Digital image classification with the help of artificial neural network by simple histogram

PubMed Central

Dey, Pranab; Banerjee, Nirmalya; Kaur, Rajwant

2016-01-01

Background: Visual image classification is a great challenge to the cytopathologist in routine day-to-day work. Artificial neural network (ANN) may be helpful in this matter. Aims and Objectives: In this study, we have tried to classify digital images of malignant and benign cells in effusion cytology smear with the help of simple histogram data and ANN. Materials and Methods: A total of 404 digital images consisting of 168 benign cells and 236 malignant cells were selected for this study. The simple histogram data was extracted from these digital images and an ANN was constructed with the help of Neurointelligence software [Alyuda Neurointelligence 2.2 (577), Cupertino, California, USA]. The network architecture was 6-3-1. The images were classified as training set (281), validation set (63), and test set (60). The on-line backpropagation training algorithm was used for this study. Result: A total of 10,000 iterations were done to train the ANN system with the speed of 609.81/s. After the adequate training of this ANN model, the system was able to identify all 34 malignant cell images and 24 out of 26 benign cells. Conclusion: The ANN model can be used for the identification of the individual malignant cells with the help of simple histogram data. This study will be helpful in the future to identify malignant cells in unknown situations. PMID:27279679

Comparison of pre-processing techniques for fluorescence microscopy images of cells labeled for actin.

PubMed

Muralidhar, Gautam S; Channappayya, Sumohana S; Slater, John H; Blinka, Ellen M; Bovik, Alan C; Frey, Wolfgang; Markey, Mia K

2008-11-06

Automated analysis of fluorescence microscopy images of endothelial cells labeled for actin is important for quantifying changes in the actin cytoskeleton. The current manual approach is laborious and inefficient. The goal of our work is to develop automated image analysis methods, thereby increasing cell analysis throughput. In this study, we present preliminary results on comparing different algorithms for cell segmentation and image denoising.

Analysis of gene expression levels in individual bacterial cells without image segmentation

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

Kwak, In Hae; Son, Minjun; Hagen, Stephen J., E-mail: sjhagen@ufl.edu

2012-05-11

Highlights: Black-Right-Pointing-Pointer We present a method for extracting gene expression data from images of bacterial cells. Black-Right-Pointing-Pointer The method does not employ cell segmentation and does not require high magnification. Black-Right-Pointing-Pointer Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. Black-Right-Pointing-Pointer We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on amore » segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.« less

Quantifying cell mono-layer cultures by video imaging.

PubMed

Miller, K S; Hook, L A

1996-04-01

A method is described in which the relative number of adherent cells in multi-well tissue-culture plates is assayed by staining the cells with Giemsa and capturing the image of the stained cells with a video camera and charged-coupled device. The resultant image is quantified using the associated video imaging software. The method is shown to be sensitive and reproducible and should be useful for studies where quantifying relative cell numbers and/or proliferation in vitro is required.

Regulation of Cell Migration in Breast Cancer

DTIC Science & Technology

2011-04-01

the wound healing, assay by scarring and Oris plate migration assay, transwell migration assay and live - cell imaging studies. Cell migration capacity...evaluated by the use of techniques that include the wound healing assay by scarring and Oris plate migration assay, transwell migration assay and live - cell imaging studies

Noninvasive Assessment of Cell Fate and Biology in Transplanted Mesenchymal Stem Cells.

PubMed

Franchi, Federico; Rodriguez-Porcel, Martin

2017-01-01

Recently, molecular imaging has become a conditio sine qua non for cell-based regenerative medicine. Developments in molecular imaging techniques, such as reporter gene technology, have increasingly enabled the noninvasive assessment of the fate and biology of cells after cardiovascular applications. In this context, bioluminescence imaging is the most commonly used imaging modality in small animal models of preclinical studies. Here, we present a detailed protocol of a reporter gene imaging approach for monitoring the viability and biology of Mesenchymal Stem Cells transplanted in a mouse model of myocardial ischemia reperfusion injury.

In vivo imaging of emerging endocrine cells reveals a requirement for PI3K-regulated motility in pancreatic islet morphogenesis

PubMed Central

Freudenblum, Julia; Iglesias, José A.; Hermann, Martin; Walsen, Tanja; Wilfinger, Armin; Meyer, Dirk

2018-01-01

ABSTRACT The three-dimensional architecture of the pancreatic islet is integral to beta cell function, but the process of islet formation remains poorly understood due to the difficulties of imaging internal organs with cellular resolution. Within transparent zebrafish larvae, the developing pancreas is relatively superficial and thus amenable to live imaging approaches. We performed in vivo time-lapse and longitudinal imaging studies to follow islet development, visualizing both naturally occurring islet cells and cells arising with an accelerated timecourse following an induction approach. These studies revealed previously unappreciated fine dynamic protrusions projecting between neighboring and distant endocrine cells. Using pharmacological compound and toxin interference approaches, and single-cell analysis of morphology and cell dynamics, we determined that endocrine cell motility is regulated by phosphoinositide 3-kinase (PI3K) and G-protein-coupled receptor (GPCR) signaling. Linking cell dynamics to islet formation, perturbation of protrusion formation disrupted endocrine cell coalescence, and correlated with decreased islet cell differentiation. These studies identified novel cell behaviors contributing to islet morphogenesis, and suggest a model in which dynamic exploratory filopodia establish cell-cell contacts that subsequently promote cell clustering. PMID:29386244

Microbial Cell Imaging

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

Doktycz, Mitchel John; Sullivan, Claretta; Mortensen, Ninell P

Atomic force microscopy (AFM) is finding increasing application in a variety of fields including microbiology. Until the emergence of AFM, techniques for ivnestigating processes in single microbes were limited. From a biologist's perspective, the fact that AFM can be used to generate high-resolution images in buffers or media is its most appealing feature as live-cell imaging can be pursued. Imaging living cells by AFM allows dynamic biological events to be studied, at the nanoscale, in real time. Few areas of biological research have as much to gain as microbiology from the application of AFM. Whereas the scale of microbes placesmore » them near the limit of resolution for light microscopy. AFM is well suited for the study of structures on the order of a micron or less. Although electron microscopy techniques have been the standard for high-resolution imaging of microbes, AFM is quickly gaining favor for several reasons. First, fixatives that impair biological activity are not required. Second, AFM is capable of detecting forces in the pN range, and precise control of the force applied to the cantilever can be maintained. This combination facilitates the evaluation of physical characteristics of microbes. Third, rather than yielding the composite, statistical average of cell populations, as is the case with many biochemical assays, the behavior of single cells can be monitored. Despite the potential of AFM in microbiology, there are several limitations that must be considered. For example, the time required to record an image allows for the study of gross events such as cell division or membrane degradation from an antibiotic but precludes the evaluation of biological reactions and events that happen in just fractions of a second. Additionally, the AFM is a topographical tool and is restricted to imaging surfaces. Therefore, it cannot be used to look inside cells as with opticla and transmission electron microscopes. other practical considerations are the limitation on the maximum scan size (roughly 100 x 100 {mu}m) and the restricted movement of the cantilever in the Z (or height) direction. In most commercial AFMs, the Z range is restricted to roughly 10 {mu}m such that the height of cells to be imaged must be seriously considered. Nevertheless, AFM can provide structural-functional information at nanometer resolution and do so in physiologically relevant environments. Further, instrumentation for scanning probe microscopy continues to advance. Systems for high-speed imaging are becoming available, and techniques for looking inside the cells are being demonstrated. The ability to combine AFM with other imaging modalities is likely to have an even greater impact on microbiological studies. AFM studies of intact microbial cells started to appear in the literature in the 1990s. For example, AFM studies of Saccharomyces cerevisiae examined buddings cars after cell division and detailed changes related to cell growth processes. Also, the first AFM studies of bacterial biofilms appeared. In the late 1990s, AFM studies of intact fungal spores described clear changes in spore surfaces upon germination, and studies of individual bacterial cells were also described. These early bacterial imaging studies examined changes in bacterial morphology due to antimicrobial peptides exposure and bacterial adhesion properties. The majority of these early studies were carried out on dried samples and took advantage of the resolving power of AFM. The lack of cell mounting procedures presented an impediment for cell imaging studies. Subsequently, several approaches to mounting microbial cells have been developed, and these techniques are described later. Also highlighted are general considerations for microbial imaging and a description of some of the various applications of AFM to microbiology.« less

Fully Hydrated Yeast Cells Imaged with Electron Microscopy

PubMed Central

Peckys, Diana B.; Mazur, Peter; Gould, Kathleen L.; de Jonge, Niels

2011-01-01

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccaromyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. PMID:21575587

Fully hydrated yeast cells imaged with electron microscopy.

PubMed

Peckys, Diana B; Mazur, Peter; Gould, Kathleen L; de Jonge, Niels

2011-05-18

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccharomyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Cell sheets image validation of phase-diversity homodyne OCT and effect of the light irradiation on cells

NASA Astrophysics Data System (ADS)

Senda, Naoko; Osawa, Kentaro

2016-04-01

Optical coherence tomography (OCT) is one of powerful 3D tissue imaging tools with no fluorescence staining. We have reported that Phase-Diversity Homodyne OCT developed in Hitachi could be useful for non-invasive regeneration tissue evaluation test. The OCT enables cell imaging because of high resolution (axial resolution; ~2.6 μm, lateral resolution; ~1 μm, in the air), whereas conventional OCT was not used for cell imaging because of low resolution (10~20 μm). Furthermore, the OCT has advantage over other 3D imaging devices in cost because the light source and the objective were originally used as an optical pickup of compact disc. In this report, we aimed to assess effectiveness and safety of Phase-Diversity Homodyne OCT cell imaging. Effectiveness of OCT was evaluated by imaging a living cell sheet of human oral mucosal epithelial cells. OCT images were compared with reflection confocal microscopy (RCM) images, because confocal optical system is the highest resolution (<1 μm) 3D in vivo imaging technique. Similar nuclei images were confirmed with OCT and RCM, which suggested the OCT has enough resolution to image nuclei inside a cell sheet. Degree of differentiation could be estimated using OCT images, which becomes possible because the size of cells depends on distribution of differentiation. Effect of the OCT light irradiation on cells was studied using NIH/3T3 cells. Light irradiation, the exposure amount of which is equivalent to OCT, had no impact on cell shape, cell viability, and proliferation rate. It suggested that the light irradiation has no cell damage under the condition.

An approach for characterising cellular polymeric foam structures using computed tomography

NASA Astrophysics Data System (ADS)

Chen, Youming; Das, Raj; Battley, Mark

2018-02-01

Global properties of foams depend on foam base materials and microstructures. Characterisation of foam microstructures is important for developing numerical foam models. In this study, the microstructures of four polymeric structural foams were imaged using a micro-CT scanner. Image processing and analysis methods were proposed to quantify the relative density, cell wall thickness and cell size of these foams from the captured CT images. Overall, the cells in these foams are fairly isotropic, and cell walls are rather straight. The measured average relative densities are in good agreement with the actual values. Relative density, cell size and cell wall thickness in these foams are found to vary along the thickness of foam panel direction. Cell walls in two of these foams are found to be filled with secondary pores. In addition, it is found that the average cell wall thickness measured from 2D images is around 1.4 times of that measured from 3D images, and the average cell size measured from 3D images is 1.16 times of that measured from 2D images. The distributions of cell wall thickness and cell size measured from 2D images exhibit lager dispersion in comparison to those measured from 3D images.

Classification of human carcinoma cells using multispectral imagery

NASA Astrophysics Data System (ADS)

Ćinar, Umut; Y. Ćetin, Yasemin; Ćetin-Atalay, Rengul; Ćetin, Enis

2016-03-01

In this paper, we present a technique for automatically classifying human carcinoma cell images using textural features. An image dataset containing microscopy biopsy images from different patients for 14 distinct cancer cell line type is studied. The images are captured using a RGB camera attached to an inverted microscopy device. Texture based Gabor features are extracted from multispectral input images. SVM classifier is used to generate a descriptive model for the purpose of cell line classification. The experimental results depict satisfactory performance, and the proposed method is versatile for various microscopy magnification options.

Quantitative phase imaging of cell division in yeast cells and E.coli using digital holographic microscopy

NASA Astrophysics Data System (ADS)

Pandiyan, Vimal Prabhu; John, Renu

2015-12-01

Digital holographic microscope (DHM) is an emerging quantitative phase imaging technique with unique imaging scales and resolutions leading to multitude of applications. DHM is promising as a novel investigational and applied tool for cell imaging, studying the morphology and real time dynamics of cells and a number of related applications. The use of numerical propagation and computational digital optics offer unique flexibility to tune the depth of focus, and compensate for image aberrations. In this work, we report imaging the dynamics of cell division in E.coli and yeast cells using a DHM platform. We demonstrate 3-D and depth imaging as well as reconstruction of phase profiles of E.coli and yeast cells using the system. We record a digital hologram of E.coli and yeast cells and reconstruct the image using Fresnel propagation algorithm. We also use aberration compensation algorithms for correcting the aberrations that are introduced by the microscope objective in the object path using linear least square fitting techniques. This work demonstrates the strong potential of a DHM platform in 3-D live cell imaging, fast clinical quantifications and pathological applications.

Comparison of segmentation algorithms for fluorescence microscopy images of cells.

PubMed

Dima, Alden A; Elliott, John T; Filliben, James J; Halter, Michael; Peskin, Adele; Bernal, Javier; Kociolek, Marcin; Brady, Mary C; Tang, Hai C; Plant, Anne L

2011-07-01

The analysis of fluorescence microscopy of cells often requires the determination of cell edges. This is typically done using segmentation techniques that separate the cell objects in an image from the surrounding background. This study compares segmentation results from nine different segmentation techniques applied to two different cell lines and five different sets of imaging conditions. Significant variability in the results of segmentation was observed that was due solely to differences in imaging conditions or applications of different algorithms. We quantified and compared the results with a novel bivariate similarity index metric that evaluates the degree of underestimating or overestimating a cell object. The results show that commonly used threshold-based segmentation techniques are less accurate than k-means clustering with multiple clusters. Segmentation accuracy varies with imaging conditions that determine the sharpness of cell edges and with geometric features of a cell. Based on this observation, we propose a method that quantifies cell edge character to provide an estimate of how accurately an algorithm will perform. The results of this study will assist the development of criteria for evaluating interlaboratory comparability. Published 2011 Wiley-Liss, Inc.

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy.

PubMed

Kadam, Parnika; McAllister, Ryan; Urbach, Jeffrey S; Sandberg, Kathryn; Mueller, Susette C

2017-03-27

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

In Vivo Imaging and Monitoring of Transplanted Stem Cells: Clinical Applications

PubMed Central

Rodriguez-Porcel, Martin

2010-01-01

Regenerative medicine using stem cells has appeared as a potential therapeutic alternative for coronary artery disease, and stem cell clinical studies are currently on their way. However, initial results of these studies have provided mixed information, in part because of the inability to correlate organ functional information with the presence/absence of transplanted stem cells. Recent advances in molecular biology and imaging have allowed the successful noninvasive monitoring of transplanted stem cells in the living subject. In this article, different imaging strategies (direct labeling, indirect labeling with reporter genes) to study the viability and biology of stem cells are discussed. In addition, the limitations of each approach and imaging modality (eg, single photon emission computed tomography, positron emission tomography, and MRI) and their requirements for clinical use are addressed. Use of these strategies will be critical as the different regenerative therapies are being tested for clinical use. PMID:20425184

AUTOMATED CELL SEGMENTATION WITH 3D FLUORESCENCE MICROSCOPY IMAGES.

PubMed

Kong, Jun; Wang, Fusheng; Teodoro, George; Liang, Yanhui; Zhu, Yangyang; Tucker-Burden, Carol; Brat, Daniel J

2015-04-01

A large number of cell-oriented cancer investigations require an effective and reliable cell segmentation method on three dimensional (3D) fluorescence microscopic images for quantitative analysis of cell biological properties. In this paper, we present a fully automated cell segmentation method that can detect cells from 3D fluorescence microscopic images. Enlightened by fluorescence imaging techniques, we regulated the image gradient field by gradient vector flow (GVF) with interpolated and smoothed data volume, and grouped voxels based on gradient modes identified by tracking GVF field. Adaptive thresholding was then applied to voxels associated with the same gradient mode where voxel intensities were enhanced by a multiscale cell filter. We applied the method to a large volume of 3D fluorescence imaging data of human brain tumor cells with (1) small cell false detection and missing rates for individual cells; and (2) trivial over and under segmentation incidences for clustered cells. Additionally, the concordance of cell morphometry structure between automated and manual segmentation was encouraging. These results suggest a promising 3D cell segmentation method applicable to cancer studies.

Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures

NASA Astrophysics Data System (ADS)

Suman, Rakesh; Smith, Gabrielle; Hazel, Kathryn E. A.; Kasprowicz, Richard; Coles, Mark; O'Toole, Peter; Chawla, Sangeeta

2016-02-01

Time-lapse imaging is a fundamental tool for studying cellular behaviours, however studies of primary cells in complex co-culture environments often requires fluorescent labelling and significant light exposure that can perturb their natural function over time. Here, we describe ptychographic phase imaging that permits prolonged label-free time-lapse imaging of microglia in the presence of neurons and astrocytes, which better resembles in vivo microenvironments. We demonstrate the use of ptychography as an assay to study the phenotypic behaviour of microglial cells in primary neuronal co-cultures through the addition of cyclosporine A, a potent immune-modulator.

[The application of stereology in radiology imaging and cell biology fields].

PubMed

Hu, Na; Wang, Yan; Feng, Yuanming; Lin, Wang

2012-08-01

Stereology is an interdisciplinary method for 3D morphological study developed from mathematics and morphology. It is widely used in medical image analysis and cell biology studies. Because of its unbiased, simple, fast, reliable and non-invasive characteristics, stereology has been widely used in biomedical areas for quantitative analysis and statistics, such as histology, pathology and medical imaging. Because the stereological parameters show distinct differences in different pathology, many scholars use stereological methods to do quantitative analysis in their studies in recent years, for example, in the areas of the condition of cancer cells, tumor grade, disease development and the patient's prognosis, etc. This paper describes the stereological concept and estimation methods, also illustrates the applications of stereology in the fields of CT images, MRI images and cell biology, and finally reflects the universality, the superiority and reliability of stereology.

Intravital microscopy: a novel tool to study cell biology in living animals.

PubMed

Weigert, Roberto; Sramkova, Monika; Parente, Laura; Amornphimoltham, Panomwat; Masedunskas, Andrius

2010-05-01

Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criterion. Indeed, first we will focus on those studies in which organs were imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures.

Stem Cells as a Tool for Breast Imaging

PubMed Central

Padín-Iruegas, Maria Elena; López López, Rafael

2012-01-01

Stem cells are a scientific field of interest due to their therapeutic potential. There are different groups, depending on the differentiation state. We can find lonely stem cells, but generally they distribute in niches. Stem cells don't survive forever. They are affected for senescence. Cancer stem cells are best defined functionally, as a subpopulation of tumor cells that can enrich for tumorigenic property and can regenerate heterogeneity of the original tumor. Circulating tumor cells are cells that have detached from a primary tumor and circulate in the bloodstream. They may constitute seeds for subsequent growth of additional tumors (metastasis) in different tissues. Advances in molecular imaging have allowed a deeper understanding of the in vivo behavior of stem cells and have proven to be indispensable in preclinical and clinical studies. One of the first imaging modalities for monitoring pluripotent stem cells in vivo, magnetic resonance imaging (MRI) offers high spatial and temporal resolution to obtain detailed morphological and functional information. Advantages of radioscintigraphic techniques include their picomolar sensitivity, good tissue penetration, and translation to clinical applications. Radionuclide imaging is the sole direct labeling technique used thus far in human studies, involving both autologous bone marrow derived and peripheral stem cells. PMID:22848220

Development of a Monitoring Method for Nonlabeled Human Pluripotent Stem Cell Growth by Time-Lapse Image Analysis.

PubMed

Suga, Mika; Kii, Hiroaki; Niikura, Keiichi; Kiyota, Yasujiro; Furue, Miho K

2015-07-01

: Cell growth is an important criterion for determining healthy cell conditions. When somatic cells or cancer cells are dissociated into single cells for passaging, the cell numbers can be counted at each passage, providing information on cell growth as an indicator of the health conditions of these cells. In the case of human pluripotent stem cells (hPSCs), because the cells are usually dissociated into cell clumps of ∼50-100 cells for passaging, cell counting is time-consuming. In the present study, using a time-lapse imaging system, we developed a method to determine the growth of hPSCs from nonlabeled live cell phase-contrast images without damaging these cells. Next, the hPSC colony areas and number of nuclei were determined and used to derive equations to calculate the cell number in hPSC colonies, which were assessed on time-lapse images acquired using a culture observation system. The relationships between the colony areas and nuclei numbers were linear, although the equation coefficients were dependent on the cell line used, colony size, colony morphology, and culture conditions. When the culture conditions became improper, the change in cell growth conditions could be detected by analysis of the phase-contrast images. This method provided real-time information on colony growth and cell growth rates without using treatments that can damage cells and could be useful for basic research on hPSCs and cell processing for hPSC-based therapy. This is the first study to use a noninvasive method using images to systemically determine the growth of human pluripotent stem cells (hPSCs) without damaging or wasting cells. This method would be useful for quality control during cell culture of clinical hPSCs. ©AlphaMed Press.

Live cell imaging of in vitro human trophoblast syncytialization.

PubMed

Wang, Rui; Dang, Yan-Li; Zheng, Ru; Li, Yue; Li, Weiwei; Lu, Xiaoyin; Wang, Li-Juan; Zhu, Cheng; Lin, Hai-Yan; Wang, Hongmei

2014-06-01

Human trophoblast syncytialization, a process of cell-cell fusion, is one of the most important yet least understood events during placental development. Investigating the fusion process in a placenta in vivo is very challenging given the complexity of this process. Application of primary cultured cytotrophoblast cells isolated from term placentas and BeWo cells derived from human choriocarcinoma formulates a biphasic strategy to achieve the mechanism of trophoblast cell fusion, as the former can spontaneously fuse to form the multinucleated syncytium and the latter is capable of fusing under the treatment of forskolin (FSK). Live-cell imaging is a powerful tool that is widely used to investigate many physiological or pathological processes in various animal models or humans; however, to our knowledge, the mechanism of trophoblast cell fusion has not been reported using a live- cell imaging manner. In this study, a live-cell imaging system was used to delineate the fusion process of primary term cytotrophoblast cells and BeWo cells. By using live staining with Hoechst 33342 or cytoplasmic dyes or by stably transfecting enhanced green fluorescent protein (EGFP) and DsRed2-Nuc reporter plasmids, we observed finger-like protrusions on the cell membranes of fusion partners before fusion and the exchange of cytoplasmic contents during fusion. In summary, this study provides the first video recording of the process of trophoblast syncytialization. Furthermore, the various live-cell imaging systems used in this study will help to yield molecular insights into the syncytialization process during placental development. © 2014 by the Society for the Study of Reproduction, Inc.

Fibered Confocal Fluorescence Microscopy for the Noninvasive Imaging of Langerhans Cells in Macaques.

PubMed

Todorova, Biliana; Salabert, Nina; Tricot, Sabine; Boisgard, Raphaël; Rathaux, Mélanie; Le Grand, Roger; Chapon, Catherine

2017-01-01

We developed a new approach to visualize skin Langerhans cells by in vivo fluorescence imaging in nonhuman primates. Macaques were intradermally injected with a monoclonal, fluorescently labeled antibody against HLA-DR molecule and were imaged for up to 5 days by fibered confocal microscopy (FCFM). The network of skin Langerhans cells was visualized by in vivo fibered confocal fluorescence microscopy. Quantification of Langerhans cells revealed no changes to cell density with time. Ex vivo experiments confirmed that injected fluorescent HLA-DR antibody specifically targeted Langerhans cells in the epidermis. This study demonstrates the feasibility of single-cell, in vivo imaging as a noninvasive technique to track Langerhans cells in nontransgenic animals.

High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection

PubMed Central

Choudhry, Priya

2016-01-01

Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays. PMID:26848849

Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

PubMed Central

Hosny, Neveen A.; Song, Mingying; Connelly, John T.; Ameer-Beg, Simon; Knight, Martin M.; Wheeler, Ann P.

2013-01-01

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging. PMID:24130668

Dual Nuclear/Fluorescence Imaging Potantial of Zinc(II) Phthalocyanine in MIA PaCa-2 Cell Line.

PubMed

Lambrecht, Fatma Yurt; Ince, Mine; Er, Ozge; Ocakoglu, Kasim; Sarı, Fatma Aslıhan; Kayabasi, Cagla; Gunduz, Cumhur

2016-01-01

Pancreatic cancer is very common and difficult to diagnose in early stage. Imaging systems for diagnosing cancer have many disadvantages. However, combining different imaging modalities offers synergistic advantages. Optical imaging is the most multidirectional and widely used imaging modality in both clinical practice and research. In present study, Zinc(II) phthalocyanine [Zn(II)Pc] was synthesized, labeled with iodine- 131 and in vitro study was carried out. The intracellular uptake studies of radiolabeled Zn(II)Pc were performed in WI-38 [ATCC CCL-75™, tissue: human fibroblast lung] and MIA PaCa-2 [ATCC CRL-1420™, tissue: human epithelial pancreas carcinoma] cell lines. The intracellular uptake efficiency of radiolabeled Zn(II)Pc in MIA PaCa-2 cells was determined two times higher than WI-38 cells. Also, fluorescence imaging (FI) efficiency of synthesized Zn(II)Pc was investigated in MIA PaCa-2 cells and significant uptake was observed. Zn(II)Pc might be used as a new agent for dual fluorescence/nuclear imaging for pancreatic cancer. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Quantitative analyses for elucidating mechanisms of cell fate commitment in the mouse blastocyst

NASA Astrophysics Data System (ADS)

Saiz, Néstor; Kang, Minjung; Puliafito, Alberto; Schrode, Nadine; Xenopoulos, Panagiotis; Lou, Xinghua; Di Talia, Stefano; Hadjantonakis, Anna-Katerina

2015-03-01

In recent years we have witnessed a shift from qualitative image analysis towards higher resolution, quantitative analyses of imaging data in developmental biology. This shift has been fueled by technological advances in both imaging and analysis software. We have recently developed a tool for accurate, semi-automated nuclear segmentation of imaging data from early mouse embryos and embryonic stem cells. We have applied this software to the study of the first lineage decisions that take place during mouse development and established analysis pipelines for both static and time-lapse imaging experiments. In this paper we summarize the conclusions from these studies to illustrate how quantitative, single-cell level analysis of imaging data can unveil biological processes that cannot be revealed by traditional qualitative studies.

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.

Progress in Cell Marking for Synchrotron X-ray Computed Tomography

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

Hall, Christopher; Sturm, Erica; Schultke, Elisabeth

2010-07-23

Recently there has been an increase in research activity into finding ways of marking cells in live animals for pre-clinical trials. Development of certain drugs and other therapies crucially depend on tracking particular cells or cell types in living systems. Therefore cell marking techniques are required which will enable longitudinal studies, where individuals can be examined several times over the course of a therapy or study. The benefits of being able to study both disease and therapy progression in individuals, rather than cohorts are clear. The need for high contrast 3-D imaging, without harming or altering the biological system requiresmore » a non-invasive yet penetrating imaging technique. The technique will also have to provide an appropriate spatial and contrast resolution. X-ray computed tomography offers rapid acquisition of 3-D images and is set to become one of the principal imaging techniques in this area. Work by our group over the last few years has shown that marking cells with gold nano-particles (GNP) is an effective means of visualising marked cells in-vivo using x-ray CT. Here we report the latest results from these studies. Synchrotron X-ray CT images of brain lesions in rats taken using the SYRMEP facility at the Elettra synchrotron in 2009 have been compared with histological examination of the tissues. Some deductions are drawn about the visibility of the gold loaded cells in both light microscopy and x-ray imaging.« less

Progress in Cell Marking for Synchrotron X-ray Computed Tomography

NASA Astrophysics Data System (ADS)

Hall, Christopher; Sturm, Erica; Schultke, Elisabeth; Arfelli, Fulvia; Menk, Ralf-Hendrik; Astolfo, Alberto; Juurlink, Bernhard H. J.

2010-07-01

Recently there has been an increase in research activity into finding ways of marking cells in live animals for pre-clinical trials. Development of certain drugs and other therapies crucially depend on tracking particular cells or cell types in living systems. Therefore cell marking techniques are required which will enable longitudinal studies, where individuals can be examined several times over the course of a therapy or study. The benefits of being able to study both disease and therapy progression in individuals, rather than cohorts are clear. The need for high contrast 3-D imaging, without harming or altering the biological system requires a non-invasive yet penetrating imaging technique. The technique will also have to provide an appropriate spatial and contrast resolution. X-ray computed tomography offers rapid acquisition of 3-D images and is set to become one of the principal imaging techniques in this area. Work by our group over the last few years has shown that marking cells with gold nano-particles (GNP) is an effective means of visualising marked cells in-vivo using x-ray CT. Here we report the latest results from these studies. Synchrotron X-ray CT images of brain lesions in rats taken using the SYRMEP facility at the Elettra synchrotron in 2009 have been compared with histological examination of the tissues. Some deductions are drawn about the visibility of the gold loaded cells in both light microscopy and x-ray imaging.

High content image analysis for human H4 neuroglioma cells exposed to CuO nanoparticles.

PubMed

Li, Fuhai; Zhou, Xiaobo; Zhu, Jinmin; Ma, Jinwen; Huang, Xudong; Wong, Stephen T C

2007-10-09

High content screening (HCS)-based image analysis is becoming an important and widely used research tool. Capitalizing this technology, ample cellular information can be extracted from the high content cellular images. In this study, an automated, reliable and quantitative cellular image analysis system developed in house has been employed to quantify the toxic responses of human H4 neuroglioma cells exposed to metal oxide nanoparticles. This system has been proved to be an essential tool in our study. The cellular images of H4 neuroglioma cells exposed to different concentrations of CuO nanoparticles were sampled using IN Cell Analyzer 1000. A fully automated cellular image analysis system has been developed to perform the image analysis for cell viability. A multiple adaptive thresholding method was used to classify the pixels of the nuclei image into three classes: bright nuclei, dark nuclei, and background. During the development of our image analysis methodology, we have achieved the followings: (1) The Gaussian filtering with proper scale has been applied to the cellular images for generation of a local intensity maximum inside each nucleus; (2) a novel local intensity maxima detection method based on the gradient vector field has been established; and (3) a statistical model based splitting method was proposed to overcome the under segmentation problem. Computational results indicate that 95.9% nuclei can be detected and segmented correctly by the proposed image analysis system. The proposed automated image analysis system can effectively segment the images of human H4 neuroglioma cells exposed to CuO nanoparticles. The computational results confirmed our biological finding that human H4 neuroglioma cells had a dose-dependent toxic response to the insult of CuO nanoparticles.

Dual-Labeled Near-Infrared/99mTc Imaging Probes Using PAMAM-Coated Silica Nanoparticles for the Imaging of HER2-Expressing Cancer Cells

PubMed Central

Yamaguchi, Haruka; Tsuchimochi, Makoto; Hayama, Kazuhide; Kawase, Tomoyuki; Tsubokawa, Norio

2016-01-01

We sought to develop dual-modality imaging probes using functionalized silica nanoparticles to target human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer cells and achieve efficient target imaging of HER2-expressing tumors. Polyamidoamine-based functionalized silica nanoparticles (PCSNs) for multimodal imaging were synthesized with near-infrared (NIR) fluorescence (indocyanine green (ICG)) and technetium-99m (99mTc) radioactivity. Anti-HER2 antibodies were bound to the labeled PCSNs. These dual-imaging probes were tested to image HER2-overexpressing breast carcinoma cells. In vivo imaging was also examined in breast tumor xenograft models in mice. SK-BR3 (HER2 positive) cells were imaged with stronger NIR fluorescent signals than that in MDA-MB231 (HER2 negative) cells. The increased radioactivity of the SK-BR3 cells was also confirmed by phosphor imaging. NIR images showed strong fluorescent signals in the SK-BR3 tumor model compared to muscle tissues and the MDA-MB231 tumor model. Automatic well counting results showed increased radioactivity in the SK-BR3 xenograft tumors. We developed functionalized silica nanoparticles loaded with 99mTc and ICG for the targeting and imaging of HER2-expressing cells. The dual-imaging probes efficiently imaged HER2-overexpressing cells. Although further studies are needed to produce efficient isotope labeling, the results suggest that the multifunctional silica nanoparticles are a promising vehicle for imaging specific components of the cell membrane in a dual-modality manner. PMID:27399687

Dual-Labeled Near-Infrared/(99m)Tc Imaging Probes Using PAMAM-Coated Silica Nanoparticles for the Imaging of HER2-Expressing Cancer Cells.

