drug development. PMID:16604537

CytometryML, an XML format based on DICOM and FCS for analytical cytology data.

PubMed

Leif, Robert C; Leif, Suzanne B; Leif, Stephanie H

2003-07-01

Flow Cytometry Standard (FCS) was initially created to standardize the software researchers use to analyze, transmit, and store data produced by flow cytometers and sorters. Because of the clinical utility of flow cytometry, it is necessary to have a standard consistent with the requirements of medical regulatory agencies. We extended the existing mapping of FCS to the Digital Imaging and Communications in Medicine (DICOM) standard to include list-mode data produced by flow cytometry, laser scanning cytometry, and microscopic image cytometry. FCS list-mode was mapped to the DICOM Waveform Information Object. We created a collection of Extensible Markup Language (XML) schemas to express the DICOM analytical cytologic text-based data types except for large binary objects. We also developed a cytometry markup language, CytometryML, in an open environment subject to continuous peer review. The feasibility of expressing the data contained in FCS, including list-mode in DICOM, was demonstrated; and a preliminary mapping for list-mode data in the form of XML schemas and documents was completed. DICOM permitted the creation of indices that can be used to rapidly locate in a list-mode file the cells that are members of a subset. DICOM and its coding schemes for other medical standards can be represented by XML schemas, which can be combined with other relevant XML applications, such as Mathematical Markup Language (MathML). The use of XML format based on DICOM for analytical cytology met most of the previously specified requirements and appears capable of meeting the others; therefore, the present FCS should be retired and replaced by an open, XML-based, standard CytometryML. Copyright 2003 Wiley-Liss, Inc.

Quantitative multiplex immunohistochemistry reveals myeloid-inflamed tumor-immune complexity associated with poor prognosis

PubMed Central

Tsujikawa, Takahiro; Kumar, Sushil; Borkar, Rohan N.; Azimi, Vahid; Thibault, Guillaume; Chang, Young Hwan; Balter, Ariel; Kawashima, Rie; Choe, Gina; Sauer, David; El Rassi, Edward; Clayburgh, Daniel R.; Kulesz-Martin, Molly F.; Lutz, Eric R.; Zheng, Lei; Jaffee, Elizabeth M.; Leyshock, Patrick; Margolin, Adam A.; Mori, Motomi; Gray, Joe W.; Flint, Paul W.; Coussens, Lisa M.

2017-01-01

SUMMARY Here we describe a multiplexed immunohistochemical platform, with computational image processing workflows including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas, and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination, and revealed that response to therapy correlated with degree of mono-myelocytic cell density, and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification, and provide digital image processing pipelines (https://github.com/multiplexIHC/cppipe) to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to thus improve biomarker discovery and assessment. PMID:28380359

Optofluidic Fluorescent Imaging Cytometry on a Cell Phone

PubMed Central

Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F.; Yaglidere, Oguzhan; Ozcan, Aydogan

2012-01-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in remote and resource-poor settings. PMID:21774454

Optofluidic fluorescent imaging cytometry on a cell phone.

PubMed

Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F; Yaglidere, Oguzhan; Ozcan, Aydogan

2011-09-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in remote and resource-poor settings.

Label-free monitoring of cell death induced by oxidative stress in living human cells using terahertz ATR spectroscopy

PubMed Central

Zou, Yi; Liu, Qiao; Yang, Xia; Huang, Hua-Chuan; Li, Jiang; Du, Liang-Hui; Li, Ze-Ren; Zhao, Jian-Heng; Zhu, Li-Guo

2017-01-01

We demonstrated that attenuated total reflectance terahertz time-domain spectroscopy (ATR THz-TDS) is able to monitor oxidative stress response of living human cells, which is proven in this work that it is an efficient non-invasive, label-free, real-time and in situ monitoring of cell death. Furthermore, the dielectric constant and dielectric loss of cultured living human breast epithelial cells, and along with their evolution under oxidative stress response induced by high concentration of H2O2, were quantitatively determined in the work. Our observation and results were finally confirmed using standard fluorescence-labeled flow cytometry measurements and visible fluorescence imaging. PMID:29359084

Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods.

PubMed

Øbro, Nina F; Ryder, Lars P; Madsen, Hans O; Andersen, Mette K; Lausen, Birgitte; Hasle, Henrik; Schmiegelow, Kjeld; Marquart, Hanne V

2012-01-01

Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring clone-specific genomic markers (53 follow-up bone marrow samples from 28 children with B-cell precursor acute lymphoblastic leukemia). Cell populations (presumed leukemic and non-leukemic) were flow-sorted during standard flow cytometry-based minimal residual disease monitoring and explored by PCR and/or fluorescence in situ hybridization. We found good concordance between flow cytometry and genomic analyses in the individual flow-sorted leukemic (93% true positive) and normal (93% true negative) cell populations. Four cases with discrepant results had plausible explanations (e.g. partly informative immunophenotype and antigen modulation) that highlight important methodological pitfalls. These findings demonstrate that with sufficient experience, flow cytometry is reliable for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia, although rare cases require supplementary PCR-based monitoring.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.

PubMed

Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

2010-08-17

We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

PubMed Central

Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

2010-01-01

We propose a unique method for cell sorting, “Ephesia,” using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples—blood, pleural effusion, and fine needle aspirates— issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost. PMID:20679245

An improved method for differentiating cell-bound from internalized particles by imaging flow cytometry.

PubMed

Smirnov, Asya; Solga, Michael D; Lannigan, Joanne; Criss, Alison K

2015-08-01

Recognition, binding, internalization, and elimination of pathogens and cell debris are important functions of professional as well as non-professional phagocytes. However, high-throughput methods for quantifying cell-associated particles and discriminating bound from internalized particles have been lacking. Here we describe a protocol for using imaging flow cytometry to quantify the attached and phagocytosed particles that are associated with a population of cells. Cells were exposed to fluorescent particles, fixed, and exposed to an antibody of a different fluorophore that recognizes the particles. The antibody is added without cell permeabilization, such that the antibody only binds extracellular particles. Cells with and without associated particles were identified by imaging flow cytometry. For each cell with associated particles, a spot count algorithm was employed to quantify the number of extracellular (double fluorescent) and intracellular (single fluorescent) particles per cell, from which the percent particle internalization was determined. The spot count algorithm was empirically validated by examining the fluorescence and phase contrast images acquired by the flow cytometer. We used this protocol to measure binding and internalization of the bacterium Neisseria gonorrhoeae by primary human neutrophils, using different bacterial variants and under different cellular conditions. The results acquired using imaging flow cytometry agreed with findings that were previously obtained using conventional immunofluorescence microscopy. This protocol provides a rapid, powerful method for measuring the association and internalization of any particle by any cell type. Copyright © 2015 Elsevier B.V. All rights reserved.

Opto-fluidics based microscopy and flow cytometry on a cell phone for blood analysis.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2015-01-01

Blood analysis is one of the most important clinical tests for medical diagnosis. Flow cytometry and optical microscopy are widely used techniques to perform blood analysis and therefore cost-effective translation of these technologies to resource limited settings is critical for various global health as well as telemedicine applications. In this chapter, we review our recent progress on the integration of imaging flow cytometry and fluorescent microscopy on a cell phone using compact, light-weight and cost-effective opto-fluidic attachments integrated onto the camera module of a smartphone. In our cell-phone based opto-fluidic imaging cytometry design, fluorescently labeled cells are delivered into the imaging area using a disposable micro-fluidic chip that is positioned above the existing camera unit of the cell phone. Battery powered light-emitting diodes (LEDs) are butt-coupled to the sides of this micro-fluidic chip without any lenses, which effectively acts as a multimode slab waveguide, where the excitation light is guided to excite the fluorescent targets within the micro-fluidic chip. Since the excitation light propagates perpendicular to the detection path, an inexpensive plastic absorption filter is able to reject most of the scattered light and create a decent dark-field background for fluorescent imaging. With this excitation geometry, the cell-phone camera can record fluorescent movies of the particles/cells as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the solution under test. With a similar opto-fluidic design, we have recently demonstrated imaging and automated counting of stationary blood cells (e.g., labeled white blood cells or unlabeled red blood cells) loaded within a disposable cell counting chamber. We tested the performance of this cell-phone based imaging cytometry and blood analysis platform by measuring the density of red and white blood cells as well as hemoglobin concentration in human blood samples, which showed a good match to our measurement results obtained using a commercially available hematology analyzer. Such a cell-phone enabled opto-fluidics microscopy, flow cytometry, and blood analysis platform could be especially useful for various telemedicine applications in remote and resource-limited settings.

Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error.

PubMed

Chan, Leo Li-Ying; Laverty, Daniel J; Smith, Tim; Nejad, Parham; Hei, Hillary; Gandhi, Roopali; Kuksin, Dmitry; Qiu, Jean

2013-02-28

Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. The viability and concentration of isolated PBMCs are traditionally measured by manual counting with trypan blue (TB) using a hemacytometer. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). The Cellometer image cytometry system has demonstrated the capability of automated concentration and viability detection in disposable counting chambers of unpurified mouse splenocytes and PBMCs stained with acridine orange (AO) and propidium iodide (PI) under fluorescence detection. In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods: (1) manual counting of trypan blue-stained PBMCs in hemacytometer, (2) manual counting of PBMCs in bright-field images, (3) manual counting of acetic acid lysing of RBCs with TB-stained PBMCs, (4) automated counting of acetic acid lysing of RBCs with PI-stained PBMCs, and (5) AO/PI dual staining method. The results show comparable total PBMC counting among all five methods, which validate the AO/PI staining method for PBMC measurement in the image cytometry method. Copyright © 2012 Elsevier B.V. All rights reserved.

Slow-Adhering Stem Cells Derived from Injured Skeletal Muscle Have Improved Regenerative Capacity

DTIC Science & Technology

2011-08-01

images. Flow Cytometry Assay of Stem Cell Markers SASCs (1 105) isolated from noninjured or injured muscle were collected and washed twice with...muscle. Results of flow cytometry further verified Sca-1 and CD34 expression in isolated SASCs, and a greater percentage of cells were positive for Sca-1...from both injured and control noninjured muscle were analyzed using flow cytometry for the immunofluorescent signal of Sca-1 and CD34. Results

Technical advance: live-imaging analysis of human dendritic cell migrating behavior under the influence of immune-stimulating reagents in an organotypic model of lung.

PubMed

Nguyen Hoang, Anh Thu; Chen, Puran; Björnfot, Sofia; Högstrand, Kari; Lock, John G; Grandien, Alf; Coles, Mark; Svensson, Mattias

2014-09-01

This manuscript describes technical advances allowing manipulation and quantitative analyses of human DC migratory behavior in lung epithelial tissue. DCs are hematopoietic cells essential for the maintenance of tissue homeostasis and the induction of tissue-specific immune responses. Important functions include cytokine production and migration in response to infection for the induction of proper immune responses. To design appropriate strategies to exploit human DC functional properties in lung tissue for the purpose of clinical evaluation, e.g., candidate vaccination and immunotherapy strategies, we have developed a live-imaging assay based on our previously described organotypic model of the human lung. This assay allows provocations and subsequent quantitative investigations of DC functional properties under conditions mimicking morphological and functional features of the in vivo parental tissue. We present protocols to set up and prepare tissue models for 4D (x, y, z, time) fluorescence-imaging analysis that allow spatial and temporal studies of human DCs in live epithelial tissue, followed by flow cytometry analysis of DCs retrieved from digested tissue models. This model system can be useful for elucidating incompletely defined pathways controlling DC functional responses to infection and inflammation in lung epithelial tissue, as well as the efficacy of locally administered candidate interventions. © 2014 Society for Leukocyte Biology.

Quantitative Methods for Measuring Repair Rates and Innate-Immune Cell Responses in Wounded Mouse Skin.

PubMed

Li, Zhi; Gothard, Elizabeth; Coles, Mark C; Ambler, Carrie A

2018-01-01

In skin wounds, innate-immune cells clear up tissue debris and microbial contamination, and also secrete cytokines and other growth factors that impact repair process such as re-epithelialization and wound closure. After injury, there is a rapid influx and efflux of immune cells at wound sites, yet the function of each innate cell population in skin repair is still under investigation. Flow cytometry is a valuable research tool for detecting and quantifying immune cells; however, in mouse back skin, the difficulty in extracting immune cells from small area of skin due to tissue complexity has made cytometric analysis an underutilized tool. In this paper, we provide detailed methods on the digestion of lesion-specific skin without disrupting antigen expression followed by multiplex cell staining that allows for identification of seven innate-immune populations, including rare subsets such as group-3 innate lymphoid cells (ILC3s), by flow-cytometry analysis. Furthermore, when studying the functions of immune cells to tissue repair an important metric to monitor is size of the wound opening. Normal wounds close steadily albeit at non-linear rates, while slow or stalled wound closure can indicate an underlying problem with the repair process. Calliper measurements are difficult and time-consuming to obtain and can require repeated sedation of experimental animals. We provide advanced methods for measuring of wound openness; digital 3D image capture and semi-automated image processing that allows for unbiased, reliable measurements that can be taken repeatedly over time.

Quantitative Methods for Measuring Repair Rates and Innate-Immune Cell Responses in Wounded Mouse Skin

PubMed Central

Li, Zhi; Gothard, Elizabeth; Coles, Mark C.; Ambler, Carrie A.

2018-01-01

In skin wounds, innate-immune cells clear up tissue debris and microbial contamination, and also secrete cytokines and other growth factors that impact repair process such as re-epithelialization and wound closure. After injury, there is a rapid influx and efflux of immune cells at wound sites, yet the function of each innate cell population in skin repair is still under investigation. Flow cytometry is a valuable research tool for detecting and quantifying immune cells; however, in mouse back skin, the difficulty in extracting immune cells from small area of skin due to tissue complexity has made cytometric analysis an underutilized tool. In this paper, we provide detailed methods on the digestion of lesion-specific skin without disrupting antigen expression followed by multiplex cell staining that allows for identification of seven innate-immune populations, including rare subsets such as group-3 innate lymphoid cells (ILC3s), by flow-cytometry analysis. Furthermore, when studying the functions of immune cells to tissue repair an important metric to monitor is size of the wound opening. Normal wounds close steadily albeit at non-linear rates, while slow or stalled wound closure can indicate an underlying problem with the repair process. Calliper measurements are difficult and time-consuming to obtain and can require repeated sedation of experimental animals. We provide advanced methods for measuring of wound openness; digital 3D image capture and semi-automated image processing that allows for unbiased, reliable measurements that can be taken repeatedly over time. PMID:29535723

Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication

PubMed Central

Degtyarev, Michael; Reichelt, Mike; Lin, Kui

2014-01-01

Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis. PMID:24489953

Progress on an implementation of MIFlowCyt in XML

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Stephanie H.

2015-03-01

Introduction: The International Society for Advancement of Cytometry (ISAC) Data Standards Task Force (DSTF) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). The CytometryML schemas, are based in part upon the Flow Cytometry Standard and Digital Imaging and Communication (DICOM) standards. CytometryML has and will be extended and adapted to include MIFlowCyt, as well as to serve as a common standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). Individual major elements of the MIFlowCyt schema were translated into XML and filled with reasonable data. A small section of the code was formatted with HTML formatting elements. Results: The differences in the amount of detail to be recorded for 1) users of standard techniques including data analysts and 2) others, such as method and device creators, laboratory and other managers, engineers, and regulatory specialists required that separate data-types be created to describe the instrument configuration and components. A very substantial part of the MIFlowCyt element that describes the Experimental Overview part of the MIFlowCyt and substantial parts of several other major elements have been developed. Conclusions: The future use of structured XML tags and web technology should facilitate searching of experimental information, its presentation, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate in publications adherence to the MIFlowCyt standard. The use of CytometryML together with XML technology should also result in the textual and numeric data being published using web technology without any change in composition. Preliminary testing indicates that CytometryML XML pages can be directly formatted with the combination of HTML and CSS.

Crosstalk-eliminated quantitative determination of aflatoxin B1-induced hepatocellular cancer stem cells based on concurrent monitoring of CD133, CD44, and aldehyde dehydrogenase1.

PubMed

Ju, Hee; Shim, Yumi; Arumugam, Parthasarathy; Song, Joon Myong

2016-01-22

Cancer stem cells (CSCs), known as tumor initiating cells, have become a critically important issue for cancer therapy. Although much research has demonstrated the induction of hepato cellular carcinoma by aflatoxin B1, the formation of hepatocellular CSCs and their quantitative determination is hardly reported. In this work, it was found that hepatocellular CSCs were produced from HepG2 cells by aflatoxin B1-induced mutation, and their amount was quantitatively determined using crosstalk-eliminated multicolor cellular imaging based on quantum dot (Qdot) nanoprobes and an acousto-optical tunable filter (AOTF). Hepatocellular CSCs were acquired via magnetic bead-based sorting and observed using concurrent detection of three different markers: CD133, CD44, and aldehyde dehydrogenase1 (ALDH1). The DNA mutation of HepG2 cells caused by aflatoxin B1 was quantitatively observed via absorbance spectra of aflatoxin B1-8, 9-epoxide-DNA adducts. The percentages of hepatocellular CSCs formed in the entire HepG2 cells were determined to be 9.77±0.65%, 10.9±1.39%, 11.4±1.32%, and 12.8±0.7%, respectively, at 0 μM, 5 μM, 10 μM, and 20 μM of aflatoxin B1. The results matched well with those obtained utilizing flow cytometry. This study demonstrates that aflatoxin mediated mutation induced the conversion of hepatic cancer cell to hepatic CSCs by using a Qdot based constructed multicolor cellular imaging system. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Single Cell Mass Cytometry for Analysis of Immune System Functional States

PubMed Central

Bjornson, Zach B.; Nolan, Garry P.; Fantl, Wendy J.

2013-01-01

Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with antibody panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the periodic table. Isotope labelled antibodies recognize surface markers to delineate cell types and intracellular signaling molecules to provide a measure of the network state—and thereby demarcating multiple cell state functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at its network state. This review will cover the basics of mass cytometry as well as outline steps already taken to allow it to stand aside traditional fluorescence based cytometry in the immunologist’s analytical arsenal in their study of immune states during infection. PMID:23999316

Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii.

PubMed

Everson, William V; Ware, Michael W; Dubey, J P; Lindquist, H D Alan

2002-01-01

Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation.

PubMed

Böttcher, S; Ritgen, M; Pott, C; Brüggemann, M; Raff, T; Stilgenbauer, S; Döhner, H; Dreger, P; Kneba, M

2004-10-01

The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.

Real-time Image Processing for Microscopy-based Label-free Imaging Flow Cytometry in a Microfluidic Chip.

PubMed

Heo, Young Jin; Lee, Donghyeon; Kang, Junsu; Lee, Keondo; Chung, Wan Kyun

2017-09-14

Imaging flow cytometry (IFC) is an emerging technology that acquires single-cell images at high-throughput for analysis of a cell population. Rich information that comes from high sensitivity and spatial resolution of a single-cell microscopic image is beneficial for single-cell analysis in various biological applications. In this paper, we present a fast image-processing pipeline (R-MOD: Real-time Moving Object Detector) based on deep learning for high-throughput microscopy-based label-free IFC in a microfluidic chip. The R-MOD pipeline acquires all single-cell images of cells in flow, and identifies the acquired images as a real-time process with minimum hardware that consists of a microscope and a high-speed camera. Experiments show that R-MOD has the fast and reliable accuracy (500 fps and 93.3% mAP), and is expected to be used as a powerful tool for biomedical and clinical applications.

Application of magnetic carriers to two examples of quantitative cell analysis

NASA Astrophysics Data System (ADS)

Zhou, Chen; Qian, Zhixi; Choi, Young Suk; David, Allan E.; Todd, Paul; Hanley, Thomas R.

2017-04-01

The use of magnetophoretic mobility as a surrogate for fluorescence intensity in quantitative cell analysis was investigated. The objectives of quantitative fluorescence flow cytometry include establishing a level of labeling for the setting of parameters in fluorescence activated cell sorters (FACS) and the determination of levels of uptake of fluorescently labeled substrates by living cells. Likewise, the objectives of quantitative magnetic cytometry include establishing a level of labeling for the setting of parameters in flowing magnetic cell sorters and the determination of levels of uptake of magnetically labeled substrates by living cells. The magnetic counterpart to fluorescence intensity is magnetophoretic mobility, defined as the velocity imparted to a suspended cell per unit of magnetic ponderomotive force. A commercial velocimeter available for making this measurement was used to demonstrate both applications. Cultured Gallus lymphoma cells were immunolabeled with commercial magnetic beads and shown to have adequate magnetophoretic mobility to be separated by a novel flowing magnetic separator. Phagocytosis of starch nanoparticles having magnetic cores by cultured Chinese hamster ovary cells, a CHO line, was quantified on the basis of magnetophoretic mobility.

Quantitative imaging of intracellular signaling for personalized pancreatic cancer therapy in an in vivo avatar (Conference Presentation)

NASA Astrophysics Data System (ADS)

Samkoe, Kimberley S.; Schultz, Emily; Park, Yeonjae; Fischer, Dawn; Pogue, Brian W.; Smith, Kerrington; Tichauer, Kenneth M.; Gibbs, Summer L.

2017-02-01

Pancreatic ductal adenocarcinomas (PDAC) are notoriously difficult to treat and in general, molecular targeted therapies have failed even when the targeted protein is overexpressed in the tumor tissue. Genetic mutations in extracellular receptors and downstream signaling proteins (i.e., RAS signaling pathway) and convoluted intracellular cross-talk between cell signaling pathways are likely reasons that these promising therapies fail. Monitoring the complex relationship between intracellular protein signaling is difficult and to-date, standard techniques that are used (Western blot, flow cytometry, immunohistochemistry, etc.) are invasive, static and do not accurately represent in vivo structure-function relationships. Here, we describe the development of an in ovo avatar using patient derived tumors grown on the chicken chorioallantoic membrane (CAM) and the novel fluorescence-based Quantitative Protein Expression Tracking (QUIET) methodology to bridge the gap between oncology, genomics and patient outcomes. Previously developed paired-agent imaging, was extended to a three-compartment model system in QUIET, which utilizes three types of imaging agents: novel fluorophore conjugated cell permeable targeted and untargeted small molecule paired-agents, in addition to a tumor perfusion agent that is not cell membrane permeable. We have demonstrated the ability to quantify the intracellular binding domain of a trans-membrane protein in vitro using cell permeable fluorescent agents (erlotinib-TRITC and control isotype-BODIPY FL). In addition, we have demonstrated imaging protocols to simultaneously image up to 6 spectrally distinct organic fluorophores in in ovo avatars using the Nuance EX (Perkin Elmer) and established proof-of-principle intracellular and extracellular protein concentrations of epidermal growth factor receptor using QUIET and traditional paired-agent imaging.

Affibody Molecules for In vivo Characterization of HER2-Positive Tumors by Near-Infrared Imaging

PubMed Central

Lee, Sang Bong; Hassan, Moinuddin; Fisher, Robert; Chertov, Oleg; Chernomordik, Victor; Kramer-Marek, Gabriela; Gandjbakhche, Amir; Capala, Jacek

2012-01-01

Purpose HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients. We are developing molecular probes for in vivo quantitative imaging of HER2 receptors using near-infrared optical imaging. The goal is to provide probes that will minimally interfere with the studied system, i.e., whose binding does not interfere with the binding of the therapeutic agents, and whose effect on the target cells is minimal. Experimental Design We used three different types of HER2-specific Affibody molecules [monomer ZHER2:342, dimer (ZHER2:477)2, and albumin-binding domain-fused-(ZHER2:342)2] as targeting agents, and labeled them with Alexa Fluor dyes. Trastuzumab was also conjugated, using commercially available kits, as a standard control. The resulting conjugates were characterized in vitro by toxicity assays, Biacore affinity measurements, flow cytometry, and confocal microscopy. Semi-uantitative in vivo near-infrared optical imaging studies were carried out using mice with subcutaneous xenografts of HER2-positive tumors. Results The HER2-specific Affibody molecules were not toxic to HER2-overexpressing cells and their binding to HER2 did interfere with neither binding nor effectives of trastuzumab. The binding affinities and specificities of the Affibody-Alexa Fluor fluorescent conjugates to HER2 were unchanged or minimally affected by the modifications. Pharmacokinetics and biodistribution studies showed the albumin-binding domain-fused-(ZHER2:342)2-Alexa Fluor 750 conjugate to be an optimal probe for optical imaging of HER2 in vivo. Conclusion Our results suggest that Affibody-Alexa Fluor conjugates may be used as a specific near-infrared probe for the non-invasive semi-quantitative imaging of HER2 expression in vivo. PMID:18559604

Routine detection of Epstein-Barr virus specific T-cells in the peripheral blood by flow cytometry

NASA Technical Reports Server (NTRS)

Crucian, B. E.; Stowe, R. P.; Pierson, D. L.; Sams, C. F.

2001-01-01

The ability to detect cytomegalovirus-specific T-cells (CD4(+)) in the peripheral blood by flow cytometry has been recently described by Picker et al. In this method, cells are incubated with viral antigen and responding (cytokine producing) T-cells are then identified by flow cytometry. To date, this technique has not been reliably used to detect Epstein-Barr virus (EBV)-specific T-cells primarily due to the superantigen/mitogenic properties of the virus which non-specifically activate T-cells. By modifying culture conditions under which the antigens are presented, we have overcome this limitation and developed an assay to detect and quantitate EBV-specific T-cells. The detection of cytokine producing T-cells by flow cytometry requires an extremely strong signal (such as culture in the presence of PMA and ionomycin). Our data indicate that in modified culture conditions (early removal of viral antigen) the non-specific activation of T-cells by EBV is reduced, but antigen presentation will continue uninhibited. Using this method, EBV-specific T-cells may be legitimately detected using flow cytometry. No reduction in the numbers of antigen-specific T-cells was observed by the early removal of target antigen when verified using cytomegalovirus antigen (a virus with no non-specific T-cell activation properties). In EBV-seropositive individuals, the phenotype of the EBV-specific cytokine producing T-cells was evaluated using four-color flow cytometry and found to be CD45(+), CD3(+), CD4(+), CD45RA(-), CD69(+), CD25(-). This phenotype indicates the stimulation of circulating previously unactivated memory T-cells. No cytokine production was observed in CD4(+) T-cells from EBV-seronegative individuals, confirming the specificity of this assay. In addition, the use of four color cytometry (CD45, CD3, CD69, IFNgamma/IL-2) allows the total quantitative assessment of EBV-specific T-cells while monitoring the interference of EBV non-specific mitogenic activity. This method may have significant utility for the monitoring of the immune response to latent virus infection/reactivation.

Modeling of cytometry data in logarithmic space: When is a bimodal distribution not bimodal?

PubMed

Erez, Amir; Vogel, Robert; Mugler, Andrew; Belmonte, Andrew; Altan-Bonnet, Grégoire

2018-02-16

Recent efforts in systems immunology lead researchers to build quantitative models of cell activation and differentiation. One goal is to account for the distributions of proteins from single-cell measurements by flow cytometry or mass cytometry as readout of biological regulation. In that context, large cell-to-cell variability is often observed in biological quantities. We show here that these readouts, viewed in logarithmic scale may result in two easily-distinguishable modes, while the underlying distribution (in linear scale) is unimodal. We introduce a simple mathematical test to highlight this mismatch. We then dissect the flow of influence of cell-to-cell variability proposing a graphical model which motivates higher-dimensional analysis of the data. Finally we show how acquiring additional biological information can be used to reduce uncertainty introduced by cell-to-cell variability, helping to clarify whether the data is uni- or bimodal. This communication has cautionary implications for manual and automatic gating strategies, as well as clustering and modeling of single-cell measurements. © 2018 International Society for Advancement of Cytometry. © 2018 International Society for Advancement of Cytometry.

An optofluidic approach for gold nanoprobes based-cancer theranostics

NASA Astrophysics Data System (ADS)

Panwar, Nishtha; Song, Peiyi; Yang, Chengbin; Yong, Ken-Tye; Tjin, Swee Chuan

2017-02-01

Suppression of overexpressed gene mutations in cancer cells through RNA interference (RNAi) technique is a therapeutically effective modality for oncogene silencing. In general, transfection agent is needed for siRNA delivery. Also, it is a tedious and time consuming process to analyze the gene transfection using current conventional flow cytometry systems and commercially available transfection kits. Therefore, there are two urgent challenges that we need to address for understanding and real time monitoring the delivery of siRNA to cancer cells more effectively. One, nontoxic, biocompatible and stable non-viral transfection agents need to be developed and investigated for gene delivery in cancer cells. Two, new, portable optofluidic methods need to be engineered for determining the transfection efficiency of the nanoformulation in real time. First, we demonstrate the feasibility of using gold nanorods (AuNRs) as nanoprobes for the delivery of Interleukin-8 (IL-8) siRNA in a pancreatic cancer cell line- MiaPaCa-2. An optimum ratio of 10:1 for the AuNRs-siRNA nanoformulation required for efficient loading has been experimentally determined. Promising transfection rates (≈88%) of the nanoprobe-assisted gene delivery are quantified by flow cytometry and fluorescence imaging, which are higher than the commercial control, Oligofectamine. The excellent gene knockdown performance (over 81%) of the proposed model support in vivo trials for RNAi-based cancer theranostics. In addition to cancer theranostics, our nanoprobe combination can be also applied for disease outbreak monitoring like MERS. Second, we present an optical fiber-integrated microfluidic chip that utilizes simple hydrodynamic and optical setups for miniaturized on-chip flow cytometry. The chip provides a powerful and convenient tool to quantitatively determine the siRNA transfection into cancer cells without using bulky flow cytometer. These studies outline the role of AuNRs as potential non-viral gene delivery vehicles, and their suitability for microfluidics-based lab-on-chip flow cytometry applications.

Hybrid Imaging Labels: Providing the Link Between Mass Spectrometry-Based Molecular Pathology and Theranostics

PubMed Central

Buckle, Tessa; van der Wal, Steffen; van Malderen, Stijn J.M.; Müller, Larissa; Kuil, Joeri; van Unen, Vincent; Peters, Ruud J.B.; van Bemmel, Margaretha E.M.; McDonnell, Liam A.; Velders, Aldrik H.; Koning, Frits; Vanhaeke, Frank; van Leeuwen, Fijs W. B.

2017-01-01

Background: Development of theranostic concepts that include inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) imaging can be hindered by the lack of a direct comparison to more standardly used methods for in vitro and in vivo evaluation; e.g. fluorescence or nuclear medicine. In this study a bimodal (or rather, hybrid) tracer that contains both a fluorescent dye and a chelate was used to evaluate the existence of a direct link between mass spectrometry (MS) and in vitro and in vivo molecular imaging findings using fluorescence and radioisotopes. At the same time, the hybrid label was used to determine whether the use of a single isotope label would allow for MS-based diagnostics. Methods: A hybrid label that contained both a DTPA chelate (that was coordinated with either 165Ho or 111In) and a Cy5 fluorescent dye was coupled to the chemokine receptor 4 (CXCR4) targeting peptide Ac-TZ14011 (hybrid-Cy5-Ac-TZ4011). This receptor targeting tracer was used to 1) validate the efficacy of (165Ho-based) mass-cytometry in determining the receptor affinity via comparison with fluorescence-based flow cytometry (Cy5), 2) evaluate the microscopic binding pattern of the tracer in tumor cells using both fluorescence confocal imaging (Cy5) and LA-ICP-MS-imaging (165Ho), 3) compare in vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) after intravenous administration of hybrid-Cy5-Ac-TZ4011 in tumor-bearing mice. Finally, LA-ICP-MS-imaging (165Ho) was linked to fluorescence-based analysis of excised tissue samples (Cy5). Results: Analysis with both mass-cytometry and flow cytometry revealed a similar receptor affinity, respectively 352 ± 141 nM and 245 ± 65 nM (p = 0.08), but with a much lower detection sensitivity for the first modality. In vitro LA-ICP-MS imaging (165Ho) enabled clear discrimination between CXCR4 positive and negative cells, but fluorescence microscopy was required to determine the intracellular distribution. In vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) of the hybrid peptide were shown to be similar. Assessment of tracer distribution in excised tissues revealed the location of tracer uptake with both LA-ICP-MS-imaging and fluorescence imaging. Conclusion: Lanthanide-isotope chelation expands the scope of fluorescent/radioactive hybrid tracers to include MS-based analytical tools such as mass-cytometry, ICP-MS and LA-ICP-MS imaging in molecular pathology. In contradiction to common expectations, MS detection using a single chelate imaging agent was shown to be feasible, enabling a direct link between nuclear medicine-based imaging and theranostic methods. PMID:28255355

In Vivo Flow Cytometry: A Horizon of Opportunities

PubMed Central

Tuchin, Valery V.; Tárnok, Attila; Zharov, Vladimir P.

2012-01-01

Flow cytometry has been a fundamental tool of biological discovery for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents studying cells in their native environment. These problems can be overcome by use of in vivo flow cytometry which provides detection and imaging of circulating normal and abnormal cells directlyin blood or lymph flow. The goal of this mini-review is to provide a brief history, features and challenges of this new generation of flow cytometry methods and instruments. Spectrum of possibilities of in vivo flow cytometry in biological science (e.g., cell metabolism, immune function, or apoptosis) and medical fields (e.g., cancer, infection, cardiovascular disorder) including integrated photoacoustic-photothermal theranostics of circulating abnormal cells are discussed with focus on recent advances of this new platform. PMID:21915991

Evaluating reporter genes of different luciferases for optimized in vivo bioluminescence imaging of transplanted neural stem cells in the brain.

PubMed

Mezzanotte, Laura; Aswendt, Markus; Tennstaedt, Annette; Hoeben, Rob; Hoehn, Mathias; Löwik, Clemens

2013-01-01

Bioluminescence imaging (BLI) has become the method of choice for optical tracking of cells in small laboratory animals. However, the use of luciferases from different species, depending on different substrates and emitting at distinct wavelengths, has not been optimized for sensitive neuroimaging. In order to identify the most suitable luciferase, this quantitative study compared the luciferases Luc2, CBG99, PpyRE9 and hRluc. Human embryonic kidney (HEK-293) cells and mouse neural stem cells were transduced by lentiviral vector-mediated transfer to express one of the four luciferases, together with copGFP. A T2A peptide linker promoted stoichiometric expression between both imaging reporters and the comparison of cell populations upon flow cytometry. Cell dilution series were used to determine highest BLI sensitivity in vitro for Luc2. However, Coelenterazine h-dependent hRluc signals clearly exceeded d-luciferin-dependent BLI in vitro. For the quantitative in vivo analysis, cells were transplanted into mouse brain and BLI was performed including the recording of emission kinetics and spectral characteristics. Differences in light kinetics were observed for d-luciferin vs Coelenterazine h. The emission spectra of Luc2 and PpyRE9 remained almost unchanged, while the emission spectrum of CBG99 became biphasic. Most importantly, photon emission decreased in the order of Luc2, CBG99, PpyRE9 to hRluc. The feasibility of combining different luciferases for dual color and dual substrate neuroimaging was tested and discussed. This investigation provides the first complete quantitative comparison of different luciferases expressed by neural stem cells. It results in a clear recommendation of Luc2 as the best luciferase selection for in vivo neuroimaging. Copyright © 2013 John Wiley & Sons, Ltd.

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Costantini, Lindsey M.; Irvin, Susan C.; Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFPmore » enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.« less

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced.

PubMed

Morinaga, Takao; Nguyễn, Thảo Thi Thanh; Zhong, Boya; Hanazono, Michiko; Shingyoji, Masato; Sekine, Ikuo; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Tagawa, Masatoshi

2017-11-10

Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity. We used type 5 Ad in which the expression of E1A gene was activated by 5'-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses. The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level. Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

PubMed

Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

2015-05-01

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.

Study of low speed flow cytometry for diffraction imaging with different chamber and nozzle designs.

PubMed

Sa, Yu; Feng, Yuanming; Jacobs, Kenneth M; Yang, Jun; Pan, Ran; Gkigkitzis, Ioannis; Lu, Jun Q; Hu, Xin-Hua

2013-11-01

Achieving effective hydrodynamic focusing and flow stability at low speed presents a challenging design task in flow cytometry for studying phenomena such as cell adhesion and diffraction imaging of cells with low-cost cameras. We have developed different designs of flow chamber and sheath nozzle to accomplish the above goal. A 3D computational model of the chambers has been established to simulate the fluid dynamics in different chamber designs and measurements have been performed to determine the velocity and size distributions of the core fluid from the nozzle. Comparison of the simulation data with experimental results shows good agreement. With the computational model significant insights were gained for optimization of the chamber design and improvement of the cell positioning accuracy for study of slow moving cells. The benefit of low flow speed has been demonstrated also by reduced blurring in the diffraction images of single cells. Based on these results, we concluded that the new designs of chamber and sheath nozzle produce stable hydrodynamic focusing of the core fluid at low speed and allow detailed study of cellular morphology under various rheological conditions using the diffraction imaging method. © 2013 International Society for Advancement of Cytometry.

Kinetics of T-cell receptor-dependent antigen recognition determined in vivo by multi-spectral normalized epifluorescence laser scanning

NASA Astrophysics Data System (ADS)

Favicchio, Rosy; Zacharakis, Giannis; Oikonomaki, Katerina; Zacharopoulos, Athanasios; Mamalaki, Clio; Ripoll, Jorge

2012-07-01

Detection of multiple fluorophores in conditions of low signal represents a limiting factor for the application of in vivo optical imaging techniques in immunology where fluorescent labels report for different functional characteristics. A noninvasive in vivo Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS) method was developed for the simultaneous and quantitative detection of multiple fluorophores in low signal to noise ratios and used to follow T-cell activation and clonal expansion. Colocalized DsRed- and GFP-labeled T cells were followed in tandem during the mounting of an immune response. Spectral unmixing was used to distinguish the overlapping fluorescent emissions representative of the two distinct cell populations and longitudinal data reported the discrete pattern of antigen-driven proliferation. Retrieved values were validated both in vitro and in vivo with flow cytometry and significant correlation between all methodologies was achieved. Noninvasive M-SNELS successfully quantified two colocalized fluorescent populations and provides a valid alternative imaging approach to traditional invasive methods for detecting T cell dynamics.

Measuring the metastatic potential of cancer cells

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.; Gratzner, Howard; Atassi, M. Z.

1993-01-01

Cancer cells must secrete proteolytic enzymes to invade adjacent tissues and migrate to a new metastatic site. Urokinase (uPA) is a key enzyme related to metastasis in cancers of the lung, colon, gastric, uterine, breast, brain, and malignant melanoma. A NASA technology utilization project has combined fluorescence microscopy, image analysis, and flow cytometry, using fluorescent dyes, and urokinase-specific antibodies to measure uPA and abnormal DNA levels (related to cancer cell proliferation) inside the cancer cells. The project is focused on developing quantitative measurements to determine if a patient's tumor cells are actively metastasizing. If a significant number of tumor cells contain large amounts of uPA (esp. membrane-bound) then the post-surgical chemotherapy or radiotherapy can be targeted for metastatic cells that have already left the primary tumor. These analytical methods have been applied to a retrospective study of biopsy tissues from 150 node negative, stage 1 breast cancer patients. Cytopathology and image analysis has shown that uPA is present in high levels in many breast cancer cells, but not found in normal breast. Significant amounts of uPA also have been measured in glioma cell lines cultured from brain tumors. Commercial applications include new diagnostic tests for metastatic cells, in different cancers, which are being developed with a company that provides a medical testing service using flow cytometry for DNA analysis and hormone receptors on tumor cells from patient biopsies. This research also may provide the basis for developing a new 'magic bullet' treatment against metastasis using chemotherapeutic drugs or radioisotopes attached to urokinase-specific monoclonal antibodies that will only bind to metastatic cells.

Imaging flow cytometry assays for quantifying pigment grade titanium dioxide particle internalization and interactions with immune cells in whole blood

PubMed Central

Vis, Bradley; Pele, Laetitia C.; Faria, Nuno; Powell, Jonathan J.

2017-01-01

Abstract Pigment grade titanium dioxide is composed of sub‐micron sized particles, including a nanofraction, and is widely utilized in food, cosmetic, pharmaceutical, and biomedical industries. Oral exposure to pigment grade titanium dioxide results in at least some material entering the circulation in humans, although subsequent interactions with blood immune cells are unknown. Pigment grade titanium dioxide is employed for its strong light scattering properties, and this work exploited that attribute to determine whether single cell–particle associations could be determined in immune cells of human whole blood at “real life” concentrations. In vitro assays, initially using isolated peripheral blood mononuclear cells, identified titanium dioxide associated with the surface of, and within, immune cells by darkfield reflectance in imaging flow cytometry. This was confirmed at the population level by side scatter measurements using conventional flow cytometry. Next, it was demonstrated that imaging flow cytometry could quantify titanium dioxide particle‐bearing cells, within the immune cell populations of fresh whole blood, down to titanium dioxide levels of 10 parts per billion, which is in the range anticipated for human blood following titanium dioxide ingestion. Moreover, surface association and internal localization of titanium dioxide particles could be discriminated in the assays. Overall, results showed that in addition to the anticipated activity of blood monocytes internalizing titanium dioxide particles, neutrophil internalization and cell membrane adhesion also occurred, the latter for both phagocytic and nonphagocytic cell types. What happens in vivo and whether this contributes to activation of one or more of these different cells types in blood merits further attention. © 2017 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC. PMID:28941170

Analysis of molecular assemblies by flow cytometry: determinants of Gi1 and by binding

NASA Astrophysics Data System (ADS)

Sarvazyan, Noune A.; Neubig, Richard R.

1998-05-01

We report here a novel application of flow cytometry for the quantitative analysis of the high affinity interaction between membrane proteins both in detergent solutions and when reconstituted into lipid vesicles. The approach is further advanced to permit the analysis of binding to expressed protein complexes in native cell membranes. The G protein heterotrimer signal transduction function links the extracellularly activated transmembrane receptors and intracellular effectors. Upon activation, (alpha) and (beta) (gamma) subunits of G protein undergo a dissociation/association cycle on the cell membrane interface. The binding parameters of solubilized G protein (alpha) and (beta) (gamma) subunits have been defined but little is known quantitatively about their interactions in the membrane. Using a novel flow cytometry approach, the binding of low nanomolar concentrations of fluorescein-labeled G(alpha) i1 (F- (alpha) ) to (beta) (gamma) both in detergent solution and in a lipid environment was quantitatively compared. Unlabeled (beta) $gama reconstituted in biotinylated phospholipid vesicles bound F-(alpha) tightly (Kd 6 - 12 nM) while the affinity for biotinylated-(beta) (gamma) in Lubrol was even higher (Kd of 2.9 nM). The application of this approach to proteins expressed in native cell membranes will advance our understanding of G protein function in context of receptor and effector interaction. More generally, this approach can be applied to study the interaction of any fluorescently labeled protein with a membrane protein which can be expressed in Sf9 plasma membranes.

Portable real-time fluorescence cytometry of microscale cell culture analog devices

NASA Astrophysics Data System (ADS)

Kim, Donghyun; Tatosian, Daniel A.; Shuler, Michael L.

2006-02-01

A portable fluorescence cytometric system that provides a modular platform for quantitative real-time image measurements has been used to explore the applicability to investigating cellular events on multiple time scales. For a short time scale, we investigated the real-time dynamics of uptake of daunorubicin, a chemotherapeutic agent, in cultured mouse L-cells in a micro cell culture analog compartment using the fluorescent cytometric system. The green fluorescent protein (GFP) expression to monitor induction of pre-specified genes, which occurs on a much longer time scale, has also been measured. Here GFP fluorescence from a doxycycline inducible promoter in a mouse L-cell line was determined. Additionally, a system based on inexpensive LEDs showed performance comparable to a broadband light source based system and reduced photobleaching compared to microscopic examination.

Clustering methods applied in the detection of Ki67 hot-spots in whole tumor slide images: an efficient way to characterize heterogeneous tissue-based biomarkers.

PubMed

Lopez, Xavier Moles; Debeir, Olivier; Maris, Calliope; Rorive, Sandrine; Roland, Isabelle; Saerens, Marco; Salmon, Isabelle; Decaestecker, Christine

2012-09-01

Whole-slide scanners allow the digitization of an entire histological slide at very high resolution. This new acquisition technique opens a wide range of possibilities for addressing challenging image analysis problems, including the identification of tissue-based biomarkers. In this study, we use whole-slide scanner technology for imaging the proliferating activity patterns in tumor slides based on Ki67 immunohistochemistry. Faced with large images, pathologists require tools that can help them identify tumor regions that exhibit high proliferating activity, called "hot-spots" (HSs). Pathologists need tools that can quantitatively characterize these HS patterns. To respond to this clinical need, the present study investigates various clustering methods with the aim of identifying Ki67 HSs in whole tumor slide images. This task requires a method capable of identifying an unknown number of clusters, which may be highly variable in terms of shape, size, and density. We developed a hybrid clustering method, referred to as Seedlink. Compared to manual HS selections by three pathologists, we show that Seedlink provides an efficient way of detecting Ki67 HSs and improves the agreement among pathologists when identifying HSs. Copyright © 2012 International Society for Advancement of Cytometry.

Evaluation of acridine orange, LysoTracker Red, and quinacrine as fluorescent probes for long-term tracking of acidic vesicles.

PubMed

Pierzyńska-Mach, Agnieszka; Janowski, Paweł A; Dobrucki, Jurek W

2014-08-01

Acidic vesicles can be imaged and tracked in live cells after staining with several low molecular weight fluorescent probes, or with fluorescently labeled proteins. Three fluorescent dyes, acridine orange, LysoTracker Red DND-99, and quinacrine, were evaluated as acidic vesicle tracers for confocal fluorescence imaging and quantitative analysis. The stability of fluorescent signals, achievable image contrast, and phototoxicity were taken into consideration. The three tested tracers exhibit different advantages and pose different problems in imaging experiments. Acridine orange makes it possible to distinguish acidic vesicles with different internal pH but is fairly phototoxic and can cause spectacular bursts of the dye-loaded vesicles. LysoTracker Red is less phototoxic but its rapid photobleaching limits the range of useful applications considerably. We demonstrate that quinacrine is most suitable for long-term imaging when a high number of frames is required. This capacity made it possible to trace acidic vesicles for several hours, during a process of drug-induced apoptosis. An ability to record the behavior of acidic vesicles over such long periods opens a possibility to study processes like autophagy or long-term effects of drugs on endocytosis and exocytosis. © 2014 International Society for Advancement of Cytometry.

Cytometry standards continuum

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Spidlen, Josef; Brinkman, Ryan R.

2008-02-01

Introduction: The International Society for Analytical Cytology, ISAC, is developing a new combined flow and image Analytical Cytometry Standard (ACS). This standard needs to serve both the research and clinical communities. The clinical medicine and clinical research communities have a need to exchange information with hospital and other clinical information systems. Methods: 1) Prototype the standard by creating CytometryML and a RAW format for binary data. 2) Join the ISAC Data Standards Task Force. 3) Create essential project documentation. 4) Cooperate with other groups by assisting in the preparation of the DICOM Supplement 122: Specimen Module and Pathology Service-Object Pair Classes. Results: CytometryML has been created and serves as a prototype and source of experience for the following: the Analytical Cytometry Standard (ACS) 1.0, the ACS container, Minimum Information about a Flow Cytometry Experiment (MIFlowCyt), and Requirements for a Data File Standard Format to Describe Flow Cytometry and Related Analytical Cytology Data. These requirements provide a means to judge the appropriateness of design elements and to develop tests for the final ACS. The requirements include providing the information required for understanding and reproducing a cytometry experiment or clinical measurement, and for a single standard for both flow and digital microscopic cytometry. Schemas proposed by other members of the ISAC Data Standards Task Force (e.g, Gating-ML) have been independently validated and have been integrated with CytometryML. The use of netCDF as an element of the ACS container has been proposed by others and a suggested method of its use is proposed.

CytometryML with DICOM and FCS

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2018-02-01

Abstract: Flow Cytometry Standard, FCS, and Digital Imaging and Communications in Medicine standard, DICOM, are based on extensive, superb domain knowledge, However, they are isolated systems, do not take advantage of data structures, require special programs to read and write the data, lack the capability to interoperate or work with other standards and FCS lacks many of the datatypes necessary for clinical laboratory data. The large overlap between imaging and flow cytometry provides strong evidence that both modalities should be covered by the same standard. Method: The XML Schema Definition Language, XSD 1.1 was used to translate FCS and/or DICOM objects. A MIFlowCyt file was tested with published values. Results: Previously, a significant part of an XML standard based upon a combination of FCS and DICOM has been implemented and validated with MIFlowCyt data. Strongly typed translations of FCS keywords have been constructed in XML. These keywords contain links to their DICOM and FCS equivalents.

Flow cytometric HyPer-based assay for hydrogen peroxide.

PubMed

Lyublinskaya, O G; Antonov, S A; Gorokhovtsev, S G; Pugovkina, N A; Kornienko, Ju S; Ivanova, Ju S; Shatrova, A N; Aksenov, N D; Zenin, V V; Nikolsky, N N

2018-05-30

HyPer is a genetically encoded fluorogenic sensor for hydrogen peroxide which is generally used for the ratiometric imaging of H 2 O 2 fluxes in living cells. Here, we demonstrate the advantages of HyPer-based ratiometric flow cytometry assay for H 2 O 2 , by using K562 and human mesenchymal stem cell lines expressing HyPer. We show that flow cytometry analysis is suitable to detect HyPer response to submicromolar concentrations of extracellularly added H 2 O 2 that is much lower than concentrations addressed previously in the other HyPer-based assays (such as cell imaging or fluorimetry). Suggested technique is also much more sensitive to hydrogen peroxide than the widespread flow cytometry assay exploiting H 2 O 2 -reactive dye H 2 DCFDA and, contrary to the H 2 DCFDA-based assay, can be employed for the kinetic studies of H 2 O 2 utilization by cells, including measurements of the rate constants of H 2 O 2 removal. In addition, flow cytometry multi-parameter ratiometric measurements enable rapid and high-throughput detection of endogenously generated H 2 O 2 in different subpopulations of HyPer-expressing cells. To sum up, HyPer can be used in multi-parameter flow cytometry studies as a highly sensitive indicator of intracellular H 2 O 2 . Copyright © 2018. Published by Elsevier Inc.

Assay validation for the assessment of adipogenesis of multipotential stromal cells—a direct comparison of four different methods

PubMed Central

Aldridge, Andrew; Kouroupis, Dimitrios; Churchman, Sarah; English, Anne; Ingham, Eileen; Jones, Elena

2013-01-01

Background aims Mesenchymal stromal cells (MSCs) are regenerative and immuno-privileged cells that are used for both tissue regeneration and treatment of severe inflammation-related disease. For quality control of manufactured MSC batches in regard to mature fat cell contamination, a quantitative method for measuring adipogenesis is needed. Methods Four previously proposed methods were validated with the use of bone marrow (BM) MSCs during a 21-day in vitro assay. Oil red staining was scored semiquantitatively; peroxisome proliferator activated receptor-γ and fatty acid binding protein (FABP)4 transcripts were measured by quantitative real-time polymerase chain reaction; FABP4 protein accumulation was evaluated by flow cytometry; and Nile red/4′,6-diamidino-2-phenylindole (DAPI) ratios were measured in fluorescent microplate assay. Skin fibroblasts and MSCs from fat pad, cartilage and umbilical cord were used as controls. Results Oil red staining indicated considerable heterogeneity between BM donors and individual cells within the same culture. FABP4 transcript levels increased 100- to 5000-fold by day 21, with large donor variability observed. Flow cytometry revealed increasing intra-culture heterogeneity over time; more granular cells accumulated more FABP4 protein and Nile red fluorescence compared with less granular cells. Nile red increase in day-21 MSCs was ∼5- and 4-fold, measured by flow cytometry or microplate assay, respectively. MSC proliferation/apoptosis was accounted through the use of Nile red/DAPI ratios; adipogenesis levels in day-21 BM MSCs increased ∼13-fold, with significant correlations with oil red scoring observed for MSC from other sources. Conclusions Flow cytometry permits the study of MSC differentiation at the single-cell level and sorting more and less mature cells from mixed cell populations. The microplate assay with the use of the Nile red/DAPI ratio provides rapid quantitative measurements and could be used as a low-cost, high-throughput method to quality-control MSC batches from different tissue sources. PMID:23260089

An Improved Flow Cytometry Method For Precise Quantitation Of Natural-Killer Cell Activity

NASA Technical Reports Server (NTRS)

Crucian, Brian; Nehlsen-Cannarella, Sandra; Sams, Clarence

2006-01-01

The ability to assess NK cell cytotoxicity using flow cytometry has been previously described and can serve as a powerful tool to evaluate effector immune function in the clinical setting. Previous methods used membrane permeable dyes to identify target cells. The use of these dyes requires great care to achieve optimal staining and results in a broad spectral emission that can make multicolor cytometry difficult. Previous methods have also used negative staining (the elimination of target cells) to identify effector cells. This makes a precise quantitation of effector NK cells impossible due to the interfering presence of T and B lymphocytes, and the data highly subjective to the variable levels of NK cells normally found in human peripheral blood. In this study an improved version of the standard flow cytometry assay for NK activity is described that has several advantages of previous methods. Fluorescent antibody staining (CD45FITC) is used to positively identify target cells in place of membranepermeable dyes. Fluorescent antibody staining of target cells is less labor intensive and more easily reproducible than membrane dyes. NK cells (true effector lymphocytes) are also positively identified by fluorescent antibody staining (CD56PE) allowing a simultaneous absolute count assessment of both NK cells and target cells. Dead cells are identified by membrane disruption using the DNA intercalating dye PI. Using this method, an exact NK:target ratio may be determined for each assessment, including quantitation of NK target complexes. Backimmunoscatter gating may be used to track live vs. dead Target cells via scatter properties. If desired, NK activity may then be normalized to standardized ratios for clinical comparisons between patients, making the determination of PBMC counts or NK cell percentages prior to testing unnecessary. This method provides an exact cytometric determination of NK activity that highly reproducible and may be suitable for routine use in the clinical setting.

High content analysis of differentiation and cell death in human adipocytes.

PubMed

Doan-Xuan, Quang Minh; Sarvari, Anitta K; Fischer-Posovszky, Pamela; Wabitsch, Martin; Balajthy, Zoltan; Fesus, Laszlo; Bacso, Zsolt

2013-10-01

Understanding adipocyte biology and its homeostasis is in the focus of current obesity research. We aimed to introduce a high-content analysis procedure for directly visualizing and quantifying adipogenesis and adipoapoptosis by laser scanning cytometry (LSC) in a large population of cell. Slide-based image cytometry and image processing algorithms were used and optimized for high-throughput analysis of differentiating cells and apoptotic processes in cell culture at high confluence. Both preadipocytes and adipocytes were simultaneously scrutinized for lipid accumulation, texture properties, nuclear condensation, and DNA fragmentation. Adipocyte commitment was found after incubation in adipogenic medium for 3 days identified by lipid droplet formation and increased light absorption, while terminal differentiation of adipocytes occurred throughout day 9-14 with characteristic nuclear shrinkage, eccentric nuclei localization, chromatin condensation, and massive lipid deposition. Preadipocytes were shown to be more prone to tumor necrosis factor alpha (TNFα)-induced apoptosis compared to mature adipocytes. Importantly, spontaneous DNA fragmentation was observed at early stage when adipocyte commitment occurs. This DNA damage was independent from either spontaneous or induced apoptosis and probably was part of the differentiation program. © 2013 International Society for Advancement of Cytometry. Copyright © 2013 International Society for Advancement of Cytometry.

Significances of red cell bound immunoglobulin G as detected by flow cytometry in patients with Coombs-negative immune hemolysis.

PubMed

Thedsawad, A; Taka, O; Wanachiwanawin, W

2016-04-01

This study was to investigate the use of flow cytometry for detection and quantitation of red blood cells (RBC) bound IgG in immune hemolysis of patients with autoimmune hemolytic anaemia (AIHA) and systematic lupus erythematosus (SLE). Two to ten percent of patients with warm-autoimmune hemolytic anaemia (WAIHA) exhibit a negative direct Coombs test. Flow cytometry has been applied to detect RBC bound IgG with high accuracy, reproducibility and sensitivity. In this study 45 and 75 patients with AIHA and SLE, respectively were evaluated for RBC bound IgG by direct Coombs test and flow cytometry. Seventy-one percent (32/45) and 31% (23/75) of patients with AIHA and SLE respectively, had laboratory evidence of hemolysis. A positive flow cytometry, as defined by mean fluorescent intensity (MFI) values >0·21 and IgG molecules >28, was found in 4 of 32 (12·5%) and 4 of 23 (17·4%) patients with AIHA and SLE who had hemolysis with a negative direct Coombs test. There were very strong and strong correlations between the strength of direct Coombs test with MFI values and IgG molecules in patients with AIHA and SLE, respectively. Flow cytometry can be applied in the diagnosis of Coombs-negative hemolytic anaemia in patients with AIHA and SLE. © 2016 British Blood Transfusion Society.

Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

PubMed

Varga, Viktor Sebestyén; Bocsi, József; Sipos, Ferenc; Csendes, Gábor; Tulassay, Zsolt; Molnár, Béla

2004-07-01

Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. SFM is a valuable method for the evaluation of fluorescently labeled cells. Copyright 2004 Wiley-Liss, Inc.

Using laser scanning cytometry to measure PPAR-mediated peroxisome proliferation and beta oxidation.

PubMed

Pruimboom-Brees, Ingrid M; Brees, Dominique J J E; Shen, Amy C; Keener, Mary; Francone, Omar; Amacher, David E; Loy, James K; Kerlin, Roy L

2005-01-01

Laser scanning cytometry (LSC) is a new technology that combines the properties and advantages of flow cytometry (FC) and immunohistochemistry (IHC), thus providing qualitative and quantitative information on protein expression with the additional perspective provided by cell and tissue localization. Formalin-fixed, paraffin embedded liver sections from rats exposed to a Peroxisome Proliferator Activated Receptor (PPAR) agonist were stained with antibodies against peroxisomal targeting signal-1 (PTS-1) (a highly conserved tripeptide contained within all peroxisomal enzymes), Acyl CoA oxidase (AOX) (the rate limiting enzyme of peroxisomal beta oxidation), and catalase (an inducible peroxisomal antioxidant enzyme) to evaluate peroxisomal beta oxidation, oxidative stress, and peroxisome proliferation. The LSC showed increased AOX, catalase, and PTS-1 expression in centrilobular hepatocytes that correlated favorably with the microscopic observation of centrilobular hepatocellular hypertrophy and with the palmitoyl CoA biochemical assay for peroxisomal beta oxidation, and provided additional morphologic information about peroxisome proliferation and tissue patterns of activation. Therefore, the LSC provides qualitative and quantitative evaluation of peroxisome activity with similar sensitivity but higher throughput than the traditional biochemical methods. The additional benefits of the LSC include the direct correlation between histopathologic observations and peroxisomal alterations and the potential utilization of archived formalin-fixed tissues from a variety of organs and species.

A Method for the Interpretation of Flow Cytometry Data Using Genetic Algorithms.

PubMed

Angeletti, Cesar

2018-01-01

Flow cytometry analysis is the method of choice for the differential diagnosis of hematologic disorders. It is typically performed by a trained hematopathologist through visual examination of bidimensional plots, making the analysis time-consuming and sometimes too subjective. Here, a pilot study applying genetic algorithms to flow cytometry data from normal and acute myeloid leukemia subjects is described. Initially, Flow Cytometry Standard files from 316 normal and 43 acute myeloid leukemia subjects were transformed into multidimensional FITS image metafiles. Training was performed through introduction of FITS metafiles from 4 normal and 4 acute myeloid leukemia in the artificial intelligence system. Two mathematical algorithms termed 018330 and 025886 were generated. When tested against a cohort of 312 normal and 39 acute myeloid leukemia subjects, both algorithms combined showed high discriminatory power with a receiver operating characteristic (ROC) curve of 0.912. The present results suggest that machine learning systems hold a great promise in the interpretation of hematological flow cytometry data.

Standardisation of DNA quantitation by image analysis: quality control of instrumentation.

PubMed

Puech, M; Giroud, F

1999-05-01

DNA image analysis is frequently performed in clinical practice as a prognostic tool and to improve diagnosis. The precision of prognosis and diagnosis depends on the accuracy of analysis and particularly on the quality of image analysis systems. It has been reported that image analysis systems used for DNA quantification differ widely in their characteristics (Thunissen et al.: Cytometry 27: 21-25, 1997). This induces inter-laboratory variations when the same sample is analysed in different laboratories. In microscopic image analysis, the principal instrumentation errors arise from the optical and electronic parts of systems. They bring about problems of instability, non-linearity, and shading and glare phenomena. The aim of this study is to establish tools and standardised quality control procedures for microscopic image analysis systems. Specific reference standard slides have been developed to control instability, non-linearity, shading and glare phenomena and segmentation efficiency. Some systems have been controlled with these tools and these quality control procedures. Interpretation criteria and accuracy limits of these quality control procedures are proposed according to the conclusions of a European project called PRESS project (Prototype Reference Standard Slide). Beyond these limits, tested image analysis systems are not qualified to realise precise DNA analysis. The different procedures presented in this work determine if an image analysis system is qualified to deliver sufficiently precise DNA measurements for cancer case analysis. If the controlled systems are beyond the defined limits, some recommendations are given to find a solution to the problem.

Normalization of mass cytometry data with bead standards

PubMed Central

Finck, Rachel; Simonds, Erin F.; Jager, Astraea; Krishnaswamy, Smita; Sachs, Karen; Fantl, Wendy; Pe’er, Dana; Nolan, Garry P.; Bendall, Sean C.

2013-01-01

Mass cytometry uses atomic mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of mass cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of mass cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the mass cytometer, subsequent extraction of the bead-based signature, and the application of an algorithm enabling correction of both short- and long-term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one-month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9-fold to 1.3-fold. PMID:23512433

Assessing FRET using Spectral Techniques

PubMed Central

Leavesley, Silas J.; Britain, Andrea L.; Cichon, Lauren K.; Nikolaev, Viacheslav O.; Rich, Thomas C.

2015-01-01

Förster resonance energy transfer (FRET) techniques have proven invaluable for probing the complex nature of protein–protein interactions, protein folding, and intracellular signaling events. These techniques have traditionally been implemented with the use of one or more fluorescence band-pass filters, either as fluorescence microscopy filter cubes, or as dichroic mirrors and band-pass filters in flow cytometry. In addition, new approaches for measuring FRET, such as fluorescence lifetime and acceptor photobleaching, have been developed. Hyperspectral techniques for imaging and flow cytometry have also shown to be promising for performing FRET measurements. In this study, we have compared traditional (filter-based) FRET approaches to three spectral-based approaches: the ratio of acceptor-to-donor peak emission, linear spectral unmixing, and linear spectral unmixing with a correction for direct acceptor excitation. All methods are estimates of FRET efficiency, except for one-filter set and three-filter set FRET indices, which are included for consistency with prior literature. In the first part of this study, spectrofluorimetric data were collected from a CFP–Epac–YFP FRET probe that has been used for intracellular cAMP measurements. All comparisons were performed using the same spectrofluorimetric datasets as input data, to provide a relevant comparison. Linear spectral unmixing resulted in measurements with the lowest coefficient of variation (0.10) as well as accurate fits using the Hill equation. FRET efficiency methods produced coefficients of variation of less than 0.20, while FRET indices produced coefficients of variation greater than 8.00. These results demonstrate that spectral FRET measurements provide improved response over standard, filter-based measurements. Using spectral approaches, single-cell measurements were conducted through hyperspectral confocal microscopy, linear unmixing, and cell segmentation with quantitative image analysis. Results from these studies confirmed that spectral imaging is effective for measuring subcellular, time-dependent FRET dynamics and that additional fluorescent signals can be readily separated from FRET signals, enabling multilabel studies of molecular interactions. PMID:23929684

A novel method for measuring cellular antibody uptake using imaging flow cytometry reveals distinct uptake rates for two different monoclonal antibodies targeting L1.

PubMed

Hazin, John; Moldenhauer, Gerhard; Altevogt, Peter; Brady, Nathan R

2015-08-01

Monoclonal antibodies (mAbs) have emerged as a promising tool for cancer therapy. Differing approaches utilize mAbs to either deliver a drug to the tumor cells or to modulate the host's immune system to mediate tumor kill. The rate by which a therapeutic antibody is being internalized by tumor cells is a decisive feature for choosing the appropriate treatment strategy. We herein present a novel method to effectively quantitate antibody uptake of tumor cells by using image-based flow cytometry, which combines image analysis with high throughput of sample numbers and sample size. The use of this method is established by determining uptake rate of an anti-EpCAM antibody (HEA125), from single cell measurements of plasma membrane versus internalized antibody, in conjunction with inhibitors of endocytosis. The method is then applied to two mAbs (L1-9.3, L1-OV52.24) targeting the neural cell adhesion molecule L1 (L1CAM) at two different epitopes. Based on median cell population responses, we find that mAb L1-OV52.24 is rapidly internalized by the ovarian carcinoma cell line SKOV3ip while L1 mAb 9.3 is mainly retained at the cell surface. These findings suggest the L1 mAb OV52.24 as a candidate to be further developed for drug-delivery to cancer cells, while L1-9.3 may be optimized to tag the tumor cells and stimulate immunogenic cancer cell killing. Furthermore, when analyzing cell-to-cell variability, we observed L1 mAb OV52.24 rapidly transition into a subpopulation with high-internalization capacity. In summary, this novel high-content method for measuring antibody internalization rate provides a high level of accuracy and sensitivity for cell population measurements and reveals further biologically relevant information when taking into account cellular heterogeneity. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantification of proteins by flow cytometry: Quantification of human hepatic transporter P-gp and OATP1B1 using flow cytometry and mass spectrometry.

PubMed

Hogg, Karen; Thomas, Jerry; Ashford, David; Cartwright, Jared; Coldwell, Ruth; Weston, Daniel J; Pillmoor, John; Surry, Dominic; O'Toole, Peter

2015-07-01

Flow cytometry is a powerful tool for the quantitation of fluorescence and is proven to be able to correlate the fluorescence intensity to the number of protein on cells surface. Mass spectroscopy can also be used to determine the number of proteins per cell. Here we have developed two methods, using flow cytometry and mass spectroscopy to quantify number of transporters in human cells. These two approaches were then used to analyse the same samples so that a direct comparison could be made. Transporters have a major impact on the behaviour of a diverse number of drugs in human systems. While active uptake studies by transmembrane protein transporters using model substrates are routinely undertaken in human cell lines and hepatocytes as part of drug discovery and development, the interpretation of these results is currently limited by the inability to quantify the number of transporters present in the test samples. Here we provide a flow cytometric method for accurate quantification of transporter levels both on the cell surface and within the cell, and compare this to a quantitative mass spectrometric approach. Two transporters were selected for the study: OATP1B1 (also known as SLCO1B1, LST-1, OATP-C, OATP2) due to its important role in hepatic drug uptake and elimination; P-gp (also known as P-glycoprotein, MDR1, ABCB1) as a well characterised system and due to its potential impact on oral bioavailability, biliary and renal clearance, and brain penetration of drugs that are substrates for this transporter. In all cases the mass spectrometric method gave higher levels than the flow cytometry method. However, the two methods showed very similar trends in the relative ratios of both transporters in the hepatocyte samples investigated. The P-gp antibody allowed quantitative discrimination between externally facing transporters located in the cytoplasmic membrane and the total number of transporters on and in the cell. The proportion of externally facing transporter varied considerably in the four hepatocyte samples analysed, ranging from only 6% to 35% of intact and viable cells. The sample with only 6% externally facing transporter was further analysed by confocal microscopy which qualitatively confirmed the low level of transporter in the membrane and the large internal population. Here we prove that flow cytometry is an important tool for future protein analysis as it can not only quantify the number of proteins that a cell express but also identify the number of proteins on the surface and it is easy to apply for routine assays. Copyright © 2015 Elsevier Inc. All rights reserved.

Role of receptor occupancy assays by flow cytometry in drug development.

PubMed

Stewart, Jennifer J; Green, Cherie L; Jones, Nicholas; Liang, Meina; Xu, Yuanxin; Wilkins, Danice E C; Moulard, Maxime; Czechowska, Kamila; Lanham, David; McCloskey, Thomas W; Ferbas, John; van der Strate, Barry W A; Högerkorp, Carl-Magnus; Wyant, Timothy; Lackey, Alan; Litwin, Virginia

2016-03-01

The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent. Data generated from RO assays can also be used to model whether given doses of an experimental therapeutic agent and their administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent. There are a variety of approaches that can be used when undertaking RO assays and with the ability to measure distinct subsets in heterogeneous populations, flow cytometry is ideally suited to RO measurements. This article highlights the importance of RO assays on the flow cytometric platform in the development of biotherapeutic agents. © 2016 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc.

FlowCam: Quantification and Classification of Phytoplankton by Imaging Flow Cytometry.

PubMed

Poulton, Nicole J

2016-01-01

The ability to enumerate, classify, and determine biomass of phytoplankton from environmental samples is essential for determining ecosystem function and their role in the aquatic community and microbial food web. Traditional micro-phytoplankton quantification methods using microscopic techniques require preservation and are slow, tedious and very laborious. The availability of more automated imaging microscopy platforms has revolutionized the way particles and cells are detected within their natural environment. The ability to examine cells unaltered and without preservation is key to providing more accurate cell concentration estimates and overall phytoplankton biomass. The FlowCam(®) is an imaging cytometry tool that was originally developed for use in aquatic sciences and provides a more rapid and unbiased method for enumerating and classifying phytoplankton within diverse aquatic environments.

Point process models for localization and interdependence of punctate cellular structures.

PubMed

Li, Ying; Majarian, Timothy D; Naik, Armaghan W; Johnson, Gregory R; Murphy, Robert F

2016-07-01

Accurate representations of cellular organization for multiple eukaryotic cell types are required for creating predictive models of dynamic cellular function. To this end, we have previously developed the CellOrganizer platform, an open source system for generative modeling of cellular components from microscopy images. CellOrganizer models capture the inherent heterogeneity in the spatial distribution, size, and quantity of different components among a cell population. Furthermore, CellOrganizer can generate quantitatively realistic synthetic images that reflect the underlying cell population. A current focus of the project is to model the complex, interdependent nature of organelle localization. We built upon previous work on developing multiple non-parametric models of organelles or structures that show punctate patterns. The previous models described the relationships between the subcellular localization of puncta and the positions of cell and nuclear membranes and microtubules. We extend these models to consider the relationship to the endoplasmic reticulum (ER), and to consider the relationship between the positions of different puncta of the same type. Our results do not suggest that the punctate patterns we examined are dependent on ER position or inter- and intra-class proximity. With these results, we built classifiers to update previous assignments of proteins to one of 11 patterns in three distinct cell lines. Our generative models demonstrate the ability to construct statistically accurate representations of puncta localization from simple cellular markers in distinct cell types, capturing the complex phenomena of cellular structure interaction with little human input. This protocol represents a novel approach to vesicular protein annotation, a field that is often neglected in high-throughput microscopy. These results suggest that spatial point process models provide useful insight with respect to the spatial dependence between cellular structures. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Connecting the dots across time: reconstruction of single-cell signalling trajectories using time-stamped data

NASA Astrophysics Data System (ADS)

Mukherjee, Sayak; Stewart, David; Stewart, William; Lanier, Lewis L.; Das, Jayajit

2017-08-01

Single-cell responses are shaped by the geometry of signalling kinetic trajectories carved in a multidimensional space spanned by signalling protein abundances. It is, however, challenging to assay a large number (more than 3) of signalling species in live-cell imaging, which makes it difficult to probe single-cell signalling kinetic trajectories in large dimensions. Flow and mass cytometry techniques can measure a large number (4 to more than 40) of signalling species but are unable to track single cells. Thus, cytometry experiments provide detailed time-stamped snapshots of single-cell signalling kinetics. Is it possible to use the time-stamped cytometry data to reconstruct single-cell signalling trajectories? Borrowing concepts of conserved and slow variables from non-equilibrium statistical physics we develop an approach to reconstruct signalling trajectories using snapshot data by creating new variables that remain invariant or vary slowly during the signalling kinetics. We apply this approach to reconstruct trajectories using snapshot data obtained from in silico simulations, live-cell imaging measurements, and, synthetic flow cytometry datasets. The application of invariants and slow variables to reconstruct trajectories provides a radically different way to track objects using snapshot data. The approach is likely to have implications for solving matching problems in a wide range of disciplines.

Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level.

PubMed

Nerada, Zsuzsanna; Hegyi, Zoltán; Szepesi, Áron; Tóth, Szilárd; Hegedüs, Csilla; Várady, György; Matula, Zsolt; Homolya, László; Sarkadi, Balázs; Telbisz, Ágnes

2016-09-01

ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-06-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-04-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

A Flow Cytometry-Based Assay for Quantifying Non-Plaque Forming Strains of Yellow Fever Virus

PubMed Central

Hammarlund, Erika; Amanna, Ian J.; Dubois, Melissa E.; Barron, Alex; Engelmann, Flora; Messaoudi, Ilhem; Slifka, Mark K.

2012-01-01

Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE) on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar) could not be measured by plaque assay (PA), focus-forming assay (FFA), or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6) and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA) to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine). Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses. PMID:23028428

A flow cytometry-based assay for quantifying non-plaque forming strains of yellow fever virus.

PubMed

Hammarlund, Erika; Amanna, Ian J; Dubois, Melissa E; Barron, Alex; Engelmann, Flora; Messaoudi, Ilhem; Slifka, Mark K

2012-01-01

Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE) on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar) could not be measured by plaque assay (PA), focus-forming assay (FFA), or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6) and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA) to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine). Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses.

Quantitative analysis of gold and carbon nanoparticles in mammalian cells by flow cytometry light scattering

NASA Astrophysics Data System (ADS)

Zhou, Gang; Liu, Naicheng; Wang, Zhenheng; Shi, Tongguo; Gan, Jingjing; Wang, Zhenzhen; Zhang, Junfeng

2017-02-01

Nanoparticle-based applications for diagnostics and therapeutics have been extensively studied. These applications require a profound understanding of the fate of nanoparticles (NPs) in cellular environments. However, until now, few analytical methods are available and most of them rely on fluorescent properties or special elements of NPs; therefore, for NPs without observable optical properties or special elements, the existing methods are hardly applicable. In this study, we introduce a flow cytometry light scattering (FCLS)-based approach that quantifies in situ NPs accurately in mammalian cells. Continuous cells of heterogeneous human epithelial colorectal adenocarcinoma (Caco-2 cells), mouse peritoneal macrophages (MPM), and human adenocarcinomic alveolar basal epithelia (A549 cells) were cultured with NPs with certain concentrations and size. The intensity of the flow cytometric side scattered light, which indicates the quantity of NPs in the cells, was analyzed. The result shows an accurate size- and dose-dependent uptake of Au NPs (5, 30, 250 nm) in Caco-2 cells. The size- and dose- dependence of Au NPs (5, 30, 250 nm) and carbon NPs (50, 500 nm) in cells was validated by transmission electron microscope (TEM). This paper demonstrates the great potential of flow cytometry light scattering in the quantitative study of the size and dose effect on in situ metallic or non-metallic NPs in mammalian cells.

An imaging flow cytometry method to assess ricin trafficking in A549 human lung epithelial cells.

PubMed

Jenner, Dominic; Chong, Damien; Walker, Nicola; Green, A Christopher

2018-02-01

The endocytosis and trafficking of ricin in mammalian cells is an important area of research for those producing ricin anti-toxins and other ricin therapeutics. Ricin trafficking is usually observed by fluorescence microscopy techniques. This gives good resolution and leads to a detailed understanding of the internal movement of ricin within cells. However, microscopy techniques are often hampered by complex analysis and quantification techniques, and the inability to look at ricin trafficking in large populations of cells. In these studies we have directly labelled ricin and assessed if its trafficking can be observed using Imaging Flow Cytometry (IFC) both to the cytoplasmic region of cells and specifically to the Golgi apparatus. Using IDEAS® data analysis software the specific fluorescence location of the ricin within the cells was analysed. Then, using cytoplasmic masking techniques to quantify the number of cells with endocytosed cytoplasmic ricin or cells with Golgi-associated ricin, kinetic endocytosis curves were generated. Here we present, to the authors' knowledge, the first example of using imaging flow cytometry for evaluating the subcellular transport of protein cargo, using the trafficking of ricin toxin in lung cells as a model. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry.

PubMed

Chan, Leo Li-Ying; Smith, Tim; Kumph, Kendra A; Kuksin, Dmitry; Kessel, Sarah; Déry, Olivier; Cribbes, Scott; Lai, Ning; Qiu, Jean

2016-10-01

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.

A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity.

PubMed

O'Brien-Simpson, Neil M; Pantarat, Namfon; Attard, Troy J; Walsh, Katrina A; Reynolds, Eric C

2016-01-01

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy.

Quantitative Analysis of Protein Translocations by Microfluidic Total Internal Reflection Fluorescence Flow Cytometry

PubMed Central

Wang, Jun; Fei, Bei; Geahlen, Robert L.

2010-01-01

Protein translocation, or the change in a protein’s location between different subcellular compartments, is a critical process by which intracellular proteins carry out their cellular functions. Aberrant translocation events contribute to various diseases ranging from metabolic disorders to cancer. In this study, we demonstrate the use of a newly developed single-cell tool, microfluidic total internal reflection fluorescence flow cytometry (TIRF-FC), for detecting both cytosol to plasma membrane and cytosol to nucleus translocations using the tyrosine kinase Syk and the transcription factor NF-κB as models. This technique detects fluorescent molecules at the plasma membrane and in the membrane-proximal cytosol in single cells. We were able to record quantitatively changes in the fluorescence density in the evanescent field associated with these translocation processes for large cell populations with single cell resolution. We envision that TIRF-FC will provide a new approach to explore the molecular biology and clinical relevance of protein translocations. PMID:20820633

In vitro interactions between splenocytes and dansylamide dye-embedded nanoparticles detected by flow cytometry

PubMed Central

Nyland, Jennifer F.; Bai, Jennifer J. K.; Katz, Howard E.; Silbergeld, Ellen K.

2009-01-01

Engineered nanoparticles (NPs) possess a range of biological activity. In vitro methods for assessing toxicity and efficacy would be enhanced by simultaneous quantitative information on the behavior of NPs in culture systems and signals of cell response. We have developed a method for visualizing NPs within cells using standard flow cytometric techniques and uniquely designed spherical siloxane NPs with an embedded (covalently bound) dansylamide dye. This method allowed NP visualization without obscuring detection of relevant biomarkers of cell subtype, activation state, and other events relevant to assessing bioactivity. We determined that NPs penetrated cells and induced a range of biological signals consistent with activation and costimulation. These results indicate that NPs may affect cell function at concentrations below those inducing cytotoxicity or apoptosis and demonstrate a novel method to image both localization of NPs and cell-level effects. PMID:19523425

Computational and experimental single cell biology techniques for the definition of cell type heterogeneity, interplay and intracellular dynamics.

PubMed

de Vargas Roditi, Laura; Claassen, Manfred

2015-08-01

Novel technological developments enable single cell population profiling with respect to their spatial and molecular setup. These include single cell sequencing, flow cytometry and multiparametric imaging approaches and open unprecedented possibilities to learn about the heterogeneity, dynamics and interplay of the different cell types which constitute tissues and multicellular organisms. Statistical and dynamic systems theory approaches have been applied to quantitatively describe a variety of cellular processes, such as transcription and cell signaling. Machine learning approaches have been developed to define cell types, their mutual relationships, and differentiation hierarchies shaping heterogeneous cell populations, yielding insights into topics such as, for example, immune cell differentiation and tumor cell type composition. This combination of experimental and computational advances has opened perspectives towards learning predictive multi-scale models of heterogeneous cell populations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development and Evaluation of Transgenic Nude Mice Expressing Ubiquitous Green Fluorescent Protein.

PubMed

Iyer, Srikanth; Arindkar, Shailendra; Mishra, Alaknanda; Manglani, Kapil; Kumar, Jerald Mahesh; Majumdar, Subeer S; Upadhyay, Pramod; Nagarajan, Perumal

2015-08-01

Researchers had developed and characterized transgenic green/red fluorescent protein (GFP/RFP) nude mouse with ubiquitous RFP or GFP expression, but none has evaluated the level of immune cells and expression levels of GFP in this model. The nude GFP mice were evaluated by imaging, hematological indices, and flow cytometry to compare the proportion of immune T cells. Quantitative real-time PCR (qRT-PCR) was done for evaluating the relative expression of GFP transcripts in few organs of the nude GFP mice. The hematological and immune cells of nude GFP were within the range of nude mice. However, the gene expression levels were relatively less in various tissues compared with B6 GFP mice. These findings suggest that nude GFP is an ideal model resembling normal nude mice; however, GFP expression in various tissues by fluorescence should be considered, as the expression of GFP differs in various organs.

[A test of screening to predict lung cancer among dust-exposed tin miners with sputum imaging cytometry].

PubMed

Liu, Yue-wei; Chen, Jing-qiong; Sun, Xiao-rong; Yang, Jian; Liu, Yong; Chen, Wei-hong

2008-04-01

To evaluate efficacy of sputum imaging cytometry in early diagnosis on lung cancer among tin miners exposed to dust and analyze possible risk factors related to lung cancer among tin miners. From a cohort of tin miners in Guangxi Province, a total of 345 male tin miners higher than 45 years old and with high exposure to crystalline silica dust were randomly selected as the objects. Imaging cytometry was used to implement the sputum analysis for the screening on lung cancer according to the experience diagnostic standard. All objects were then followed up to the end of 2006. Clinical diagnosis of lung cancer was used as the golden standard to evaluate the efficacy of screening. From 1998 to 2006, 11 new cases were diagnosed as clinical lung cancer. Except of age and exposure to occupational hazards, smoking status (P = 0.0384) and mean smoking dose (P = 0.0078) were significantly associated with lung cancer, and the adjusted odds ratio of high level to the low was 18.21 (2.15 approximately 154.39). The sensitivity, specificity and Youden's index of the sputum imaging cytometry for the experience diagnosis were 27.3%, 83.9% and 11.2% respectively. According to the ROC curve analysis, area under ROC Curve (AUC) of C2.5 (the percentage when the DNA index ranged from 1.25 to 2.50) was 0.647 (0.525 approximately 0.768), with the optimal operating point (OOP) of 1.70%. Sensitivity, specificity, agreement rate, positive predictive value, negative predictive value and Youden's index for predicting lung cancers in high-exposure tin miners were found to be 72.7%, 62.3%, 62.6%, 6.0%, 98.6% and 35.0% respectively. Smoking is confirmed as an important risk factor of lung cancer in tin miners. The diagnostic efficiency can be improved if the diagnostic point of C2.5 is adjusted to 1.70%.

[Flow cytometry in datecting lymph node micrometastasis in colorectal cancer].

PubMed

Sun, Q; Ding, Y; Zhang, J

2001-01-25

To study the methodology and significance of flow cytometry in detecting lymph node micrometastasis of colorectal cancer. One hundred sixty-two cellular suspensions were prepared with lymph nodes which were resected radically on 25 patients with colorectal cancer and in which no cancer cells were found by HE staining. Different concentrations of cultured Lovo colorectal cancer cells were added into the celular suspension prepared from lymph node tissue of persons without colorectal cancer in order to prepare a control model. Dual staining with CK/FTTC and PI was made to the sedimetns from those 2 kinds of suspension. Flow cytometry was used to detect cancer cells. An ideal correlation was obtained between the detection value and the theoretical value of cancer cells in the specimen suspensions and control models (r = 0.097 6) with a sensitivity rate of 10/10(5). Cancer cells were detected from 7 out of the 25 patients and 30 of the 162 cellular suspensions. The detection rate was correlated with the size and infiltrating depth of the cancer. Flow cytometry is a reliable, rapid, and quantitative method for detecting lymph node micrometastasis in colorectal cancer.

Highly multiparametric analysis by mass cytometry.

PubMed

Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Nitz, Mark; Winnik, Mitchell A; Tanner, Scott

2010-09-30

This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays. Copyright © 2010 Elsevier B.V. All rights reserved.

An open-source solution for advanced imaging flow cytometry data analysis using machine learning.

PubMed

Hennig, Holger; Rees, Paul; Blasi, Thomas; Kamentsky, Lee; Hung, Jane; Dao, David; Carpenter, Anne E; Filby, Andrew

2017-01-01

Imaging flow cytometry (IFC) enables the high throughput collection of morphological and spatial information from hundreds of thousands of single cells. This high content, information rich image data can in theory resolve important biological differences among complex, often heterogeneous biological samples. However, data analysis is often performed in a highly manual and subjective manner using very limited image analysis techniques in combination with conventional flow cytometry gating strategies. This approach is not scalable to the hundreds of available image-based features per cell and thus makes use of only a fraction of the spatial and morphometric information. As a result, the quality, reproducibility and rigour of results are limited by the skill, experience and ingenuity of the data analyst. Here, we describe a pipeline using open-source software that leverages the rich information in digital imagery using machine learning algorithms. Compensated and corrected raw image files (.rif) data files from an imaging flow cytometer (the proprietary .cif file format) are imported into the open-source software CellProfiler, where an image processing pipeline identifies cells and subcellular compartments allowing hundreds of morphological features to be measured. This high-dimensional data can then be analysed using cutting-edge machine learning and clustering approaches using "user-friendly" platforms such as CellProfiler Analyst. Researchers can train an automated cell classifier to recognize different cell types, cell cycle phases, drug treatment/control conditions, etc., using supervised machine learning. This workflow should enable the scientific community to leverage the full analytical power of IFC-derived data sets. It will help to reveal otherwise unappreciated populations of cells based on features that may be hidden to the human eye that include subtle measured differences in label free detection channels such as bright-field and dark-field imagery. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Hyperchromatic laser scanning cytometry

NASA Astrophysics Data System (ADS)

Tárnok, Attila; Mittag, Anja

2007-02-01

In the emerging fields of high-content and high-throughput single cell analysis for Systems Biology and Cytomics multi- and polychromatic analysis of biological specimens has become increasingly important. Combining different technologies and staining methods polychromatic analysis (i.e. using 8 or more fluorescent colors at a time) can be pushed forward to measure anything stainable in a cell, an approach termed hyperchromatic cytometry. For cytometric cell analysis microscope based Slide Based Cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide. Based on the feature of relocation identical cells can be subsequently reanalyzed. In this manner data on the single cell level after manipulation steps can be collected. In this overview various components for hyperchromatic cytometry are demonstrated for a SBC instrument, the Laser Scanning Cytometer (Compucyte Corp., Cambridge, MA): 1) polychromatic cytometry, 2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating fluorochromes by their different photostability), 4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and 5) photodestruction (destruction of FRET dyes). With the intelligent combination of several of these techniques hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on the identical cell. The combination of high-throughput and high-content SBC analysis with high-resolution confocal imaging allows clear verification of phenotypically distinct subpopulations of cells with structural information. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

Cutting-edge analysis of extracellular microparticles using ImageStream(X) imaging flow cytometry.

PubMed

Headland, Sarah E; Jones, Hefin R; D'Sa, Adelina S V; Perretti, Mauro; Norling, Lucy V

2014-06-10

Interest in extracellular vesicle biology has exploded in the past decade, since these microstructures seem endowed with multiple roles, from blood coagulation to inter-cellular communication in pathophysiology. In order for microparticle research to evolve as a preclinical and clinical tool, accurate quantification of microparticle levels is a fundamental requirement, but their size and the complexity of sample fluids present major technical challenges. Flow cytometry is commonly used, but suffers from low sensitivity and accuracy. Use of Amnis ImageStream(X) Mk II imaging flow cytometer afforded accurate analysis of calibration beads ranging from 1 μm to 20 nm; and microparticles, which could be observed and quantified in whole blood, platelet-rich and platelet-free plasma and in leukocyte supernatants. Another advantage was the minimal sample preparation and volume required. Use of this high throughput analyzer allowed simultaneous phenotypic definition of the parent cells and offspring microparticles along with real time microparticle generation kinetics. With the current paucity of reliable techniques for the analysis of microparticles, we propose that the ImageStream(X) could be used effectively to advance this scientific field.

White blood cell segmentation by color-space-based k-means clustering.

PubMed

Zhang, Congcong; Xiao, Xiaoyan; Li, Xiaomei; Chen, Ying-Jie; Zhen, Wu; Chang, Jun; Zheng, Chengyun; Liu, Zhi

2014-09-01

White blood cell (WBC) segmentation, which is important for cytometry, is a challenging issue because of the morphological diversity of WBCs and the complex and uncertain background of blood smear images. This paper proposes a novel method for the nucleus and cytoplasm segmentation of WBCs for cytometry. A color adjustment step was also introduced before segmentation. Color space decomposition and k-means clustering were combined for segmentation. A database including 300 microscopic blood smear images were used to evaluate the performance of our method. The proposed segmentation method achieves 95.7% and 91.3% overall accuracy for nucleus segmentation and cytoplasm segmentation, respectively. Experimental results demonstrate that the proposed method can segment WBCs effectively with high accuracy.

In vitro and in vivo characterization of a dual-function green fluorescent protein--HSV1-thymidine kinase reporter gene driven by the human elongation factor 1 alpha promoter.

PubMed

Luker, Gary D; Luker, Kathryn E; Sharma, Vijay; Pica, Christina M; Dahlheimer, Julie L; Ocheskey, Joe A; Fahrner, Timothy J; Milbrandt, Jeffrey; Piwnica-Worms, David

2002-01-01

Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP) and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK) as a reporter gene driven by the promoter for human elongation factor 1 alpha (EF-1 alpha-EGFP-TK). Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV). As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK) in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP) and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) approximately 8-[3H]penciclovir (8-[3H]PCV) < 2'-fluoro-2'-deoxy-5-iodouracil-beta-D-arabinofuranoside (2-[14C]FIAU). Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.

Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment.

PubMed

Mietke, Alexander; Otto, Oliver; Girardo, Salvatore; Rosendahl, Philipp; Taubenberger, Anna; Golfier, Stefan; Ulbricht, Elke; Aland, Sebastian; Guck, Jochen; Fischer-Friedrich, Elisabeth

2015-11-17

Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment

PubMed Central

Mietke, Alexander; Otto, Oliver; Girardo, Salvatore; Rosendahl, Philipp; Taubenberger, Anna; Golfier, Stefan; Ulbricht, Elke; Aland, Sebastian; Guck, Jochen; Fischer-Friedrich, Elisabeth

2015-01-01

Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible. PMID:26588562

Principles and applications of flow cytometry and cell sorting in companion animal medicine.

PubMed

Wilkerson, Melinda J

2012-01-01

Flow cytometry measures multiple characteristic of single cells using light scatter properties and fluorescence properties of fluorescent probes with specificity to cellular constituents. The use of flow cytometry in the veterinary clinical laboratory has become more routine in veterinary diagnostic laboratories and institutions (http://www.vet.k-state.edu/depts/dmp/service/immunology/index.htm), and reference laboratories. The most common applications in small animal medicine includes quantitation of erythrocytes and leukocytes in automated hematology instruments, detection of antibodies to erythrocytes and platelets in cases of immune-mediated diseases, immunophenotyping of leukocytes and lymphocytes in immunodeficiency syndromes, or leukemias and lymphomas. DNA content analysis to identify aneuploidy or replicating cells in tumor preparations has not gained routine acceptance because of the variability of prognostic results. Other applications including cell sorting and multiplexing using microspheres are potential assays of the future once they become validated and the instrumentation footprint becomes more and more compact, less expensive, and easier to use.

High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry

PubMed Central

Bonar, Micha M.; Tilton, John C.

2017-01-01

Detection of viruses by flow cytometry is complicated by their small size. Here, we characterized the ability of a standard (FACSAria II) and a sub-micron flow cytometer (A50 Micro) to resolve HIV-1 viruses. The A50 was superior at resolving small particles but did not reliably distinguish HIV-1, extracellular vesicles, and laser noise by light scatter properties alone. However, single fluorescent HIV-1 particles could readily be detected by both cytometers. Fluorescent particles were sorted and retained infectivity, permitting further exploration of the functional consequences of HIV-1 heterogeneity. Finally, flow cytometry had a limit of detection of 80 viruses/ml, nearly equal to PCR assays. These studies demonstrate the power of flow cytometry to detect and sort viral particles and provide a critical toolkit to validate methods to label wild-type HIV-1; quantitatively assess integrity and aggregation of viruses and virus-based therapeutics; and efficiently screen drugs inhibiting viral assembly and release. PMID:28235684

High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry.

PubMed

Bonar, Michał M; Tilton, John C

2017-05-01

Detection of viruses by flow cytometry is complicated by their small size. Here, we characterized the ability of a standard (FACSAria II) and a sub-micron flow cytometer (A50 Micro) to resolve HIV-1 viruses. The A50 was superior at resolving small particles but did not reliably distinguish HIV-1, extracellular vesicles, and laser noise by light scatter properties alone. However, single fluorescent HIV-1 particles could readily be detected by both cytometers. Fluorescent particles were sorted and retained infectivity, permitting further exploration of the functional consequences of HIV-1 heterogeneity. Finally, flow cytometry had a limit of detection of 80 viruses/ml, nearly equal to PCR assays. These studies demonstrate the power of flow cytometry to detect and sort viral particles and provide a critical toolkit to validate methods to label wild-type HIV-1; quantitatively assess integrity and aggregation of viruses and virus-based therapeutics; and efficiently screen drugs inhibiting viral assembly and release. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

PubMed Central

Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

2013-01-01

The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

Detection of antibody to Purpureocillium lilacinum by immunofluorescent assay and flow cytometry in serum of infected C57BL/6 mice.

PubMed

de Sequeira, Danielly C M; Peixoto, Mariana L P; De Luca, Paula M; Oliveira-Ferreira, Joseli; Antas, Paulo R Z; Borba, Cintia M

2013-10-31

Purpureocillium lilacinum is an emerging pathogenic fungus that can cause different clinical manifestations ranging from cutaneous and sub-cutaneous infections to severe oculomycosis. In this study, using both conventional indirect immunofluorescence and non-conventional flow cytometry approaches, IgG antibodies were readily detected in both C57BL/6 immunocompetent and immunosuppressed mice after i.v. infection with P. lilacinum. The humoral immune response was specific, since virtually no antibodies were detected in the serum of control mice. Flow cytometry assays also showed both quantitative and qualitative differences in total IgG and its isotypes in sera of immunocompetent and immunosupressed infected mice. Although a good starting point, it is clear that the effectiveness of serological assays for P. lilacinum hyalohyphomycosis identification in clinical studies still requires further standardization. Upon further validation in humans, these techniques have the potential to be suitable to detect P. lilacinum infection in patients, thereby avoiding current laborious and time-consuming culture techniques. © 2013.

Multiplex immunoassay for persistent organic pollutants in tilapia: Comparison of imaging- and flow cytometry-based platforms using spectrally encoded paramagnetic microspheres

USDA-ARS?s Scientific Manuscript database

Recent developments in spectrally encoded microspheres (SEMs)-based technologies provide high multiplexing possibilities. Most SEMs-based assays required a flow cytometer with sophisticated fluidics and optics. The new imaging superparamagnetic SEMs-based platform transports SEMs with considerably ...

Quantitative Magnetic Separation of Particles and Cells using Gradient Magnetic Ratcheting

PubMed Central

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-01-01

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting (MACS), are robust but perform coarse, qualitative separations based on surface antigen expression. We report a quantitative magnetic separation technology using high-force magnetic ratcheting over arrays of magnetically soft micro-pillars with gradient spacing, and use the system to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micro-pillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic-field. Particles with higher IOC separate and equilibrate along the miro-pillar array at larger pitches. We develop a semi-analytical model that predicts behavior for particles and cells. Using the system, LNCaP cells were separated based on the bound quantity of 1μm anti-EpCAM particles as a metric for expression. The ratcheting cytometry system was able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof of concept, EpCAM-labeled cells from patient blood were isolated with 74% purity, demonstrating potential towards a quantitative magnetic separation instrument. PMID:26890496

[Clinical usefulness of urine-formed elements' information obtained from bacteria detection by flow cytometry method that uses nucleic acid staining].

PubMed

Nakagawa, Hiroko; Yuno, Tomoji; Itho, Kiichi

2009-03-01

Recently, specific detection method for Bacteria, by flow cytometry method using nucleic acid staining, was developed as a function of automated urine formed elements analyzer for routine urine testing. Here, we performed a basic study on this bacteria analysis method. In addition, we also have a comparison among urine sediment analysis, urine Gram staining and urine quantitative cultivation, the conventional methods performed up to now. As a result, the bacteria analysis with flow cytometry method that uses nucleic acid staining was excellent in reproducibility, and higher sensitivity compared with microscopic urinary sediment analysis. Based on the ROC curve analysis, which settled urine culture method as standard, cut-off level of 120/microL was defined and its sensitivity = 85.7%, specificity = 88.2%. In the analysis of scattergram, accompanied with urine culture method, among 90% of rod positive samples, 80% of dots were appeared in the area of 30 degrees from axis X. In addition, one case even indicated that analysis of bacteria by flow cytometry and scattergram of time series analysis might be helpful to trace the progress of causative bacteria therefore the information supposed to be clinically significant. Reporting bacteria information with nucleic acid staining flow cytometry method is expected to contribute to a rapid diagnostics and treatment of urinary tract infections. Besides, the contribution to screening examination of microbiology and clinical chemistry, will deliver a more efficient solution to urine analysis.

Quantitative evaluation of morphological changes in activated platelets in vitro using digital holographic microscopy.

PubMed

Kitamura, Yutaka; Isobe, Kazushige; Kawabata, Hideo; Tsujino, Tetsuhiro; Watanabe, Taisuke; Nakamura, Masayuki; Toyoda, Toshihisa; Okudera, Hajime; Okuda, Kazuhiro; Nakata, Koh; Kawase, Tomoyuki

2018-06-18

Platelet activation and aggregation have been conventionally evaluated using an aggregometer. However, this method is suitable for short-term but not long-term quantitative evaluation of platelet aggregation, morphological changes, and/or adhesion to specific materials. The recently developed digital holographic microscopy (DHM) has enabled the quantitative evaluation of cell size and morphology without labeling or destruction. Thus, we aim to validate its applicability in quantitatively evaluating changes in cell morphology, especially in the aggregation and spreading of activated platelets, thus modifying typical image analysis procedures to suit aggregated platelets. Freshly prepared platelet-rich plasma was washed with phosphate-buffered saline and treated with 0.1% CaCl 2 . Platelets were then fixed and subjected to DHM, scanning electron microscopy (SEM), atomic force microscopy, optical microscopy, and flow cytometry (FCM). Tightly aggregated platelets were identified as single cells. Data obtained from time-course experiments were plotted two-dimensionally according to the average optical thickness versus attachment area and divided into four regions. The majority of the control platelets, which supposedly contained small and round platelets, were distributed in the lower left region. As activation time increased, however, this population dispersed toward the upper right region. The distribution shift demonstrated by DHM was essentially consistent with data obtained from SEM and FCM. Therefore, DHM was validated as a promising device for testing platelet function given that it allows for the quantitative evaluation of activation-dependent morphological changes in platelets. DHM technology will be applicable to the quality assurance of platelet concentrates, as well as diagnosis and drug discovery related to platelet functions. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Means: Cytometry and Mass Spectrometry Converge in a Single Cell Deep Profiling Platform

PubMed Central

Weis-Garcia, Frances; Bandura, Dmitry; Baranov, Vladimir; Ornatsky, Olga; Tanner, Scott

2013-01-01

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a distinct flavor of mass spectrometry that has had little association with cell biology: it remains the state of the art for the determination of the atomic composition of materials. Unrelatedly, flow cytometry is the superior method for distinguishing the heterogeneity of cells through the determination of antigen signatures using tagged antibodies. Simply replacing fluorophore tags with stable isotopes of the heavy metals, and measuring these cell-by-cell with ICP-MS, dramatically increases the number of probes that can be simultaneously measured in cytometry and enables a transformative increase in the resolution of rare cell populations in complex biological samples. While this can be thought of as a novel incarnation of single-cell targeted proteomics, the metal-labeling reagents, ICP-MS of single cells, and accompanying informatics comprise a new field of technology termed Mass Cytometry. While the conception of mass cytometry is simple the embodiment to address the issues of multi-parameter flow cytometry has been far more challenging. There are many elements, and many more stable isotopes of those elements, that might be used as distinct reporter tags. Still, there are many approaches to conjugating metals to antibodies (or other affinity reagents) and work in this area along with developing new applications is ongoing. The mass resolution and linear (quantitative) dynamic range of ICP-MS allows those many stable isotopes to be measured simultaneously and without the spectral overlap issues that limit fluorescence assay. However, the adaptation of ICP-MS to allow high-speed simultaneous measurement with single cell distinction at high throughput required innovation of the cell introduction system, ion optics (sampling, transmission and beam-shaping), mass analysis, and signal handling and processing. An overview of “the nuts and bolts” of Mass Cytometry is presented.

Clinical and laboratory applications of slide-based cytometry with the LSC, SFM, and the iCYTE imaging cytometer instruments

NASA Astrophysics Data System (ADS)

Bocsi, Jozsef; Luther, Ed; Mittag, Anja; Jensen, Ingo; Sack, Ulrich; Lenz, Dominik; Trezl, Lajos; Varga, Viktor S.; Molnar, Beea; Tarnok, Attila

2004-06-01

Background: Slide based cytometry (SBC) is a technology for the rapid stoichiometric analysis of cells fixed to surfaces. Its applications are highly versatile and ranges from the clinics to high throughput drug discovery. SBC is realized in different instruments such as the Laser Scanning Cytometer (LSC) and Scanning Fluorescent Microscope (SFM) and the novel inverted microscope based iCyte image cytometer (Compucyte Corp.). Methods: Fluorochrome labeled specimens were immobilized on microscopic slides. They were placed on a conventional fluorescence microscope and analyzed by photomultiplayers or digital camera. Data comparable to flow cytometry were generated. In addition, each individual event could be visualized. Applications: The major advantage of instruments is the combination of two features: a) the minimal sample volume needed, and b) the connection of fluorescence data and morphological information. Rare cells were detected, frequency of apoptosis by myricetin formaldehyde and H2O2 mixtures was determined;. Conclusion: LSC, SFM and the novel iCyte have a wide spectrum of applicability in SBC and can be introduced as a standard technology for multiple settings. In addition, the iCyte and SFM instrument is suited for high throughput screening by automation and may be in future adapted to telepathology due to their high quality images. (This study was supported by the IZKF-Leipzig, Germany and T 034245 OTKA, Hungary)

Mean frequency and relative fluorescence intensity measurement of γ-H2AX foci dose response in PBL exposed to γ-irradiation: An inter- and intra-laboratory comparison and its relevance for radiation triage.

PubMed

Venkateswarlu, Raavi; Tamizh, Selvan G; Bhavani, Manivannan; Kumar, Arun; Alok, Amit; Karthik, Kanagaraj; Kalra, Namita; Vijayalakshmi, J; Paul, Solomon F D; Chaudhury, N K; Venkatachalam, Perumal

2015-12-01

Measurement of γ-H2AX protein changes in the peripheral blood lymphocytes (PBL) of individuals exposed to ionizing radiation is a simple, sensitive, and rapid assay for radiation triage and early marker of dose estimation. The qualitative and quantitative measurements of the protein changes were examined using flow cytometry and microscopy. Whole blood and isolated lymphocytes were exposed in vitro between 0.1 and 5 Gy doses of (60) Co γ-radiation at a dose rate of 1 Gy/min. Radiation induced γ-H2AX foci frequency (n = 3) and relative fluorescence intensity (n = 7) in PBL was measured at 0.5 and 2 hrs postexposure. The observed dose response for γ-H2AX foci frequency at both time points, for whole blood and isolated lymphocytes did not show any significant (P > 0.05) differences. However, when compared with γ-H2AX foci frequency scored manually (microscopy), the semiautomated analysis (captured images) showed a better correlation (r(2) = 0.918) than that obtained with automated (Metafer) scoring (r(2) = 0.690). It is noteworthy to mention that, the γ-H2AX foci frequency quantified using microscopy showed a dose dependent increase up to 2 Gy and the relative fluorescence intensity (RFI) measured with flow cytometry revealed an increase up to 5 Gy in the PBL exposed in vitro. Moreover, a better correlation was observed between the γ-H2AX foci frequency obtained by manual scoring and RFI (r(2) = 0.910). Kinetic studies showed that the γ-H2AX foci remain more or less unchanged up to 4 hrs and reduces gradually over 48 hrs of postexposure at 37°C. Further, inter and intra-laboratory comparisons showed consistency in the scoring of γ-H2AX foci frequency by manual and semiautomated scoring. The overall results suggest that measurement of γ-H2AX (microscopy and flow cytometry) should be employed within 4 to 6 hrs for a reliable dosimetry either by sharing the work load between the laboratories or investing more manpower; however, triage can be possible even up to 48 hrs of postirradiation. © 2015 International Society for Advancement of Cytometry.

Semantic segmentation of mFISH images using convolutional networks.

PubMed

Pardo, Esteban; Morgado, José Mário T; Malpica, Norberto

2018-04-30

Multicolor in situ hybridization (mFISH) is a karyotyping technique used to detect major chromosomal alterations using fluorescent probes and imaging techniques. Manual interpretation of mFISH images is a time consuming step that can be automated using machine learning; in previous works, pixel or patch wise classification was employed, overlooking spatial information which can help identify chromosomes. In this work, we propose a fully convolutional semantic segmentation network for the interpretation of mFISH images, which uses both spatial and spectral information to classify each pixel in an end-to-end fashion. The semantic segmentation network developed was tested on samples extracted from a public dataset using cross validation. Despite having no labeling information of the image it was tested on, our algorithm yielded an average correct classification ratio (CCR) of 87.41%. Previously, this level of accuracy was only achieved with state of the art algorithms when classifying pixels from the same image in which the classifier has been trained. These results provide evidence that fully convolutional semantic segmentation networks may be employed in the computer aided diagnosis of genetic diseases with improved performance over the current image analysis methods. © 2018 International Society for Advancement of Cytometry. © 2018 International Society for Advancement of Cytometry.

Design of point-of-care (POC) microfluidic medical diagnostic devices

NASA Astrophysics Data System (ADS)

Leary, James F.

2018-02-01

Design of inexpensive and portable hand-held microfluidic flow/image cytometry devices for initial medical diagnostics at the point of initial patient contact by emergency medical personnel in the field requires careful design in terms of power/weight requirements to allow for realistic portability as a hand-held, point-of-care medical diagnostics device. True portability also requires small micro-pumps for high-throughput capability. Weight/power requirements dictate use of super-bright LEDs and very small silicon photodiodes or nanophotonic sensors that can be powered by batteries. Signal-to-noise characteristics can be greatly improved by appropriately pulsing the LED excitation sources and sampling and subtracting noise in between excitation pulses. The requirements for basic computing, imaging, GPS and basic telecommunications can be simultaneously met by use of smartphone technologies, which become part of the overall device. Software for a user-interface system, limited real-time computing, real-time imaging, and offline data analysis can be accomplished through multi-platform software development systems that are well-suited to a variety of currently available cellphone technologies which already contain all of these capabilities. Microfluidic cytometry requires judicious use of small sample volumes and appropriate statistical sampling by microfluidic cytometry or imaging for adequate statistical significance to permit real-time (typically < 15 minutes) medical decisions for patients at the physician's office or real-time decision making in the field. One or two drops of blood obtained by pin-prick should be able to provide statistically meaningful results for use in making real-time medical decisions without the need for blood fractionation, which is not realistic in the field.

In vivo, label-free, and noninvasive detection of melanoma metastasis by photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Liu, Rongrong; Wang, Cheng; Hu, Cheng; Wang, Xueding; Wei, Xunbin

2014-02-01

Melanoma, a malignant tumor of melanocytes, is the most serious type of skin cancer in the world. It accounts for about 80% of deaths of all skin cancer. For cancer detection, circulating tumor cells (CTCs) serve as a marker for metastasis development, cancer recurrence, and therapeutic efficacy. Melanoma tumor cells have high content of melanin, which has high light absorption and can serve as endogenous biomarker for CTC detection without labeling. Here, we have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of melanoma cancer by counting CTCs of melanoma tumor bearing mice in vivo. To test in vivo PAFC's capability of detecting melanoma cancer, we have constructed a melanoma tumor model by subcutaneous inoculation of highly metastatic murine melanoma cancer cells, B16F10. In order to effectively distinguish the targeting PA signals from background noise, we have used the algorithm of Wavelet denoising method to reduce the background noise. The in vivo flow cytometry (IVFC) has shown a great potential for detecting circulating tumor cells quantitatively in the blood stream. Compared with fluorescence-based in vivo flow cytometry (IVFC), PAFC technique can be used for in vivo, label-free, and noninvasive detection of circulating tumor cells (CTCs).

Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo.

PubMed

Rice, G C; Bump, E A; Shrieve, D C; Lee, W; Kovacs, M

1986-12-01

An assay using a bimane derivative has been developed to detect free glutathione (GSH) in individual viable cells by flow cytometry. Monochlorobimane [syn-(ClCH2CH3)-1,5-diazabicycla[3.30]acta-3,6-diene-2,8-dio ne], itself nonfluorescent, reacts with GSH to form a highly fluorescent derivative. High pressure liquid chromatography analysis showed that, using specific staining conditions, the only low molecular weight fluorescent derivative formed in Chinese hamster ovary cells was that formed with GSH. Very little reaction with protein sulfhydryls was observed. Rates of GSH depletion in Chinese hamster ovary cells exposed to diethylmaleate were essentially the same, whether measured by relative fluorescence intensity, by flow cytometry or by enzymatic assay on cellular extracts. This method was shown to be useful for measurement of GSH resynthesis, uptake, and depletion by prolonged hypoxia and misonidazole treatment. Since measurements are made on individual cells, cell-to-cell variation and populational heterogeneity in GSH content are revealed by flow cytometry. Although under most conditions in vitro GSH content is relatively homogeneous, under certain circumstances, such as release from hypoxia, heterogeneity in populational GSH levels was observed. The significance of this heterogeneity is discussed in regard to the induction of gene amplification and drug resistance by transient hypoxia. Numerous subclones of Chinese hamster ovary cells selected by growth in Adriamycin or methotrexate-containing medium express elevated levels of GSH per cell. The method was extended to quantitate the GSH content of cells excised from EMT-6/SF mouse tumors that had been treated in vivo with L-buthionine-S-R-sulfoximine, an inhibitor of GSH synthesis. The bivariate analysis (forward angle light scatter versus monochlorobimane fluorescence) of cells derived from these tumors gave excellent resolution of normal and tumor cells and demonstrated extensive heterogeneity in the tumor cell population with respect to GSH content per cell.

Human Adipose-Derived Mesenchymal Stem/Stromal Cells Handling Protocols. Lipid Droplets and Proteins Double-Staining

PubMed Central

Gojanovich, Aldana D.; Gimenez, María C.; Masone, Diego; Rodriguez, Tania M.; Dewey, Ricardo A.; Delgui, Laura R.; Bustos, Diego M.; Uhart, Marina

2018-01-01

Human Adipose-derived mesenchymal stem/stromal cells (hASCs) are of great interest because of their potential for therapeutic approaches. The method described here covers every single step necessary for hASCs isolation from subcutaneous abdominal adipose tissue, multicolor phenotyping by flow cytometry, and quantitative determination of adipogenic differentiation status by means of lipid droplets (LDs) accumulation, and Western blot analysis. Moreover, to simultaneously analyze both LDs accumulation and cellular proteins localization by fluorescence microscopy, we combined Oil Red O (ORO) staining with immunofluorescence detection. For LDs quantification we wrote a program for automatic ORO-stained digital image processing implemented in Octave, a freely available software package. Our method is based on the use of the traditional low cost neutral lipids dye ORO, which can be imaged both by bright-field and fluorescence microscopy. The utilization of ORO instead of other more expensive lipid-specific dyes, together with the fact that the whole method has been designed employing cost-effective culture reagents (standard culture medium and serum), makes it affordable for tight-budget research laboratories. These may be replaced, if necessary or desired, by defined xeno-free reagents for clinical research and applications. PMID:29670879

Human Adipose-Derived Mesenchymal Stem/Stromal Cells Handling Protocols. Lipid Droplets and Proteins Double-Staining.

PubMed

Gojanovich, Aldana D; Gimenez, María C; Masone, Diego; Rodriguez, Tania M; Dewey, Ricardo A; Delgui, Laura R; Bustos, Diego M; Uhart, Marina

2018-01-01

Human Adipose-derived mesenchymal stem/stromal cells (hASCs) are of great interest because of their potential for therapeutic approaches. The method described here covers every single step necessary for hASCs isolation from subcutaneous abdominal adipose tissue, multicolor phenotyping by flow cytometry, and quantitative determination of adipogenic differentiation status by means of lipid droplets (LDs) accumulation, and Western blot analysis. Moreover, to simultaneously analyze both LDs accumulation and cellular proteins localization by fluorescence microscopy, we combined Oil Red O (ORO) staining with immunofluorescence detection. For LDs quantification we wrote a program for automatic ORO-stained digital image processing implemented in Octave, a freely available software package. Our method is based on the use of the traditional low cost neutral lipids dye ORO, which can be imaged both by bright-field and fluorescence microscopy. The utilization of ORO instead of other more expensive lipid-specific dyes, together with the fact that the whole method has been designed employing cost-effective culture reagents (standard culture medium and serum), makes it affordable for tight-budget research laboratories. These may be replaced, if necessary or desired, by defined xeno-free reagents for clinical research and applications.

MPQ-cytometry: a magnetism-based method for quantification of nanoparticle-cell interactions

NASA Astrophysics Data System (ADS)

Shipunova, V. O.; Nikitin, M. P.; Nikitin, P. I.; Deyev, S. M.

2016-06-01

Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method called MPQ-cytometry is developed, which measures the integral non-linear response produced by magnetically labeled nanoparticles in a cell sample with an original magnetic particle quantification (MPQ) technique. MPQ-cytometry provides a sensitivity limit 0.33 ng of nanoparticles and is devoid of a background signal present in many label-based assays. Each measurement takes only a few seconds, and no complicated sample preparation or data processing is required. The capabilities of the method have been demonstrated by quantification of interactions of iron oxide nanoparticles with eukaryotic cells. The total amount of targeted nanoparticles that specifically recognized the HER2/neu oncomarker on the human cancer cell surface was successfully measured, the specificity of interaction permitting the detection of HER2/neu positive cells in a cell mixture. Moreover, it has been shown that MPQ-cytometry analysis of a HER2/neu-specific iron oxide nanoparticle interaction with six cell lines of different tissue origins quantitatively reflects the HER2/neu status of the cells. High correlation of MPQ-cytometry data with those obtained by three other commonly used in molecular and cell biology methods supports consideration of this method as a prospective alternative for both quantifying cell-bound nanoparticles and estimating the expression level of cell surface antigens. The proposed method does not require expensive sophisticated equipment or highly skilled personnel and it can be easily applied for rapid diagnostics, especially under field conditions.Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method called MPQ-cytometry is developed, which measures the integral non-linear response produced by magnetically labeled nanoparticles in a cell sample with an original magnetic particle quantification (MPQ) technique. MPQ-cytometry provides a sensitivity limit 0.33 ng of nanoparticles and is devoid of a background signal present in many label-based assays. Each measurement takes only a few seconds, and no complicated sample preparation or data processing is required. The capabilities of the method have been demonstrated by quantification of interactions of iron oxide nanoparticles with eukaryotic cells. The total amount of targeted nanoparticles that specifically recognized the HER2/neu oncomarker on the human cancer cell surface was successfully measured, the specificity of interaction permitting the detection of HER2/neu positive cells in a cell mixture. Moreover, it has been shown that MPQ-cytometry analysis of a HER2/neu-specific iron oxide nanoparticle interaction with six cell lines of different tissue origins quantitatively reflects the HER2/neu status of the cells. High correlation of MPQ-cytometry data with those obtained by three other commonly used in molecular and cell biology methods supports consideration of this method as a prospective alternative for both quantifying cell-bound nanoparticles and estimating the expression level of cell surface antigens. The proposed method does not require expensive sophisticated equipment or highly skilled personnel and it can be easily applied for rapid diagnostics, especially under field conditions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr03507h

A pathologist-designed imaging system for anatomic pathology signout, teaching, and research.