PubMed

Yamaguchi, Haruka; Tsuchimochi, Makoto; Hayama, Kazuhide; Kawase, Tomoyuki; Tsubokawa, Norio

2016-07-07

We sought to develop dual-modality imaging probes using functionalized silica nanoparticles to target human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer cells and achieve efficient target imaging of HER2-expressing tumors. Polyamidoamine-based functionalized silica nanoparticles (PCSNs) for multimodal imaging were synthesized with near-infrared (NIR) fluorescence (indocyanine green (ICG)) and technetium-99m ((99m)Tc) radioactivity. Anti-HER2 antibodies were bound to the labeled PCSNs. These dual-imaging probes were tested to image HER2-overexpressing breast carcinoma cells. In vivo imaging was also examined in breast tumor xenograft models in mice. SK-BR3 (HER2 positive) cells were imaged with stronger NIR fluorescent signals than that in MDA-MB231 (HER2 negative) cells. The increased radioactivity of the SK-BR3 cells was also confirmed by phosphor imaging. NIR images showed strong fluorescent signals in the SK-BR3 tumor model compared to muscle tissues and the MDA-MB231 tumor model. Automatic well counting results showed increased radioactivity in the SK-BR3 xenograft tumors. We developed functionalized silica nanoparticles loaded with (99m)Tc and ICG for the targeting and imaging of HER2-expressing cells. The dual-imaging probes efficiently imaged HER2-overexpressing cells. Although further studies are needed to produce efficient isotope labeling, the results suggest that the multifunctional silica nanoparticles are a promising vehicle for imaging specific components of the cell membrane in a dual-modality manner.

Multiplex Quantitative Histologic Analysis of Human Breast Cancer Cell Signaling and Cell Fate

DTIC Science & Technology

2008-05-01

stains. 15. SUBJECT TERMS Breast cancer, cell signaling, cell proliferation, histology, image analysis 16. SECURITY CLASSIFICATION OF: 17...fluorescence, and these DAPI-stained nuclei are often not counted during subsequent image analysis ). To study two analytes in the same tumor section or...analytes (p-ERK, p-AKT, Ki67) and for epithelial cytokeratin (CK), so that tumor cells may be identified during subsequent automated image analysis (as

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.

Live CLEM imaging to analyze nuclear structures at high resolution.

PubMed

Haraguchi, Tokuko; Osakada, Hiroko; Koujin, Takako

2015-01-01

Fluorescence microscopy (FM) and electron microscopy (EM) are powerful tools for observing molecular components in cells. FM can provide temporal information about cellular proteins and structures in living cells. EM provides nanometer resolution images of cellular structures in fixed cells. We have combined FM and EM to develop a new method of correlative light and electron microscopy (CLEM), called "Live CLEM." In this method, the dynamic behavior of specific molecules of interest is first observed in living cells using fluorescence microscopy (FM) and then cellular structures in the same cell are observed using electron microscopy (EM). Following image acquisition, FM and EM images are compared to enable the fluorescent images to be correlated with the high-resolution images of cellular structures obtained using EM. As this method enables analysis of dynamic events involving specific molecules of interest in the context of specific cellular structures at high resolution, it is useful for the study of nuclear structures including nuclear bodies. Here we describe Live CLEM that can be applied to the study of nuclear structures in mammalian cells.

Replication of Muscle Cell Using Bioimprint

NASA Astrophysics Data System (ADS)

Samsuri, Fahmi; Mitchell, John S.; Alkaisi, Maan M.; Evans, John J.

2009-07-01

In our earlier study a heat-curable PDMS or a UV curable elastomer, was used as the replicating material to introduce Bioimprint methodology to facilitate cell imaging [1-2] But, replicating conditions for thermal polymerization is known to cause cell dehydration during curing. In this study, a new type of polymer was developed for use in living cell replica formation, and it was tested on human muscle cells. The cells were incubated and cultured according to standard biological culturing procedures, and they were grown for about 10 days. The replicas were then separated from the muscle cells and taken for analysis under an Atomic Force Microscope (AFM). The new polymer was designed to be biocompatible with higher resolution and fast curing process compared to other types of silicon-based organic polymers such as polydimethylsiloxane (PDMS). Muscle cell imprints were achieved and higher resolution images were able to show the micro structures of the muscle cells, including the cellular fibers and cell membranes. The AFM is able to image features at nanoscale resolution. This capacity enables a number of characteristics of biological cells to be visualized in a unique manner. Polymer and muscle cells preparations were developed at Hamilton, in collaboration between Plant and Food Research and the Department of Electrical and Computer Engineering, University of Canterbury. Tapping mode was used for the AFM image analysis as it has low tip-sample forces and non-destructive imaging capability. We will be presenting the bioimprinting processes of muscle cells, their AFM imaging and characterization of the newly developed polymer.

Spirally-patterned pinhole arrays for long-term fluorescence cell imaging.

PubMed

Koo, Bon Ung; Kang, YooNa; Moon, SangJun; Lee, Won Gu

2015-11-07

Fluorescence cell imaging using a fluorescence microscope is an extensively used technique to examine the cell nucleus, internal structures, and other cellular molecules with fluorescence response time and intensity. However, it is difficult to perform high resolution cell imaging for a long period of time with this technique due to necrosis and apoptosis depending on the type and subcellular location of the damage caused by phototoxicity. A large number of studies have been performed to resolve this problem, but researchers have struggled to meet the challenge between cellular viability and image resolution. In this study, we employ a specially designed disc to reduce cell damage by controlling total fluorescence exposure time without deterioration of the image resolution. This approach has many advantages such as, the apparatus is simple, cost-effective, and easily integrated into the optical pathway through a conventional fluorescence microscope.

Live-Cell Imaging of the Adult Drosophila Ovary Using Confocal Microscopy.

PubMed

Shalaby, Nevine A; Buszczak, Michael

2017-01-01

The Drosophila ovary represents a key in vivo model used to study germline stem cell (GSC) maintenance and stem cell daughter differentiation because these cells and their somatic cell neighbors can be identified at single-cell resolution within their native environment. Here we describe a fluorescent-based technique for the acquisition of 4D datasets of the Drosophila ovariole for periods that can exceed 12 consecutive hours. Live-cell imaging facilitates the investigation of molecular and cellular dynamics that were not previously possible using still images.

Imaging cell picker: A morphology-based automated cell separation system on a photodegradable hydrogel culture platform.

PubMed

Shibuta, Mayu; Tamura, Masato; Kanie, Kei; Yanagisawa, Masumi; Matsui, Hirofumi; Satoh, Taku; Takagi, Toshiyuki; Kanamori, Toshiyuki; Sugiura, Shinji; Kato, Ryuji

2018-06-09

Cellular morphology on and in a scaffold composed of extracellular matrix generally represents the cellular phenotype. Therefore, morphology-based cell separation should be interesting method that is applicable to cell separation without staining surface markers in contrast to conventional cell separation methods (e.g., fluorescence activated cell sorting and magnetic activated cell sorting). In our previous study, we have proposed a cloning technology using a photodegradable gelatin hydrogel to separate the individual cells on and in hydrogels. To further expand the applicability of this photodegradable hydrogel culture platform, we here report an image-based cell separation system imaging cell picker for the morphology-based cell separation on a photodegradable hydrogel. We have developed the platform which enables the automated workflow of image acquisition, image processing and morphology analysis, and collection of a target cells. We have shown the performance of the morphology-based cell separation through the optimization of the critical parameters that determine the system's performance, such as (i) culture conditions, (ii) imaging conditions, and (iii) the image analysis scheme, to actually clone the cells of interest. Furthermore, we demonstrated the morphology-based cloning performance of cancer cells in the mixture of cells by automated hydrogel degradation by light irradiation and pipetting. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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.

Alterations of the cytoskeleton in human cells in space proved by life-cell imaging.

PubMed

Corydon, Thomas J; Kopp, Sascha; Wehland, Markus; Braun, Markus; Schütte, Andreas; Mayer, Tobias; Hülsing, Thomas; Oltmann, Hergen; Schmitz, Burkhard; Hemmersbach, Ruth; Grimm, Daniela

2016-01-28

Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24(th) DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31(st) parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization.

Alterations of the cytoskeleton in human cells in space proved by life-cell imaging

PubMed Central

Corydon, Thomas J.; Kopp, Sascha; Wehland, Markus; Braun, Markus; Schütte, Andreas; Mayer, Tobias; Hülsing, Thomas; Oltmann, Hergen; Schmitz, Burkhard; Hemmersbach, Ruth; Grimm, Daniela

2016-01-01

Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24th DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31st parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization. PMID:26818711

An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy

NASA Astrophysics Data System (ADS)

Wollman, Adam J. M.; Miller, Helen; Foster, Simon; Leake, Mark C.

2016-10-01

Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules. We demonstrate this approach using a fluorescent reporter GFP fused to the protein EzrA that localises to a mid-cell plane during division and is involved in regulation of cell size and division. This image analysis framework presents a valuable platform from which to study candidate new antimicrobials which target the cell division machinery, but may also have more general application in detecting morphologically complex structures of fluorescently labelled proteins present in clusters of other types of cells.

CARS hyperspectral imaging of cartilage aiming for state discrimination of cell

NASA Astrophysics Data System (ADS)

Shiozawa, Manabu; Shirai, Masataka; Izumisawa, Junko; Tanabe, Maiko; Watanabe, Koichi

2016-03-01

Non-invasive cell analyses are increasingly important for medical field. A CARS microscope is one of the non-invasive imaging equipments and enables to obtain images indicating molecular distribution. Some studies on discrimination of cell state by using CARS images of lipid are reported. However, due to low signal intensity, it is still challenging to obtain images of the fingerprint region (800~1800 cm-1), in which many spectrum peaks correspond to compositions of a cell. Here, to identify cell differentiation by using multiplex CARS, we investigated hyperspectral imaging of fingerprint region of living cells. To perform multiplex CARS, we used a prototype of a compact light source, which consists of a microchip laser, a single-mode fiber, and a photonic crystal fiber to generate supercontinuum light. Assuming application to regenerative medicine, we chose a cartilage cell, whose differentiation is difficult to be identified by change of the cell morphology. Because one of the major components of cartilage is collagen, we focused on distribution of proline, which accounts for approximately 20% of collagen in general. The spectrum quality was improved by optical adjustments about power branching ratio and divergence of broadband Stokes light. Hyperspectral images were successfully obtained by the improvement. Periphery of a cartilage cell was highlighted in CARS image of proline, and this result suggests correspondence with collagen generated as extracellular matrix. A possibility of cell analyses by using CARS hyperspectral imaging was indicated.

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

PubMed

Peng, Tao; Hang, Howard C

2016-11-02

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing.

PubMed

Moo, Eng Kuan; Abusara, Ziad; Abu Osman, Noor Azuan; Pingguan-Murphy, Belinda; Herzog, Walter

2013-08-09

Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in cell responses to mechanical stimuli that depend on the microscopic approach and the thresholding methods used and may result in contradictory interpretations. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

PubMed

Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2017-10-01

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Nanofiber Nerve Guide for Peripheral Nerve Repair and Regeneration

DTIC Science & Technology

2014-01-01

observing cell migration using live - cell imaging microscopy, and analyzing cell migration with our MATLAB-based programs. Our studies...are then pipetted into the chamber and their path of migration is observed using a live - cell imaging microscope (Fig. 6d). Utilizing this migration

Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy

NASA Astrophysics Data System (ADS)

Taik Lim, Yong; Cho, Mi Young; Noh, Young-Woock; Chung, Jin Woong; Chung, Bong Hyun

2009-11-01

This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN- γ) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

Three-Dimensional Unstained Live-Cell Imaging Using Stimulated Parametric Emission Microscopy

NASA Astrophysics Data System (ADS)

Dang, Hieu M.; Kawasumi, Takehito; Omura, Gen; Umano, Toshiyuki; Kajiyama, Shin'ichiro; Ozeki, Yasuyuki; Itoh, Kazuyoshi; Fukui, Kiichi

2009-09-01

The ability to perform high-resolution unstained live imaging is very important to in vivo study of cell structures and functions. Stimulated parametric emission (SPE) microscopy is a nonlinear-optical microscopy based on ultra-fast electronic nonlinear-optical responses. For the first time, we have successfully applied this technique to archive three-dimensional (3D) images of unstained sub-cellular structures, such as, microtubules, nuclei, nucleoli, etc. in live cells. Observation of a complete cell division confirms the ability of SPE microscopy for long time-scale imaging.

Fourier transform infrared spectroscopy imaging of live epithelial cancer cells under non-aqueous media.

PubMed

Soh, JunYi; Chueng, Adeline; Adio, Aminat; Cooper, Alan J; Birch, Brian R; Lwaleed, Bashir A

2013-04-01

Fourier transform infrared (FT-IR) imaging is increasingly being applied to biomedical specimens, but strong IR absorption by water complicates live cell imaging. This study investigates the viability of adherent epithelial cells maintained for short periods under mineral oils in order to facilitate live cell spectroscopy using FT-IR with subsequent imaging. The MGH-U1 urothelial or CaCo2 colorectal cancer cell lines were grown on plastic surfaces or mid-range infrared transparent windows. Medium in established cultures was replaced with paraffin mineral oil, or Fluorolube, for up to 2 h, and viability assessed by supravital staining. Drug handling characteristics were also assessed. Imaging of preparations was attempted by reflectance and transmission using a Varian FT-IR microscope. Cells covered by mineral oil remained viable for 2 h, with recovery into normal medium possible. MTT ((3-(4,5-dimethylthlazol-2-yl)-2,5-diphenyl tetrazolium) conversion to crystalline formazan and differential patterns of drug uptake were maintained. The combination of a calcium fluoride substrate, Fluorolube oil, and transmission optics proved best for spectroscopy. Spectral features were used to obtain images of live cells. The viability of cells overlaid with IR transparent oils was assessed as part of a technique to optimise conditions for FT-IR imaging. Images of untreated cells were obtained using both reflectance and transmission. This represents an effective means of imaging live cells by IR spectroscopy, and also means that imaging is not necessarily a terminal event. It also increases options for producing images based on real-time biochemistry in a range of in vitro experimental and 'optical biopsy' contexts.

Time-lapse contact microscopy of cell cultures based on non-coherent illumination

NASA Astrophysics Data System (ADS)

Gabriel, Marion; Balle, Dorothée; Bigault, Stéphanie; Pornin, Cyrille; Gétin, Stéphane; Perraut, François; Block, Marc R.; Chatelain, François; Picollet-D'Hahan, Nathalie; Gidrol, Xavier; Haguet, Vincent

2015-10-01

Video microscopy offers outstanding capabilities to investigate the dynamics of biological and pathological mechanisms in optimal culture conditions. Contact imaging is one of the simplest imaging architectures to digitally record images of cells due to the absence of any objective between the sample and the image sensor. However, in the framework of in-line holography, other optical components, e.g., an optical filter or a pinhole, are placed underneath the light source in order to illuminate the cells with a coherent or quasi-coherent incident light. In this study, we demonstrate that contact imaging with an incident light of both limited temporal and spatial coherences can be achieved with sufficiently high quality for most applications in cell biology, including monitoring of cell sedimentation, rolling, adhesion, spreading, proliferation, motility, death and detachment. Patterns of cells were recorded at various distances between 0 and 1000 μm from the pixel array of the image sensors. Cells in suspension, just deposited or at mitosis focalise light into photonic nanojets which can be visualised by contact imaging. Light refraction by cells significantly varies during the adhesion process, the cell cycle and among the cell population in connection with every modification in the tridimensional morphology of a cell.

Cell nuclei and cytoplasm joint segmentation using the sliding band filter.

PubMed

Quelhas, Pedro; Marcuzzo, Monica; Mendonça, Ana Maria; Campilho, Aurélio

2010-08-01

Microscopy cell image analysis is a fundamental tool for biological research. In particular, multivariate fluorescence microscopy is used to observe different aspects of cells in cultures. It is still common practice to perform analysis tasks by visual inspection of individual cells which is time consuming, exhausting and prone to induce subjective bias. This makes automatic cell image analysis essential for large scale, objective studies of cell cultures. Traditionally the task of automatic cell analysis is approached through the use of image segmentation methods for extraction of cells' locations and shapes. Image segmentation, although fundamental, is neither an easy task in computer vision nor is it robust to image quality changes. This makes image segmentation for cell detection semi-automated requiring frequent tuning of parameters. We introduce a new approach for cell detection and shape estimation in multivariate images based on the sliding band filter (SBF). This filter's design makes it adequate to detect overall convex shapes and as such it performs well for cell detection. Furthermore, the parameters involved are intuitive as they are directly related to the expected cell size. Using the SBF filter we detect cells' nucleus and cytoplasm location and shapes. Based on the assumption that each cell has the same approximate shape center in both nuclei and cytoplasm fluorescence channels, we guide cytoplasm shape estimation by the nuclear detections improving performance and reducing errors. Then we validate cell detection by gathering evidence from nuclei and cytoplasm channels. Additionally, we include overlap correction and shape regularization steps which further improve the estimated cell shapes. The approach is evaluated using two datasets with different types of data: a 20 images benchmark set of simulated cell culture images, containing 1000 simulated cells; a 16 images Drosophila melanogaster Kc167 dataset containing 1255 cells, stained for DNA and actin. Both image datasets present a difficult problem due to the high variability of cell shapes and frequent cluster overlap between cells. On the Drosophila dataset our approach achieved a precision/recall of 95%/69% and 82%/90% for nuclei and cytoplasm detection respectively and an overall accuracy of 76%.

Liquid scanning transmission electron microscopy: imaging protein complexes in their native environment in whole eukaryotic cells.

PubMed

Peckys, Diana B; de Jonge, Niels

2014-04-01

Scanning transmission electron microscopy (STEM) of specimens in liquid, so-called Liquid STEM, is capable of imaging the individual subunits of macromolecular complexes in whole eukaryotic cells in liquid. This paper discusses this new microscopy modality within the context of state-of-the-art microscopy of cells. The principle of operation and equations for the resolution are described. The obtained images are different from those acquired with standard transmission electron microscopy showing the cellular ultrastructure. Instead, contrast is obtained on specific labels. Images can be recorded in two ways, either via STEM at 200 keV electron beam energy using a microfluidic chamber enclosing the cells, or via environmental scanning electron microscopy at 30 keV of cells in a wet environment. The first series of experiments involved the epidermal growth factor receptor labeled with gold nanoparticles. The labels were imaged in whole fixed cells with nanometer resolution. Since the cells can be kept alive in the microfluidic chamber, it is also feasible to detect the labels in unfixed, live cells. The rapid sample preparation and imaging allows studies of multiple whole cells.

Evaluating the extent of cell death in 3D high frequency ultrasound by registration with whole-mount tumor histopathology

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

Vlad, Roxana M.; Kolios, Michael C.; Moseley, Joanne L.

Purpose: High frequency ultrasound imaging, 10-30 MHz, has the capability to assess tumor response to radiotherapy in mouse tumors as early as 24 h after treatment administration. The advantage of this technique is that the image contrast is generated by changes in the physical properties of dying cells. Therefore, a subject can be imaged before and multiple times during the treatment without the requirement of injecting specialized contrast agents. This study is motivated by a need to provide metrics of comparison between the volume and localization of cell death, assessed from histology, with the volume and localization of cell deathmore » surrogate, assessed as regions with increased echogeneity from ultrasound images. Methods: The mice were exposed to radiation doses of 2, 4, and 8 Gy. Ultrasound images were collected from each tumor before and 24 h after exposure to radiation using a broadband 25 MHz center frequency transducer. After radiotherapy, tumors exhibited hyperechoic regions in ultrasound images that corresponded to areas of cell death in histology. The ultrasound and histological images were rigidly registered. The tumors and regions of cell death were manually outlined on histological images. Similarly, the tumors and hyperechoic regions were outlined on the ultrasound images. Each set of contours was converted to a volumetric mesh in order to compare the volumes and the localization of cell death in histological and ultrasound images. Results: A shrinkage factor of 17{+-}2% was calculated from the difference in the tumor volumes evaluated from histological and ultrasound images. This was used to correct the tumor and cell death volumes assessed from histology. After this correction, the average absolute difference between the volume of cell death assessed from ultrasound and histological images was 11{+-}14% and the volume overlap was 70{+-}12%. Conclusions: The method provided metrics of comparison between the volume of cell death assessed from histology and that assessed from ultrasound images. It was applied here to evaluate the capability of ultrasound imaging to assess early tumor response to radiotherapy in mouse tumors. Similarly, it can be applied in the future to evaluate the capability of ultrasound imaging to assess early tumor response to other modalities of cancer treatment. The study contributes to an understanding of the capabilities and limitation of ultrasound imaging at noninvasively detecting cell death. This provides a foundation for future developments regarding the use of ultrasound in preclinical and clinical applications to adapt treatments based on tumor response to cancer therapy.« less

Radionuclide and Fluorescence Imaging of Clear Cell Renal Cell Carcinoma Using Dual Labeled Anti-Carbonic Anhydrase IX Antibody G250.

PubMed

Muselaers, Constantijn H J; Rijpkema, Mark; Bos, Desirée L; Langenhuijsen, Johan F; Oyen, Wim J G; Mulders, Peter F A; Oosterwijk, Egbert; Boerman, Otto C

2015-08-01

Tumor targeted optical imaging using antibodies labeled with near infrared fluorophores is a sensitive imaging modality that might be used during surgery to assure complete removal of malignant tissue. We evaluated the feasibility of dual modality imaging and image guided surgery with the dual labeled anti-carbonic anhydrase IX antibody preparation (111)In-DTPA-G250-IRDye800CW in mice with intraperitoneal clear cell renal cell carcinoma. BALB/c nu/nu mice with intraperitoneal SK-RC-52 lesions received 10 μg DTPA-G250-IRDye800CW labeled with 15 MBq (111)In or 10 μg of the dual labeled irrelevant control antibody NUH-82 (20 mice each). To evaluate when tumors could be detected, 4 mice per group were imaged weekly during 5 weeks with single photon emission computerized tomography/computerized tomography and the fluorescence imaging followed by ex vivo biodistribution studies. As early as 1 week after tumor cell inoculation single photon emission computerized tomography and fluorescence images showed clear delineation of intraperitoneal clear cell renal cell carcinoma with good concordance between single photon emission computerized tomography/computerized tomography and fluorescence images. The high and specific accumulation of the dual labeled antibody conjugate in tumors was confirmed in the biodistribution studies. Maximum tumor uptake was observed 1 week after inoculation (mean ± SD 58.5% ± 18.7% vs 5.6% ± 2.3% injected dose per gm for DTPA-G250-IRDye800CW vs NUH-82, respectively). High tumor uptake was also observed at other time points. This study demonstrates the feasibility of dual modality imaging with dual labeled antibody (111)In-DTPA-G250-IRDye800CW in a clear cell renal cell carcinoma model. Results indicate that preoperative and intraoperative detection of carbonic anhydrase IX expressing tumors, positive resection margins and metastasis might be feasible with this approach. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Nanoscale live cell imaging using hopping probe ion conductance microscopy

PubMed Central

Novak, Pavel; Li, Chao; Shevchuk, Andrew I.; Stepanyan, Ruben; Caldwell, Matthew; Hughes, Simon; Smart, Trevor G.; Gorelik, Julia; Ostanin, Victor P.; Lab, Max J.; Moss, Guy W. J.; Frolenkov, Gregory I.; Klenerman, David; Korchev, Yuri E.

2009-01-01

We describe a major advance in scanning ion conductance microscopy: a new hopping mode that allows non-contact imaging of the complex surfaces of live cells with resolution better than 20 nm. The effectiveness of this novel technique was demonstrated by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allows studying nanoscale phenomena on the surface of live cells under physiological conditions. PMID:19252505

Lens-free shadow image based high-throughput continuous cell monitoring technique.

PubMed

Jin, Geonsoo; Yoo, In-Hwa; Pack, Seung Pil; Yang, Ji-Woon; Ha, Un-Hwan; Paek, Se-Hwan; Seo, Sungkyu

2012-01-01

A high-throughput continuous cell monitoring technique which does not require any labeling reagents or destruction of the specimen is demonstrated. More than 6000 human alveolar epithelial A549 cells are monitored for up to 72 h simultaneously and continuously with a single digital image within a cost and space effective lens-free shadow imaging platform. In an experiment performed within a custom built incubator integrated with the lens-free shadow imaging platform, the cell nucleus division process could be successfully characterized by calculating the signal-to-noise ratios (SNRs) and the shadow diameters (SDs) of the cell shadow patterns. The versatile nature of this platform also enabled a single cell viability test followed by live cell counting. This study firstly shows that the lens-free shadow imaging technique can provide a continuous cell monitoring without any staining/labeling reagent and destruction of the specimen. This high-throughput continuous cell monitoring technique based on lens-free shadow imaging may be widely utilized as a compact, low-cost, and high-throughput cell monitoring tool in the fields of drug and food screening or cell proliferation and viability testing. Copyright © 2012 Elsevier B.V. All rights reserved.

Studying the relationship between redox and cell growth using quantitative phase imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Sridharan, Shamira; Leslie, Matthew T.; Bapst, Natalya; Smith, John; Gaskins, H. Rex; Popescu, Gabriel

2016-03-01

Quantitative phase imaging has been used in the past to study the dry mass of cells and study cell growth under various treatment conditions. However, the relationship between cellular redox and growth rates has not yet been studied in this context. This study employed the recombinant Glrx-roGFP2 redox biosensor targeted to the mitochondrial matrix or cytosolic compartments of A549 lung epithelial carcinoma cells. The Glrx-roGFP2s biosensor consists of a modified GFP protein containing internal cysteine residues sensitive to the local redox environment. The formation/dissolution of sulfide bridges contorts the internal chromophore, dictating corresponding changes in florescence emission that provide direct measures of the local redox potential. Combining 2-channel florescent imaging of the redox sensor with quantitative phase imaging allowed observation of redox homeostasis alongside measurements of cellular mass during full cycles of cellular division. The results indicate that mitochondrial redox showed a stronger inverse correlation with cell growth than cytoplasmic redox states; although redox changes are restricted to a 5% range. We are now studying the relationship between mitochondrial redox and cell growth in an isogenic series of breast cell lines built upon the MCF-10A genetic background that vary both in malignancy and metastatic potential.

Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope.

PubMed

Peckys, Diana B; Veith, Gabriel M; Joy, David C; de Jonge, Niels

2009-12-14

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.

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

Anna-Liisa Brownell

Adult progenitor cells hold promise for therapeutic treatment where there has been a disabling loss of function due to death of cells from trauma, disease or aging. However, it will be essential in clinical application to be able to follow the fate of the transplanted cells over time using in vivo tracking methods. We have developed protocol for labeling of progenitor cells to monitor cell trafficking by high resolution magnetic resonance imaging (MRI) and super high resolution positron emission tomography (PET). We have transfected rat subventricular zone stem cells (SVZ, progenitor cell line) and another control cell line (PC12, pheochromocytomamore » cells) utilizing super paramagnetic iron oxide and poly-L-lysine complex for MR imaging or radiolabeling with 18F-fluor deoxy-D- glucose for PET imaging. The labeled cells were transplanted into the rostral migratory stream (RMS) or striatum of normal or 6-hydroxydopamine lesioned Spraque-Dawley rats. Longitudinal MRI studies (up to 40 days) showed that transplantation site has significant impact to the fate of the cells; when SVZ cells were transplanted into the RMS, cells migrated several centimeter into the olfactory bulb; after transplantation into the striatum, the migration was minimal, only 2 mm. PC 12 cells grew a massive tumor after the striatal implantation and significantly smaller tumor after the RMS implantation. PET studies conducted immediately after transplantation verified the transplantation site. MRI studies were able to show the whole path of migration in one image, since part of the cells die during migration and will get detected because of iron content. Endpoint histological studies verified the cell survival and immunohistochemical studies revealed the differentiation of the transplanted cells into astrocytes and neurons.« less

Ferritin heavy chain as a molecular imaging reporter gene in glioma xenografts.

PubMed

Cheng, Sen; Mi, Ruifang; Xu, Yu; Jin, Guishan; Zhang, Junwen; Zhou, Yiqiang; Chen, Zhengguang; Liu, Fusheng

2017-06-01

The development of glioma therapy in clinical practice (e.g., gene therapy) calls for efficiently visualizing and tracking glioma cells in vivo. Human ferritin heavy chain is a novel gene reporter in magnetic resonance imaging. This study proposes hFTH as a reporter gene for MR molecular imaging in glioma xenografts. Rat C6 glioma cells were infected by packaged lentivirus carrying hFTH and EGFP genes and obtained by fluorescence-activated cell sorting. The iron-loaded ability was analyzed by the total iron reagent kit. Glioma nude mouse models were established subcutaneously and intracranially. Then, in vivo tumor bioluminescence was performed via the IVIS spectrum imaging system. The MR imaging analysis was analyzed on a 7T animal MRI scanner. Finally, the expression of hFTH was analyzed by western blotting and histological analysis. Stable glioma cells carrying hFTH and EGFP reporter genes were successfully obtained. The intracellular iron concentration was increased without impairing the cell proliferation rate. Glioma cells overexpressing hFTH showed significantly decreased signal intensity on T 2 -weighted MRI both in vitro and in vivo. EGFP fluorescent imaging could also be detected in the subcutaneous and intracranial glioma xenografts. Moreover, the expression of the transferritin receptor was significantly increased in glioma cells carrying the hFTH reporter gene. Our study illustrated that hFTH generated cellular MR imaging contrast efficiently in glioma via regulating the expression of transferritin receptor. This might be a useful reporter gene in cell tracking and MR molecular imaging for glioma diagnosis, gene therapy and tumor metastasis.

Imaging of oxygenation in 3D tissue models with multi-modal phosphorescent probes

NASA Astrophysics Data System (ADS)

Papkovsky, Dmitri B.; Dmitriev, Ruslan I.; Borisov, Sergei

2015-03-01

Cell-penetrating phosphorescence based probes allow real-time, high-resolution imaging of O2 concentration in respiring cells and 3D tissue models. We have developed a panel of such probes, small molecule and nanoparticle structures, which have different spectral characteristics, cell penetrating and tissue staining behavior. The probes are compatible with conventional live cell imaging platforms and can be used in different detection modalities, including ratiometric intensity and PLIM (Phosphorescence Lifetime IMaging) under one- or two-photon excitation. Analytical performance of these probes and utility of the O2 imaging method have been demonstrated with different types of samples: 2D cell cultures, multi-cellular spheroids from cancer cell lines and primary neurons, excised slices from mouse brain, colon and bladder tissue, and live animals. They are particularly useful for hypoxia research, ex-vivo studies of tissue physiology, cell metabolism, cancer, inflammation, and multiplexing with many conventional fluorophors and markers of cellular function.

Photoacoustic imaging of single circulating melanoma cells in vivo

NASA Astrophysics Data System (ADS)

Wang, Lidai; Yao, Junjie; Zhang, Ruiying; Xu, Song; Li, Guo; Zou, Jun; Wang, Lihong V.