PubMed

Schubert, E; Gross, W; Siderits, R H; Deckenbaugh, L; He, F; Becich, M J

1994-11-01

Pathology images are derived from gross surgical specimens, light microscopy, immunofluorescence, electron microscopy, molecular diagnostic gels, flow cytometry, image analysis data, and clinical laboratory data in graphic form. We have implemented a network of desktop personal computers (PCs) that allow us to easily capture, store, and retrieve gross and microscopic, anatomic, and research pathology images. System architecture involves multiple image acquisition and retrieval sites and a central file server for storage. The digitized images are conveyed via a local area network to and from image capture or display stations. Acquisition sites consist of a high-resolution camera connected to a frame grabber card in a 486-type personal computer, equipped with 16 MB (Table 1) RAM, a 1.05-gigabyte hard drive, and a 32-bit ethernet card for access to our anatomic pathology reporting system. We have designed a push-button workstation for acquiring and indexing images that does not significantly interfere with surgical pathology sign-out. Advantages of the system include the following: (1) Improving patient care: the availability of gross images at time of microscopic sign-out, verification of recurrence of malignancy from archived images, monitoring of bone marrow engraftment and immunosuppressive intervention after bone marrow/solid organ transplantation on repeat biopsies, and ability to seek instantaneous consultation with any pathologist on the network; (2) enhancing the teaching environment: building a digital surgical pathology atlas, improving the availability of images for conference support, and sharing cases across the network; (3) enhancing research: case study compilation, metastudy analysis, and availability of digitized images for quantitative analysis and permanent/reusable image records for archival study; and (4) other practical and economic considerations: storing case requisition images and hand-drawn diagrams deters the spread of gross room contaminants and results in considerable cost savings in photographic media for conferences, improved quality assurance by porting control stains across the network, and a multiplicity of other advantages that enhance image and information management in pathology.

Fountain Flow cytometry, a new technique for the rapid detection and enumeration of microorganisms in aqueous samples.

PubMed

Johnson, Paul E; Deromedi, Anthony J; Lebaron, Philippe; Catala, Philippe; Cash, Jennifer

2006-12-01

Pathogenic microorganisms are known to cause widespread waterborne disease worldwide. There is an urgent need to develop a technique for the real-time detection of pathogens in environmental samples at low concentrations, <10 microorganisms/ml, in large sample volumes, > or =100 ml. A novel method, Fountain Flowtrade mark cytometry, for the rapid and sensitive detection of individual microorganisms in aqueous samples is presented. Each sample is first incubated with a fluorescent label and then passed as a stream in front of a laser, which excites the label. The fluorescence is detected with a CCD imager as the sample flows toward the imager along its optical axis. The feasibility of Fountain Flow cytometry (FFC) is demonstrated by the detection of Escherichia coli labeled with ChemChrome CV6 and SYBR Gold in buffer and natural river water. Detections of labeled E. coli were made in aqueous suspensions with an efficiency of 96% +/- 14% down to a concentration approximately 200 bacteria/ml. The feasibility of FFC is demonstrated by the detection of E. coli in buffer and natural river water. FFC should apply to the detection of a wide range of pathogenic microorganisms including amoebae.

Feasibility study of stain-free classification of cell apoptosis based on diffraction imaging flow cytometry and supervised machine learning techniques.

PubMed

Feng, Jingwen; Feng, Tong; Yang, Chengwen; Wang, Wei; Sa, Yu; Feng, Yuanming

2018-06-01

This study was to explore the feasibility of prediction and classification of cells in different stages of apoptosis with a stain-free method based on diffraction images and supervised machine learning. Apoptosis was induced in human chronic myelogenous leukemia K562 cells by cis-platinum (DDP). A newly developed technique of polarization diffraction imaging flow cytometry (p-DIFC) was performed to acquire diffraction images of the cells in three different statuses (viable, early apoptotic and late apoptotic/necrotic) after cell separation through fluorescence activated cell sorting with Annexin V-PE and SYTOX® Green double staining. The texture features of the diffraction images were extracted with in-house software based on the Gray-level co-occurrence matrix algorithm to generate datasets for cell classification with supervised machine learning method. Therefore, this new method has been verified in hydrogen peroxide induced apoptosis model of HL-60. Results show that accuracy of higher than 90% was achieved respectively in independent test datasets from each cell type based on logistic regression with ridge estimators, which indicated that p-DIFC system has a great potential in predicting and classifying cells in different stages of apoptosis.

Global gray-level thresholding based on object size.

PubMed

Ranefall, Petter; Wählby, Carolina

2016-04-01

In this article, we propose a fast and robust global gray-level thresholding method based on object size, where the selection of threshold level is based on recall and maximum precision with regard to objects within a given size interval. The method relies on the component tree representation, which can be computed in quasi-linear time. Feature-based segmentation is especially suitable for biomedical microscopy applications where objects often vary in number, but have limited variation in size. We show that for real images of cell nuclei and synthetic data sets mimicking fluorescent spots the proposed method is more robust than all standard global thresholding methods available for microscopy applications in ImageJ and CellProfiler. The proposed method, provided as ImageJ and CellProfiler plugins, is simple to use and the only required input is an interval of the expected object sizes. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Enumeration of Circulating Tumor Cells and Disseminated Tumor Cells in Blood and Bone Marrow by Immunomagnetic Enrichment and Flow Cytometry (IE/FC).

PubMed

Magbanua, Mark Jesus M; Solanki, Tulasi I; Ordonez, Andrea D; Hsiao, Feng; Park, John W

2017-01-01

Enumerating circulating tumor cells (CTCs) in blood and disseminated tumor cells (DTCs) in bone marrow has shown to be clinically useful, as elevated numbers of these cells predict poor clinical outcomes. Accurate detection and quantification is, however, difficult and technically challenging because CTCs and DTCs are extremely rare. We have developed a novel quantitative detection method for enumeration of CTCs and DTCs. Our approach consists of two steps: (1) EPCAM-based immunomagnetic enrichment followed by (2) flow cytometry (IE/FC). The assay takes approximately 2 h to complete. In addition to tumor cell enumeration, IE/FC offers opportunities for direct isolation of highly pure tumor cells for downstream molecular characterization.

SCENERY: a web application for (causal) network reconstruction from cytometry data

PubMed Central

Papoutsoglou, Georgios; Athineou, Giorgos; Lagani, Vincenzo; Xanthopoulos, Iordanis; Schmidt, Angelika; Éliás, Szabolcs; Tegnér, Jesper

2017-01-01

Abstract Flow and mass cytometry technologies can probe proteins as biological markers in thousands of individual cells simultaneously, providing unprecedented opportunities for reconstructing networks of protein interactions through machine learning algorithms. The network reconstruction (NR) problem has been well-studied by the machine learning community. However, the potentials of available methods remain largely unknown to the cytometry community, mainly due to their intrinsic complexity and the lack of comprehensive, powerful and easy-to-use NR software implementations specific for cytometry data. To bridge this gap, we present Single CEll NEtwork Reconstruction sYstem (SCENERY), a web server featuring several standard and advanced cytometry data analysis methods coupled with NR algorithms in a user-friendly, on-line environment. In SCENERY, users may upload their data and set their own study design. The server offers several data analysis options categorized into three classes of methods: data (pre)processing, statistical analysis and NR. The server also provides interactive visualization and download of results as ready-to-publish images or multimedia reports. Its core is modular and based on the widely-used and robust R platform allowing power users to extend its functionalities by submitting their own NR methods. SCENERY is available at scenery.csd.uoc.gr or http://mensxmachina.org/en/software/. PMID:28525568

Effect of Quantitative Nuclear Image Features on Recurrence of Ductal Carcinoma In Situ (DCIS) of the Breast

PubMed Central

Axelrod, David E.; Miller, Naomi A.; Lickley, H. Lavina; Qian, Jin; Christens-Barry, William A.; Yuan, Yan; Fu, Yuejiao; Chapman, Judith-Anne W.

2008-01-01

Background Nuclear grade has been associated with breast DCIS recurrence and progression to invasive carcinoma; however, our previous study of a cohort of patients with breast DCIS did not find such an association with outcome. Fifty percent of patients had heterogeneous DCIS with more than one nuclear grade. The aim of the current study was to investigate the effect of quantitative nuclear features assessed with digital image analysis on ipsilateral DCIS recurrence. Methods Hematoxylin and eosin stained slides for a cohort of 80 patients with primary breast DCIS were reviewed and two fields with representative grade (or grades) were identified by a Pathologist and simultaneously used for acquisition of digital images for each field. Van Nuys worst nuclear grade was assigned, as was predominant grade, and heterogeneous grading when present. Patients were grouped by heterogeneity of their nuclear grade: Group A: nuclear grade 1 only, nuclear grades 1 and 2, or nuclear grade 2 only (32 patients), Group B: nuclear grades 1, 2 and 3, or nuclear grades 2 and 3 (31 patients), Group 3: nuclear grade 3 only (17 patients). Nuclear fine structure was assessed by software which captured thirty-nine nuclear feature values describing nuclear morphometry, densitometry, and texture. Step-wise forward Cox regressions were performed with previous clinical and pathologic factors, and the new image analysis features. Results Duplicate measurements were similar for 89.7% to 97.4% of assessed image features. The rate of correct classification of nuclear grading with digital image analysis features was similar in the two fields, and pooled assessment across both fields. In the pooled assessment, a discriminant function with one nuclear morphometric and one texture feature was significantly (p = 0.001) associated with nuclear grading, and provided correct jackknifed classification of a patient’s nuclear grade for Group A (78.1%), Group B (48.4%), and Group C (70.6%). The factors significantly associated with DCIS recurrence were those previously found, type of initial presentation (p = 0.03) and amount of parenchymal involvement (p = 0.05), along with the morphometry image feature of ellipticity (p = 0.04). Conclusion Analysis of nuclear features measured by image cytometry may contribute to the classification and prognosis of breast DCIS patients with more than one nuclear grade. PMID:18779878

Automation of sample preparation for mass cytometry barcoding in support of clinical research: protocol optimization.

PubMed

Nassar, Ala F; Wisnewski, Adam V; Raddassi, Khadir

2017-03-01

Analysis of multiplexed assays is highly important for clinical diagnostics and other analytical applications. Mass cytometry enables multi-dimensional, single-cell analysis of cell type and state. In mass cytometry, the rare earth metals used as reporters on antibodies allow determination of marker expression in individual cells. Barcode-based bioassays for CyTOF are able to encode and decode for different experimental conditions or samples within the same experiment, facilitating progress in producing straightforward and consistent results. Herein, an integrated protocol for automated sample preparation for barcoding used in conjunction with mass cytometry for clinical bioanalysis samples is described; we offer results of our work with barcoding protocol optimization. In addition, we present some points to be considered in order to minimize the variability of quantitative mass cytometry measurements. For example, we discuss the importance of having multiple populations during titration of the antibodies and effect of storage and shipping of labelled samples on the stability of staining for purposes of CyTOF analysis. Data quality is not affected when labelled samples are stored either frozen or at 4 °C and used within 10 days; we observed that cell loss is greater if cells are washed with deionized water prior to shipment or are shipped in lower concentration. Once the labelled samples for CyTOF are suspended in deionized water, the analysis should be performed expeditiously, preferably within the first hour. Damage can be minimized if the cells are resuspended in phosphate-buffered saline (PBS) rather than deionized water while waiting for data acquisition.

In vivo photoacoustic flow cytometry for early malaria diagnosis.

PubMed

Cai, Chengzhong; Carey, Kai A; Nedosekin, Dmitry A; Menyaev, Yulian A; Sarimollaoglu, Mustafa; Galanzha, Ekaterina I; Stumhofer, Jason S; Zharov, Vladimir P

2016-06-01

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Development of an unbiased, semi-automated approach for classifying plasma cell immunophenotype following multicolor flow cytometry of bone marrow aspirates.

PubMed

Post, Steven R; Post, Ginell R; Nikolic, Dejan; Owens, Rebecca; Insuasti-Beltran, Giovanni

2018-03-24

Despite increased usage of multiparameter flow cytometry (MFC) to assess diagnosis, prognosis, and therapeutic efficacy (minimal residual disease, MRD) in plasma cell neoplasms (PCNs), standardization of methodology and data analysis is suboptimal. We investigated the utility of using the mean and median fluorescence intensities (FI) obtained from MFC to objectively describe parameters that distinguish plasma cell (PC) phenotypes. In this retrospective study, flow cytometry results from bone marrow aspirate specimens from 570 patients referred to the Myeloma Institute at UAMS were evaluated. Mean and median FI data were obtained from 8-color MFC of non-neoplastic, malignant, and mixed PC populations using antibodies to CD38, CD138, CD19, CD20, CD27, CD45, CD56, and CD81. Of 570 cases, 252 cases showed only non-neoplastic PCs, 168 showed only malignant PCs, and 150 showed mixed PC populations. Statistical analysis of median FI data for each CD marker showed no difference in expression intensity on non-neoplastic and malignant PCs, between pure and mixed PC populations. ROC analysis of the median FI of CD expression in non-neoplastic and malignant PCs was used to develop an algorithm to convert quantitative FI values to qualitative assessments including "negative," "positive," "dim," and "heterogeneous" expression. FI data derived from 8-color MFC can be used to define marker expression on PCs. Translation of FI data from Infinicyt software to an Excel worksheet streamlines workflow and eliminates transcriptional errors when generating flow reports. © 2018 International Clinical Cytometry Society. © 2018 International Clinical Cytometry Society.

Multicellular tumor spheroids as an in vivo-like tumor model for three-dimensional imaging of chemotherapeutic and nano material cellular penetration.

PubMed

Ma, Hui-li; Jiang, Qiao; Han, Siyuan; Wu, Yan; Cui Tomshine, Jin; Wang, Dongliang; Gan, Yaling; Zou, Guozhang; Liang, Xing-Jie

2012-01-01

We present a flexible and highly reproducible method using three-dimensional (3D) multicellular tumor spheroids to quantify chemotherapeutic and nanoparticle penetration properties in vitro. We generated HeLa cell-derived spheroids using the liquid overlay method. To properly characterize HeLa spheroids, scanning electron microscopy, transmission electron microscopy, and multiphoton microscopy were used to obtain high-resolution 3D images of HeLa spheroids. Next, pairing high-resolution optical characterization techniques with flow cytometry, we quantitatively compared the penetration of doxorubicin, quantum dots, and synthetic micelles into 3D HeLa spheroid versus HeLa cells grown in a traditional two-dimensional culturing system. Our data revealed that 3D cultured HeLa cells acquired several clinically relevant morphologic and cellular characteristics (such as resistance to chemotherapeutics) often found in human solid tumors. These characteristic, however, could not be captured using conventional two-dimensional cell culture techniques. This study demonstrated the remarkable versatility of HeLa spheroid 3D imaging. In addition, our results revealed the capability of HeLa spheroids to function as a screening tool for nanoparticles or synthetic micelles that, due to their inherent size, charge, and hydrophobicity, can penetrate into solid tumors and act as delivery vehicles for chemotherapeutics. The development of this image-based, reproducible, and quantifiable in vitro HeLa spheroid screening tool will greatly aid future exploration of chemotherapeutics and nanoparticle delivery into solid tumors.

Quantitative identification of senescent cells in aging and disease.

PubMed

Biran, Anat; Zada, Lior; Abou Karam, Paula; Vadai, Ezra; Roitman, Lior; Ovadya, Yossi; Porat, Ziv; Krizhanovsky, Valery

2017-08-01

Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single-cell basis. The method combines a senescence-associated beta-galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high-content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

High throughput image cytometry for detection of suspicious lesions in the oral cavity

NASA Astrophysics Data System (ADS)

MacAulay, Calum; Poh, Catherine F.; Guillaud, Martial; Michele Williams, Pamela; Laronde, Denise M.; Zhang, Lewei; Rosin, Miriam P.

2012-08-01

The successful management of oral cancer depends upon early detection, which relies heavily on the clinician's ability to discriminate sometimes subtle alterations of the infrequent premalignant lesions from the more common reactive and inflammatory conditions in the oral mucosa. Even among experienced oral specialists this can be challenging, particularly when using new wide field-of-view direct fluorescence visualization devices clinically introduced for the recognition of at-risk tissue. The objective of this study is to examine if quantitative cytometric analysis of oral brushing samples could facilitate the assessment of the risk of visually ambiguous lesions. About 369 cytological samples were collected and analyzed: (1) 148 samples from pathology-proven sites of SCC, carcinoma in situ or severe dysplasia; (2) 77 samples from sites with inflammation, infection, or trauma, and (3) 144 samples from normal sites. These were randomly separated into training and test sets. The best algorithm correctly recognized 92.5% of the normal samples, 89.4% of the abnormal samples, 86.2% of the confounders in the training set as well as 100% of the normal samples, and 94.4% of the abnormal samples in the test set. These data suggest that quantitative cytology could reduce by more than 85% the number of visually suspect lesions requiring further assessment by biopsy.

DICOM-compatible format for analytical cytology data

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Suzanne B.

1998-04-01

The addition of a list mode data type to the Digital Imaging and Communications in Medicine standard, DICOM will enhance the storage and transmission of digital microscopy data and extend DICOM to include flow cytometry data. This would permit the present International Society for analytical Cytology Flow Cytometry Standard to be retired. DICOM includes: image graphics objects, specifications for describing: studies, reports, the acquisition of the data, work list management, and the individuals involved (physician, patient, etc.) The glossary of terms (objects) suitable for use with DICOM has been extended to include the collaborative effort of Logical Observation Identifier Names and Codes (LOINC) and Systematized Nomenclature of Human and Veterinary Medicine (SNOMED) to create a consistent, unambiguous clinical reference terminology. It also appears that DICOM will be a significant part of the Common Object Request Broker Architecture, CORBA.

Multi-parametric analysis of phagocyte antimicrobial responses using imaging flow cytometry.

PubMed

Havixbeck, Jeffrey J; Wong, Michael E; More Bayona, Juan A; Barreda, Daniel R

2015-08-01

We feature a multi-parametric approach based on an imaging flow cytometry platform for examining phagocyte antimicrobial responses against the gram-negative bacterium Aeromonas veronii. This pathogen is known to induce strong inflammatory responses across a broad range of animal species, including humans. We examined the contribution of A. veronii to the induction of early phagocyte inflammatory processes in RAW 264.7 murine macrophages in vitro. We found that A. veronii, both in live or heat-killed forms, induced similar levels of macrophage activation based on NF-κB translocation. Although these macrophages maintained high levels of viability following heat-killed or live challenges with A. veronii, we identified inhibition of macrophage proliferation as early as 1h post in vitro challenge. The characterization of phagocytic responses showed a time-dependent increase in phagocytosis upon A. veronii challenge, which was paired with a robust induction of intracellular respiratory burst responses. Interestingly, despite the overall increase in the production of reactive oxygen species (ROS) among RAW 264.7 macrophages, we found a significant reduction in the production of ROS among the macrophage subset that had bound A. veronii. Phagocytic uptake of the pathogen further decreased ROS production levels, even beyond those of unstimulated controls. Overall, this multi-parametric imaging flow cytometry-based approach allowed for segregation of unique phagocyte sub-populations and examination of their downstream antimicrobial responses, and should contribute to improved understanding of phagocyte responses against Aeromonas and other pathogens. Copyright © 2015 Elsevier B.V. All rights reserved.

Facile and quantitative electrochemical detection of yeast cell apoptosis

NASA Astrophysics Data System (ADS)

Yue, Qiulin; Xiong, Shiquan; Cai, Dongqing; Wu, Zhengyan; Zhang, Xin

2014-03-01

An electrochemical method based on square wave anodic stripping voltammetry (SWASV) was developed to detect the apoptosis of yeast cells conveniently and quantitatively through the high affinity between Cu2+ and phosphatidylserine (PS) translocated from the inner to the outer plasma membrane of the apoptotic cells. The combination of negatively charged PS and Cu2+ could decrease the electrochemical response of Cu2+ on the electrode. The results showed that the apoptotic rates of cells could be detected quantitatively through the variations of peak currents of Cu2+ by SWASV, and agreed well with those obtained through traditional flow cytometry detection. This work thus may provide a novel, simple, immediate and accurate detection method for cell apoptosis.

Design of portable ultraminiature flow cytometers for medical diagnostics

NASA Astrophysics Data System (ADS)

Leary, James F.

2018-02-01

Design of portable microfluidic flow/image cytometry devices for measurements in the field (e.g. initial medical diagnostics) requires careful design in terms of power requirements and weight to allow for realistic portability. True portability with high-throughput microfluidic systems also requires sampling systems without the need for sheath hydrodynamic focusing both to avoid the need for sheath fluid and to enable higher volumes of actual sample, rather than sheath/sample combinations. Weight/power requirements dictate use of super-bright LEDs with top-hat excitation beam architectures and very small silicon photodiodes or nanophotonic sensors that can both be powered by small batteries. Signal-to-noise characteristics can be greatly improved by appropriately pulsing the LED excitation sources and sampling and subtracting noise in between excitation pulses. Microfluidic cytometry also requires judicious use of small sample volumes and appropriate statistical sampling by microfluidic cytometry or imaging for adequate statistical significance to permit real-time (typically in less than 15 minutes) initial medical decisions for patients in the field. This is not something conventional cytometry traditionally worries about, but is very important for development of small, portable microfluidic devices with small-volume throughputs. It also provides a more reasonable alternative to conventional tubes of blood when sampling geriatric and newborn patients for whom a conventional peripheral blood draw can be problematical. Instead one or two drops of blood obtained by pin-prick should be able to provide statistically meaningful results for use in making real-time medical decisions without the need for blood fractionation, which is not realistic in the doctor's office or field.

Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting.

PubMed

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-04-13

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 μm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry.

PubMed

Guo, Ling; Wang, Zhen; Anderson, Courtney M; Doolittle, Emerald; Kernag, Siobhan; Cotta, Claudiu V; Ondrejka, Sarah L; Ma, Xiao-Jun; Cook, James R

2018-03-01

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin-fixed, paraffin-embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells but are often insufficiently sensitive to detect the much lower abundance of light chains present in B-cells. We describe an ultrasensitive RNA in situ hybridization assay that has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain-restricted B-cells in 85 (42%) vs 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified restricted B-cells in 74 (89%) vs 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases owing to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphological features in formalin-fixed, paraffin-embedded tissues with a clinical sensitivity similar or superior to flow cytometry.

Ultrasensitive Automated RNA in situ Hybridization for Kappa and Lambda Light Chain mRNA Detects B-cell Clonality in Tissue Biopsies with Performance Comparable or Superior to Flow Cytometry

PubMed Central

Guo, Ling; Wang, Zhen; Anderson, Courtney M.; Doolittle, Emerald; Kernag, Siobhan; Cotta, Claudiu V.; Ondrejka, Sarah L.; Ma, Xiao-Jun; Cook, James R.

2017-01-01

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin fixed paraffin embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells, but are often insufficiently sensitive to detect the much lower abundance of light chains present in B cells. We describe an ultrasensitive RNA in situ hybridization assay which has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain restricted B-cells in 85 (42%) vs. 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified a restricted B-cells in 74 (89%) vs. 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases due to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry, and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphologic features in formalin fixed, paraffin embedded tissues with a clinical sensitivity similar or superior to flow cytometry. PMID:29052600

Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells.

PubMed

Lee, Hyang Yeon; Lee, Jae Jeong; Park, Jongmin; Park, Seung Bum

2011-01-03

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ultrafast Microfluidic Cellular Imaging by Optical Time-Stretch.

PubMed

Lau, Andy K S; Wong, Terence T W; Shum, Ho Cheung; Wong, Kenneth K Y; Tsia, Kevin K

2016-01-01

There is an unmet need in biomedicine for measuring a multitude of parameters of individual cells (i.e., high content) in a large population efficiently (i.e., high throughput). This is particularly driven by the emerging interest in bringing Big-Data analysis into this arena, encompassing pathology, drug discovery, rare cancer cell detection, emulsion microdroplet assays, to name a few. This momentum is particularly evident in recent advancements in flow cytometry. They include scaling of the number of measurable colors from the labeled cells and incorporation of imaging capability to access the morphological information of the cells. However, an unspoken predicament appears in the current technologies: higher content comes at the expense of lower throughput, and vice versa. For example, accessing additional spatial information of individual cells, imaging flow cytometers only achieve an imaging throughput ~1000 cells/s, orders of magnitude slower than the non-imaging flow cytometers. In this chapter, we introduce an entirely new imaging platform, namely optical time-stretch microscopy, for ultrahigh speed and high contrast label-free single-cell (in a ultrafast microfluidic flow up to 10 m/s) imaging and analysis with an ultra-fast imaging line-scan rate as high as tens of MHz. Based on this technique, not only morphological information of the individual cells can be obtained in an ultrafast manner, quantitative evaluation of cellular information (e.g., cell volume, mass, refractive index, stiffness, membrane tension) at nanometer scale based on the optical phase is also possible. The technology can also be integrated with conventional fluorescence measurements widely adopted in the non-imaging flow cytometers. Therefore, these two combinatorial and complementary measurement capabilities in long run is an attractive platform for addressing the pressing need for expanding the "parameter space" in high-throughput single-cell analysis. This chapter provides the general guidelines of constructing the optical system for time stretch imaging, fabrication and design of the microfluidic chip for ultrafast fluidic flow, as well as the image acquisition and processing.

A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

NASA Astrophysics Data System (ADS)

Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.; Muldoon, Timothy J.

2015-09-01

Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min-1 with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels-1.

A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.

Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated aftermore » imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min{sup −1} with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels{sup −1}.« less

Impaired coordination between signaling pathways is revealed in human colorectal cancer using single-cell mass cytometry of archival tissue blocks

PubMed Central

Simmons, Alan J.; Scurrah, Cherie’ R.; McKinley, Eliot T.; Herring, Charles A.; Irish, Jonathan M.; Washington, Mary K.; Coffey, Robert J.; Lau, Ken S.

2016-01-01

Cellular heterogeneity poses a significant challenge to understanding tissue level phenotypes and confounds conventional bulk analyses. To facilitate the analysis of signaling at the single-cell level in human tissues, we applied mass cytometry using CyTOF (Cytometry Time-of-Flight) to formalin-fixed paraffin-embedded (FFPE) normal and diseased intestinal specimens. We developed and validated a technique called FFPE-DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue), a single-cell approach for characterizing native signaling states from embedded solid tissue samples. We applied FFPE-DISSECT coupled to mass cytometry and found differential signaling by tumor necrosis factor α (TNF-α) in intestinal enterocytes, goblet cells and enteroendocrine cells, implicating the role of the downstream RAS-RAF-MEK-ERK signaling pathway in dictating goblet cell identity. In addition, application of FFPE-DISSECT, mass cytometry, and data-driven computational analyses to human colon specimens confirmed reduced differentiation in colorectal cancer (CRC) compared to normal colon, and revealed quantitative increases in inter- and intra-tissue heterogeneity in CRC with regards to the modular regulation of signaling pathways. Specifically, modular co-regulation of the kinases P38 and ERK, the translation regulator 4EBP1, and the transcription factor CREB in the proliferative compartment of the normal colon was loss in CRC, as evidenced by their impaired coordination over samplings of single cells in tissue. Our data suggest that this single-cell approach, applied in conjunction with genomic annotation, such as microsatellite instability and mutations in KRAS and BRAF, allows rapid and detailed characterization of cellular heterogeneity from clinical repositories of embedded human tissues. FFPE-DISSECT coupled of mass cytometry can be used for deriving cellular landscapes from archived patient samples, beyond CRC, and as a high resolution tool for disease characterization and subtyping. PMID:27729552

SCENERY: a web application for (causal) network reconstruction from cytometry data.

PubMed

Papoutsoglou, Georgios; Athineou, Giorgos; Lagani, Vincenzo; Xanthopoulos, Iordanis; Schmidt, Angelika; Éliás, Szabolcs; Tegnér, Jesper; Tsamardinos, Ioannis

2017-07-03

Flow and mass cytometry technologies can probe proteins as biological markers in thousands of individual cells simultaneously, providing unprecedented opportunities for reconstructing networks of protein interactions through machine learning algorithms. The network reconstruction (NR) problem has been well-studied by the machine learning community. However, the potentials of available methods remain largely unknown to the cytometry community, mainly due to their intrinsic complexity and the lack of comprehensive, powerful and easy-to-use NR software implementations specific for cytometry data. To bridge this gap, we present Single CEll NEtwork Reconstruction sYstem (SCENERY), a web server featuring several standard and advanced cytometry data analysis methods coupled with NR algorithms in a user-friendly, on-line environment. In SCENERY, users may upload their data and set their own study design. The server offers several data analysis options categorized into three classes of methods: data (pre)processing, statistical analysis and NR. The server also provides interactive visualization and download of results as ready-to-publish images or multimedia reports. Its core is modular and based on the widely-used and robust R platform allowing power users to extend its functionalities by submitting their own NR methods. SCENERY is available at scenery.csd.uoc.gr or http://mensxmachina.org/en/software/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Novel Methods of Determining Urinary Calculi Composition: Petrographic Thin Sectioning of Calculi and Nanoscale Flow Cytometry Urinalysis

PubMed Central

Gavin, Carson T; Ali, Sohrab N; Tailly, Thomas; Olvera-Posada, Daniel; Alenezi, Husain; Power, Nicholas E; Hou, Jinqiang; St. Amant, Andre H; Luyt, Leonard G; Wood, Stephen; Wu, Charles; Razvi, Hassan; Leong, Hon S

2016-01-01

Accurate determination of urinary stone composition has significant bearing on understanding pathophysiology, choosing treatment modalities and preventing recurrence. A need exists for improved methods to determine stone composition. Urine of 31 patients with known renal calculi was examined with nanoscale flow cytometry and the calculi collected during surgery subsequently underwent petrographic thin sectioning with polarized and fluorescent microscopy. Fluorescently labeled bisphosphonate probes (Alendronate-fluorescein/Alendronate-Cy5) were developed for nanoscale flow cytometry to enumerate nanocrystals that bound the fluorescent probes. Petrographic sections of stones were also imaged by fluorescent and polarized light microscopy with composition analysis correlated to alendronate +ve nanocrystal counts in corresponding urine samples. Urine samples from patients with Ca2+ and Mg2+ based calculi exhibited the highest alendronate +ve nanocrystal counts, ranging from 100–1000 nm in diameter. This novel urine based assay was in agreement with composition determined by petrographic thin sections with Alendronate probes. In some cases, high alendronate +ve nanocrystal counts indicated a Ca2+ or Mg2+ composition, as confirmed by petrographic analysis, overturning initial spectrophotometric diagnosis of stone composition. The combination of nanoscale flow cytometry and petrographic thin sections offer an alternative means for determining stone composition. Nanoscale flow cytometry of alendronate +ve nanocrystals alone may provide a high-throughput means of evaluating stone burden. PMID:26771074

High-Throughput Particle Uptake Analysis by Imaging Flow Cytometry

PubMed Central

Smirnov, Asya; Solga, Michael D.; Lannigan, Joanne; Criss, Alison K.

2017-01-01

Quantifying the efficiency of particle uptake by host cells is important in fields including infectious diseases, autoimmunity, cancer, developmental biology, and drug delivery. Here we present a protocol for high-throughput analysis of particle uptake using imaging flow cytometry, using the bacterium Neisseria gonorrhoeae attached and internalized to neutrophils as an example. Cells are exposed to fluorescently labeled bacteria, fixed, and stained with a bacteria-specific antibody of a different fluorophore. Thus in the absence of a permeabilizing agent, extracellular bacteria are double-labeled with two fluorophores while intracellular bacteria remain single-labeled. A spot count algorithm is used to determine the number of single- and double-labeled bacteria in individual cells, to calculate the percent of cells associated with bacteria, percent of cells with internalized bacteria, and percent of cell-associated bacteria that are internalized. These analyses quantify bacterial association and internalization across thousands of cells and can be applied to diverse experimental systems. PMID:28369762

Single cell analysis using surface enhanced Raman scattering (SERS) tags

PubMed Central

Nolan, John P.; Duggan, Erika; Liu, Er; Condello, Danilo; Dave, Isha; Stoner, Samuel A.

2013-01-01

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis. PMID:22498143

Evaluation of brush cytology and DNA image cytometry for the detection of cancer of the oral cavity.

PubMed

Kaur, Manveen; Handa, Uma; Mohan, Harsh; Dass, Arjun

2016-03-01

Cancer of the oral cavity is the sixth most common malignancy reported worldwide. This study was undertaken to evaluate the efficacy of brush cytology and the adjunctive role of DNA image cytometry (ICM) in the diagnosis of oral cancer. Oral brush smears and biopsy were obtained from 100 consecutive patients presenting with suspicious oral lesions. DNA-ICM was performed on 96 cytology smears which had adequate cellularity. On cytology, 54 cases were benign, 36 were malignant, 6 were suspicious for malignancy, and 4 were inadequate due to scanty cellularity. On histopathologic examination, 49 cases were diagnosed as squamous cell carcinoma and 51 cases as benign. The sensitivity of brush cytology for the detection of cancer was 83.3% and the specificity was 95.8%. The positive and negative predictive values were 95.2% and 85.2%, respectively, with a diagnostic accuracy of 86%. Out of 96 cases analyzed by image analysis to assess DNA ploidy, 33 cases were aneuploid and 63 were diploid. The sensitivity, specificity, positive predictive value, and negative predictive value of DNA-ICM were 68.7%, 100%, 100%, and 76.1%, respectively, giving a diagnostic accuracy of 81%. The combination of cytology and DNA cytometry increased the sensitivity to 92% and specificity to 100%. The study demonstrates the usefulness of DNA-ICM as an adjunct to brush cytology to diagnose oral cancer. It reduces the false negative cases on cytology and also adds to objectivity in cytologically doubtful cases. © 2016 Wiley Periodicals, Inc.

Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry

PubMed Central

2013-01-01

Background The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface. Results Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells. Conclusion The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs. PMID:23388071

CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

NASA Astrophysics Data System (ADS)

Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

2011-07-01

Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ~ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ~ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ~ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ~ 12 nm retained bright fluorescence over an extended duration of ~ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ~ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ~ 8.2% in human peripheral blood cells (PBMCs) which are CD33low. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

Calibration procedures for the quantitative determination of membrane potential in human cells using anionic dyes.

PubMed

Klapperstück, Thomas; Glanz, Dagobert; Hanitsch, Stefan; Klapperstück, Manuela; Markwardt, Fritz; Wohlrab, Johannes

2013-07-01

Quantitative determinations of the cell membrane potential of lymphocytes (Wilson et al., J Cell Physiol 1985;125:72-81) and thymocytes (Krasznai et al., J Photochem Photobiol B 1995;28:93-99) using the anionic dye DiBAC4 (3) proved that dye depletion in the extracellular medium as a result of cellular uptake can be negligible over a wide range of cell densities. In contrast, most flow cytometric studies have not verified this condition but rather assumed it from the start. Consequently, the initially prepared extracellular dye concentration has usually been used for the calculation of the Nernst potential of the dye. In this study, however, external dye depletion could be observed in both large IGR-1 and small LCL-HO cells under experimental conditions, which have often been applied routinely in spectrofluorimetry and flow cytometry. The maximum cell density at which dye depletion could be virtually avoided was dependent on cell size and membrane potential and definitely needed to be taken into account to ensure reliable results. In addition, accepted calibration procedures based on the partition of sodium and potassium (Goldman-Hodgkin-Katz equation) or potassium alone (Nernst equation) were performed by flow cytometry on cell suspensions with an appropriately low cell density. The observed extensive lack of concordance between the correspondingly calculated membrane potential and the equilibrium potential of DiBAC4 (3) revealed that these methods require the additional measurement of cation parameters (membrane permeability and/or intracellular concentration). In contrast, due to the linear relation between fluorescence and low DiBAC4 (3) concentrations, the Nernst potential of the dye for totally depolarized cells can be reliably used for calibration with an essentially lower effort and expense. Copyright © 2013 International Society for Advancement of Cytometry.

Spectroscopic imaging system for high-throughput viability assessment of ovarian spheroids or microdissected tumor tissues (MDTs) in a microfluidic chip

NASA Astrophysics Data System (ADS)

St-Georges-Robillard, A.; Masse, M.; Kendall-Dupont, J.; Strupler, M.; Patra, B.; Jermyn, M.; Mes-Masson, A.-M.; Leblond, F.; Gervais, T.

2016-02-01

There is a growing effort in the biomicrosystems community to develop a personalized treatment response assay for cancer patients using primary cells, patient-derived spheroids, or live tissues on-chip. Recently, our group has developed a technique to cut tumors in 350 μm diameter microtissues and keep them alive on-chip, enabling multiplexed in vitro drug assays on primary tumor tissue. Two-photon microscopy, confocal microscopy and flow cytometry are the current standard to assay tissue chemosensitivity on-chip. While these techniques provide microscopic and molecular information, they are not adapted for high-throughput analysis of microtissues. We present a spectroscopic imaging system that allows rapid quantitative measurements of multiple fluorescent viability markers simultaneously by using a liquid crystal tunable filter to record fluorescence and transmittance spectra. As a proof of concept, 24 spheroids composed of ovarian cancer cell line OV90 were formed in a microfluidic chip, stained with two live cell markers (CellTrackerTM Green and Orange), and imaged. Fluorescence images acquired were normalized to the acquisition time and gain of the camera, dark noise was removed, spectral calibration was applied, and spatial uniformity was corrected. Spectral un-mixing was applied to separate each fluorophore's contribution. We have demonstrated that rapid and simultaneous viability measurements on multiple spheroids can be achieved, which will have a significant impact on the prediction of a tumor's response to multiple treatment options. This technique may be applied as well in drug discovery to assess the potential of a drug candidate directly on human primary tissue.

Molecular imaging and sensing using plasmonic nanoparticles

NASA Astrophysics Data System (ADS)

Crow, Matthew James

Noble metal nanoparticles exhibit unique optical properties that are beneficial to a variety of applications, including molecular imaging. The large scattering cross sections of nanoparticles provide high contrast necessary for biomarkers. Unlike alternative contrast agents, nanoparticles provide refractive index sensitivity revealing information regarding the local cellular environment. Altering the shape and composition of the nanoparticle shifts the peak resonant wavelength of scattered light, allowing for implementation of multiple spectrally distinct tags. In this project, nanoparticles that scatter in different spectral windows are functionalized with various antibodies recognizing extra-cellular receptors integral to cancer progression. A hyperspectral imaging system is developed, allowing for visualization and spectral characterization of cells labeled with these conjugates. Various molecular imaging and microspectroscopy applications of plasmonic nanoparticles are then investigated. First, anti-EGFR gold nanospheres are shown to quantitatively measure receptor expression with similar performance to fluorescence assays. Second, anti-EGFR gold nanorods and novel anti-IGF-1R silver nanospheres are implemented to indicate local cellular refractive indices. Third, because biosensing capabilities of nanoparticle tags may be limited by plasmonic coupling, polarization mapping is investigated as a method to discern these effects. Fourth, plasmonic coupling is tested to monitor HER-2 dimerization. Experiments reveal the interparticle conformation of proximal HER-2 bound labels, required for plasmonic coupling-enhanced dielectric sensing. Fifth, all three functionalized plasmonic tags are implemented simultaneously to indicate clinically relevant cell immunophenotype information and changes in the cellular dielectric environment. Finally, flow cytometry experiments are conducted utilizing the anti-EGFR nanorod tag to demonstrate profiling of receptor expression distribution and potential increased multiplexing capability.

Segmentation and detection of fluorescent 3D spots.

PubMed

Ram, Sundaresh; Rodríguez, Jeffrey J; Bosco, Giovanni

2012-03-01

The 3D spatial organization of genes and other genetic elements within the nucleus is important for regulating gene expression. Understanding how this spatial organization is established and maintained throughout the life of a cell is key to elucidating the many layers of gene regulation. Quantitative methods for studying nuclear organization will lead to insights into the molecular mechanisms that maintain gene organization as well as serve as diagnostic tools for pathologies caused by loss of nuclear structure. However, biologists currently lack automated and high throughput methods for quantitative and qualitative global analysis of 3D gene organization. In this study, we use confocal microscopy and fluorescence in-situ hybridization (FISH) as a cytogenetic technique to detect and localize the presence of specific DNA sequences in 3D. FISH uses probes that bind to specific targeted locations on the chromosomes, appearing as fluorescent spots in 3D images obtained using fluorescence microscopy. In this article, we propose an automated algorithm for segmentation and detection of 3D FISH spots. The algorithm is divided into two stages: spot segmentation and spot detection. Spot segmentation consists of 3D anisotropic smoothing to reduce the effect of noise, top-hat filtering, and intensity thresholding, followed by 3D region-growing. Spot detection uses a Bayesian classifier with spot features such as volume, average intensity, texture, and contrast to detect and classify the segmented spots as either true or false spots. Quantitative assessment of the proposed algorithm demonstrates improved segmentation and detection accuracy compared to other techniques. Copyright © 2012 International Society for Advancement of Cytometry.

A high throughput spectral image microscopy system

NASA Astrophysics Data System (ADS)

Gesley, M.; Puri, R.

2018-01-01

A high throughput spectral image microscopy system is configured for rapid detection of rare cells in large populations. To overcome flow cytometry rates and use of fluorophore tags, a system architecture integrates sample mechanical handling, signal processors, and optics in a non-confocal version of light absorption and scattering spectroscopic microscopy. Spectral images with native contrast do not require the use of exogeneous stain to render cells with submicron resolution. Structure may be characterized without restriction to cell clusters of differentiation.

Near infrared lasers in flow cytometry.

PubMed

Telford, William G

2015-07-01

Technology development in flow cytometry has closely tracked laser technology, the light source that flow cytometers almost exclusively use to excite fluorescent probes. The original flow cytometers from the 1970s and 1980s used large water-cooled lasers to produce only one or two laser lines at a time. Modern cytometers can take advantage of the revolution in solid state laser technology to use almost any laser wavelength ranging from the ultraviolet to the near infrared. Commercial cytometers can now be equipped with many small solid state lasers, providing almost any wavelength needed for cellular analysis. Flow cytometers are now equipped to analyze 20 or more fluorescent probes simultaneously, requiring multiple laser wavelengths. Instrument developers are now trying to increase this number by designing fluorescent probes that can be excited by laser wavelength at the "edges" of the visible light range, in the near ultraviolet and near-infrared region. A variety of fluorescent probes have been developed that excite with violet and long wavelength ultraviolet light; however, the near-infrared range (660-800 nm) has yet seen only exploitation in flow cytometry. Fortunately, near-infrared laser diodes and other solid state laser technologies appropriate for flow cytometry have been in existence for some time, and can be readily incorporated into flow cytometers to accelerate fluorescent probe development. The near infrared region represents one of the last "frontiers" to maximize the number of fluorescent probes that can be analyzed by flow cytometry. In addition, near infrared fluorescent probes used in biomedical tracking and imaging could also be employed for flow cytometry with the correct laser wavelengths. This review describes the available technology, including lasers, fluorescent probes and detector technology optimal for near infrared signal detection. Published by Elsevier Inc.

One-dimensional acoustic standing waves in rectangular channels for flow cytometry.

PubMed

Austin Suthanthiraraj, Pearlson P; Piyasena, Menake E; Woods, Travis A; Naivar, Mark A; Lόpez, Gabriel P; Graves, Steven W

2012-07-01

Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry. Copyright © 2012 Elsevier Inc. All rights reserved.

Epithelial invasion outcompetes hypha development during Candida albicans infection as revealed by an image-based systems biology approach.

PubMed

Mech, Franziska; Wilson, Duncan; Lehnert, Teresa; Hube, Bernhard; Thilo Figge, Marc

2014-02-01

Candida albicans is the most common opportunistic fungal pathogen of the human mucosal flora, frequently causing infections. The fungus is responsible for invasive infections in immunocompromised patients that can lead to sepsis. The yeast to hypha transition and invasion of host-tissue represent major determinants in the switch from benign colonizer to invasive pathogen. A comprehensive understanding of the infection process requires analyses at the quantitative level. Utilizing fluorescence microscopy with differential staining, we obtained images of C. albicans undergoing epithelial invasion during a time course of 6 h. An image-based systems biology approach, combining image analysis and mathematical modeling, was applied to quantify the kinetics of hyphae development, hyphal elongation, and epithelial invasion. The automated image analysis facilitates high-throughput screening and provided quantities that allow for the time-resolved characterization of the morphological and invasive state of fungal cells. The interpretation of these data was supported by two mathematical models, a kinetic growth model and a kinetic transition model, that were developed using differential equations. The kinetic growth model describes the increase in hyphal length and revealed that hyphae undergo mass invasion of epithelial cells following primary hypha formation. We also provide evidence that epithelial cells stimulate the production of secondary hyphae by C. albicans. Based on the kinetic transition model, the route of invasion was quantified in the state space of non-invasive and invasive fungal cells depending on their number of hyphae. This analysis revealed that the initiation of hyphae formation represents an ultimate commitment to invasive growth and suggests that in vivo, the yeast to hypha transition must be under exquisitely tight negative regulation to avoid the transition from commensal to pathogen invading the epithelium. © 2013 International Society for Advancement of Cytometry.

Heterogeneity Between Ducts of the Same Nuclear Grade Involved by Duct Carcinoma In Situ (DCIS) of the Breast

PubMed Central

Miller, Naomi A.; Chapman, Judith-Anne W.; Qian, Jin; Christens-Barry, William A.; Fu, Yuejiao; Yuan, Yan; Lickley, H. Lavina A.; Axelrod, David E.

2010-01-01

Purpose Nuclear grade of breast DCIS is considered during patient management decision-making although it may have only a modest prognostic association with therapeutic outcome. We hypothesized that visual inspection may miss substantive differences in nuclei classified as having the same nuclear grade. To test this hypothesis, we measured subvisual nuclear features by quantitative image cytometry for nuclei with the same grade, and tested for statistical differences in these features. Experimental design and statistical analysis Thirty-nine nuclear digital image features of about 100 nuclei were measured in digital images of H&E stained slides of 81 breast biopsy specimens. One field with at least 5 ducts was evaluated for each patient. We compared features of nuclei with the same grade in multiple ducts of the same patient with ANOVA (or Welch test), and compared features of nuclei with the same grade in two ducts of different patients using 2-sided t-tests (P ≤ 0.05). Also, we compared image features for nuclei in patients with single grade to those with the same grade in patients with multiple grades using t-tests. Results Statistically significant differences were detected in nuclear features between ducts with the same nuclear grade, both in different ducts of the same patient, and between ducts in different patients with DCIS of more than one grade. Conclusion Nuclei in ducts visually described as having the same nuclear grade had significantly different subvisual digital image features. These subvisual differences may be considered additional manifestations of heterogeneity over and above differences that can be observed microscopically. This heterogeneity may explain the inconsistency of nuclear grading as a prognostic factor. PMID:20981137

A novel measure and significance testing in data analysis of cell image segmentation.

PubMed

Wu, Jin Chu; Halter, Michael; Kacker, Raghu N; Elliott, John T; Plant, Anne L

2017-03-14

Cell image segmentation (CIS) is an essential part of quantitative imaging of biological cells. Designing a performance measure and conducting significance testing are critical for evaluating and comparing the CIS algorithms for image-based cell assays in cytometry. Many measures and methods have been proposed and implemented to evaluate segmentation methods. However, computing the standard errors (SE) of the measures and their correlation coefficient is not described, and thus the statistical significance of performance differences between CIS algorithms cannot be assessed. We propose the total error rate (TER), a novel performance measure for segmenting all cells in the supervised evaluation. The TER statistically aggregates all misclassification error rates (MER) by taking cell sizes as weights. The MERs are for segmenting each single cell in the population. The TER is fully supported by the pairwise comparisons of MERs using 106 manually segmented ground-truth cells with different sizes and seven CIS algorithms taken from ImageJ. Further, the SE and 95% confidence interval (CI) of TER are computed based on the SE of MER that is calculated using the bootstrap method. An algorithm for computing the correlation coefficient of TERs between two CIS algorithms is also provided. Hence, the 95% CI error bars can be used to classify CIS algorithms. The SEs of TERs and their correlation coefficient can be employed to conduct the hypothesis testing, while the CIs overlap, to determine the statistical significance of the performance differences between CIS algorithms. A novel measure TER of CIS is proposed. The TER's SEs and correlation coefficient are computed. Thereafter, CIS algorithms can be evaluated and compared statistically by conducting the significance testing.

The role of multiparametric flow cytometry in the detection of minimal residual disease in acute leukaemia.

PubMed

Lee, Denise; Grigoriadis, George; Westerman, David

2015-12-01

Flow cytometry is the most accessible method for minimal residual disease (MRD) detection due to its availability in most haematological centres. Using a precise combination of different antibodies, immunophenotypic detection of MRD in acute leukaemia can be performed by identifying abnormal combinations or expressions of antigens on malignant cells at diagnosis, during and post treatment. These abnormal phenotypes, referred to as leukaemia-associated immunophenotypes (LAIPs) are either absent or expressed at low frequency in normal bone marrow (BM) cells and are used to monitor the behaviour and quantitate the amount of residual disease following treatment. In paediatric acute lymphoblastic leukaemia (ALL), the level of MRD by multiparametric flow cytometry (MPFC) during therapy is recognised as an important predictor of outcome. Although less extensively studied, adult ALL and adult and paediatric acute myeloid leukaemia (AML) have also demonstrated similar findings. The challenge now is incorporating this information for risk-stratification so that therapy can be tailored individually and ultimately improve outcome while also limiting treatment-related toxicity. In this review we will elaborate on the current and future role of MPFC in MRD in acute leukaemia while also addressing its limitations.

Opportunities of using Stimulated Emission from Biological Tissue

NASA Astrophysics Data System (ADS)

Yun, S. H. Andy

Fluorescence or spontaneous emission has been a powerful tool in biomedical applications ranging from biochemical assays and cytometry to microscopy and medical imaging. Here I present the opportunities in the generation and applications of coherent stimulated emission within biological samples.

The impact of digital imaging in the field of cytopathology.

PubMed

Pantanowitz, Liron; Hornish, Maryanne; Goulart, Robert A

2009-03-06

With the introduction of digital imaging, pathology is undergoing a digital transformation. In the field of cytology, digital images are being used for telecytology, automated screening of Pap test slides, training and education (e.g. online digital atlases), and proficiency testing. To date, there has been no systematic review on the impact of digital imaging on the practice of cytopathology. This article critically addresses the emerging role of computer-assisted screening and the application of digital imaging to the field of cytology, including telecytology, virtual microscopy, and the impact of online cytology resources. The role of novel diagnostic techniques like image cytometry is also reviewed.

Development of a diffraction imaging flow cytometer

PubMed Central

Jacobs, Kenneth M.; Lu, Jun Q.

2013-01-01

Diffraction images record angle-resolved distribution of scattered light from a particle excited by coherent light and can correlate highly with the 3D morphology of a particle. We present a jet-in-fluid design of flow chamber for acquisition of clear diffraction images in a laminar flow. Diffraction images of polystyrene spheres of different diameters were acquired and found to correlate highly with the calculated ones based on the Mie theory. Fast Fourier transform analysis indicated that the measured images can be used to extract sphere diameter values. These results demonstrate the significant potentials of high-throughput diffraction imaging flow cytometry for extracting 3D morphological features of cells. PMID:19794790

Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging

PubMed Central

Jochums, André; Friehs, Elsa; Sambale, Franziska; Lavrentieva, Antonina; Bahnemann, Detlef; Scheper, Thomas

2017-01-01

The uptake of nanomaterials into different cell types is a central pharmacological issue for the determination of nanotoxicity as well as for the development of drug delivery strategies. Most responses of the cells depend on their intracellular interactions with nanoparticles (NPs). Uptake behavior can be precisely investigated in vitro, with sensitive high throughput methods such as flow cytometry. In this study, we investigated two different standard cell lines, human lung carcinoma (A549) and mouse fibroblast (NIH/3T3) cells, regarding their uptake behavior of titanium dioxide NPs. Cells were incubated with different concentrations of TiO2 NPs and samples were taken at certain time points to compare the uptake kinetics of both cell lines. Samples were analyzed with the help of flow cytometry by studying changes in the side and forward scattering signal. To additionally enable a detection via fluorescence, NPs were labeled with the fluorescent dye fluorescein isothiocyanate (FITC) and propidium iodide (PI). We found that NIH/3T3 cells take up the studied NPs more efficiently than A549 cells. These findings were supported by time-lapse microscopic imaging of the cells incubated with TiO2 NPs. Our results confirm that the uptake behavior of individual cell types has to be considered before interpreting any results of nanomaterial studies. PMID:29051447

Characterization of functional variables in epididymal alpaca (Vicugna pacos) sperm using imaging flow cytometry.

PubMed

Santiani, Alexei; Ugarelli, Alejandra; Evangelista-Vargas, Shirley

2016-10-01

Epididymal alpaca sperm represent an alternative model for the study of alpaca semen. The objective of this study was to characterize the normal values of some functional variables in epididymal alpaca sperm using imaging flow cytometry. Alpaca testicles (n=150) were processed and sperm were recovered from the cauda epididymides. Only 76 samples with acceptable motility and sperm count were considered for assessment by imaging flow cytometry. Acrosome integrity and integrity/viability were assessed by FITC-PSA/PI and FITC-PNA/PI. Mitochondrial membrane potential (MMP) was assessed by MitoTracker CMXRos and MitoTracker Deep Red FM. Lipid peroxidation was evaluated using BODIPY 581/591 C11. Results show that the mean values for acrosome-intact sperm were 95.03±6.39% and 93.34±7.96%, using FITC-PSA and FITC-PNA, respectively. The mean values for acrosome-intact viable sperm were 60.58±12.12% with FITC-PSA/PI and 58.81±12.94% with FITC-PNA/PI. Greater MMP was detected in 65.03±15.92% and 59.52±19.19%, using MitoTracker CMXRos and MitoTracker Deep Red FM, respectively. Lipid peroxidation was 0.84±0.95%. Evaluation of acrosome-intact and acrosome-intact viable sperm with FITC-PSA/PI compared with. FITC-PNA/PI or MMP with MitoTracker CMXRos compared with MitoTracker Deep Red FM were correlated (P<0.05). The MMP using MitoTracker CMXRos was the only variable correlated (P<0.05) with sperm motility (r=0.3979). This report provides a basis for future research related to alpaca semen using the epididymal sperm model. Copyright © 2016 Elsevier B.V. All rights reserved.

The use of the decision tree technique and image cytometry to characterize aggressiveness in World Health Organization (WHO) grade II superficial transitional cell carcinomas of the bladder.

PubMed

Decaestecker, C; van Velthoven, R; Petein, M; Janssen, T; Salmon, I; Pasteels, J L; van Ham, P; Schulman, C; Kiss, R

1996-03-01

The aggressiveness of human bladder tumours can be assessed by means of various classification systems, including the one proposed by the World Health Organization (WHO). According to the WHO classification, three levels of malignancy are identified as grades I (low), II (intermediate), and III (high). This classification system operates satisfactorily for two of the three grades in forecasting clinical progression, most grade I tumours being associated with good prognoses and most grade III with bad. In contrast, the grade II group is very heterogeneous in terms of their clinical behaviour. The present study used two computer-assisted methods to investigate whether it is possible to sub-classify grade II tumours: computer-assisted microscope analysis (image cytometry) of Feulgen-stained nuclei and the Decision Tree Technique. This latter technique belongs to the Supervised Learning Algorithm and enables an objective assessment to be made of the diagnostic value associated with a given parameter. The combined use of these two methods in a series of 292 superficial transitional cell carcinomas shows that it is possible to identify one subgroup of grade II tumours which behave clinically like grade I tumours and a second subgroup which behaves clinically like grade III tumours. Of the nine ploidy-related parameters computed by means of image cytometry [the DNA index (DI), DNA histogram type (DHT), and the percentages of diploid, hyperdiploid, triploid, hypertriploid, tetraploid, hypertetraploid, and polyploid cell nuclei], it was the percentage of hyperdiploid and hypertetraploid cell nuclei which enabled identification, rather than conventional parameters such as the DI or the DHT.

Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

PubMed

Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

2016-07-01

Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections. © 2016 Society for Laboratory Automation and Screening.

Capture of Fluorescence Decay Times by Flow Cytometry

PubMed Central

Naivar, Mark A.; Jenkins, Patrick; Freyer, James P.

2012-01-01

In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction. PMID:25419263

Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification

NASA Astrophysics Data System (ADS)

Illien, Françoise; Rodriguez, Nicolas; Amoura, Mehdi; Joliot, Alain; Pallerla, Manjula; Cribier, Sophie; Burlina, Fabienne; Sagan, Sandrine

2016-11-01

The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.

CMOS based image cytometry for detection of phytoplankton in ballast water.

PubMed

Pérez, J M; Jofre, M; Martínez, P; Yáñez, M A; Catalan, V; Parker, A; Veldhuis, M; Pruneri, V

2017-02-01

We introduce an image cytometer (I-CYT) for the analysis of phytoplankton in fresh and marine water environments. A linear quantification of cell numbers was observed covering several orders of magnitude using cultures of Tetraselmis and Nannochloropsis measured by autofluorescence in a laboratory environment. We assessed the functionality of the system outside the laboratory by phytoplankton quantification of samples taken from a marine water environment (Dutch Wadden Sea, The Netherlands) and a fresh water environment (Lake Ijssel, The Netherlands). The I-CYT was also employed to study the effects of two ballast water treatment systems (BWTS), based on chlorine electrolysis and UV sterilization, with the analysis including the vitality of the phytoplankton. For comparative study and benchmarking of the I-CYT, a standard flow cytometer was used. Our results prove a limit of detection (LOD) of 10 cells/ml with an accuracy between 0.7 and 0.5 log, and a correlation of 88.29% in quantification and 96.21% in vitality, with respect to the flow cytometry results.

Contribution of high-content imaging technologies to the development of anti-infective drugs.