2015-03-01

Melanoma, one of the most common types of skin cancer, has a high mortality rate, mainly due to a high propensity for tumor metastasis. The presence of circulating tumor cells (CTCs) is a potential predictor for metastasis. Label-free imaging of single circulating melanoma cells in vivo provides rich information on tumor progress. Here we present photoacoustic microscopy of single melanoma cells in living animals. We used a fast-scanning optical-resolution photoacoustic microscope to image the microvasculature in mouse ears. The imaging system has sub-cellular spatial resolution and works in reflection mode. A fast-scanning mirror allows the system to acquire fast volumetric images over a large field of view. A 500-kHz pulsed laser was used to image blood and CTCs. Single circulating melanoma cells were imaged in both capillaries and trunk vessels in living animals. These high-resolution images may be used in early detection of CTCs with potentially high sensitivity. In addition, this technique enables in vivo study of tumor cell extravasation from a primary tumor, which addresses an urgent pre-clinical need.

Quantitative photothermal phase imaging of red blood cells using digital holographic photothermal microscope.

PubMed

Vasudevan, Srivathsan; Chen, George C K; Lin, Zhiping; Ng, Beng Koon

2015-05-10

Photothermal microscopy (PTM), a noninvasive pump-probe high-resolution microscopy, has been applied as a bioimaging tool in many biomedical studies. PTM utilizes a conventional phase contrast microscope to obtain highly resolved photothermal images. However, phase information cannot be extracted from these photothermal images, as they are not quantitative. Moreover, the problem of halos inherent in conventional phase contrast microscopy needs to be tackled. Hence, a digital holographic photothermal microscopy technique is proposed as a solution to obtain quantitative phase images. The proposed technique is demonstrated by extracting phase values of red blood cells from their photothermal images. These phase values can potentially be used to determine the temperature distribution of the photothermal images, which is an important study in live cell monitoring applications.

In vivo imaging of the retinal pigment epithelial cells

NASA Astrophysics Data System (ADS)

Morgan, Jessica Ijams Wolfing

The retinal pigment epithelial (RPE) cells form an important layer of the retina because they are responsible for providing metabolic support to the photoreceptors. Techniques to image the RPE layer include autofluorescence imaging with a scanning laser ophthalmoscope (SLO). However, previous studies were unable to resolve single RPE cells in vivo. This thesis describes the technique of combining autofluorescence, SLO, adaptive optics (AO), and dual-wavelength simultaneous imaging and registration to visualize the individual cells in the RPE mosaic in human and primate retina for the first time in vivo. After imaging the RPE mosaic non-invasively, the cell layer's structure and regularity were characterized using quantitative metrics of cell density, spacing, and nearest neighbor distances. The RPE mosaic was compared to the cone mosaic, and RPE imaging methods were confirmed using histology. The ability to image the RPE mosaic led to the discovery of a novel retinal change following light exposure; 568 nm exposures caused an immediate reduction in autofluorescence followed by either full recovery or permanent damage in the RPE layer. A safety study was conducted to determine the range of exposure irradiances that caused permanent damage or transient autofluorescence reductions. Additionally, the threshold exposure causing autofluorescence reduction was determined and reciprocity of radiant exposure was confirmed. Light exposures delivered by the AOSLO were not significantly different than those delivered by a uniform source. As all exposures tested were near or below the permissible light levels of safety standards, this thesis provides evidence that the current light safety standards need to be revised. Finally, with the retinal damage and autofluorescence reduction thresholds identified, the methods of RPE imaging were modified to allow successful imaging of the individual cells in the RPE mosaic while still ensuring retinal safety. This thesis has provided a highly sensitive method for studying the in vivo morphology of individual RPE cells in normal, diseased, and damaged retinas. The methods presented here also will allow longitudinal studies for tracking disease progression and assessing treatment efficacy in human patients and animal models of retinal diseases affecting the RPE.

Single-Molecule Light-Sheet Imaging of Suspended T Cells.

PubMed

Ponjavic, Aleks; McColl, James; Carr, Alexander R; Santos, Ana Mafalda; Kulenkampff, Klara; Lippert, Anna; Davis, Simon J; Klenerman, David; Lee, Steven F

2018-05-08

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose. Copyright © 2018. Published by Elsevier Inc.

Time lapse video recordings of highly purified human hematopoietic progenitor cells in culture.

PubMed

Denkers, I A; Dragowska, W; Jaggi, B; Palcic, B; Lansdorp, P M

1993-05-01

Major hurdles in studies of stem cell biology include the low frequency and heterogeneity of human hematopoietic precursor cells in bone marrow and the difficulty of directly studying the effect of various culture conditions and growth factors on such cells. We have adapted the cell analyzer imaging system for monitoring and recording the morphology of limited numbers of cells under various culture conditions. Hematopoietic progenitor cells with a CD34+ CD45RAlo CD71lo phenotype were purified from previously frozen organ donor bone marrow by fluorescence activated cell sorting. Cultures of such cells were analyzed with the imaging system composed of an inverted microscope contained in an incubator, a video camera, an optical memory disk recorder and a computer-controlled motorized microscope XYZ precision stage. Fully computer-controlled video images at defined XYZ positions were captured at selected time intervals and recorded at a predetermined sequence on an optical memory disk. In this study, the cell analyzer system was used to obtain descriptions and measurements of hematopoietic cell behavior, like cell motility, cell interactions, cell shape, cell division, cell cycle time and cell size changes under different culture conditions.

Image analysis for the automated estimation of clonal growth and its application to the growth of smooth muscle cells.

PubMed

Gavino, V C; Milo, G E; Cornwell, D G

1982-03-01

Image analysis was used for the automated measurement of colony frequency (f) and colony diameter (d) in cultures of smooth muscle cells, Initial studies with the inverted microscope showed that number of cells (N) in a colony varied directly with d: log N = 1.98 log d - 3.469 Image analysis generated the complement of a cumulative distribution for f as a function of d. The number of cells in each segment of the distribution function was calculated by multiplying f and the average N for the segment. These data were displayed as a cumulative distribution function. The total number of colonies (fT) and the total number of cells (NT) were used to calculate the average colony size (NA). Population doublings (PD) were then expressed as log2 NA. Image analysis confirmed previous studies in which colonies were sized and counted with an inverted microscope. Thus, image analysis is a rapid and automated technique for the measurement of clonal growth.

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

PubMed

Krtolica, Ana; Ortiz de Solorzano, Carlos; Lockett, Stephen; Campisi, Judith

2002-10-01

To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies. Copyright 2002 Wiley-Liss, Inc.

Detecting cells in time varying intensity images in confocal microscopy for gene expression studies in living cells

NASA Astrophysics Data System (ADS)

Mitra, Debasis; Boutchko, Rostyslav; Ray, Judhajeet; Nilsen-Hamilton, Marit

2015-03-01

In this work we present a time-lapsed confocal microscopy image analysis technique for an automated gene expression study of multiple single living cells. Fluorescence Resonance Energy Transfer (FRET) is a technology by which molecule-to-molecule interactions are visualized. We analyzed a dynamic series of ~102 images obtained using confocal microscopy of fluorescence in yeast cells containing RNA reporters that give a FRET signal when the gene promoter is activated. For each time frame, separate images are available for three spectral channels and the integrated intensity snapshot of the system. A large number of time-lapsed frames must be analyzed to identify each cell individually across time and space, as it is moving in and out of the focal plane of the microscope. This makes it a difficult image processing problem. We have proposed an algorithm here, based on scale-space technique, which solves the problem satisfactorily. The algorithm has multiple directions for even further improvement. The ability to rapidly measure changes in gene expression simultaneously in many cells in a population will open the opportunity for real-time studies of the heterogeneity of genetic response in a living cell population and the interactions between cells that occur in a mixed population, such as the ones found in the organs and tissues of multicellular organisms.

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.

Pathological proof of cellular death in radiofrequency ablation therapy and correlation with flash echo imaging--an experiment study.

PubMed

Fujiki, Kei

2004-01-01

The aims of this study were to clarify the geographic distribution of complete cell death in the radiofrequency ablated area in a porcine liver experiment, and to evaluate the efficacy of ultrasonography using contrast media in detecting the area of Radiofrequency-induced cell death. Radiofrequency ablation was performed at 3 sites in each liver in seven swine with a RF2000TM radiofrequency generator using an expandable type needle electrode. The ablation area was investigated histologically by Hematoxylin-Eosin staining and NADH staining. The area of radiofrequency-induced cell death was correlated to the ultrasonographic findings using contrast media, by means of contrast harmonic imaging, flash echo imaging-subtraction and flash echo imaging-power Doppler. The ablation area showed three distinct regions. Although the HE staining did not indicate necrosis, the NADH staining showed a complete loss of cellular activity in the inner and middle layers of the ablation area. However, in the outer layer cells displaying cellular integrity were intermingled with the necrotic cells, indicating that some of the cells in this layer had a chance to survive. Further, in some cases the outer layer of the ablated area had irregular margins. The flash-echo power-doppler images were accurately correlated in size and shape to the pathologically proved region of complete cell death in the radiofrequency-induced lesions. In the marginal part of the radiofrequency ablation area, cell death was incomplete. Flash echo imaging-power doppler was a useful and sensitive real time imaging technique for accurate evaluation of the region of complete cell death.

Multiphoton autofluorescence lifetime imaging of induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada

2017-06-01

The multiphoton fluorescence lifetime imaging tomograph MPTflex with its flexible 360-deg scan head, articulated arm, and tunable femtosecond laser source was employed to study induced pluripotent stem cell (iPS) cultures. Autofluorescence (AF) lifetime imaging was performed with 250-ps temporal resolution and submicron spatial resolution using time-correlated single-photon counting. The two-photon excited AF was based on the metabolic coenzymes NAD(P)H and flavin adenine dinucleotide/flavoproteins. iPS cells generated from mouse embryonic fibroblasts (MEFs) and cocultured with growth-arrested MEFs as feeder cells have been studied. Significant differences on AF lifetime signatures were identified between iPS and feeder cells as well as between their differentiating counterparts.

Targeting Phosphatidylserine with a 64Cu-Labeled Peptide for Molecular Imaging of Apoptosis.

PubMed

Perreault, Amanda; Richter, Susan; Bergman, Cody; Wuest, Melinda; Wuest, Frank

2016-10-03

Molecular imaging of programmed cell death (apoptosis) in vivo is an innovative strategy for early assessment of treatment response and treatment efficacy in cancer patients. Externalization of phosphatidylserine (PS) to the cell membrane surface of dying cells makes this phospholipid an attractive molecular target for the development of apoptosis imaging probes. In this study, we have radiolabeled PS-binding 14-mer peptide FNFRLKAGAKIRFG (PSBP-6) with positron-emitter copper-64 ( 64 Cu) for PET imaging of apoptosis. Peptide PSBP-6 was conjugated with radiometal chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through an aminovaleric acid (Ava) linker for subsequent radiolabeling with 64 Cu to prepare radiotracer 64 Cu-NOTA-Ava-PSBP-6. PS-binding potencies of PSBP-6, NOTA-Ava-PSBP-6, and nat Cu-NOTA-Ava-PSBP-6 were determined in a competitive radiometric PS-binding assay. Radiotracer 64 Cu-NOTA-Ava-PSBP-6 was studied in camptothecin-induced apoptotic EL4 mouse lymphoma cells and in a murine EL4 tumor model of apoptosis using dynamic PET imaging. Peptide PSBP-6 was also conjugated via an Ava linker with fluorescein isothiocyanate (FITC). FITC-Ava-PSBP-6 was evaluated in flow cytometry and fluorescence confocal microscopy experiments. Radiopeptide 64 Cu-NOTA-Ava-PSBP-6 was synthesized in high radiochemical yields of >95%. The IC 50 values for PS-binding potency of PSBP-6, NOTA-Ava-PSBP-6, and nat Cu-NOTA-PSBP-6 were 600 μM, 30 μM, and 23 μM, respectively. A competitive radiometric cell binding assay confirmed binding of 64 Cu-NOTA-Ava-PSBP-6 to camptothecin-induced apoptotic EL4 cells in a Ca 2+ -independent manner. PET imaging studies demonstrated significantly higher uptake of 64 Cu-NOTA-Ava-PSBP-6 in apoptotic EL4 tumors (SUV 5min 0.95 ± 0.04) compared to control tumors (SUV 5min 0.74 ± 0.03). Flow cytometry studies showed significantly higher binding of FITC-Ava-PSBP-6 to EL4 cells treated with camptothecin compared to untreated cells. Fluorescence microscopy studies revealed that FITC-Ava-PSBP-6 was binding to cell membranes of early apoptotic cells, but was internalized in late apoptotic and necrotic cells. The present study showed that radiotracer 64 Cu-NOTA-Ava-PSBP-6 holds promise as a first peptide-based PET imaging agent for molecular imaging of apoptosis. However, additional "fine-tuning" of 64 Cu-NOTA-Ava-PSBP-6 is required to enhance PS-binding potency and in vivo stability to improve tumor uptake and retention.

Three-dimensional morphological imaging of human induced pluripotent stem cells by using low-coherence quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Kakuno, Yumi; Goto, Kentaro; Fukami, Tadashi; Sugiyama, Norikazu; Iwai, Hidenao; Mizuguchi, Yoshinori; Yamashita, Yutaka

2014-03-01

There is an increasing need for non-invasive imaging techniques in the field of stem cell research. Label-free techniques are the best choice for assessment of stem cells because the cells remain intact after imaging and can be used for further studies such as differentiation induction. To develop a high-resolution label-free imaging system, we have been working on a low-coherence quantitative phase microscope (LC-QPM). LC-QPM is a Linnik-type interference microscope equipped with nanometer-resolution optical-path-length control and capable of obtaining three-dimensional volumetric images. The lateral and vertical resolutions of our system are respectively 0.5 and 0.93 μm and this performance allows capturing sub-cellular morphological features of live cells without labeling. Utilizing LC-QPM, we reported on three-dimensional imaging of membrane fluctuations, dynamics of filopodia, and motions of intracellular organelles. In this presentation, we report three-dimensional morphological imaging of human induced pluripotent stem cells (hiPS cells). Two groups of monolayer hiPS cell cultures were prepared so that one group was cultured in a suitable culture medium that kept the cells undifferentiated, and the other group was cultured in a medium supplemented with retinoic acid, which forces the stem cells to differentiate. The volumetric images of the 2 groups show distinctive differences, especially in surface roughness. We believe that our LC-QPM system will prove useful in assessing many other stem cell conditions.

Applying image quality in cell phone cameras: lens distortion

NASA Astrophysics Data System (ADS)

Baxter, Donald; Goma, Sergio R.; Aleksic, Milivoje

2009-01-01

This paper describes the framework used in one of the pilot studies run under the I3A CPIQ initiative to quantify overall image quality in cell-phone cameras. The framework is based on a multivariate formalism which tries to predict overall image quality from individual image quality attributes and was validated in a CPIQ pilot program. The pilot study focuses on image quality distortions introduced in the optical path of a cell-phone camera, which may or may not be corrected in the image processing path. The assumption is that the captured image used is JPEG compressed and the cellphone camera is set to 'auto' mode. As the used framework requires that the individual attributes to be relatively perceptually orthogonal, in the pilot study, the attributes used are lens geometric distortion (LGD) and lateral chromatic aberrations (LCA). The goal of this paper is to present the framework of this pilot project starting with the definition of the individual attributes, up to their quantification in JNDs of quality, a requirement of the multivariate formalism, therefore both objective and subjective evaluations were used. A major distinction in the objective part from the 'DSC imaging world' is that the LCA/LGD distortions found in cell-phone cameras, rarely exhibit radial behavior, therefore a radial mapping/modeling cannot be used in this case.

Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

2016-03-01

Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

Imaging the beating heart in the mouse using intravital microscopy techniques

PubMed Central

Vinegoni, Claudio; Aguirre, Aaron D; Lee, Sungon; Weissleder, Ralph

2017-01-01

Real-time microscopic imaging of moving organs at single-cell resolution represents a major challenge in studying complex biology in living systems. Motion of the tissue from the cardiac and respiratory cycles severely limits intravital microscopy by compromising ultimate spatial and temporal imaging resolution. However, significant recent advances have enabled single-cell resolution imaging to be achieved in vivo. In this protocol, we describe experimental procedures for intravital microscopy based on a combination of thoracic surgery, tissue stabilizers and acquisition gating methods, which enable imaging at the single-cell level in the beating heart in the mouse. Setup of the model is typically completed in 1 h, which allows 2 h or more of continuous cardiac imaging. This protocol can be readily adapted for the imaging of other moving organs, and it will therefore broadly facilitate in vivo high-resolution microscopy studies. PMID:26492138

Live-cell imaging of budding yeast telomerase RNA and TERRA.

PubMed

Laprade, Hadrien; Lalonde, Maxime; Guérit, David; Chartrand, Pascal

2017-02-01

In most eukaryotes, the ribonucleoprotein complex telomerase is responsible for maintaining telomere length. In recent years, single-cell microscopy techniques such as fluorescent in situ hybridization and live-cell imaging have been developed to image the RNA subunit of the telomerase holoenzyme. These techniques are now becoming important tools for the study of telomerase biogenesis, its association with telomeres and its regulation. Here, we present detailed protocols for live-cell imaging of the Saccharomyces cerevisiae telomerase RNA subunit, called TLC1, and also of the non-coding telomeric repeat-containing RNA TERRA. We describe the approach used for genomic integration of MS2 stem-loops in these transcripts, and provide information for optimal live-cell imaging of these non-coding RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Opportunities for Live Cell FT-Infrared Imaging: Macromolecule Identification with 2D and 3D Localization

PubMed Central

Mattson, Eric C.; Aboualizadeh, Ebrahim; Barabas, Marie E.; Stucky, Cheryl L.; Hirschmugl, Carol J.

2013-01-01

Infrared (IR) spectromicroscopy, or chemical imaging, is an evolving technique that is poised to make significant contributions in the fields of biology and medicine. Recent developments in sources, detectors, measurement techniques and speciman holders have now made diffraction-limited Fourier transform infrared (FTIR) imaging of cellular chemistry in living cells a reality. The availability of bright, broadband IR sources and large area, pixelated detectors facilitate live cell imaging, which requires rapid measurements using non-destructive probes. In this work, we review advances in the field of FTIR spectromicroscopy that have contributed to live-cell two and three-dimensional IR imaging, and discuss several key examples that highlight the utility of this technique for studying the structure and chemistry of living cells. PMID:24256815

Correlation of live-cell imaging with volume scanning electron microscopy.

PubMed

Lucas, Miriam S; Günthert, Maja; Bittermann, Anne Greet; de Marco, Alex; Wepf, Roger

2017-01-01

Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods. Copyright © 2017 Elsevier Inc. All rights reserved.

Imaging immune surveillance of individual natural killer cells confined in microwell arrays.

PubMed

Guldevall, Karolin; Vanherberghen, Bruno; Frisk, Thomas; Hurtig, Johan; Christakou, Athanasia E; Manneberg, Otto; Lindström, Sara; Andersson-Svahn, Helene; Wiklund, Martin; Önfelt, Björn

2010-11-12

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.

Inter-chromosomal Contact Properties in Live-Cell Imaging and in Hi-C.

PubMed

Maass, Philipp G; Barutcu, A Rasim; Weiner, Catherine L; Rinn, John L

2018-03-15

Imaging (fluorescence in situ hybridization [FISH]) and genome-wide chromosome conformation capture (Hi-C) are two major approaches to the study of higher-order genome organization in the nucleus. Intra-chromosomal and inter-chromosomal interactions (referred to as non-homologous chromosomal contacts [NHCCs]) have been observed by several FISH-based studies, but locus-specific NHCCs have not been detected by Hi-C. Due to crosslinking, neither of these approaches assesses spatiotemporal properties. Toward resolving the discrepancies between imaging and Hi-C, we sought to understand the spatiotemporal properties of NHCCs in living cells by CRISPR/Cas9 live-cell imaging (CLING). In mammalian cells, we find that NHCCs are stable and occur as frequently as intra-chromosomal interactions, but NHCCs occur at farther spatial distance that could explain their lack of detection in Hi-C. By revealing the spatiotemporal properties in living cells, our study provides fundamental insights into the biology of NHCCs. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.

PubMed

Mahen, Robert; Koch, Birgit; Wachsmuth, Malte; Politi, Antonio Z; Perez-Gonzalez, Alexis; Mergenthaler, Julia; Cai, Yin; Ellenberg, Jan

2014-11-05

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells. © 2014 Mahen et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

A study of glasses-type color CGH using a color filter considering reduction of blurring

NASA Astrophysics Data System (ADS)

Iwami, Saki; Sakamoto, Yuji

2009-02-01

We have developed a glasses-type color computer generated hologram (CGH) by using a color filter. The proposed glasses consist of two "lenses" made of overlapping holograms and color filters. The holograms, which are calculated to reconstruct images in each primary color, are divided to small areas, which we called cells, and superimposed on one hologram. In the same way, colors of the filter correspond to the hologram cells. We can configure it very simply without a complex optical system, and the configuration yields a small and light weight system suitable for glasses. When the cell is small enough, the colors are mixed and reconstructed color images are observed. In addition, color expression of reconstruction images improves, too. However, using small cells blurrs reconstructed images because of the following reasons: (1) interference between cells because of the correlation with the cells, and (2) reduction of resolution caused by the size of the cell hologram. We are investigating in order to make a hologram that has high resolution reconstructed color images without ghost images. In this paper, we discuss (1) the details of the proposed glasses-type color CGH, (2) appropriate cell size for an eye system, (3) effects of cell shape on the reconstructed images, and (4) a new method to reduce the blurring of the images.

Long lifetime near-infrared-emitting quantum dots for time-gated in vivo imaging of rare circulating cells (Conference Presentation)

NASA Astrophysics Data System (ADS)

Fragola, Alexandra; Bouccara, Sophie; Pezet, Sophie; Lequeux, Nicolas; Loriette, Vincent; Pons, Thomas

2017-02-01

The in vivo detection of rare circulating cells using non invasive fluorescence imaging would provide a key tool to study migration of eg. tumoral or immunological cells. Fluorescence detection is however currently limited by a lack of contrast between the small emission of isolated, fast circulating cells and the strong autofluorescence background of the surrounding tissues. We present the development of near infrared emitting quantum dots (NIR-QDs) with long fluorescence lifetime for sensitive time-gated in vivo imaging of circulating cells. These QDs are composed of low toxicity ZnCuInSe/ZnS materials and made biocompatible using a novel multidentate imidazole zwitterionic block copolymer, ensuring their long term intracellular stability. Cells of interest can thus be labeled ex vivo with QDs, injected intravenously and imaged in the near infrared range. Excitation using a pulsed laser coupled to time-gated detection enables the efficient rejection of short lifetime (≈ ns) autofluorescence background and detection of long lifetime (≈ 150 ns) fluorescence from QD-labeled cells. We demonstrate efficient in vivo imaging of single fast-flowing cells, which opens opportunities for future biological studies. [1] M. Tasso et al, "Sulfobetaine-Vinylimidazole block copolymers: a robust quantum dot surface chemistry expanding bioimaging's horizons", ACS Nano, 9(11), 2015 [2] S. Bouccara et al, "Time-gated cell imaging using long lifetime near-infrared-emitting quantum dots for autofluorescence rejection", J Biomed Optc, 19(5), 2014

Imaging Stem Cell Therapy for the Treatment of Peripheral Arterial Disease

PubMed Central

Ransohoff, Julia D.; Wu, Joseph C.

2013-01-01

Arteriosclerotic cardiovascular diseases are among the leading causes of morbidity and mortality worldwide. Therapeutic angiogenesis aims to treat ischemic myocardial and peripheral tissues by delivery of recombinant proteins, genes, or cells to promote neoangiogenesis. Concerns regarding the safety, side effects, and efficacy of protein and gene transfer studies have led to the development of cell-based therapies as alternative approaches to induce vascular regeneration and to improve function of damaged tissue. Cell-based therapies may be improved by the application of imaging technologies that allow investigators to track the location, engraftment, and survival of the administered cell population. The past decade of investigations has produced promising clinical data regarding cell therapy, but design of trials and evaluation of treatments stand to be improved by emerging insight from imaging studies. Here, we provide an overview of pre-clinical and clinical experience using cell-based therapies to promote vascular regeneration in the treatment of peripheral arterial disease. We also review four major imaging modalities and underscore the importance of in vivo analysis of cell fate for a full understanding of functional outcomes. PMID:22239638

In Vivo Imaging of the Human Retinal Pigment Epithelial Mosaic Using Adaptive Optics Enhanced Indocyanine Green Ophthalmoscopy

PubMed Central

Tam, Johnny; Liu, Jianfei; Dubra, Alfredo; Fariss, Robert

2016-01-01

Purpose The purpose of this study was to establish that retinal pigment epithelial (RPE) cells take up indocyanine green (ICG) dye following systemic injection and that adaptive optics enhanced indocyanine green ophthalmoscopy (AO-ICG) enables direct visualization of the RPE mosaic in the living human eye. Methods A customized adaptive optics scanning light ophthalmoscope (AOSLO) was used to acquire high-resolution retinal fluorescence images of residual ICG dye in human subjects after intravenous injection at the standard clinical dose. Simultaneously, multimodal AOSLO images were also acquired, which included confocal reflectance, nonconfocal split detection, and darkfield. Imaging was performed in 6 eyes of three healthy subjects with no history of ocular or systemic diseases. In addition, histologic studies in mice were carried out. Results The AO-ICG channel successfully resolved individual RPE cells in human subjects at various time points, including 20 minutes and 2 hours after dye administration. Adaptive optics-ICG images of RPE revealed detail which could be correlated with AO dark-field images of the same cells. Interestingly, there was a marked heterogeneity in the fluorescence of individual RPE cells. Confirmatory histologic studies in mice corroborated the specific uptake of ICG by the RPE layer at a late time point after systemic ICG injection. Conclusions Adaptive optics-enhanced imaging of ICG dye provides a novel way to visualize and assess the RPE mosaic in the living human eye alongside images of the overlying photoreceptors and other cells. PMID:27564519

Pulsed magneto-motive ultrasound imaging to detect intracellular trafficking of magnetic nanoparticles

PubMed Central

Mehrmohamamdi, Mohammad; Qu, Min; Ma, Li L.; Romanovicz, Dwight K.; Johnston, Keith P.; Sokolov, Konstantin V.; Emelianov, Stanislav Y.

2012-01-01

As applications of nanoparticles in medical imaging and biomedicine rapidly expand, the interactions of nanoparticles with living cells have become an area of active interest. For example, intracellular trafficking of nanoparticles – an important part of cell-nanoparticle interaction, has been well studied using plasmonic nanoparticles and optical or optics-based techniques due to the change in optical properties of the nanoparticle aggregates. However, magnetic nanoparticles, despite their wide range of clinical applications, do not exhibit plasmonic-resonant properties and therefore their intracellular aggregation cannot be detected by optics-based imaging techniques. In this study, we investigated the feasibility of a novel imaging technique – pulsed magneto-motive ultrasound (pMMUS), to identify intracellular trafficking of endocytosed magnetic nanoparticles. In pulsed magneto-motive ultrasound imaging a focused, high intensity, pulsed magnetic field is used to excite the cells labeled with magnetic nanoparticles, and ultrasound imaging is then used to monitor the mechanical response of the tissue. We demonstrated previously that clusters of magnetic nanoparticles amplify the pMMUS signal in comparison to signal from individual nanoparticles. Here we further demonstrate that pMMUS imaging can identify interaction between magnetic nanoparticles and living cells, i.e. intracellular aggregation of nanoparticles within the cells. The results of our study suggest that pMMUS imaging can not only detect the presence of magnetic nanoparticles but also provides information about their intracellular trafficking non-invasively and in real-time. PMID:21926454

In Vivo Imaging of the Human Retinal Pigment Epithelial Mosaic Using Adaptive Optics Enhanced Indocyanine Green Ophthalmoscopy.

PubMed

Tam, Johnny; Liu, Jianfei; Dubra, Alfredo; Fariss, Robert

2016-08-01

The purpose of this study was to establish that retinal pigment epithelial (RPE) cells take up indocyanine green (ICG) dye following systemic injection and that adaptive optics enhanced indocyanine green ophthalmoscopy (AO-ICG) enables direct visualization of the RPE mosaic in the living human eye. A customized adaptive optics scanning light ophthalmoscope (AOSLO) was used to acquire high-resolution retinal fluorescence images of residual ICG dye in human subjects after intravenous injection at the standard clinical dose. Simultaneously, multimodal AOSLO images were also acquired, which included confocal reflectance, nonconfocal split detection, and darkfield. Imaging was performed in 6 eyes of three healthy subjects with no history of ocular or systemic diseases. In addition, histologic studies in mice were carried out. The AO-ICG channel successfully resolved individual RPE cells in human subjects at various time points, including 20 minutes and 2 hours after dye administration. Adaptive optics-ICG images of RPE revealed detail which could be correlated with AO dark-field images of the same cells. Interestingly, there was a marked heterogeneity in the fluorescence of individual RPE cells. Confirmatory histologic studies in mice corroborated the specific uptake of ICG by the RPE layer at a late time point after systemic ICG injection. Adaptive optics-enhanced imaging of ICG dye provides a novel way to visualize and assess the RPE mosaic in the living human eye alongside images of the overlying photoreceptors and other cells.

Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye

PubMed Central

Guevara-Torres, A.; Joseph, A.; Schallek, J. B.

2016-01-01

Measuring blood cell dynamics within the capillaries of the living eye provides crucial information regarding the health of the microvascular network. To date, the study of single blood cell movement in this network has been obscured by optical aberrations, hindered by weak optical contrast, and often required injection of exogenous fluorescent dyes to perform measurements. Here we present a new strategy to non-invasively image single blood cells in the living mouse eye without contrast agents. Eye aberrations were corrected with an adaptive optics camera coupled with a fast, 15 kHz scanned beam orthogonal to a capillary of interest. Blood cells were imaged as they flowed past a near infrared imaging beam to which the eye is relatively insensitive. Optical contrast of cells was optimized using differential scatter of blood cells in the split-detector imaging configuration. Combined, these strategies provide label-free, non-invasive imaging of blood cells in the retina as they travel in single file in capillaries, enabling determination of cell flux, morphology, class, velocity, and rheology at the single cell level. PMID:27867728

Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye.

PubMed

Guevara-Torres, A; Joseph, A; Schallek, J B

2016-10-01

Measuring blood cell dynamics within the capillaries of the living eye provides crucial information regarding the health of the microvascular network. To date, the study of single blood cell movement in this network has been obscured by optical aberrations, hindered by weak optical contrast, and often required injection of exogenous fluorescent dyes to perform measurements. Here we present a new strategy to non-invasively image single blood cells in the living mouse eye without contrast agents. Eye aberrations were corrected with an adaptive optics camera coupled with a fast, 15 kHz scanned beam orthogonal to a capillary of interest. Blood cells were imaged as they flowed past a near infrared imaging beam to which the eye is relatively insensitive. Optical contrast of cells was optimized using differential scatter of blood cells in the split-detector imaging configuration. Combined, these strategies provide label-free, non-invasive imaging of blood cells in the retina as they travel in single file in capillaries, enabling determination of cell flux, morphology, class, velocity, and rheology at the single cell level.

Imaging Lung Clearance of Radiolabeled Tumor Cells to Study Mice with Normal, Activated or Depleted Natural Killer (NK) Cells

NASA Astrophysics Data System (ADS)

Kulkarni, P. V.; Bennett, M.; Constantinescu, A.; Arora, V.; Viguet, M.; Antich, P.; Parkey, R. W.; Mathews, D.; Mason, R. P.; Oz, O. K.