PubMed

Ang, Michelle Lay Teng; Pethe, Kevin

2016-08-01

Originally developed to study fundamental aspects of cellular biology, high-content imaging (HCI) was rapidly adapted to study host-pathogen interactions at the cellular level and adopted as a technology of choice to unravel disease biology. HCI platforms allow for the visualization and quantification of discrete phenotypes that cannot be captured using classical screening approaches. A key advantage of high-content screening technologies lies in the possibility to develop and interrogate physiologically significant, predictive ex vivo disease models that reproduce complex conditions relevant for infection. Here we review and discuss recent advances in HCI technologies and chemical biology approaches that are contributing to an increased understanding of the intricate host-pathogen interrelationship on the cellular level, and which will foster the development of novel therapeutic approaches for the treatment of human bacterial and protozoan infections. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

Recent advances in applying mass spectrometry and systems biology to determine brain dynamics.

PubMed

Scifo, Enzo; Calza, Giulio; Fuhrmann, Martin; Soliymani, Rabah; Baumann, Marc; Lalowski, Maciej

2017-06-01

Neurological disorders encompass various pathologies which disrupt normal brain physiology and function. Poor understanding of their underlying molecular mechanisms and their societal burden argues for the necessity of novel prevention strategies, early diagnostic techniques and alternative treatment options to reduce the scale of their expected increase. Areas covered: This review scrutinizes mass spectrometry based approaches used to investigate brain dynamics in various conditions, including neurodegenerative and neuropsychiatric disorders. Different proteomics workflows for isolation/enrichment of specific cell populations or brain regions, sample processing; mass spectrometry technologies, for differential proteome quantitation, analysis of post-translational modifications and imaging approaches in the brain are critically deliberated. Future directions, including analysis of cellular sub-compartments, targeted MS platforms (selected/parallel reaction monitoring) and use of mass cytometry are also discussed. Expert commentary: Here, we summarize and evaluate current mass spectrometry based approaches for determining brain dynamics in health and diseases states, with a focus on neurological disorders. Furthermore, we provide insight on current trends and new MS technologies with potential to improve this analysis.

Quantum-dot-based quantitative identification of pathogens in complex mixture

NASA Astrophysics Data System (ADS)

Lim, Sun Hee; Bestwater, Felix; Buchy, Philippe; Mardy, Sek; Yu, Alexey Dan Chin

2010-02-01

In the present study we describe sandwich design hybridization probes consisting of magnetic particles (MP) and quantum dots (QD) with target DNA, and their application in the detection of avian influenza virus (H5N1) sequences. Hybridization of 25-, 40-, and 100-mer target DNA with both probes was analyzed and quantified by flow cytometry and fluorescence microscopy on the scale of single particles. The following steps were used in the assay: (i) target selection by MP probes and (ii) target detection by QD probes. Hybridization efficiency between MP conjugated probes and target DNA hybrids was controlled by a fluorescent dye specific for nucleic acids. Fluorescence was detected by flow cytometry to distinguish differences in oligo sequences as short as 25-mer capturing in target DNA and by gel-electrophoresis in the case of QD probes. This report shows that effective manipulation and control of micro- and nanoparticles in hybridization assays is possible.

Waveguide detection of right-angle-scattered light in flow cytometry

DOEpatents

Mariella, Jr., Raymond P.

2000-01-01

A transparent flow cell is used as an index-guided optical waveguide. A detector for the flow cell but not the liquid stream detects the Right-Angle-Scattered (RAS) Light exiting from one end of the flow cell. The detector(s) could view the trapped RAS light from the flow cell either directly or through intermediate optical light guides. If the light exits one end of the flow cell, then the other end of the flow cell can be given a high-reflectivity coating to approximately double the amount of light collected. This system is more robust in its alignment than the traditional flow cytometry systems which use imaging optics, such as microscope objectives.

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.

PubMed

Trivedi, Vikas; Choi, Harry M T; Fraser, Scott E; Pierce, Niles A

2018-01-08

For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization. © 2018. Published by The Company of Biologists Ltd.

Variables affecting the quantitation of CD22 in neoplastic B cells.

PubMed

Jasper, Gregory A; Arun, Indu; Venzon, David; Kreitman, Robert J; Wayne, Alan S; Yuan, Constance M; Marti, Gerald E; Stetler-Stevenson, Maryalice

2011-03-01

Quantitative flow cytometry (QFCM) is being applied in the clinical flow cytometry laboratory for diagnosis, prognosis, and assessment of patients receiving antibody-based therapy. ABC values and the effect of technical variables on CD22 quantitation in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FCL), hairy cell leukemia (HCL) and normal B cells were studied. The QuantiBrite System® was used to determine the level of CD22 expression (mean antibody bound per cell, ABC) by malignant and normal B cells. The intra-assay variability, number of cells required for precision, effect of delayed processing as well as shipment of peripheral blood specimens (delayed processing and exposure to noncontrolled environments), and the effect of paraformaldehyde fixation on assay results were studied. The QuantiBRITE method of measuring CD22 ABC is precise (median CV 1.6%, 95% confidence interval, 1.2-2.3%) but a threshold of 250 malignant cells is required for reliable CD22 ABC values. Delayed processing and overnight shipment of specimens resulted in significantly different ABC values whereas fixation for up to 12 h had no significant effect. ABC measurements determined that CD22 expression is lower than normal in ALL, CLL, FCL, and MCL but higher than normal in HCL. CD22 expression was atypical in the hematolymphoid malignancies studied and may have diagnostic utility. Technical variables such as cell number analyzed and delayed processing or overnight shipment of specimens impact significantly on the measurement of antigen expression by QFCM in the clinical laboratory. Published 2010 Wiley-Liss, Inc.

FlowCal: A user-friendly, open source software tool for automatically converting flow cytometry data from arbitrary to calibrated units

PubMed Central

Castillo-Hair, Sebastian M.; Sexton, John T.; Landry, Brian P.; Olson, Evan J.; Igoshin, Oleg A.; Tabor, Jeffrey J.

2017-01-01

Flow cytometry is widely used to measure gene expression and other molecular biological processes with single cell resolution via fluorescent probes. Flow cytometers output data in arbitrary units (a.u.) that vary with the probe, instrument, and settings. Arbitrary units can be converted to the calibrated unit molecules of equivalent fluorophore (MEF) using commercially available calibration particles. However, there is no convenient, non-proprietary tool available to perform this calibration. Consequently, most researchers report data in a.u., limiting interpretation. Here, we report a software tool named FlowCal to overcome current limitations. FlowCal can be run using an intuitive Microsoft Excel interface, or customizable Python scripts. The software accepts Flow Cytometry Standard (FCS) files as inputs and is compatible with different calibration particles, fluorescent probes, and cell types. Additionally, FlowCal automatically gates data, calculates common statistics, and produces publication quality plots. We validate FlowCal by calibrating a.u. measurements of E. coli expressing superfolder GFP (sfGFP) collected at 10 different detector sensitivity (gain) settings to a single MEF value. Additionally, we reduce day-to-day variability in replicate E. coli sfGFP expression measurements due to instrument drift by 33%, and calibrate S. cerevisiae mVenus expression data to MEF units. Finally, we demonstrate a simple method for using FlowCal to calibrate fluorescence units across different cytometers. FlowCal should ease the quantitative analysis of flow cytometry data within and across laboratories and facilitate the adoption of standard fluorescence units in synthetic biology and beyond. PMID:27110723

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part II - preanalytical issues.

PubMed

Davis, Bruce H; Dasgupta, Amar; Kussick, Steven; Han, Jin-Yeong; Estrellado, Annalee

2013-01-01

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part II - Preanalytical issues. © 2013 International Clinical Cytometry Society. © 2013 International Clinical Cytometry Society.

Distinction between asymptomatic monoclonal B-cell lymphocytosis with cyclin D1 overexpression and mantle cell lymphoma: from molecular profiling to flow cytometry.

PubMed

Espinet, Blanca; Ferrer, Ana; Bellosillo, Beatriz; Nonell, Lara; Salar, Antonio; Fernández-Rodríguez, Concepción; Puigdecanet, Eulàlia; Gimeno, Javier; Garcia-Garcia, Mar; Vela, Maria Carmen; Luño, Elisa; Collado, Rosa; Navarro, José Tomás; de la Banda, Esmeralda; Abrisqueta, Pau; Arenillas, Leonor; Serrano, Cristina; Lloreta, Josep; Miñana, Belén; Cerutti, Andrea; Florensa, Lourdes; Orfao, Alberto; Sanz, Ferran; Solé, Francesc; Dominguez-Sola, David; Serrano, Sergio

2014-02-15

According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1-positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment. ©2013 AACR

Distinction between Asymptomatic Monoclonal B-cell Lymphocytosis with Cyclin D1 Overexpression and Mantle Cell Lymphoma: From Molecular Profiling to Flow Cytometry

PubMed Central

Espinet, Blanca; Ferrer, Ana; Bellosillo, Beatriz; Nonell, Lara; Salar, Antonio; Fernández-Rodríguez, Concepción; Puigdecanet, Eulàlia; Gimeno, Javier; Garcia-Garcia, Mar; Carmen Vela, Maria; Luño, Elisa; Collado, Rosa; Navarro, José Tomás; de la Banda, Esmeralda; Abrisqueta, Pau; Arenillas, Leonor; Serrano, Cristina; Lloreta, Josep; Miñana, Belén; Cerutti, Andrea; Florensa, Lourdes; Orfao, Alberto; Sanz, Ferran; Solé, Francesc; Dominguez-Sola, David; Serrano, Sergio

2015-01-01

Purpose According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1–positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. Experimental Design We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. Results We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. Conclusion We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment. PMID:24352646

Evaluating mononuclear cells as nanoparticle delivery vehicles for the treatment of breast tumors

NASA Astrophysics Data System (ADS)

Murton, Jaclyn K.; Hu, Chelin; Ahmed, Mona M.; Hathaway, Helen J.; Nysus, Monique; Anderson Daniels, Tamara; Norenberg, Jeffrey P.; Adolphi, Natalie L.

2015-08-01

In breast cancer, certain types of circulating immune cells respond to long-range chemical signals from tumors by leaving the blood stream to actively infiltrate tumor tissue. The aim of this study was to evaluate whether immune cells could be used to deliver therapeutic nanoparticles into breast tumors in mice. Mononuclear splenocytes (MS) were harvested from donor mice, labeled with Indium-111, injected intravenously into immune-competent recipient mice (3 tumor-bearing and 3 control), and imaged longitudinally by SPECT/CT. For comparison, the biodistribution of bonemarrow derived macrophages (BMDM) in one pair of mice was also imaged. Quantitative analysis of the SPECT images demonstrates that, after 24 hours, the concentration of MS detected in mammary tumors is more than 3-fold higher than the concentration detected in normal mammary glands. The ratio of MS concentration in mammary tissue to MS concentration in non-target tissues (muscle, lung, heart, liver, spleen, and kidney) was enhanced in tumor-bearing mice (compared to controls), with statistical significance achieved for mammary/muscle (p<0.01), mammary/lung (p<0.05), and mammary/kidney (p<0.05). By contrast, BMDM did not show a different affinity for tumors relative to normal mammary tissue. MS were incubated with 100 nm red fluorescent nanoparticles, and flow cytometry demonstrated that ~35% of the MS population exhibited strong phagocytic uptake of the nanoparticles. After intravenous injection into tumor-bearing mice, fluorescence microscopy images of tumor sections show qualitatively that nanoparticle-loaded MS retain the ability to infiltrate mammary tumors. Taken together, these results suggest that MS carriers are capable of actively targeting therapeutic nanoparticles to breast tumors.

Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry.

PubMed

Reis, Monica; McDonald, David; Nicholson, Lindsay; Godthardt, Kathrin; Knobel, Sebastian; Dickinson, Anne M; Filby, Andrew; Wang, Xiao-Nong

2018-03-02

Mesenchymal stromal cells (MSCs) are a promising cell source to develop cell therapy for many diseases. Human platelet lysate (PLT) is increasingly used as an alternative to foetal calf serum (FCS) for clinical-scale MSC production. To date, the global surface protein expression of PLT-expended MSCs (MSC-PLT) is not known. To investigate this, paired MSC-PLT and MSC-FCS were analysed in parallel using high-throughput flow cytometry for the expression of 356 cell surface proteins. MSC-PLT showed differential surface protein expression compared to their MSC-FCS counterpart. Higher percentage of positive cells was observed in MSC-PLT for 48 surface proteins, of which 13 were significantly enriched on MSC-PLT. This finding was validated using multiparameter flow cytometry and further confirmed by quantitative staining intensity analysis. The enriched surface proteins are relevant to increased proliferation and migration capacity, as well as enhanced chondrogenic and osteogenic differentiation properties. In silico network analysis revealed that these enriched surface proteins are involved in three distinct networks that are associated with inflammatory responses, carbohydrate metabolism and cellular motility. This is the first study reporting differential cell surface protein expression between MSC-PLT and MSC-FSC. Further studies are required to uncover the impact of those enriched proteins on biological functions of MSC-PLT.

Immense random colocalization, revealed by automated high content image cytometry, seriously questions FISH as gold standard for detecting EML4-ALK fusion.

PubMed

Smuk, Gábor; Tornóczky, Tamás; Pajor, László; Chudoba, Ilse; Kajtár, Béla; Sárosi, Veronika; Pajor, Gábor

2018-05-19

EML4-ALK gene fusion (inv2(p21p23)) of non-small cell lung cancer (NSCLC) predisposes to tyrosine kinase inhibitor treatment. One of the gold standard diagnostics is the dual color (DC) break-apart (BA) FISH technique, however, the unusual closeness of the involved genes has been suggested to raise likelihood of random co-localization (RCL) of signals. Although this is suspected to decrease sensitivity (often to as low as 40-70%), the exact level and effect of RCL has not been revealed thus far. Signal distances were analyzed to the 0.1 µm precision in more than 25,000 nuclei, via automated high content-image cytometry. Negative and positive controls were created using conventional DC BA-, and inv2(p21p23) mimicking probe-sets, respectively. Average distance between red and green signals was 9.72 pixels (px) (±5.14px) and 3.28px (±2.44px), in positives and negatives, respectively; overlap in distribution being 41%. Specificity and sensitivity of correctly determining ALK status was 97% and 29%, respectively. When investigating inv2(p21p23) with DC BA FISH, specificity is high, but seven out of ten aberrant nuclei are inevitably falsely classified as negative, due to the extreme level of RCL. Together with genetic heterogeneity and dilution effect of non-tumor cells in NSCLC, this immense analytical false negativity is the primary cause behind the often described low diagnostic sensitivity. These results convincingly suggest that if FISH is to remain a gold standard for detecting the therapy relevant inv(2), either a modified evaluation protocol, or a more reliable probe-design should be considered than the current DC BA one. © 2018 International Society for Advancement of Cytometry. © 2018 International Society for Advancement of Cytometry.

Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis

PubMed Central

Schaub, Franz X; Reza, Md Shamim; Flaveny, Colin A; Li, Weimin; Musicant, Adele M; Hoxha, Sany; Guo, Min; Cleveland, John L; Amelio, Antonio L

2015-01-01

Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer (BRET) that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bi-functional nature of these LumiFluor reporters enables greatly improved spatio-temporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, non-invasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes. PMID:26424696

Dual-Color Fluorescence Imaging of Magnetic Nanoparticles in Live Cancer Cells Using Conjugated Polymer Probes

PubMed Central

Sun, Minjie; Sun, Bin; Liu, Yun; Shen, Qun-Dong; Jiang, Shaojun

2016-01-01

Rapid growth in biological applications of nanomaterials brings about pressing needs for exploring nanomaterial-cell interactions. Cationic blue-emissive and anionic green-emissive conjugated polymers are applied as dual-color fluorescence probes to the surface of negatively charged magnetic nanoparticles through sequentially electrostatic adsorption. These conjugated polymers have large extinction coefficients and high fluorescence quantum yield (82% for PFN and 62% for ThPFS). Thereby, one can visualize trace amount (2.7 μg/mL) of fluorescence-labeled nanoparticles within cancer cells by confocal laser scanning microscopy. Fluorescence labeling by the conjugated polymers is also validated for quantitative determination of the internalized nanoparticles in each individual cell by flow cytometry analysis. Extensive overlap of blue and green fluorescence signals in the cytoplasm indicates that both conjugated polymer probes tightly bind to the surface of the nanoparticles during cellular internalization. The highly charged and fluorescence-labeled nanoparticles non-specifically bind to the cell membranes, followed by cellular uptake through endocytosis. The nanoparticles form aggregates inside endosomes, which yields a punctuated staining pattern. Cellular internalization of the nanoparticles is dependent on the dosage and time. Uptake efficiency can be enhanced three-fold by application of an external magnetic field. The nanoparticles are low cytotoxicity and suitable for simultaneously noninvasive fluorescence and magnetic resonance imaging application. PMID:26931282

Quantitative in vivo immunohistochemistry of epidermal growth factor receptor using a receptor concentration imaging approach

PubMed Central

Samkoe, Kimberley S.; Tichauer, Kenneth M.; Gunn, Jason R.; Wells, Wendy A.; Hasan, Tayyaba; Pogue, Brian W.

2014-01-01

As receptor-targeted therapeutics become increasingly used in clinical oncology, the ability to quantify protein expression and pharmacokinetics in vivo is imperative to ensure successful individualized treatment plans. Current standards for receptor analysis are performed on extracted tissues. These measurements are static and often physiologically irrelevant, therefore, only a partial picture of available receptors for drug targeting in vivo is provided. Until recently, in vivo measurements were limited by the inability to separate delivery, binding, and retention effects but this can be circumvented by a dual-tracer approach for referencing the detected signal. We hypothesized that in vivo receptor concentration imaging (RCI) would be superior to ex vivo immunohistochemistry. Using multiple xenograft tumor models with varying epidermal growth factor receptor (EGFR) expression, we determined the EGFR concentration in each model using a novel targeted agent (anti-EGFR affibody-IRDye800CW conjugate) along with a simultaneously delivered reference agent (control affibody-IRDye680RD conjugate). The RCI-calculated in vivo receptor concentration was strongly correlated with ex vivo pathologist-scored immunohistochemistry and computer-quantified ex vivo immunofluorescence. In contrast, no correlation was observed with ex vivo Western blot or in vitro flow cytometry assays. Overall, our results argue that in vivo RCI provides a robust measure of receptor expression equivalent to ex vivo immuno-staining, with implications for use in non-invasive monitoring of therapy or therapeutic guidance during surgery. PMID:25344226

Invasion of Peripheral Immune Cells into Brain Parenchyma after Cardiac Arrest and Resuscitation.

PubMed

Zhang, Can; Brandon, Nicole R; Koper, Kerryann; Tang, Pei; Xu, Yan; Dou, Huanyu

2018-06-01

Although a direct link has long been suspected between systemic immune responses and neuronal injuries after stroke, it is unclear which immune cells play an important role. A question remains as to whether the blood brain barrier (BBB) is transiently disrupted after circulatory arrest to allow peripheral immune cells to enter brain parenchyma. Here, we developed a clinically relevant cardiac arrest and resuscitation model in mice to investigate the BBB integrity using noninvasive magnetic resonance imaging. Changes in immune signals in the brain and periphery were assayed by immunohistochemistry and flow cytometry. Quantitative variance maps from T1-weighted difference images before and after blood-pool contrast clearance revealed BBB disruptions immediately after resuscitation and one day after reperfusion. Time profiles of hippocampal CA1 neuronal injuries correlated with the morphological changes of microglia activation. Cytotoxic T cells, CD11b + CD11c + dendritic cells, and CD11b + CD45 +hi monocytes and macrophages were significantly increased in the brain three days after cardiac arrest and resuscitation, suggesting direct infiltration of these cells following the BBB disruption. Importantly, these immune cell changes were coupled with a parallel increase in the same subset of immune cell populations in the bone marrow and blood. We conclude that neurovascular breakdown during the initial reperfusion phase contributes to the systemic immune cell invasion and subsequent neuropathogenesis affecting the long-term outcome after cardiac arrest and resuscitation.

Establishment and quantitative imaging of a 3D lung organotypic model of mammary tumor outgrowth.

PubMed

Martin, Michelle D; Fingleton, Barbara; Lynch, Conor C; Wells, Sam; McIntyre, J Oliver; Piston, David W; Matrisian, Lynn M

2008-01-01

The lung is the second most common site of metastatic spread in breast cancer and experimental evidence has been provided in many systems for the importance of an organ-specific microenvironment in the development of metastasis. To better understand the interaction between tumor and host cells in this important secondary site, we have developed a 3D in vitro organotypic model of breast tumor metastatic growth in the lung. In our model, cells isolated from mouse lungs are placed in a collagen sponge to serve as a scaffold and co-cultured with a green fluorescent protein-labeled polyoma virus middle T antigen (PyVT) mammary tumor cell line. Analysis of the co-culture system was performed using flow cytometry to determine the relative constitution of the co-cultures over time. This analysis determined that the cultures consisted of viable lung and breast cancer cells over a 5-day period. Confocal microscopy was then used to perform live cell imaging of the co-cultures over time. Our studies determined that host lung cells influence the ability of tumor cells to grow, as the presence of lung parenchyma positively affected the proliferation of the mammary tumor cells in culture. In summary, we have developed a novel in vitro model of breast tumor cells in a common metastatic site that can be used to study tumor/host interactions in an important microenvironment.

Entropy-Based Adaptive Nuclear Texture Features are Independent Prognostic Markers in a Total Population of Uterine Sarcomas

PubMed Central

Nielsen, Birgitte; Hveem, Tarjei Sveinsgjerd; Kildal, Wanja; Abeler, Vera M; Kristensen, Gunnar B; Albregtsen, Fritz; Danielsen, Håvard E; Rohde, Gustavo K

2015-01-01

Nuclear texture analysis measures the spatial arrangement of the pixel gray levels in a digitized microscopic nuclear image and is a promising quantitative tool for prognosis of cancer. The aim of this study was to evaluate the prognostic value of entropy-based adaptive nuclear texture features in a total population of 354 uterine sarcomas. Isolated nuclei (monolayers) were prepared from 50 µm tissue sections and stained with Feulgen-Schiff. Local gray level entropy was measured within small windows of each nuclear image and stored in gray level entropy matrices, and two superior adaptive texture features were calculated from each matrix. The 5-year crude survival was significantly higher (P < 0.001) for patients with high texture feature values (72%) than for patients with low feature values (36%). When combining DNA ploidy classification (diploid/nondiploid) and texture (high/low feature value), the patients could be stratified into three risk groups with 5-year crude survival of 77, 57, and 34% (Hazard Ratios (HR) of 1, 2.3, and 4.1, P < 0.001). Entropy-based adaptive nuclear texture was an independent prognostic marker for crude survival in multivariate analysis including relevant clinicopathological features (HR = 2.1, P = 0.001), and should therefore be considered as a potential prognostic marker in uterine sarcomas. © The Authors. Published 2014 International Society for Advancement of Cytometry PMID:25483227

STATISTICAL EVALUATION OF CONFOCAL MICROSCOPY IMAGES

EPA Science Inventory

Abstract

In this study the CV is defined as the Mean/SD of the population of beads or pixels. Flow cytometry uses the CV of beads to determine if the machine is aligned correctly and performing properly. This CV concept to determine machine performance has been adapted to...

High throughput, real-time detection of Naegleria lovaniensis in natural river water using LED-illuminated Fountain Flow Cytometry.

PubMed

Johnson, P E; Deromedi, A J; Lebaron, P; Catala, P; Havens, C; Pougnard, C

2007-09-01

To test Fountain Flow Cytometry (FFC) for the rapid and sensitive detection of Naegleria lovaniensis amoebae (an analogue for Naegleria fowleri) in natural river waters. Samples were incubated with one of two fluorescent labels to facilitate detection: ChemChrome V6, a viability indicator, and an R-phycoerytherin (RPE) immunolabel to detect N. lovaniensis specifically. The resulting aqueous sample was passed as a stream in front of a light-emitting diode, which excited the fluorescent labels. The fluorescence was detected with a digital camera as the sample flowed toward the imager. Detections of N. lovaniensis were made in inoculated samples of natural water from eight rivers in France and the United States. FFC enumeration yielded results that are consistent with other counting methods: solid-phase cytometry, flow cytometry, and hemocytometry, down to concentrations of 0.06 amoebae ml(-1), using a flow rate of 15 ml min(-1). This study supports the efficacy of using FFC for the detection of viable protozoa in natural waters and indicates that use of RPE illuminated at 530 nm and detected at 585 nm provides a satisfactory means of attenuating background. Because of the severe global public health issues with drinking water and sanitation, there is an urgent need to develop a technique for the real-time detection of viable pathogens in environmental samples at low concentrations. FFC addresses this need.

Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy.

PubMed

Keilholz, Ulrich; Weber, Jeffrey; Finke, James H; Gabrilovich, Dmitry I; Kast, W Martin; Disis, Mary L; Kirkwood, John M; Scheibenbogen, Carmen; Schlom, Jeff; Maino, Vernon C; Lyerly, H Kim; Lee, Peter P; Storkus, Walter; Marincola, Franceso; Worobec, Alexandra; Atkins, Michael B

2002-01-01

The Society for Biological Therapy held a Workshop last fall devoted to immune monitoring for cancer immunotherapy trials. Participants included members of the academic and pharmaceutical communities as well as the National Cancer Institute and the Food and Drug Administration. Discussion focused on the relative merits and appropriate use of various immune monitoring tools. Six breakout groups dealt with assays of T-cell function, serologic and proliferation assays to assess B cell and T helper cell activity, and enzyme-linked immunospot assay, tetramer, cytokine flow cytometry, and reverse transcription polymerase chain reaction assays of T-cell immunity. General conclusions included: (1) future vaccine studies should be designed to determine whether T-cell dysfunction (tumor-specific and nonspecific) correlated with clinical outcome; (2) tetramer-based assays yield quantitative but not functional data (3) enzyme-linked immunospot assays have the lowest limit of detection (4) cytokine flow cytometry have a higher limit of detection than enzyme-linked immunospot assay, but offer the advantages of speed and the ability to identify subsets of reactive cells; (5) antibody tests are simple and accurate and should be incorporated to a greater extent in monitoring plans; (6) proliferation assays are imprecise and should not be emphasized in future studies; (7) the reverse transcription polymerase chain reaction assay is a promising research approach that is not ready for widespread application; and (8)there is a critical need to validate these assays as surrogates for vaccine potency and clinical effect. Current data and opinion support the use of a functional assay like the enzyme-linked immunospot assay or cytokine flow cytometry in combination with a quantitative assay like tetramers for immune monitoring. At present, assays appear to be most useful as measures of vaccine potency. Careful immune monitoring in association with larger scale clinical trials ultimately may enable the correlation of monitoring results with clinical benefit.

Brain Injury Lesion Imaging Using Preconditioned Quantitative Susceptibility Mapping without Skull Stripping.

PubMed

Soman, S; Liu, Z; Kim, G; Nemec, U; Holdsworth, S J; Main, K; Lee, B; Kolakowsky-Hayner, S; Selim, M; Furst, A J; Massaband, P; Yesavage, J; Adamson, M M; Spincemallie, P; Moseley, M; Wang, Y

2018-04-01

Identifying cerebral microhemorrhage burden can aid in the diagnosis and management of traumatic brain injury, stroke, hypertension, and cerebral amyloid angiopathy. MR imaging susceptibility-based methods are more sensitive than CT for detecting cerebral microhemorrhage, but methods other than quantitative susceptibility mapping provide results that vary with field strength and TE, require additional phase maps to distinguish blood from calcification, and depict cerebral microhemorrhages as bloom artifacts. Quantitative susceptibility mapping provides universal quantification of tissue magnetic property without these constraints but traditionally requires a mask generated by skull-stripping, which can pose challenges at tissue interphases. We evaluated the preconditioned quantitative susceptibility mapping MR imaging method, which does not require skull-stripping, for improved depiction of brain parenchyma and pathology. Fifty-six subjects underwent brain MR imaging with a 3D multiecho gradient recalled echo acquisition. Mask-based quantitative susceptibility mapping images were created using a commonly used mask-based quantitative susceptibility mapping method, and preconditioned quantitative susceptibility images were made using precondition-based total field inversion. All images were reviewed by a neuroradiologist and a radiology resident. Ten subjects (18%), all with traumatic brain injury, demonstrated blood products on 3D gradient recalled echo imaging. All lesions were visible on preconditioned quantitative susceptibility mapping, while 6 were not visible on mask-based quantitative susceptibility mapping. Thirty-one subjects (55%) demonstrated brain parenchyma and/or lesions that were visible on preconditioned quantitative susceptibility mapping but not on mask-based quantitative susceptibility mapping. Six subjects (11%) demonstrated pons artifacts on preconditioned quantitative susceptibility mapping and mask-based quantitative susceptibility mapping; they were worse on preconditioned quantitative susceptibility mapping. Preconditioned quantitative susceptibility mapping MR imaging can bring the benefits of quantitative susceptibility mapping imaging to clinical practice without the limitations of mask-based quantitative susceptibility mapping, especially for evaluating cerebral microhemorrhage-associated pathologies, such as traumatic brain injury. © 2018 by American Journal of Neuroradiology.

Measuring sickle cell morphology in flow using spectrally encoded flow cytometry (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kviatkovsky, Inna; Zeidan, Adel; Yeheskely-Hayon, Daniella; Dann, Eldad J.; Yelin, Dvir

2017-02-01

During a sickle cell crisis in sickle cell anemia patients, deoxygenated red blood cells may change their mechanical properties and block small blood vessels, causing pain, local tissue damage and even organ failure. Measuring these cellular structural and morphological changes is important for understanding the factors contributing to vessel blockage and developing an effective treatment. In this work, we use spectrally encoded flow cytometry for confocal, high-resolution imaging of flowing blood cells from sickle cell anemia patients. A wide variety of cell morphologies were observed by analyzing the interference patterns resulting from reflections from the front and back faces of the cells' membrane. Using numerical simulation for calculating the two-dimensional reflection pattern from the cells, we propose an analytical expression for the three-dimensional shape of a characteristic sickle cell and compare it to a previous from the literature. In vitro spectrally encoded flow cytometry offers new means for analyzing the morphology of sickle cells in stress-free environment, and could provide an effective tool for studying the unique physiological properties of these cells.

Photothermal and photoacoustic Raman cytometry in vitro and in vivo

PubMed Central

Shashkov, Evgeny V.; Galanzha, Ekaterina I.; Zharov, Vladimir P.

2010-01-01

An integrated Raman-based cytometry was developed with photothermal (PT) and photoacoustic (PA) detection of Raman-induced thermal and acoustic signals in biological samples with Raman-active vibrational modes. The two-frequency, spatially and temporally overlapping pump–Stokes excitation in counterpropagating geometry was provided by a nanosecond tunable (420–2300 nm) optical parametric oscillator and a Raman shifter (639 nm) pumped by a double-pulsed Q-switched Nd:YAG laser using microscopic and fiberoptic delivery of laser radiation. The PA and PT Raman detection and imaging technique was tested in vitro with benzene, acetone, olive oil, carbon nanotubes, chylomicron phantom, and cancer cells, and in vivo in single adipocytes in mouse mesentery model. The integration of linear and nonlinear PA and PT Raman scanning and flow cytometry has the potential to enhance its chemical specificity and sensitivity including nanobubble-based amplification (up to 10- fold) for detection of absorbing and nonabsorbing targets that are important for both basic and clinically relevant studies of lymph and blood biochemistry, cancer, and fat distribution at the single-cell level. PMID:20389713

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part I - rationale and aims.

PubMed

Davis, Bruce H; Wood, Brent; Oldaker, Teri; Barnett, David

2013-01-01

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part I - Rationale and aims. © 2013 International Clinical Cytometry Society. © 2013 International Clinical Cytometry Society.

Characterization of Amniotic Stem Cells

PubMed Central

Koike, Chika; Zhou, Kaixuan; Takeda, Yuji; Fathy, Moustafa; Okabe, Motonori; Yoshida, Toshiko; Nakamura, Yukio; Kato, Yukio

2014-01-01

Abstract The amnion membrane is developed from embryo-derived cells, and amniotic cells have been shown to exhibit multidifferentiation potential. These cells represent a desirable source for stem cells for a variety of reasons. However, to date very few molecular analyses of amnion-derived cells have been reported, and efficient markers for isolating the stem cells remain unclear. This paper assesses the characterization of amnion-derived cells as stem cells by examining stemness marker expressions for amnion-derived epithelial cells and mesenchymal cells by flow cytometry, immunocytochemistry, and quantitative PCR. Flow cytometry revealed that amnion epithelial cells expressed CD133, CD 271, and TRA-1-60, whereas mecenchymal cells expressed CD44, CD73, CD90, and CD105. Immunohistochemistry showed that both cells expressed the stemness markers Oct3/4, Sox2, Klf4, and SSEA4. Stemness genes' expression in amnion epithelial cells, mesenchymal cells, fibroblast, bone marrow–derived mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) was compared by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Amnion-derived epithelial cells and mesenchymal cells expressed Oct3/4, Nanog, and Klf4 more than bone marrow–derived MSCs. The sorted TRA1-60–positive cells expressed Oct3/4, Nanog, and Klf4 more than unsorted cells or TRA1-60–negative cells. TRA1-60 can be a marker for isolating amnion epithelial stem cells. PMID:25068631

Red and near-infrared fluorophores inspired by chlorophylls: consideration of practical brightness in multicolor flow cytometry and biomedical sciences

NASA Astrophysics Data System (ADS)

Taniguchi, Masahiko; Hu, Gongfang; Liu, Rui; Du, Hai; Lindsey, Jonathan S.

2018-02-01

Demands in flow cytometry for increased multiplexing (for detection of multiple antigens) and brightness (for detection of rare entities) require new fluorophores (i.e., "colors") with spectrally distinct fluorescence outside the relatively congested visible spectral region. Flow cytometry fluorophores typically must function in aqueous solution upon bioconjugation and ideally should exhibit a host of photophysical features: (i) strong absorption, (ii) sizable Stokes shift, (iii) modest if not strong fluorescence, and (iv) narrow fluorescence band. Tandem dyes have long been pursued to achieve a large effective Stokes shift, increased brightness, and better control over the excitation and emission wavelengths. Here, the attractive photophysical features of chlorophylls and bacteriochlorophylls - Nature's chosen photoactive pigments for photosynthesis - are described with regards to use in flow cytometry. A chlorophyll (or bacteriochlorophyll) constitutes an intrinsic tandem dye given the red (or near-infrared) fluorescence upon excitation in the higher energy ultraviolet (UV) or visible absorption bands (due to rapid internal conversion to the lowest energy state). Synthetic (bacterio)chlorins are available with strong absorption (near-UV molar absorption coefficient ɛ(λexc) 105 M-1cm-1), modest fluorescence quantum yield (Φf = 0.05-0.30), and narrow fluorescence band (10-25 nm) tunable from 600-900 nm depending on synthetic design. The "relative practical brightness" is given by intrinsic brightness [ɛ(λexc) x Φf] times ηf, the fraction of the fluorescence band that is captured by an emission filter in a multicolor experiment. The spectroscopic features of (bacterio)chlorins are evaluated quantitatively to illustrate practical brightness for this novel class of fluorophores in a prospective 8-color panel.

Comparison between the effects of ultrasound and gamma-rays on the inactivation of Saccharomyces cerevisiae: analyses of cell membrane permeability and DNA or RNA synthesis by flow cytometry.

PubMed

Oyane, Ikuko; Takeda, Tomo; Oda, Yasunori; Sakata, Takashi; Furuta, Masakazu; Okitsu, Kenji; Maeda, Yasuaki; Nishimura, Rokuro

2009-04-01

The effects of 200 kHz ultrasonic irradiation on DNA or RNA formation and membrane permeability of yeast cells were investigated by flow cytometry and compared with those of (60)Co gamma-ray radiation. Colony counting analyses were also performed for comparison. It was observed that the colony-forming activity of yeast cells was not affected by small doses of ultrasonic irradiation, but was closely related to the amounts of sonolytically formed hydrogen peroxide at concentrations of more than 80 microM. On the other hand, gamma-rays directly retarded colony-forming ability in addition to the effects of radiolytically formed hydrogen peroxide. The results obtained by flow cytometry also indicated that the amounts of DNA or RNA formed decreased with an increase in ultrasonic irradiation time without any threshold. These results indicated that flow cytometry can show early growth activities, but that colony counting analyses are insufficient to evaluate continuous and quantitative changes in these activities. In addition, by analyzing the amounts of DNA or RNA formed in the presence of the same amount of hydrogen peroxide, it was found that DNA or RNA formation behavior in the presence of hydrogen peroxide with no irradiation was similar to that following ultrasonic irradiation. These results suggested that similar chemical effects due to the formation of hydrogen peroxide were produced during ultrasonic irradiation. In addition, physical effects of ultrasound, such as shock wave, hardly contributed to cell inactivation and cell membrane damage, because relatively high frequency ultrasound was used here. In the case of gamma-ray radiation, direct physical effects on the cells were clearly observed.

Phaedra, a protocol-driven system for analysis and validation of high-content imaging and flow cytometry.

PubMed

Cornelissen, Frans; Cik, Miroslav; Gustin, Emmanuel

2012-04-01

High-content screening has brought new dimensions to cellular assays by generating rich data sets that characterize cell populations in great detail and detect subtle phenotypes. To derive relevant, reliable conclusions from these complex data, it is crucial to have informatics tools supporting quality control, data reduction, and data mining. These tools must reconcile the complexity of advanced analysis methods with the user-friendliness demanded by the user community. After review of existing applications, we realized the possibility of adding innovative new analysis options. Phaedra was developed to support workflows for drug screening and target discovery, interact with several laboratory information management systems, and process data generated by a range of techniques including high-content imaging, multicolor flow cytometry, and traditional high-throughput screening assays. The application is modular and flexible, with an interface that can be tuned to specific user roles. It offers user-friendly data visualization and reduction tools for HCS but also integrates Matlab for custom image analysis and the Konstanz Information Miner (KNIME) framework for data mining. Phaedra features efficient JPEG2000 compression and full drill-down functionality from dose-response curves down to individual cells, with exclusion and annotation options, cell classification, statistical quality controls, and reporting.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding.

PubMed

Shahi, Payam; Kim, Samuel C; Haliburton, John R; Gartner, Zev J; Abate, Adam R

2017-03-14

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

NASA Astrophysics Data System (ADS)

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-03-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

PubMed Central

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-01-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing. PMID:28290550

Accurate quantitation of D+ fetomaternal hemorrhage by flow cytometry using a novel reagent to eliminate granulocytes from analysis.

PubMed

Kumpel, Belinda; Hazell, Matthew; Guest, Alan; Dixey, Jonathan; Mushens, Rosey; Bishop, Debbie; Wreford-Bush, Tim; Lee, Edmond

2014-05-01

Quantitation of fetomaternal hemorrhage (FMH) is performed to determine the dose of prophylactic anti-D (RhIG) required to prevent D immunization of D- women. Flow cytometry (FC) is the most accurate method. However, maternal white blood cells (WBCs) can give high background by binding anti-D nonspecifically, compromising accuracy. Maternal blood samples (69) were sent for FC quantitation of FMH after positive Kleihauer-Betke test (KBT) analysis and RhIG administration. Reagents used were BRAD-3-fluorescein isothiocyanate (FITC; anti-D), AEVZ5.3-FITC (anti-varicella zoster [anti-VZ], negative control), anti-fetal hemoglobin (HbF)-FITC, blended two-color reagents, BRAD-3-FITC/anti-CD45-phycoerythrin (PE; anti-D/L), and BRAD-3-FITC/anti-CD66b-PE (anti-D/G). PE-positive WBCs were eliminated from analysis by gating. Full blood counts were performed on maternal samples and female donors. Elevated numbers of neutrophils were present in 80% of patients. Red blood cell (RBC) indices varied widely in maternal blood. D+ FMH values obtained with anti-D/L, anti-D/G, and anti-HbF-FITC were very similar (r = 0.99, p < 0.001). Correlation between KBT and anti-HbF-FITC FMH results was low (r = 0.716). Inaccurate FMH quantitation using the current method (anti-D minus anti-VZ) occurred with 71% samples having less than 15 mL of D+ FMH (RBCs) and insufficient RhIG calculated for 9%. Using two-color reagents and anti-HbF-FITC, approximately 30% patients had elevated F cells, 26% had no fetal cells, 6% had D- FMH, 26% had 4 to 15 mL of D+ FMH, and 12% patients had more than 15 mL of D+ FMH (RBCs) requiring more than 300 μg of RhIG. Without accurate quantitation of D+ FMH by FC, some women would receive inappropriate or inadequate anti-D prophylaxis. The latter may be at risk of immunization leading to hemolytic disease of the newborn. © 2013 American Association of Blood Banks.

Principles of Quantitative MR Imaging with Illustrated Review of Applicable Modular Pulse Diagrams.

PubMed

Mills, Andrew F; Sakai, Osamu; Anderson, Stephan W; Jara, Hernan

2017-01-01

Continued improvements in diagnostic accuracy using magnetic resonance (MR) imaging will require development of methods for tissue analysis that complement traditional qualitative MR imaging studies. Quantitative MR imaging is based on measurement and interpretation of tissue-specific parameters independent of experimental design, compared with qualitative MR imaging, which relies on interpretation of tissue contrast that results from experimental pulse sequence parameters. Quantitative MR imaging represents a natural next step in the evolution of MR imaging practice, since quantitative MR imaging data can be acquired using currently available qualitative imaging pulse sequences without modifications to imaging equipment. The article presents a review of the basic physical concepts used in MR imaging and how quantitative MR imaging is distinct from qualitative MR imaging. Subsequently, the article reviews the hierarchical organization of major applicable pulse sequences used in this article, with the sequences organized into conventional, hybrid, and multispectral sequences capable of calculating the main tissue parameters of T1, T2, and proton density. While this new concept offers the potential for improved diagnostic accuracy and workflow, awareness of this extension to qualitative imaging is generally low. This article reviews the basic physical concepts in MR imaging, describes commonly measured tissue parameters in quantitative MR imaging, and presents the major available pulse sequences used for quantitative MR imaging, with a focus on the hierarchical organization of these sequences. © RSNA, 2017.

Hyperspectral Imaging, Flow cytometry and Microscopic Morphology of Silver Nanoparticle within Cells

EPA Science Inventory

The ability to detect and track silver nanoparticles (AgNP) that enter cells is important to understand the potential biological and toxicological actions of AgNP. The uptake and fate in cells of four different types of AgNP was studied in a retinal pigment epithelial cell line ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

PubMed Central

Beaumont, Kimberley A.; Anfosso, Andrea; Ahmed, Farzana

2015-01-01

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions. PMID:26779761

Quantitative imaging biomarkers: the application of advanced image processing and analysis to clinical and preclinical decision making.

PubMed

Prescott, Jeffrey William

2013-02-01

The importance of medical imaging for clinical decision making has been steadily increasing over the last four decades. Recently, there has also been an emphasis on medical imaging for preclinical decision making, i.e., for use in pharamaceutical and medical device development. There is also a drive towards quantification of imaging findings by using quantitative imaging biomarkers, which can improve sensitivity, specificity, accuracy and reproducibility of imaged characteristics used for diagnostic and therapeutic decisions. An important component of the discovery, characterization, validation and application of quantitative imaging biomarkers is the extraction of information and meaning from images through image processing and subsequent analysis. However, many advanced image processing and analysis methods are not applied directly to questions of clinical interest, i.e., for diagnostic and therapeutic decision making, which is a consideration that should be closely linked to the development of such algorithms. This article is meant to address these concerns. First, quantitative imaging biomarkers are introduced by providing definitions and concepts. Then, potential applications of advanced image processing and analysis to areas of quantitative imaging biomarker research are described; specifically, research into osteoarthritis (OA), Alzheimer's disease (AD) and cancer is presented. Then, challenges in quantitative imaging biomarker research are discussed. Finally, a conceptual framework for integrating clinical and preclinical considerations into the development of quantitative imaging biomarkers and their computer-assisted methods of extraction is presented.

Toward Quantitative Small Animal Pinhole SPECT: Assessment of Quantitation Accuracy Prior to Image Compensations

PubMed Central

Chen, Chia-Lin; Wang, Yuchuan; Lee, Jason J. S.; Tsui, Benjamin M. W.

2011-01-01

Purpose We assessed the quantitation accuracy of small animal pinhole single photon emission computed tomography (SPECT) under the current preclinical settings, where image compensations are not routinely applied. Procedures The effects of several common image-degrading factors and imaging parameters on quantitation accuracy were evaluated using Monte-Carlo simulation methods. Typical preclinical imaging configurations were modeled, and quantitative analyses were performed based on image reconstructions without compensating for attenuation, scatter, and limited system resolution. Results Using mouse-sized phantom studies as examples, attenuation effects alone degraded quantitation accuracy by up to −18% (Tc-99m or In-111) or −41% (I-125). The inclusion of scatter effects changed the above numbers to −12% (Tc-99m or In-111) and −21% (I-125), respectively, indicating the significance of scatter in quantitative I-125 imaging. Region-of-interest (ROI) definitions have greater impacts on regional quantitation accuracy for small sphere sources as compared to attenuation and scatter effects. For the same ROI, SPECT acquisitions using pinhole apertures of different sizes could significantly affect the outcome, whereas the use of different radii-of-rotation yielded negligible differences in quantitation accuracy for the imaging configurations simulated. Conclusions We have systematically quantified the influence of several factors affecting the quantitation accuracy of small animal pinhole SPECT. In order to consistently achieve accurate quantitation within 5% of the truth, comprehensive image compensation methods are needed. PMID:19048346

Interleukin-12 in patients with cancer is synthesized by peripheral helper T lymphocytes.

PubMed

Michelin, Marcia A; Montes, Leticia; Nomelini, Rosekeila S; Abdalla, Douglas R; Aleixo, Andre A R; Murta, Eddie F C

2015-09-01

The production of cytokines by helper T lymphocytes is a critical event in the immune response, as alterations in the regulation of this process may result in an appropriate immune response, persistent infection or the development of autoimmune disease. Previously, this group has used flow cytometry to demonstrate the expression of interleukin-12 (IL-12) in peripheral blood CD4+ T lymphocytes from patients and mice with advanced cancer. The aim of the present study was to investigate whether CD4+ T lymphocytes from the peripheral blood (PB) of patients with cancer produce IL-12, using molecular approaches, flow cytometry and cellular imaging techniques. CD3+ and CD4+ cells, and cells producing IL-12, were isolated from the PB obtained from patients with cancer, using a cell sorting flow cytometry technique. The positivity of cells for CD3, CD4 and IL-12, which were identified by cell sorting, was visualized using immunofluorescent cellular imaging. Total RNA was extracted from the CD3+CD4+IL-12+ cells, obtained by cell sorting, for confirmation of the presence of IL-12 mRNA, using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR demonstrated the presence of IL-12 mRNA in all patients (n=14), in contrast to the control group, in whom IL-12 expression was not detected. Immunofluorescent analysis of CD4+ T lymphocytes showed positive intracytoplasmatic IL-12 staining. These results demonstrated that CD3+CD4+ T lymphocytes in the PB of patients with cancer have the capacity to synthesize and express IL-12.

Interleukin-12 in patients with cancer is synthesized by peripheral helper T lymphocytes

PubMed Central

MICHELIN, MARCIA A.; MONTES, LETICIA; NOMELINI, ROSEKEILA S.; ABDALLA, DOUGLAS R.; ALEIXO, ANDRE A. R.; MURTA, EDDIE F. C.

2015-01-01

The production of cytokines by helper T lymphocytes is a critical event in the immune response, as alterations in the regulation of this process may result in an appropriate immune response, persistent infection or the development of autoimmune disease. Previously, this group has used flow cytometry to demonstrate the expression of interleukin-12 (IL-12) in peripheral blood CD4+ T lymphocytes from patients and mice with advanced cancer. The aim of the present study was to investigate whether CD4+ T lymphocytes from the peripheral blood (PB) of patients with cancer produce IL-12, using molecular approaches, flow cytometry and cellular imaging techniques. CD3+ and CD4+ cells, and cells producing IL-12, were isolated from the PB obtained from patients with cancer, using a cell sorting flow cytometry technique. The positivity of cells for CD3, CD4 and IL-12, which were identified by cell sorting, was visualized using immunofluorescent cellular imaging. Total RNA was extracted from the CD3+CD4+IL-12+ cells, obtained by cell sorting, for confirmation of the presence of IL-12 mRNA, using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR demonstrated the presence of IL-12 mRNA in all patients (n=14), in contrast to the control group, in whom IL-12 expression was not detected. Immunofluorescent analysis of CD4+ T lymphocytes showed positive intracytoplasmatic IL-12 staining. These results demonstrated that CD3+CD4+ T lymphocytes in the PB of patients with cancer have the capacity to synthesize and express IL-12. PMID:26622702

Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements.

PubMed

Guérin, Frédéric; Arnaiz, Olivier; Boggetto, Nicole; Denby Wilkes, Cyril; Meyer, Eric; Sperling, Linda; Duharcourt, Sandra

2017-04-26

DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.

Sizing up arthropod genomes: an evaluation of the impact of environmental variation on genome size estimates by flow cytometry and the use of qPCR as a method of estimation.

PubMed

Gregory, T Ryan; Nathwani, Paula; Bonnett, Tiffany R; Huber, Dezene P W

2013-09-01

A study was undertaken to evaluate both a pre-existing method and a newly proposed approach for the estimation of nuclear genome sizes in arthropods. First, concerns regarding the reliability of the well-established method of flow cytometry relating to impacts of rearing conditions on genome size estimates were examined. Contrary to previous reports, a more carefully controlled test found negligible environmental effects on genome size estimates in the fly Drosophila melanogaster. Second, a more recently touted method based on quantitative real-time PCR (qPCR) was examined in terms of ease of use, efficiency, and (most importantly) accuracy using four test species: the flies Drosophila melanogaster and Musca domestica and the beetles Tribolium castaneum and Dendroctonus ponderosa. The results of this analysis demonstrated that qPCR has the tendency to produce substantially different genome size estimates from other established techniques while also being far less efficient than existing methods.

Flow Cytometry of Extracellular Vesicles: Potential, Pitfalls, and Prospects.

PubMed

Nolan, John P

2015-07-01

Evidence suggests that extracellular vesicles (EVs) can play roles in physiology and pathology, providing impetus to explore their use as diagnostic and therapeutic targets. However, EVs are also small, heterogeneous, and difficult to measure, and so this potential has not yet been realized. The development of improved approaches to EV detection and characterization will be critical to further understanding their roles in physiology and disease. Flow cytometry has been a popular tool for measuring cell-derived EVs, but has often been used in an uncritical manner in which fundamental principles and limitations of the instrument are ignored. Recent efforts to standardize procedures and document the effects of different methodologies have helped to address this shortcoming, but much work remains. In this paper, I address some of the instrument, reagent, and analysis considerations relevant to measurement of individual EVs in flow, with the aim of clarifying a path to quantitative and standardized measurement of these interesting and potentially important biological nanoparticles. Copyright © 2015 John Wiley & Sons, Inc.

A rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites using fluorescence flow cytometry.

PubMed

Davis, W C; Wyatt, C R; Hamilton, M J; Goff, W L

1992-01-01

Fluorescence flow cytometry was employed to assess the potential of a vital dye, hydroethidine, for use in the detection and monitoring of the viability of hemoparasites in infected erythrocytes, using Babesia bovis as a model parasite. The studies demonstrated that hydroethidine is taken up by B. bovis and metabolically converted to the DNA binding fluorochrome, ethidium. Following uptake of the dye, erythrocytes containing viable parasites were readily distinguished and quantitated. Timed studies with the parasiticidal drug, Ganaseg, showed that it is possible to use the fluorochrome assay to monitor the effects of the drug on the rate of replication and viability of B. bovis in culture. The assay provides a rapid method for evaluation of the in vitro effect of drugs on hemoparasites and for analysis of the effect of various components of the immune response, such as lymphokines, monocyte products, antibodies, and effector cells (T, NK, LAK, ADCC) on the growth and viability of intraerythrocytic parasites.

Temporal lobe epilepsy: quantitative MR volumetry in detection of hippocampal atrophy.

PubMed

Farid, Nikdokht; Girard, Holly M; Kemmotsu, Nobuko; Smith, Michael E; Magda, Sebastian W; Lim, Wei Y; Lee, Roland R; McDonald, Carrie R

2012-08-01

To determine the ability of fully automated volumetric magnetic resonance (MR) imaging to depict hippocampal atrophy (HA) and to help correctly lateralize the seizure focus in patients with temporal lobe epilepsy (TLE). This study was conducted with institutional review board approval and in compliance with HIPAA regulations. Volumetric MR imaging data were analyzed for 34 patients with TLE and 116 control subjects. Structural volumes were calculated by using U.S. Food and Drug Administration-cleared software for automated quantitative MR imaging analysis (NeuroQuant). Results of quantitative MR imaging were compared with visual detection of atrophy, and, when available, with histologic specimens. Receiver operating characteristic analyses were performed to determine the optimal sensitivity and specificity of quantitative MR imaging for detecting HA and asymmetry. A linear classifier with cross validation was used to estimate the ability of quantitative MR imaging to help lateralize the seizure focus. Quantitative MR imaging-derived hippocampal asymmetries discriminated patients with TLE from control subjects with high sensitivity (86.7%-89.5%) and specificity (92.2%-94.1%). When a linear classifier was used to discriminate left versus right TLE, hippocampal asymmetry achieved 94% classification accuracy. Volumetric asymmetries of other subcortical structures did not improve classification. Compared with invasive video electroencephalographic recordings, lateralization accuracy was 88% with quantitative MR imaging and 85% with visual inspection of volumetric MR imaging studies but only 76% with visual inspection of clinical MR imaging studies. Quantitative MR imaging can depict the presence and laterality of HA in TLE with accuracy rates that may exceed those achieved with visual inspection of clinical MR imaging studies. Thus, quantitative MR imaging may enhance standard visual analysis, providing a useful and viable means for translating volumetric analysis into clinical practice.

In vivo flow cytometry of circulating clots using negative photothermal and photoacoustic contrasts.

PubMed

Galanzha, Ekaterina I; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A; Keyrouz, Salah G; Mehta, Jawahar L; Zharov, Vladimir P

2011-10-01

Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of thromboembolism including ischemia at strokes and myocardial infarction. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red, and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 μm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting themby negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted. Copyright © 2011 International Society for Advancement of Cytometry.

Increasing lanthanide luminescence by use of the RETEL effect.

PubMed

Leif, Robert C; Vallarino, Lidia M; Becker, Margie C; Yang, Sean

2006-08-01

Luminescent lanthanide complexes produce emissions with the narrowest-known width at half maximum; however, their significant use in cytometry required an increase in luminescence intensity. The companion review, Leif et al., Cytometry 2006;69A:767-778, described a new technique for the enhancement of lanthanide luminescence, the Resonance Energy Transfer Enhanced Luminescence (RETEL) effect, which increases luminescence and is compatible with standard slide microscopy. The luminescence of the europium ion macrocyclic complex, EuMac, was increased by employing the RETEL effect. After adding the nonluminescent gadolinium ion complex of the thenoyltrifluoroacetonate (TTFA) ligand or the sodium salt of TTFA in ethanol solution, the EuMac-labeled sample was allowed to dry. Both a conventional arc lamp and a time-gated UV LED served as light sources for microscopic imaging. The emission intensity was measured with a CCD camera. Multiple time-gated images were summed with special software to permit analysis and effective presentation of the final image. With the RETEL effect, the luminescence of the EuMac-streptavidin conjugate increased at least six-fold upon drying. Nuclei of apoptotic cells were stained with DAPI and tailed with 5BrdUrd to which a EuMac-anti-5BrdU conjugate was subsequently attached. Time-gated images showed the long-lived EuMac luminescence but did not show the short-lived DAPI fluorescence. Imaging of DNA-synthesizing cells with an arc lamp showed that both S phase and apoptotic cells were labeled, and that their labeling patterns were different. The images of the luminescent EuMac and fluorescent DAPI were combined to produce a color image on a white background. This combination of simple chemistry, instrumentation, and presentation should make possible the inexpensive use of the lanthanide macrocycles, Quantum Dyes, as molecular diagnostics for cytological and histopathological microscopic imaging. (c) 2006 International Society for Analytical Cytology.

Semiautomated analysis of embryoscope images: Using localized variance of image intensity to detect embryo developmental stages.

PubMed

Mölder, Anna; Drury, Sarah; Costen, Nicholas; Hartshorne, Geraldine M; Czanner, Silvester

2015-02-01

Embryo selection in in vitro fertilization (IVF) treatment has traditionally been done manually using microscopy at intermittent time points during embryo development. Novel technique has made it possible to monitor embryos using time lapse for long periods of time and together with the reduced cost of data storage, this has opened the door to long-term time-lapse monitoring, and large amounts of image material is now routinely gathered. However, the analysis is still to a large extent performed manually, and images are mostly used as qualitative reference. To make full use of the increased amount of microscopic image material, (semi)automated computer-aided tools are needed. An additional benefit of automation is the establishment of standardization tools for embryo selection and transfer, making decisions more transparent and less subjective. Another is the possibility to gather and analyze data in a high-throughput manner, gathering data from multiple clinics and increasing our knowledge of early human embryo development. In this study, the extraction of data to automatically select and track spatio-temporal events and features from sets of embryo images has been achieved using localized variance based on the distribution of image grey scale levels. A retrospective cohort study was performed using time-lapse imaging data derived from 39 human embryos from seven couples, covering the time from fertilization up to 6.3 days. The profile of localized variance has been used to characterize syngamy, mitotic division and stages of cleavage, compaction, and blastocoel formation. Prior to analysis, focal plane and embryo location were automatically detected, limiting precomputational user interaction to a calibration step and usable for automatic detection of region of interest (ROI) regardless of the method of analysis. The results were validated against the opinion of clinical experts. © 2015 International Society for Advancement of Cytometry. © 2015 International Society for Advancement of Cytometry.

Development of a two-parameter slit-scan flow cytometer for screening of normal and aberrant chromosomes: application to a karyotype of Sus scrofa domestica (pig)

NASA Astrophysics Data System (ADS)

Hausmann, Michael; Doelle, Juergen; Arnold, Armin; Stepanow, Boris; Wickert, Burkhard; Boscher, Jeannine; Popescu, Paul C.; Cremer, Christoph

1992-07-01

Laser fluorescence activated slit-scan flow cytometry offers an approach to a fast, quantitative characterization of chromosomes due to morphological features. It can be applied for screening of chromosomal abnormalities. We give a preliminary report on the development of the Heidelberg slit-scan flow cytometer. Time-resolved measurement of the fluorescence intensity along the chromosome axis can be registered simultaneously for two parameters when the chromosome axis can be registered simultaneously for two parameters when the chromosome passes perpendicularly through a narrowly focused laser beam combined by a detection slit in the image plane. So far automated data analysis has been performed off-line on a PC. In its final performance, the Heidelberg slit-scan flow cytometer will achieve on-line data analysis that allows an electro-acoustical sorting of chromosomes of interest. Interest is high in the agriculture field to study chromosome aberrations that influence the size of litters in pig (Sus scrofa domestica) breeding. Slit-scan measurements have been performed to characterize chromosomes of pigs; we present results for chromosome 1 and a translocation chromosome 6/15.

Enhanced Cellular Uptake and Pharmacokinetic Characteristics of Doxorubicin-Valine Amide Prodrug.

PubMed

Park, Yohan; Park, Ju-Hwan; Park, Suryeon; Lee, Song Yi; Cho, Kwan Hyung; Kim, Dae-Duk; Shim, Won-Sik; Yoon, In-Soo; Cho, Hyun-Jong; Maeng, Han-Joo

2016-09-22

In this study, we synthesized the valine (Val)-conjugated amide prodrug of doxorubicin (DOX) by the formation of amide bonds between DOX and Val. The synthesis of the DOX-Val prodrug was identified by a proton nuclear magnetic resonance (¹H-NMR) assay. In the MCF-7 cells (human breast adenocarcinoma cell; amino acid transporter-positive cell), the cellular accumulation efficiency of DOX-Val was higher than that of DOX according to the flow cytometry analysis data. Using confocal laser scanning microscopy (CLSM) imaging, it was confirmed that DOX-Val as well as DOX was mainly distributed in the nucleus of cancer cells. DOX-Val was intravenously administered to rats at a dose of 4 mg/kg, and the plasma concentrations of DOX-Val (prodrug) and DOX (formed metabolite) were quantitatively determined. Based on the systemic exposure (represented as area under the curve (AUC) values) of DOX-Val (prodrug) and DOX (formed metabolite), approximately half of DOX-Val seemed to be metabolized into DOX. However, it is expected that the remaining DOX-Val may exert improved cellular uptake efficiency in cancer cells after its delivery to the cancer region.

Novel Peptide Sequence (“IQ-tag”) with High Affinity for NIR Fluorochromes Allows Protein and Cell Specific Labeling for In Vivo Imaging

PubMed Central

McCarthy, Jason R.; Weissleder, Ralph

2007-01-01

Background Probes that allow site-specific protein labeling have become critical tools for visualizing biological processes. Methods Here we used phage display to identify a novel peptide sequence with nanomolar affinity for near infrared (NIR) (benz)indolium fluorochromes. The developed peptide sequence (“IQ-tag”) allows detection of NIR dyes in a wide range of assays including ELISA, flow cytometry, high throughput screens, microscopy, and optical in vivo imaging. Significance The described method is expected to have broad utility in numerous applications, namely site-specific protein imaging, target identification, cell tracking, and drug development. PMID:17653285

Shedding quantitative fluorescence light on novel regulatory mechanisms in skeletal biomedicine and biodentistry.

PubMed

Lee, Ji-Won; Iimura, Tadahiro

2017-02-01

Digitalized fluorescence images contain numerical information such as color (wavelength), fluorescence intensity and spatial position. However, quantitative analyses of acquired data and their validation remained to be established. Our research group has applied quantitative fluorescence imaging on tissue sections and uncovered novel findings in skeletal biomedicine and biodentistry. This review paper includes a brief background of quantitative fluorescence imaging and discusses practical applications by introducing our previous research. Finally, the future perspectives of quantitative fluorescence imaging are discussed.

Magnetic Resonance-based Motion Correction for Quantitative PET in Simultaneous PET-MR Imaging.

PubMed

Rakvongthai, Yothin; El Fakhri, Georges

2017-07-01

Motion degrades image quality and quantitation of PET images, and is an obstacle to quantitative PET imaging. Simultaneous PET-MR offers a tool that can be used for correcting the motion in PET images by using anatomic information from MR imaging acquired concurrently. Motion correction can be performed by transforming a set of reconstructed PET images into the same frame or by incorporating the transformation into the system model and reconstructing the motion-corrected image. Several phantom and patient studies have validated that MR-based motion correction strategies have great promise for quantitative PET imaging in simultaneous PET-MR. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantitative imaging biomarker ontology (QIBO) for knowledge representation of biomedical imaging biomarkers.

PubMed

Buckler, Andrew J; Liu, Tiffany Ting; Savig, Erica; Suzek, Baris E; Ouellette, M; Danagoulian, J; Wernsing, G; Rubin, Daniel L; Paik, David

2013-08-01

A widening array of novel imaging biomarkers is being developed using ever more powerful clinical and preclinical imaging modalities. These biomarkers have demonstrated effectiveness in quantifying biological processes as they occur in vivo and in the early prediction of therapeutic outcomes. However, quantitative imaging biomarker data and knowledge are not standardized, representing a critical barrier to accumulating medical knowledge based on quantitative imaging data. We use an ontology to represent, integrate, and harmonize heterogeneous knowledge across the domain of imaging biomarkers. This advances the goal of developing applications to (1) improve precision and recall of storage and retrieval of quantitative imaging-related data using standardized terminology; (2) streamline the discovery and development of novel imaging biomarkers by normalizing knowledge across heterogeneous resources; (3) effectively annotate imaging experiments thus aiding comprehension, re-use, and reproducibility; and (4) provide validation frameworks through rigorous specification as a basis for testable hypotheses and compliance tests. We have developed the Quantitative Imaging Biomarker Ontology (QIBO), which currently consists of 488 terms spanning the following upper classes: experimental subject, biological intervention, imaging agent, imaging instrument, image post-processing algorithm, biological target, indicated biology, and biomarker application. We have demonstrated that QIBO can be used to annotate imaging experiments with standardized terms in the ontology and to generate hypotheses for novel imaging biomarker-disease associations. Our results established the utility of QIBO in enabling integrated analysis of quantitative imaging data.

A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery.

PubMed

Joslin, John; Gilligan, James; Anderson, Paul; Garcia, Catherine; Sharif, Orzala; Hampton, Janice; Cohen, Steven; King, Miranda; Zhou, Bin; Jiang, Shumei; Trussell, Christopher; Dunn, Robert; Fathman, John W; Snead, Jennifer L; Boitano, Anthony E; Nguyen, Tommy; Conner, Michael; Cooke, Mike; Harris, Jennifer; Ainscow, Ed; Zhou, Yingyao; Shaw, Chris; Sipes, Dan; Mainquist, James; Lesley, Scott

2018-05-01

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.

Performance of computer vision in vivo flow cytometry with low fluorescence contrast

NASA Astrophysics Data System (ADS)

Markovic, Stacey; Li, Siyuan; Niedre, Mark

2015-03-01

Detection and enumeration of circulating cells in the bloodstream of small animals are important in many areas of preclinical biomedical research, including cancer metastasis, immunology, and reproductive medicine. Optical in vivo flow cytometry (IVFC) represents a class of technologies that allow noninvasive and continuous enumeration of circulating cells without drawing blood samples. We recently developed a technique termed computer vision in vivo flow cytometry (CV-IVFC) that uses a high-sensitivity fluorescence camera and an automated computer vision algorithm to interrogate relatively large circulating blood volumes in the ear of a mouse. We detected circulating cells at concentrations as low as 20 cells/mL. In the present work, we characterized the performance of CV-IVFC with low-contrast imaging conditions with (1) weak cell fluorescent labeling using cell-simulating fluorescent microspheres with varying brightness and (2) high background tissue autofluorescence by varying autofluorescence properties of optical phantoms. Our analysis indicates that CV-IVFC can robustly track and enumerate circulating cells with at least 50% sensitivity even in conditions with two orders of magnitude degraded contrast than our previous in vivo work. These results support the significant potential utility of CV-IVFC in a wide range of in vivo biological models.

Growth fraction in non-small cell lung cancer estimated by proliferating cell nuclear antigen and comparison with Ki-67 labeling and DNA flow cytometry data.

PubMed Central

Fontanini, G.; Pingitore, R.; Bigini, D.; Vignati, S.; Pepe, S.; Ruggiero, A.; Macchiarini, P.

1992-01-01

Results generated by the immunohistochemical staining with PC10, a new monoclonal antibody recognizing PCNA (a nuclear protein associated with cell proliferation) in formalin-fixed and paraffin-embedded tissue were compared with those of Ki-67 labeling and DNA flow cytometry in 47 consecutive non-small cell lung cancer (NSCLC). PCNA reactivity was observed in all samples and confined to the nuclei of cancer cells. Its frequency ranged from 0 to 80% (37.7 +/- 23.6) and larger sized, early-staged and DNA aneuploid tumors expressed a significant higher number of PCNA-reactive cells. The PCNA and Ki-67 labeling rates were closely correlated (r = 0.383, P = 0.009). By flow cytometry, we observed a good correlation among PCNA labeling and S-phase fraction (r = 0.422, P = .0093) and G1 phase (r = 0.303, P = .051) of the cell cycle. Results indicate that PCNA labeling with PC10 is a simple method for assessing the proliferative activity in formalin-fixed, paraffin-embedded tissue of NSCLC and correlates well with Ki-67 labeling and S-phase fraction of the cell cycle. Images Figure 2 PMID:1361306

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

PubMed Central

2012-01-01

Background Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Methods Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. Results A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Discussion Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene-expression profiling and analysis. The approach outlined here results in substantially improved yield of GFP-expressing parasites, and requires decreased sorting time in comparison to standard methods. It is anticipated that this protocol will be useful for a wide range of applications involving rare events. PMID:22950515

Prospects and challenges of quantitative phase imaging in tumor cell biology

NASA Astrophysics Data System (ADS)

Kemper, Björn; Götte, Martin; Greve, Burkhard; Ketelhut, Steffi

2016-03-01

Quantitative phase imaging (QPI) techniques provide high resolution label-free quantitative live cell imaging. Here, prospects and challenges of QPI in tumor cell biology are presented, using the example of digital holographic microscopy (DHM). It is shown that the evaluation of quantitative DHM phase images allows the retrieval of different parameter sets for quantification of cellular motion changes in migration and motility assays that are caused by genetic modifications. Furthermore, we demonstrate simultaneously label-free imaging of cell growth and morphology properties.

Nuclear DNA Methylation and Chromatin Condensation Phenotypes Are Distinct Between Normally Proliferating/Aging, Rapidly Growing/Immortal, and Senescent Cells

PubMed Central

Gertych, Arkadiusz; Tajbakhsh, Jian

2013-01-01

This study reports on probing the utility of in situ chromatin texture features such as nuclear DNA methylation and chromatin condensation patterns — visualized by fluorescent staining and evaluated by dedicated three-dimensional (3D) quantitative and high-throughput cell-by-cell image analysis — in assessing the proliferative capacity, i.e. growth behavior of cells: to provide a more dynamic picture of a cell population with potential implications in basic science, cancer diagnostics/prognostics and therapeutic drug development. Two types of primary cells and four different cancer cell lines were propagated and subjected to cell-counting, flow cytometry, confocal imaging, and 3D image analysis at various points in culture. Additionally a subset of primary and cancer cells was accelerated into senescence by oxidative stress. DNA methylation and chromatin condensation levels decreased with declining doubling times when primary cells aged in culture with the lowest levels reached at the stage of proliferative senescence. In comparison, immortal cancer cells with constant but higher doubling times mostly displayed lower and constant levels of the two in situ-derived features. However, stress-induced senescent primary and cancer cells showed similar levels of these features compared with primary cells that had reached natural growth arrest. With regards to global DNA methylation and chromatin condensation levels, aggressively growing cancer cells seem to take an intermediate level between normally proliferating and senescent cells. Thus, normal cells apparently reach cancer-cell equivalent stages of the two parameters at some point in aging, which might challenge phenotypic distinction between these two types of cells. Companion high-resolution molecular profiling could provide information on possible underlying differences that would explain benign versus malign cell growth behaviors. PMID:23562889

Nuclear DNA methylation and chromatin condensation phenotypes are distinct between normally proliferating/aging, rapidly growing/immortal, and senescent cells.

PubMed

Oh, Jin Ho; Gertych, Arkadiusz; Tajbakhsh, Jian

2013-03-01

This study reports on probing the utility of in situ chromatin texture features such as nuclear DNA methylation and chromatin condensation patterns - visualized by fluorescent staining and evaluated by dedicated three-dimensional (3D) quantitative and high-throughput cell-by-cell image analysis - in assessing the proliferative capacity, i.e. growth behavior of cells: to provide a more dynamic picture of a cell population with potential implications in basic science, cancer diagnostics/prognostics and therapeutic drug development. Two types of primary cells and four different cancer cell lines were propagated and subjected to cell-counting, flow cytometry, confocal imaging, and 3D image analysis at various points in culture. Additionally a subset of primary and cancer cells was accelerated into senescence by oxidative stress. DNA methylation and chromatin condensation levels decreased with declining doubling times when primary cells aged in culture with the lowest levels reached at the stage of proliferative senescence. In comparison, immortal cancer cells with constant but higher doubling times mostly displayed lower and constant levels of the two in situ-derived features. However, stress-induced senescent primary and cancer cells showed similar levels of these features compared with primary cells that had reached natural growth arrest. With regards to global DNA methylation and chromatin condensation levels, aggressively growing cancer cells seem to take an intermediate level between normally proliferating and senescent cells. Thus, normal cells apparently reach cancer-cell equivalent stages of the two parameters at some point in aging, which might challenge phenotypic distinction between these two types of cells. Companion high-resolution molecular profiling could provide information on possible underlying differences that would explain benign versus malign cell growth behaviors.

Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images.

PubMed

Zhang, Zhiqing; Kuzmin, Nikolay V; Groot, Marie Louise; de Munck, Jan C

2018-01-01

Third harmonic generation (THG) microscopy is a label-free imaging technique that shows great potential for rapid pathology of brain tissue during brain tumor surgery. However, the interpretation of THG brain images should be quantitatively linked to images of more standard imaging techniques, which so far has been done qualitatively only. We establish here such a quantitative link between THG images of mouse brain tissue and all-nuclei-highlighted fluorescence images, acquired simultaneously from the same tissue area. For quantitative comparison of a substantial pair of images, we present here a segmentation workflow that is applicable for both THG and fluorescence images, with a precision of 91.3 % and 95.8 % achieved respectively. We find that the correspondence between the main features of the two imaging modalities amounts to 88.9 %, providing quantitative evidence of the interpretation of dark holes as brain cells. Moreover, 80 % bright objects in THG images overlap with nuclei highlighted in the fluorescence images, and they are 2 times smaller than the dark holes, showing that cells of different morphologies can be recognized in THG images. We expect that the described quantitative comparison is applicable to other types of brain tissue and with more specific staining experiments for cell type identification. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The emerging science of quantitative imaging biomarkers terminology and definitions for scientific studies and regulatory submissions.

PubMed

Kessler, Larry G; Barnhart, Huiman X; Buckler, Andrew J; Choudhury, Kingshuk Roy; Kondratovich, Marina V; Toledano, Alicia; Guimaraes, Alexander R; Filice, Ross; Zhang, Zheng; Sullivan, Daniel C

2015-02-01

The development and implementation of quantitative imaging biomarkers has been hampered by the inconsistent and often incorrect use of terminology related to these markers. Sponsored by the Radiological Society of North America, an interdisciplinary group of radiologists, statisticians, physicists, and other researchers worked to develop a comprehensive terminology to serve as a foundation for quantitative imaging biomarker claims. Where possible, this working group adapted existing definitions derived from national or international standards bodies rather than invent new definitions for these terms. This terminology also serves as a foundation for the design of studies that evaluate the technical performance of quantitative imaging biomarkers and for studies of algorithms that generate the quantitative imaging biomarkers from clinical scans. This paper provides examples of research studies and quantitative imaging biomarker claims that use terminology consistent with these definitions as well as examples of the rampant confusion in this emerging field. We provide recommendations for appropriate use of quantitative imaging biomarker terminological concepts. It is hoped that this document will assist researchers and regulatory reviewers who examine quantitative imaging biomarkers and will also inform regulatory guidance. More consistent and correct use of terminology could advance regulatory science, improve clinical research, and provide better care for patients who undergo imaging studies. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Real-time quantitative fluorescence imaging using a single snapshot optical properties technique for neurosurgical guidance

NASA Astrophysics Data System (ADS)

Valdes, Pablo A.; Angelo, Joseph; Gioux, Sylvain

2015-03-01

Fluorescence imaging has shown promise as an adjunct to improve the extent of resection in neurosurgery and oncologic surgery. Nevertheless, current fluorescence imaging techniques do not account for the heterogeneous attenuation effects of tissue optical properties. In this work, we present a novel imaging system that performs real time quantitative fluorescence imaging using Single Snapshot Optical Properties (SSOP) imaging. We developed the technique and performed initial phantom studies to validate the quantitative capabilities of the system for intraoperative feasibility. Overall, this work introduces a novel real-time quantitative fluorescence imaging method capable of being used intraoperatively for neurosurgical guidance.

Untangling cell tracks: Quantifying cell migration by time lapse image data analysis.

PubMed

Svensson, Carl-Magnus; Medyukhina, Anna; Belyaev, Ivan; Al-Zaben, Naim; Figge, Marc Thilo

2018-03-01

Automated microscopy has given researchers access to great amounts of live cell imaging data from in vitro and in vivo experiments. Much focus has been put on extracting cell tracks from such data using a plethora of segmentation and tracking algorithms, but further analysis is normally required to draw biologically relevant conclusions. Such relevant conclusions may be whether the migration is directed or not, whether the population has homogeneous or heterogeneous migration patterns. This review focuses on the analysis of cell migration data that are extracted from time lapse images. We discuss a range of measures and models used to analyze cell tracks independent of the biological system or the way the tracks were obtained. For single-cell migration, we focus on measures and models giving examples of biological systems where they have been applied, for example, migration of bacteria, fibroblasts, and immune cells. For collective migration, we describe the model systems wound healing, neural crest migration, and Drosophila gastrulation and discuss methods for cell migration within these systems. We also discuss the role of the extracellular matrix and subsequent differences between track analysis in vitro and in vivo. Besides methods and measures, we are putting special focus on the need for openly available data and code, as well as a lack of common vocabulary in cell track analysis. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Macrophagic and microglial responses after focal traumatic brain injury in the female rat

PubMed Central

2014-01-01

Background After central nervous system injury, inflammatory macrophages (M1) predominate over anti-inflammatory macrophages (M2). The temporal profile of M1/M2 phenotypes in macrophages and microglia after traumatic brain injury (TBI) in rats is unknown. We subjected female rats to severe controlled cortical impact (CCI) and examined the postinjury M1/M2 time course in their brains. Methods The motor cortex (2.5 mm left laterally and 1.0 mm anteriorly from the bregma) of anesthetized female Wistar rats (ages 8 to 10 weeks; N = 72) underwent histologically moderate to severe CCI with a 5-mm impactor tip. Separate cohorts of rats had their brains dissociated into cells for flow cytometry, perfusion-fixed for immunohistochemistry (IHC) and ex vivo magnetic resonance imaging or flash-frozen for RNA and protein analysis. For each analytical method used, separate postinjury times were included for 24 hours; 3 or 5 days; or 1, 2, 4 or 8 weeks. Results By IHC, we found that the macrophagic and microglial responses peaked at 5 to 7 days post-TBI with characteristics of mixed populations of M1 and M2 phenotypes. Upon flow cytometry examination of immunological cells isolated from brain tissue, we observed that peak M2-associated staining occurred at 5 days post-TBI. Chemokine analysis by multiplex assay showed statistically significant increases in macrophage inflammatory protein 1α and keratinocyte chemoattractant/growth-related oncogene on the ipsilateral side within the first 24 hours after injury relative to controls and to the contralateral side. Quantitative RT-PCR analysis demonstrated expression of both M1- and M2-associated markers, which peaked at 5 days post-TBI. Conclusions The responses of macrophagic and microglial cells to histologically severe CCI in the female rat are maximal between days 3 and 7 postinjury. The response to injury is a mixture of M1 and M2 phenotypes. PMID:24761998

Associations Between Fibrocytes and Postcontrast Myocardial T1 Times in Hypertrophic Cardiomyopathy

PubMed Central

Fang, Lu; Beale, Anna; Ellims, Andris H.; Moore, Xiao‐lei; Ling, Liang‐han; Taylor, Andrew J.; Chin‐Dusting, Jaye; Dart, Anthony M.

2013-01-01

Background Fibrocytes are bone marrow‐derived mesenchymal progenitors that have been linked to various fibrotic disorders. This study was undertaken to investigate whether fibrocytes are increased in diffuse myocardial fibrosis in humans. Methods and Results Thirty‐seven patients with hypertrophic cardiomyopathy (HCM) and 20 healthy controls were recruited. Cardiac magnetic resonance imaging with postcontrast T1 mapping was performed to non‐invasively quantify diffuse myocardial fibrosis and these patients were classified into 2 groups (T1<470 ms or T1≥470 ms, as likely or unlikely to have diffuse fibrosis, respectively). Circulating fibrocytes (CD45+/CD34+/collagen I+) were measured by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were cultured for 13 days and fibrocytes were quantitated by flow cytometry (CD45+/collagen I+) and real‐time PCR (gene expression of matrix proteins). Plasma cytokines/chemokines mediating fibrocyte trafficking and differentiation were measured by multiplex assays. Circulating fibrocytes were decreased in HCM patients compared to controls. The proportion of fibrocytes derived from PBMCs was increased in patients with diffuse fibrosis compared with those without or controls (31.1±4.1% versus 18.9±3.9% and 10.9±2.0%, P<0.05 and P<0.001, respectively), and the proportion of fibrocytes was inversely correlated with T1 time (r=−0.37, P=0.03). Plasma levels of stromal cell‐derived factor‐1 were elevated in patients with diffuse fibrosis compared with those without or controls (5131±271 pg/mL versus 3893±356 pg/mL and 4172±185 pg/mL, respectively, both P<0.05). Conclusions HCM patients with diffuse fibrosis as assessed by postcontrast T1 mapping have elevated plasma SDF and an enhanced ability of PBMCs to differentiate into fibrocytes, suggesting that fibrocytes may contribute to the pathogenesis of myocardial fibrosis. PMID:24125844

High levels of circulating extracellular vesicles with altered expression and function during pregnancy.

PubMed

Nardi, Fabiola da Silva; Michelon, Tatiana Ferreira; Neumann, Jorge; Manvailer, Luis Felipe Santos; Wagner, Bettina; Horn, Peter A; Bicalho, Maria da Graça; Rebmann, Vera

2016-07-01

Extracellular vesicles (EVs) are widely considered important modulators of cell-cell communication and may interact with target cells locally and on a systemic level. Several studies had shown that circulating EVs' levels are increased during pregnancy. However, EVs characteristics, composition and biological functions in pregnancy still need to be clarified. This study aims to determine if circulating EVs during pregnancy are modified regarding levels, markers and cytokine profile as well as their reactivity towards peripheral blood cells. 26 pregnant women (PW) being in the second gestational trimester and 59 non-pregnant women (NPW) were investigated. EVs enrichment was performed by ExoQuick™ or ultracentrifugation; nanoparticle tracking analysis, SDS-PAGE followed by Western Blotting and densitometry, and IFN-γ, IL-10 and TGF-β1 ELISA for EVs characterization; imaging flow cytometry to analyze EVs' uptake by peripheral blood cells and flow cytometry were performed to analyze EVs function regarding induction of caspase-3 activity. Circulating EVs' levels were increased during pregnancy [26.9×10(6)EVs/ml (range: 6.4-46.3); p=0.003] vs NPW [18.9×10(6)EVs/ml (range: 2.5-61.3)]. Importantly, the immunosuppressive TGF-β1 and IL-10 cytokine cargo were increased in EVs of PW even after normalization to 1 million EVs [TGF-β1: 0.25pg/10(6)EVs (range: 0.0-2.0); p<0.0001] and [IL-10: 0.21pg/10(6)EVs (range: 0.0-16.8); p=0.006] vs NPW. Although EVs derived from non-pregnant and pregnant women were taken up by NK cells, the latter exclusively enhanced the caspase-3 activity in CD56(dim) NK cells (8.2±0.9; p=0.02). The qualitative and quantitative pregnancy-related alterations of circulating EVs provide first hints for an immune modulating role of circulating EVs during pregnancy. Copyright © 2016 Elsevier GmbH. All rights reserved.

Primary porcine Kupffer cell phagocytosis of human platelets involves the CD18 receptor.

PubMed

Chihara, Ray K; Paris, Leela L; Reyes, Luz M; Sidner, Richard A; Estrada, Jose L; Downey, Susan M; Wang, Zheng-Yu; Tector, A Joseph; Burlak, Christopher

2011-10-15

Hepatic failure has been treated successfully with clinical extracorporeal perfusions of porcine livers. However, dog-to-pig and pig-to-baboon liver xenotransplant models have resulted in severe bleeding secondary to liver xenograft-induced thrombocytopenia. Kupffer cells (KC) are abundant phagocytic cells in the liver. KC express the CD11b/CD18 receptor, which has been implicated in chilled platelet binding and phagocytosis through interaction with platelet surface proteins and carbohydrates. We sought to identify the role of KC CD18 in liver xenograft-induced thrombocytopenia. Primary pig KC were characterized by flow cytometry, immunoblots, and quantitative polymerase chain reaction. Pig KC were used in inhibition assays with fluorescently labeled human platelets. The CD18 receptor was targeted for siRNA knockdown. Domestic and α1,3-galactosyltransferase double knockout porcine KC cultures were approximately 92% positive for CD18 as detected by quantitative polymerase chain reaction and flow cytometry. Use of CD18 blocking antibodies resulted in reduction of human platelet binding and phagocytosis. Additionally, asialofetuin, not fetuin, inhibited platelet phagocytosis suggesting the involvement of an oligosaccharide-binding site. Furthermore, reduced CD18 expression by siRNA resulted in decreased human platelet binding. Our data suggest that primary pig KC bind and phagocytose human platelets with involvement of CD18. Further understanding and modification of CD18 expression in pigs may result in a liver xenograft with reduced thrombocytopenic effects, which could be used as a bridge to allogeneic liver transplantation.

Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cuneo, Kyle C.; Mito, Jeffrey K.; Javid, Melodi P.

2013-05-01

Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activatedmore » fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.« less

Impact of the accuracy of automatic segmentation of cell nuclei clusters on classification of thyroid follicular lesions.

PubMed

Jung, Chanho; Kim, Changick

2014-08-01

Automatic segmentation of cell nuclei clusters is a key building block in systems for quantitative analysis of microscopy cell images. For that reason, it has received a great attention over the last decade, and diverse automatic approaches to segment clustered nuclei with varying levels of performance under different test conditions have been proposed in literature. To the best of our knowledge, however, so far there is no comparative study on the methods. This study is a first attempt to fill this research gap. More precisely, the purpose of this study is to present an objective performance comparison of existing state-of-the-art segmentation methods. Particularly, the impact of their accuracy on classification of thyroid follicular lesions is also investigated "quantitatively" under the same experimental condition, to evaluate the applicability of the methods. Thirteen different segmentation approaches are compared in terms of not only errors in nuclei segmentation and delineation, but also their impact on the performance of system to classify thyroid follicular lesions using different metrics (e.g., diagnostic accuracy, sensitivity, specificity, etc.). Extensive experiments have been conducted on a total of 204 digitized thyroid biopsy specimens. Our study demonstrates that significant diagnostic errors can be avoided using more advanced segmentation approaches. We believe that this comprehensive comparative study serves as a reference point and guide for developers and practitioners in choosing an appropriate automatic segmentation technique adopted for building automated systems for specifically classifying follicular thyroid lesions. © 2014 International Society for Advancement of Cytometry.

STING-Dependent Interferon-λ1 Induction in HT29 Cells, a Human Colorectal Cancer Cell Line, After Gamma-Radiation.

PubMed

Chen, Jianzhou; Markelc, Bostjan; Kaeppler, Jakob; Ogundipe, Vivian M L; Cao, Yunhong; McKenna, W Gillies; Muschel, Ruth J

2018-05-01

To investigate the induction of type III interferons (IFNs) in human cancer cells by gamma-rays. Type III IFN expression in human cancer cell lines after gamma-ray irradiation in vitro was assessed by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Signaling pathways mediating type III IFN induction were examined by a variety of means, including immunoblotting, flow cytometry, confocal imaging, and reverse transcription-quantitative polymerase chain reaction. Key mediators in these pathways were further explored and validated using gene CRISPR knockout or short hairpin RNA knockdown. Exposure to gamma-rays directly induced type III IFNs (mainly IFNL1) in human cancer cell lines in dose- and time-dependent fashions. The induction of IFNL1 was primarily mediated by the cytosolic DNA sensors-STING-TBK1-IRF1 signaling axis, with a lesser contribution from the nuclear factor kappa b signaling in HT29 cells. In addition, type III IFN signaling through its receptors serves as a positive feedback loop, further enhancing IFN expression via up-regulation of the kinases in the STING-TBK1 signaling axis. Our results suggest that IFNL1 can be up-regulated in human cancer cell lines after gamma-ray treatment. In HT29 cells this induction occurs via the STING pathway, adding another layer of complexity to the understanding of radiation-induced antitumor immunity, and may provide novel insights into IFN-based cancer treatment. Copyright © 2018 Elsevier Inc. All rights reserved.

Streak Imaging Flow Cytometer for Rare Cell Analysis.

PubMed

Balsam, Joshua; Bruck, Hugh Alan; Ossandon, Miguel; Prickril, Ben; Rasooly, Avraham

2017-01-01

There is a need for simple and affordable techniques for cytology for clinical applications, especially for point-of-care (POC) medical diagnostics in resource-poor settings. However, this often requires adapting expensive and complex laboratory-based techniques that often require significant power and are too massive to transport easily. One such technique is flow cytometry, which has great potential for modification due to the simplicity of the principle of optical tracking of cells. However, it is limited in that regard due to the flow focusing technique used to isolate cells for optical detection. This technique inherently reduces the flow rate and is therefore unsuitable for rapid detection of rare cells which require large volume for analysis.To address these limitations, we developed a low-cost, mobile flow cytometer based on streak imaging. In our new configuration we utilize a simple webcam for optical detection over a large area associated with a wide-field flow cell. The new flow cell is capable of larger volume and higher throughput fluorescence detection of rare cells than the flow cells with hydrodynamic focusing used in conventional flow cytometry. The webcam is an inexpensive, commercially available system, and for fluorescence analysis we use a 1 W 450 nm blue laser to excite Syto-9 stained cells with emission at 535 nm. We were able to detect low concentrations of stained cells at high flow rates of 10 mL/min, which is suitable for rapidly analyzing larger specimen volumes to detect rare cells at appropriate concentration levels. The new rapid detection capabilities, combined with the simplicity and low cost of this device, suggest a potential for clinical POC flow cytometry in resource-poor settings associated with global health.

Photoacoustic and photothermal detection of circulating tumor cells, bacteria and nanoparticles in cerebrospinal fluid in vivo and ex vivo.

PubMed

Nedosekin, Dmitry A; Juratli, Mazen A; Sarimollaoglu, Mustafa; Moore, Christopher L; Rusch, Nancy J; Smeltzer, Mark S; Zharov, Vladimir P; Galanzha, Ekaterina I

2013-06-01

Circulating cells, bacteria, proteins, microparticles, and DNA in cerebrospinal fluid (CSF) are excellent biomarkers of many diseases, including cancer and infections. However, the sensitivity of existing methods is limited in their ability to detect rare CSF biomarkers at the treatable, early-stage of diseases. Here, we introduce novel CSF tests based on in vivo photoacoustic flow cytometry (PAFC) and ex vivo photothermal scanning cytometry. In the CSF of tumor-bearing mice, we molecularly detected in vivo circulating tumor cells (CTCs) before the development of breast cancer brain metastasis with 20-times higher sensitivity than with current assays. For the first time, we demonstrated assessing three pathways (i.e., blood, lymphatic, and CSF) of CTC dissemination, tracking nanoparticles in CSF in vivo and their imaging ex vivo. In label-free CSF samples, we counted leukocytes, erythrocytes, melanoma cells, and bacteria and imaged intracellular cytochromes, hemoglobin, melanin, and carotenoids, respectively. Taking into account the safety of PAFC, its translation for use in humans is expected to improve disease diagnosis beyond conventional detection limits. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cell dynamic morphology classification using deep convolutional neural networks.

PubMed

Li, Heng; Pang, Fengqian; Shi, Yonggang; Liu, Zhiwen

2018-05-15

Cell morphology is often used as a proxy measurement of cell status to understand cell physiology. Hence, interpretation of cell dynamic morphology is a meaningful task in biomedical research. Inspired by the recent success of deep learning, we here explore the application of convolutional neural networks (CNNs) to cell dynamic morphology classification. An innovative strategy for the implementation of CNNs is introduced in this study. Mouse lymphocytes were collected to observe the dynamic morphology, and two datasets were thus set up to investigate the performances of CNNs. Considering the installation of deep learning, the classification problem was simplified from video data to image data, and was then solved by CNNs in a self-taught manner with the generated image data. CNNs were separately performed in three installation scenarios and compared with existing methods. Experimental results demonstrated the potential of CNNs in cell dynamic morphology classification, and validated the effectiveness of the proposed strategy. CNNs were successfully applied to the classification problem, and outperformed the existing methods in the classification accuracy. For the installation of CNNs, transfer learning was proved to be a promising scheme. © 2018 International Society for Advancement of Cytometry. © 2018 International Society for Advancement of Cytometry.

Monitoring Extracellular Vesicle Cargo Active Uptake by Imaging Flow Cytometry.

PubMed

Ofir-Birin, Yifat; Abou Karam, Paula; Rudik, Ariel; Giladi, Tal; Porat, Ziv; Regev-Rudzki, Neta

2018-01-01

Extracellular vesicles are essential for long distance cell-cell communication. They function as carriers of different compounds, including proteins, lipids and nucleic acids. Pathogens, like malaria parasites ( Plasmodium falciparum, Pf ), excel in employing vesicle release to mediate cell communication in diverse processes, particularly in manipulating the host response. Establishing research tools to study the interface between pathogen-derived vesicles and their host recipient cells will greatly benefit the scientific community. Here, we present an imaging flow cytometry (IFC) method for monitoring the uptake of malaria-derived vesicles by host immune cells. By staining different cargo components, we were able to directly track the cargo's internalization over time and measure the kinetics of its delivery. Impressively, we demonstrate that this method can be used to specifically monitor the translocation of a specific protein within the cellular milieu upon internalization of parasitic cargo; namely, we were able to visually observe how uptaken parasitic Pf -DNA cargo leads to translocation of transcription factor IRF3 from the cytosol to the nucleus within the recipient immune cell. Our findings demonstrate that our method can be used to study cellular dynamics upon vesicle uptake in different host-pathogen and pathogen-pathogen systems.

Quantitative imaging test approval and biomarker qualification: interrelated but distinct activities.

PubMed

Buckler, Andrew J; Bresolin, Linda; Dunnick, N Reed; Sullivan, Daniel C; Aerts, Hugo J W L; Bendriem, Bernard; Bendtsen, Claus; Boellaard, Ronald; Boone, John M; Cole, Patricia E; Conklin, James J; Dorfman, Gary S; Douglas, Pamela S; Eidsaunet, Willy; Elsinger, Cathy; Frank, Richard A; Gatsonis, Constantine; Giger, Maryellen L; Gupta, Sandeep N; Gustafson, David; Hoekstra, Otto S; Jackson, Edward F; Karam, Lisa; Kelloff, Gary J; Kinahan, Paul E; McLennan, Geoffrey; Miller, Colin G; Mozley, P David; Muller, Keith E; Patt, Rick; Raunig, David; Rosen, Mark; Rupani, Haren; Schwartz, Lawrence H; Siegel, Barry A; Sorensen, A Gregory; Wahl, Richard L; Waterton, John C; Wolf, Walter; Zahlmann, Gudrun; Zimmerman, Brian

2011-06-01

Quantitative imaging biomarkers could speed the development of new treatments for unmet medical needs and improve routine clinical care. However, it is not clear how the various regulatory and nonregulatory (eg, reimbursement) processes (often referred to as pathways) relate, nor is it clear which data need to be collected to support these different pathways most efficiently, given the time- and cost-intensive nature of doing so. The purpose of this article is to describe current thinking regarding these pathways emerging from diverse stakeholders interested and active in the definition, validation, and qualification of quantitative imaging biomarkers and to propose processes to facilitate the development and use of quantitative imaging biomarkers. A flexible framework is described that may be adapted for each imaging application, providing mechanisms that can be used to develop, assess, and evaluate relevant biomarkers. From this framework, processes can be mapped that would be applicable to both imaging product development and to quantitative imaging biomarker development aimed at increasing the effectiveness and availability of quantitative imaging. http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.10100800/-/DC1. RSNA, 2011

Evaluation of quantitative image analysis criteria for the high-resolution microendoscopic detection of neoplasia in Barrett's esophagus

NASA Astrophysics Data System (ADS)

Muldoon, Timothy J.; Thekkek, Nadhi; Roblyer, Darren; Maru, Dipen; Harpaz, Noam; Potack, Jonathan; Anandasabapathy, Sharmila; Richards-Kortum, Rebecca

2010-03-01

Early detection of neoplasia in patients with Barrett's esophagus is essential to improve outcomes. The aim of this ex vivo study was to evaluate the ability of high-resolution microendoscopic imaging and quantitative image analysis to identify neoplastic lesions in patients with Barrett's esophagus. Nine patients with pathologically confirmed Barrett's esophagus underwent endoscopic examination with biopsies or endoscopic mucosal resection. Resected fresh tissue was imaged with fiber bundle microendoscopy; images were analyzed by visual interpretation or by quantitative image analysis to predict whether the imaged sites were non-neoplastic or neoplastic. The best performing pair of quantitative features were chosen based on their ability to correctly classify the data into the two groups. Predictions were compared to the gold standard of histopathology. Subjective analysis of the images by expert clinicians achieved average sensitivity and specificity of 87% and 61%, respectively. The best performing quantitative classification algorithm relied on two image textural features and achieved a sensitivity and specificity of 87% and 85%, respectively. This ex vivo pilot trial demonstrates that quantitative analysis of images obtained with a simple microendoscope system can distinguish neoplasia in Barrett's esophagus with good sensitivity and specificity when compared to histopathology and to subjective image interpretation.

Quantitative phase imaging and complex field reconstruction by pupil modulation differential phase contrast

PubMed Central

Lu, Hangwen; Chung, Jaebum; Ou, Xiaoze; Yang, Changhuei

2016-01-01

Differential phase contrast (DPC) is a non-interferometric quantitative phase imaging method achieved by using an asymmetric imaging procedure. We report a pupil modulation differential phase contrast (PMDPC) imaging method by filtering a sample’s Fourier domain with half-circle pupils. A phase gradient image is captured with each half-circle pupil, and a quantitative high resolution phase image is obtained after a deconvolution process with a minimum of two phase gradient images. Here, we introduce PMDPC quantitative phase image reconstruction algorithm and realize it experimentally in a 4f system with an SLM placed at the pupil plane. In our current experimental setup with the numerical aperture of 0.36, we obtain a quantitative phase image with a resolution of 1.73μm after computationally removing system aberrations and refocusing. We also extend the depth of field digitally by 20 times to ±50μm with a resolution of 1.76μm. PMID:27828473

Analysis of airborne MAIS imaging spectrometric data for mineral exploration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Jinnian; Zheng Lanfen; Tong Qingxi

1996-11-01

The high spectral resolution imaging spectrometric system made quantitative analysis and mapping of surface composition possible. The key issue will be the quantitative approach for analysis of surface parameters for imaging spectrometer data. This paper describes the methods and the stages of quantitative analysis. (1) Extracting surface reflectance from imaging spectrometer image. Lab. and inflight field measurements are conducted for calibration of imaging spectrometer data, and the atmospheric correction has also been used to obtain ground reflectance by using empirical line method and radiation transfer modeling. (2) Determining quantitative relationship between absorption band parameters from the imaging spectrometer data andmore » chemical composition of minerals. (3) Spectral comparison between the spectra of spectral library and the spectra derived from the imagery. The wavelet analysis-based spectrum-matching techniques for quantitative analysis of imaging spectrometer data has beer, developed. Airborne MAIS imaging spectrometer data were used for analysis and the analysis results have been applied to the mineral and petroleum exploration in Tarim Basin area china. 8 refs., 8 figs.« less

Temporal Lobe Epilepsy: Quantitative MR Volumetry in Detection of Hippocampal Atrophy

PubMed Central

Farid, Nikdokht; Girard, Holly M.; Kemmotsu, Nobuko; Smith, Michael E.; Magda, Sebastian W.; Lim, Wei Y.; Lee, Roland R.

2012-01-01

Purpose: To determine the ability of fully automated volumetric magnetic resonance (MR) imaging to depict hippocampal atrophy (HA) and to help correctly lateralize the seizure focus in patients with temporal lobe epilepsy (TLE). Materials and Methods: This study was conducted with institutional review board approval and in compliance with HIPAA regulations. Volumetric MR imaging data were analyzed for 34 patients with TLE and 116 control subjects. Structural volumes were calculated by using U.S. Food and Drug Administration–cleared software for automated quantitative MR imaging analysis (NeuroQuant). Results of quantitative MR imaging were compared with visual detection of atrophy, and, when available, with histologic specimens. Receiver operating characteristic analyses were performed to determine the optimal sensitivity and specificity of quantitative MR imaging for detecting HA and asymmetry. A linear classifier with cross validation was used to estimate the ability of quantitative MR imaging to help lateralize the seizure focus. Results: Quantitative MR imaging–derived hippocampal asymmetries discriminated patients with TLE from control subjects with high sensitivity (86.7%–89.5%) and specificity (92.2%–94.1%). When a linear classifier was used to discriminate left versus right TLE, hippocampal asymmetry achieved 94% classification accuracy. Volumetric asymmetries of other subcortical structures did not improve classification. Compared with invasive video electroencephalographic recordings, lateralization accuracy was 88% with quantitative MR imaging and 85% with visual inspection of volumetric MR imaging studies but only 76% with visual inspection of clinical MR imaging studies. Conclusion: Quantitative MR imaging can depict the presence and laterality of HA in TLE with accuracy rates that may exceed those achieved with visual inspection of clinical MR imaging studies. Thus, quantitative MR imaging may enhance standard visual analysis, providing a useful and viable means for translating volumetric analysis into clinical practice. © RSNA, 2012 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12112638/-/DC1 PMID:22723496

Mobile flow cytometer for mHealth.

PubMed

Balsam, Joshua; Bruck, Hugh Alan; Rasooly, Avraham

2015-01-01

Flow cytometry is used for cell counting and analysis in numerous clinical and environmental applications. However flow cytometry is not used in mHealth mainly because current flow cytometers are large, expensive, power-intensive devices designed to operate in a laboratory. Their design results in a lack of portability and makes them unsuitable for mHealth applications. Another limitation of current technology is the low volumetric throughput rates that are not suitable for rapid detection of rare cells.To address these limitations, we describe here a novel, low-cost, mobile flow cytometer based on wide-field imaging with a webcam for large volume and high throughput fluorescence detection of rare cells as a simulation for circulating tumor cells (CTCs) detection. The mobile flow cytometer uses a commercially available webcam capable of 187 frames per second video capture at a resolution of 320 × 240 pixels. For fluorescence detection, a 1 W 450 nm blue laser is used for excitation of Syto-9 fluorescently stained cells detected at 535 nm. A wide-field flow cell was developed for large volume analysis that allows for the linear velocity of target cells to be lower than in conventional hydrodynamic focusing flow cells typically used in cytometry. The mobile flow cytometer was found to be capable of detecting low concentrations at flow rates of 500 μL/min, suitable for rare cell detection in large volumes. The simplicity and low cost of this device suggests that it may have a potential clinical use for mHealth flow cytometry for resource-poor settings associated with global health.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part IV - postanalytic considerations.

PubMed

Barnett, David; Louzao, Raaul; Gambell, Peter; De, Jitakshi; Oldaker, Teri; Hanson, Curtis A

2013-01-01

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called home brew assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part IV - Postanalytic considerations. © 2013 International Clinical Cytometry Society.

Preparation of genomic DNA from a single species of uncultured magnetotactic bacterium by multiple-displacement amplification.

PubMed

Arakaki, Atsushi; Shibusawa, Mie; Hosokawa, Masahito; Matsunaga, Tadashi

2010-03-01

Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using phi29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, "Magnetospirillum magneticum AMB-1," whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.

Antitumor effect of CXCR4 antagonist AMD3100 on the tumorigenic cell line of BHP10-3 papillary thyroid cancer cells.

PubMed

Jung, Young Ho; Lee, Doh Young; Cha, Wonjae; Kim, Bo Hae; Sung, Myung-Whun; Kim, Kwang Hyun; Ahn, Soon-Hyun

2016-10-01

A tumorigenic cell line (BHP10-3M) derived from nontumorigenic papillary thyroid carcinoma (PTC) cells (BHP10-3) having rearranged during transfection (RET)/PTC1 gene rearrangement might have a higher expression of CXCR4, either quantitatively or functionally. The authors also postulated that CXCR4-mediated invasion or tumorigenesis could be blocked by CXCR4 antagonists, including AMD3100. The expression of CXCR4 in BHP10-3 and BHP10-3M cells was assessed using immunoblot analysis, flow cytometry, and quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The effect of AMD3100 on BHP10-3 and BHP10-3M cell lines was evaluated using cell proliferation assay, invasion assay, and tumor growth experiment in nude mice. Immunoblotting, flow cytometry, and quantitative RT-PCR proved that BHP10-3M cells expressed a higher level of CXCR4 than BHP10-3 cells. Although blocking CXCR4 with AMD3100 did not suppress cell proliferation in both cell lines from 1 ng/mL to 100 ng/mL concentration, AMD3100 suppressed invasion of BHP10-3M cells in vitro in a dose-dependent manner. At higher concentrations from 10(3) ng/mL to 10(5) ng/mL, the proliferation of BHP10-3M cells was inhibited more strongly by AMD3100 than that of BHP10-3 cells. Intraperitoneal injection of AMD3100 inhibited tumor formation by BHP10-3M cells in the thyroid of nude mice. A tumorigenic cell line (BHP10-3M) of PTC showed higher expression of CXCR4 quantitatively and functionally than a nontumorigenic cell line (BHP10-3). The CXCR4 antagonist (AMD3100) showed a significant antitumor effect on the tumorigenic cell line of PTC BHP10-3 cells both in vitro and in vivo. CXCR4 antagonist can be expected to have an adjuvant role in the management of PTC. © 2016 Wiley Periodicals, Inc. Head Neck, 2016 © 2016 Wiley Periodicals, Inc. Head Neck 38: First-1486, 2016. © 2016 Wiley Periodicals, Inc.

Multimodal quantitative phase and fluorescence imaging of cell apoptosis

NASA Astrophysics Data System (ADS)

Fu, Xinye; Zuo, Chao; Yan, Hao

2017-06-01

Fluorescence microscopy, utilizing fluorescence labeling, has the capability to observe intercellular changes which transmitted and reflected light microscopy techniques cannot resolve. However, the parts without fluorescence labeling are not imaged. Hence, the processes simultaneously happen in these parts cannot be revealed. Meanwhile, fluorescence imaging is 2D imaging where information in the depth is missing. Therefore the information in labeling parts is also not complete. On the other hand, quantitative phase imaging is capable to image cells in 3D in real time through phase calculation. However, its resolution is limited by the optical diffraction and cannot observe intercellular changes below 200 nanometers. In this work, fluorescence imaging and quantitative phase imaging are combined to build a multimodal imaging system. Such system has the capability to simultaneously observe the detailed intercellular phenomenon and 3D cell morphology. In this study the proposed multimodal imaging system is used to observe the cell behavior in the cell apoptosis. The aim is to highlight the limitations of fluorescence microscopy and to point out the advantages of multimodal quantitative phase and fluorescence imaging. The proposed multimodal quantitative phase imaging could be further applied in cell related biomedical research, such as tumor.

Using Cell-ID 1.4 with R for Microscope-Based Cytometry

PubMed Central

Bush, Alan; Chernomoretz, Ariel; Yu, Richard; Gordon, Andrew

2012-01-01

This unit describes a method for quantifying various cellular features (e.g., volume, total and subcellular fluorescence localization) from sets of microscope images of individual cells. It includes procedures for tracking cells over time. One purposefully defocused transmission image (sometimes referred to as bright-field or BF) is acquired to segment the image and locate each cell. Fluorescent images (one for each of the color channels to be analyzed) are then acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses the image processing capabilities of Cell-ID (Gordon et al., 2007, as updated here) and data analysis by the statistical programming framework R (R-Development-Team, 2008), which we have supplemented with a package of routines for analyzing Cell-ID output. Both Cell-ID and the analysis package are open-source. PMID:23026908

Logarithmic amplifiers.

PubMed

Gandler, W; Shapiro, H

1990-01-01

Logarithmic amplifiers (log amps), which produce an output signal proportional to the logarithm of the input signal, are widely used in cytometry for measurements of parameters that vary over a wide dynamic range, e.g., cell surface immunofluorescence. Existing log amp circuits all deviate to some extent from ideal performance with respect to dynamic range and fidelity to the logarithmic curve; accuracy in quantitative analysis using log amps therefore requires that log amps be individually calibrated. However, accuracy and precision may be limited by photon statistics and system noise when very low level input signals are encountered.

Alternative fluorescent labeling strategies for characterizing gram-positive pathogenic bacteria: Flow cytometry supported counting, sorting, and proteome analysis of Staphylococcus aureus retrieved from infected host cells.

PubMed

Hildebrandt, Petra; Surmann, Kristin; Salazar, Manuela Gesell; Normann, Nicole; Völker, Uwe; Schmidt, Frank

2016-10-01

Staphylococcus aureus is a Gram-positive opportunistic pathogen that is able to cause a broad range of infectious diseases in humans. Furthermore, S. aureus is able to survive inside nonprofessional phagocytic host cell which serve as a niche for the pathogen to hide from the immune system and antibiotics therapies. Modern OMICs technologies provide valuable tools to investigate host-pathogen interactions upon internalization. However, these experiments are often hampered by limited capabilities to retrieve bacteria from such an experimental setting. Thus, the aim of this study was to develop a labeling strategy allowing fast detection and quantitation of S. aureus in cell lysates or infected cell lines by flow cytometry for subsequent proteome analyses. Therefore, S. aureus cells were labeled with the DNA stain SYTO ® 9, or Vancomycin BODIPY ® FL (VMB), a glycopeptide antibiotic binding to most Gram-positive bacteria which was conjugated to a fluorescent dye. Staining of S. aureus HG001 with SYTO 9 allowed counting of bacteria from pure cultures but not in cell lysates from infection experiments. In contrast, with VMB it was feasible to stain bacteria from pure cultures as well as from samples of infection experiments. VMB can also be applied for histocytochemistry analysis of formaldehyde fixed cell layers grown on coverslips. Proteome analyses of S. aureus labeled with VMB revealed that the labeling procedure provoked only minor changes on proteome level and allowed cell sorting and analysis of S. aureus from infection settings with sensitivity similar to continuous gfp expression. Furthermore, VMB labeling allowed precise counting of internalized bacteria and can be employed for downstream analyses, e.g., proteomics, of strains not easily amendable to genetic manipulation such as clinical isolates. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Quantitative imaging methods in osteoporosis.

PubMed

Oei, Ling; Koromani, Fjorda; Rivadeneira, Fernando; Zillikens, M Carola; Oei, Edwin H G

2016-12-01

Osteoporosis is characterized by a decreased bone mass and quality resulting in an increased fracture risk. Quantitative imaging methods are critical in the diagnosis and follow-up of treatment effects in osteoporosis. Prior radiographic vertebral fractures and bone mineral density (BMD) as a quantitative parameter derived from dual-energy X-ray absorptiometry (DXA) are among the strongest known predictors of future osteoporotic fractures. Therefore, current clinical decision making relies heavily on accurate assessment of these imaging features. Further, novel quantitative techniques are being developed to appraise additional characteristics of osteoporosis including three-dimensional bone architecture with quantitative computed tomography (QCT). Dedicated high-resolution (HR) CT equipment is available to enhance image quality. At the other end of the spectrum, by utilizing post-processing techniques such as the trabecular bone score (TBS) information on three-dimensional architecture can be derived from DXA images. Further developments in magnetic resonance imaging (MRI) seem promising to not only capture bone micro-architecture but also characterize processes at the molecular level. This review provides an overview of various quantitative imaging techniques based on different radiological modalities utilized in clinical osteoporosis care and research.

Quantitation of Fine Displacement in Echography

NASA Astrophysics Data System (ADS)

Masuda, Kohji; Ishihara, Ken; Yoshii, Ken; Furukawa, Toshiyuki; Kumagai, Sadatoshi; Maeda, Hajime; Kodama, Shinzo

1993-05-01

A High-speed Digital Subtraction Echography was developed to visualize the fine displacement of human internal organs. This method indicates differences in position through time series images of high-frame-rate echography. Fine displacement less than ultrasonic wavelength can be observed. This method, however, lacks the ability to quantitatively measure displacement length. The subtraction between two successive images was affected by displacement direction in spite of the displacement length being the same. To solve this problem, convolution of an echogram with Gaussian distribution was used. To express displacement length as brightness quantitatively, normalization using a brightness gradient was applied. The quantitation algorithm was applied to successive B-mode images. Compared to the simply subtracted images, quantitated images express more precisely the motion of organs. Expansion of the carotid artery and fine motion of ventricular walls can be visualized more easily. Displacement length can be quantitated with wavelength. Under more static conditions, this system quantitates displacement length that is much less than wavelength.

Efficient blue conversion from a 1064 nm microchip laser in long photonic crystal fiber tapers for fluorescence microscopy.

PubMed

Kudlinski, A; Lelek, M; Barviau, B; Audry, L; Mussot, A

2010-08-02

Using a low-cost microchip laser and a long photonic crystal fiber taper, we report a supercontinuum source with a very efficient visible conversion, especially in the blue region (around 420 nm). About 30 % of the total average output power is located in the 350-600 nm band, which is of primary importance in a number of biophotonics applications such as flow cytometry or fluorescence imaging microscopy for instance. We successfully demonstrate the use of this visible-enhanced source for a three-color imaging of HeLa cells in wide-field microscopy.

Imaging System and Method for Biomedical Analysis

DTIC Science & Technology

2013-03-11

biological particles and items of interest. Broadly, Padmanabhan et al. utilize the diffraction of a laser light source in flow cytometry to count...spread of light from multiple LED devices over the entire sample surface. Preferably, light source 308 projects a full spectrum white light. Light...for example, red blood cells, white blood cells (which may include lymphocytes which are relatively large and easily detectable), T-helper cells

Patient Stratification with a Novel Molecular Imaging Agent

DTIC Science & Technology

2014-12-01

policy or decision unless so designated by other documentation. REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188 Public reporting...ester of biotin and removed unreacted biotin with a desalting column. 0.7 biotins per protein were achieved as measured by matrix-assisted laser...translation to flow cytometry assays has been inconsistent. In addition to the next two advances (cellular panning and improved combinatorial library design

Performance of computer vision in vivo flow cytometry with low fluorescence contrast

PubMed Central

Markovic, Stacey; Li, Siyuan; Niedre, Mark

2015-01-01

Abstract. Detection and enumeration of circulating cells in the bloodstream of small animals are important in many areas of preclinical biomedical research, including cancer metastasis, immunology, and reproductive medicine. Optical in vivo flow cytometry (IVFC) represents a class of technologies that allow noninvasive and continuous enumeration of circulating cells without drawing blood samples. We recently developed a technique termed computer vision in vivo flow cytometry (CV-IVFC) that uses a high-sensitivity fluorescence camera and an automated computer vision algorithm to interrogate relatively large circulating blood volumes in the ear of a mouse. We detected circulating cells at concentrations as low as 20  cells/mL. In the present work, we characterized the performance of CV-IVFC with low-contrast imaging conditions with (1) weak cell fluorescent labeling using cell-simulating fluorescent microspheres with varying brightness and (2) high background tissue autofluorescence by varying autofluorescence properties of optical phantoms. Our analysis indicates that CV-IVFC can robustly track and enumerate circulating cells with at least 50% sensitivity even in conditions with two orders of magnitude degraded contrast than our previous in vivo work. These results support the significant potential utility of CV-IVFC in a wide range of in vivo biological models. PMID:25822954

Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.

PubMed

Shackney, Stanley; Emlet, David R; Pollice, Agnese; Smith, Charles; Brown, Kathryn; Kociban, Deborah

2006-01-01

Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications. Copyright (c) 2005 Wiley-Liss, Inc.

Frapbot: An open-source application for FRAP data.

PubMed

Kohze, Robin; Dieteren, Cindy E J; Koopman, Werner J H; Brock, Roland; Schmidt, Samuel

2017-08-01

We introduce Frapbot, a free-of-charge open source software web application written in R, which provides manual and automated analyses of fluorescence recovery after photobleaching (FRAP) datasets. For automated operation, starting from data tables containing columns of time-dependent intensity values for various regions of interests within the images, a pattern recognition algorithm recognizes the relevant columns and identifies the presence or absence of prebleach values and the time point of photobleaching. Raw data, residuals, normalization, and boxplots indicating the distribution of half times of recovery (t 1/2 ) of all uploaded files are visualized instantly in a batch-wise manner using a variety of user-definable fitting options. The fitted results are provided as .zip file, which contains .csv formatted output tables. Alternatively, the user can manually control any of the options described earlier. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Genome size and chromosome number in velvet worms (Onychophora).