2003-08-01

Lung clearance of 51CR and 125I iododeoxyuridine (IUDR) labeled cancer cells assess NK cell activity. It is desirable to develop noninvasive imaging technique to assess NK activity in mice. We labeled target YAC-1 tumor cells with 125I, 111In, 99mTc, or 67Ga and injected I.V. into three groups of BALB/c mice. Animals were treated with medium (group I), 300mg/kg cyclophosmamide (CY) to kill NK cell (group II), or anti-LY49C/1) (ab')2 mAb to augment NK function (group III). Lungs were removed 15 min or 2 h later for tissue counting. Control and treated mice were imaged every 5 min with a scintillating camera for 1 h after 15 min of infusion of the 111In labeled cells. Lung clearance increased after 15 min (lodging: 60-80%) and (2 h retention: 3-7%). Similar results were obtained with all the isotopes studied. Images distinguished the control and treated mice for lung activity. Cells labeled with 111In, 99mTc or 67Ga are cleared similar to those labeled with 51Cr or 125I. NK cell destruction of tumor cells may be assessed by noninvasive imaging method either by SPECT (99mTc, 111In, 67Ga) or by PET (68Ga).

Imaging and characterizing cells using tomography

PubMed Central

Do, Myan; Isaacson, Samuel A.; McDermott, Gerry; Le Gros, Mark A.; Larabell, Carolyn A.

2015-01-01

We can learn much about cell function by imaging and quantifying sub-cellular structures, especially if this is done non-destructively without altering said structures. Soft x-ray tomography (SXT) is a high-resolution imaging technique for visualizing cells and their interior structure in 3D. A tomogram of the cell, reconstructed from a series of 2D projection images, can be easily segmented and analyzed. SXT has a very high specimen throughput compared to other high-resolution structure imaging modalities; for example, tomographic data for reconstructing an entire eukaryotic cell is acquired in a matter of minutes. SXT visualizes cells without the need for chemical fixation, dehydration, or staining of the specimen. As a result, the SXT reconstructions are close representations of cells in their native state. SXT is applicable to most cell types. The deep penetration of soft x-rays allows cells, even mammalian cells, to be imaged without being sectioned. Image contrast in SXT is generated by the differential attenuation soft x-ray illumination as it passes through the specimen. Accordingly, each voxel in the tomographic reconstruction has a measured linear absorption coefficient (LAC) value. LAC values are quantitative and give rise to each sub-cellular component having a characteristic LAC profile, allowing organelles to be identified and segmented from the milieu of other cell contents. In this chapter, we describe the fundamentals of SXT imaging and how this technique can answer real world questions in the study of the nucleus. We also describe the development of correlative methods for the localization of specific molecules in a SXT reconstruction. The combination of fluorescence and SXT data acquired from the same specimen produces composite 3D images, rich with detailed information on the inner workings of cells. PMID:25602704

Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging.

PubMed

Shuai, Hongyan; Xu, Yunjian; Yu, Qian; Gylfe, Erik; Tengholm, Anders

2016-10-01

The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca(2+) and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca(2+) signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca(2+) oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.

NanoLuc reporter for dual luciferase imaging in living animals.

PubMed

Stacer, Amanda C; Nyati, Shyam; Moudgil, Pranav; Iyengar, Rahul; Luker, Kathryn E; Rehemtulla, Alnawaz; Luker, Gary D

2013-10-01

Bioluminescence imaging is widely used for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on the high signal to background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events, as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, adenosine triphosphate–independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc [NL]) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although the detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in transforming growth factor β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as a new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development.

NanoLuc Reporter for Dual Luciferase Imaging in Living Animals

PubMed Central

Stacer, Amanda C.; Nyati, Shyam; Moudgil, Pranav; Iyengar, Rahul; Luker, Kathryn E.; Rehemtulla, Alnawaz; Luker, Gary D.

2014-01-01

Bioluminescence imaging is utilized widely for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on high signal-to-background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, ATP-independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc, NL) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in TGF-β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development. PMID:24371848

In vivo fluorescence imaging of primate retinal ganglion cells and retinal pigment epithelial cells

NASA Astrophysics Data System (ADS)

Gray, Daniel C.; Merigan, William; Wolfing, Jessica I.; Gee, Bernard P.; Porter, Jason; Dubra, Alfredo; Twietmeyer, Ted H.; Ahamd, Kamran; Tumbar, Remy; Reinholz, Fred; Williams, David R.

2006-08-01

The ability to resolve single cells noninvasively in the living retina has important applications for the study of normal retina, diseased retina, and the efficacy of therapies for retinal disease. We describe a new instrument for high-resolution, in vivo imaging of the mammalian retina that combines the benefits of confocal detection, adaptive optics, multispectral, and fluorescence imaging. The instrument is capable of imaging single ganglion cells and their axons through retrograde transport in ganglion cells of fluorescent dyes injected into the monkey lateral geniculate nucleus (LGN). In addition, we demonstrate a method involving simultaneous imaging in two spectral bands that allows the integration of very weak signals across many frames despite inter-frame movement of the eye. With this method, we are also able to resolve the smallest retinal capillaries in fluorescein angiography and the mosaic of retinal pigment epithelium (RPE) cells with lipofuscin autofluorescence.

A Novel Technique to Follow Consequences of Exogenous Factors, Including Therapeutic Drugs, on Living Human Breast Epithelial Cells

DTIC Science & Technology

1999-07-01

and lipid vectors, are being tested. Concurrent with the development of procedures for live - cell imaging , we are examining the distribution of proteins...dimensional matrix. These studies have not yet begun. There are a number of procedures that must be developed and perfected in the live - cell imaging , as...components of the Wnt signaling pathway are too preliminary and require additional research prior to publication. (9) CONCLUSIONS Live cell imaging of

High-resolution imaging of basal cell carcinoma: a comparison between multiphoton microscopy with fluorescence lifetime imaging and reflectance confocal microscopy.

PubMed

Manfredini, Marco; Arginelli, Federica; Dunsby, Christopher; French, Paul; Talbot, Clifford; König, Karsten; Pellacani, Giovanni; Ponti, Giovanni; Seidenari, Stefania

2013-02-01

The aim of this study was to compare morphological aspects of basal cell carcinoma (BCC) as assessed by two different imaging methods: in vivo reflectance confocal microscopy (RCM) and multiphoton tomography with fluorescence lifetime imaging implementation (MPT-FLIM). The study comprised 16 BCCs for which a complete set of RCM and MPT-FLIM images were available. The presence of seven MPT-FLIM descriptors was evaluated. The presence of seven RCM equivalent parameters was scored in accordance to their extension. Chi-squared test with Fisher's exact test and Spearman's rank correlation coefficient were determined between MPT-FLIM scores and adjusted-RCM scores. MPT-FLIM and RCM descriptors of BCC were coupled to match the descriptors that define the same pathological structures. The comparison included: Streaming and Aligned elongated cells, Streaming with multiple directions and Double alignment, Palisading (RCM) and Palisading (MPT-FLIM), Typical tumor islands, and Cell islands surrounded by fibers, Dark silhouettes and Phantom islands, Plump bright cells and Melanophages, Vessels (RCM), and Vessels (MPT-FLIM). The parameters that were significantly correlated were Melanophages/Plump Bright Cells, Aligned elongated cells/Streaming, Double alignment/Streaming with multiple directions, and Palisading (MPT-FLIM)/Palisading (RCM). According to our data, both methods are suitable to image BCC's features. The concordance between MPT-FLIM and RCM is high, with some limitations due to the technical differences between the two devices. The hardest difficulty when comparing the images generated by the two imaging modalities is represented by their different field of view. © 2012 John Wiley & Sons A/S.

Multifunctional ferritin cage nanostructures for fluorescence and MR imaging of tumor cells

NASA Astrophysics Data System (ADS)

Li, Ke; Zhang, Zhi-Ping; Luo, Ming; Yu, Xiang; Han, Yu; Wei, Hong-Ping; Cui, Zong-Qiang; Zhang, Xian-En

2011-12-01

Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging αvβ3 integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging αvβ3 integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging. Electronic supplementary information (ESI) available. See DOI: 10.1039/c1nr11132a

Prolonged Dye Release from Mesoporous Silica-Based Imaging Probes Facilitates Long-Term Optical Tracking of Cell Populations In Vivo.

PubMed

Rosenholm, Jessica M; Gulin-Sarfraz, Tina; Mamaeva, Veronika; Niemi, Rasmus; Özliseli, Ezgi; Desai, Diti; Antfolk, Daniel; von Haartman, Eva; Lindberg, Desiré; Prabhakar, Neeraj; Näreoja, Tuomas; Sahlgren, Cecilia

2016-03-23

Nanomedicine is gaining ground worldwide in therapy and diagnostics. Novel nanoscopic imaging probes serve as imaging tools for studying dynamic biological processes in vitro and in vivo. To allow detectability in the physiological environment, the nanostructure-based probes need to be either inherently detectable by biomedical imaging techniques, or serve as carriers for existing imaging agents. In this study, the potential of mesoporous silica nanoparticles carrying commercially available fluorochromes as self-regenerating cell labels for long-term cellular tracking is investigated. The particle surface is organically modified for enhanced cellular uptake, the fluorescence intensity of labeled cells is followed over time both in vitro and in vivo. The particles are not exocytosed and particles which escaped cells due to cell injury or death are degraded and no labeling of nontargeted cell populations are observed. The labeling efficiency is significantly improved as compared to that of quantum dots of similar emission wavelength. Labeled human breast cancer cells are xenotransplanted in nude mice, and the fluorescent cells can be detected in vivo for a period of 1 month. Moreover, ex vivo analysis reveals fluorescently labeled metastatic colonies in lymph node and rib, highlighting the capability of the developed probes for tracking of metastasis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Imaging mammalian cells with soft x rays: The importance of specimen preparation

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

Brown, J.T.; Meyer-Ilse, W.

1997-04-01

Studies of mammalian cell structure and spatial organization are a very prominent part of modern cell biology. The interest in them as well as their size make them very accommodating subject specimens for imaging with soft x-rays using the XM-1 transmission microscope built and operated by The Center for X-ray Optics on Beam Line 6.1 at the Advanced Light Source. The purpose of these experiments was to determine if the fixative protocols normally used in electron or visible light microscopy were adequate to allow imaging cells, either fibroblasts or neurons, with minimal visible radiation damage due to imaging with softmore » x-rays at 2.4 nm. Two cell types were selected. Fibroblasts are easily cultured but fragile cells which are commonly used as models for the detailed study of cell physiology. Neurons are complex and sensitive cells which are difficult to prepare and to culture for study in isolation from their connections with surrounding cells. These cell types pose problems in their preparation for any microscopy. To improve the contrast and to prevent postmortem alteration of the chemistry and hence the structure of cells extracted from culture or from living organisms, fixation and staining techniques are employed in electron and visible light microscopy. It has been accepted by biologists for years that these treatments create artifacts and false structure. The authors have begun to develop protocols for specimens of each of these two cell types for soft x-ray microscopy which will preserve them in as near normal state as possible using minimal fixation, and make it possible to image them in either a hydrated or dried state free of secondary addition of stains or other labels.« less

Cell lineage and cell cycling analyses of the 4d micromere using live imaging in the marine annelid Platynereis dumerilii

PubMed Central

Handberg-Thorsager, Mette; Vervoort, Michel

2017-01-01

Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids. We also identified the precise cellular origins of the larval mesodermal posterior growth zone. We found that differentially-fated progeny of 4d (germline, segmental mesoderm, growth zone) display significantly different cell cycling. This work has evolutionary implications, sets up the foundation for functional studies in annelid stem cells, and presents newly established techniques for live imaging marine embryos. PMID:29231816

Modular low-light microscope for imaging cellular bioluminescence and radioluminescence

PubMed Central

Kim, Tae Jin; Türkcan, Silvan; Pratx, Guillem

2017-01-01

Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy, such as bioluminescence, chemiluminescence, or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1-4 d) by coupling two microscope objective lenses, back-to-back from each other, using standard optomechanical components. We also provide directions on how to image dim signals such as radioluminescence (1-1.5 h), bioluminescence (∼30 min) and low-excitation fluorescence (∼15 min). In particular, radioluminescence microscopy is explained in detail as it is a newly developed technique, which enables the study of small molecule transport (eg. radiolabeled drugs, metabolic precursors, and nuclear medicine contrast agents) by single cells without perturbing endogenous biochemical processes. In this imaging technique, a scintillator crystal (eg. CdWO4) is placed in close proximity to the radiolabeled cells, where it converts the radioactive decays into optical flashes detectable using a sensitive camera. Using the image reconstruction toolkit provided in this protocol, the flashes can be reconstructed to yield high-resolution image of the radiotracer distribution. With appropriate timing, the three aforementioned imaging modalities may be performed altogether on a population of live cells, allowing the user to perform parallel functional studies of cell heterogeneity at the single-cell level. PMID:28426025

Nanoscale Imaging of Whole Cells Using a Liquid Enclosure and a Scanning Transmission Electron Microscope

PubMed Central

Peckys, Diana B.; Veith, Gabriel M.; Joy, David C.; de Jonge, Niels

2009-01-01

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory. PMID:20020038

In Situ Probing of Cholesterol in Astrocytes at the Single Cell Level using Laser Desorption Ionization Mass Spectrometric Imaging with Colloidal Silver

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

Perdian, D.C.; Cha, Sangwon; Oh, Jisun

2010-03-18

Mass spectrometric imaging has been utilized to localize individual astrocytes and to obtain cholesterol populations at the single-cell level in laser desorption ionization (LDI) with colloidal silver. The silver ion adduct of membrane-bound cholesterol was monitored to detect individual cells. Good correlation between mass spectrometric and optical images at different cell densities indicates the ability to perform single-cell studies of cholesterol abundance. The feasibility of quantification is confirmed by the agreement between the LDI-MS ion signals and the results from a traditional enzymatic fluorometric assay. We propose that this approach could be an effective tool to study chemical populations atmore » the cellular level.« less

3D X-Ray Nanotomography of Cells Grown on Electrospun Scaffolds.

PubMed

Bradley, Robert S; Robinson, Ian K; Yusuf, Mohammed

2017-02-01

Here, it is demonstrated that X-ray nanotomography with Zernike phase contrast can be used for 3D imaging of cells grown on electrospun polymer scaffolds. The scaffold fibers and cells are simultaneously imaged, enabling the influence of scaffold architecture on cell location and morphology to be studied. The high resolution enables subcellular details to be revealed. The X-ray imaging conditions were optimized to reduce scan times, making it feasible to scan multiple regions of interest in relatively large samples. An image processing procedure is presented which enables scaffold characteristics and cell location to be quantified. The procedure is demonstrated by comparing the ingrowth of cells after culture for 3 and 6 days. © 2016 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Live-cell imaging for the assessment of the dynamics of autophagosome formation: focus on early steps.

PubMed

Karanasios, Eleftherios; Ktistakis, Nicholas T

2015-03-01

Autophagy is a cytosolic degradative pathway, which through a series of complicated membrane rearrangements leads to the formation of a unique double membrane vesicle, the autophagosome. The use of fluorescent proteins has allowed visualizing the autophagosome formation in live cells and in real time, almost 40 years after electron microscopy studies observed these structures for the first time. In the last decade, live-cell imaging has been extensively used to study the dynamics of autophagosome formation in cultured mammalian cells. Hereby we will discuss how the live-cell imaging studies have tried to settle the debate about the origin of the autophagosome membrane and how they have described the way different autophagy proteins coordinate in space and time in order to drive autophagosome formation. Copyright © 2014 Elsevier Inc. All rights reserved.

Ultrasound and photoacoustic imaging to monitor ocular stem cell delivery and tissue regeneration (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kubelick, Kelsey; Snider, Eric; Yoon, Heechul; Ethier, C. Ross; Emelianov, Stanislav Y.

2017-03-01

Glaucoma is associated with dysfunction of the trabecular meshwork (TM), a fluid drainage tissue in the anterior eye. A promising treatment involves delivery of stem cells to the TM to restore tissue function. Currently histology is the gold standard for tracking stem cell delivery and differentiation. To expedite clinical translation, non-invasive longitudinal monitoring in vivo is desired. Our current research explores a technique combining ultrasound (US) and photoacoustic (PA) imaging to track mesenchymal stem cells (MSCs) after intraocular injection. Adipose-derived MSCs were incubated with gold nanospheres to label cells (AuNS-MSCs) for PA imaging. Successful labeling was first verified with in vitro phantom studies. Next, MSC delivery was imaged ex vivo in porcine eyes, while intraocular pressure was hydrostatically clamped to maintain a physiological flow rate through the TM. US/PA imaging was performed before, during, and after AuNS-MSC delivery. Additionally, spectroscopic PA imaging was implemented to isolate PA signals from AuNS-MSCs. In vitro cell imaging showed AuNS-MSCs produce strong PA signals, suggesting that MSCs can be tracked using PA imaging. While the cornea, sclera, iris, and TM region can be visualized with US imaging, pigmented tissues also produce PA signals. Both modalities provide valuable anatomical landmarks for MSC localization. During delivery, PA imaging can visualize AuNS-MSC motion and location, creating a unique opportunity to guide ocular cell delivery. Lastly, distinct spectral signatures of AuNS-MSCs allow unmixing, with potential for quantitative PA imaging. In conclusion, results show proof-of-concept for monitoring MSC ocular delivery, raising opportunities for in vivo image-guided cell delivery.

Quantitative analysis of phosphoinositide 3-kinase (PI3K) signaling using live-cell total internal reflection fluorescence (TIRF) microscopy.

PubMed

Johnson, Heath E; Haugh, Jason M

2013-12-02

This unit focuses on the use of total internal reflection fluorescence (TIRF) microscopy and image analysis methods to study the dynamics of signal transduction mediated by class I phosphoinositide 3-kinases (PI3Ks) in mammalian cells. The first four protocols cover live-cell imaging experiments, image acquisition parameters, and basic image processing and segmentation. These methods are generally applicable to live-cell TIRF experiments. The remaining protocols outline more advanced image analysis methods, which were developed in our laboratory for the purpose of characterizing the spatiotemporal dynamics of PI3K signaling. These methods may be extended to analyze other cellular processes monitored using fluorescent biosensors. Copyright © 2013 John Wiley & Sons, Inc.

A method for prolonged imaging of motile lymphocytes.

PubMed

Day, Daniel; Pham, Kim; Ludford-Menting, Mandy J; Oliaro, Jane; Izon, David; Russell, Sarah M; Gu, Min

2009-02-01

With new imaging technologies and fluorescent probes, live imaging of cells in vitro has revolutionized many aspects of cell biology. A key goal now is to develop systems to optimize in vitro imaging, which do not compromise the physiological relevance of the study. We have developed a methodology that contains non-adherent cells within the field of view. 'Cell paddocks' are created by generating an array of microgrids using polydimethylsiloxane. Each microgrid is up to 250 x 250 microm(2) with a height of 60 microm. Overlayed cells settle into the grids and the walls restrict their lateral movement, but a contiguous supply of medium between neighboring microgrids facilitates the exchange of cytokines and growth factors. This allows culture over at least 6 days with no impact upon viability and proliferation. Adaptations of the microgrids have enabled imaging and tracking of lymphocyte division through multiple generations of long-term interactions between T lymphocytes and dendritic cells, and of thymocyte-stromal cell interactions.

Automated segmentation of retinal pigment epithelium cells in fluorescence adaptive optics images.

PubMed

Rangel-Fonseca, Piero; Gómez-Vieyra, Armando; Malacara-Hernández, Daniel; Wilson, Mario C; Williams, David R; Rossi, Ethan A

2013-12-01

Adaptive optics (AO) imaging methods allow the histological characteristics of retinal cell mosaics, such as photoreceptors and retinal pigment epithelium (RPE) cells, to be studied in vivo. The high-resolution images obtained with ophthalmic AO imaging devices are rich with information that is difficult and/or tedious to quantify using manual methods. Thus, robust, automated analysis tools that can provide reproducible quantitative information about the cellular mosaics under examination are required. Automated algorithms have been developed to detect the position of individual photoreceptor cells; however, most of these methods are not well suited for characterizing the RPE mosaic. We have developed an algorithm for RPE cell segmentation and show its performance here on simulated and real fluorescence AO images of the RPE mosaic. Algorithm performance was compared to manual cell identification and yielded better than 91% correspondence. This method can be used to segment RPE cells for morphometric analysis of the RPE mosaic and speed the analysis of both healthy and diseased RPE mosaics.

Imaging molecular dynamics in vivo--from cell biology to animal models.

PubMed

Timpson, Paul; McGhee, Ewan J; Anderson, Kurt I

2011-09-01

Advances in fluorescence microscopy have enabled the study of membrane diffusion, cell adhesion and signal transduction at the molecular level in living cells grown in culture. By contrast, imaging in living organisms has primarily been restricted to the localization and dynamics of cells in tissues. Now, imaging of molecular dynamics is on the cusp of progressing from cell culture to living tissue. This transition has been driven by the understanding that the microenvironment critically determines many developmental and pathological processes. Here, we review recent progress in fluorescent protein imaging in vivo by drawing primarily on cancer-related studies in mice. We emphasize the need for techniques that can be easily combined with genetic models and complement fluorescent protein imaging by providing contextual information about the cellular environment. In this Commentary we will consider differences between in vitro and in vivo experimental design and argue for an approach to in vivo imaging that is built upon the use of intermediate systems, such as 3-D and explant culture models, which offer flexibility and control that is not always available in vivo. Collectively, these methods present a paradigm shift towards the molecular-level investigation of disease and therapy in animal models of disease.

Arraycount, an algorithm for automatic cell counting in microwell arrays.

PubMed

Kachouie, Nezamoddin; Kang, Lifeng; Khademhosseini, Ali

2009-09-01

Microscale technologies have emerged as a powerful tool for studying and manipulating biological systems and miniaturizing experiments. However, the lack of software complementing these techniques has made it difficult to apply them for many high-throughput experiments. This work establishes Arraycount, an approach to automatically count cells in microwell arrays. The procedure consists of fluorescent microscope imaging of cells that are seeded in microwells of a microarray system and then analyzing images via computer to recognize the array and count cells inside each microwell. To start counting, green and red fluorescent images (representing live and dead cells, respectively) are extracted from the original image and processed separately. A template-matching algorithm is proposed in which pre-defined well and cell templates are matched against the red and green images to locate microwells and cells. Subsequently, local maxima in the correlation maps are determined and local maxima maps are thresholded. At the end, the software records the cell counts for each detected microwell on the original image in high-throughput. The automated counting was shown to be accurate compared with manual counting, with a difference of approximately 1-2 cells per microwell: based on cell concentration, the absolute difference between manual and automatic counting measurements was 2.5-13%.

Mapping owl's eye cells of patients with cytomegalovirus corneal endotheliitis using in vivo laser confocal microscopy.

PubMed

Yokogawa, Hideaki; Kobayashi, Akira; Sugiyama, Kazuhisa

2013-01-01

To produce a two-dimensional reconstruction map of owl's eye cells using in vivo laser confocal microscopy in patients with cytomegalovirus (CMV) corneal endotheliitis, and to demonstrate any association between owl's eye cells and coin-shaped lesions observed with slit-lamp biomicroscopy. Two patients (75- and 77-year-old men) with polymerase chain reaction-proven CMV corneal endotheliitis were evaluated in this study. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Images of owl's eye cells in the endothelial cell layer were arranged and mapped into subconfluent montages. Montage images of owl's eye cells were then superimposed on a slit-lamp photo of the corresponding coin-shaped lesion. Degree of concordance between the confocal microscopic images and slit-lamp photos was evaluated. In both eyes, a two-dimensional reconstruction map of the owl's eye cells was created by computer software using acquired confocal images; the maps showed circular patterns. Superimposing montage images of owl's eye cells onto the photos of a coin-shaped lesion showed good concordance in the two eyes. This study suggests that there is an association between owl's eye cells observed by confocal microscopy and coin-shaped lesions observed by slit-lamp biomicroscopy in patients with CMV corneal endotheliitis. The use of in vivo laser confocal microscopy may provide clues as to the underlying causes of CMV corneal endotheliitis.

Dynamic Assessment of the Endothelialization of Tissue-Engineered Blood Vessels Using an Optical Coherence Tomography Catheter-Based Fluorescence Imaging System.

PubMed

Gurjarpadhye, Abhijit Achyut; DeWitt, Matthew R; Xu, Yong; Wang, Ge; Rylander, Marissa Nichole; Rylander, Christopher G

2015-07-01

Lumen endothelialization of bioengineered vascular scaffolds is essential to maintain small-diameter graft patency and prevent thrombosis postimplantation. Unfortunately, nondestructive imaging methods to visualize this dynamic process are lacking, thus slowing development and clinical translation of these potential tissue-engineering approaches. To meet this need, a fluorescence imaging system utilizing a commercial optical coherence tomography (OCT) catheter was designed to visualize graft endothelialization. C7 DragonFly™ intravascular OCT catheter was used as a channel for delivery and collection of excitation and emission spectra. Poly-dl-lactide (PDLLA) electrospun scaffolds were seeded with endothelial cells (ECs). Seeded cells were exposed to Calcein AM before imaging, causing the living cells to emit green fluorescence in response to blue laser. By positioning the catheter tip precisely over a specimen using high-fidelity electromechanical components, small regions of the specimen were excited selectively. The resulting fluorescence intensities were mapped on a two-dimensional digital grid to generate spatial distribution of fluorophores at single-cell-level resolution. Fluorescence imaging of endothelialization on glass and PDLLA scaffolds was performed using the OCT catheter-based imaging system as well as with a commercial fluorescence microscope. Cell coverage area was calculated for both image sets for quantitative comparison of imaging techniques. Tubular PDLLA scaffolds were maintained in a bioreactor on seeding with ECs, and endothelialization was monitored over 5 days using the OCT catheter-based imaging system. No significant difference was observed in images obtained using our imaging system to those acquired with the fluorescence microscope. Cell area coverage calculated using the images yielded similar values. Nondestructive imaging of endothelialization on tubular scaffolds showed cell proliferation with cell coverage area increasing from 15 ± 4% to 89 ± 6% over 5 days. In this study, we showed the capability of an OCT catheter-based imaging system to obtain single-cell resolution and to quantify endothelialization in tubular electrospun scaffolds. We also compared the resulting images with traditional microscopy, showing high fidelity in image capability. This imaging system, used in conjunction with OCT, could potentially be a powerful tool for in vitro optimization of scaffold cellularization, ensuring long-term graft patency postimplantation.

Digital optical imaging of green fluorescent proteins for tracking vascular gene expression: feasibility study in rabbit and human cell models.

PubMed

Yang, X; Liu, H; Li, D; Zhou, X; Jung, W C; Deans, A E; Cui, Y; Cheng, L

2001-04-01

To investigate the feasibility of using a sensitive digital optical imaging technique to detect green fluorescent protein (GFP) expressed in rabbit vasculature and human arterial smooth muscle cells. A GFP plasmid was transfected into human arterial smooth muscle cells to obtain a GFP-smooth muscle cell solution. This solution was imaged in cell phantoms by using a prototype digital optical imaging system. For in vivo validation, a GFP-lentivirus vector was transfected during surgery into the carotid arteries of two rabbits, and GFP-targeted vessels were harvested for digital optical imaging ex vivo. Optical imaging of cell phantoms resulted in a spatial resolution of 25 microm/pixel. Fluorescent signals were detected as diffusely distributed bright spots. At ex vivo optical imaging of arterial tissues, the average fluorescent signal was significantly higher (P <.05) in GFP-targeted tissues (mean +/- SD, 9,357.3 absolute units of density +/- 1,001.3) than in control tissues (5,633.7 absolute units of density +/- 985.2). Both fluorescence microscopic and immunohistochemical findings confirmed these differences between GFP-targeted and control vessels. The digital optical imaging system was sensitive to GFPs and may potentially provide an in vivo imaging tool to monitor and track vascular gene transfer and expression in experimental investigations.

Unraveling Cell Processes: Interference Imaging Interwoven with Data Analysis

PubMed Central

Brazhe, A. R.; Pavlov, A. N.; Erokhova, L. A.; Yusipovich, A. I.; Maksimov, G. V.; Mosekilde, E.; Sosnovtseva, O. V.

2006-01-01

The paper presents results on the application of interference microscopy and wavelet-analysis for cell visualization and studies of cell dynamics. We demonstrate that interference imaging of erythrocytes can reveal reorganization of the cytoskeleton and inhomogenity in the distribution of hemoglobin, and that interference imaging of neurons can show intracellular compartmentalization and submembrane structures. We investigate temporal and spatial variations of the refractive index for different cell types: isolated neurons, mast cells and erythrocytes. We show that the refractive dynamical properties differ from cell type to cell type and depend on the cellular compartment. Our results suggest that low frequency variations (0.1–0.6 Hz) result from plasma membrane processes and that higher frequency variations (20–26 Hz) are related to the movement of vesicles. Using double-wavelet analysis, we study the modulation of the 1 Hz rhythm in neurons and reveal its changes under depolarization and hyperpolarization of the plasma membrane. We conclude that interference microscopy combined with wavelet analysis is a useful technique for non-invasive cell studies, cell visualization, and investigation of plasma membrane properties. PMID:19669463

Imaging Tumor Cell Movement In Vivo

PubMed Central

Entenberg, David; Kedrin, Dmitriy; Wyckoff, Jeffrey; Sahai, Erik; Condeelis, John; Segall, Jeffrey E.

2013-01-01

This unit describes the methods that we have been developing for analyzing tumor cell motility in mouse and rat models of breast cancer metastasis. Rodents are commonly used both to provide a mammalian system for studying human tumor cells (as xenografts in immunocompromised mice) as well as for following the development of tumors from a specific tissue type in transgenic lines. The Basic Protocol in this unit describes the standard methods used for generation of mammary tumors and imaging them. Additional protocols for labeling macrophages, blood vessel imaging, and image analysis are also included. PMID:23456602

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

PubMed Central

Maynard, Carrie; Plaks, Vicki

2016-01-01

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment. PMID:26862704

Developmental imaging: the avian embryo hatches to the challenge.

PubMed

Kulesa, Paul M; McKinney, Mary C; McLennan, Rebecca

2013-06-01

The avian embryo provides a multifaceted model to study developmental mechanisms because of its accessibility to microsurgery, fluorescence cell labeling, in vivo imaging, and molecular manipulation. Early two-dimensional planar growth of the avian embryo mimics human development and provides unique access to complex cell migration patterns using light microscopy. Later developmental events continue to permit access to both light and other imaging modalities, making the avian embryo an excellent model for developmental imaging. For example, significant insights into cell and tissue behaviors within the primitive streak, craniofacial region, and cardiovascular and peripheral nervous systems have come from avian embryo studies. In this review, we provide an update to recent advances in embryo and tissue slice culture and imaging, fluorescence cell labeling, and gene profiling. We focus on how technical advances in the chick and quail provide a clearer understanding of how embryonic cell dynamics are beautifully choreographed in space and time to sculpt cells into functioning structures. We summarize how these technical advances help us to better understand basic developmental mechanisms that may lead to clinical research into human birth defects and tissue repair. Copyright © 2013 Wiley Periodicals, Inc.

Optical computed tomography for spatially isotropic four-dimensional imaging of live single cells

PubMed Central

Kelbauskas, Laimonas; Shetty, Rishabh; Cao, Bin; Wang, Kuo-Chen; Smith, Dean; Wang, Hong; Chao, Shi-Hui; Gangaraju, Sandhya; Ashcroft, Brian; Kritzer, Margaret; Glenn, Honor; Johnson, Roger H.; Meldrum, Deirdre R.

2017-01-01

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field. PMID:29226240

Raman Spectroscopic Imaging of the Whole Ciona intestinalis Embryo during Development

PubMed Central

Nakamura, Mitsuru J.; Hotta, Kohji; Oka, Kotaro

2013-01-01

Intracellular composition and the distribution of bio-molecules play central roles in the specification of cell fates and morphogenesis during embryogenesis. Consequently, investigation of changes in the expression and distribution of bio-molecules, especially mRNAs and proteins, is an important challenge in developmental biology. Raman spectroscopic imaging, a non-invasive and label-free technique, allows simultaneous imaging of the intracellular composition and distribution of multiple bio-molecules. In this study, we explored the application of Raman spectroscopic imaging in the whole Ciona intestinalis embryo during development. Analysis of Raman spectra scattered from C. intestinalis embryos revealed a number of localized patterns of high Raman intensity within the embryo. Based on the observed distribution of bio-molecules, we succeeded in identifying the location and structure of differentiated muscle and endoderm within the whole embryo, up to the tailbud stage, in a label-free manner. Furthermore, during cell differentiation, we detected significant differences in cell state between muscle/endoderm daughter cells and daughter cells with other fates that had divided from the same mother cells; this was achieved by focusing on the Raman intensity of single Raman bands at 1002 or 1526 cm−1, respectively. This study reports the first application of Raman spectroscopic imaging to the study of identifying and characterizing differentiating tissues in a whole chordate embryo. Our results suggest that Raman spectroscopic imaging is a feasible label-free technique for investigating the developmental process of the whole embryo of C. intestinalis. PMID:23977129

Visualization and correction of automated segmentation, tracking and lineaging from 5-D stem cell image sequences.

PubMed

Wait, Eric; Winter, Mark; Bjornsson, Chris; Kokovay, Erzsebet; Wang, Yue; Goderie, Susan; Temple, Sally; Cohen, Andrew R

2014-10-03

Neural stem cells are motile and proliferative cells that undergo mitosis, dividing to produce daughter cells and ultimately generating differentiated neurons and glia. Understanding the mechanisms controlling neural stem cell proliferation and differentiation will play a key role in the emerging fields of regenerative medicine and cancer therapeutics. Stem cell studies in vitro from 2-D image data are well established. Visualizing and analyzing large three dimensional images of intact tissue is a challenging task. It becomes more difficult as the dimensionality of the image data increases to include time and additional fluorescence channels. There is a pressing need for 5-D image analysis and visualization tools to study cellular dynamics in the intact niche and to quantify the role that environmental factors play in determining cell fate. We present an application that integrates visualization and quantitative analysis of 5-D (x,y,z,t,channel) and large montage confocal fluorescence microscopy images. The image sequences show stem cells together with blood vessels, enabling quantification of the dynamic behaviors of stem cells in relation to their vascular niche, with applications in developmental and cancer biology. Our application automatically segments, tracks, and lineages the image sequence data and then allows the user to view and edit the results of automated algorithms in a stereoscopic 3-D window while simultaneously viewing the stem cell lineage tree in a 2-D window. Using the GPU to store and render the image sequence data enables a hybrid computational approach. An inference-based approach utilizing user-provided edits to automatically correct related mistakes executes interactively on the system CPU while the GPU handles 3-D visualization tasks. By exploiting commodity computer gaming hardware, we have developed an application that can be run in the laboratory to facilitate rapid iteration through biological experiments. We combine unsupervised image analysis algorithms with an interactive visualization of the results. Our validation interface allows for each data set to be corrected to 100% accuracy, ensuring that downstream data analysis is accurate and verifiable. Our tool is the first to combine all of these aspects, leveraging the synergies obtained by utilizing validation information from stereo visualization to improve the low level image processing tasks.

Giant cell arteritis: diagnostic accuracy of MR imaging of superficial cranial arteries in initial diagnosis-results from a multicenter trial.

PubMed

Klink, Thorsten; Geiger, Julia; Both, Marcus; Ness, Thomas; Heinzelmann, Sonja; Reinhard, Matthias; Holl-Ulrich, Konstanze; Duwendag, Dirk; Vaith, Peter; Bley, Thorsten Alexander

2014-12-01

To assess the diagnostic accuracy of contrast material-enhanced magnetic resonance (MR) imaging of superficial cranial arteries in the initial diagnosis of giant cell arteritis ( GCA giant cell arteritis ). Following institutional review board approval and informed consent, 185 patients suspected of having GCA giant cell arteritis were included in a prospective three-university medical center trial. GCA giant cell arteritis was diagnosed or excluded clinically in all patients (reference standard [final clinical diagnosis]). In 53.0% of patients (98 of 185), temporal artery biopsy ( TAB temporal artery biopsy ) was performed (diagnostic standard [ TAB temporal artery biopsy ]). Two observers independently evaluated contrast-enhanced T1-weighted MR images of superficial cranial arteries by using a four-point scale. Diagnostic accuracy, involvement pattern, and systemic corticosteroid ( sCS systemic corticosteroid ) therapy effects were assessed in comparison with the reference standard (total study cohort) and separately in comparison with the diagnostic standard TAB temporal artery biopsy ( TAB temporal artery biopsy subcohort). Statistical analysis included diagnostic accuracy parameters, interobserver agreement, and receiver operating characteristic analysis. Sensitivity of MR imaging was 78.4% and specificity was 90.4% for the total study cohort, and sensitivity was 88.7% and specificity was 75.0% for the TAB temporal artery biopsy subcohort (first observer). Diagnostic accuracy was comparable for both observers, with good interobserver agreement ( TAB temporal artery biopsy subcohort, κ = 0.718; total study cohort, κ = 0.676). MR imaging scores were significantly higher in patients with GCA giant cell arteritis -positive results than in patients with GCA giant cell arteritis -negative results ( TAB temporal artery biopsy subcohort and total study cohort, P < .001). Diagnostic accuracy of MR imaging was high in patients without and with sCS systemic corticosteroid therapy for 5 days or fewer (area under the curve, ≥0.9) and was decreased in patients receiving sCS systemic corticosteroid therapy for 6-14 days. In 56.5% of patients with TAB temporal artery biopsy -positive results (35 of 62), MR imaging displayed symmetrical and simultaneous inflammation of arterial segments. MR imaging of superficial cranial arteries is accurate in the initial diagnosis of GCA giant cell arteritis . Sensitivity probably decreases after more than 5 days of sCS systemic corticosteroid therapy; thus, imaging should not be delayed. Clinical trial registration no. DRKS00000594 . © RSNA, 2014.

High MRI performance fluorescent mesoporous silica-coated magnetic nanoparticles for tracking neural progenitor cells in an ischemic mouse model

NASA Astrophysics Data System (ADS)

Zhang, Lu; Wang, Yao; Tang, Yaohui; Jiao, Zheng; Xie, Chengying; Zhang, Haijiao; Gu, Ping; Wei, Xunbin; Yang, Guo-Yuan; Gu, Hongchen; Zhang, Chunfu

2013-05-01

Multifunctional probes with high MRI sensitivity and high efficiency for cell labeling are desirable for MR cell imaging. Herein, we have fabricated fluorescent mesoporous silica-coated superparamagnetic iron oxide nanoparticles (fmSiO4@SPIONs) for neural progenitor cell (C17.2) MR imaging. FmSiO4@SPIONs were discrete and uniform in size, and had a clear core-shell structure. The magnetic core size was about 10 nm and the fluorescent mesoporous silica coating layer was around 20 nm. Compared with fluorescent dense silica-coated SPIONs (fdSiO4@SPIONs) with a similar size, fmSiO4@SPIONs demonstrated higher MR sensitivity and cell labeling efficiency. When implanted into the right hemisphere of stroke mice, contralateral to the ischemic territory, a small amount of labeled cells were able to be tracked migrating to the lesion sites using a clinical MRI scanner (3 T). More impressively, even when administered intravenously, the labeled cells could also be monitored homing to the ischemic area. MRI observations were corroborated by histological studies of the brain tissues. Our study demonstrated that fmSiO4@SPIONs are highly effective for cell imaging and hold great promise for MRI cell tracking in future.Multifunctional probes with high MRI sensitivity and high efficiency for cell labeling are desirable for MR cell imaging. Herein, we have fabricated fluorescent mesoporous silica-coated superparamagnetic iron oxide nanoparticles (fmSiO4@SPIONs) for neural progenitor cell (C17.2) MR imaging. FmSiO4@SPIONs were discrete and uniform in size, and had a clear core-shell structure. The magnetic core size was about 10 nm and the fluorescent mesoporous silica coating layer was around 20 nm. Compared with fluorescent dense silica-coated SPIONs (fdSiO4@SPIONs) with a similar size, fmSiO4@SPIONs demonstrated higher MR sensitivity and cell labeling efficiency. When implanted into the right hemisphere of stroke mice, contralateral to the ischemic territory, a small amount of labeled cells were able to be tracked migrating to the lesion sites using a clinical MRI scanner (3 T). More impressively, even when administered intravenously, the labeled cells could also be monitored homing to the ischemic area. MRI observations were corroborated by histological studies of the brain tissues. Our study demonstrated that fmSiO4@SPIONs are highly effective for cell imaging and hold great promise for MRI cell tracking in future. Electronic supplementary information (ESI) available: Details of cell internalization of fmSiO4@SPIONs compared with SHU555A, immunofluorescence image of the immature phenotype of labeled C17.2. See DOI: 10.1039/c3nr00119a

Combining fluorescence imaging with Hi-C to study 3D genome architecture of the same single cell.

PubMed

Lando, David; Basu, Srinjan; Stevens, Tim J; Riddell, Andy; Wohlfahrt, Kai J; Cao, Yang; Boucher, Wayne; Leeb, Martin; Atkinson, Liam P; Lee, Steven F; Hendrich, Brian; Klenerman, Dave; Laue, Ernest D

2018-05-01

Fluorescence imaging and chromosome conformation capture assays such as Hi-C are key tools for studying genome organization. However, traditionally, they have been carried out independently, making integration of the two types of data difficult to perform. By trapping individual cell nuclei inside a well of a 384-well glass-bottom plate with an agarose pad, we have established a protocol that allows both fluorescence imaging and Hi-C processing to be carried out on the same single cell. The protocol identifies 30,000-100,000 chromosome contacts per single haploid genome in parallel with fluorescence images. Contacts can be used to calculate intact genome structures to better than 100-kb resolution, which can then be directly compared with the images. Preparation of 20 single-cell Hi-C libraries using this protocol takes 5 d of bench work by researchers experienced in molecular biology techniques. Image acquisition and analysis require basic understanding of fluorescence microscopy, and some bioinformatics knowledge is required to run the sequence-processing tools described here.

Emerging roles for integrated imaging modalities in cardiovascular cell-based therapeutics: a clinical perspective

PubMed Central

Psaltis, Peter J.; Simari, Robert D.

2012-01-01

Despite preclinical promise, the progress of cell-based therapy to clinical cardiovascular practice has been slowed by several challenges and uncertainties that have been highlighted by the conflicting results of human trials. Most telling has been the revelation that current strategies fall short of achieving sufficient retention and engraftment of cells to meet the ambitious objective of myocardial regeneration. This has sparked novel research into the refinement of cell biology and delivery to overcome these shortcomings. Within this context, molecular imaging has emerged as a valuable tool for providing noninvasive surveillance of cell fate in vivo. Direct and indirect labelling of cells can be coupled with clinically relevant imaging modalities, such as radionuclide single photon emission computed tomography and positron emission tomography, and magnetic resonance imaging, to assess their short- and long-term distributions, along with their viability, proliferation and functional interaction with the host myocardium. This review details the strengths and limitations of the different cell labelling and imaging techniques and their potential application to the clinical realm. We also consider the broader, multifaceted utility of imaging throughout the cell therapy process, providing a discussion of its considerable value during cell delivery and its importance during the evaluation of cardiac outcomes in clinical studies. PMID:21901381

Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells

NASA Astrophysics Data System (ADS)

Han, Meng; Blindewald-Wittich, Almut; Holz, Frank G.; Giese, Günter; Niemz, Markolf H.; Snyder, Sarah; Sun, Hui; Yu, Jiayi; Agopov, Michael; La Schiazza, Olivier; Bille, Josef F.

2006-01-01

Degeneration of retinal pigment epithelial (RPE) cells severely impairs the visual function of retina photoreceptors. However, little is known about the events that trigger the death of RPE cells at the subcellular level. Two-photon excited autofluorescence (TPEF) imaging of RPE cells proves to be well suited to investigate both the morphological and the spectral characteristics of the human RPE cells. The dominant fluorophores of autofluorescence derive from lipofuscin (LF) granules that accumulate in the cytoplasm of the RPE cells with increasing age. Spectral TPEF imaging reveals the existence of abnormal LF granules with blue shifted autofluorescence in RPE cells of aging patients and brings new insights into the complicated composition of the LF granules. Based on a proposed two-photon laser scanning ophthalmoscope, TPEF imaging of the living retina may be valuable for diagnostic and pathological studies of age related eye diseases.

Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms.

PubMed

Liu, Tsung-Li; Upadhyayula, Srigokul; Milkie, Daniel E; Singh, Ved; Wang, Kai; Swinburne, Ian A; Mosaliganti, Kishore R; Collins, Zach M; Hiscock, Tom W; Shea, Jamien; Kohrman, Abraham Q; Medwig, Taylor N; Dambournet, Daphne; Forster, Ryan; Cunniff, Brian; Ruan, Yuan; Yashiro, Hanako; Scholpp, Steffen; Meyerowitz, Elliot M; Hockemeyer, Dirk; Drubin, David G; Martin, Benjamin L; Matus, David Q; Koyama, Minoru; Megason, Sean G; Kirchhausen, Tom; Betzig, Eric

2018-04-20

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Advantages of indium-tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells.

PubMed

Pluk, H; Stokes, D J; Lich, B; Wieringa, B; Fransen, J

2009-03-01

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium-tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.

Unravel lipid accumulation mechanism in oleaginous yeast through single cell systems biology study

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

Xie, Xiaoliang; Ding, Shiyou

Searching for alternative and clean energy is one of the most important tasks today. Our research aimed at finding the best living condition for certain types of oleaginous yeasts for efficient lipid production. We found that R. glutinis yeast cells has great variability in lipid production among cells while Y. lipolytica cells has similar oil production ability. We found some individual cells shows much higher level of oil production. In order to further study these cases, we employed a label-free chemical sensitive microscopy method call stimulated Raman scattering (SRS). With SRS, we could measure the lipid content in each cell.more » We combined SRS microscopy with microfluidic device so that we can isolate cells with high fat content. We also developed SRS imaging technique that has higher imaging speed, which is highly desirable for high throughput cell screening and sorting. Since these cells has similar genome, it must be the transcriptome caused their difference in oil production. We developed a single cell transcriptome sequencing method to study which genes are responsible for elevated oil production. These methods that are developed for this project can easily be applied for many other areas of research. For example, the single transcriptome can be used to study the transcriptomes of other cell types. The high-speed SRS microscopy techniques can be used to speed up chemical imaging for lablefree histology or imaging distribution of chemicals in tissues of live mice or in humans. The developed microfluidic platform can be used to sort other type of cells, e.g., white blood cells for diagnosis of cancer or other blood diseases.« less

Structural and elemental changes in glioblastoma cells in situ: complementary imaging with high resolution visible light- and X-ray microscopy

DOE PAGES

Ducic, Tanja; Paunesku, Tatjana; Chen, Si; ...

2016-12-09

The glioblastoma (GBM) is characterized by a short median survival and an almost 100% tumor related mortality. GBM cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores application of X-ray and visible light microscopy to display the elemental and structural images of cells from 3 patient derived GMB samples and an established GMB cell line. Slight differences in elemental concentrations, in actin cytoskeleton organization and cell morphology were noted between all cells types by X-ray fluorescence and full field soft X-ray microscopy, as well as the Structured Illumination Super-resolution Microscope (SIM). Different samplemore » preparation approaches were used to match each imaging technique. While preparation for SIM included cell fixation and staining, intact frozen hydrated cells were used for the trace element imaging by hard X-ray fluorescence and exploration of the structural features by soft X-ray absorption tomography. In conclusion, each technique documented differences between samples with regard to morphology and elemental composition and underscored the importance of use of multiple patient derived samples for detailed GBM study.« less

Structural and elemental changes in glioblastoma cells in situ: complementary imaging with high resolution visible light- and X-ray microscopy

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

Ducic, Tanja; Paunesku, Tatjana; Chen, Si

The glioblastoma (GBM) is characterized by a short median survival and an almost 100% tumor related mortality. GBM cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores application of X-ray and visible light microscopy to display the elemental and structural images of cells from 3 patient derived GMB samples and an established GMB cell line. Slight differences in elemental concentrations, in actin cytoskeleton organization and cell morphology were noted between all cells types by X-ray fluorescence and full field soft X-ray microscopy, as well as the Structured Illumination Super-resolution Microscope (SIM). Different samplemore » preparation approaches were used to match each imaging technique. While preparation for SIM included cell fixation and staining, intact frozen hydrated cells were used for the trace element imaging by hard X-ray fluorescence and exploration of the structural features by soft X-ray absorption tomography. In conclusion, each technique documented differences between samples with regard to morphology and elemental composition and underscored the importance of use of multiple patient derived samples for detailed GBM study.« less

Long-Term Live Cell Imaging of Cell Migration: Effects of Pathogenic Fungi on Human Epithelial Cell Migration.

PubMed

Wöllert, Torsten; Langford, George M

2016-01-01

Long-term live cell imaging was used in this study to determine the responses of human epithelial cells to pathogenic biofilms formed by Candida albicans. Epithelial cells of the skin represent the front line of defense against invasive pathogens such as C. albicans but under certain circumstances, especially when the host's immune system is compromised, the skin barrier is breached. The mechanisms by which the fungal pathogen penetrates the skin and invade the deeper layers are not fully understood. In this study we used keratinocytes grown in culture as an in vitro model system to determine changes in host cell migration and the actin cytoskeleton in response to virulence factors produced by biofilms of pathogenic C. albicans. It is clear that changes in epithelial cell migration are part of the response to virulence factors secreted by biofilms of C. albicans and the actin cytoskeleton is the downstream effector that mediates cell migration. Our goal is to understand the mechanism by which virulence factors hijack the signaling pathways of the actin cytoskeleton to alter cell migration and thereby invade host tissues. To understand the dynamic changes of the actin cytoskeleton during infection, we used long-term live cell imaging to obtain spatial and temporal information of actin filament dynamics and to identify signal transduction pathways that regulate the actin cytoskeleton and its associated proteins. Long-term live cell imaging was achieved using a high resolution, multi-mode epifluorescence microscope equipped with specialized light sources, high-speed cameras with high sensitivity detectors, and specific biocompatible fluorescent markers. In addition to the multi-mode epifluorescence microscope, a spinning disk confocal long-term live cell imaging system (Olympus CV1000) equipped with a stage incubator to create a stable in vitro environment for long-term real-time and time-lapse microscopy was used. Detailed descriptions of these two long-term live cell imaging systems are provided.

SERS imaging of cell-surface biomolecules metabolically labeled with bioorthogonal Raman reporters.

PubMed

Xiao, Ming; Lin, Liang; Li, Zefan; Liu, Jie; Hong, Senlian; Li, Yaya; Zheng, Meiling; Duan, Xuanming; Chen, Xing

2014-08-01

Live imaging of biomolecules with high specificity and sensitivity as well as minimal perturbation is essential for studying cellular processes. Here, we report the development of a bioorthogonal surface-enhanced Raman scattering (SERS) imaging approach that exploits small Raman reporters for visualizing cell-surface biomolecules. The cells were cultured and imaged by SERS microscopy on arrays of Raman-enhancing nanoparticles coated on silicon wafers or glass slides. The Raman reporters including azides, alkynes, and carbondeuterium bonds are small in size and spectroscopically bioorthogonal (background-free). We demonstrated that various cell-surface biomolecules including proteins, glycans, and lipids were metabolically incorporated with the corresponding precursors bearing a Raman reporter and visualized by SERS microscopy. The coupling of SERS microscopy with bioorthogonal Raman reporters expands the capabilities of live-cell microscopy beyond the modalities of fluorescence and label-free imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Strain Library Imaging Protocol for high-throughput, automated single-cell microscopy of large bacterial collections arrayed on multiwell plates.

PubMed

Shi, Handuo; Colavin, Alexandre; Lee, Timothy K; Huang, Kerwyn Casey

2017-02-01

Single-cell microscopy is a powerful tool for studying gene functions using strain libraries, but it suffers from throughput limitations. Here we describe the Strain Library Imaging Protocol (SLIP), which is a high-throughput, automated microscopy workflow for large strain collections that requires minimal user involvement. SLIP involves transferring arrayed bacterial cultures from multiwell plates onto large agar pads using inexpensive replicator pins and automatically imaging the resulting single cells. The acquired images are subsequently reviewed and analyzed by custom MATLAB scripts that segment single-cell contours and extract quantitative metrics. SLIP yields rich data sets on cell morphology and gene expression that illustrate the function of certain genes and the connections among strains in a library. For a library arrayed on 96-well plates, image acquisition can be completed within 4 min per plate.

Self-assembled dual-modality contrast agents for non-invasive stem cell tracking via near-infrared fluorescence and magnetic resonance imaging.

PubMed

Liu, Hong; Tan, Yan; Xie, Lisi; Yang, Lei; Zhao, Jing; Bai, Jingxuan; Huang, Ping; Zhan, Wugen; Wan, Qian; Zou, Chao; Han, Yali; Wang, Zhiyong

2016-09-15

Stem cells hold great promise for treating various diseases. However, one of the main drawbacks of stem cell therapy is the lack of non-invasive image-tracking technologies. Although magnetic resonance imaging (MRI) and near-infrared fluorescence (NIRF) imaging have been employed to analyse cellular and subcellular events via the assistance of contrast agents, the sensitivity and temporal resolution of MRI and the spatial resolution of NIRF are still shortcomings. In this study, superparamagnetic iron oxide nanocrystals and IR-780 dyes were co-encapsulated in stearic acid-modified polyethylenimine to form a dual-modality contrast agent with nano-size and positive charge. These resulting agents efficiently labelled stem cells and did not influence the cellular viability and differentiation. Moreover, the labelled cells showed the advantages of dual-modality imaging in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

3D nanoscale imaging of the yeast, Schizosaccharomyces pombe, by full-field transmission X-ray microscopy at 5.4 keV.

PubMed

Chen, Jie; Yang, Yunhao; Zhang, Xiaobo; Andrews, Joy C; Pianetta, Piero; Guan, Yong; Liu, Gang; Xiong, Ying; Wu, Ziyu; Tian, Yangchao

2010-07-01

Three-dimensional (3D) nanoscale structures of the fission yeast, Schizosaccharomyces pombe, can be obtained by full-field transmission hard X-ray microscopy with 30 nm resolution using synchrotron radiation sources. Sample preparation is relatively simple and the samples are portable across various imaging environments, allowing for high-throughput sample screening. The yeast cells were fixed and double-stained with Reynold's lead citrate and uranyl acetate. We performed both absorption contrast and Zernike phase contrast imaging on these cells in order to test this method. The membranes, nucleus, and subcellular organelles of the cells were clearly visualized using absorption contrast mode. The X-ray images of the cells could be used to study the spatial distributions of the organelles in the cells. These results show unique structural information, demonstrating that hard X-ray microscopy is a complementary method for imaging and analyzing biological samples.

In situ probing of cholesterol in astrocytes at the single-cell level using laser desorption ionization mass spectrometric imaging with colloidal silver.

PubMed

Perdian, D C; Cha, Sangwon; Oh, Jisun; Sakaguchi, Donald S; Yeung, Edward S; Lee, Young Jin

2010-04-30

Mass spectrometric imaging has been utilized to localize individual astrocytes and to obtain cholesterol populations at the single-cell level in laser desorption ionization (LDI) with colloidal silver. The silver ion adduct of membrane-bound cholesterol was monitored to detect individual cells. Good correlation between mass spectrometric and optical images at different cell densities indicates the ability to perform single-cell studies of cholesterol abundance. The feasibility of quantification is confirmed by the agreement between the LDI-MS ion signals and the results from a traditional enzymatic fluorometric assay. We propose that this approach could be an effective tool to study chemical populations at the cellular level. Published in 2010 by John Wiley & Sons, Ltd.

Imaging of single liver tumor cells intoxicated by heavy metals using ToF-SIMS

NASA Astrophysics Data System (ADS)

Mai, Fu-Der; Chen, Bo-Jung; Wu, Li-Chen; Li, Feng-Yin; Chen, Wen-Kang

2006-07-01

Human liver tumor cells intoxicated with five different Cd, Cu, Cr, Hg and Zn metals were analyzed using imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) to visualize the metal distributions in a single cell basis. A protocol was developed by combining rapid freezing, freeze-fracture and imprinting for transferring the intoxicated cells to a silicon wafer. As shown in the ToF-SIMS images, the cellular morphology was preserved indicating that this protocol can be used to prepare a representative cell for ToF-SIMS imaging analysis. Among the five metal ions investigated in this study, only Cr and Cu ions show preferential diffusion into the cell after simulated intoxication while the signals of the other three ions are either too low to be detected or unable to be distinguished from background intensity.

Hollow fiber: a biophotonic implant for live cells

NASA Astrophysics Data System (ADS)

Silvestre, Oscar F.; Holton, Mark D.; Summers, Huw D.; Smith, Paul J.; Errington, Rachel J.

2009-02-01

The technical objective of this study has been to design, build and validate biocompatible hollow fiber implants based on fluorescence with integrated biophotonics components to enable in fiber kinetic cell based assays. A human osteosarcoma in vitro cell model fiber system has been established with validation studies to determine in fiber cell growth, cell cycle analysis and organization in normal and drug treated conditions. The rationale for implant development have focused on developing benchmark concepts in standard monolayer tissue culture followed by the development of in vitro hollow fiber designs; encompassing imaging with and without integrated biophotonics. Furthermore the effect of introducing targetable biosensors into the encapsulated tumor implant such as quantum dots for informing new detection readouts and possible implant designs have been evaluated. A preliminary micro/macro imaging approach has been undertaken, that could provide a mean to track distinct morphological changes in cells growing in a 3D matrix within the fiber which affect the light scattering properties of the implant. Parallel engineering studies have showed the influence of the optical properties of the fiber polymer wall in all imaging modes. Taken all together, we show the basic foundation and the opportunities for multi-modal imaging within an in vitro implant format.

Tumor-stem cells interactions by fluorescence imaging

NASA Astrophysics Data System (ADS)

Meleshina, Aleksandra V.; Cherkasova, Elena I.; Sergeeva, Ekaterina; Turchin, Ilya V.; Kiseleva, Ekaterina V.; Dashinimaev, Erdem B.; Shirmanova, Marina V.; Zagaynova, Elena V.

2013-02-01

Recently, great deal of interest is investigation the function of the stem cells (SC) in tumors. In this study, we studied «recipient-tumor- fluorescent stem cells » system using the methods of in vivo imaging and laser scanning microscopy (LSM). We used adipose-derived adult stem (ADAS) cells of human lentiviral transfected with the gene of fluorescent protein Turbo FP635. ADAS cells were administrated into nude mice with transplanted tumor HeLa Kyoto (human cervical carcinoma) at different stages of tumor growth (0-8 days) intravenously or into tumor. In vivo imaging was performed on the experimental setup for epi - luminescence bioimaging (IAP RAS, Nizhny Novgorod). The results of the imaging showed localization of fluorophore tagged stem cells in the spleen on day 5-9 after injection. The sensitivity of the technique may be improved by spectral separation autofluorescence and fluorescence of stem cells. We compared the results of in vivo imaging and confocal laser scanning microscopy (LSM 510 META, Carl Zeiss, Germany). Internal organs of the animals and tumor tissue were investigated. It was shown that with i.v. injection of ADAS, bright fluorescent structures with spectral characteristics corresponding to TurboFP635 protein are locally accumulated in the marrow, lungs and tumors of animals. These findings indicate that ADAS cells integrate in the animal body with transplanted tumor and can be identified by fluorescence bioimaging techniques in vivo and ex vivo.

Development of an ultralow-light-level luminescence image analysis system for dynamic measurements of transcriptional activity in living and migrating cells.

PubMed

Maire, E; Lelièvre, E; Brau, D; Lyons, A; Woodward, M; Fafeur, V; Vandenbunder, B

2000-04-10

We have developed an approach to study in single living epithelial cells both cell migration and transcriptional activation, which was evidenced by the detection of luminescence emission from cells transfected with luciferase reporter vectors. The image acquisition chain consists of an epifluorescence inverted microscope, connected to an ultralow-light-level photon-counting camera and an image-acquisition card associated to specialized image analysis software running on a PC computer. Using a simple method based on a thin calibrated light source, the image acquisition chain has been optimized following comparisons of the performance of microscopy objectives and photon-counting cameras designed to observe luminescence. This setup allows us to measure by image analysis the luminescent light emitted by individual cells stably expressing a luciferase reporter vector. The sensitivity of the camera was adjusted to a high value, which required the use of a segmentation algorithm to eliminate the background noise. Following mathematical morphology treatments, kinetic changes of luminescent sources were analyzed and then correlated with the distance and speed of migration. Our results highlight the usefulness of our image acquisition chain and mathematical morphology software to quantify the kinetics of luminescence changes in migrating cells.

A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis.

PubMed

Schmitter, Daniel; Wachowicz, Paulina; Sage, Daniel; Chasapi, Anastasia; Xenarios, Ioannis; Simanis; Unser, Michael

2013-01-01

The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. (Continued on next page) (Continued from previous page). "RodCellJ" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells. RodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html.

Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

PubMed

Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

2014-01-01

Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

Imaging of bioluminescent LNCaP-luc-M6 tumors: a new animal model for the study of metastatic human prostate cancer.

PubMed

Scatena, Caroline D; Hepner, Mischa A; Oei, Yoko A; Dusich, Joan M; Yu, Shang-Fan; Purchio, Tony; Contag, Pamela R; Jenkins, Darlene E

2004-05-15

Animal experiments examining hormone-sensitive metastatic prostate cancer using the human LNCaP cell line have been limited to endpoint analyses. To permit longitudinal studies, we generated a luciferase-expressing cell line and used bioluminescent imaging (BLI) to non-invasively monitor the in vivo growth of primary LNCaP tumors and metastasis. LNCaP.FGC cells were transfected to constitutively express firefly luciferase. LNCaP-luc-M6 cells were tested for bioluminescent signal intensity and hormone responsiveness in vitro. The cells were implanted in subcutaneous and orthotopic sites in SCID-bg mice and imaged over time. The LNCaP-luc-M6 cells formed subcutaneous and orthotopic tumors in SCID-bg mice, and nearly all tumor-bearing animals developed pulmonary metastases. Early detection and temporal growth of primary tumors and metastatic lesions was successfully monitored by BLI. The LNCaP-luc-M6 cell line is a bioluminescent, hormone-sensitive prostate cancer cell line applicable for BLI studies to non-invasively monitor subcutaneous and orthotopic prostate tumor growth and metastasis in vivo. Copyright 2004 Wiley-Liss, Inc.

Confocal Raman imaging for cancer cell classification

NASA Astrophysics Data System (ADS)

Mathieu, Evelien; Van Dorpe, Pol; Stakenborg, Tim; Liu, Chengxun; Lagae, Liesbet

2014-05-01

We propose confocal Raman imaging as a label-free single cell characterization method that can be used as an alternative for conventional cell identification techniques that typically require labels, long incubation times and complex sample preparation. In this study it is investigated whether cancer and blood cells can be distinguished based on their Raman spectra. 2D Raman scans are recorded of 114 single cells, i.e. 60 breast (MCF-7), 5 cervix (HeLa) and 39 prostate (LNCaP) cancer cells and 10 monocytes (from healthy donors). For each cell an average spectrum is calculated and principal component analysis is performed on all average cell spectra. The main features of these principal components indicate that the information for cell identification based on Raman spectra mainly comes from the fatty acid composition in the cell. Based on the second and third principal component, blood cells could be distinguished from cancer cells; and prostate cancer cells could be distinguished from breast and cervix cancer cells. However, it was not possible to distinguish breast and cervix cancer cells. The results obtained in this study, demonstrate the potential of confocal Raman imaging for cell type classification and identification purposes.