PubMed

Jeffery, Nicholas W; Oliveira, Ivo S; Gregory, T Ryan; Rowell, David M; Mayer, Georg

2012-12-01

The Onychophora (velvet worms) represents a small group of invertebrates (~180 valid species), which is commonly united with Tardigrada and Arthropoda in a clade called Panarthropoda. As with the majority of invertebrate taxa, genome size data are very limited for the Onychophora, with only one previously published estimate. Here we use both flow cytometry and Feulgen image analysis densitometry to provide genome size estimates for seven species of velvet worms from both major subgroups, Peripatidae and Peripatopsidae, along with karyotype data for each species. Genome sizes in these species range from roughly 5-19 pg, with densitometric estimates being slightly larger than those obtained by flow cytometry for all species. Chromosome numbers range from 2n = 8 to 2n = 54. No relationship is evident between genome size, chromosome number, or reproductive mode. Various avenues for future genomic research are presented based on these results.

Detection of ASC Speck Formation by Flow Cytometry and Chemical Cross-linking.

PubMed

Hoss, Florian; Rolfes, Verena; Davanso, Mariana R; Braga, Tarcio T; Franklin, Bernardo S

2018-01-01

Assembly of a relatively large protein aggregate or "speck" formed by the adaptor protein ASC is a common downstream step in the activation of most inflammasomes. This unique feature of ASC allows its visualization by several imaging techniques and constitutes a reliable and feasible readout for inflammasome activation in cells and tissues. We have previously described step-by-step protocols to generate immortalized cell lines stably expressing ASC fused to a fluorescent protein for measuring inflammasome activation by confocal microscopy, and immunofluorescence of endogenous ASC in primary cells. Here, we present two more methods to detect ASC speck formation: (1) Assessment of ASC speck formation by flow cytometry; and (2) Chemical cross-linking of ASC followed by immunoblotting. These methods allow for the discrimination of inflammasome-activated versus non-activated cells, the identification of lineage-specific inflammasome activation in complex cell mixtures, and sorting of inflammasome-activated cells for further analysis.

Differential Interaction of Platelet-Derived Extracellular Vesicles with Leukocyte Subsets in Human Whole Blood.

PubMed

Weiss, René; Gröger, Marion; Rauscher, Sabine; Fendl, Birgit; Eichhorn, Tanja; Fischer, Michael B; Spittler, Andreas; Weber, Viktoria

2018-04-26

Secretion and exchange of biomolecules via extracellular vesicles (EVs) are crucial mechanisms in intercellular communication, and the roles of EVs in infection, inflammation, or thrombosis have been increasingly recognized. EVs have emerged as central players in immune regulation and can enhance or suppress the immune response, depending on the state of donor and recipient cells. We investigated the interaction of blood cell-derived EVs with leukocyte subpopulations (monocytes and their subsets, granulocytes, B cells, T cells, and NK cells) directly in whole blood using a combination of flow cytometry, imaging flow cytometry, cell sorting, and high resolution confocal microscopy. Platelet-derived EVs constituted the majority of circulating EVs and were preferentially associated with granulocytes and monocytes, while they scarcely interacted with lymphocytes. Further flow cytometric differentiation of monocyte subsets provided clear indications for a preferential association of platelet-derived EVs with intermediate (CD14 ++ CD16 + ) monocytes in whole blood.

In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography

PubMed Central

Genevois, Coralie; Loiseau, Hugues; Couillaud, Franck

2016-01-01

Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT®). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors. PMID:27809256

In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography.

PubMed

Genevois, Coralie; Loiseau, Hugues; Couillaud, Franck

2016-10-31

Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT ® ). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors.

Preoperative Cerebral Oxygen Extraction Fraction Imaging Generated from 7T MR Quantitative Susceptibility Mapping Predicts Development of Cerebral Hyperperfusion following Carotid Endarterectomy.

PubMed

Nomura, J-I; Uwano, I; Sasaki, M; Kudo, K; Yamashita, F; Ito, K; Fujiwara, S; Kobayashi, M; Ogasawara, K

2017-12-01

Preoperative hemodynamic impairment in the affected cerebral hemisphere is associated with the development of cerebral hyperperfusion following carotid endarterectomy. Cerebral oxygen extraction fraction images generated from 7T MR quantitative susceptibility mapping correlate with oxygen extraction fraction images on positron-emission tomography. The present study aimed to determine whether preoperative oxygen extraction fraction imaging generated from 7T MR quantitative susceptibility mapping could identify patients at risk for cerebral hyperperfusion following carotid endarterectomy. Seventy-seven patients with unilateral internal carotid artery stenosis (≥70%) underwent preoperative 3D T2*-weighted imaging using a multiple dipole-inversion algorithm with a 7T MR imager. Quantitative susceptibility mapping images were then obtained, and oxygen extraction fraction maps were generated. Quantitative brain perfusion single-photon emission CT was also performed before and immediately after carotid endarterectomy. ROIs were automatically placed in the bilateral middle cerebral artery territories in all images using a 3D stereotactic ROI template, and affected-to-contralateral ratios in the ROIs were calculated on quantitative susceptibility mapping-oxygen extraction fraction images. Ten patients (13%) showed post-carotid endarterectomy hyperperfusion (cerebral blood flow increases of ≥100% compared with preoperative values in the ROIs on brain perfusion SPECT). Multivariate analysis showed that a high quantitative susceptibility mapping-oxygen extraction fraction ratio was significantly associated with the development of post-carotid endarterectomy hyperperfusion (95% confidence interval, 33.5-249.7; P = .002). Sensitivity, specificity, and positive- and negative-predictive values of the quantitative susceptibility mapping-oxygen extraction fraction ratio for the prediction of the development of post-carotid endarterectomy hyperperfusion were 90%, 84%, 45%, and 98%, respectively. Preoperative oxygen extraction fraction imaging generated from 7T MR quantitative susceptibility mapping identifies patients at risk for cerebral hyperperfusion following carotid endarterectomy. © 2017 by American Journal of Neuroradiology.

Quantitative imaging biomarkers: a review of statistical methods for technical performance assessment.

PubMed

Raunig, David L; McShane, Lisa M; Pennello, Gene; Gatsonis, Constantine; Carson, Paul L; Voyvodic, James T; Wahl, Richard L; Kurland, Brenda F; Schwarz, Adam J; Gönen, Mithat; Zahlmann, Gudrun; Kondratovich, Marina V; O'Donnell, Kevin; Petrick, Nicholas; Cole, Patricia E; Garra, Brian; Sullivan, Daniel C

2015-02-01

Technological developments and greater rigor in the quantitative measurement of biological features in medical images have given rise to an increased interest in using quantitative imaging biomarkers to measure changes in these features. Critical to the performance of a quantitative imaging biomarker in preclinical or clinical settings are three primary metrology areas of interest: measurement linearity and bias, repeatability, and the ability to consistently reproduce equivalent results when conditions change, as would be expected in any clinical trial. Unfortunately, performance studies to date differ greatly in designs, analysis method, and metrics used to assess a quantitative imaging biomarker for clinical use. It is therefore difficult or not possible to integrate results from different studies or to use reported results to design studies. The Radiological Society of North America and the Quantitative Imaging Biomarker Alliance with technical, radiological, and statistical experts developed a set of technical performance analysis methods, metrics, and study designs that provide terminology, metrics, and methods consistent with widely accepted metrological standards. This document provides a consistent framework for the conduct and evaluation of quantitative imaging biomarker performance studies so that results from multiple studies can be compared, contrasted, or combined. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Simultaneous automatic scoring and co-registration of hormone receptors in tumor areas in whole slide images of breast cancer tissue slides.

PubMed

Trahearn, Nicholas; Tsang, Yee Wah; Cree, Ian A; Snead, David; Epstein, David; Rajpoot, Nasir

2017-06-01

Automation of downstream analysis may offer many potential benefits to routine histopathology. One area of interest for automation is in the scoring of multiple immunohistochemical markers to predict the patient's response to targeted therapies. Automated serial slide analysis of this kind requires robust registration to identify common tissue regions across sections. We present an automated method for co-localized scoring of Estrogen Receptor and Progesterone Receptor (ER/PR) in breast cancer core biopsies using whole slide images. Regions of tumor in a series of fifty consecutive breast core biopsies were identified by annotation on H&E whole slide images. Sequentially cut immunohistochemical stained sections were scored manually, before being digitally scanned and then exported into JPEG 2000 format. A two-stage registration process was performed to identify the annotated regions of interest in the immunohistochemistry sections, which were then scored using the Allred system. Overall correlation between manual and automated scoring for ER and PR was 0.944 and 0.883, respectively, with 90% of ER and 80% of PR scores within in one point or less of agreement. This proof of principle study indicates slide registration can be used as a basis for automation of the downstream analysis for clinically relevant biomarkers in the majority of cases. The approach is likely to be improved by implantation of safeguarding analysis steps post registration. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Iterative h-minima-based marker-controlled watershed for cell nucleus segmentation.

PubMed

Koyuncu, Can Fahrettin; Akhan, Ece; Ersahin, Tulin; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem

2016-04-01

Automated microscopy imaging systems facilitate high-throughput screening in molecular cellular biology research. The first step of these systems is cell nucleus segmentation, which has a great impact on the success of the overall system. The marker-controlled watershed is a technique commonly used by the previous studies for nucleus segmentation. These studies define their markers finding regional minima on the intensity/gradient and/or distance transform maps. They typically use the h-minima transform beforehand to suppress noise on these maps. The selection of the h value is critical; unnecessarily small values do not sufficiently suppress the noise, resulting in false and oversegmented markers, and unnecessarily large ones suppress too many pixels, causing missing and undersegmented markers. Because cell nuclei show different characteristics within an image, the same h value may not work to define correct markers for all the nuclei. To address this issue, in this work, we propose a new watershed algorithm that iteratively identifies its markers, considering a set of different h values. In each iteration, the proposed algorithm defines a set of candidates using a particular h value and selects the markers from those candidates provided that they fulfill the size requirement. Working with widefield fluorescence microscopy images, our experiments reveal that the use of multiple h values in our iterative algorithm leads to better segmentation results, compared to its counterparts. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Adenomatous tumors of the middle ear and temporal bone: clinical, morphological and tumor biological characteristics of challenging neoplastic lesions.

PubMed

Duderstadt, M; Förster, Christine; Welkoborsky, H-J; Ostertag, H

2012-03-01

Adenomatous tumors of the middle ear and temporal bone are rare tumors. In this retrospective study, we examined nine patients who underwent surgery for an adenomatous tumor of the middle ear, mastoid cavity or eustachian tube. In seven patients, a middle ear adenoma (MEA) and in two patients an aggressive papillary tumor (APT) was diagnosed. We report the clinical, radiologic, morphologic, immunohistochemical and DNA image cytometrical characteristics that can help to correctly classify these tumors. Therapy consisted of surgical excision of the tumors in eight cases. In one elderly patient, only a large biopsy was taken, because this patient suffered from cardial and kidney disorders and was not suitable for an extended surgical approach. This patient received stereotactic radiotherapy. Seven patients underwent planned second look operation. Recurrences occurred in three patients (one with APT, two with MEA), whereas in two of these cases rather a residual tumor due to initial incomplete tumor resection occurred. By image analysis, DNA cytometry MEA were considered benign, whereas the appearance of aneuploid tumor cells in APT confirmed these tumors as low grade malignant lesions. The proliferation rates were equally low in both entities. APT and MEA are tumor entities which can only be correctly classified by a synopsis of histopathology, immunohistochemistry and DNA image cytometry. The recommended therapy is the complete tumor excision. In cases of APT, von Hippel-Lindau syndrome has to be excluded.

Asymmetric-detection time-stretch optical microscopy (ATOM) for ultrafast high-contrast cellular imaging in flow

PubMed Central

Wong, Terence T. W.; Lau, Andy K. S.; Ho, Kenneth K. Y.; Tang, Matthew Y. H.; Robles, Joseph D. F.; Wei, Xiaoming; Chan, Antony C. S.; Tang, Anson H. L.; Lam, Edmund Y.; Wong, Kenneth K. Y.; Chan, Godfrey C. F.; Shum, Ho Cheung; Tsia, Kevin K.

2014-01-01

Accelerating imaging speed in optical microscopy is often realized at the expense of image contrast, image resolution, and detection sensitivity – a common predicament for advancing high-speed and high-throughput cellular imaging. We here demonstrate a new imaging approach, called asymmetric-detection time-stretch optical microscopy (ATOM), which can deliver ultrafast label-free high-contrast flow imaging with well delineated cellular morphological resolution and in-line optical image amplification to overcome the compromised imaging sensitivity at high speed. We show that ATOM can separately reveal the enhanced phase-gradient and absorption contrast in microfluidic live-cell imaging at a flow speed as high as ~10 m/s, corresponding to an imaging throughput of ~100,000 cells/sec. ATOM could thus be the enabling platform to meet the pressing need for intercalating optical microscopy in cellular assay, e.g. imaging flow cytometry – permitting high-throughput access to the morphological information of the individual cells simultaneously with a multitude of parameters obtained in the standard assay. PMID:24413677

Quantitative radiomics studies for tissue characterization: a review of technology and methodological procedures.

PubMed

Larue, Ruben T H M; Defraene, Gilles; De Ruysscher, Dirk; Lambin, Philippe; van Elmpt, Wouter

2017-02-01

Quantitative analysis of tumour characteristics based on medical imaging is an emerging field of research. In recent years, quantitative imaging features derived from CT, positron emission tomography and MR scans were shown to be of added value in the prediction of outcome parameters in oncology, in what is called the radiomics field. However, results might be difficult to compare owing to a lack of standardized methodologies to conduct quantitative image analyses. In this review, we aim to present an overview of the current challenges, technical routines and protocols that are involved in quantitative imaging studies. The first issue that should be overcome is the dependency of several features on the scan acquisition and image reconstruction parameters. Adopting consistent methods in the subsequent target segmentation step is evenly crucial. To further establish robust quantitative image analyses, standardization or at least calibration of imaging features based on different feature extraction settings is required, especially for texture- and filter-based features. Several open-source and commercial software packages to perform feature extraction are currently available, all with slightly different functionalities, which makes benchmarking quite challenging. The number of imaging features calculated is typically larger than the number of patients studied, which emphasizes the importance of proper feature selection and prediction model-building routines to prevent overfitting. Even though many of these challenges still need to be addressed before quantitative imaging can be brought into daily clinical practice, radiomics is expected to be a critical component for the integration of image-derived information to personalize treatment in the future.

Digitized hand-wrist radiographs: comparison of subjective and software-derived image quality at various compression ratios.

PubMed

McCord, Layne K; Scarfe, William C; Naylor, Rachel H; Scheetz, James P; Silveira, Anibal; Gillespie, Kevin R

2007-05-01

The objectives of this study were to compare the effect of JPEG 2000 compression of hand-wrist radiographs on observer image quality qualitative assessment and to compare with a software-derived quantitative image quality index. Fifteen hand-wrist radiographs were digitized and saved as TIFF and JPEG 2000 images at 4 levels of compression (20:1, 40:1, 60:1, and 80:1). The images, including rereads, were viewed by 13 orthodontic residents who determined the image quality rating on a scale of 1 to 5. A quantitative analysis was also performed by using a readily available software based on the human visual system (Image Quality Measure Computer Program, version 6.2, Mitre, Bedford, Mass). ANOVA was used to determine the optimal compression level (P < or =.05). When we compared subjective indexes, JPEG compression greater than 60:1 significantly reduced image quality. When we used quantitative indexes, the JPEG 2000 images had lower quality at all compression ratios compared with the original TIFF images. There was excellent correlation (R2 >0.92) between qualitative and quantitative indexes. Image Quality Measure indexes are more sensitive than subjective image quality assessments in quantifying image degradation with compression. There is potential for this software-based quantitative method in determining the optimal compression ratio for any image without the use of subjective raters.

A quantitative reconstruction software suite for SPECT imaging

NASA Astrophysics Data System (ADS)

Namías, Mauro; Jeraj, Robert

2017-11-01

Quantitative Single Photon Emission Tomography (SPECT) imaging allows for measurement of activity concentrations of a given radiotracer in vivo. Although SPECT has usually been perceived as non-quantitative by the medical community, the introduction of accurate CT based attenuation correction and scatter correction from hybrid SPECT/CT scanners has enabled SPECT systems to be as quantitative as Positron Emission Tomography (PET) systems. We implemented a software suite to reconstruct quantitative SPECT images from hybrid or dedicated SPECT systems with a separate CT scanner. Attenuation, scatter and collimator response corrections were included in an Ordered Subset Expectation Maximization (OSEM) algorithm. A novel scatter fraction estimation technique was introduced. The SPECT/CT system was calibrated with a cylindrical phantom and quantitative accuracy was assessed with an anthropomorphic phantom and a NEMA/IEC image quality phantom. Accurate activity measurements were achieved at an organ level. This software suite helps increasing quantitative accuracy of SPECT scanners.

PET Imaging of VEGFR-2 Expression in Lung Cancer with 64Cu-Labeled Ramucirumab.

PubMed

Luo, Haiming; England, Christopher G; Graves, Stephen A; Sun, Haiyan; Liu, Glenn; Nickles, Robert J; Cai, Weibo

2016-02-01

Lung cancer accounts for 17% of cancer-related deaths worldwide, and most patients present with locally advanced or metastatic disease. Novel PET imaging agents for assessing vascular endothelial growth factor receptor-2 (VEGFR-2) expression can be used for detecting VEGFR-2-positive malignancies and subsequent monitoring of therapeutic response to VEGFR-2-targeted therapies. Here, we report the synthesis and characterization of an antibody-based imaging agent for PET imaging of VEGFR-2 expression in vivo. Ramucirumab (named RamAb), a fully humanized IgG1 monoclonal antibody, was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (64)Cu. Flow cytometry analysis and microscopy studies were performed to compare the VEGFR-2 binding affinity of RamAb and NOTA-RamAb. PET imaging and biodistribution studies were performed in nude mice bearing HCC4006 and A549 xenograft tumors. Ex vivo histopathology was performed to elucidate the expression patterns of VEGFR-2 in different tissues and organs to validate in vivo results. Flow cytometry examination revealed the specific binding capacity of fluorescein isothiocyanate-RamAb to VEGFR-2, and no difference in VEGFR-2 binding affinity was seen between RamAb and NOTA-RamAb. After being labeled with (64)Cu, PET imaging revealed specific and prominent uptake of (64)Cu-NOTA-RamAb in VEGFR-2-positive HCC4006 tumors (9.4 ± 0.5 percentage injected dose per gram at 48 h after injection; n = 4) and significantly lower uptake in VEGFR-2-negative A549 tumors (4.3 ± 0.2 percentage injected dose per gram at 48 h after injection; n = 3). Blocking experiments revealed significantly lower uptake in HCC4006 tumors, along with histology analysis, further confirming the VEGFR-2 specificity of (64)Cu-NOTA-RamAb. This study provides initial evidence that (64)Cu-NOTA-RamAb can function as a PET imaging agent for visualizing VEGFR-2 expression in vivo, which may also find potential applications in monitoring the treatment response of VEGFR-2-targeted cancer therapy. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Evaluation of slide based cytometry (SBC) for concentration measurements of fluorescent dyes in solution

NASA Astrophysics Data System (ADS)

Pierzchalski, Arkadiusz; Marecka, Monika; Müller, Hans-Willy; Bocsi, József; Tárnok, Attila

2009-02-01

Flow cytometers (FCM) are built for particle measurements. In principle, concentration measurement of a homogeneous solution is not possible with FCM due to the lack of a trigger signal. In contrast to FCM slide based cytometry systems could act as tools for the measurement of concentrations using volume defined cell counting chambers. These chambers enable to analyze a well defined volume. Sensovation AG (Stockach, Germany) introduced an automated imaging system that combines imaging with cytometric features analysis. Aim of this study was to apply this imaging system to quantify the fluorescent molecule concentrations. The Lumisens (Sensovation AG) slide-based technology based on fluorescence digital imaging microscopy was used. The instrument is equipped with an inverted microscope, blue and red LEDs, double band-pass filters and a high-resolution cooled 16-bit digital camera. The instrument was focussed on the bottom of 400μm deep 6 chamber slides (IBIDI GmbH, Martinsried, Germany) or flat bottom 96 well plates (Greiner Bio One GmbH, Frickenhausen, Germany). Fluorescent solutions were imaged under 90% pixel saturation in a broad concentration range (FITC: 0.0002-250 μg/ml, methylene blue (MethB): 0.0002-250 μg/ml). Exposition times were recorded. Images were analysed by the iCys (CompuCyte Corp., Cambridge, MA, USA) image analysis software with the phantom contour function. Relative fluorescence intensities were calculated from mean fluorescence intensities per phantom contours divided by the exposition time. Solution concentrations could be distinguished over a broad dynamic range of 3.5 to 5.5 decades log (range FITC: 0.0002-31.25μg/ml, MethB: 0.0076-31.25μg/ml) with a good linear relationship between dye concentration and relative fluorescence intensity. The minimal number of fluorescent molecules per pixel as determined by the mean fluorescence intensity and the molecular weight of the fluorochrome were about 800 molecules FITC and ~2.000 MethB. The novel slide-based imaging system is suitable for detection of fluorescence differences over a broad range of concentrations. This approach may lead to novel assays for measuring concentration differences in cell free solutions and cell cultures e.g. in secretion assays.

Imaging Cerebral Microhemorrhages in Military Service Members with Chronic Traumatic Brain Injury

PubMed Central

Liu, Wei; Soderlund, Karl; Senseney, Justin S.; Joy, David; Yeh, Ping-Hong; Ollinger, John; Sham, Elyssa B.; Liu, Tian; Wang, Yi; Oakes, Terrence R.; Riedy, Gerard

2017-01-01

Purpose To detect cerebral microhemorrhages in military service members with chronic traumatic brain injury by using susceptibility-weighted magnetic resonance (MR) imaging. The longitudinal evolution of microhemorrhages was monitored in a subset of patients by using quantitative susceptibility mapping. Materials and Methods The study was approved by the Walter Reed National Military Medical Center institutional review board and is compliant with HIPAA guidelines. All participants underwent two-dimensional conventional gradient-recalled-echo MR imaging and three-dimensional flow-compensated multi-echo gradient-recalled-echo MR imaging (processed to generate susceptibility-weighted images and quantitative susceptibility maps), and a subset of patients underwent follow-up imaging. Microhemorrhages were identified by two radiologists independently. Comparisons of microhemorrhage number, size, and magnetic susceptibility derived from quantitative susceptibility maps between baseline and follow-up imaging examinations were performed by using the paired t test. Results Among the 603 patients, cerebral microhemorrhages were identified in 43 patients, with six excluded for further analysis owing to artifacts. Seventy-seven percent (451 of 585) of the microhemorrhages on susceptibility-weighted images had a more conspicuous appearance than on gradient-recalled-echo images. Thirteen of the 37 patients underwent follow-up imaging examinations. In these patients, a smaller number of microhemorrhages were identified at follow-up imaging compared with baseline on quantitative susceptibility maps (mean ± standard deviation, 9.8 microhemorrhages ± 12.8 vs 13.7 microhemorrhages ± 16.6; P = .019). Quantitative susceptibility mapping–derived quantitative measures of microhemorrhages also decreased over time: −0.85 mm3 per day ± 1.59 for total volume (P = .039) and −0.10 parts per billion per day ± 0.14 for mean magnetic susceptibility (P = .016). Conclusion The number of microhemorrhages and quantitative susceptibility mapping–derived quantitative measures of microhemorrhages all decreased over time, suggesting that hemosiderin products undergo continued, subtle evolution in the chronic stage. PMID:26371749

Imaging Cerebral Microhemorrhages in Military Service Members with Chronic Traumatic Brain Injury.

PubMed

Liu, Wei; Soderlund, Karl; Senseney, Justin S; Joy, David; Yeh, Ping-Hong; Ollinger, John; Sham, Elyssa B; Liu, Tian; Wang, Yi; Oakes, Terrence R; Riedy, Gerard

2016-02-01

To detect cerebral microhemorrhages in military service members with chronic traumatic brain injury by using susceptibility-weighted magnetic resonance (MR) imaging. The longitudinal evolution of microhemorrhages was monitored in a subset of patients by using quantitative susceptibility mapping. The study was approved by the Walter Reed National Military Medical Center institutional review board and is compliant with HIPAA guidelines. All participants underwent two-dimensional conventional gradient-recalled-echo MR imaging and three-dimensional flow-compensated multiecho gradient-recalled-echo MR imaging (processed to generate susceptibility-weighted images and quantitative susceptibility maps), and a subset of patients underwent follow-up imaging. Microhemorrhages were identified by two radiologists independently. Comparisons of microhemorrhage number, size, and magnetic susceptibility derived from quantitative susceptibility maps between baseline and follow-up imaging examinations were performed by using the paired t test. Among the 603 patients, cerebral microhemorrhages were identified in 43 patients, with six excluded for further analysis owing to artifacts. Seventy-seven percent (451 of 585) of the microhemorrhages on susceptibility-weighted images had a more conspicuous appearance than on gradient-recalled-echo images. Thirteen of the 37 patients underwent follow-up imaging examinations. In these patients, a smaller number of microhemorrhages were identified at follow-up imaging compared with baseline on quantitative susceptibility maps (mean ± standard deviation, 9.8 microhemorrhages ± 12.8 vs 13.7 microhemorrhages ± 16.6; P = .019). Quantitative susceptibility mapping-derived quantitative measures of microhemorrhages also decreased over time: -0.85 mm(3) per day ± 1.59 for total volume (P = .039) and -0.10 parts per billion per day ± 0.14 for mean magnetic susceptibility (P = .016). The number of microhemorrhages and quantitative susceptibility mapping-derived quantitative measures of microhemorrhages all decreased over time, suggesting that hemosiderin products undergo continued, subtle evolution in the chronic stage. © RSNA, 2015.

Webcam-based flow cytometer using wide-field imaging for low cell number detection at high throughput.

PubMed

Balsam, Joshua; Bruck, Hugh Alan; Rasooly, Avraham

2014-09-07

Here we describe a novel low-cost flow cytometer based on a webcam capable of low cell number detection in a large volume which may overcome the limitations of current flow cytometry. Several key elements have been combined to yield both high throughput and high sensitivity. The first element is a commercially available webcam capable of 187 frames per second video capture at a resolution of 320 × 240 pixels. The second element in this design is a 1 W 450 nm laser module for area-excitation, which combined with the webcam allows for rapid interrogation of a flow field. The final element is a 2D flow-cell which overcomes the flow limitation of hydrodynamic focusing and allows for higher sample throughput in a wider flow field. This cell allows for the linear velocity of target cells to be lower than in a conventional "1D" hydrodynamic focusing flow-cells typically used in cytometry at similar volumetric flow rates. It also allows cells to be imaged at the full frame rate of the webcam. Using this webcam-based flow cytometer with wide-field imaging, it was confirmed that the detection of fluorescently tagged 5 μm polystyrene beads in "1D" hydrodynamic focusing flow-cells was not practical for low cell number detection due to streaking from the motion of the beads, which did not occur with the 2D flow-cell design. The sensitivity and throughput of this webcam-based flow cytometer was then investigated using THP-1 human monocytes stained with SYTO-9 florescent dye in the 2D flow-cell. The flow cytometer was found to be capable of detecting fluorescently tagged cells at concentrations as low as 1 cell per mL at flow rates of 500 μL min(-1) in buffer and in blood. The effectiveness of detection was concentration dependent: at 100 cells per mL 84% of the cells were detected compared to microscopy, 10 cells per mL 79% detected and 1 cell per mL 59% of the cells were detected. With the blood samples spiked to 100 cells per mL, the average concentration for all samples was 91.4 cells per mL, with a 95% confidence interval of 86-97 cells per mL. These low cell concentrations and the large volume capabilities of the system may overcome the limitations of current cytometry, and are applicable to rare cell (such as circulating tumor cell) detection The simplicity and low cost of this device suggests that it may have a potential use in developing point-of-care clinical flow cytometry for resource-poor settings associated with global health.

Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients.

PubMed

Zhai, Juping; Ding, Mengyuan; Yang, Tianjie; Zuo, Bin; Weng, Zhen; Zhao, Yunxiao; He, Jun; Wu, Qingyu; Ruan, Changgeng; He, Yang

2017-10-23

Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation. Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs. The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%. A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.

Cancer imaging phenomics toolkit: quantitative imaging analytics for precision diagnostics and predictive modeling of clinical outcome.

PubMed

Davatzikos, Christos; Rathore, Saima; Bakas, Spyridon; Pati, Sarthak; Bergman, Mark; Kalarot, Ratheesh; Sridharan, Patmaa; Gastounioti, Aimilia; Jahani, Nariman; Cohen, Eric; Akbari, Hamed; Tunc, Birkan; Doshi, Jimit; Parker, Drew; Hsieh, Michael; Sotiras, Aristeidis; Li, Hongming; Ou, Yangming; Doot, Robert K; Bilello, Michel; Fan, Yong; Shinohara, Russell T; Yushkevich, Paul; Verma, Ragini; Kontos, Despina

2018-01-01

The growth of multiparametric imaging protocols has paved the way for quantitative imaging phenotypes that predict treatment response and clinical outcome, reflect underlying cancer molecular characteristics and spatiotemporal heterogeneity, and can guide personalized treatment planning. This growth has underlined the need for efficient quantitative analytics to derive high-dimensional imaging signatures of diagnostic and predictive value in this emerging era of integrated precision diagnostics. This paper presents cancer imaging phenomics toolkit (CaPTk), a new and dynamically growing software platform for analysis of radiographic images of cancer, currently focusing on brain, breast, and lung cancer. CaPTk leverages the value of quantitative imaging analytics along with machine learning to derive phenotypic imaging signatures, based on two-level functionality. First, image analysis algorithms are used to extract comprehensive panels of diverse and complementary features, such as multiparametric intensity histogram distributions, texture, shape, kinetics, connectomics, and spatial patterns. At the second level, these quantitative imaging signatures are fed into multivariate machine learning models to produce diagnostic, prognostic, and predictive biomarkers. Results from clinical studies in three areas are shown: (i) computational neuro-oncology of brain gliomas for precision diagnostics, prediction of outcome, and treatment planning; (ii) prediction of treatment response for breast and lung cancer, and (iii) risk assessment for breast cancer.

Flow Cytometry of Spinach Chloroplasts 1

PubMed Central

Schröder, Wolfgang P.; Petit, Patrice X.

1992-01-01

Intact spinach (Spinacia oleracea) chloroplasts, thylakoid membranes, and inside-out or right-side-out thylakoid vesicles have been characterized by flow cytometry with respect to forward angle light scatter, right angle light scatter, and chlorophyll fluorescence. Analysis of intact chloroplasts with respect to forward light scatter and the chlorophyll fluorescence parameter revealed the presence of truly “intact” and “disrupted” chloroplasts. The forward light scatter parameter, normally considered to reflect object size, was instead found to reflect the particle density. One essential advantage of flow cytometry is that additional parameters such as Ricinus communis agglutinin (linked to fluorescein isothiocyanate) fluorescence can be determined through logical conditions placed on bit-maps, amounting to an analytical purification procedure. In the present case, chloroplast subpopulations with fully preserved envelopes, thylakoid membrane, and inside-out or right-side-out thylakoid membranes vesicles can be distinguished. Flow cytometry is also a useful tool to address the question of availability of glycosyl moities on the membrane surfaces if one keeps in mind that organelle-to-organelle interactions could be partially mediated through a recognition process. A high specific binding of R. communis agglutinin and peanut lectin to the chloroplast envelope was detected. This showed that galactose residues were exposed and accessible to specific lectins on the chloroplast surface. No exposed glucose, fucose, or mannose residues could be detected by the appropriate lectins. Ricin binding to the intact chloroplasts caused a strong aggregation. Disruption of these aggregates by resuspension or during passage in the flow cytometer induced partial breakage of the chloroplasts. Only minor binding of R. communis agglutinin and peanut lectin to the purified thylakoid membranes was detected; the binding was found to be low for both inside-out and right-side-out vesicles of the thylakoid membranes. Images Figure 1 Figure 1 Figure 1 PMID:16653090

Col-F, a fluorescent probe for ex vivo confocal imaging of collagen and elastin in animal tissues.

PubMed

Biela, Ewa; Galas, Jerzy; Lee, Brian; Johnson, Gary L; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W

2013-06-01

A new low-molecular-weight fluorescent probe, Col-F, that exhibits affinity to collagen and elastin, was used successfully in imaging of extracellular matrix in freshly excised animal tissues. Col-F readily penetrates between live cells into tissues and binds to fibers of collagen and elastin by a noncovalent mechanism. Fibers of collagen and elastin have been stained in a variety of tissues, including tendon, skeletal muscle, connective tissue, and arteries. Cells migrating in a Col-F-stained collagenous biomaterial were also imaged. No phototoxic effects were detected when live keratocytes were imaged in the in vitro culture in the presence of Col-F. In conclusion, Col-F provides a simple and convenient tool for fluorescence three-dimensional imaging of intricate collagenous and elastic structures in live and fixed animal tissues, as well as in collagen-containing biomaterials. Copyright © 2013 International Society for Advancement of Cytometry.

Semi-automated scoring of triple-probe FISH in human sperm using confocal microscopy.

PubMed

Branch, Francesca; Nguyen, GiaLinh; Porter, Nicholas; Young, Heather A; Martenies, Sheena E; McCray, Nathan; Deloid, Glen; Popratiloff, Anastas; Perry, Melissa J

2017-09-01

Structural and numerical sperm chromosomal aberrations result from abnormal meiosis and are directly linked to infertility. Any live births that arise from aneuploid conceptuses can result in syndromes such as Kleinfelter, Turners, XYY and Edwards. Multi-probe fluorescence in situ hybridization (FISH) is commonly used to study sperm aneuploidy, however manual FISH scoring in sperm samples is labor-intensive and introduces errors. Automated scoring methods are continuously evolving. One challenging aspect for optimizing automated sperm FISH scoring has been the overlap in excitation and emission of the fluorescent probes used to enumerate the chromosomes of interest. Our objective was to demonstrate the feasibility of combining confocal microscopy and spectral imaging with high-throughput methods for accurately measuring sperm aneuploidy. Our approach used confocal microscopy to analyze numerical chromosomal abnormalities in human sperm using enhanced slide preparation and rigorous semi-automated scoring methods. FISH for chromosomes X, Y, and 18 was conducted to determine sex chromosome disomy in sperm nuclei. Application of online spectral linear unmixing was used for effective separation of four fluorochromes while decreasing data acquisition time. Semi-automated image processing, segmentation, classification, and scoring were performed on 10 slides using custom image processing and analysis software and results were compared with manual methods. No significant differences in disomy frequencies were seen between the semi automated and manual methods. Samples treated with pepsin were observed to have reduced background autofluorescence and more uniform distribution of cells. These results demonstrate that semi-automated methods using spectral imaging on a confocal platform are a feasible approach for analyzing numerical chromosomal aberrations in sperm, and are comparable to manual methods. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Improving the Sensitivity and Functionality of Mobile Webcam-Based Fluorescence Detectors for Point-of-Care Diagnostics in Global Health.

PubMed

Rasooly, Reuven; Bruck, Hugh Alan; Balsam, Joshua; Prickril, Ben; Ossandon, Miguel; Rasooly, Avraham

2016-05-17

Resource-poor countries and regions require effective, low-cost diagnostic devices for accurate identification and diagnosis of health conditions. Optical detection technologies used for many types of biological and clinical analysis can play a significant role in addressing this need, but must be sufficiently affordable and portable for use in global health settings. Most current clinical optical imaging technologies are accurate and sensitive, but also expensive and difficult to adapt for use in these settings. These challenges can be mitigated by taking advantage of affordable consumer electronics mobile devices such as webcams, mobile phones, charge-coupled device (CCD) cameras, lasers, and LEDs. Low-cost, portable multi-wavelength fluorescence plate readers have been developed for many applications including detection of microbial toxins such as C. Botulinum A neurotoxin, Shiga toxin, and S. aureus enterotoxin B (SEB), and flow cytometry has been used to detect very low cell concentrations. However, the relatively low sensitivities of these devices limit their clinical utility. We have developed several approaches to improve their sensitivity presented here for webcam based fluorescence detectors, including (1) image stacking to improve signal-to-noise ratios; (2) lasers to enable fluorescence excitation for flow cytometry; and (3) streak imaging to capture the trajectory of a single cell, enabling imaging sensors with high noise levels to detect rare cell events. These approaches can also help to overcome some of the limitations of other low-cost optical detection technologies such as CCD or phone-based detectors (like high noise levels or low sensitivities), and provide for their use in low-cost medical diagnostics in resource-poor settings.

Improving the Sensitivity and Functionality of Mobile Webcam-Based Fluorescence Detectors for Point-of-Care Diagnostics in Global Health

PubMed Central

Rasooly, Reuven; Bruck, Hugh Alan; Balsam, Joshua; Prickril, Ben; Ossandon, Miguel; Rasooly, Avraham

2016-01-01

Resource-poor countries and regions require effective, low-cost diagnostic devices for accurate identification and diagnosis of health conditions. Optical detection technologies used for many types of biological and clinical analysis can play a significant role in addressing this need, but must be sufficiently affordable and portable for use in global health settings. Most current clinical optical imaging technologies are accurate and sensitive, but also expensive and difficult to adapt for use in these settings. These challenges can be mitigated by taking advantage of affordable consumer electronics mobile devices such as webcams, mobile phones, charge-coupled device (CCD) cameras, lasers, and LEDs. Low-cost, portable multi-wavelength fluorescence plate readers have been developed for many applications including detection of microbial toxins such as C. Botulinum A neurotoxin, Shiga toxin, and S. aureus enterotoxin B (SEB), and flow cytometry has been used to detect very low cell concentrations. However, the relatively low sensitivities of these devices limit their clinical utility. We have developed several approaches to improve their sensitivity presented here for webcam based fluorescence detectors, including (1) image stacking to improve signal-to-noise ratios; (2) lasers to enable fluorescence excitation for flow cytometry; and (3) streak imaging to capture the trajectory of a single cell, enabling imaging sensors with high noise levels to detect rare cell events. These approaches can also help to overcome some of the limitations of other low-cost optical detection technologies such as CCD or phone-based detectors (like high noise levels or low sensitivities), and provide for their use in low-cost medical diagnostics in resource-poor settings. PMID:27196933

The impact of "omic" and imaging technologies on assessing the host immune response to biodefence agents.

PubMed

Tree, Julia A; Flick-Smith, Helen; Elmore, Michael J; Rowland, Caroline A

2014-01-01

Understanding the interactions between host and pathogen is important for the development and assessment of medical countermeasures to infectious agents, including potential biodefence pathogens such as Bacillus anthracis, Ebola virus, and Francisella tularensis. This review focuses on technological advances which allow this interaction to be studied in much greater detail. Namely, the use of "omic" technologies (next generation sequencing, DNA, and protein microarrays) for dissecting the underlying host response to infection at the molecular level; optical imaging techniques (flow cytometry and fluorescence microscopy) for assessing cellular responses to infection; and biophotonic imaging for visualising the infectious disease process. All of these technologies hold great promise for important breakthroughs in the rational development of vaccines and therapeutics for biodefence agents.

Quantitative comparison of bright field and annular bright field imaging modes for characterization of oxygen octahedral tilts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Young-Min; Pennycook, Stephen J.; Borisevich, Albina Y.

Octahedral tilt behavior is increasingly recognized as an important contributing factor to the physical behavior of perovskite oxide materials and especially their interfaces, necessitating the development of high-resolution methods of tilt mapping. There are currently two major approaches for quantitative imaging of tilts in scanning transmission electron microscopy (STEM), bright field (BF) and annular bright field (ABF). In this study, we show that BF STEM can be reliably used for measurements of oxygen octahedral tilts. While optimal conditions for BF imaging are more restricted with respect to sample thickness and defocus, we find that BF imaging with an aberration-corrected microscopemore » with the accelerating voltage of 300 kV gives us the most accurate quantitative measurement of the oxygen column positions. Using the tilted perovskite structure of BiFeO 3 (BFO) as our test sample, we simulate BF and ABF images in a wide range of conditions, identifying the optimal imaging conditions for each mode. Finally, we show that unlike ABF imaging, BF imaging remains directly quantitatively interpretable for a wide range of the specimen mistilt, suggesting that it should be preferable to the ABF STEM imaging for quantitative structure determination.« less

Quantitative comparison of bright field and annular bright field imaging modes for characterization of oxygen octahedral tilts

DOE PAGES

Kim, Young-Min; Pennycook, Stephen J.; Borisevich, Albina Y.

2017-04-29

Octahedral tilt behavior is increasingly recognized as an important contributing factor to the physical behavior of perovskite oxide materials and especially their interfaces, necessitating the development of high-resolution methods of tilt mapping. There are currently two major approaches for quantitative imaging of tilts in scanning transmission electron microscopy (STEM), bright field (BF) and annular bright field (ABF). In this study, we show that BF STEM can be reliably used for measurements of oxygen octahedral tilts. While optimal conditions for BF imaging are more restricted with respect to sample thickness and defocus, we find that BF imaging with an aberration-corrected microscopemore » with the accelerating voltage of 300 kV gives us the most accurate quantitative measurement of the oxygen column positions. Using the tilted perovskite structure of BiFeO 3 (BFO) as our test sample, we simulate BF and ABF images in a wide range of conditions, identifying the optimal imaging conditions for each mode. Finally, we show that unlike ABF imaging, BF imaging remains directly quantitatively interpretable for a wide range of the specimen mistilt, suggesting that it should be preferable to the ABF STEM imaging for quantitative structure determination.« less

Profiling pleural effusion cells by a diffraction imaging method

NASA Astrophysics Data System (ADS)

Al-Qaysi, Safaa; Hong, Heng; Wen, Yuhua; Lu, Jun Q.; Feng, Yuanming; Hu, Xin-Hua

2018-02-01

Assay of cells in pleural effusion (PE) is an important means of disease diagnosis. Conventional cytology of effusion samples, however, has low sensitivity and depends heavily on the expertise of cytopathologists. We applied a polarization diffraction imaging flow cytometry method on effusion cells to investigate their features. Diffraction imaging of the PE cell samples has been performed on 6000 to 12000 cells for each effusion cell sample of three patients. After prescreening to remove images by cellular debris and aggregated non-cellular particles, the image textures were extracted with a gray level co-occurrence matrix (GLCM) algorithm. The distribution of the imaged cells in the GLCM parameters space was analyzed by a Gaussian Mixture Model (GMM) to determine the number of clusters among the effusion cells. These results yield insight on textural features of diffraction images and related cellular morphology in effusion samples and can be used toward the development of a label-free method for effusion cells assay.

Clinical application of DNA ploidy to cervical cancer screening: A review.

PubMed

Garner, David

2014-12-10

Screening for cervical cancer with DNA ploidy assessment by automated quantitative image cytometry has spread throughout China over the past decade and now an estimated 1 million tests per year are done there. Compared to conventional liquid based cytology, DNA ploidy has competitive accuracy with much higher throughput per technician. DNA ploidy has the enormous advantage that it is an objective technology that can be taught in typically 2 or 3 wk, unlike qualitative cytology, and so it can enable screening in places that lack sufficient qualified cytotechnologists and cytopathologists for conventional cytology. Most papers on experience with application of the technology to cervical cancer screening over the past decade were published in the Chinese language. This review aims to provide a consistent framework for analysis of screening data and to summarize some of the work published from 2005 to the end of 2013. Of particular interest are a few studies comparing DNA ploidy with testing for high risk human papilloma virus (hrHPV) which suggest that DNA ploidy is at least equivalent, easier and less expensive than hrHPV testing. There may also be patient management benefits to combining hrHPV testing with DNA ploidy. Some knowledge gaps are identified and some suggestions are made for future research directions.

Clinical application of DNA ploidy to cervical cancer screening: A review

PubMed Central

Garner, David

2014-01-01

Screening for cervical cancer with DNA ploidy assessment by automated quantitative image cytometry has spread throughout China over the past decade and now an estimated 1 million tests per year are done there. Compared to conventional liquid based cytology, DNA ploidy has competitive accuracy with much higher throughput per technician. DNA ploidy has the enormous advantage that it is an objective technology that can be taught in typically 2 or 3 wk, unlike qualitative cytology, and so it can enable screening in places that lack sufficient qualified cytotechnologists and cytopathologists for conventional cytology. Most papers on experience with application of the technology to cervical cancer screening over the past decade were published in the Chinese language. This review aims to provide a consistent framework for analysis of screening data and to summarize some of the work published from 2005 to the end of 2013. Of particular interest are a few studies comparing DNA ploidy with testing for high risk human papilloma virus (hrHPV) which suggest that DNA ploidy is at least equivalent, easier and less expensive than hrHPV testing. There may also be patient management benefits to combining hrHPV testing with DNA ploidy. Some knowledge gaps are identified and some suggestions are made for future research directions. PMID:25493231

Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells.

PubMed

Phetsouphanh, Chansavath; Zaunders, John James; Kelleher, Anthony Dominic

2015-08-12

A new generation of sensitive T cell-based assays facilitates the direct quantitation and characterization of antigen-specific T cell responses. Single-cell analyses have focused on measuring the quality and breadth of a response. Accumulating data from these studies demonstrate that there is considerable, previously-unrecognized, heterogeneity. Standard assays, such as the ICS, are often insufficient for characterization of rare subsets of cells. Enhanced flow cytometry with imaging capabilities enables the determination of cell morphology, as well as the spatial localization of the protein molecules within a single cell. Advances in both microfluidics and digital PCR have improved the efficiency of single-cell sorting and allowed multiplexed gene detection at the single-cell level. Delving further into the transcriptome of single-cells using RNA-seq is likely to reveal the fine-specificity of cellular events such as alternative splicing (i.e., splice variants) and allele-specific expression, and will also define the roles of new genes. Finally, detailed analysis of clonally related antigen-specific T cells using single-cell TCR RNA-seq will provide information on pathways of differentiation of memory T cells. With these state of the art technologies the transcriptomics and genomics of Ag-specific T cells can be more definitively elucidated.

Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells

PubMed Central

Phetsouphanh, Chansavath; Zaunders, John James; Kelleher, Anthony Dominic

2015-01-01

A new generation of sensitive T cell-based assays facilitates the direct quantitation and characterization of antigen-specific T cell responses. Single-cell analyses have focused on measuring the quality and breadth of a response. Accumulating data from these studies demonstrate that there is considerable, previously-unrecognized, heterogeneity. Standard assays, such as the ICS, are often insufficient for characterization of rare subsets of cells. Enhanced flow cytometry with imaging capabilities enables the determination of cell morphology, as well as the spatial localization of the protein molecules within a single cell. Advances in both microfluidics and digital PCR have improved the efficiency of single-cell sorting and allowed multiplexed gene detection at the single-cell level. Delving further into the transcriptome of single-cells using RNA-seq is likely to reveal the fine-specificity of cellular events such as alternative splicing (i.e., splice variants) and allele-specific expression, and will also define the roles of new genes. Finally, detailed analysis of clonally related antigen-specific T cells using single-cell TCR RNA-seq will provide information on pathways of differentiation of memory T cells. With these state of the art technologies the transcriptomics and genomics of Ag-specific T cells can be more definitively elucidated. PMID:26274954

A system for counting fetal and maternal red blood cells.

PubMed

Ge, Ji; Gong, Zheng; Chen, Jun; Liu, Jun; Nguyen, John; Yang, Zongyi; Wang, Chen; Sun, Yu

2014-12-01

The Kleihauer-Betke (KB) test is the standard method for quantitating fetal-maternal hemorrhage in maternal care. In hospitals, the KB test is performed by a certified technologist to count a minimum of 2000 fetal and maternal red blood cells (RBCs) on a blood smear. Manual counting suffers from inherent inconsistency and unreliability. This paper describes a system for automated counting and distinguishing fetal and maternal RBCs on clinical KB slides. A custom-adapted hardware platform is used for KB slide scanning and image capturing. Spatial-color pixel classification with spectral clustering is proposed to separate overlapping cells. Optimal clustering number and total cell number are obtained through maximizing cluster validity index. To accurately identify fetal RBCs from maternal RBCs, multiple features including cell size, roundness, gradient, and saturation difference between cell and whole slide are used in supervised learning to generate feature vectors, to tackle cell color, shape, and contrast variations across clinical KB slides. The results show that the automated system is capable of completing the counting of over 60,000 cells (versus ∼2000 by technologists) within 5 min (versus ∼15 min by technologists). The throughput is improved by approximately 90 times compared to manual reading by technologists. The counting results are highly accurate and correlate strongly with those from benchmarking flow cytometry measurement.

Studies of the Tumor Microenvironment in Pathogenesis of Neuroblastoma

DTIC Science & Technology

2012-07-01

DISTRIBUTION / AVAILABILITY STATEMENT 13. SUPPLEMENTARY NOTES 14. ABSTRACT The NBL -Tag neuroblastoma mice were crossed with B-cell... NBL -Tag mice were established. The NBL -Tag/B-cell deficient mice lacked B-cells as expected using flow cytometry analyses and immunohistochemistry...However, the lack of B-cells did not alter the growth patterns of NBL -Tag tumor formation as imaged by MRI. Studies using anti-B cell therapy were

Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells.

PubMed

Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

2017-07-01

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only a few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. 3D-magnetic twisting cytometry (3D-MTC) is a technique for applying local mechanical stresses to living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors, followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic-field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super-resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real-time acquisition of a living cell's mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC-microscopy platform takes ∼20 d to construct, and the experimental procedures require ∼4 d when carried out by a life sciences graduate student.

Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells

PubMed Central

Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

2017-01-01

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. Three dimensional-Magnetic Twisting Cytometry (3D-MTC) is a technique for applying local mechanical stresses on living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real time acquisition of a living cell’s mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC – microscopy platform takes around 20 days to construct and the experimental procedures require ~4 days when carried out by a life sciences graduate student. PMID:28686583

Improvements in Diagnostic Accuracy with Quantitative Dynamic Contrast-Enhanced MRI

DTIC Science & Technology

2011-12-01

Magnetic   Resonance   Imaging  during  the  Menstrual  Cylce:  Perfusion   Imaging  Signal   Enhanceent,  and  Influence  of...acquisition of quantitative images displaying the concentration of contrast media as well as MRI -detectable proton density. To date 21 patients have...truly  quantitative   images  of  a  dynamic  contrast-­‐enhanced  (DCE)   MRI  of  the

Developing the Quantitative Histopathology Image Ontology (QHIO): A case study using the hot spot detection problem.

PubMed

Gurcan, Metin N; Tomaszewski, John; Overton, James A; Doyle, Scott; Ruttenberg, Alan; Smith, Barry

2017-02-01

Interoperability across data sets is a key challenge for quantitative histopathological imaging. There is a need for an ontology that can support effective merging of pathological image data with associated clinical and demographic data. To foster organized, cross-disciplinary, information-driven collaborations in the pathological imaging field, we propose to develop an ontology to represent imaging data and methods used in pathological imaging and analysis, and call it Quantitative Histopathological Imaging Ontology - QHIO. We apply QHIO to breast cancer hot-spot detection with the goal of enhancing reliability of detection by promoting the sharing of data between image analysts. Copyright © 2016 Elsevier Inc. All rights reserved.

A novel iris transillumination grading scale allowing flexible assessment with quantitative image analysis and visual matching.

PubMed

Wang, Chen; Brancusi, Flavia; Valivullah, Zaheer M; Anderson, Michael G; Cunningham, Denise; Hedberg-Buenz, Adam; Power, Bradley; Simeonov, Dimitre; Gahl, William A; Zein, Wadih M; Adams, David R; Brooks, Brian

2018-01-01

To develop a sensitive scale of iris transillumination suitable for clinical and research use, with the capability of either quantitative analysis or visual matching of images. Iris transillumination photographic images were used from 70 study subjects with ocular or oculocutaneous albinism. Subjects represented a broad range of ocular pigmentation. A subset of images was subjected to image analysis and ranking by both expert and nonexpert reviewers. Quantitative ordering of images was compared with ordering by visual inspection. Images were binned to establish an 8-point scale. Ranking consistency was evaluated using the Kendall rank correlation coefficient (Kendall's tau). Visual ranking results were assessed using Kendall's coefficient of concordance (Kendall's W) analysis. There was a high degree of correlation among the image analysis, expert-based and non-expert-based image rankings. Pairwise comparisons of the quantitative ranking with each reviewer generated an average Kendall's tau of 0.83 ± 0.04 (SD). Inter-rater correlation was also high with Kendall's W of 0.96, 0.95, and 0.95 for nonexpert, expert, and all reviewers, respectively. The current standard for assessing iris transillumination is expert assessment of clinical exam findings. We adapted an image-analysis technique to generate quantitative transillumination values. Quantitative ranking was shown to be highly similar to a ranking produced by both expert and nonexpert reviewers. This finding suggests that the image characteristics used to quantify iris transillumination do not require expert interpretation. Inter-rater rankings were also highly similar, suggesting that varied methods of transillumination ranking are robust in terms of producing reproducible results.

Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

PubMed Central

Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

2015-01-01

This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications. PMID:25938973

Hyperspectral cytometry.

PubMed

Grégori, Gérald; Rajwa, Bartek; Patsekin, Valery; Jones, James; Furuki, Motohiro; Yamamoto, Masanobu; Paul Robinson, J

2014-01-01

Hyperspectral cytometry is an emerging technology for single-cell analysis that combines ultrafast optical spectroscopy and flow cytometry. Spectral cytometry systems utilize diffraction gratings or prism-based monochromators to disperse fluorescence signals from multiple labels (organic dyes, nanoparticles, or fluorescent proteins) present in each analyzed bioparticle onto linear detector arrays such as multianode photomultipliers or charge-coupled device sensors. The resultant data, consisting of a series of characterizing every analyzed cell, are not compensated by employing the traditional cytometry approach, but rather are spectrally unmixed utilizing algorithms such as constrained Poisson regression or non-negative matrix factorization. Although implementations of spectral cytometry were envisioned as early as the 1980s, only recently has the development of highly sensitive photomultiplier tube arrays led to design and construction of functional prototypes and subsequently to introduction of commercially available systems. This chapter summarizes the historical efforts and work in the field of spectral cytometry performed at Purdue University Cytometry Laboratories and describes the technology developed by Sony Corporation that resulted in release of the first commercial spectral cytometry system-the Sony SP6800. A brief introduction to spectral data analysis is also provided, with emphasis on the differences between traditional polychromatic and spectral cytometry approaches.

Free-living amoebae: Health concerns in the indoor environment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tyndall, R.L.; Ironside, K.S.

1990-01-01

Free-living amoebae are the most likely protozoa implicated in health concerns of the indoor environment. These amoebae can be the source of allergic reactions, eye infections or, on rare occasions, encephalitis. While too large to be effectively aerosolized, free- living amoebae can support the multiplication of pathogens such as Legionella which are easily aerosolized and infectious via the pulmonary route. Traditional detection methods for free-living amoebae are laborious and time consuming. Newer techniques for rapidly detecting and quantitating free-living amoebae such as monoclonal antibodies, flow cytometry, gene probes, and laser optics have or could be employed. 25 refs.

[Quantitative data analysis for live imaging of bone.

PubMed

Seno, Shigeto

Bone tissue is a hard tissue, it was difficult to observe the interior of the bone tissue alive. With the progress of microscopic technology and fluorescent probe technology in recent years, it becomes possible to observe various activities of various cells forming bone society. On the other hand, the quantitative increase in data and the diversification and complexity of the images makes it difficult to perform quantitative analysis by visual inspection. It has been expected to develop a methodology for processing microscopic images and data analysis. In this article, we introduce the research field of bioimage informatics which is the boundary area of biology and information science, and then outline the basic image processing technology for quantitative analysis of live imaging data of bone.

The Use of Quantitative SPECT/CT Imaging to Assess Residual Limb Health

DTIC Science & Technology

2016-10-01

AWARD NUMBER: W81XWH-15-1-0669 TITLE: The Use of Quantitative SPECT/CT Imaging to Assess Residual Limb Health PRINCIPAL INVESTIGATOR...3. DATES COVERED 30 Sep 2015 - 29 Sep 2016 4. TITLE AND SUBTITLE The Use of Quantitative SPECT/CT Imaging to Assess Residual Limb Health 5a...amputation and subsequently evaluate the utility of non-invasive imaging for evaluating the impact of next-generation socket technologies on the health of

Quantitative fractography by digital image processing: NIH Image macro tools for stereo pair analysis and 3-D reconstruction.

PubMed

Hein, L R

2001-10-01

A set of NIH Image macro programs was developed to make qualitative and quantitative analyses from digital stereo pictures produced by scanning electron microscopes. These tools were designed for image alignment, anaglyph representation, animation, reconstruction of true elevation surfaces, reconstruction of elevation profiles, true-scale elevation mapping and, for the quantitative approach, surface area and roughness calculations. Limitations on time processing, scanning techniques and programming concepts are also discussed.

Comparison of qualitative and quantitative evaluation of diffusion-weighted MRI and chemical-shift imaging in the differentiation of benign and malignant vertebral body fractures.

PubMed

Geith, Tobias; Schmidt, Gerwin; Biffar, Andreas; Dietrich, Olaf; Dürr, Hans Roland; Reiser, Maximilian; Baur-Melnyk, Andrea

2012-11-01

The objective of our study was to compare the diagnostic value of qualitative diffusion-weighted imaging (DWI), quantitative DWI, and chemical-shift imaging in a single prospective cohort of patients with acute osteoporotic and malignant vertebral fractures. The study group was composed of patients with 26 osteoporotic vertebral fractures (18 women, eight men; mean age, 69 years; age range, 31 years 6 months to 86 years 2 months) and 20 malignant vertebral fractures (nine women, 11 men; mean age, 63.4 years; age range, 24 years 8 months to 86 years 4 months). T1-weighted, STIR, and T2-weighted sequences were acquired at 1.5 T. A DW reverse fast imaging with steady-state free precession (PSIF) sequence at different delta values was evaluated qualitatively. A DW echo-planar imaging (EPI) sequence and a DW single-shot turbo spin-echo (TSE) sequence at different b values were evaluated qualitatively and quantitatively using the apparent diffusion coefficient. Opposed-phase sequences were used to assess signal intensity qualitatively. The signal loss between in- and opposed-phase images was determined quantitatively. Two-tailed Fisher exact test, Mann-Whitney test, and receiver operating characteristic analysis were performed. Sensitivities, specificities, and accuracies were determined. Qualitative DW-PSIF imaging (delta = 3 ms) showed the best performance for distinguishing between benign and malignant fractures (sensitivity, 100%; specificity, 88.5%; accuracy, 93.5%). Qualitative DW-EPI (b = 50 s/mm(2) [p = 1.00]; b = 250 s/mm(2) [p = 0.50]) and DW single-shot TSE imaging (b = 100 s/mm(2) [p = 1.00]; b = 250 s/mm(2) [p = 0.18]; b = 400 s/mm(2) [p = 0.18]; b = 600 s/mm(2) [p = 0.39]) did not indicate significant differences between benign and malignant fractures. DW-EPI using a b value of 500 s/mm(2) (p = 0.01) indicated significant differences between benign and malignant vertebral fractures. Quantitative DW-EPI (p = 0.09) and qualitative opposed-phase imaging (p = 0.06) did not exhibit significant differences, quantitative DW single-shot TSE imaging (p = 0.002) and quantitative chemical-shift imaging (p = 0.01) showed significant differences between benign and malignant fractures. The DW-PSIF sequence (delta = 3 ms) had the highest accuracy in differentiating benign from malignant vertebral fractures. Quantitative chemical-shift imaging and quantitative DW single-shot TSE imaging had a lower accuracy than DW-PSIF imaging because of a large overlap. Qualitative assessment of opposed-phase, DW-EPI, and DW single-shot TSE sequences and quantitative assessment of the DW-EPI sequence were not suitable for distinguishing between benign and malignant vertebral fractures.