A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D-Printed Parts, a Smartphone, and Off-The-Shelf Electronics

PubMed Central

Schwan, Emil; Fatsis-Kavalopoulos, Nikos; Kreuger, Johan

2016-01-01

Time-lapse imaging is a powerful tool for studying cellular dynamics and cell behavior over long periods of time to acquire detailed functional information. However, commercially available time-lapse imaging systems are expensive and this has limited a broader implementation of this technique in low-resource environments. Further, the availability of time-lapse imaging systems often present workflow bottlenecks in well-funded institutions. To address these limitations we have designed a modular and affordable time-lapse imaging and incubation system (ATLIS). The ATLIS enables the transformation of simple inverted microscopes into live cell imaging systems using custom-designed 3D-printed parts, a smartphone, and off-the-shelf electronic components. We demonstrate that the ATLIS provides stable environmental conditions to support normal cell behavior during live imaging experiments in both traditional and evaporation-sensitive microfluidic cell culture systems. Thus, the system presented here has the potential to increase the accessibility of time-lapse microscopy of living cells for the wider research community. PMID:28002463

A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D-Printed Parts, a Smartphone, and Off-The-Shelf Electronics.

PubMed

Hernández Vera, Rodrigo; Schwan, Emil; Fatsis-Kavalopoulos, Nikos; Kreuger, Johan

2016-01-01

Time-lapse imaging is a powerful tool for studying cellular dynamics and cell behavior over long periods of time to acquire detailed functional information. However, commercially available time-lapse imaging systems are expensive and this has limited a broader implementation of this technique in low-resource environments. Further, the availability of time-lapse imaging systems often present workflow bottlenecks in well-funded institutions. To address these limitations we have designed a modular and affordable time-lapse imaging and incubation system (ATLIS). The ATLIS enables the transformation of simple inverted microscopes into live cell imaging systems using custom-designed 3D-printed parts, a smartphone, and off-the-shelf electronic components. We demonstrate that the ATLIS provides stable environmental conditions to support normal cell behavior during live imaging experiments in both traditional and evaporation-sensitive microfluidic cell culture systems. Thus, the system presented here has the potential to increase the accessibility of time-lapse microscopy of living cells for the wider research community.

Effects of Image Compression on Automatic Count of Immunohistochemically Stained Nuclei in Digital Images

PubMed Central

López, Carlos; Lejeune, Marylène; Escrivà, Patricia; Bosch, Ramón; Salvadó, Maria Teresa; Pons, Lluis E.; Baucells, Jordi; Cugat, Xavier; Álvaro, Tomás; Jaén, Joaquín

2008-01-01

This study investigates the effects of digital image compression on automatic quantification of immunohistochemical nuclear markers. We examined 188 images with a previously validated computer-assisted analysis system. A first group was composed of 47 images captured in TIFF format, and other three contained the same images converted from TIFF to JPEG format with 3×, 23× and 46× compression. Counts of TIFF format images were compared with the other three groups. Overall, differences in the count of the images increased with the percentage of compression. Low-complexity images (≤100 cells/field, without clusters or with small-area clusters) had small differences (<5 cells/field in 95–100% of cases) and high-complexity images showed substantial differences (<35–50 cells/field in 95–100% of cases). Compression does not compromise the accuracy of immunohistochemical nuclear marker counts obtained by computer-assisted analysis systems for digital images with low complexity and could be an efficient method for storing these images. PMID:18755997

Cells from icons to symbols: molecularizing cell biology in the 1980s.

PubMed

Serpente, Norberto

2011-12-01

Over centuries cells have been the target of optical and electronic microscopes as well as others technologies, with distinctive types of visual output. Whilst optical technologies produce images 'evident to the eye', the electronic and especially the molecular create images that are more elusive to conceptualization and assessment. My study applies the semiotic approach to the production of images in cell biology to capture the shift from microscopic images to non-traditional visual technologies around 1980. Here I argue that the visual shift that coincides with the growing dominance of molecular biology involves a change from iconic to symbolic forms. Copyright © 2011 Elsevier Ltd. All rights reserved.

Imaging Study of Multi-Crystalline Silicon Wafers Throughout the Manufacturing Process: Preprint

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

Johnston, S.; Yan, F.; Zaunbracher, K.

2011-07-01

Imaging techniques are applied to multi-crystalline silicon bricks, wafers at various process steps, and finished solar cells. Photoluminescence (PL) imaging is used to characterize defects and material quality on bricks and wafers. Defect regions within the wafers are influenced by brick position within an ingot and height within the brick. The defect areas in as-cut wafers are compared to imaging results from reverse-bias electroluminescence and dark lock-in thermography and cell parameters of near-neighbor finished cells. Defect areas are also characterized by defect band emissions. The defect areas measured by these techniques on as-cut wafers are shown to correlate to finishedmore » cell performance.« less

In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine.

PubMed

Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W; Cai, Jiye

2014-11-07

The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In this review, we attempt to summarize the characteristics of these advanced techniques for use in the in situ single molecule imaging of cell membranes. We believe that this work will help to promote the technological and methodological developments of super-resolution techniques for the single molecule imaging of cell membranes and help researchers better understand which technique is most suitable for their future exploring of membrane biomolecules; ultimately promoting further developments in cell biology, immunology and medicine.

In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine

NASA Astrophysics Data System (ADS)

Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W.; Cai, Jiye

2014-10-01

The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In this review, we attempt to summarize the characteristics of these advanced techniques for use in the in situ single molecule imaging of cell membranes. We believe that this work will help to promote the technological and methodological developments of super-resolution techniques for the single molecule imaging of cell membranes and help researchers better understand which technique is most suitable for their future exploring of membrane biomolecules; ultimately promoting further developments in cell biology, immunology and medicine.

Study of living single cells in culture: automated recognition of cell behavior.

PubMed

Bodin, P; Papin, S; Meyer, C; Travo, P

1988-07-01

An automated system capable of analyzing the behavior, in real time, of single living cells in culture, in a noninvasive and nondestructive way, has been developed. A large number of cell positions in single culture dishes were recorded using a computer controlled, robotized microscope. During subsequent observations, binary images obtained from video image analysis of the microscope visual field allowed the identification of the recorded cells. These cells could be revisited automatically every few minutes. Long-term studies of the behavior of cells make possible the analysis of cellular locomotary and mitotic activities as well as determination of cell shape (chosen from a defined library) for several hours or days in a fully automated way with observations spaced up to 30 minutes. Short-term studies of the behavior of cells permit the study, in a semiautomatic way, of acute effects of drugs (5 to 15 minutes) on changes of surface area and length of cells.

Imaging of polysaccharides in the tomato cell wall with Raman microspectroscopy

PubMed Central

2014-01-01

Background The primary cell wall of fruits and vegetables is a structure mainly composed of polysaccharides (pectins, hemicelluloses, cellulose). Polysaccharides are assembled into a network and linked together. It is thought that the percentage of components and of plant cell wall has an important influence on mechanical properties of fruits and vegetables. Results In this study the Raman microspectroscopy technique was introduced to the visualization of the distribution of polysaccharides in cell wall of fruit. The methodology of the sample preparation, the measurement using Raman microscope and multivariate image analysis are discussed. Single band imaging (for preliminary analysis) and multivariate image analysis methods (principal component analysis and multivariate curve resolution) were used for the identification and localization of the components in the primary cell wall. Conclusions Raman microspectroscopy supported by multivariate image analysis methods is useful in distinguishing cellulose and pectins in the cell wall in tomatoes. It presents how the localization of biopolymers was possible with minimally prepared samples. PMID:24917885

Multispectral Live-Cell Imaging.

PubMed

Cohen, Sarah; Valm, Alex M; Lippincott-Schwartz, Jennifer

2018-06-01

Fluorescent proteins and vital dyes are invaluable tools for studying dynamic processes within living cells. However, the ability to distinguish more than a few different fluorescent reporters in a single sample is limited by the spectral overlap of available fluorophores. Here, we present a protocol for imaging live cells labeled with six fluorophores simultaneously. A confocal microscope with a spectral detector is used to acquire images, and linear unmixing algorithms are applied to identify the fluorophores present in each pixel of the image. We describe the application of this method to visualize the dynamics of six different organelles, and to quantify the contacts between organelles. However, this method can be used to image any molecule amenable to tagging with a fluorescent probe. Thus, multispectral live-cell imaging is a powerful tool for systems-level analysis of cellular organization and dynamics. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

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.

Imaging elemental distribution and ion transport in cultured cells with ion microscopy.

PubMed

Chandra, S; Morrison, G H

1985-06-28

Both elemental distribution and ion transport in cultured cells have been imaged by ion microscopy. Morphological and chemical information was obtained with a spatial resolution of approximately 0.5 micron for sodium, potassium, calcium, and magnesium in freeze-fixed, cryofractured, and freeze-dried normal rat kidney cells and Chinese hamster ovary cells. Ion transport was successfully demonstrated by imaging Na+-K+ fluxes after the inhibition of Na+- and K+ -dependent adenosine triphosphatase with ouabain. This method allows measurements of elemental (isotopic) distribution to be related to cell morphology, thereby providing the means for studying ion distribution and ion transport under different physiological, pathological, and toxicological conditions in cell culture systems.

Molecular imaging promotes progress in orthopedic research.

PubMed

Mayer-Kuckuk, Philipp; Boskey, Adele L

2006-11-01

Modern orthopedic research is directed towards the understanding of molecular mechanisms that determine development, maintenance and health of musculoskeletal tissues. In recent years, many genetic and proteomic discoveries have been made which necessitate investigation under physiological conditions in intact, living tissues. Molecular imaging can meet this demand and is, in fact, the only strategy currently available for noninvasive, quantitative, real-time biology studies in living subjects. In this review, techniques of molecular imaging are summarized, and applications to bone and joint biology are presented. The imaging modality most frequently used in the past was optical imaging, particularly bioluminescence and near-infrared fluorescence imaging. Alternate technologies including nuclear and magnetic resonance imaging were also employed. Orthopedic researchers have applied molecular imaging to murine models including transgenic mice to monitor gene expression, protein degradation, cell migration and cell death. Within the bone compartment, osteoblasts and their stem cells have been investigated, and the organic and mineral bone phases have been assessed. These studies addressed malignancy and injury as well as repair, including fracture healing and cell/gene therapy for skeletal defects. In the joints, molecular imaging has focused on the inflammatory and tissue destructive processes that cause arthritis. As described in this review, the feasibility of applying molecular imaging to numerous areas of orthopedic research has been demonstrated and will likely result in an increase in research dedicated to this powerful strategy. Molecular imaging holds great promise in the future for preclinical orthopedic research as well as next-generation clinical musculoskeletal diagnostics.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences.

PubMed

Dudczig, Stefanie; Currie, Peter D; Poggi, Lucia; Jusuf, Patricia R

2017-03-22

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments. In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

Phase correlation imaging of unlabeled cell dynamics

NASA Astrophysics Data System (ADS)

Ma, Lihong; Rajshekhar, Gannavarpu; Wang, Ru; Bhaduri, Basanta; Sridharan, Shamira; Mir, Mustafa; Chakraborty, Arindam; Iyer, Rajashekar; Prasanth, Supriya; Millet, Larry; Gillette, Martha U.; Popescu, Gabriel

2016-09-01

We present phase correlation imaging (PCI) as a novel approach to study cell dynamics in a spatially-resolved manner. PCI relies on quantitative phase imaging time-lapse data and, as such, functions in label-free mode, without the limitations associated with exogenous markers. The correlation time map outputted in PCI informs on the dynamics of the intracellular mass transport. Specifically, we show that PCI can extract quantitatively the diffusion coefficient map associated with live cells, as well as standard Brownian particles. Due to its high sensitivity to mass transport, PCI can be applied to studying the integrity of actin polymerization dynamics. Our results indicate that the cyto-D treatment blocking the actin polymerization has a dominant effect at the large spatial scales, in the region surrounding the cell. We found that PCI can distinguish between senescent and quiescent cells, which is extremely difficult without using specific markers currently. We anticipate that PCI will be used alongside established, fluorescence-based techniques to enable valuable new studies of cell function.

Imaging and Scattering Measurements for Diesel Spray Combustion: Optical Development and Phenomenological Studies

DTIC Science & Technology

2012-09-30

the CS2 was contained in a rectangular colorimeter cell with a custom built Teflon cap to alleviate the evaporation of the hazardous chemical...6: A comparison of the image quality between the older colorimeter cell (a) and the new containment cell (b). 2.5 Autocorrelation-Based Pulse Length

Improved defect analysis of Gallium Arsenide solar cells using image enhancement

NASA Technical Reports Server (NTRS)

Kilmer, Louis C.; Honsberg, Christiana; Barnett, Allen M.; Phillips, James E.

1989-01-01

A new technique has been developed to capture, digitize, and enhance the image of light emission from a forward biased direct bandgap solar cell. Since the forward biased light emission from a direct bandgap solar cell has been shown to display both qualitative and quantitative information about the solar cell's performance and its defects, signal processing techniques can be applied to the light emission images to identify and analyze shunt diodes. Shunt diodes are of particular importance because they have been found to be the type of defect which is likely to cause failure in a GaAs solar cell. The presence of a shunt diode can be detected from the light emission by using a photodetector to measure the quantity of light emitted at various current densities. However, to analyze how the shunt diodes affect the quality of the solar cell the pattern of the light emission must be studied. With the use of image enhancement routines, the light emission can be studied at low light emission levels where shunt diode effects are dominant.

Image classification of human carcinoma cells using complex wavelet-based covariance descriptors.

PubMed

Keskin, Furkan; Suhre, Alexander; Kose, Kivanc; Ersahin, Tulin; Cetin, A Enis; Cetin-Atalay, Rengul

2013-01-01

Cancer cell lines are widely used for research purposes in laboratories all over the world. Computer-assisted classification of cancer cells can alleviate the burden of manual labeling and help cancer research. In this paper, we present a novel computerized method for cancer cell line image classification. The aim is to automatically classify 14 different classes of cell lines including 7 classes of breast and 7 classes of liver cancer cells. Microscopic images containing irregular carcinoma cell patterns are represented by subwindows which correspond to foreground pixels. For each subwindow, a covariance descriptor utilizing the dual-tree complex wavelet transform (DT-[Formula: see text]WT) coefficients and several morphological attributes are computed. Directionally selective DT-[Formula: see text]WT feature parameters are preferred primarily because of their ability to characterize edges at multiple orientations which is the characteristic feature of carcinoma cell line images. A Support Vector Machine (SVM) classifier with radial basis function (RBF) kernel is employed for final classification. Over a dataset of 840 images, we achieve an accuracy above 98%, which outperforms the classical covariance-based methods. The proposed system can be used as a reliable decision maker for laboratory studies. Our tool provides an automated, time- and cost-efficient analysis of cancer cell morphology to classify different cancer cell lines using image-processing techniques, which can be used as an alternative to the costly short tandem repeat (STR) analysis. The data set used in this manuscript is available as supplementary material through http://signal.ee.bilkent.edu.tr/cancerCellLineClassificationSampleImages.html.

Image Classification of Human Carcinoma Cells Using Complex Wavelet-Based Covariance Descriptors

PubMed Central

Keskin, Furkan; Suhre, Alexander; Kose, Kivanc; Ersahin, Tulin; Cetin, A. Enis; Cetin-Atalay, Rengul

2013-01-01

Cancer cell lines are widely used for research purposes in laboratories all over the world. Computer-assisted classification of cancer cells can alleviate the burden of manual labeling and help cancer research. In this paper, we present a novel computerized method for cancer cell line image classification. The aim is to automatically classify 14 different classes of cell lines including 7 classes of breast and 7 classes of liver cancer cells. Microscopic images containing irregular carcinoma cell patterns are represented by subwindows which correspond to foreground pixels. For each subwindow, a covariance descriptor utilizing the dual-tree complex wavelet transform (DT-WT) coefficients and several morphological attributes are computed. Directionally selective DT-WT feature parameters are preferred primarily because of their ability to characterize edges at multiple orientations which is the characteristic feature of carcinoma cell line images. A Support Vector Machine (SVM) classifier with radial basis function (RBF) kernel is employed for final classification. Over a dataset of 840 images, we achieve an accuracy above 98%, which outperforms the classical covariance-based methods. The proposed system can be used as a reliable decision maker for laboratory studies. Our tool provides an automated, time- and cost-efficient analysis of cancer cell morphology to classify different cancer cell lines using image-processing techniques, which can be used as an alternative to the costly short tandem repeat (STR) analysis. The data set used in this manuscript is available as supplementary material through http://signal.ee.bilkent.edu.tr/cancerCellLineClassificationSampleImages.html. PMID:23341908

Imaging, biodistribution and in vitro study of smart 99mTc-PAMAM G4 dendrimer as novel nano-complex.

PubMed

Narmani, Asghar; Yavari, Kamal; Mohammadnejad, Javad

2017-11-01

Overexpression of folic acid receptor in various human tumors cells makes it as good candidate for targeting delivery of chemotherapeutic and radiopharmaceutical agents. In this research, FA used for functionalization of PEG modified PAMAM G4 dendrimer as a smart delivery of 5-FU and 99m Tc for the breast carcinoma in order to chemotherapeutic and imaging goals. One aim of this research was assess the FA-mediated cell viability assay of PEG-PAMAM G4-FA-5FU- 99m Tc and in vitro uptake of PEG-PAMAM G4-FA- 99m Tc as the novel nano-complex determined on C2Cl2 (normal cell) and MCF-7 (breast cancer cell) cell lines. Other main goals were studied. Morover, an investigation in to in vivo imaging and biodistribution was carried out via a novel radio tracer by which tumor accumulation and site were obviously detected. The targeted tumor images taken by tail intravenous injection demonstrated that nano-complex can be smartly used in imaging study of the clinical practices. Also, the biodistribution of this nano-complex was investigated and the organ predestination of 99m Tc labeled nano-complex (%ID/g) was ascertained. Copyright © 2017 Elsevier B.V. All rights reserved.

Morphology and Topography of Retinal Pericytes in the Living Mouse Retina Using In Vivo Adaptive Optics Imaging and Ex Vivo Characterization

PubMed Central

Schallek, Jesse; Geng, Ying; Nguyen, HoanVu; Williams, David R.

2013-01-01

Purpose. To noninvasively image retinal pericytes in the living eye and characterize NG2-positive cell topography and morphology in the adult mouse retina. Methods. Transgenic mice expressing fluorescent pericytes (NG2, DsRed) were imaged using a two-channel, adaptive optics scanning laser ophthalmoscope (AOSLO). One channel imaged vascular perfusion with near infrared light. A second channel simultaneously imaged fluorescent retinal pericytes. Mice were also imaged using wide-field ophthalmoscopy. To confirm in vivo imaging, five eyes were enucleated and imaged in flat mount with conventional fluorescent microscopy. Cell topography was quantified relative to the optic disc. Results. We observed strong DsRed fluorescence from NG2-positive cells. AOSLO revealed fluorescent vascular mural cells enveloping all vessels in the living retina. Cells were stellate on larger venules, and showed banded morphology on arterioles. NG2-positive cells indicative of pericytes were found on the smallest capillaries of the retinal circulation. Wide-field SLO enabled quick assessment of NG2-positive distribution, but provided insufficient resolution for cell counts. Ex vivo microscopy showed relatively even topography of NG2-positive capillary pericytes at eccentricities more than 0.3 mm from the optic disc (515 ± 94 cells/mm2 of retinal area). Conclusions. We provide the first high-resolution images of retinal pericytes in the living animal. Subcellular resolution enabled morphological identification of NG2-positive cells on capillaries showing classic features and topography of retinal pericytes. This report provides foundational basis for future studies that will track and quantify pericyte topography, morphology, and function in the living retina over time, especially in the progression of microvascular disease. PMID:24150762

Cell segmentation in phase contrast microscopy images via semi-supervised classification over optics-related features.

PubMed

Su, Hang; Yin, Zhaozheng; Huh, Seungil; Kanade, Takeo

2013-10-01

Phase-contrast microscopy is one of the most common and convenient imaging modalities to observe long-term multi-cellular processes, which generates images by the interference of lights passing through transparent specimens and background medium with different retarded phases. Despite many years of study, computer-aided phase contrast microscopy analysis on cell behavior is challenged by image qualities and artifacts caused by phase contrast optics. Addressing the unsolved challenges, the authors propose (1) a phase contrast microscopy image restoration method that produces phase retardation features, which are intrinsic features of phase contrast microscopy, and (2) a semi-supervised learning based algorithm for cell segmentation, which is a fundamental task for various cell behavior analysis. Specifically, the image formation process of phase contrast microscopy images is first computationally modeled with a dictionary of diffraction patterns; as a result, each pixel of a phase contrast microscopy image is represented by a linear combination of the bases, which we call phase retardation features. Images are then partitioned into phase-homogeneous atoms by clustering neighboring pixels with similar phase retardation features. Consequently, cell segmentation is performed via a semi-supervised classification technique over the phase-homogeneous atoms. Experiments demonstrate that the proposed approach produces quality segmentation of individual cells and outperforms previous approaches. Copyright © 2013 Elsevier B.V. All rights reserved.

Image-Based Single Cell Profiling: High-Throughput Processing of Mother Machine Experiments

PubMed Central

Sachs, Christian Carsten; Grünberger, Alexander; Helfrich, Stefan; Probst, Christopher; Wiechert, Wolfgang; Kohlheyer, Dietrich; Nöh, Katharina

2016-01-01

Background Microfluidic lab-on-chip technology combined with live-cell imaging has enabled the observation of single cells in their spatio-temporal context. The mother machine (MM) cultivation system is particularly attractive for the long-term investigation of rod-shaped bacteria since it facilitates continuous cultivation and observation of individual cells over many generations in a highly parallelized manner. To date, the lack of fully automated image analysis software limits the practical applicability of the MM as a phenotypic screening tool. Results We present an image analysis pipeline for the automated processing of MM time lapse image stacks. The pipeline supports all analysis steps, i.e., image registration, orientation correction, channel/cell detection, cell tracking, and result visualization. Tailored algorithms account for the specialized MM layout to enable a robust automated analysis. Image data generated in a two-day growth study (≈ 90 GB) is analyzed in ≈ 30 min with negligible differences in growth rate between automated and manual evaluation quality. The proposed methods are implemented in the software molyso (MOther machine AnaLYsis SOftware) that provides a new profiling tool to analyze unbiasedly hitherto inaccessible large-scale MM image stacks. Conclusion Presented is the software molyso, a ready-to-use open source software (BSD-licensed) for the unsupervised analysis of MM time-lapse image stacks. molyso source code and user manual are available at https://github.com/modsim/molyso. PMID:27661996

Cryo-image Analysis of Tumor Cell Migration, Invasion, and Dispersal in a Mouse Xenograft Model of Human Glioblastoma Multiforme

PubMed Central

Qutaish, Mohammed Q.; Sullivant, Kristin E.; Burden-Gulley, Susan M.; Lu, Hong; Roy, Debashish; Wang, Jing; Basilion, James P.; Brady-Kalnay, Susann M.; Wilson, David L.

2012-01-01

Purpose The goals of this study were to create cryo-imaging methods to quantify characteristics (size, dispersal, and blood vessel density) of mouse orthotopic models of glioblastoma multiforme (GBM) and to enable studies of tumor biology, targeted imaging agents, and theranostic nanoparticles. Procedures Green fluorescent protein-labeled, human glioma LN-229 cells were implanted into mouse brain. At 20–38 days, cryo-imaging gave whole brain, 4-GB, 3D microscopic images of bright field anatomy, including vasculature, and fluorescent tumor. Image analysis/visualization methods were developed. Results Vessel visualization and segmentation methods successfully enabled analyses. The main tumor mass volume, the number of dispersed clusters, the number of cells/cluster, and the percent dispersed volume all increase with age of the tumor. Histograms of dispersal distance give a mean and median of 63 and 56 μm, respectively, averaged over all brains. Dispersal distance tends to increase with age of the tumors. Dispersal tends to occur along blood vessels. Blood vessel density did not appear to increase in and around the tumor with this cell line. Conclusion Cryo-imaging and software allow, for the first time, 3D, whole brain, microscopic characterization of a tumor from a particular cell line. LN-229 exhibits considerable dispersal along blood vessels, a characteristic of human tumors that limits treatment success. PMID:22125093

Single-Molecule and Superresolution Imaging in Live Bacteria Cells

PubMed Central

Biteen, Julie S.; Moerner, W.E.

2010-01-01

Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells. PMID:20300204

Superparamagnetic iron oxide nanoparticle-labeled cells as an effective vehicle for tracking the GFP gene marker using magnetic resonance imaging

PubMed Central

Zhang, Z; Mascheri, N; Dharmakumar, R; Fan, Z; Paunesku, T; Woloschak, G; Li, D

2010-01-01

Background Detection of a gene using magnetic resonance imaging (MRI) is hindered by the magnetic resonance (MR) targeting gene technique. Therefore it may be advantageous to image gene-expressing cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles by MRI. Methods The GFP-R3230Ac (GFP) cell line was incubated for 24 h using SPIO nanoparticles at a concentration of 20 μg Fe/mL. Cell samples were prepared for iron content analysis and cell function evaluation. The labeled cells were imaged using fluorescent microscopy and MRI. Results SPIO was used to label GFP cells effectively, with no effects on cell function and GFP expression. Iron-loaded GFP cells were successfully imaged with both fluorescent microscopy and T2*-weighted MRI. Prussian blue staining showed intracellular iron accumulation in the cells. All cells were labeled (100% labeling efficiency). The average iron content per cell was 4.75±0.11 pg Fe/cell (P<0.05 versus control). Discussion This study demonstrates that the GFP expression of cells is not altered by the SPIO labeling process. SPIO-labeled GFP cells can be visualized by MRI; therefore, GFP, a gene marker, was tracked indirectly with the SPIO-loaded cells using MRI. The technique holds promise for monitoring the temporal and spatial migration of cells with a gene marker and enhancing the understanding of cell- and gene-based therapeutic strategies. PMID:18956269

Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

NASA Astrophysics Data System (ADS)

Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

2015-07-01

Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ˜16. Ten animals showed no residual tumor cells in the wound bed (mean SBR<2, P<0.001). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

Photothermal optical coherence tomography for depth-resolved imaging of mesenchymal stem cells via single wall carbon nanotubes

NASA Astrophysics Data System (ADS)

Subhash, Hrebesh M.; Connolly, Emma; Murphy, Mary; Barron, Valerie; Leahy, Martin

2014-03-01

The progress in stem cell research over the past decade holds promise and potential to address many unmet clinical therapeutic needs. Tracking stem cell with modern imaging modalities are critically needed for optimizing stem cell therapy, which offers insight into various underlying biological processes such as cell migration, engraftment, homing, differentiation, and functions etc. In this study we report the feasibility of photothermal optical coherence tomography (PT-OCT) to image human mesenchymal stem cells (hMSCs) labeled with single-walled carbon nanotubes (SWNTs) for in vitro cell tracking in three dimensional scaffolds. PT-OCT is a functional extension of conventional OCT with extended capability of localized detection of absorbing targets from scattering background to provide depth-resolved molecular contrast imaging. A 91 kHz line rate, spectral domain PT-OCT system at 1310nm was developed to detect the photothermal signal generated by 800nm excitation laser. In general, MSCs do not have obvious optical absorption properties and cannot be directly visualized using PT-OCT imaging. However, the optical absorption properties of hMSCs can me modified by labeling with SWNTs. Using this approach, MSC were labeled with SWNT and the cell distribution imaged in a 3D polymer scaffold using PT-OCT.

Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

PubMed Central

Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

2015-01-01

Abstract. Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16. Ten animals showed no residual tumor cells in the wound bed (mean SBR<2, P<0.001). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations. PMID:26160347

Imaging cell competition in Drosophila imaginal discs.

PubMed

Ohsawa, Shizue; Sugimura, Kaoru; Takino, Kyoko; Igaki, Tatsushi

2012-01-01

Cell competition is a process in which cells with higher fitness ("winners") survive and proliferate at the expense of less fit neighbors ("losers"). It has been suggested that cell competition is involved in a variety of biological processes such as organ size control, tissue homeostasis, cancer progression, and the maintenance of stem cell population. By advent of a genetic mosaic technique, which enables to generate fluorescently marked somatic clones in Drosophila imaginal discs, recent studies have presented some aspects of molecular mechanisms underlying cell competition. Now, with a live-imaging technique using ex vivo-cultured imaginal discs, we can dissect the spatiotemporal nature of competitive cell behaviors within multicellular communities. Here, we describe procedures and tips for live imaging of cell competition in Drosophila imaginal discs. Copyright © 2012 Elsevier Inc. All rights reserved.

Enhanced Fluorescence Imaging of Live Cells by Effective Cytosolic Delivery of Probes

PubMed Central

Massignani, Marzia; Canton, Irene; Sun, Tao; Hearnden, Vanessa; MacNeil, Sheila; Blanazs, Adam; Armes, Steven P.; Lewis, Andrew; Battaglia, Giuseppe

2010-01-01

Background Microscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery. Principal Findings We show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes) that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots. Significance We show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation. PMID:20454666

Structure-activity relationships of succinimidyl-Cys-C(O)-Glu derivatives with different near-infrared fluorophores as optical imaging probes for prostate-specific membrane antigen.

PubMed

Matsuoka, Daiko; Watanabe, Hiroyuki; Shimizu, Yoichi; Kimura, Hiroyuki; Yagi, Yusuke; Kawai, Ryoko; Ono, Masahiro; Saji, Hideo

2018-05-15

Prostate-specific membrane antigen (PSMA), which is overexpressed in malignant prostate cancer (PCa), is an ideal target for imaging and therapy of PCa. We previously reported a PSMA imaging probe, 800CW-SCE, based on succinimidyl-Cys-C(O)-Glu (SCE) for optical imaging of PCa. In this study, we investigated the structure-activity relationships of novel SCE derivatives with five different near-infrared (NIR) fluorophores (IRDye 680LT, IRDye 750, Indocyanine Green, Cyanine 5.5, and Cyanine 7) as optical imaging probes targeting PSMA. An in vitro binding assay revealed that 800CW-SCE, 680LT-SCE, and 750-SCE exhibited higher binding affinity than 2-PMPA, which is known as a PSMA inhibitor. These three SCE derivatives were internalized into PSMA-positive cells (LNCaP cells) but not into PSMA-negative cells (PC-3 cells). In the in vivo imaging study, 800CW-SCE and 750-SCE were highly accumulated in LNCaP tumors but not in PC-3 tumors, and the ratio of LNCaP/PC-3 accumulation of 800CW-SCE was higher than that of 750-SCE. The present study may provide valuable molecular design information for the future development of new PSMA imaging probes based on the SCE scaffold. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biodegradable polymer based theranostic agents for photoacoustic imaging and cancer therapy

NASA Astrophysics Data System (ADS)

Wang, Yan J.; Strohm, Eric M.; Kolios, Michael C.

2016-03-01

In this study, multifunctional theranostic agents for photoacoustic (PA), ultrasound (US), fluorescent imaging, and for therapeutic drug delivery were developed and tested. These agents consisted of a shell made from a biodegradable Poly(lactide-co-glycolic acid) (PLGA) polymer, loaded with perfluorohexane (PFH) liquid and gold nanoparticles (GNPs) in the core, and lipophilic carbocyanines fluorescent dye DiD and therapeutic drug Paclitaxel (PAC) in the shell. Their multifunctional capacity was investigated in an in vitro study. The PLGA/PFH/DiD-GNPs particles were synthesized by a double emulsion technique. The average PLGA particle diameter was 560 nm, with 50 nm diameter silica-coated gold nano-spheres in the shell. MCF7 human breast cancer cells were incubated with PLGA/PFH/DiDGNPs for 24 hours. Fluorescent and PA images were recorded using a fluorescent/PA microscope using a 1000 MHz transducer and a 532 nm pulsed laser. For the particle vaporization and drug delivery test, MCF7 cells were incubated with the PLGA/PFH-GNPs-PAC or PLGA/PFH-GNPs particles for 6, 12 and 24 hours. The effects of particle vaporization and drug delivery inside the cells were examined by irradiating the cells with a laser fluence of 100 mJ/cm2, and cell viability quantified using the MTT assay. The PA images of MCF7 cells containing PLGA/PFH/DiD-GNPs were spatially coincident with the fluorescent images, and confirmed particle uptake. After exposure to the PLGA/PFHGNP- PAC for 6, 12 and 24 hours, the cell survival rate was 43%, 38%, and 36% respectively compared with the control group, confirming drug delivery and release inside the cells. Upon vaporization, cell viability decreased to 20%. The particles show potential as imaging agents and drug delivery vehicles.