High-speed bioimaging with frequency-division-multiplexed fluorescence confocal microscopy

NASA Astrophysics Data System (ADS)

Mikami, Hideharu; Harmon, Jeffrey; Ozeki, Yasuyuki; Goda, Keisuke

2017-04-01

We present methods of fluorescence confocal microscopy that enable unprecedentedly high frame rate of > 10,000 fps. The methods are based on a frequency-division multiplexing technique, which was originally developed in the field of communication engineering. Specifically, we achieved a broad bandwidth ( 400 MHz) of detection signals using a dual- AOD method and overcame limitations in frame rate, due to a scanning device, by using a multi-line focusing method, resulting in a significant increase in frame rate. The methods have potential biomedical applications such as observation of sub-millisecond dynamics in biological tissues, in-vivo three-dimensional imaging, and fluorescence imaging flow cytometry.

Semi-quantitative methods yield greater inter- and intraobserver agreement than subjective methods for interpreting 99m technetium-hydroxymethylene-diphosphonate uptake in equine thoracic processi spinosi.

PubMed

van Zadelhoff, Claudia; Ehrle, Anna; Merle, Roswitha; Jahn, Werner; Lischer, Christoph

2018-05-09

Scintigraphy is a standard diagnostic method for evaluating horses with back pain due to suspected thoracic processus spinosus pathology. Lesion detection is based on subjective or semi-quantitative assessments of increased uptake. This retrospective, analytical study is aimed to compare semi-quantitative and subjective methods in the evaluation of scintigraphic images of the processi spinosi in the equine thoracic spine. Scintigraphic images of 20 Warmblood horses, presented for assessment of orthopedic conditions between 2014 and 2016, were included in the study. Randomized, blinded image evaluation was performed by 11 veterinarians using subjective and semi-quantitative methods. Subjective grading was performed for the analysis of red-green-blue and grayscale scintigraphic images, which were presented in full-size or as masked images. For the semi-quantitative assessment, observers placed regions of interest over each processus spinosus. The uptake ratio of each processus spinosus in comparison to a reference region of interest was determined. Subsequently, a modified semi-quantitative calculation was developed whereby only the highest counts-per-pixel for a specified number of pixels was processed. Inter- and intraobserver agreement was calculated using intraclass correlation coefficients. Inter- and intraobserver intraclass correlation coefficients were 41.65% and 71.39%, respectively, for the subjective image assessment. Additionally, a correlation between intraobserver agreement, experience, and grayscale images was identified. The inter- and intraobserver agreement was significantly increased when using semi-quantitative analysis (97.35% and 98.36%, respectively) or the modified semi-quantitative calculation (98.61% and 98.82%, respectively). The proposed modified semi-quantitative technique showed a higher inter- and intraobserver agreement when compared to other methods, which makes it a useful tool for the analysis of scintigraphic images. The association of the findings from this study with clinical and radiological examinations requires further investigation. © 2018 American College of Veterinary Radiology.

Respiratory trace feature analysis for the prediction of respiratory-gated PET quantification.

PubMed

Wang, Shouyi; Bowen, Stephen R; Chaovalitwongse, W Art; Sandison, George A; Grabowski, Thomas J; Kinahan, Paul E

2014-02-21

The benefits of respiratory gating in quantitative PET/CT vary tremendously between individual patients. Respiratory pattern is among many patient-specific characteristics that are thought to play an important role in gating-induced imaging improvements. However, the quantitative relationship between patient-specific characteristics of respiratory pattern and improvements in quantitative accuracy from respiratory-gated PET/CT has not been well established. If such a relationship could be estimated, then patient-specific respiratory patterns could be used to prospectively select appropriate motion compensation during image acquisition on a per-patient basis. This study was undertaken to develop a novel statistical model that predicts quantitative changes in PET/CT imaging due to respiratory gating. Free-breathing static FDG-PET images without gating and respiratory-gated FDG-PET images were collected from 22 lung and liver cancer patients on a PET/CT scanner. PET imaging quality was quantified with peak standardized uptake value (SUV(peak)) over lesions of interest. Relative differences in SUV(peak) between static and gated PET images were calculated to indicate quantitative imaging changes due to gating. A comprehensive multidimensional extraction of the morphological and statistical characteristics of respiratory patterns was conducted, resulting in 16 features that characterize representative patterns of a single respiratory trace. The six most informative features were subsequently extracted using a stepwise feature selection approach. The multiple-regression model was trained and tested based on a leave-one-subject-out cross-validation. The predicted quantitative improvements in PET imaging achieved an accuracy higher than 90% using a criterion with a dynamic error-tolerance range for SUV(peak) values. The results of this study suggest that our prediction framework could be applied to determine which patients would likely benefit from respiratory motion compensation when clinicians quantitatively assess PET/CT for therapy target definition and response assessment.

Respiratory trace feature analysis for the prediction of respiratory-gated PET quantification

NASA Astrophysics Data System (ADS)

Wang, Shouyi; Bowen, Stephen R.; Chaovalitwongse, W. Art; Sandison, George A.; Grabowski, Thomas J.; Kinahan, Paul E.

2014-02-01

The benefits of respiratory gating in quantitative PET/CT vary tremendously between individual patients. Respiratory pattern is among many patient-specific characteristics that are thought to play an important role in gating-induced imaging improvements. However, the quantitative relationship between patient-specific characteristics of respiratory pattern and improvements in quantitative accuracy from respiratory-gated PET/CT has not been well established. If such a relationship could be estimated, then patient-specific respiratory patterns could be used to prospectively select appropriate motion compensation during image acquisition on a per-patient basis. This study was undertaken to develop a novel statistical model that predicts quantitative changes in PET/CT imaging due to respiratory gating. Free-breathing static FDG-PET images without gating and respiratory-gated FDG-PET images were collected from 22 lung and liver cancer patients on a PET/CT scanner. PET imaging quality was quantified with peak standardized uptake value (SUVpeak) over lesions of interest. Relative differences in SUVpeak between static and gated PET images were calculated to indicate quantitative imaging changes due to gating. A comprehensive multidimensional extraction of the morphological and statistical characteristics of respiratory patterns was conducted, resulting in 16 features that characterize representative patterns of a single respiratory trace. The six most informative features were subsequently extracted using a stepwise feature selection approach. The multiple-regression model was trained and tested based on a leave-one-subject-out cross-validation. The predicted quantitative improvements in PET imaging achieved an accuracy higher than 90% using a criterion with a dynamic error-tolerance range for SUVpeak values. The results of this study suggest that our prediction framework could be applied to determine which patients would likely benefit from respiratory motion compensation when clinicians quantitatively assess PET/CT for therapy target definition and response assessment.

A novel concentration and viability detection method for Brettanomyces using the Cellometer image cytometry.

PubMed

Martyniak, Brian; Bolton, Jason; Kuksin, Dmitry; Shahin, Suzanne M; Chan, Leo Li-Ying

2017-01-01

Brettanomyces spp. can present unique cell morphologies comprised of excessive pseudohyphae and budding, leading to difficulties in enumerating cells. The current cell counting methods include manual counting of methylene blue-stained yeasts or measuring optical densities using a spectrophotometer. However, manual counting can be time-consuming and has high operator-dependent variations due to subjectivity. Optical density measurement can also introduce uncertainties where instead of individual cells counted, an average of a cell population is measured. In contrast, by utilizing the fluorescence capability of an image cytometer to detect acridine orange and propidium iodide viability dyes, individual cell nuclei can be counted directly in the pseudohyphae chains, which can improve the accuracy and efficiency of cell counting, as well as eliminating the subjectivity from manual counting. In this work, two experiments were performed to demonstrate the capability of Cellometer image cytometer to monitor Brettanomyces concentrations, viabilities, and budding/pseudohyphae percentages. First, a yeast propagation experiment was conducted to optimize software counting parameters for monitoring the growth of Brettanomyces clausenii, Brettanomyces bruxellensis, and Brettanomyces lambicus, which showed increasing cell concentrations, and varying pseudohyphae percentages. The pseudohyphae formed during propagation were counted either as multiple nuclei or a single multi-nuclei organism, where the results of counting the yeast as a single multi-nuclei organism were directly compared to manual counting. Second, a yeast fermentation experiment was conducted to demonstrate that the proposed image cytometric analysis method can monitor the growth pattern of B. lambicus and B. clausenii during beer fermentation. The results from both experiments displayed different growth patterns, viability, and budding/pseudohyphae percentages for each Brettanomyces species. The proposed Cellometer image cytometry method can improve efficiency and eliminate operator-dependent variations of cell counting compared with the traditional methods, which can potentially improve the quality of beverage products employing Brettanomyces yeasts.

Quantitative comparison of OSEM and penalized likelihood image reconstruction using relative difference penalties for clinical PET

NASA Astrophysics Data System (ADS)

Ahn, Sangtae; Ross, Steven G.; Asma, Evren; Miao, Jun; Jin, Xiao; Cheng, Lishui; Wollenweber, Scott D.; Manjeshwar, Ravindra M.

2015-08-01

Ordered subset expectation maximization (OSEM) is the most widely used algorithm for clinical PET image reconstruction. OSEM is usually stopped early and post-filtered to control image noise and does not necessarily achieve optimal quantitation accuracy. As an alternative to OSEM, we have recently implemented a penalized likelihood (PL) image reconstruction algorithm for clinical PET using the relative difference penalty with the aim of improving quantitation accuracy without compromising visual image quality. Preliminary clinical studies have demonstrated visual image quality including lesion conspicuity in images reconstructed by the PL algorithm is better than or at least as good as that in OSEM images. In this paper we evaluate lesion quantitation accuracy of the PL algorithm with the relative difference penalty compared to OSEM by using various data sets including phantom data acquired with an anthropomorphic torso phantom, an extended oval phantom and the NEMA image quality phantom; clinical data; and hybrid clinical data generated by adding simulated lesion data to clinical data. We focus on mean standardized uptake values and compare them for PL and OSEM using both time-of-flight (TOF) and non-TOF data. The results demonstrate improvements of PL in lesion quantitation accuracy compared to OSEM with a particular improvement in cold background regions such as lungs.

Informatics methods to enable sharing of quantitative imaging research data.

PubMed

Levy, Mia A; Freymann, John B; Kirby, Justin S; Fedorov, Andriy; Fennessy, Fiona M; Eschrich, Steven A; Berglund, Anders E; Fenstermacher, David A; Tan, Yongqiang; Guo, Xiaotao; Casavant, Thomas L; Brown, Bartley J; Braun, Terry A; Dekker, Andre; Roelofs, Erik; Mountz, James M; Boada, Fernando; Laymon, Charles; Oborski, Matt; Rubin, Daniel L

2012-11-01

The National Cancer Institute Quantitative Research Network (QIN) is a collaborative research network whose goal is to share data, algorithms and research tools to accelerate quantitative imaging research. A challenge is the variability in tools and analysis platforms used in quantitative imaging. Our goal was to understand the extent of this variation and to develop an approach to enable sharing data and to promote reuse of quantitative imaging data in the community. We performed a survey of the current tools in use by the QIN member sites for representation and storage of their QIN research data including images, image meta-data and clinical data. We identified existing systems and standards for data sharing and their gaps for the QIN use case. We then proposed a system architecture to enable data sharing and collaborative experimentation within the QIN. There are a variety of tools currently used by each QIN institution. We developed a general information system architecture to support the QIN goals. We also describe the remaining architecture gaps we are developing to enable members to share research images and image meta-data across the network. As a research network, the QIN will stimulate quantitative imaging research by pooling data, algorithms and research tools. However, there are gaps in current functional requirements that will need to be met by future informatics development. Special attention must be given to the technical requirements needed to translate these methods into the clinical research workflow to enable validation and qualification of these novel imaging biomarkers. Copyright © 2012 Elsevier Inc. All rights reserved.

Calibration of Wide-Field Deconvolution Microscopy for Quantitative Fluorescence Imaging

PubMed Central

Lee, Ji-Sook; Wee, Tse-Luen (Erika); Brown, Claire M.

2014-01-01

Deconvolution enhances contrast in fluorescence microscopy images, especially in low-contrast, high-background wide-field microscope images, improving characterization of features within the sample. Deconvolution can also be combined with other imaging modalities, such as confocal microscopy, and most software programs seek to improve resolution as well as contrast. Quantitative image analyses require instrument calibration and with deconvolution, necessitate that this process itself preserves the relative quantitative relationships between fluorescence intensities. To ensure that the quantitative nature of the data remains unaltered, deconvolution algorithms need to be tested thoroughly. This study investigated whether the deconvolution algorithms in AutoQuant X3 preserve relative quantitative intensity data. InSpeck Green calibration microspheres were prepared for imaging, z-stacks were collected using a wide-field microscope, and the images were deconvolved using the iterative deconvolution algorithms with default settings. Afterwards, the mean intensities and volumes of microspheres in the original and the deconvolved images were measured. Deconvolved data sets showed higher average microsphere intensities and smaller volumes than the original wide-field data sets. In original and deconvolved data sets, intensity means showed linear relationships with the relative microsphere intensities given by the manufacturer. Importantly, upon normalization, the trend lines were found to have similar slopes. In original and deconvolved images, the volumes of the microspheres were quite uniform for all relative microsphere intensities. We were able to show that AutoQuant X3 deconvolution software data are quantitative. In general, the protocol presented can be used to calibrate any fluorescence microscope or image processing and analysis procedure. PMID:24688321

Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes

PubMed Central

Telford, William G.; Shcherbakova, Daria M.; Buschke, David; Hawley, Teresa S.; Verkhusha, Vladislav V.

2015-01-01

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo. PMID:25811854

Multiparametric flow cytometry using near-infrared fluorescent proteins engineered from bacterial phytochromes.

PubMed

Telford, William G; Shcherbakova, Daria M; Buschke, David; Hawley, Teresa S; Verkhusha, Vladislav V

2015-01-01

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo.

Generation and characterization of monoclonal antibodies against Giardia muris trophozoites.

PubMed Central

Heyworth, M F; Ho, K E; Pappo, J

1989-01-01

Mouse monoclonal antibodies (mAb) were produced against Giardia muris trophozoite surface antigens. To generate B-cell hybridomas, P3/NS1/1-Ag4-1 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized parenterally with G. muris trophozoites. Hybridoma culture supernatants were screened for mAb by flow cytometry of G. muris trophozoites incubated with culture supernatant followed by fluorescein-conjugated anti-mouse IgG and IgM. Flow cytometry showed three types of trophozoite staining by mAb: (i) bright staining of greater than 90% of trophozoites, with aggregation of the organisms; (ii) bright staining of approximately 90% of trophozoites, with little or no aggregation; (iii) dull staining of approximately 20% of trophozoites, without aggregation. Western blotting of mAb on G. muris trophozoite antigens separated by polyacrylamide gel electrophoresis showed that a mAb exhibiting the third of these flow cytometry staining patterns recognized trophozoite antigens of MW approximately 31,000 and 35,000. Immunoprecipitation studies indicated that the same mAb specifically precipitated two 125I-labelled trophozoite surface antigens of MW approximately 30,000. Monoclonal antibodies generated in this study may facilitate the purification and biochemical characterization of trophozoite antigens that are targets for protective intestinal antibody in G. muris-infected mice. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:2592009

Quantitative analysis of single-molecule superresolution images

PubMed Central

Coltharp, Carla; Yang, Xinxing; Xiao, Jie

2014-01-01

This review highlights the quantitative capabilities of single-molecule localization-based superresolution imaging methods. In addition to revealing fine structural details, the molecule coordinate lists generated by these methods provide the critical ability to quantify the number, clustering, and colocalization of molecules with 10 – 50 nm resolution. Here we describe typical workflows and precautions for quantitative analysis of single-molecule superresolution images. These guidelines include potential pitfalls and essential control experiments, allowing critical assessment and interpretation of superresolution images. PMID:25179006

ggCyto: Next Generation Open-Source Visualization Software for Cytometry.

PubMed

Van, Phu; Jiang, Wenxin; Gottardo, Raphael; Finak, Greg

2018-06-01

Open source software for computational cytometry has gained in popularity over the past few years. Efforts such as FlowCAP, the Lyoplate and Euroflow projects have highlighted the importance of efforts to standardize both experimental and computational aspects of cytometry data analysis. The R/BioConductor platform hosts the largest collection of open source cytometry software covering all aspects of data analysis and providing infrastructure to represent and analyze cytometry data with all relevant experimental, gating, and cell population annotations enabling fully reproducible data analysis. Data visualization frameworks to support this infrastructure have lagged behind. ggCyto is a new open-source BioConductor software package for cytometry data visualization built on ggplot2 that enables ggplot-like functionality with the core BioConductor flow cytometry data structures. Amongst its features are the ability to transform data and axes on-the-fly using cytometry-specific transformations, plot faceting by experimental meta-data variables, and partial matching of channel, marker and cell populations names to the contents of the BioConductor cytometry data structures. We demonstrate the salient features of the package using publicly available cytometry data with complete reproducible examples in a supplementary material vignette. https://bioconductor.org/packages/devel/bioc/html/ggcyto.html. gfinak@fredhutch.org. Supplementary data are available at Bioinformatics online and at http://rglab.org/ggcyto/.

Digital Imaging

NASA Technical Reports Server (NTRS)

1986-01-01

Digital Imaging is the computer processed numerical representation of physical images. Enhancement of images results in easier interpretation. Quantitative digital image analysis by Perceptive Scientific Instruments, locates objects within an image and measures them to extract quantitative information. Applications are CAT scanners, radiography, microscopy in medicine as well as various industrial and manufacturing uses. The PSICOM 327 performs all digital image analysis functions. It is based on Jet Propulsion Laboratory technology, is accurate and cost efficient.

Quantitative magnetic resonance imaging phantoms: A review and the need for a system phantom.

PubMed

Keenan, Kathryn E; Ainslie, Maureen; Barker, Alex J; Boss, Michael A; Cecil, Kim M; Charles, Cecil; Chenevert, Thomas L; Clarke, Larry; Evelhoch, Jeffrey L; Finn, Paul; Gembris, Daniel; Gunter, Jeffrey L; Hill, Derek L G; Jack, Clifford R; Jackson, Edward F; Liu, Guoying; Russek, Stephen E; Sharma, Samir D; Steckner, Michael; Stupic, Karl F; Trzasko, Joshua D; Yuan, Chun; Zheng, Jie

2018-01-01

The MRI community is using quantitative mapping techniques to complement qualitative imaging. For quantitative imaging to reach its full potential, it is necessary to analyze measurements across systems and longitudinally. Clinical use of quantitative imaging can be facilitated through adoption and use of a standard system phantom, a calibration/standard reference object, to assess the performance of an MRI machine. The International Society of Magnetic Resonance in Medicine AdHoc Committee on Standards for Quantitative Magnetic Resonance was established in February 2007 to facilitate the expansion of MRI as a mainstream modality for multi-institutional measurements, including, among other things, multicenter trials. The goal of the Standards for Quantitative Magnetic Resonance committee was to provide a framework to ensure that quantitative measures derived from MR data are comparable over time, between subjects, between sites, and between vendors. This paper, written by members of the Standards for Quantitative Magnetic Resonance committee, reviews standardization attempts and then details the need, requirements, and implementation plan for a standard system phantom for quantitative MRI. In addition, application-specific phantoms and implementation of quantitative MRI are reviewed. Magn Reson Med 79:48-61, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

High throughput image cytometry micronucleus assay to investigate the presence or absence of mutagenic effects of cold physical plasma.

PubMed

Bekeschus, Sander; Schmidt, Anke; Kramer, Axel; Metelmann, Hans-Robert; Adler, Frank; von Woedtke, Thomas; Niessner, Felix; Weltmann, Klaus-Dieter; Wende, Kristian

2018-05-01

Promising cold physical plasma sources have been developed in the field of plasma medicine. An important prerequisite to their clinical use is lack of genotoxic effects in cells. During optimization of one or even different plasma sources for a specific application, large numbers of samples need to be analyzed. There are soft and easy-to-assess markers for genotoxic stress such as phosphorylation of histone H2AX (γH2AX) but only few tests are accredited by the OECD with regard to mutagenicity detection. The micronucleus (MN) assay is among them but often requires manual counting of many thousands of cells per sample under the microscope. A high-throughput MN assay is presented using image flow cytometry and image analysis software. A human lymphocyte cell line was treated with plasma generated with ten different feed gas conditions corresponding to distinct reactive species patterns that were investigated for their genotoxic potential. Several millions of cells were automatically analyzed by a MN quantification strategy outlined in detail in this work. Our data demonstrates the absence of newly formed MN in any feed gas condition using the atmospheric pressure plasma jet kINPen. As positive control, ionizing radiation gave a significant 5-fold increase in micronucleus frequency. Thus, this assay is suitable to assess the genotoxic potential in large sample sets of cells exposed chemical or physical agents including plasmas in an efficient, reliable, and semiautomated manner. Environ. Mol. Mutagen. 59:268-277, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Comparison and optimization of machine learning methods for automated classification of circulating tumor cells.

PubMed

Lannin, Timothy B; Thege, Fredrik I; Kirby, Brian J

2016-10-01

Advances in rare cell capture technology have made possible the interrogation of circulating tumor cells (CTCs) captured from whole patient blood. However, locating captured cells in the device by manual counting bottlenecks data processing by being tedious (hours per sample) and compromises the results by being inconsistent and prone to user bias. Some recent work has been done to automate the cell location and classification process to address these problems, employing image processing and machine learning (ML) algorithms to locate and classify cells in fluorescent microscope images. However, the type of machine learning method used is a part of the design space that has not been thoroughly explored. Thus, we have trained four ML algorithms on three different datasets. The trained ML algorithms locate and classify thousands of possible cells in a few minutes rather than a few hours, representing an order of magnitude increase in processing speed. Furthermore, some algorithms have a significantly (P < 0.05) higher area under the receiver operating characteristic curve than do other algorithms. Additionally, significant (P < 0.05) losses to performance occur when training on cell lines and testing on CTCs (and vice versa), indicating the need to train on a system that is representative of future unlabeled data. Optimal algorithm selection depends on the peculiarities of the individual dataset, indicating the need of a careful comparison and optimization of algorithms for individual image classification tasks. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Automated sample area definition for high-throughput microscopy.

PubMed

Zeder, M; Ellrott, A; Amann, R

2011-04-01

High-throughput screening platforms based on epifluorescence microscopy are powerful tools in a variety of scientific fields. Although some applications are based on imaging geometrically defined samples such as microtiter plates, multiwell slides, or spotted gene arrays, others need to cope with inhomogeneously located samples on glass slides. The analysis of microbial communities in aquatic systems by sample filtration on membrane filters followed by multiple fluorescent staining, or the investigation of tissue sections are examples. Therefore, we developed a strategy for flexible and fast definition of sample locations by the acquisition of whole slide overview images and automated sample recognition by image analysis. Our approach was tested on different microscopes and the computer programs are freely available (http://www.technobiology.ch). Copyright © 2011 International Society for Advancement of Cytometry.

Quantitative, spectrally-resolved intraoperative fluorescence imaging

PubMed Central

Valdés, Pablo A.; Leblond, Frederic; Jacobs, Valerie L.; Wilson, Brian C.; Paulsen, Keith D.; Roberts, David W.

2012-01-01

Intraoperative visual fluorescence imaging (vFI) has emerged as a promising aid to surgical guidance, but does not fully exploit the potential of the fluorescent agents that are currently available. Here, we introduce a quantitative fluorescence imaging (qFI) approach that converts spectrally-resolved data into images of absolute fluorophore concentration pixel-by-pixel across the surgical field of view (FOV). The resulting estimates are linear, accurate, and precise relative to true values, and spectral decomposition of multiple fluorophores is also achieved. Experiments with protoporphyrin IX in a glioma rodent model demonstrate in vivo quantitative and spectrally-resolved fluorescence imaging of infiltrating tumor margins for the first time. Moreover, we present images from human surgery which detect residual tumor not evident with state-of-the-art vFI. The wide-field qFI technique has broad implications for intraoperative surgical guidance because it provides near real-time quantitative assessment of multiple fluorescent biomarkers across the operative field. PMID:23152935

A Novel Quantitative Kinase Assay Using Bacterial Surface Display and Flow Cytometry

PubMed Central

Henriques, Sónia Troeira; Thorstholm, Louise; Huang, Yen-Hua; Getz, Jennifer A.; Daugherty, Patrick S.; Craik, David J.

2013-01-01

The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase, an oncogenic target and a model protein for cancer studies, we describe a novel cost-effective high-throughput screening kinase assay. In this approach, named the BacKin assay, substrates displayed on a Bacterial cell surface are incubated with Kinase and their phosphorylation is examined and quantified by flow cytometry. This approach has several advantages over existing approaches, as using bacteria (i.e. Escherichia coli) to display peptide substrates provides a self renewing solid support that does not require laborious chemical strategies. Here we show that the BacKin approach can be used for kinetic and mechanistic studies, as well as a platform to characterize and identify small-molecule or peptide-based kinase inhibitors with potential applications in drug development. PMID:24260399

A critical review of published methods for analysis of red cell antigen-antibody reactions by flow cytometry, and approaches for resolving problems with red cell agglutination.

PubMed

Arndt, Patricia A; Garratty, George

2010-07-01

Flow cytometry operators often apply familiar white blood cell (WBC) methods when studying red blood cell (RBC) antigens and antibodies. Some WBC methods are not appropriate for RBCs, as the analysis of RBCs requires special considerations, for example, avoidance of agglutination. One hundred seventy-six published articles from 88 groups studying RBC interactions were reviewed. Three fourths of groups used at least one unnecessary WBC procedure for RBCs, and about one fourth did not use any method to prevent/disperse RBC agglutination. Flow cytometric studies were performed to determine the effect of RBC agglutination on results and compare different methods of preventing and/or dispersing agglutination. The presence of RBC agglutinates have been shown to be affected by the type of pipette tip used for mixing RBC suspensions, the number of antigen sites/RBC, the type and concentration of primary antibody, and the type of secondary antibody. For quantitation methods, for example, fetal maternal hemorrhage, the presence of agglutinates have been shown to adversely affect results (fewer fetal D+ RBCs detected). Copyright 2010 Elsevier Inc. All rights reserved.

DNA-based probes for flow cytometry analysis of endocytosis and recycling.

PubMed

Dumont, Claire; Czuba, Ewa; Chen, Moore; Villadangos, Jose A; Johnston, Angus P R; Mintern, Justine D

2017-04-01

The internalization of proteins plays a key role in cell development, cell signaling and immunity. We have previously developed a specific hybridization internalization probe (SHIP) to quantitate the internalization of proteins and particles into cells. Herein, we extend the utility of SHIP to examine both the endocytosis and recycling of surface receptors using flow cytometry. SHIP was used to monitor endocytosis of membrane-bound transferrin receptor (TFR) and its soluble ligand transferrin (TF). SHIP enabled measurements of the proportion of surface molecules internalized, the internalization kinetics and the proportion and rate of internalized molecules that recycle to the cell surface with time. Using this method, we have demonstrated the internalization and recycling of holo-TF and an antibody against the TFR behave differently. This assay therefore highlights the implications of receptor internalization and recycling, where the internalization of the receptor-antibody complex behaves differently to the receptor-ligand complex. In addition, we observe distinct internalization patterns for these molecules expressed by different subpopulations of primary cells. SHIP provides a convenient and high throughput technique for analysis of trafficking parameters for both cell surface receptors and their ligands. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A model for cytoplasmic rheology consistent with magnetic twisting cytometry.

PubMed

Butler, J P; Kelly, S M

1998-01-01

Magnetic twisting cytometry is gaining wide applicability as a tool for the investigation of the rheological properties of cells and the mechanical properties of receptor-cytoskeletal interactions. Current technology involves the application and release of magnetically induced torques on small magnetic particles bound to or inside cells, with measurements of the resulting angular rotation of the particles. The properties of purely elastic or purely viscous materials can be determined by the angular strain and strain rate, respectively. However, the cytoskeleton and its linkage to cell surface receptors display elastic, viscous, and even plastic deformation, and the simultaneous characterization of these properties using only elastic or viscous models is internally inconsistent. Data interpretation is complicated by the fact that in current technology, the applied torques are not constant in time, but decrease as the particles rotate. This paper describes an internally consistent model consisting of a parallel viscoelastic element in series with a parallel viscoelastic element, and one approach to quantitative parameter evaluation. The unified model reproduces all essential features seen in data obtained from a wide variety of cell populations, and contains the pure elastic, viscoelastic, and viscous cases as subsets.

Spot counting on fluorescence in situ hybridization in suspension images using Gaussian mixture model

NASA Astrophysics Data System (ADS)

Liu, Sijia; Sa, Ruhan; Maguire, Orla; Minderman, Hans; Chaudhary, Vipin

2015-03-01

Cytogenetic abnormalities are important diagnostic and prognostic criteria for acute myeloid leukemia (AML). A flow cytometry-based imaging approach for FISH in suspension (FISH-IS) was established that enables the automated analysis of several log-magnitude higher number of cells compared to the microscopy-based approaches. The rotational positioning can occur leading to discordance between spot count. As a solution of counting error from overlapping spots, in this study, a Gaussian Mixture Model based classification method is proposed. The Akaike information criterion (AIC) and Bayesian information criterion (BIC) of GMM are used as global image features of this classification method. Via Random Forest classifier, the result shows that the proposed method is able to detect closely overlapping spots which cannot be separated by existing image segmentation based spot detection methods. The experiment results show that by the proposed method we can obtain a significant improvement in spot counting accuracy.

Flow cytometry and real-time quantitative PCR as tools for assessing plasmid persistence.

PubMed

Loftie-Eaton, Wesley; Tucker, Allison; Norton, Ann; Top, Eva M

2014-09-01

The maintenance of a plasmid in the absence of selection for plasmid-borne genes is not guaranteed. However, plasmid persistence can evolve under selective conditions. Studying the molecular mechanisms behind the evolution of plasmid persistence is key to understanding how plasmids are maintained under nonselective conditions. Given the current crisis of rapid antibiotic resistance spread by multidrug resistance plasmids, this insight is of high medical relevance. The conventional method for monitoring plasmid persistence (i.e., the fraction of plasmid-containing cells in a population over time) is based on cultivation and involves differentiating colonies of plasmid-containing and plasmid-free cells on agar plates. However, this technique is time-consuming and does not easily lend itself to high-throughput applications. Here, we present flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative tools for monitoring plasmid persistence. For this, we measured the persistence of a model plasmid, pB10::gfp, in three Pseudomonas hosts and in known mixtures of plasmid-containing and -free cells. We also compared three performance criteria: dynamic range, resolution, and variance. Although not without exceptions, both techniques generated estimates of overall plasmid loss rates that were rather similar to those generated by the conventional plate count (PC) method. They also were able to resolve differences in loss rates between artificial plasmid persistence assays. Finally, we briefly discuss the advantages and disadvantages for each technique and conclude that, overall, both FCM and real-time qPCR are suitable alternatives to cultivation-based methods for routine measurement of plasmid persistence, thereby opening avenues for high-throughput analyses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

3D Slicer as an Image Computing Platform for the Quantitative Imaging Network

PubMed Central

Fedorov, Andriy; Beichel, Reinhard; Kalpathy-Cramer, Jayashree; Finet, Julien; Fillion-Robin, Jean-Christophe; Pujol, Sonia; Bauer, Christian; Jennings, Dominique; Fennessy, Fiona; Sonka, Milan; Buatti, John; Aylward, Stephen; Miller, James V.; Pieper, Steve; Kikinis, Ron

2012-01-01

Quantitative analysis has tremendous but mostly unrealized potential in healthcare to support objective and accurate interpretation of the clinical imaging. In 2008, the National Cancer Institute began building the Quantitative Imaging Network (QIN) initiative with the goal of advancing quantitative imaging in the context of personalized therapy and evaluation of treatment response. Computerized analysis is an important component contributing to reproducibility and efficiency of the quantitative imaging techniques. The success of quantitative imaging is contingent on robust analysis methods and software tools to bring these methods from bench to bedside. 3D Slicer is a free open source software application for medical image computing. As a clinical research tool, 3D Slicer is similar to a radiology workstation that supports versatile visualizations but also provides advanced functionality such as automated segmentation and registration for a variety of application domains. Unlike a typical radiology workstation, 3D Slicer is free and is not tied to specific hardware. As a programming platform, 3D Slicer facilitates translation and evaluation of the new quantitative methods by allowing the biomedical researcher to focus on the implementation of the algorithm, and providing abstractions for the common tasks of data communication, visualization and user interface development. Compared to other tools that provide aspects of this functionality, 3D Slicer is fully open source and can be readily extended and redistributed. In addition, 3D Slicer is designed to facilitate the development of new functionality in the form of 3D Slicer extensions. In this paper, we present an overview of 3D Slicer as a platform for prototyping, development and evaluation of image analysis tools for clinical research applications. To illustrate the utility of the platform in the scope of QIN, we discuss several use cases of 3D Slicer by the existing QIN teams, and we elaborate on the future directions that can further facilitate development and validation of imaging biomarkers using 3D Slicer. PMID:22770690

Biomarkers and Surrogate Endpoints in Uveitis: The Impact of Quantitative Imaging.

PubMed

Denniston, Alastair K; Keane, Pearse A; Srivastava, Sunil K

2017-05-01

Uveitis is a major cause of sight loss across the world. The reliable assessment of intraocular inflammation in uveitis ('disease activity') is essential in order to score disease severity and response to treatment. In this review, we describe how 'quantitative imaging', the approach of using automated analysis and measurement algorithms across both standard and emerging imaging modalities, can develop objective instrument-based measures of disease activity. This is a narrative review based on searches of the current world literature using terms related to quantitative imaging techniques in uveitis, supplemented by clinical trial registry data, and expert knowledge of surrogate endpoints and outcome measures in ophthalmology. Current measures of disease activity are largely based on subjective clinical estimation, and are relatively insensitive, with poor discrimination and reliability. The development of quantitative imaging in uveitis is most established in the use of optical coherence tomographic (OCT) measurement of central macular thickness (CMT) to measure severity of macular edema (ME). The transformative effect of CMT in clinical assessment of patients with ME provides a paradigm for the development and impact of other forms of quantitative imaging. Quantitative imaging approaches are now being developed and validated for other key inflammatory parameters such as anterior chamber cells, vitreous haze, retinovascular leakage, and chorioretinal infiltrates. As new forms of quantitative imaging in uveitis are proposed, the uveitis community will need to evaluate these tools against the current subjective clinical estimates and reach a new consensus for how disease activity in uveitis should be measured. The development, validation, and adoption of sensitive and discriminatory measures of disease activity is an unmet need that has the potential to transform both drug development and routine clinical care for the patient with uveitis.

Quantitative SIMS Imaging of Agar-Based Microbial Communities.

PubMed

Dunham, Sage J B; Ellis, Joseph F; Baig, Nameera F; Morales-Soto, Nydia; Cao, Tianyuan; Shrout, Joshua D; Bohn, Paul W; Sweedler, Jonathan V

2018-05-01

After several decades of widespread use for mapping elemental ions and small molecular fragments in surface science, secondary ion mass spectrometry (SIMS) has emerged as a powerful analytical tool for molecular imaging in biology. Biomolecular SIMS imaging has primarily been used as a qualitative technique; although the distribution of a single analyte can be accurately determined, it is difficult to map the absolute quantity of a compound or even to compare the relative abundance of one molecular species to that of another. We describe a method for quantitative SIMS imaging of small molecules in agar-based microbial communities. The microbes are cultivated on a thin film of agar, dried under nitrogen, and imaged directly with SIMS. By use of optical microscopy, we show that the area of the agar is reduced by 26 ± 2% (standard deviation) during dehydration, but the overall biofilm morphology and analyte distribution are largely retained. We detail a quantitative imaging methodology, in which the ion intensity of each analyte is (1) normalized to an external quadratic regression curve, (2) corrected for isomeric interference, and (3) filtered for sample-specific noise and lower and upper limits of quantitation. The end result is a two-dimensional surface density image for each analyte. The sample preparation and quantitation methods are validated by quantitatively imaging four alkyl-quinolone and alkyl-quinoline N-oxide signaling molecules (including Pseudomonas quinolone signal) in Pseudomonas aeruginosa colony biofilms. We show that the relative surface densities of the target biomolecules are substantially different from values inferred through direct intensity comparison and that the developed methodologies can be used to quantitatively compare as many ions as there are available standards.

Quantitative analysis of γ-oryzanol content in cold pressed rice bran oil by TLC-image analysis method.

PubMed

Sakunpak, Apirak; Suksaeree, Jirapornchai; Monton, Chaowalit; Pathompak, Pathamaporn; Kraisintu, Krisana

2014-02-01

To develop and validate an image analysis method for quantitative analysis of γ-oryzanol in cold pressed rice bran oil. TLC-densitometric and TLC-image analysis methods were developed, validated, and used for quantitative analysis of γ-oryzanol in cold pressed rice bran oil. The results obtained by these two different quantification methods were compared by paired t-test. Both assays provided good linearity, accuracy, reproducibility and selectivity for determination of γ-oryzanol. The TLC-densitometric and TLC-image analysis methods provided a similar reproducibility, accuracy and selectivity for the quantitative determination of γ-oryzanol in cold pressed rice bran oil. A statistical comparison of the quantitative determinations of γ-oryzanol in samples did not show any statistically significant difference between TLC-densitometric and TLC-image analysis methods. As both methods were found to be equal, they therefore can be used for the determination of γ-oryzanol in cold pressed rice bran oil.

Quantitative analysis of γ-oryzanol content in cold pressed rice bran oil by TLC-image analysis method

PubMed Central

Sakunpak, Apirak; Suksaeree, Jirapornchai; Monton, Chaowalit; Pathompak, Pathamaporn; Kraisintu, Krisana

2014-01-01

Objective To develop and validate an image analysis method for quantitative analysis of γ-oryzanol in cold pressed rice bran oil. Methods TLC-densitometric and TLC-image analysis methods were developed, validated, and used for quantitative analysis of γ-oryzanol in cold pressed rice bran oil. The results obtained by these two different quantification methods were compared by paired t-test. Results Both assays provided good linearity, accuracy, reproducibility and selectivity for determination of γ-oryzanol. Conclusions The TLC-densitometric and TLC-image analysis methods provided a similar reproducibility, accuracy and selectivity for the quantitative determination of γ-oryzanol in cold pressed rice bran oil. A statistical comparison of the quantitative determinations of γ-oryzanol in samples did not show any statistically significant difference between TLC-densitometric and TLC-image analysis methods. As both methods were found to be equal, they therefore can be used for the determination of γ-oryzanol in cold pressed rice bran oil. PMID:25182282

Altered immunophenotypic features of peripheral blood platelets in myelodysplastic syndromes

PubMed Central

Sandes, Alex F.; Yamamoto, Mihoko; Matarraz, Sergio; Chauffaille, Maria de Lourdes L.F.; Quijano, Sandra; López, Antonio; Oguro, Tsutomu; Kimura, Eliza Y. S.; Orfao, Alberto

2012-01-01

Background Multiparameter flow cytometric analysis of bone marrow and peripheral blood cells has proven to be of help in the diagnostic workup of myelodysplastic syndromes. However, the usefulness of flow cytometry for the detection of megakaryocytic and platelet dysplasia has not yet been investigated. The aim of this pilot study was to evaluate by flow cytometry the diagnostic and prognostic value of platelet dysplasia in myelodysplastic syndromes. Design and Methods We investigated the pattern of expression of distinct surface glycoproteins on peripheral blood platelets from a series of 44 myelodysplastic syndrome patients, 20 healthy subjects and 19 patients with platelet alterations associated to disease conditions other than myelodysplastic syndromes. Quantitative expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b and CD61 glycoproteins together with the PAC-1, CD62-P, fibrinogen and CD63 platelet activation-associated markers and platelet light scatter properties were systematically evaluated. Results Overall, flow cytometry identified multiple immunophenotypic abnormalities on platelets of myelodysplastic syndrome patients, including altered light scatter characteristics, over-and under expression of specific platelet glycoproteins and asynchronous expression of CD34; decreased expression of CD36 (n=5), CD42a (n=1) and CD61 (n=2), together with reactivity for CD34 (n=1) were only observed among myelodysplastic syndrome cases, while other alterations were also found in other platelet disorders. Based on the overall platelet alterations detected for each patient, an immunophenotypic score was built which identified a subgroup of myelodysplastic syndrome patients with a high rate of moderate to severe alterations (score>1.5; n=16) who more frequently showed thrombocytopenia, megakaryocytic dysplasia and high-risk disease, together with a shorter overall survival. Conclusions Our results show the presence of altered phenotypes by flow cytometry on platelets from around half of the myelodysplastic syndrome patients studied. If confirmed in larger series of patients, these findings may help refine the diagnostic and prognostic assessment of this group of disorders. PMID:22271903

Increased expression of brother of the regulator of imprinted sites in peripheral blood neutrophils is associated with both benign and malignant breast lesions.

PubMed

El-Sharkawy, Nahla M; Radwan, Wafaa M; Essa, Enas S; Kandeel, Eman Z; Abd El-Fattah, Eman K; Kandil, Samia H; Kamel, Azza M

2017-09-01

BORIS, a paralog of the multifunctional CCCTC-binding factor (CTCF) gene is restricted to testis and normally not present in females. It is aberrantly activated in various human cancers including cancer breast. Using immunohistochemistry, western blot and/or RT-PCR, significantly higher levels of BORIS expression were reported in the neutrophils of cancer breast patients. We hypothesized that Flow Cytometry might be a better technique for objective quantitative evaluation of BORIS in neutrophils and we wanted to investigate if BORIS would discriminate between benign and malignant breast lesions. The study included 85 females; 52 breast cancer, 13 benign breast lesions and 20 age-matched healthy controls. BORIS expression in the neutrophils was detected by Flow Cytometry. High level of BORIS was detected in all malignant (64.4 ± 16.6%) and benign cases (67 ± 12.3), mean florescent intensity ratio (MFIR) of 7.2 ± 4.1 and 7 ± 3.5, median 5.8 and 6.6%; and staining index (SI) 8.3 ± 3.9 and 8.2 ± 3.4, median 7.6 and 7.9 respectively vs.13.4 ± 11.5% MFI 1.8 ± 0.7, median1.6 and SI 2.6 ± 0.69, median 2.5 for the control. BORIS level was comparable in the malignant and benign group (P = 0.934) and significantly higher than control (P = 0.0001). There was no correlation between neutrophil BORIS expression and ER/PR status, HER-2/neu expression or tumor stage or size. Increased BORIS expression in peripheral blood neutrophils is associated with both benign and malignant breast lesions; apparently, increased proliferation of breast tissue is the determining factor. This excludes BORIS as a tumor marker but it does not jeopardize its value as a potential therapeutic target. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

Detection of Prostate Cancer: Quantitative Multiparametric MR Imaging Models Developed Using Registered Correlative Histopathology.

PubMed

Metzger, Gregory J; Kalavagunta, Chaitanya; Spilseth, Benjamin; Bolan, Patrick J; Li, Xiufeng; Hutter, Diane; Nam, Jung W; Johnson, Andrew D; Henriksen, Jonathan C; Moench, Laura; Konety, Badrinath; Warlick, Christopher A; Schmechel, Stephen C; Koopmeiners, Joseph S

2016-06-01

Purpose To develop multiparametric magnetic resonance (MR) imaging models to generate a quantitative, user-independent, voxel-wise composite biomarker score (CBS) for detection of prostate cancer by using coregistered correlative histopathologic results, and to compare performance of CBS-based detection with that of single quantitative MR imaging parameters. Materials and Methods Institutional review board approval and informed consent were obtained. Patients with a diagnosis of prostate cancer underwent multiparametric MR imaging before surgery for treatment. All MR imaging voxels in the prostate were classified as cancer or noncancer on the basis of coregistered histopathologic data. Predictive models were developed by using more than one quantitative MR imaging parameter to generate CBS maps. Model development and evaluation of quantitative MR imaging parameters and CBS were performed separately for the peripheral zone and the whole gland. Model accuracy was evaluated by using the area under the receiver operating characteristic curve (AUC), and confidence intervals were calculated with the bootstrap procedure. The improvement in classification accuracy was evaluated by comparing the AUC for the multiparametric model and the single best-performing quantitative MR imaging parameter at the individual level and in aggregate. Results Quantitative T2, apparent diffusion coefficient (ADC), volume transfer constant (K(trans)), reflux rate constant (kep), and area under the gadolinium concentration curve at 90 seconds (AUGC90) were significantly different between cancer and noncancer voxels (P < .001), with ADC showing the best accuracy (peripheral zone AUC, 0.82; whole gland AUC, 0.74). Four-parameter models demonstrated the best performance in both the peripheral zone (AUC, 0.85; P = .010 vs ADC alone) and whole gland (AUC, 0.77; P = .043 vs ADC alone). Individual-level analysis showed statistically significant improvement in AUC in 82% (23 of 28) and 71% (24 of 34) of patients with peripheral-zone and whole-gland models, respectively, compared with ADC alone. Model-based CBS maps for cancer detection showed improved visualization of cancer location and extent. Conclusion Quantitative multiparametric MR imaging models developed by using coregistered correlative histopathologic data yielded a voxel-wise CBS that outperformed single quantitative MR imaging parameters for detection of prostate cancer, especially when the models were assessed at the individual level. (©) RSNA, 2016 Online supplemental material is available for this article.

Wallace H. Coulter: decades of invention and discovery.

PubMed

Robinson, J Paul

2013-05-01

Only a few inventors can be said to have made as great an impact on mankind as Wallace Coulter. His inquisitive mind and ability to see well beyond what existed served him well for 40 years of inventing. So many of the fundamental tools that exist today in the area of hematology were derived from or driven by Coulter's inventions that he could be called the most technological innovator in the field of modern hematology. In achieving these discoveries Wallace Coulter was clearly capable of visualizing future opportunities that few others recognized. His vision was combined with an uncanny ability to translate his ideas into products. He developed a large number of tools that shaped the fields of cytometry, image analysis, and industrial materials. His understanding of the future power of computation drove him to link these technologies in a unique way. In the end, Coulter shaped the technologies that ultimately drove hematology in a new direction, one that remains on a critical pathway linking technology innovation all the way to true translational impact. It was said of Henry Ford that "[h]e has no notion that wealth has made him great, and any one who is imprest merely by his wealth bores him. In his personal contacts he likes to dodge the subject. He would prefer to talk with a machinist about machinery, or with somebody who likes birds about birds. In these contacts, he asks no deference; and if he gets it, he suspects it is mere deference to wealth, and that ends his interest."(1) The same could be said of Wallace Coulter, who, like Ford, understood the concepts of mass production and customer service. Coulter had the ability to recognize the opportunity and fulfill the need for development of a blood-cell counter that could be placed in every pathology laboratory, and in so doing transformed a field from a qualitative to a quantitative environment. Every person who has ever entered a medical lab, hospital, or doctor's office has felt the impact of Coulter's discovery. Copyright © 2013 International Society for Advancement of Cytometry.

High-throughput linear optical stretcher for mechanical characterization of blood cells.

PubMed

Roth, Kevin B; Neeves, Keith B; Squier, Jeff; Marr, David W M

2016-04-01

This study describes a linear optical stretcher as a high-throughput mechanical property cytometer. Custom, inexpensive, and scalable optics image a linear diode bar source into a microfluidic channel, where cells are hydrodynamically focused into the optical stretcher. Upon entering the stretching region, antipodal optical forces generated by the refraction of tightly focused laser light at the cell membrane deform each cell in flow. Each cell relaxes as it flows out of the trap and is compared to the stretched state to determine deformation. The deformation response of untreated red blood cells and neutrophils were compared to chemically treated cells. Statistically significant differences were observed between normal, diamide-treated, and glutaraldehyde-treated red blood cells, as well as between normal and cytochalasin D-treated neutrophils. Based on the behavior of the pure, untreated populations of red cells and neutrophils, a mixed population of these cells was tested and the discrete populations were identified by deformability. © 2015 International Society for Advancement of Cytometry. © 2015 International Society for Advancement of Cytometry.

In Vivo Cytometry of Antigen-Specific T Cells Using 19F MRI

PubMed Central

Srinivas, Mangala; Turner, Michael S.; Janjic, Jelena M.; Morel, Penelope A.; Laidlaw, David H.; Ahrens, Eric T.

2009-01-01

Noninvasive methods to image the trafficking of phenotypically defined immune cells are paramount as we attempt to understand adaptive immunity. A 19F MRI-based methodology for tracking and quantifying cells of a defined phenotype is presented. These methods were applied to a murine inflammation model using antigen-specific T cells. The T cells that were intracellularly labeled ex vivo with a perfluoropolyether (PFPE) nanoemulsion and cells were transferred to a host receiving a localized inoculation of antigen. Longitudinal 19F MRI over 21 days revealed a dynamic accumulation and clearance of T cells in the lymph node (LN) draining the antigen. The apparent T-cell numbers were calculated in the LN from the time-lapse 19F MRI data. The effect of in vivo T-cell division on the 19F MRI cell quantification accuracy was investigated using fluorescence assays. Overall, in vivo cytometry using PFPE labeling and 19F MRI is broadly applicable to studies of whole-body cell biodistribution. PMID:19585593

Human immune cell targeting of protein nanoparticles - caveospheres

NASA Astrophysics Data System (ADS)

Glass, Joshua J.; Yuen, Daniel; Rae, James; Johnston, Angus P. R.; Parton, Robert G.; Kent, Stephen J.; de Rose, Robert

2016-04-01

Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

In vivo visualisation of different modes of action of biological DMARDs inhibiting osteoclastic bone resorption.

PubMed

Matsuura, Yoshinobu; Kikuta, Junichi; Kishi, Yuika; Hasegawa, Tetsuo; Okuzaki, Daisuke; Hirano, Toru; Minoshima, Masafumi; Kikuchi, Kazuya; Kumanogoh, Atsushi; Ishii, Masaru

2018-04-28

Osteoclasts play critical roles in inflammatory bone destruction. Precursor cell migration, cell differentiation, and functional cell activation are all in play. Biological disease-modifying antirheumatic drugs (DMARDs) have been shown to significantly inhibit both bone erosion as well as synovitis, although how such agents reduce osteoclastic bone destruction in vivo has not been fully explained. Here, we used an intravital time-lapse imaging technique to directly visualise mature osteoclasts and their precursors, and explored how different biological DMARDs acted in vivo . Lipopolysaccharide (LPS) was injected into the calvarial periosteum of fluorescent reporter mice to induce inflammatory bone destruction. Time-lapse imaging was performed via intravital multiphoton microscopy 5 days after LPS injection. Biological DMARDs, including monoclonal antibodies (mAbs) against the interleukin (IL) 6 receptor (IL-6R) and tumour necrosis factor α (TNFα), or cytotoxic T-lymphocyte-associated protein 4 (CTLA4)-Ig, were intraperitoneally administered at the time of LPS injection. We determined CD80/86 expression levels in mature osteoclasts and their precursors by flow cytometry, quantitative PCR and immunohistochemistry. Of the biologicals tested, anti-IL-6R and anti-TNFα mAbs affected mature osteoclasts and switched bone-resorbing osteoclasts to non-resorbing cells. CTLA4-Ig had no action on mature osteoclasts but mobilised osteoclast precursors, eliminating their firm attachment to bone surfaces. In agreement with these results, CD80/86 (the target molecules of CTLA4-Ig) were prominently expressed only in osteoclast precursor cells, being suppressed during osteoclast maturation. Intravital imaging revealed that various biological DMARDs acted at specific therapeutic time points during osteoclastic bone destruction, with different efficacies. These results enable us to grasp the real modes of action of drugs, optimising the usage of drug regimens. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

A non-invasive in vivo imaging system to study dissemination of bioluminescent Yersinia pestis CO92 in a mouse model of pneumonic plague

PubMed Central

Sha, Jian; Rosenzweig, Jason A.; Kirtley, Michelle L.; van Lier, Christina J.; Fitts, Eric C.; Kozlova, Elena V.; Erova, Tatiana E.; Tiner, Bethany L.; Chopra, Ashok K.

2012-01-01

The gold standard in microbiology for monitoring bacterial dissemination in infected animals has always been viable plate counts. This method, despite being quantitative, requires sacrificing the infected animals. Recently, however, an alternative method of in vivo imaging of bioluminescent bacteria (IVIBB) for monitoring microbial dissemination within the host has been employed. Yersina pestis is a Gram-negative bacterium capable of causing bubonic, septicemic, and pneumonic plague. In this study, we compared the conventional counting of bacterial colony forming units (cfu) in the various infected tissues to IVIBB in monitoring Y. pestis dissemination in a mouse model of pneumonic plague. By using a transposon mutagenesis system harboring the luciferase (luc) gene, we screened approximately 4000 clones and obtained a fully virulent, luc-positive Y. pestis CO92 (Y. pestis-luc2) reporter strain in which transposition occurred within the largest pMT1 plasmid which possesses murine toxin and capsular antigen encoding genes. The aforementioned reporter strain and the wild-type CO92 exhibited similar growth curves, formed capsule based on immunofluorescence microscopy and flow cytometry, and had a similar LD50. Intranasal infection of mice with 15 LD50 of CO92-luc2 resulted in animal mortality by 72 h, and an increasing number of bioluminescent bacteria were observed in various mouse organs over a 24–72 h period when whole animals were imaged. However, following levofloxacin treatment (10 mg/kg/day) for 6 days 24 h post infection, no luminescence was observed after 72 h of infection, indicating that the tested antimicrobial killed bacteria preventing their detection in host peripheral tissues. Overall, we demonstrated that IVIBB is an effective and non-invasive way of monitoring bacterial dissemination in animals following pneumonic plague having strong correlation with cfu, and our reporter CO92-luc2 strain can be employed as a useful tool to monitor the efficacy of antimicrobial countermeasures in real time. PMID:23063826

Quantitative imaging biomarkers: Effect of sample size and bias on confidence interval coverage.

PubMed

Obuchowski, Nancy A; Bullen, Jennifer

2017-01-01

Introduction Quantitative imaging biomarkers (QIBs) are being increasingly used in medical practice and clinical trials. An essential first step in the adoption of a quantitative imaging biomarker is the characterization of its technical performance, i.e. precision and bias, through one or more performance studies. Then, given the technical performance, a confidence interval for a new patient's true biomarker value can be constructed. Estimating bias and precision can be problematic because rarely are both estimated in the same study, precision studies are usually quite small, and bias cannot be measured when there is no reference standard. Methods A Monte Carlo simulation study was conducted to assess factors affecting nominal coverage of confidence intervals for a new patient's quantitative imaging biomarker measurement and for change in the quantitative imaging biomarker over time. Factors considered include sample size for estimating bias and precision, effect of fixed and non-proportional bias, clustered data, and absence of a reference standard. Results Technical performance studies of a quantitative imaging biomarker should include at least 35 test-retest subjects to estimate precision and 65 cases to estimate bias. Confidence intervals for a new patient's quantitative imaging biomarker measurement constructed under the no-bias assumption provide nominal coverage as long as the fixed bias is <12%. For confidence intervals of the true change over time, linearity must hold and the slope of the regression of the measurements vs. true values should be between 0.95 and 1.05. The regression slope can be assessed adequately as long as fixed multiples of the measurand can be generated. Even small non-proportional bias greatly reduces confidence interval coverage. Multiple lesions in the same subject can be treated as independent when estimating precision. Conclusion Technical performance studies of quantitative imaging biomarkers require moderate sample sizes in order to provide robust estimates of bias and precision for constructing confidence intervals for new patients. Assumptions of linearity and non-proportional bias should be assessed thoroughly.

Generalized PSF modeling for optimized quantitation in PET imaging.

PubMed

Ashrafinia, Saeed; Mohy-Ud-Din, Hassan; Karakatsanis, Nicolas A; Jha, Abhinav K; Casey, Michael E; Kadrmas, Dan J; Rahmim, Arman

2017-06-21

Point-spread function (PSF) modeling offers the ability to account for resolution degrading phenomena within the PET image generation framework. PSF modeling improves resolution and enhances contrast, but at the same time significantly alters image noise properties and induces edge overshoot effect. Thus, studying the effect of PSF modeling on quantitation task performance can be very important. Frameworks explored in the past involved a dichotomy of PSF versus no-PSF modeling. By contrast, the present work focuses on quantitative performance evaluation of standard uptake value (SUV) PET images, while incorporating a wide spectrum of PSF models, including those that under- and over-estimate the true PSF, for the potential of enhanced quantitation of SUVs. The developed framework first analytically models the true PSF, considering a range of resolution degradation phenomena (including photon non-collinearity, inter-crystal penetration and scattering) as present in data acquisitions with modern commercial PET systems. In the context of oncologic liver FDG PET imaging, we generated 200 noisy datasets per image-set (with clinically realistic noise levels) using an XCAT anthropomorphic phantom with liver tumours of varying sizes. These were subsequently reconstructed using the OS-EM algorithm with varying PSF modelled kernels. We focused on quantitation of both SUV mean and SUV max , including assessment of contrast recovery coefficients, as well as noise-bias characteristics (including both image roughness and coefficient of-variability), for different tumours/iterations/PSF kernels. It was observed that overestimated PSF yielded more accurate contrast recovery for a range of tumours, and typically improved quantitative performance. For a clinically reasonable number of iterations, edge enhancement due to PSF modeling (especially due to over-estimated PSF) was in fact seen to lower SUV mean bias in small tumours. Overall, the results indicate that exactly matched PSF modeling does not offer optimized PET quantitation, and that PSF overestimation may provide enhanced SUV quantitation. Furthermore, generalized PSF modeling may provide a valuable approach for quantitative tasks such as treatment-response assessment and prognostication.

Separating the signal from the noise: Expanding flow cytometry into the sub-micron range.

EPA Science Inventory

Cytometry Part A Special Section: Separating the signal from the noise: Expanding flow cytometry into the sub-micron range. The current Cytometry Part A Special Section presents three studies that utilize cytometers to study sub-micron particles. The three studies involve the 1...

Evaluation of a web based informatics system with data mining tools for predicting outcomes with quantitative imaging features in stroke rehabilitation clinical trials

NASA Astrophysics Data System (ADS)

Wang, Ximing; Kim, Bokkyu; Park, Ji Hoon; Wang, Erik; Forsyth, Sydney; Lim, Cody; Ravi, Ragini; Karibyan, Sarkis; Sanchez, Alexander; Liu, Brent

2017-03-01

Quantitative imaging biomarkers are used widely in clinical trials for tracking and evaluation of medical interventions. Previously, we have presented a web based informatics system utilizing quantitative imaging features for predicting outcomes in stroke rehabilitation clinical trials. The system integrates imaging features extraction tools and a web-based statistical analysis tool. The tools include a generalized linear mixed model(GLMM) that can investigate potential significance and correlation based on features extracted from clinical data and quantitative biomarkers. The imaging features extraction tools allow the user to collect imaging features and the GLMM module allows the user to select clinical data and imaging features such as stroke lesion characteristics from the database as regressors and regressands. This paper discusses the application scenario and evaluation results of the system in a stroke rehabilitation clinical trial. The system was utilized to manage clinical data and extract imaging biomarkers including stroke lesion volume, location and ventricle/brain ratio. The GLMM module was validated and the efficiency of data analysis was also evaluated.

Ptychography: use of quantitative phase information for high-contrast label free time-lapse imaging of living cells

NASA Astrophysics Data System (ADS)

Suman, Rakesh; O'Toole, Peter

2014-03-01

Here we report a novel label free, high contrast and quantitative method for imaging live cells. The technique reconstructs an image from overlapping diffraction patterns using a ptychographical algorithm. The algorithm utilises both amplitude and phase data from the sample to report on quantitative changes related to the refractive index (RI) and thickness of the specimen. We report the ability of this technique to generate high contrast images, to visualise neurite elongation in neuronal cells, and to provide measure of cell proliferation.

Ultra-fast quantitative imaging using ptychographic iterative engine based digital micro-mirror device

NASA Astrophysics Data System (ADS)

Sun, Aihui; Tian, Xiaolin; Kong, Yan; Jiang, Zhilong; Liu, Fei; Xue, Liang; Wang, Shouyu; Liu, Cheng

2018-01-01

As a lensfree imaging technique, ptychographic iterative engine (PIE) method can provide both quantitative sample amplitude and phase distributions avoiding aberration. However, it requires field of view (FoV) scanning often relying on mechanical translation, which not only slows down measuring speed, but also introduces mechanical errors decreasing both resolution and accuracy in retrieved information. In order to achieve high-accurate quantitative imaging with fast speed, digital micromirror device (DMD) is adopted in PIE for large FoV scanning controlled by on/off state coding by DMD. Measurements were implemented using biological samples as well as USAF resolution target, proving high resolution in quantitative imaging using the proposed system. Considering its fast and accurate imaging capability, it is believed the DMD based PIE technique provides a potential solution for medical observation and measurements.

Congenital hypothyroidism: diagnostic scintigraphic evaluation of an organification defect

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cone, L.; Oates, E.; Vazquez, R.

1988-06-01

Quantitative Tc-99m pertechnetate thyroid imaging was performed on a hypothyroid neonate. The image revealed markedly increased trapping in an enlarged, bilobed, eutopic gland. A perchlorate washout test using quantitative imaging with I-123 confirmed an organification problem.

Super-resolved calibration-free flow cytometric characterization of platelets and cell-derived microparticles in platelet-rich plasma.

PubMed

Konokhova, Anastasiya I; Chernova, Darya N; Moskalensky, Alexander E; Strokotov, Dmitry I; Yurkin, Maxim A; Chernyshev, Andrei V; Maltsev, Valeri P

2016-02-01

Importance of microparticles (MPs), also regarded as extracellular vesicles, in many physiological processes and clinical conditions motivates one to use the most informative and precise methods for their characterization. Methods based on individual particle analysis provide statistically reliable distributions of MP population over characteristics. Although flow cytometry is one of the most powerful technologies of this type, the standard forward-versus-side-scattering plots of MPs and platelets (PLTs) overlap considerably because of similarity of their morphological characteristics. Moreover, ordinary flow cytometry is not capable of measurement of size and refractive index (RI) of MPs. In this study, we 1) employed the potential of the scanning flow cytometer (SFC) for identification and characterization of MPs from light scattering; 2) suggested the reference method to characterize MP morphology (size and RI) with high precision; and 3) determined the lowest size of a MP that can be characterized from light scattering with the SFC. We equipped the SFC with 405 and 488 nm lasers to measure the light-scattering profiles and side scattering from MPs, respectively. The developed two-stage method allowed accurate separation of PLTs and MPs in platelet-rich plasma. We used two optical models for MPs, a sphere and a bisphere, in the solution of the inverse light-scattering problem. This solution provides unprecedented precision in determination of size and RI of individual spherical MPs-median uncertainties (standard deviations) were 6 nm and 0.003, respectively. The developed method provides instrument-independent quantitative information on MPs, which can be used in studies of various factors affecting MP population. © 2015 International Society for Advancement of Cytometry.

Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.

PubMed Central

Davey, H M; Kell, D B

1996-01-01

The most fundamental questions such as whether a cell is alive, in the sense of being able to divide or to form a colony, may sometimes be very hard to answer, since even axenic microbial cultures are extremely heterogeneous. Analyses that seek to correlate such things as viability, which is a property of an individual cell, with macroscopic measurements of culture variables such as ATP content, respiratory activity, and so on, must inevitably fail. It is therefore necessary to make physiological measurements on individual cells. Flow cytometry is such a technique, which allows one to analyze cells rapidly and individually and permits the quantitative analysis of microbial heterogeneity. It therefore offers many advantages over conventional measurements for both routine and more exploratory analyses of microbial properties. While the technique has been widely applied to the study of mammalian cells, is use in microbiology has until recently been much more limited, largely because of the smaller size of microbes and the consequently smaller optical signals obtainable from them. Since these technical barriers no longer hold, flow cytometry with appropriate stains has been used for the rapid discrimination and identification of microbial cells, for the rapid assessment of viability and of the heterogeneous distributions of a wealth of other more detailed physiological properties, for the analysis of antimicrobial drug-cell interactions, and for the isolation of high-yielding strains of biotechnological interest. Flow cytometric analyses provide an abundance of multivariate data, and special methods have been devised to exploit these. Ongoing advances mean that modern flow cytometers may now be used by nonspecialists to effect a renaissance in our understanding of microbial heterogeneity. PMID:8987359

Differentiation potential of human CD133 positive hematopoietic stem cells into motor neuron- like cells, in vitro.

PubMed

Moghaddam, Sepideh Alavi; Yousefi, Behnam; Sanooghi, Davood; Faghihi, Faezeh; Hayati Roodbari, Nasim; Bana, Nikoo; Joghataei, Mohammad Taghi; Pooyan, Paria; Arjmand, Babak

2017-12-01

Spinal cord injuries and motor neuron-related disorders impact on life of many patients around the world. Since pharmacotherapy and surgical approaches were not efficient to regenerate these types of defects; stem cell therapy as a good strategy to restore the lost cells has become the focus of interest among the scientists. Umbilical cord blood CD133 + hematopoietic stem cells (UCB- CD133 + HSCs) with self- renewal property and neural lineage differentiation capacity are ethically approved cell candidate for use in regenerative medicine. In this regard the aim of this study was to quantitatively evaluate the capability of these cells to differentiate into motor neuron-like cells (MNL), in vitro. CD133 + HSCs were isolated from human UCB using MACS system. After cell characterization using flow cytometry, the cells were treated with a combination of Retinoic acid, Sonic hedgehog, Brain derived neurotrophic factor, and B27 through a 2- step procedure for two weeks. The expression of MN-specific markers was examined using qRT- PCR, flow cytometry and immunocytochemistry. By the end of the two-week differentiation protocol, CD133 + cells acquired unipolar MNL morphology with thin and long neurites. The expression of Isl-1(62.15%), AChE (41.83%), SMI-32 (21.55%) and Nestin (17.46%) was detected using flow cytometry and immunocytochemistry. The analysis of the expression of PAX6, ISL-1, ACHE, CHAT and SMI-32 revealed that MNLs present these neural markers at levels comparable with undifferentiated cells. In Conclusion Human UCB- CD133 + HSCs are remarkably potent cell candidates to transdifferentiate into motor neuron-like cells, in vitro. Copyright © 2017. Published by Elsevier B.V.

[Fiat Lux. May be no more true in cytometry! Go to mass and spectrum but still stay classic].

PubMed

Idziorek, Thierry; Cazareth, Julie; Blanc, Catherine; Jouy, Nathalie; Bourdely, Pierre; Corneau, Aurélien

2018-05-01

The last decade has been an era of accelerated technological progress for flow cytometry. New technologies have been developed such as mass cytometry in which standard fluorochromes have been replaced by lanthanide-based non-radioactive metals and by spectral cytometry that measures the complete fluorescence spectrum. In this review, we schematically describe conventional, mass and spectral cytometry and present the plus and minus of each technology. © 2018 médecine/sciences – Inserm.

In Vivo Fluorescence Imaging and Tracking of Circulating Cells and Therapeutic Nanoparticles

NASA Astrophysics Data System (ADS)

Markovic, Stacey

Noninvasive enumeration of rare circulating cells in small animals is of great importance in many areas of biomedical research, but most existing enumeration techniques involve drawing and enriching blood which is known to be problematic. Recently, small animal "in vivo flow cytometry" (IVFC) techniques have been developed, where cells flowing through small arterioles are counted continuously and noninvasively in vivo. However, higher sensitivity IVFC techniques are needed for studying low-abundance (<100/mL) circulating cells. To this end, we developed a macroscopic fluorescence imaging system and automated computer vision algorithm that allows in vivo detection, enumeration and tracking of circulating fluorescently labeled cells from multiple large blood vessels in the ear of a mouse. This technique ---"computer vision IVFC" (CV-IVFC) --- allows cell detection and enumeration at concentrations of 20 cells/mL. Performance of CV-IVFC was also characterized for low-contrast imaging scenarios, representing conditions of weak cell fluorescent labeling or high background tissue autofluorescence, and showed efficient tracking and enumeration of circulating cells with 50% sensitivity in contrast conditions degraded 2 orders of magnitude compared to in vivo testing supporting the potential utility of CV-IVFC in a range of biological models. Refinement of prior work in our lab of a separate rare-cell detection platform - "diffuse fluorescence flow cytometry" (DFFC) --- implemented a "frequency encoding" scheme by modulating two excitation lasers. Fluorescent light from both lasers can be simultaneously detected and split by frequency allowing for better discrimination of noise, sensitivity, and cell localization. The system design is described in detail and preliminary data is shown. Last, we developed a broad-field transmission fluorescence imaging system to observe nanoparticle (NP) diffusion in bulk biological tissue. Novel, implantable NP spacers allow controlled, long-term release of drugs. However, kinetics of NP (drug) diffusion over time is still poorly understood. Our imaging system allowed us to quantify diffusion of free dye and NPs of different sizes in vitro and in vivo. Subsequent analysis verified that there was continuous diffusion which could be controlled based on particle size. Continued use of this imaging system will aid optimization of NP spacers.

An introduction to mass cytometry: fundamentals and applications.

PubMed

Tanner, Scott D; Baranov, Vladimir I; Ornatsky, Olga I; Bandura, Dmitry R; George, Thaddeus C

2013-05-01

Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometry-compatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a high-level review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.

Statistical prediction of nanoparticle delivery: from culture media to cell

NASA Astrophysics Data System (ADS)

Rowan Brown, M.; Hondow, Nicole; Brydson, Rik; Rees, Paul; Brown, Andrew P.; Summers, Huw D.

2015-04-01

The application of nanoparticles (NPs) within medicine is of great interest; their innate physicochemical characteristics provide the potential to enhance current technology, diagnostics and therapeutics. Recently a number of NP-based diagnostic and therapeutic agents have been developed for treatment of various diseases, where judicious surface functionalization is exploited to increase efficacy of administered therapeutic dose. However, quantification of heterogeneity associated with absolute dose of a nanotherapeutic (NP number), how this is trafficked across biological barriers has proven difficult to achieve. The main issue being the quantitative assessment of NP number at the spatial scale of the individual NP, data which is essential for the continued growth and development of the next generation of nanotherapeutics. Recent advances in sample preparation and the imaging fidelity of transmission electron microscopy (TEM) platforms provide information at the required spatial scale, where individual NPs can be individually identified. High spatial resolution however reduces the sample frequency and as a result dynamic biological features or processes become opaque. However, the combination of TEM data with appropriate probabilistic models provide a means to extract biophysical information that imaging alone cannot. Previously, we demonstrated that limited cell sampling via TEM can be statistically coupled to large population flow cytometry measurements to quantify exact NP dose. Here we extended this concept to link TEM measurements of NP agglomerates in cell culture media to that encapsulated within vesicles in human osteosarcoma cells. By construction and validation of a data-driven transfer function, we are able to investigate the dynamic properties of NP agglomeration through endocytosis. In particular, we statistically predict how NP agglomerates may traverse a biological barrier, detailing inter-agglomerate merging events providing the basis for predictive modelling of nanopharmacology.

Spontaneous anaplasia in pilocytic astrocytoma of cerebellum.

PubMed

Lach, B; Al Shail, E; Patay, Z

2003-06-01

We report a cystic cerebellar astrocytoma with a mural nodule that contained an additional focus of astrocytoma with the histological features of anaplasia, and showed up to 48% of aneuploid and 3% S-phase cells on flow cytometry. This focus was detectable on the enhanced, as well as non-enhanced T1 and T2 images. This appears to be the first case of pilocytic astrocytoma of cerebellum with focal anaplasia detected on histological and radiological studies.

Intracellular Detection of Viral Transcription and Replication Using RNA FISH

DTIC Science & Technology

2016-05-26

sequence and how it can be rapidly performed to minimize time spent in high containment. We have adapted existing protocols for mRNA detection with...We and others have been expanded this technique to use in virus-infected tissue sections, high -throughput imaging, and for flow-cytometry based...free water. Add 1 gram of dextran sulfate and mix for 10 minutes at room temperature . Aliquot and store the hybridization buffer at 4ºC. Wash

Chip-Based Dynamic Real-Time Quantification of Drug-Induced Cytotoxicity in Human Tumor Cells

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; McGuinness, Dagmara; Faley, Shannon; Kolch, Walter; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell cytotoxicity tests are among the most common bioassays using flow cytometry and fluorescence imaging analysis. The permeability of plasma membranes to charged fluorescent probes serves, in these assays, as a marker distinguishing live from dead cells. Since it is generally assumed that probes, such as propidium iodide (PI) or 7-amino-actinomycin D (7-AAD), are themselves cytotoxic, they are currently generally used only as the end-point markers of assays for live versus dead cells. In the current study, we provide novel insights into potential applications of these classical plasma membrane integrity markers in the dynamic tracking of drug-induced cytotoxicity. We show that treatment of a number of different human tumor cell lines in cultures for up to 72 h with the PI, 7-AAD, SYTOX Green (SY-G), SYTOX Red (SYR), TO-PRO, and YO-PRO had no effect on cell viability assessed by the integrity of plasma membrane, cell cycle progression, and rate of proliferation. We subsequently explore the potential of dynamic labeling with these markers in real-time analysis, by comparing results from both conventional cytometry and microfluidic chips. Considering the simplicity of the staining protocols and their low cost combined with the potential for real-time data collection, we show how that real-time fluorescent imaging and Lab-on-a-Chip platforms have the potential to be used for automated drug screening routines. PMID:19572560

Subsurface imaging and cell refractometry using quantitative phase/ shear-force feedback microscopy

NASA Astrophysics Data System (ADS)

Edward, Kert; Farahi, Faramarz

2009-10-01

Over the last few years, several novel quantitative phase imaging techniques have been developed for the study of biological cells. However, many of these techniques are encumbered by inherent limitations including 2π phase ambiguities and diffraction limited spatial resolution. In addition, subsurface information in the phase data is not exploited. We hereby present a novel quantitative phase imaging system without 2 π ambiguities, which also allows for subsurface imaging and cell refractometry studies. This is accomplished by utilizing simultaneously obtained shear-force topography information. We will demonstrate how the quantitative phase and topography data can be used for subsurface and cell refractometry analysis and will present results for a fabricated structure and a malaria infected red blood cell.

Signal amplification of FISH for automated detection using image cytometry.

PubMed

Truong, K; Boenders, J; Maciorowski, Z; Vielh, P; Dutrillaux, B; Malfoy, B; Bourgeois, C A

1997-05-01

The purpose of this study was to improve the detection of FISH signals, in order that spot counting by a fully automated image cytometer be comparable to that obtained visually under the microscope. Two systems of spot scoring, visual and automated counting, were investigated in parallel on stimulated human lymphocytes with FISH using a biotinylated centromeric probe for chromosome 3. Signal characteristics were first analyzed on images recorded with a coupled charge device (CCD) camera. Number of spots per nucleus were scored visually on these recorded images versus automatically with a DISCOVERY image analyzer. Several fluochromes, amplification and pretreatments were tested. Our results for both visual and automated scoring show that the tyramide amplification system (TSA) gives the best amplification of signal if pepsin treatment is applied prior to FISH. Accuracy of the automated scoring, however, remained low (58% of nuclei containing two spots) compared to the visual scoring because of the high intranuclear variation between FISH spots.

Widefield quantitative multiplex surface enhanced Raman scattering imaging in vivo

NASA Astrophysics Data System (ADS)

McVeigh, Patrick Z.; Mallia, Rupananda J.; Veilleux, Israel; Wilson, Brian C.

2013-04-01

In recent years numerous studies have shown the potential advantages of molecular imaging in vitro and in vivo using contrast agents based on surface enhanced Raman scattering (SERS), however the low throughput of traditional point-scanned imaging methodologies have limited their use in biological imaging. In this work we demonstrate that direct widefield Raman imaging based on a tunable filter is capable of quantitative multiplex SERS imaging in vivo, and that this imaging is possible with acquisition times which are orders of magnitude lower than achievable with comparable point-scanned methodologies. The system, designed for small animal imaging, has a linear response from (0.01 to 100 pM), acquires typical in vivo images in <10 s, and with suitable SERS reporter molecules is capable of multiplex imaging without compensation for spectral overlap. To demonstrate the utility of widefield Raman imaging in biological applications, we show quantitative imaging of four simultaneous SERS reporter molecules in vivo with resulting probe quantification that is in excellent agreement with known quantities (R2>0.98).

Organizing the Cellular and Molecular Heterogeneity in High-Grade Serous Ovarian Cancer by Mass Cytometry

DTIC Science & Technology

2014-10-01

Bendall SC, Sung P, Nolan GP, Arvin AM. Single-cell mass cytometry analysis of human tonsil T cell remodeling by varicella zoster virus. Cell Rep...Perspectives on Flow Cytometry 2013, September 20, 2013, Mass Cytometry and Cell Cycle, Mexico City, Mexico (by Web Conference) Nolan: Nuclear

Quantitative photoacoustic elasticity and viscosity imaging for cirrhosis detection

NASA Astrophysics Data System (ADS)

Wang, Qian; Shi, Yujiao; Yang, Fen; Yang, Sihua

2018-05-01

Elasticity and viscosity assessments are essential for understanding and characterizing the physiological and pathological states of tissue. In this work, by establishing a photoacoustic (PA) shear wave model, an approach for quantitative PA elasticity imaging based on measurement of the rise time of the thermoelastic displacement was developed. Thus, using an existing PA viscoelasticity imaging method that features a phase delay measurement, quantitative PA elasticity imaging and viscosity imaging can be obtained in a simultaneous manner. The method was tested and validated by imaging viscoelastic agar phantoms prepared at different agar concentrations, and the imaging data were in good agreement with rheometry results. Ex vivo experiments on liver pathological models demonstrated the capability for cirrhosis detection, and the results were consistent with the corresponding histological results. This method expands the scope of conventional PA imaging and has potential to become an important alternative imaging modality.

Quantitative Image Restoration in Bright Field Optical Microscopy.

PubMed

Gutiérrez-Medina, Braulio; Sánchez Miranda, Manuel de Jesús

2017-11-07

Bright field (BF) optical microscopy is regarded as a poor method to observe unstained biological samples due to intrinsic low image contrast. We introduce quantitative image restoration in bright field (QRBF), a digital image processing method that restores out-of-focus BF images of unstained cells. Our procedure is based on deconvolution, using a point spread function modeled from theory. By comparing with reference images of bacteria observed in fluorescence, we show that QRBF faithfully recovers shape and enables quantify size of individual cells, even from a single input image. We applied QRBF in a high-throughput image cytometer to assess shape changes in Escherichia coli during hyperosmotic shock, finding size heterogeneity. We demonstrate that QRBF is also applicable to eukaryotic cells (yeast). Altogether, digital restoration emerges as a straightforward alternative to methods designed to generate contrast in BF imaging for quantitative analysis. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Standardizing Quality Assessment of Fused Remotely Sensed Images

NASA Astrophysics Data System (ADS)

Pohl, C.; Moellmann, J.; Fries, K.

2017-09-01

The multitude of available operational remote sensing satellites led to the development of many image fusion techniques to provide high spatial, spectral and temporal resolution images. The comparison of different techniques is necessary to obtain an optimized image for the different applications of remote sensing. There are two approaches in assessing image quality: 1. Quantitatively by visual interpretation and 2. Quantitatively using image quality indices. However an objective comparison is difficult due to the fact that a visual assessment is always subject and a quantitative assessment is done by different criteria. Depending on the criteria and indices the result varies. Therefore it is necessary to standardize both processes (qualitative and quantitative assessment) in order to allow an objective image fusion quality evaluation. Various studies have been conducted at the University of Osnabrueck (UOS) to establish a standardized process to objectively compare fused image quality. First established image fusion quality assessment protocols, i.e. Quality with No Reference (QNR) and Khan's protocol, were compared on varies fusion experiments. Second the process of visual quality assessment was structured and standardized with the aim to provide an evaluation protocol. This manuscript reports on the results of the comparison and provides recommendations for future research.

Simple and fast spectral domain algorithm for quantitative phase imaging of living cells with digital holographic microscopy

NASA Astrophysics Data System (ADS)

Min, Junwei; Yao, Baoli; Ketelhut, Steffi; Kemper, Björn

2017-02-01

The modular combination of optical microscopes with digital holographic microscopy (DHM) has been proven to be a powerful tool for quantitative live cell imaging. The introduction of condenser and different microscope objectives (MO) simplifies the usage of the technique and makes it easier to measure different kinds of specimens with different magnifications. However, the high flexibility of illumination and imaging also causes variable phase aberrations that need to be eliminated for high resolution quantitative phase imaging. The existent phase aberrations compensation methods either require add additional elements into the reference arm or need specimen free reference areas or separate reference holograms to build up suitable digital phase masks. These inherent requirements make them unpractical for usage with highly variable illumination and imaging systems and prevent on-line monitoring of living cells. In this paper, we present a simple numerical method for phase aberration compensation based on the analysis of holograms in spatial frequency domain with capabilities for on-line quantitative phase imaging. From a single shot off-axis hologram, the whole phase aberration can be eliminated automatically without numerical fitting or pre-knowledge of the setup. The capabilities and robustness for quantitative phase imaging of living cancer cells are demonstrated.

Cardiac imaging: working towards fully-automated machine analysis & interpretation.

PubMed

Slomka, Piotr J; Dey, Damini; Sitek, Arkadiusz; Motwani, Manish; Berman, Daniel S; Germano, Guido

2017-03-01

Non-invasive imaging plays a critical role in managing patients with cardiovascular disease. Although subjective visual interpretation remains the clinical mainstay, quantitative analysis facilitates objective, evidence-based management, and advances in clinical research. This has driven developments in computing and software tools aimed at achieving fully automated image processing and quantitative analysis. In parallel, machine learning techniques have been used to rapidly integrate large amounts of clinical and quantitative imaging data to provide highly personalized individual patient-based conclusions. Areas covered: This review summarizes recent advances in automated quantitative imaging in cardiology and describes the latest techniques which incorporate machine learning principles. The review focuses on the cardiac imaging techniques which are in wide clinical use. It also discusses key issues and obstacles for these tools to become utilized in mainstream clinical practice. Expert commentary: Fully-automated processing and high-level computer interpretation of cardiac imaging are becoming a reality. Application of machine learning to the vast amounts of quantitative data generated per scan and integration with clinical data also facilitates a move to more patient-specific interpretation. These developments are unlikely to replace interpreting physicians but will provide them with highly accurate tools to detect disease, risk-stratify, and optimize patient-specific treatment. However, with each technological advance, we move further from human dependence and closer to fully-automated machine interpretation.

Dependence of quantitative accuracy of CT perfusion imaging on system parameters

NASA Astrophysics Data System (ADS)

Li, Ke; Chen, Guang-Hong

2017-03-01

Deconvolution is a popular method to calculate parametric perfusion parameters from four dimensional CT perfusion (CTP) source images. During the deconvolution process, the four dimensional space is squeezed into three-dimensional space by removing the temporal dimension, and a prior knowledge is often used to suppress noise associated with the process. These additional complexities confound the understanding about deconvolution-based CTP imaging system and how its quantitative accuracy depends on parameters and sub-operations involved in the image formation process. Meanwhile, there has been a strong clinical need in answering this question, as physicians often rely heavily on the quantitative values of perfusion parameters to make diagnostic decisions, particularly during an emergent clinical situation (e.g. diagnosis of acute ischemic stroke). The purpose of this work was to develop a theoretical framework that quantitatively relates the quantification accuracy of parametric perfusion parameters with CTP acquisition and post-processing parameters. This goal was achieved with the help of a cascaded systems analysis for deconvolution-based CTP imaging systems. Based on the cascaded systems analysis, the quantitative relationship between regularization strength, source image noise, arterial input function, and the quantification accuracy of perfusion parameters was established. The theory could potentially be used to guide developments of CTP imaging technology for better quantification accuracy and lower radiation dose.

Quantitative phase imaging of arthropods

PubMed Central

Sridharan, Shamira; Katz, Aron; Soto-Adames, Felipe; Popescu, Gabriel

2015-01-01

Abstract. Classification of arthropods is performed by characterization of fine features such as setae and cuticles. An unstained whole arthropod specimen mounted on a slide can be preserved for many decades, but is difficult to study since current methods require sample manipulation or tedious image processing. Spatial light interference microscopy (SLIM) is a quantitative phase imaging (QPI) technique that is an add-on module to a commercial phase contrast microscope. We use SLIM to image a whole organism springtail Ceratophysella denticulata mounted on a slide. This is the first time, to our knowledge, that an entire organism has been imaged using QPI. We also demonstrate the ability of SLIM to image fine structures in addition to providing quantitative data that cannot be obtained by traditional bright field microscopy. PMID:26334858

Quantitative Metrics in Clinical Radiology Reporting: A Snapshot Perspective from a Single Mixed Academic-Community Practice

PubMed Central

Abramson, Richard G.; Su, Pei-Fang; Shyr, Yu

2012-01-01

Quantitative imaging has emerged as a leading priority on the imaging research agenda, yet clinical radiology has traditionally maintained a skeptical attitude toward numerical measurement in diagnostic interpretation. To gauge the extent to which quantitative reporting has been incorporated into routine clinical radiology practice, and to offer preliminary baseline data against which the evolution of quantitative imaging can be measured, we obtained all clinical computed tomography (CT) and magnetic resonance imaging (MRI) reports from two randomly selected weekdays in 2011 at a single mixed academic-community practice and evaluated those reports for the presence of quantitative descriptors. We found that 44% of all reports contained at least one “quantitative metric” (QM), defined as any numerical descriptor of a physical property other than quantity, but only 2% of reports contained an “advanced quantitative metric” (AQM), defined as a numerical parameter reporting on lesion function or composition, excluding simple size and distance measurements. Possible reasons for the slow translation of AQMs into routine clinical radiology reporting include perceptions that the primary clinical question may be qualitative in nature or that a qualitative answer may be sufficient; concern that quantitative approaches may obscure important qualitative information, may not be adequately validated, or may not allow sufficient expression of uncertainty; the feeling that “gestalt” interpretation may be superior to quantitative paradigms; and practical workflow limitations. We suggest that quantitative imaging techniques will evolve primarily as dedicated instruments for answering specific clinical questions requiring precise and standardized interpretation. Validation in real-world settings, ease of use, and reimbursement economics will all play a role in determining the rate of translation of AQMs into broad practice. PMID:22795791

Applying quantitative benefit-risk analysis to aid regulatory decision making in diagnostic imaging: methods, challenges, and opportunities.

PubMed

Agapova, Maria; Devine, Emily Beth; Bresnahan, Brian W; Higashi, Mitchell K; Garrison, Louis P

2014-09-01

Health agencies making regulatory marketing-authorization decisions use qualitative and quantitative approaches to assess expected benefits and expected risks associated with medical interventions. There is, however, no universal standard approach that regulatory agencies consistently use to conduct benefit-risk assessment (BRA) for pharmaceuticals or medical devices, including for imaging technologies. Economics, health services research, and health outcomes research use quantitative approaches to elicit preferences of stakeholders, identify priorities, and model health conditions and health intervention effects. Challenges to BRA in medical devices are outlined, highlighting additional barriers in radiology. Three quantitative methods--multi-criteria decision analysis, health outcomes modeling and stated-choice survey--are assessed using criteria that are important in balancing benefits and risks of medical devices and imaging technologies. To be useful in regulatory BRA, quantitative methods need to: aggregate multiple benefits and risks, incorporate qualitative considerations, account for uncertainty, and make clear whose preferences/priorities are being used. Each quantitative method performs differently across these criteria and little is known about how BRA estimates and conclusions vary by approach. While no specific quantitative method is likely to be the strongest in all of the important areas, quantitative methods may have a place in BRA of medical devices and radiology. Quantitative BRA approaches have been more widely applied in medicines, with fewer BRAs in devices. Despite substantial differences in characteristics of pharmaceuticals and devices, BRA methods may be as applicable to medical devices and imaging technologies as they are to pharmaceuticals. Further research to guide the development and selection of quantitative BRA methods for medical devices and imaging technologies is needed. Copyright © 2014 AUR. Published by Elsevier Inc. All rights reserved.

Phase calibration target for quantitative phase imaging with ptychography.

PubMed

Godden, T M; Muñiz-Piniella, A; Claverley, J D; Yacoot, A; Humphry, M J

2016-04-04

Quantitative phase imaging (QPI) utilizes refractive index and thickness variations that lead to optical phase shifts. This gives contrast to images of transparent objects. In quantitative biology, phase images are used to accurately segment cells and calculate properties such as dry mass, volume and proliferation rate. The fidelity of the measured phase shifts is of critical importance in this field. However to date, there has been no standardized method for characterizing the performance of phase imaging systems. Consequently, there is an increasing need for protocols to test the performance of phase imaging systems using well-defined phase calibration and resolution targets. In this work, we present a candidate for a standardized phase resolution target, and measurement protocol for the determination of the transfer of spatial frequencies, and sensitivity of a phase imaging system. The target has been carefully designed to contain well-defined depth variations over a broadband range of spatial frequencies. In order to demonstrate the utility of the target, we measure quantitative phase images on a ptychographic microscope, and compare the measured optical phase shifts with Atomic Force Microscopy (AFM) topography maps and surface profile measurements from coherence scanning interferometry. The results show that ptychography has fully quantitative nanometer sensitivity in optical path differences over a broadband range of spatial frequencies for feature sizes ranging from micrometers to hundreds of micrometers.

An Improved Method for Measuring Quantitative Resistance to the Wheat Pathogen Zymoseptoria tritici Using High-Throughput Automated Image Analysis.

PubMed

Stewart, Ethan L; Hagerty, Christina H; Mikaberidze, Alexey; Mundt, Christopher C; Zhong, Ziming; McDonald, Bruce A

2016-07-01

Zymoseptoria tritici causes Septoria tritici blotch (STB) on wheat. An improved method of quantifying STB symptoms was developed based on automated analysis of diseased leaf images made using a flatbed scanner. Naturally infected leaves (n = 949) sampled from fungicide-treated field plots comprising 39 wheat cultivars grown in Switzerland and 9 recombinant inbred lines (RIL) grown in Oregon were included in these analyses. Measures of quantitative resistance were percent leaf area covered by lesions, pycnidia size and gray value, and pycnidia density per leaf and lesion. These measures were obtained automatically with a batch-processing macro utilizing the image-processing software ImageJ. All phenotypes in both locations showed a continuous distribution, as expected for a quantitative trait. The trait distributions at both sites were largely overlapping even though the field and host environments were quite different. Cultivars and RILs could be assigned to two or more statistically different groups for each measured phenotype. Traditional visual assessments of field resistance were highly correlated with quantitative resistance measures based on image analysis for the Oregon RILs. These results show that automated image analysis provides a promising tool for assessing quantitative resistance to Z. tritici under field conditions.

TH-A-207B-00: Shear-Wave Imaging and a QIBA US Biomarker Update

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

Imaging of tissue elastic properties is a relatively new and powerful approach to one of the oldest and most important diagnostic tools. Imaging of shear wave speed with ultrasound is has been added to most high-end ultrasound systems. Understanding this exciting imaging mode aiding its most effective use in medicine can be a rewarding effort for medical physicists and other medical imaging and treatment professionals. Assuring consistent, quantitative measurements across the many ultrasound systems in a typical imaging department will constitute a major step toward realizing the great potential of this technique and other quantitative imaging. This session will targetmore » these two goals with two presentations. A. Basics and Current Implementations of Ultrasound Imaging of Shear Wave Speed and Elasticity - Shigao Chen, Ph.D. Learning objectives-To understand: Introduction: Importance of tissue elasticity measurement Strain vs. shear wave elastography (SWE), beneficial features of SWE The link between shear wave speed and material properties, influence of viscosity Generation of shear waves External vibration (Fibroscan) ultrasound radiation force Point push Supersonic push (Aixplorer) Comb push (GE Logiq E9) Detection of shear waves Motion detection from pulse-echo ultrasound Importance of frame rate for shear wave imaging Plane wave imaging detection How to achieve high effective frame rate using line-by-line scanners Shear wave speed calculation Time to peak Random sample consensus (RANSAC) Cross correlation Sources of bias and variation in SWE Tissue viscosity Transducer compression or internal pressure of organ Reflection of shear waves at boundaries B. Elasticity Imaging System Biomarker Qualification and User Testing of Systems – Brian Garra, M.D. Learning objectives-To understand: Goals Review the need for quantitative medical imaging Provide examples of quantitative imaging biomarkers Acquaint the participant with the purpose of the RSNA Quantitative Imaging Biomarker Alliance and the need for such an organization Review the QIBA process for creating a quantitative biomarker Summarize steps needed to verify adherence of site, operators, and imaging systems to a QIBA profile Underlying Premise and Assumptions Objective, quantifiable results are needed to enhance the value of diagnostic imaging in clinical practice Reasons for quantification Evidence based medicine requires objective, not subjective observer data Computerized decision support tools (eg CAD) generally require quantitative input. Quantitative, reproducible measures are more easily used to develop personalized molecular medical diagnostic and treatment systems What is quantitative imaging? Definition from Imaging Metrology Workshop The Quantitative Imaging Biomarker Alliance Formation 2008 Mission Structure Example Imaging Biomarkers Being Explored Biomarker Selection Groundwork Draft Protocol for imaging and data evaluation QIBA Profile Drafting Equipment and Site Validation Technical Clinical Site and Equipment QA and Compliance Checking Ultrasound Elasticity Estimation Biomarker US Elasticity Estimation Background Current Status and Problems Biomarker Selection-process and outcome US SWS for Liver Fibrosis Biomarker Work Groundwork Literature search and analysis results Phase I phantom testing-Elastic phantoms Phase II phantom testing-Viscoelastic phantoms Digital Simulated Data Protocol and Profile Drafting Protocol: based on UPICT and existing literature and standards bodies protocols Profile-Current claims, Manufacturer specific appendices What comes after the profile Profile Validation Technical validation Clinical validation QA and Compliance Possible approaches Site Operator testing Site protocol re-evaluation Imaging system Manufacturer testing and attestation User acceptance testing and periodic QA Phantom Tests Digital Phantom Based Testing Standard QA Testing Remediation Schemes Profile Evolution Towards additional applications Towards higher accuracy and precision Supported in part by NIH contract HHSN268201300071C from NIBIB. Collaboration with GE Global Research, no personal support.; S. Chen, Some technologies described in this presentation have been licensed. Mayo Clinic and Dr. Chen have financial interests these technologies.« less

TH-A-207B-01: Basics and Current Implementations of Ultrasound Imaging of Shear Wave Speed and Elasticity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, S.

Imaging of tissue elastic properties is a relatively new and powerful approach to one of the oldest and most important diagnostic tools. Imaging of shear wave speed with ultrasound is has been added to most high-end ultrasound systems. Understanding this exciting imaging mode aiding its most effective use in medicine can be a rewarding effort for medical physicists and other medical imaging and treatment professionals. Assuring consistent, quantitative measurements across the many ultrasound systems in a typical imaging department will constitute a major step toward realizing the great potential of this technique and other quantitative imaging. This session will targetmore » these two goals with two presentations. A. Basics and Current Implementations of Ultrasound Imaging of Shear Wave Speed and Elasticity - Shigao Chen, Ph.D. Learning objectives-To understand: Introduction: Importance of tissue elasticity measurement Strain vs. shear wave elastography (SWE), beneficial features of SWE The link between shear wave speed and material properties, influence of viscosity Generation of shear waves External vibration (Fibroscan) ultrasound radiation force Point push Supersonic push (Aixplorer) Comb push (GE Logiq E9) Detection of shear waves Motion detection from pulse-echo ultrasound Importance of frame rate for shear wave imaging Plane wave imaging detection How to achieve high effective frame rate using line-by-line scanners Shear wave speed calculation Time to peak Random sample consensus (RANSAC) Cross correlation Sources of bias and variation in SWE Tissue viscosity Transducer compression or internal pressure of organ Reflection of shear waves at boundaries B. Elasticity Imaging System Biomarker Qualification and User Testing of Systems – Brian Garra, M.D. Learning objectives-To understand: Goals Review the need for quantitative medical imaging Provide examples of quantitative imaging biomarkers Acquaint the participant with the purpose of the RSNA Quantitative Imaging Biomarker Alliance and the need for such an organization Review the QIBA process for creating a quantitative biomarker Summarize steps needed to verify adherence of site, operators, and imaging systems to a QIBA profile Underlying Premise and Assumptions Objective, quantifiable results are needed to enhance the value of diagnostic imaging in clinical practice Reasons for quantification Evidence based medicine requires objective, not subjective observer data Computerized decision support tools (eg CAD) generally require quantitative input. Quantitative, reproducible measures are more easily used to develop personalized molecular medical diagnostic and treatment systems What is quantitative imaging? Definition from Imaging Metrology Workshop The Quantitative Imaging Biomarker Alliance Formation 2008 Mission Structure Example Imaging Biomarkers Being Explored Biomarker Selection Groundwork Draft Protocol for imaging and data evaluation QIBA Profile Drafting Equipment and Site Validation Technical Clinical Site and Equipment QA and Compliance Checking Ultrasound Elasticity Estimation Biomarker US Elasticity Estimation Background Current Status and Problems Biomarker Selection-process and outcome US SWS for Liver Fibrosis Biomarker Work Groundwork Literature search and analysis results Phase I phantom testing-Elastic phantoms Phase II phantom testing-Viscoelastic phantoms Digital Simulated Data Protocol and Profile Drafting Protocol: based on UPICT and existing literature and standards bodies protocols Profile-Current claims, Manufacturer specific appendices What comes after the profile Profile Validation Technical validation Clinical validation QA and Compliance Possible approaches Site Operator testing Site protocol re-evaluation Imaging system Manufacturer testing and attestation User acceptance testing and periodic QA Phantom Tests Digital Phantom Based Testing Standard QA Testing Remediation Schemes Profile Evolution Towards additional applications Towards higher accuracy and precision Supported in part by NIH contract HHSN268201300071C from NIBIB. Collaboration with GE Global Research, no personal support.; S. Chen, Some technologies described in this presentation have been licensed. Mayo Clinic and Dr. Chen have financial interests these technologies.« less

TH-A-207B-02: QIBA Ultrasound Elasticity Imaging System Biomarker Qualification and User Testing of Systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garra, B.

Imaging of tissue elastic properties is a relatively new and powerful approach to one of the oldest and most important diagnostic tools. Imaging of shear wave speed with ultrasound is has been added to most high-end ultrasound systems. Understanding this exciting imaging mode aiding its most effective use in medicine can be a rewarding effort for medical physicists and other medical imaging and treatment professionals. Assuring consistent, quantitative measurements across the many ultrasound systems in a typical imaging department will constitute a major step toward realizing the great potential of this technique and other quantitative imaging. This session will targetmore » these two goals with two presentations. A. Basics and Current Implementations of Ultrasound Imaging of Shear Wave Speed and Elasticity - Shigao Chen, Ph.D. Learning objectives-To understand: Introduction: Importance of tissue elasticity measurement Strain vs. shear wave elastography (SWE), beneficial features of SWE The link between shear wave speed and material properties, influence of viscosity Generation of shear waves External vibration (Fibroscan) ultrasound radiation force Point push Supersonic push (Aixplorer) Comb push (GE Logiq E9) Detection of shear waves Motion detection from pulse-echo ultrasound Importance of frame rate for shear wave imaging Plane wave imaging detection How to achieve high effective frame rate using line-by-line scanners Shear wave speed calculation Time to peak Random sample consensus (RANSAC) Cross correlation Sources of bias and variation in SWE Tissue viscosity Transducer compression or internal pressure of organ Reflection of shear waves at boundaries B. Elasticity Imaging System Biomarker Qualification and User Testing of Systems – Brian Garra, M.D. Learning objectives-To understand: Goals Review the need for quantitative medical imaging Provide examples of quantitative imaging biomarkers Acquaint the participant with the purpose of the RSNA Quantitative Imaging Biomarker Alliance and the need for such an organization Review the QIBA process for creating a quantitative biomarker Summarize steps needed to verify adherence of site, operators, and imaging systems to a QIBA profile Underlying Premise and Assumptions Objective, quantifiable results are needed to enhance the value of diagnostic imaging in clinical practice Reasons for quantification Evidence based medicine requires objective, not subjective observer data Computerized decision support tools (eg CAD) generally require quantitative input. Quantitative, reproducible measures are more easily used to develop personalized molecular medical diagnostic and treatment systems What is quantitative imaging? Definition from Imaging Metrology Workshop The Quantitative Imaging Biomarker Alliance Formation 2008 Mission Structure Example Imaging Biomarkers Being Explored Biomarker Selection Groundwork Draft Protocol for imaging and data evaluation QIBA Profile Drafting Equipment and Site Validation Technical Clinical Site and Equipment QA and Compliance Checking Ultrasound Elasticity Estimation Biomarker US Elasticity Estimation Background Current Status and Problems Biomarker Selection-process and outcome US SWS for Liver Fibrosis Biomarker Work Groundwork Literature search and analysis results Phase I phantom testing-Elastic phantoms Phase II phantom testing-Viscoelastic phantoms Digital Simulated Data Protocol and Profile Drafting Protocol: based on UPICT and existing literature and standards bodies protocols Profile-Current claims, Manufacturer specific appendices What comes after the profile Profile Validation Technical validation Clinical validation QA and Compliance Possible approaches Site Operator testing Site protocol re-evaluation Imaging system Manufacturer testing and attestation User acceptance testing and periodic QA Phantom Tests Digital Phantom Based Testing Standard QA Testing Remediation Schemes Profile Evolution Towards additional applications Towards higher accuracy and precision Supported in part by NIH contract HHSN268201300071C from NIBIB. Collaboration with GE Global Research, no personal support.; S. Chen, Some technologies described in this presentation have been licensed. Mayo Clinic and Dr. Chen have financial interests these technologies.« less

Magnetic Field Gradient Calibration as an Experiment to Illustrate Magnetic Resonance Imaging

ERIC Educational Resources Information Center

Seedhouse, Steven J.; Hoffmann, Markus M.

2008-01-01

A nuclear magnetic resonance (NMR) spectroscopy experiment for the undergraduate physical chemistry laboratory is described that encompasses both qualitative and quantitative pedagogical goals. Qualitatively, the experiment illustrates how images are obtained in magnetic resonance imaging (MRI). Quantitatively, students experience the…

Qualitative and quantitative effects of harmonic echocardiographic imaging on endocardial edge definition and side-lobe artifacts

NASA Technical Reports Server (NTRS)

Rubin, D. N.; Yazbek, N.; Garcia, M. J.; Stewart, W. J.; Thomas, J. D.

2000-01-01

Harmonic imaging is a new ultrasonographic technique that is designed to improve image quality by exploiting the spontaneous generation of higher frequencies as ultrasound propagates through tissue. We studied 51 difficult-to-image patients with blinded side-by-side cineloop evaluation of endocardial border definition by harmonic versus fundamental imaging. In addition, quantitative intensities from cavity versus wall were compared for harmonic versus fundamental imaging. Harmonic imaging improved left ventricular endocardial border delineation over fundamental imaging (superior: harmonic = 71.1%, fundamental = 18.7%; similar: 10.2%; P <.001). Quantitative analysis of 100 wall/cavity combinations demonstrated brighter wall segments and more strikingly darker cavities during harmonic imaging (cavity intensity on a 0 to 255 scale: fundamental = 15.6 +/- 8.6; harmonic = 6.0 +/- 5.3; P <.0001), which led to enhanced contrast between the wall and cavity (1.89 versus 1.19, P <.0001). Harmonic imaging reduces side-lobe artifacts, resulting in a darker cavity and brighter walls, thereby improving image contrast and endocardial delineation.

TH-AB-209-09: Quantitative Imaging of Electrical Conductivity by VHF-Induced Thermoacoustics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patch, S; Hull, D; See, W

Purpose: To demonstrate that very high frequency (VHF) induced thermoacoustics has the potential to provide quantitative images of electrical conductivity in Siemens/meter, much as shear wave elastography provides tissue stiffness in kPa. Quantitatively imaging a large organ requires exciting thermoacoustic pulses throughout the volume and broadband detection of those pulses because tomographic image reconstruction preserves frequency content. Applying the half-wavelength limit to a 200-micron inclusion inside a 7.5 cm diameter organ requires measurement sensitivity to frequencies ranging from 4 MHz down to 10 kHz, respectively. VHF irradiation provides superior depth penetration over near infrared used in photoacoustics. Additionally, VHF signalmore » production is proportional to electrical conductivity, and prostate cancer is known to suppress electrical conductivity of prostatic fluid. Methods: A dual-transducer system utilizing a P4-1 array connected to a Verasonics V1 system augmented by a lower frequency focused single element transducer was developed. Simultaneous acquisition of VHF-induced thermoacoustic pulses by both transducers enabled comparison of transducer performance. Data from the clinical array generated a stack of 96-images with separation of 0.3 mm, whereas the single element transducer imaged only in a single plane. In-plane resolution and quantitative accuracy were measured at isocenter. Results: The array provided volumetric imaging capability with superior resolution whereas the single element transducer provided superior quantitative accuracy. Combining axial images from both transducers preserved resolution of the P4-1 array and improved image contrast. Neither transducer was sensitive to frequencies below 50 kHz, resulting in a DC offset and low-frequency shading over fields of view exceeding 15 mm. Fresh human prostates were imaged ex vivo and volumetric reconstructions reveal structures rarely seen in diagnostic images. Conclusion: Quantitative whole-organ thermoacoustic tomography will be feasible by sparsely interspersing transducer elements sensitive to the low end of the ultrasonic range.« less

Quantitative analysis for peripheral vascularity assessment based on clinical photoacoustic and ultrasound images

NASA Astrophysics Data System (ADS)

Murakoshi, Dai; Hirota, Kazuhiro; Ishii, Hiroyasu; Hashimoto, Atsushi; Ebata, Tetsurou; Irisawa, Kaku; Wada, Takatsugu; Hayakawa, Toshiro; Itoh, Kenji; Ishihara, Miya

2018-02-01

Photoacoustic (PA) imaging technology is expected to be applied to clinical assessment for peripheral vascularity. We started a clinical evaluation with the prototype PA imaging system we recently developed. Prototype PA imaging system was composed with in-house Q-switched Alexandrite laser system which emits short-pulsed laser with 750 nm wavelength, handheld ultrasound transducer where illumination optics were integrated and signal processing for PA image reconstruction implemented in the clinical ultrasound (US) system. For the purpose of quantitative assessment of PA images, an image analyzing function has been developed and applied to clinical PA images. In this analyzing function, vascularity derived from PA signal intensity ranged for prescribed threshold was defined as a numerical index of vessel fulfillment and calculated for the prescribed region of interest (ROI). Skin surface was automatically detected by utilizing B-mode image acquired simultaneously with PA image. Skinsurface position is utilized to place the ROI objectively while avoiding unwanted signals such as artifacts which were imposed due to melanin pigment in the epidermal layer which absorbs laser emission and generates strong PA signals. Multiple images were available to support the scanned image set for 3D viewing. PA images for several fingers of patients with systemic sclerosis (SSc) were quantitatively assessed. Since the artifact region is trimmed off in PA images, the visibility of vessels with rather low PA signal intensity on the 3D projection image was enhanced and the reliability of the quantitative analysis was improved.

Quantitative Analysis of Rat Dorsal Root Ganglion Neurons Cultured on Microelectrode Arrays Based on Fluorescence Microscopy Image Processing.

PubMed

Mari, João Fernando; Saito, José Hiroki; Neves, Amanda Ferreira; Lotufo, Celina Monteiro da Cruz; Destro-Filho, João-Batista; Nicoletti, Maria do Carmo

2015-12-01

Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.

Quantitative Imaging in Cancer Clinical Trials

PubMed Central

Yankeelov, Thomas E.; Mankoff, David A.; Schwartz, Lawrence H.; Lieberman, Frank S.; Buatti, John M.; Mountz, James M.; Erickson, Bradley J.; Fennessy, Fiona M.M.; Huang, Wei; Kalpathy-Cramer, Jayashree; Wahl, Richard L.; Linden, Hannah M.; Kinahan, Paul; Zhao, Binsheng; Hylton, Nola M.; Gillies, Robert J.; Clarke, Laurence; Nordstrom, Robert; Rubin, Daniel L.

2015-01-01

As anti-cancer therapies designed to target specific molecular pathways have been developed, it has become critical to develop methods to assess the response induced by such agents. While traditional, anatomic CT and MRI exams are useful in many settings, there is increasing evidence that these methods cannot answer the fundamental biological and physiological questions essential for assessment and, eventually, prediction of treatment response in the clinical trial setting, especially in the critical period soon after treatment is initiated. To optimally apply advances in quantitative imaging methods to trials of targeted cancer therapy, new infrastructure improvements are needed that incorporate these emerging techniques into the settings where they are most likely to have impact. In this review, we first elucidate the needs for therapeutic response assessment in the era of molecularly targeted therapy and describe how quantitative imaging can most effectively provide scientifically and clinically relevant data. We then describe the tools and methods required to apply quantitative imaging and provide concrete examples of work making these advances practically available for routine application in clinical trials. We conclude by proposing strategies to surmount barriers to wider incorporation of these quantitative imaging methods into clinical trials and, eventually, clinical practice. Our goal is to encourage and guide the oncology community to deploy standardized quantitative imaging techniques in clinical trials to further personalize care for cancer patients, and to provide a more efficient path for the development of improved targeted therapies. PMID:26773162

Evaluation of imaging biomarkers for identification of single cancer cells in blood

NASA Astrophysics Data System (ADS)

Odaka, Masao; Kim, Hyonchol; Girault, Mathias; Hattori, Akihiro; Terazono, Hideyuki; Matsuura, Kenji; Yasuda, Kenji

2015-06-01

A method of discriminating single cancer cells from whole blood cells based on their morphological visual characteristics (i.e., “imaging biomarker”) was examined. Cells in healthy rat blood, a cancer cell line (MAT-LyLu), and cells in cancer-cell-implanted rat blood were chosen as models, and their bright-field (BF, whole-cell morphology) and fluorescence (FL, nucleus morphology) images were taken by an on-chip multi-imaging flow cytometry system and compared. Eight imaging biomarker indices, i.e., cellular area in a BF image, nucleus area in an FL image, area ratio of a whole cell and its nucleus, distance of the mass center between a whole cell and nucleus, cellular and nucleus perimeter, and perimeter ratios were calculated and analyzed using the BF and FL images taken. Results show that cancer cells can be clearly distinguished from healthy blood cells using correlation diagrams for cellular and nucleus areas as two different categories. Moreover, a portion of cancer cells showed a low nucleus perimeter ratio less than 0.9 because of the irregular nucleus morphologies of cancer cells. These results indicate that the measurements of imaging biomarkers are practically applicable to identifying cancer cells in blood.

Listening to light scattering in turbid media: quantitative optical scattering imaging using photoacoustic measurements with one-wavelength illumination

NASA Astrophysics Data System (ADS)

Yuan, Zhen; Li, Xiaoqi; Xi, Lei

2014-06-01

Biomedical photoacoustic tomography (PAT), as a potential imaging modality, can visualize tissue structure and function with high spatial resolution and excellent optical contrast. It is widely recognized that the ability of quantitatively imaging optical absorption and scattering coefficients from photoacoustic measurements is essential before PAT can become a powerful imaging modality. Existing quantitative PAT (qPAT), while successful, has been focused on recovering absorption coefficient only by assuming scattering coefficient a constant. An effective method for photoacoustically recovering optical scattering coefficient is presently not available. Here we propose and experimentally validate such a method for quantitative scattering coefficient imaging using photoacoustic data from one-wavelength illumination. The reconstruction method developed combines conventional PAT with the photon diffusion equation in a novel way to realize the recovery of scattering coefficient. We demonstrate the method using various objects having scattering contrast only or both absorption and scattering contrasts embedded in turbid media. The listening-to-light-scattering method described will be able to provide high resolution scattering imaging for various biomedical applications ranging from breast to brain imaging.

Quantitative Imaging in Laboratory: Fast Kinetics and Fluorescence Quenching

ERIC Educational Resources Information Center

Cumberbatch, Tanya; Hanley, Quentin S.

2007-01-01

The process of quantitative imaging, which is very commonly used in laboratory, is shown to be very useful for studying the fast kinetics and fluorescence quenching of many experiments. The imaging technique is extremely cheap and hence can be used in many absorption and luminescence experiments.

qF-SSOP: real-time optical property corrected fluorescence imaging

PubMed Central

Valdes, Pablo A.; Angelo, Joseph P.; Choi, Hak Soo; Gioux, Sylvain

2017-01-01

Fluorescence imaging is well suited to provide image guidance during resections in oncologic and vascular surgery. However, the distorting effects of tissue optical properties on the emitted fluorescence are poorly compensated for on even the most advanced fluorescence image guidance systems, leading to subjective and inaccurate estimates of tissue fluorophore concentrations. Here we present a novel fluorescence imaging technique that performs real-time (i.e., video rate) optical property corrected fluorescence imaging. We perform full field of view simultaneous imaging of tissue optical properties using Single Snapshot of Optical Properties (SSOP) and fluorescence detection. The estimated optical properties are used to correct the emitted fluorescence with a quantitative fluorescence model to provide quantitative fluorescence-Single Snapshot of Optical Properties (qF-SSOP) images with less than 5% error. The technique is rigorous, fast, and quantitative, enabling ease of integration into the surgical workflow with the potential to improve molecular guidance intraoperatively. PMID:28856038

3D tissue-engineered bone marrow as a novel model to study pathophysiology and drug resistance in multiple myeloma

PubMed Central

de la Puente, Pilar; Muz, Barbara; Gilson, Rebecca C; Azab, Feda; Luderer, Micah; King, Justin; Achilefu, Samuel; Vij, Ravi; Azab, Abdel Kareem

2016-01-01

Purpose Multiple myeloma (MM) is the second most prevalent hematological malignancy and it remains incurable despite the introduction of several novel drugs. The discrepancy between preclinical and clinical outcomes can be attributed to the failure of classic two-dimensional (2D) culture models to accurately recapitulate the complex biology of MM and drug responses observed in patients. Experimental design We developed 3D tissue engineered bone marrow (3DTEBM) cultures derived from the BM supernatant of MM patients to incorporate different BM components including MM cells, stromal cells, and endothelial cells. Distribution and growth were analyzed by confocal imaging, and cell proliferation of cell lines and primary MM cells was tested by flow cytometry. Oxygen and drug gradients were evaluated by immunohistochemistry and flow cytometry, and drug resistance was studied by flow cytometry. Results 3DTEBM cultures allowed proliferation of MM cells, recapitulated their interaction with the microenvironment, recreated 3D aspects observed in the bone marrow niche (such as oxygen and drug gradients), and induced drug resistance in MM cells more than 2D or commercial 3D tissue culture systems. Conclusions 3DTEBM cultures not only provide a better model for investigating the pathophysiology of MM, but also serve as a tool for drug development and screening in MM. In the future, we will use the 3DTEBM cultures for developing personalized therapeutic strategies for individual MM patients. PMID:26402156