Force-activatable coating enables high-resolution cellular force imaging directly on regular cell culture surfaces.

PubMed

Sarkar, Anwesha; Zhao, Yuanchang; Wang, Yongliang; Wang, Xuefeng

2018-06-25

Integrin-transmitted cellular forces are crucial mechanical signals regulating a vast range of cell functions. Although various methods have been developed to visualize and quantify cellular forces at the cell-matrix interface, a method with high performance and low technical barrier is still in demand. Here we developed a force-activatable coating (FAC), which can be simply coated on regular cell culture apparatus' surfaces by physical adsorption, and turn these surfaces to force reporting platforms that enable cellular force mapping directly by fluorescence imaging. The FAC molecule consists of an adhesive domain for surface coating and a force-reporting domain which can be activated to fluoresce by integrin molecular tension. The tension threshold required for FAC activation is tunable in 10-60 piconewton (pN), allowing the selective imaging of cellular force contributed by integrin tension at different force levels. We tested the performance of two FACs with tension thresholds of 12 and 54 pN (nominal values), respectively, on both glass and polystyrene surfaces. Cellular forces were successfully mapped by fluorescence imaging on all the surfaces. FAC-coated surfaces also enable co-imaging of cellular forces and cell structures in both live cells and immunostained cells, therefore opening a new avenue for the study of the interplay of force and structure. We demonstrated the co-imaging of integrin tension and talin clustering in live cells, and concluded that talin clustering always occurs before the generation of integrin tension above 54 pN, reinforcing the notion that talin is an important adaptor protein for integrin tension transmission. Overall, FAC provides a highly convenient approach that is accessible to general biological laboratories for the study of cellular forces with high sensitivity and resolution, thus holding the potential to greatly boost the research of cell mechanobiology.

Contribution of macrophages in the contrast loss in iron oxide-based MRI cancer cell tracking studies

PubMed Central

Danhier, Pierre; Deumer, Gladys; Joudiou, Nicolas; Bouzin, Caroline; Levêque, Philippe; Haufroid, Vincent; Jordan, Bénédicte F.; Feron, Olivier; Sonveaux, Pierre; Gallez, Bernard

2017-01-01

Magnetic resonance imaging (MRI) cell tracking of cancer cells labeled with superparamagnetic iron oxides (SPIO) allows visualizing metastatic cells in preclinical models. However, previous works showed that the signal void induced by SPIO on T2(*)-weighted images decreased over time. Here, we aim at characterizing the fate of iron oxide nanoparticles used in cell tracking studies and the role of macrophages in SPIO metabolism. In vivo MRI cell tracking of SPIO positive 4T1 breast cancer cells revealed a quick loss of T2* contrast after injection. We next took advantage of electron paramagnetic resonance (EPR) spectroscopy and inductively coupled plasma mass spectroscopy (ICP-MS) for characterizing the evolution of superparamagnetic and non-superparamagnetic iron pools in 4T1 breast cancer cells and J774 macrophages after SPIO labeling. These in vitro experiments and histology studies performed on 4T1 tumors highlighted the quick degradation of iron oxides by macrophages in SPIO-based cell tracking experiments. In conclusion, the release of SPIO by dying cancer cells and the subsequent uptake of iron oxides by tumor macrophages are limiting factors in MRI cell tracking experiments that plead for the use of (MR) reporter-gene based imaging methods for the long-term tracking of metastatic cells. PMID:28467814

Dynamic Assessment of the Endothelialization of Tissue-Engineered Blood Vessels Using an Optical Coherence Tomography Catheter-Based Fluorescence Imaging System

PubMed Central

Gurjarpadhye, Abhijit Achyut; DeWitt, Matthew R.; Xu, Yong; Wang, Ge; Rylander, Marissa Nichole

2015-01-01

Background: Lumen endothelialization of bioengineered vascular scaffolds is essential to maintain small-diameter graft patency and prevent thrombosis postimplantation. Unfortunately, nondestructive imaging methods to visualize this dynamic process are lacking, thus slowing development and clinical translation of these potential tissue-engineering approaches. To meet this need, a fluorescence imaging system utilizing a commercial optical coherence tomography (OCT) catheter was designed to visualize graft endothelialization. Methods: C7 DragonFly™ intravascular OCT catheter was used as a channel for delivery and collection of excitation and emission spectra. Poly-dl-lactide (PDLLA) electrospun scaffolds were seeded with endothelial cells (ECs). Seeded cells were exposed to Calcein AM before imaging, causing the living cells to emit green fluorescence in response to blue laser. By positioning the catheter tip precisely over a specimen using high-fidelity electromechanical components, small regions of the specimen were excited selectively. The resulting fluorescence intensities were mapped on a two-dimensional digital grid to generate spatial distribution of fluorophores at single-cell-level resolution. Fluorescence imaging of endothelialization on glass and PDLLA scaffolds was performed using the OCT catheter-based imaging system as well as with a commercial fluorescence microscope. Cell coverage area was calculated for both image sets for quantitative comparison of imaging techniques. Tubular PDLLA scaffolds were maintained in a bioreactor on seeding with ECs, and endothelialization was monitored over 5 days using the OCT catheter-based imaging system. Results: No significant difference was observed in images obtained using our imaging system to those acquired with the fluorescence microscope. Cell area coverage calculated using the images yielded similar values. Nondestructive imaging of endothelialization on tubular scaffolds showed cell proliferation with cell coverage area increasing from 15±4% to 89±6% over 5 days. Conclusion: In this study, we showed the capability of an OCT catheter-based imaging system to obtain single-cell resolution and to quantify endothelialization in tubular electrospun scaffolds. We also compared the resulting images with traditional microscopy, showing high fidelity in image capability. This imaging system, used in conjunction with OCT, could potentially be a powerful tool for in vitro optimization of scaffold cellularization, ensuring long-term graft patency postimplantation. PMID:25539889

Study of Fluorescent Imaging of Se (IV) in Living Cells Using a Turn-on Fluorescent Probe Based on a Rhodamine Spirolactame Derivative.

PubMed

Guan, Mingming; Mi, Hongyu; Xu, Hui; Fei, Qiang; Shan, Hongyan; Huan, Yanfu; Lv, Shaowu; Feng, Guodong

2017-03-01

A highly selective fluorescent probe 2-(2-(2-aminoethylamino)ethyl)-3',6'-bis(ethylamino)-2',7'-dimethylspiro[isoindoline-1,9'-xanthen]-3-one (ABDO) for Se (IV) had been synthesized in our earlier report. In this study, this fluorescent sensor is applied on analysis fluorescent imaging of Se (IV) in Hela cells. The experiment conditions, such as the MTT assay, different concentration of saline, incubated time of Hela cells with ABDO and Se (IV), and intracellular action position of Se (IV), are investigated. Through a series of experiments, the fluorescent image of Se (IV) in Hela cells can be observed when the cells cultured by 2 μM ABDO and 2 μM Se (IV) for 210 min. And the intracellular action position of Se (IV) is verified after the co-localization experiments are done. It is mitochondria. These experimental results show that ABDO will be an eagerly anticipated sensor for fluorescent imaging analysis of selenium ion in living cells. Besides, we also can use the complexes of ABDO-Se to observe morphology and distribution of mitochondria in cells like JG-B.

Image processing and machine learning in the morphological analysis of blood cells.

PubMed

Rodellar, J; Alférez, S; Acevedo, A; Molina, A; Merino, A

2018-05-01

This review focuses on how image processing and machine learning can be useful for the morphological characterization and automatic recognition of cell images captured from peripheral blood smears. The basics of the 3 core elements (segmentation, quantitative features, and classification) are outlined, and recent literature is discussed. Although red blood cells are a significant part of this context, this study focuses on malignant lymphoid cells and blast cells. There is no doubt that these technologies may help the cytologist to perform efficient, objective, and fast morphological analysis of blood cells. They may also help in the interpretation of some morphological features and may serve as learning and survey tools. Although research is still needed, it is important to define screening strategies to exploit the potential of image-based automatic recognition systems integrated in the daily routine of laboratories along with other analysis methodologies. © 2018 John Wiley & Sons Ltd.

Magnetic resonance imaging (MRI) of PEM dehydration and gas manifold flooding during continuous fuel cell operation

NASA Astrophysics Data System (ADS)

Minard, Kevin R.; Viswanathan, Vilayanur V.; Majors, Paul D.; Wang, Li-Qiong; Rieke, Peter C.

Magnetic resonance imaging (MRI) was employed for visualizing water inside a proton exchange membrane (PEM) fuel cell during 11.4 h of continuous operation with a constant load. Two-dimensional images acquired every 128 s revealed the formation of a dehydration front that propagated slowly over the surface of the fuel cell membrane-starting from gas inlets and progressing toward gas outlets. After traversing the entire PEM surface, channels in the gas manifold began to flood on the cathode side. To establish a qualitative understanding of these observations, acquired images were correlated to the current output and the operating characteristics of the fuel cell. Results demonstrate the power of MRI for visualizing changing water distributions during PEM fuel cell operation, and highlight its potential utility for studying the causes of cell failure and/or strategies of water management.

A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

PubMed Central

Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T. C.; Tseng, Hubert; Souza, Glauco R.

2013-01-01

There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures. PMID:24141454

Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets

PubMed Central

Saitoh, Sei; Ohno, Nobuhiko; Saitoh, Yurika; Terada, Nobuo; Shimo, Satoshi; Aida, Kaoru; Fujii, Hideki; Kobayashi, Tetsuro; Ohno, Shinichi

2018-01-01

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets. PMID:29622846

Comparison of parameter-adapted segmentation methods for fluorescence micrographs.

PubMed

Held, Christian; Palmisano, Ralf; Häberle, Lothar; Hensel, Michael; Wittenberg, Thomas

2011-11-01

Interpreting images from fluorescence microscopy is often a time-consuming task with poor reproducibility. Various image processing routines that can help investigators evaluate the images are therefore useful. The critical aspect for a reliable automatic image analysis system is a robust segmentation algorithm that can perform accurate segmentation for different cell types. In this study, several image segmentation methods were therefore compared and evaluated in order to identify the most appropriate segmentation schemes that are usable with little new parameterization and robustly with different types of fluorescence-stained cells for various biological and biomedical tasks. The study investigated, compared, and enhanced four different methods for segmentation of cultured epithelial cells. The maximum-intensity linking (MIL) method, an improved MIL, a watershed method, and an improved watershed method based on morphological reconstruction were used. Three manually annotated datasets consisting of 261, 817, and 1,333 HeLa or L929 cells were used to compare the different algorithms. The comparisons and evaluations showed that the segmentation performance of methods based on the watershed transform was significantly superior to the performance of the MIL method. The results also indicate that using morphological opening by reconstruction can improve the segmentation of cells stained with a marker that exhibits the dotted surface of cells. Copyright © 2011 International Society for Advancement of Cytometry.

In vivo dark-field imaging of the retinal pigment epithelium cell mosaic

PubMed Central

Scoles, Drew; Sulai, Yusufu N.; Dubra, Alfredo

2013-01-01

Non-invasive reflectance imaging of the human RPE cell mosaic is demonstrated using a modified confocal adaptive optics scanning light ophthalmoscope (AOSLO). The confocal circular aperture in front of the imaging detector was replaced with a combination of a circular aperture 4 to 16 Airy disks in diameter and an opaque filament, 1 or 3 Airy disks thick. This arrangement reveals the RPE cell mosaic by dramatically attenuating the light backscattered by the photoreceptors. The RPE cell mosaic was visualized in all 7 recruited subjects at multiple retinal locations with varying degrees of contrast and cross-talk from the photoreceptors. Various experimental settings were explored for improving the visualization of the RPE cell boundaries including: pinhole diameter, filament thickness, illumination and imaging pupil apodization, unmatched imaging and illumination focus, wavelength and polarization. None of these offered an obvious path for enhancing image contrast. The demonstrated implementation of dark-field AOSLO imaging using 790 nm light requires low light exposures relative to light safety standards and it is more comfortable for the subject than the traditional autofluorescence RPE imaging with visible light. Both these factors make RPE dark-field imaging appealing for studying mechanisms of eye disease, as well as a clinical tool for screening and monitoring disease progression. PMID:24049692

Breakdown voltage mapping through voltage dependent ReBEL intensity imaging of multi-crystalline Si solar cells

NASA Astrophysics Data System (ADS)

Dix-Peek, RM.; van Dyk, EE.; Vorster, FJ.; Pretorius, CJ.

2018-04-01

Device material quality affects both the efficiency and the longevity of photovoltaic (PV) cells. Therefore, identifying these defects can be beneficial in the development of more efficient and longer lasting PV cells. In this study, a combination of spatially-resolved, electroluminescence (EL), and light beam induced current (LBIC) measurements, were used to identify specific defects and features of a multi-crystalline Si PV cells. In this study, a novel approach is used to map the breakdown voltage of a PV cell through voltage dependent Reverse Bias EL (ReBEL) intensity imaging.

Automated high resolution full-field spatial coherence tomography for quantitative phase imaging of human red blood cells

NASA Astrophysics Data System (ADS)

Singla, Neeru; Dubey, Kavita; Srivastava, Vishal; Ahmad, Azeem; Mehta, D. S.

2018-02-01

We developed an automated high-resolution full-field spatial coherence tomography (FF-SCT) microscope for quantitative phase imaging that is based on the spatial, rather than the temporal, coherence gating. The Red and Green color laser light was used for finding the quantitative phase images of unstained human red blood cells (RBCs). This study uses morphological parameters of unstained RBCs phase images to distinguish between normal and infected cells. We recorded the single interferogram by a FF-SCT microscope for red and green color wavelength and average the two phase images to further reduced the noise artifacts. In order to characterize anemia infected from normal cells different morphological features were extracted and these features were used to train machine learning ensemble model to classify RBCs with high accuracy.

Multifunctional ferritin cage nanostructures for fluorescence and MR imaging of tumor cells.

PubMed

Li, Ke; Zhang, Zhi-Ping; Luo, Ming; Yu, Xiang; Han, Yu; Wei, Hong-Ping; Cui, Zong-Qiang; Zhang, Xian-En

2012-01-07

Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging α(v)β(3) integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.

Mapping the Complex Morphology of Cell Interactions with Nanowire Substrates Using FIB-SEM

PubMed Central

Jensen, Mikkel R. B.; Łopacińska, Joanna; Schmidt, Michael S.; Skolimowski, Maciej; Abeille, Fabien; Qvortrup, Klaus; Mølhave, Kristian

2013-01-01

Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells’ interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered. PMID:23326412

Atomic force-multi-optical imaging integrated microscope for monitoring molecular dynamics in live cells.

PubMed

Trache, Andreea; Meininger, Gerald A

2005-01-01

A novel hybrid imaging system is constructed integrating atomic force microscopy (AFM) with a combination of optical imaging techniques that offer high spatial resolution. The main application of this instrument (the NanoFluor microscope) is the study of mechanotransduction with an emphasis on extracellular matrix-integrin-cytoskeletal interactions and their role in the cellular responses to changes in external chemical and mechanical factors. The AFM allows the quantitative assessment of cytoskeletal changes, binding probability, adhesion forces, and micromechanical properties of the cells, while the optical imaging applications allow thin sectioning of the cell body at the coverslip-cell interface, permitting the study of focal adhesions using total internal reflection fluorescence (TIRF) and internal reflection microscopy (IRM). Combined AFM-optical imaging experiments show that mechanical stimulation at the apical surface of cells induces a force-generating cytoskeletal response, resulting in focal contact reorganization on the basal surface that can be monitored in real time. The NanoFluor system is also equipped with a novel mechanically aligned dual camera acquisition system for synthesized Forster resonance energy transfer (FRET). The integrated NanoFluor microscope system is described, including its characteristics, applications, and limitations.

Protocol for Biomarker Ratio Imaging Microscopy with Specific Application to Ductal Carcinoma In situ of the Breast

PubMed Central

Clark, Andrea J.; Petty, Howard R.

2016-01-01

This protocol describes the methods and steps involved in performing biomarker ratio imaging microscopy (BRIM) using formalin fixed paraffin-embedded (FFPE) samples of human breast tissue. The technique is based on the acquisition of two fluorescence images of the same microscopic field using two biomarkers and immunohistochemical tools. The biomarkers are selected such that one biomarker correlates with breast cancer aggressiveness while the second biomarker anti-correlates with aggressiveness. When the former image is divided by the latter image, a computed ratio image is formed that reflects the aggressiveness of tumor cells while increasing contrast and eliminating path-length and other artifacts from the image. For example, the aggressiveness of epithelial cells may be assessed by computing ratio images of N-cadherin and E-cadherin images or CD44 and CD24 images, which specifically reflect the mesenchymal or stem cell nature of the constituent cells, respectively. This methodology is illustrated for tissue samples of ductal carcinoma in situ (DCIS) and invasive breast cancer. This tool should be useful in tissue studies of experimental cancer as well as the management of cancer patients. PMID:27857940

Two-photon microscopy of fungal keratitis-affected rabbit cornea ex vivo using moxifloxacin as a labeling agent.

PubMed

Lee, Jun Ho; Le, Viet-Hoan; Lee, Seunghun; Park, Jin Hyoung; Lee, Jin Ah; Tchah, Hungwon; Kim, Sungjee; Kim, Myoung Joon; Kim, Ki Hean

2018-05-19

Two-photon microscopy (TPM) is a three dimensional (3D) microscopic technique based on nonlinear two-photon fluorescence, which has been tested as an alternative to reflectance confocal microscopy (RCM) for detecting fungal keratitis via optical imaging. Although TPM provided images with better contrast than RCM for fungal keratitis, its imaging speed was relatively low because of weak intrinsic signal. Moxifloxacin, a Food and Drug Administration (FDA)-approved antibiotic, was recently used as a cell-labeling agent for TPM. In this study, moxifloxacin was used to label fungal cells for TPM imaging of fungal keratitis models. Fungal cell suspensions and ex vivo fungal keratitis-affected rabbit corneas were prepared using two types of fungal pathogens, Aspergillus fumigatus and Candida albicans, and TPM imaging was performed both with and without moxifloxacin treatment. Fungal cells with enhanced fluorescence were clearly visible by TPM of moxifloxacin-treated fungal cell suspensions. TPM of moxifloxacin-treated fungal keratitis rabbit corneas revealed both the infecting fungal cells and corneal cells similar to those observed in TPM without moxifloxacin treatment, albeit with approximately 10-times enhanced fluorescence. Fungal cells were distinguished from corneal cells on the basis of their distinct morphologies. Thus, TPM with moxifloxacin labeling might be useful for the detection of fungal keratitis at the improved imaging speed. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Associative image analysis: a method for automated quantification of 3D multi-parameter images of brain tissue

PubMed Central

Bjornsson, Christopher S; Lin, Gang; Al-Kofahi, Yousef; Narayanaswamy, Arunachalam; Smith, Karen L; Shain, William; Roysam, Badrinath

2009-01-01

Brain structural complexity has confounded prior efforts to extract quantitative image-based measurements. We present a systematic ‘divide and conquer’ methodology for analyzing three-dimensional (3D) multi-parameter images of brain tissue to delineate and classify key structures, and compute quantitative associations among them. To demonstrate the method, thick (~100 μm) slices of rat brain tissue were labeled using 3 – 5 fluorescent signals, and imaged using spectral confocal microscopy and unmixing algorithms. Automated 3D segmentation and tracing algorithms were used to delineate cell nuclei, vasculature, and cell processes. From these segmentations, a set of 23 intrinsic and 8 associative image-based measurements was computed for each cell. These features were used to classify astrocytes, microglia, neurons, and endothelial cells. Associations among cells and between cells and vasculature were computed and represented as graphical networks to enable further analysis. The automated results were validated using a graphical interface that permits investigator inspection and corrective editing of each cell in 3D. Nuclear counting accuracy was >89%, and cell classification accuracy ranged from 81–92% depending on cell type. We present a software system named FARSIGHT implementing our methodology. Its output is a detailed XML file containing measurements that may be used for diverse quantitative hypothesis-driven and exploratory studies of the central nervous system. PMID:18294697

Cellular Level Brain Imaging in Behaving Mammals: An Engineering Approach

PubMed Central

Hamel, Elizabeth J.O.; Grewe, Benjamin F.; Parker, Jones G.; Schnitzer, Mark J.

2017-01-01

Fluorescence imaging offers expanding capabilities for recording neural dynamics in behaving mammals, including the means to monitor hundreds of cells targeted by genetic type or connectivity, track cells over weeks, densely sample neurons within local microcircuits, study cells too inactive to isolate in extracellular electrical recordings, and visualize activity in dendrites, axons, or dendritic spines. We discuss recent progress and future directions for imaging in behaving mammals from a systems engineering perspective, which seeks holistic consideration of fluorescent indicators, optical instrumentation, and computational analyses. Today, genetically encoded indicators of neural Ca2+ dynamics are widely used, and those of trans-membrane voltage are rapidly improving. Two complementary imaging paradigms involve conventional microscopes for studying head-restrained animals and head-mounted miniature microscopes for imaging in freely behaving animals. Overall, the field has attained sufficient sophistication that increased cooperation between those designing new indicators, light sources, microscopes, and computational analyses would greatly benefit future progress. PMID:25856491

3D Imaging of Retinal Pigment Epithelial Cells in the Living Human Retina

PubMed Central

Liu, Zhuolin; Kocaoglu, Omer P.; Miller, Donald T.

2016-01-01

Purpose Dysfunction of the retinal pigment epithelium (RPE) underlies numerous retinal pathologies, but biomarkers sensitive to RPE change at the cellular level are limited. In this study, we used adaptive optics optical coherence tomography (AO-OCT) in conjunction with organelle motility as a novel contrast mechanism to visualize RPE cells and characterize their 3-dimensional (3D) reflectance profile. Methods Using the Indiana AO-OCT imaging system (λc = 790 nm), volumes were acquired in the macula of six normal subjects (25–61 years). Volumes were registered in 3D with subcellular accuracy, layers segmented, and RPE and photoreceptor en face images extracted and averaged. Voronoi and two-dimensional (2D) power spectra analyses were applied to the images to quantify RPE and cone packing and cone-to-RPE ratio. Results Adaptive optics OCT revealed two distinct reflectance patterns at the depth of the RPE. One is characterized by the RPE interface with rod photoreceptor tips, the second by the RPE cell nuclei and surrounding organelles, likely melanin. Increasing cell contrast by averaging proved critical for observing the RPE cell mosaic, successful in all subjects and retinal eccentricities imaged. Retinal pigment epithelium mosaic packing and cell thickness generally agreed with that of histology and in vivo studies using other imaging modalities. Conclusions We have presented, to our knowledge, the first detailed characterization of the 3D reflectance profile of individual RPE cells and their relation to cones and rods in the living human retina. Success in younger and older eyes establishes a path for testing aging effects in larger populations. Because the technology is based on OCT, our measurements will aid in interpreting clinical OCT images. PMID:27472277

Multi-scale Gaussian representation and outline-learning based cell image segmentation.

PubMed

Farhan, Muhammad; Ruusuvuori, Pekka; Emmenlauer, Mario; Rämö, Pauli; Dehio, Christoph; Yli-Harja, Olli

2013-01-01

High-throughput genome-wide screening to study gene-specific functions, e.g. for drug discovery, demands fast automated image analysis methods to assist in unraveling the full potential of such studies. Image segmentation is typically at the forefront of such analysis as the performance of the subsequent steps, for example, cell classification, cell tracking etc., often relies on the results of segmentation. We present a cell cytoplasm segmentation framework which first separates cell cytoplasm from image background using novel approach of image enhancement and coefficient of variation of multi-scale Gaussian scale-space representation. A novel outline-learning based classification method is developed using regularized logistic regression with embedded feature selection which classifies image pixels as outline/non-outline to give cytoplasm outlines. Refinement of the detected outlines to separate cells from each other is performed in a post-processing step where the nuclei segmentation is used as contextual information. We evaluate the proposed segmentation methodology using two challenging test cases, presenting images with completely different characteristics, with cells of varying size, shape, texture and degrees of overlap. The feature selection and classification framework for outline detection produces very simple sparse models which use only a small subset of the large, generic feature set, that is, only 7 and 5 features for the two cases. Quantitative comparison of the results for the two test cases against state-of-the-art methods show that our methodology outperforms them with an increase of 4-9% in segmentation accuracy with maximum accuracy of 93%. Finally, the results obtained for diverse datasets demonstrate that our framework not only produces accurate segmentation but also generalizes well to different segmentation tasks.

Multi-scale Gaussian representation and outline-learning based cell image segmentation

PubMed Central

2013-01-01

Background High-throughput genome-wide screening to study gene-specific functions, e.g. for drug discovery, demands fast automated image analysis methods to assist in unraveling the full potential of such studies. Image segmentation is typically at the forefront of such analysis as the performance of the subsequent steps, for example, cell classification, cell tracking etc., often relies on the results of segmentation. Methods We present a cell cytoplasm segmentation framework which first separates cell cytoplasm from image background using novel approach of image enhancement and coefficient of variation of multi-scale Gaussian scale-space representation. A novel outline-learning based classification method is developed using regularized logistic regression with embedded feature selection which classifies image pixels as outline/non-outline to give cytoplasm outlines. Refinement of the detected outlines to separate cells from each other is performed in a post-processing step where the nuclei segmentation is used as contextual information. Results and conclusions We evaluate the proposed segmentation methodology using two challenging test cases, presenting images with completely different characteristics, with cells of varying size, shape, texture and degrees of overlap. The feature selection and classification framework for outline detection produces very simple sparse models which use only a small subset of the large, generic feature set, that is, only 7 and 5 features for the two cases. Quantitative comparison of the results for the two test cases against state-of-the-art methods show that our methodology outperforms them with an increase of 4-9% in segmentation accuracy with maximum accuracy of 93%. Finally, the results obtained for diverse datasets demonstrate that our framework not only produces accurate segmentation but also generalizes well to different segmentation tasks. PMID:24267488

Phase contrast microscopy of living cells within the whole lens: spatial correlations and morphological dynamics

PubMed Central

Kong, Zhiying; Zhu, Xiangjia; Zhang, Shenghai; Wu, Jihong

2012-01-01

Purpose Images from cultured lens cells do not convey enough spatial information, and imaging of fixed lens specimens cannot reveal dynamic changes in the cells. As such, a real-time, convenient approach for monitoring label-free imaging of dynamic processes of living cells within the whole lens is urgently needed. Methods Female Wistar rat lenses were kept in organ culture. Insulin-like growth factor-I was added to the culture medium to induce cell mitosis. A novel method of ultraviolet (UV) irradiation was used to induce cell apoptosis and fiber damage. The cellular morphological dynamics within the whole lens were monitored by inverted phase contrast microscopy. Apoptosis was assessed using a commercial kit with Hoechst 33342/YO-PRO®-1/propidium iodide (PI). Results The intrinsic transparency and low-light scattering property of the rat lens permitted direct imaging of the lens epithelial cells (LECs) and the superficial fiber cells. We visualized the processes of mitosis and apoptosis of the LECs, and we obtained dynamic images of posterior fiber cells following UVA irradiation. Conclusions This method opens a new window for observing lens cells in their physiologic location, and it can be readily applied in studies on lens physiology and pathology. PMID:22879736

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.

Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer

PubMed Central

Elliott, Amicia D.; Gao, Liang; Ustione, Alessandro; Bedard, Noah; Kester, Robert; Piston, David W.; Tkaczyk, Tomasz S.

2012-01-01

Summary The development of multi-colored fluorescent proteins, nanocrystals and organic fluorophores, along with the resulting engineered biosensors, has revolutionized the study of protein localization and dynamics in living cells. Hyperspectral imaging has proven to be a useful approach for such studies, but this technique is often limited by low signal and insufficient temporal resolution. Here, we present an implementation of a snapshot hyperspectral imaging device, the image mapping spectrometer (IMS), which acquires full spectral information simultaneously from each pixel in the field without scanning. The IMS is capable of real-time signal capture from multiple fluorophores with high collection efficiency (∼65%) and image acquisition rate (up to 7.2 fps). To demonstrate the capabilities of the IMS in cellular applications, we have combined fluorescent protein (FP)-FRET and [Ca2+]i biosensors to measure simultaneously intracellular cAMP and [Ca2+]i signaling in pancreatic β-cells. Additionally, we have compared quantitatively the IMS detection efficiency with a laser-scanning confocal microscope. PMID:22854044

Tryptophan autofluorescence imaging of neoplasms of the human colon

NASA Astrophysics Data System (ADS)

Banerjee, Bhaskar; Renkoski, Timothy; Graves, Logan R.; Rial, Nathaniel S.; Tsikitis, Vassiliki Liana; Nfonsom, Valentine; Pugh, Judith; Tiwari, Piyush; Gavini, Hemanth; Utzinger, Urs

2012-01-01

Detection of flat neoplasia is a major challenge in colorectal cancer screening, as missed lesions can lead to the development of an unexpected `incident' cancer prior to the subsequent endoscopy. The use of a tryptophan-related autofluorescence has been reported to be increased in murine intestinal dysplasia. The emission spectra of cells isolated from human adenocarcinoma and normal mucosa of the colon were studied and showed markedly greater emission intensity from cancerous cells compared to cells obtained from the surrounding normal mucosa. A proto-type multispectral imaging system optimized for ultraviolet macroscopic imaging of tissue was used to obtain autofluorescence images of surgical specimens of colonic neoplasms and normal mucosa after resection. Fluorescence images did not display the expected greater emission from the tumor as compared to the normal mucosa, most probably due to increased optical absorption and scattering in the tumors. Increased fluorescence intensity in neoplasms was observed however, once fluorescence images were corrected using reflectance images. Tryptophan fluorescence alone may be useful in differentiating normal and cancerous cells, while in tissues its autofluorescence image divided by green reflectance may be useful in displaying neoplasms.

Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics

PubMed Central

Jang, Joon Hee; Huang, Yu; Zheng, Peilin; Jo, Myeong Chan; Bertolet, Grant; Qin, Lidong; Liu, Dongfang

2015-01-01

The immunological synapse (IS) is one of the most pivotal communication strategies in immune cells. Understanding the molecular basis of the IS provides critical information regarding how immune cells mount an effective immune response. Fluorescence microscopy provides a fundamental tool to study the IS. However, current imaging techniques for studying the IS cannot sufficiently achieve high resolution in real cell-cell conjugates. Here we present a new device that allows for high-resolution imaging of the IS with conventional confocal microscopy in a high-throughput manner. Combining micropits and single cell trap arrays, we have developed a new microfluidic platform that allows visualization of the IS in vertically “stacked” cells. Using this vertical cell pairing (VCP) system, we investigated the dynamics of the inhibitory synapse mediated by an inhibitory receptor, programed death protein-1 (PD-1) and the cytotoxic synapse at the single cell level. In addition to the technique innovation, we demonstrated novel biological findings by this VCP device, including novel distribution of F-actin and cytolytic granules at the IS, PD-1 microclusters in the NK IS, and kinetics of cytotoxicity. We propose that this high-throughput, cost-effective, easy-to-use VCP system, along with conventional imaging techniques, can be used to address a number of significant biological questions in a variety of disciplines. PMID:26123352

Recent advances in live cell imaging of hepatoma cells

PubMed Central

2014-01-01

Live cell imaging enables the study of dynamic processes of living cells in real time by use of suitable reporter proteins and the staining of specific cellular structures and/or organelles. With the availability of advanced optical devices and improved cell culture protocols it has become a rapidly growing research methodology. The success of this technique relies mainly on the selection of suitable reporter proteins, construction of recombinant plasmids possessing cell type specific promoters as well as reliable methods of gene transfer. This review aims to provide an overview of the recent developments in the field of marker proteins (bioluminescence and fluorescent) and methodologies (fluorescent resonance energy transfer, fluorescent recovery after photobleaching and proximity ligation assay) employed as to achieve an improved imaging of biological processes in hepatoma cells. Moreover, different expression systems of marker proteins and the modes of gene transfer are discussed with emphasis on the study of lipid droplet formation in hepatocytes as an example. PMID:25005127

Multicolor fluorescence microscopic imaging of cancer cells on the plasmonic chip (Presentation Recording)

NASA Astrophysics Data System (ADS)

Tawa, Keiko; Sasakawa, Chisato; Yamamura, Shohei; Shibata, Izumi; Kataoka, Masatoshi

2015-09-01

A plasmonic chip which is a metal coated substrate with grating structure can provide the enhanced fluorescence by the grating-coupled surface plasmon field. In our previous studies, bright epi-fluorescence microscopic imaging of neuron cells and sensitive immunosesnsing have been reported. In this study, two kinds of breast cancer cells, MCF-7 and MDA-MB231, were observed with epi-fluorescence microscope on the plasmonic chip with 2D hole-arrays . They were multicolor stained with 4', 6-diamidino-2-phenylindole (DAPI) and allophycocyanin (APC)-labeled anti-epithelial cell adhesion molecule (EpCAM) antibody. Our plasmonic chip provided the brighter fluorescence images of these cells compared with the glass slide. Even in the cells including few EpCAM, the distribution of EpCAM was clearly observed in the cell membrane. It was found that the plasmonic chip can be one of the powerful tools to detect the marker protein existing around the chip surface even at low concentration.

Live Cell Imaging of a Fluorescent Gentamicin Conjugate

PubMed Central

Escobedo, Jorge O.; Chu, Yu-Hsuan; Wang, Qi; Steyger, Peter S.; Strongin, Robert M.

2012-01-01

Understanding cellular mechanisms of ototoxic and nephrotoxic drug uptake, intracellular distribution, and molecular trafficking across cellular barrier systems aids the study of potential uptake blockers that preserve sensory and renal function during critical life-saving therapy. Herein we report the design, synthesis characterization and evaluation of a fluorescent conjugate of the aminoglycoside antibiotic gentamicin. Live cell imaging results show the potential utility of this new material. Related gentamicin conjugates studied to date quench in live kindney cells, and have been largely restricted to use in fixed (delipidated) cells. PMID:22545403

Measurement of time-varying displacement fields in cell culture for traction force optical coherence microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Mulligan, Jeffrey A.; Adie, Steven G.

2017-02-01

Mechanobiology is an emerging field which seeks to link mechanical forces and properties to the behaviors of cells and tissues in cancer, stem cell growth, and other processes. Traction force microscopy (TFM) is an imaging technique that enables the study of traction forces exerted by cells on their environment to migrate as well as sense and manipulate their surroundings. To date, TFM research has been performed using incoherent imaging modalities and, until recently, has been largely confined to the study of cell-induced tractions within two-dimensions using highly artificial and controlled environments. As the field of mechanobiology advances, and demand grows for research in physiologically relevant 3D culture and in vivo models, TFM will require imaging modalities that support such settings. Optical coherence microscopy (OCM) is an interferometric imaging modality which enables 3D cellular resolution imaging in highly scattering environments. Moreover, optical coherence elastography (OCE) enables the measurement of tissue mechanical properties. OCE relies on the principle of measuring material deformations in response to artificially applied stress. By extension, similar techniques can enable the measurement of cell-induced deformations, imaged with OCM. We propose traction force optical coherence microscopy (TF-OCM) as a natural extension and partner to existing OCM and OCE methods. We report the first use of OCM data and digital image correlation to track temporally varying displacement fields exhibited within a 3D culture setting. These results mark the first steps toward the realization of TF-OCM in 2D and 3D settings, bolstering OCM as a platform for advancing research in mechanobiology.

Automated recognition of cell phenotypes in histology images based on membrane- and nuclei-targeting biomarkers

PubMed Central

Karaçalı, Bilge; Vamvakidou, Alexandra P; Tözeren, Aydın

2007-01-01

Background Three-dimensional in vitro culture of cancer cells are used to predict the effects of prospective anti-cancer drugs in vivo. In this study, we present an automated image analysis protocol for detailed morphological protein marker profiling of tumoroid cross section images. Methods Histologic cross sections of breast tumoroids developed in co-culture suspensions of breast cancer cell lines, stained for E-cadherin and progesterone receptor, were digitized and pixels in these images were classified into five categories using k-means clustering. Automated segmentation was used to identify image regions composed of cells expressing a given biomarker. Synthesized images were created to check the accuracy of the image processing system. Results Accuracy of automated segmentation was over 95% in identifying regions of interest in synthesized images. Image analysis of adjacent histology slides stained, respectively, for Ecad and PR, accurately predicted regions of different cell phenotypes. Image analysis of tumoroid cross sections from different tumoroids obtained under the same co-culture conditions indicated the variation of cellular composition from one tumoroid to another. Variations in the compositions of cross sections obtained from the same tumoroid were established by parallel analysis of Ecad and PR-stained cross section images. Conclusion Proposed image analysis methods offer standardized high throughput profiling of molecular anatomy of tumoroids based on both membrane and nuclei markers that is suitable to rapid large scale investigations of anti-cancer compounds for drug development. PMID:17822559

A simple microfluidic device for live cell imaging of Arabidopsis cotyledons, leaves, and seedlings.

PubMed

Vang, Shia; Seitz, Kati; Krysan, Patrick J

2018-06-01

One of the challenges of performing live-cell imaging in plants is establishing a system for securing the sample during imaging that allows for the rapid addition of treatments. Here we report how a commercially available device called a HybriWell ™ can be repurposed to create an imaging chamber suitable for Arabidopsis seedlings, cotyledons and leaves. Liquid in the imaging chamber can be rapidly exchanged to introduce chemical treatments via microfluidic passive pumping. When used in conjunction with fluorescent biosensors, this system can facilitate live-cell imaging studies of signal transduction pathways triggered by different treatments. As a demonstration, we show how the HybriWell can be used to monitor flg22-induced calcium transients using the R-GECO1 calcium indicator in detached Arabidopsis leaves.

Lipid tethering of breast tumor cells enables real-time imaging of free-floating cell dynamics and drug response

PubMed Central

Whipple, Rebecca A.; Zhang, Peipei; Sooklal, Elisabeth L.; Martin, Stuart S.; Jewell, Christopher M.

2016-01-01

Free-floating tumor cells located in the blood of cancer patients, known as circulating tumor cells (CTCs), have become key targets for studying metastasis. However, effective strategies to study the free-floating behavior of tumor cells in vitro have been a major barrier limiting the understanding of the functional properties of CTCs. Upon extracellular-matrix (ECM) detachment, breast tumor cells form tubulin-based protrusions known as microtentacles (McTNs) that play a role in the aggregation and re-attachment of tumor cells to increase their metastatic efficiency. In this study, we have designed a strategy to spatially immobilize ECM-detached tumor cells while maintaining their free-floating character. We use polyelectrolyte multilayers deposited on microfluidic substrates to prevent tumor cell adhesion and the addition of lipid moieties to tether tumor cells to these surfaces through interactions with the cell membranes. This coating remains optically clear, allowing capture of high-resolution images and videos of McTNs on viable free-floating cells. In addition, we show that tethering allows for the real-time analysis of McTN dynamics on individual tumor cells and in response to tubulin-targeting drugs. The ability to image detached tumor cells can vastly enhance our understanding of CTCs under conditions that better recapitulate the microenvironments they encounter during metastasis. PMID:26871289

Cysteamine-based cell-permeable Zn(2+)-specific molecular bioimaging materials: from animal to plant cells.

PubMed

Sinha, Sougata; Dey, Gourab; Kumar, Sunil; Mathew, Jomon; Mukherjee, Trinetra; Mukherjee, Subhrakanti; Ghosh, Subrata

2013-11-27

Structure-interaction/fluorescence relationship studies led to the development of a small chemical library of Zn(2+)-specific cysteamine-based molecular probes. The probe L5 with higher excitation/emission wavelengths, which absorbs in the visible region and emits in the green, was chosen as a model imaging material for biological studies. After successful imaging of intracellular zinc in four different kinds of cells including living organisms, plant, and animal cells, in vivo imaging potential of L5 was evaluated using plant systems. In vivo imaging of translocation of zinc through the stem of a small herb with a transparent stem, Peperomia pellucida, confirmed the stability of L5 inside biological systems and the suitability of L5 for real-time analysis. Similarly, fluorescence imaging of zinc in gram sprouts revealed the efficacy of the probe in the detection and localization of zinc in cereal crops. This imaging technique will help in knowing the efficiency of various techniques used for zinc enrichment of cereal crops. Computational analyses were carried out to better understand the structure, the formation of probe-Zn(2+) complexes, and the emission properties of these complexes.

Magneto-optical labeling of fetal neural stem cells for in vivo MRI tracking.

PubMed

Flexman, J A; Minoshima, S; Kim, Y; Cross, D J

2006-01-01

Neural stem cell therapy for neurological pathologies, such as Alzheimer's and Parkinson's disease, may delay the onset of symptoms, replace damaged neurons and/or support the survival of endogenous cells. Magnetic resonance imaging (MRI) can be used to track magnetically labeled cells in vivo to observe migration. Prior to transplantation, labeled cells must be characterized to show that they retain their intrinsic properties, such as cell proliferation into neurospheres in a supplemented environment. In vivo images must also be correlated to sensitive, histological markers. In this study, we show that fetus-derived neural stem cells can be co-labeled with superparamagnetic iron oxide and PKH26, a fluorescent dye. Labeled cells retain the ability to proliferate into neurospheres in culture, but labeling prevents neurospheres from merging in a non-adherent culture environment. After labeled NSCs were transplantation into the rat brain, their location and subsequent migration along the corpus callosum was detected using MRI. This study demonstrates an imaging paradigm with which to develop an in vivo assay for quantitatively evaluating fetal neural stem cell migration.

In silico characterization of cell-cell interactions using a cellular automata model of cell culture.

PubMed

Kihara, Takanori; Kashitani, Kosuke; Miyake, Jun

2017-07-14

Cell proliferation is a key characteristic of eukaryotic cells. During cell proliferation, cells interact with each other. In this study, we developed a cellular automata model to estimate cell-cell interactions using experimentally obtained images of cultured cells. We used four types of cells; HeLa cells, human osteosarcoma (HOS) cells, rat mesenchymal stem cells (MSCs), and rat smooth muscle A7r5 cells. These cells were cultured and stained daily. The obtained cell images were binarized and clipped into squares containing about 10 4 cells. These cells showed characteristic cell proliferation patterns. The growth curves of these cells were generated from the cell proliferation images and we determined the doubling time of these cells from the growth curves. We developed a simple cellular automata system with an easily accessible graphical user interface. This system has five variable parameters, namely, initial cell number, doubling time, motility, cell-cell adhesion, and cell-cell contact inhibition (of proliferation). Within these parameters, we obtained initial cell numbers and doubling times experimentally. We set the motility at a constant value because the effect of the parameter for our simulation was restricted. Therefore, we simulated cell proliferation behavior with cell-cell adhesion and cell-cell contact inhibition as variables. By comparing growth curves and proliferation cell images, we succeeded in determining the cell-cell interaction properties of each cell. Simulated HeLa and HOS cells exhibited low cell-cell adhesion and weak cell-cell contact inhibition. Simulated MSCs exhibited high cell-cell adhesion and positive cell-cell contact inhibition. Simulated A7r5 cells exhibited low cell-cell adhesion and strong cell-cell contact inhibition. These simulated results correlated with the experimental growth curves and proliferation images. Our simulation approach is an easy method for evaluating the cell-cell interaction properties of cells.

Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

PubMed

Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

2016-10-01

This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

White blood cell differential count of maturation stages in bone marrow smear using dual-stage convolutional neural networks.

PubMed

Choi, Jin Woo; Ku, Yunseo; Yoo, Byeong Wook; Kim, Jung-Ah; Lee, Dong Soon; Chai, Young Jun; Kong, Hyoun-Joong; Kim, Hee Chan

2017-01-01

The white blood cell differential count of the bone marrow provides information concerning the distribution of immature and mature cells within maturation stages. The results of such examinations are important for the diagnosis of various diseases and for follow-up care after chemotherapy. However, manual, labor-intensive methods to determine the differential count lead to inter- and intra-variations among the results obtained by hematologists. Therefore, an automated system to conduct the white blood cell differential count is highly desirable, but several difficulties hinder progress. There are variations in the white blood cells of each maturation stage, small inter-class differences within each stage, and variations in images because of the different acquisition and staining processes. Moreover, a large number of classes need to be classified for bone marrow smear analysis, and the high density of touching cells in bone marrow smears renders difficult the segmentation of single cells, which is crucial to traditional image processing and machine learning. Few studies have attempted to discriminate bone marrow cells, and even these have either discriminated only a few classes or yielded insufficient performance. In this study, we propose an automated white blood cell differential counting system from bone marrow smear images using a dual-stage convolutional neural network (CNN). A total of 2,174 patch images were collected for training and testing. The dual-stage CNN classified images into 10 classes of the myeloid and erythroid maturation series, and achieved an accuracy of 97.06%, a precision of 97.13%, a recall of 97.06%, and an F-1 score of 97.1%. The proposed method not only showed high classification performance, but also successfully classified raw images without single cell segmentation and manual feature extraction by implementing CNN. Moreover, it demonstrated rotation and location invariance. These results highlight the promise of the proposed method as an automated white blood cell differential count system.

White blood cell differential count of maturation stages in bone marrow smear using dual-stage convolutional neural networks

PubMed Central

Choi, Jin Woo; Ku, Yunseo; Yoo, Byeong Wook; Kim, Jung-Ah; Lee, Dong Soon; Chai, Young Jun; Kong, Hyoun-Joong

2017-01-01

The white blood cell differential count of the bone marrow provides information concerning the distribution of immature and mature cells within maturation stages. The results of such examinations are important for the diagnosis of various diseases and for follow-up care after chemotherapy. However, manual, labor-intensive methods to determine the differential count lead to inter- and intra-variations among the results obtained by hematologists. Therefore, an automated system to conduct the white blood cell differential count is highly desirable, but several difficulties hinder progress. There are variations in the white blood cells of each maturation stage, small inter-class differences within each stage, and variations in images because of the different acquisition and staining processes. Moreover, a large number of classes need to be classified for bone marrow smear analysis, and the high density of touching cells in bone marrow smears renders difficult the segmentation of single cells, which is crucial to traditional image processing and machine learning. Few studies have attempted to discriminate bone marrow cells, and even these have either discriminated only a few classes or yielded insufficient performance. In this study, we propose an automated white blood cell differential counting system from bone marrow smear images using a dual-stage convolutional neural network (CNN). A total of 2,174 patch images were collected for training and testing. The dual-stage CNN classified images into 10 classes of the myeloid and erythroid maturation series, and achieved an accuracy of 97.06%, a precision of 97.13%, a recall of 97.06%, and an F-1 score of 97.1%. The proposed method not only showed high classification performance, but also successfully classified raw images without single cell segmentation and manual feature extraction by implementing CNN. Moreover, it demonstrated rotation and location invariance. These results highlight the promise of the proposed method as an automated white blood cell differential count system. PMID:29228051

Simple Perfusion Apparatus (SPA) for Manipulation, Tracking and Study of Oocytes and Embryos

PubMed Central

Angione, Stephanie L.; Oulhen, Nathalie; Brayboy, Lynae M.; Tripathi, Anubhav; Wessel, Gary M.

2016-01-01

Objective To develop and implement a device and protocol for oocyte analysis at a single cell level. The device must be capable of high resolution imaging, temperature control, perfusion of media, drugs, sperm, and immunolabeling reagents all at defined flow-rates. Each oocyte and resultant embryo must remain spatially separated and defined. Design Experimental laboratory study Setting University and Academic Center for reproductive medicine. Patients/Animals Women with eggs retrieved for ICSI cycles, adult female FVBN and B6C3F1 mouse strains, sea stars. Intervention Real-time, longitudinal imaging of oocytes following fluorescent labeling, insemination, and viability tests. Main outcome measure(s) Cell and embryo viability, immunolabeling efficiency, live cell endocytosis quantitation, precise metrics of fertilization and embryonic development. Results Single oocytes were longitudinally imaged following significant changes in media, markers, endocytosis quantitation, and development, all with supreme control by microfluidics. Cells remained viable, enclosed, and separate for precision measurements, repeatability, and imaging. Conclusions We engineered a simple device to load, visualize, experiment, and effectively record individual oocytes and embryos, without loss of cells. Prolonged incubation capabilities provide longitudinal studies without need for transfer and potential loss of cells. This simple perfusion apparatus (SPA) provides for careful, precise, and flexible handling of precious samples facilitating clinical in vitro fertilization approaches. PMID:25450296

Label-free imaging of the dynamics of cell-to-cell string-like structure bridging in the free-space by low-coherent quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Iwai, Hidenao; Yamashita, Yutaka

2013-03-01

We succeeded in utilizing our low-coherent quantitative phase microscopy (LC-QPM) to achieve label-free and three-dimensional imaging of string-like structures bridging the free-space between live cells. In past studies, three dimensional morphology of the string-like structures between cells had been investigated by electron microscopies and fluorescence microscopies and these structures were called "membrane nanotubes" or "tunneling nanotubes." However, use of electron microscopy inevitably kills these cells and fluorescence microscopy is itself a potentially invasive method. To achieve noninvasive imaging of live cells, we applied our LC-QPM which is a reflection-type, phase resolved and full-field interference microscope employing a low-coherent light source. LC-QPM is able to visualize the three-dimensional morphology of live cells without labeling by means of low-coherence interferometry. The lateral (diffraction limit) and longitudinal (coherence-length) spatial resolution of LC-QPM were respectively 0.49 and 0.93 micrometers and the repeatability of the phase measurement was 0.02 radians (1.0 nm). We successfully obtained three-dimensional morphology of live cultured epithelial cells (cell type: HeLa, derived from cervix cancer) and were able to clearly observe the individual string-like structures interconnecting the cells. When we performed volumetric imaging, a 80 micrometer by 60 micrometer by 6.5 micrometer volume was scanned every 5.67 seconds and 70 frames of a three-dimensional movie were recorded for a duration of 397 seconds. Moreover, the optical phase images gave us detailed information about the three-dimensional morphology of the string-like structure at sub-wavelength resolution. We believe that our LC-QPM will be a useful tool for the study of three-dimensional morphology of live cells.

Atomic Force Microscopy Based Cell Shape Index

NASA Astrophysics Data System (ADS)

Adia-Nimuwa, Usienemfon; Mujdat Tiryaki, Volkan; Hartz, Steven; Xie, Kan; Ayres, Virginia

2013-03-01

Stellation is a measure of cell physiology and pathology for several cell groups including neural, liver and pancreatic cells. In the present work, we compare the results of a conventional two-dimensional shape index study of both atomic force microscopy (AFM) and fluorescent microscopy images with the results obtained using a new three-dimensional AFM-based shape index similar to sphericity index. The stellation of astrocytes is investigated on nanofibrillar scaffolds composed of electrospun polyamide nanofibers that has demonstrated promise for central nervous system (CNS) repair. Recent work by our group has given us the ability to clearly segment the cells from nanofibrillar scaffolds in AFM images. The clear-featured AFM images indicated that the astrocyte processes were longer than previously identified at 24h. It was furthermore shown that cell spreading could vary significantly as a function of environmental parameters, and that AFM images could record these variations. The new three-dimensional AFM-based shape index incorporates the new information: longer stellate processes and cell spreading. The support of NSF PHY-095776 is acknowledged.

Low-cost motility tracking system (LOCOMOTIS) for time-lapse microscopy applications and cell visualisation.

PubMed

Lynch, Adam E; Triajianto, Junian; Routledge, Edwin

2014-01-01

Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81 ± 0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17 ± 0.004 (MDA-MB-231 breast cancer cells), 1.24 ± 0.006 (SC5 mouse Sertoli cells) and 2.21 ± 0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers.

Low-Cost Motility Tracking System (LOCOMOTIS) for Time-Lapse Microscopy Applications and Cell Visualisation

PubMed Central

Lynch, Adam E.; Triajianto, Junian; Routledge, Edwin

2014-01-01

Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81±0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17±0.004 (MDA-MB-231 breast cancer cells), 1.24±0.006 (SC5 mouse Sertoli cells) and 2.21±0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers. PMID:25121722

Luminescent single-walled carbon nanotube-sensitized europium nanoprobes for cellular imaging

PubMed Central

Avti, Pramod K; Sitharaman, Balaji

2012-01-01

Lanthanoid-based optical probes with excitation wavelengths in the ultra-violet (UV) range (300–325 nm) have been widely developed as imaging probes. Efficient cellular imaging requires that lanthanoid optical probes be excited at visible wavelengths, to avoid UV damage to cells. The efficacy of europium-catalyzed single-walled carbon nanotubes (Eu-SWCNTs), as visible nanoprobes for cellular imaging, is reported in this study. Confocal fluorescence microscopy images of breast cancer cells (SK-BR-3 and MCF-7) and normal cells (NIH 3T3), treated with Eu-SWCNT at 0.2 μg/mL concentration, showed bright red luminescence after excitation at 365 nm and 458 nm wavelengths. Cell viability analysis showed no cytotoxic effects after the incubation of cells with Eu-SWCNTs at this concentration. Eu-SWCNT uptake is via the endocytosis mechanism. Labeling efficiency, defined as the percentage of incubated cells that uptake Eu-SWCNT, was 95%–100% for all cell types. The average cellular uptake concentration was 6.68 ng Eu per cell. Intracellular localization was further corroborated by transmission electron microscopy and Raman microscopy. The results indicate that Eu-SWCNT shows potential as a novel cellular imaging probe, wherein SWCNT sensitizes Eu3+ ions to allow excitation at visible wavelengths, and stable time-resolved red emission. The ability to functionalize biomolecules on the exterior surface of Eu-SWCNT makes it an excellent candidate for targeted cellular imaging. PMID:22619533

A plasmid-based reporter system for live cell imaging of dengue virus infected cells.

PubMed

Medin, Carey L; Valois, Sierra; Patkar, Chinmay G; Rothman, Alan L

2015-01-01

Cell culture models are used widely to study the effects of dengue virus (DENV) on host cell function. Current methods of identification of cells infected with an unmodified DENV requires fixation and permeablization of cells to allow DENV-specific antibody staining. This method does not permit imaging of viable cells over time. In this report, a plasmid-based reporter was developed to allow non-destructive identification of DENV-infected cells. The plasmid-based reporter was demonstrated to be broadly applicable to the four DENV serotypes, including low-passaged strains, and was specifically cleaved by the viral protease with minimal interference on viral production. This study reveals the potential for this novel reporter system to advance the studies of virus-host interactions during DENV infection. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell segmentation in histopathological images with deep learning algorithms by utilizing spatial relationships.

PubMed

Hatipoglu, Nuh; Bilgin, Gokhan

2017-10-01

In many computerized methods for cell detection, segmentation, and classification in digital histopathology that have recently emerged, the task of cell segmentation remains a chief problem for image processing in designing computer-aided diagnosis (CAD) systems. In research and diagnostic studies on cancer, pathologists can use CAD systems as second readers to analyze high-resolution histopathological images. Since cell detection and segmentation are critical for cancer grade assessments, cellular and extracellular structures should primarily be extracted from histopathological images. In response, we sought to identify a useful cell segmentation approach with histopathological images that uses not only prominent deep learning algorithms (i.e., convolutional neural networks, stacked autoencoders, and deep belief networks), but also spatial relationships, information of which is critical for achieving better cell segmentation results. To that end, we collected cellular and extracellular samples from histopathological images by windowing in small patches with various sizes. In experiments, the segmentation accuracies of the methods used improved as the window sizes increased due to the addition of local spatial and contextual information. Once we compared the effects of training sample size and influence of window size, results revealed that the deep learning algorithms, especially convolutional neural networks and partly stacked autoencoders, performed better than conventional methods in cell segmentation.

Imaging approaches for the study of cell based cardiac therapies

PubMed Central

Lau, Joe F.; Anderson, Stasia A.; Adler, Eric; Frank, Joseph A.

2009-01-01

Despite promising preclinical data, the treatment of cardiovascular diseases using embryonic, bone-marrow-derived, and skeletal myoblast stem cells has not yet come to fruition within mainstream clinical practice. Major obstacles in cardiac stem cell investigations include the ability to monitor cell engraftment and survival following implantation within the myocardium. Several cellular imaging modalities, including reporter gene and MRI-based tracking approaches, have emerged that provide the means to identify, localize and monitor stem cells longitudinally in vivo following implantation. This Review will examine the various cardiac cellular tracking modalities, including the combinatorial use of several probes in multimodality imaging, with a focus on data from the last five years. PMID:20027188

A multiplexed method for kinetic measurements of apoptosis and proliferation using live-content imaging.

PubMed

Artymovich, Katherine; Appledorn, Daniel M

2015-01-01

In vitro cell proliferation and apoptosis assays are widely used to study cancer cell biology. Commonly used methodologies are however performed at a single, user-defined endpoint. We describe a kinetic multiplex assay incorporating the CellPlayer(TM) NucLight Red reagent to measure proliferation and the CellPlayer(TM) Caspase-3/7 reagent to measure apoptosis using the two-color, live-content imaging platform, IncuCyte(TM) ZOOM. High-definition phase-contrast images provide an additional qualitative validation of cell death based on morphological characteristics. The kinetic data generated using this strategy can be used to derive informed pharmacology measurements to screen potential cancer therapeutics.

Subnuclear foci quantification using high-throughput 3D image cytometry

NASA Astrophysics Data System (ADS)

Wadduwage, Dushan N.; Parrish, Marcus; Choi, Heejin; Engelward, Bevin P.; Matsudaira, Paul; So, Peter T. C.

2015-07-01

Ionising radiation causes various types of DNA damages including double strand breaks (DSBs). DSBs are often recognized by DNA repair protein ATM which forms gamma-H2AX foci at the site of the DSBs that can be visualized using immunohistochemistry. However most of such experiments are of low throughput in terms of imaging and image analysis techniques. Most of the studies still use manual counting or classification. Hence they are limited to counting a low number of foci per cell (5 foci per nucleus) as the quantification process is extremely labour intensive. Therefore we have developed a high throughput instrumentation and computational pipeline specialized for gamma-H2AX foci quantification. A population of cells with highly clustered foci inside nuclei were imaged, in 3D with submicron resolution, using an in-house developed high throughput image cytometer. Imaging speeds as high as 800 cells/second in 3D were achieved by using HiLo wide-field depth resolved imaging and a remote z-scanning technique. Then the number of foci per cell nucleus were quantified using a 3D extended maxima transform based algorithm. Our results suggests that while most of the other 2D imaging and manual quantification studies can count only up to about 5 foci per nucleus our method is capable of counting more than 100. Moreover we show that 3D analysis is significantly superior compared to the 2D techniques.

Three-dimensional image analysis as a tool for embryology

NASA Astrophysics Data System (ADS)

Verweij, Andre

1992-06-01

In the study of cell fate, cell lineage, and morphogenetic transformation it is necessary to obtain 3-D data. Serial sections of glutaraldehyde fixed and glycol methacrylate embedded material provide high resolution data. Clonal spread during germ layer formation in the mouse embryo has been followed by labeling a progenitor epiblast cell with horseradish peroxidase and staining its descendants one or two days later, followed by histological processing. Reconstruction of a 3-D image from histological sections must provide a solution for the alignment problem. As we want to study images at different magnification levels, we have chosen a method in which the sections are aligned under the microscope. Positioning is possible through a translation and a rotation stage. The first step for reconstruction is a coarse alignment on the basis of the moments in a binary, low magnification image of the embedding block. Thereafter, images of higher magnification levels are aligned by optimizing a similarity measure between the images. To analyze, first a global 3-D second order surface is fitted on the image to obtain the orientation of the embryo. The coefficients of this fit are used to normalize the size of the different embryos. Thereafter, the image is resampled with respect to the surface to create a 2-D mapping of the embryo and to guide the segmentation of the different cell layers which make up the embryo.

3D nanoscale imaging of the yeast, Schizosaccharomyces pombe, by full-field transmission x-ray microscopy at 5.4 keV

PubMed Central

Chen, Jie; Yang, Yunhao; Zhang, Xiaobo; Andrews, Joy C.; Pianetta, Piero; Guan, Yong; Liu, Gang; Xiong, Ying; Wu, Ziyu; Tian, Yangchao

2010-01-01

Three-dimensional (3D) nanoscale structures of the fission yeast, Schizosaccharomyces pombe, can be obtained by full-field transmission hard x-ray microscopy with 30 nm resolution using synchrotron radiation sources. Sample preparation is relatively simple and the samples are portable across various imaging environments, allowing for high throughput sample screening. The yeast cells were fixed and double stained with Reynold’s lead citrate and uranyl acetate. We performed both absorption contrast and Zernike phase contrast imaging on these cells in order to test this method. The membranes, nucleus and subcellular organelles of the cells were clearly visualized using absorption contrast mode. The x-ray images of the cells could be used to study the spatial distributions of the organelles in the cells. These results show unique structural information, demonstrating that hard x-ray microscopy is a complementary method for imaging and analyzing biological samples. PMID:20349228

An Advanced Preclinical Mouse Model for Acute Myeloid Leukemia Using Patients' Cells of Various Genetic Subgroups and In Vivo Bioluminescence Imaging

PubMed Central

Vick, Binje; Rothenberg, Maja; Sandhöfer, Nadine; Carlet, Michela; Finkenzeller, Cornelia; Krupka, Christina; Grunert, Michaela; Trumpp, Andreas; Corbacioglu, Selim; Ebinger, Martin; André, Maya C.; Hiddemann, Wolfgang; Schneider, Stephanie; Subklewe, Marion; Metzeler, Klaus H.; Spiekermann, Karsten; Jeremias, Irmela

2015-01-01

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups. PMID:25793878

Biodistribution and Clearance of Human Mesenchymal Stem Cells by Quantitative Three-Dimensional Cryo-Imaging After Intravenous Infusion in a Rat Lung Injury Model.

PubMed

Schmuck, Eric G; Koch, Jill M; Centanni, John M; Hacker, Timothy A; Braun, Rudolf K; Eldridge, Marlowe; Hei, Derek J; Hematti, Peiman; Raval, Amish N

2016-12-01

: Cell tracking is a critical component of the safety and efficacy evaluation of therapeutic cell products. To date, cell-tracking modalities have been hampered by poor resolution, low sensitivity, and inability to track cells beyond the shortterm. Three-dimensional (3D) cryo-imaging coregisters fluorescent and bright-field microcopy images and allows for single-cell quantification within a 3D organ volume. We hypothesized that 3D cryo-imaging could be used to measure cell biodistribution and clearance after intravenous infusion in a rat lung injury model compared with normal rats. A bleomycin lung injury model was established in Sprague-Dawley rats (n = 12). Human mesenchymal stem cells (hMSCs) labeled with QTracker655 were infused via jugular vein. After 2, 4, or 8 days, a second dose of hMSCs labeled with QTracker605 was infused, and animals were euthanized after 60, 120, or 240 minutes. Lungs, liver, spleen, heart, kidney, testis, and intestine were cryopreserved, followed by 3D cryo-imaging of each organ. At 60 minutes, 82% ± 9.7% of cells were detected; detection decreased to 60% ± 17% and 66% ± 22% at 120 and 240 minutes, respectively. At day 2, 0.06% of cells were detected, and this level remained constant at days 4 and 8 postinfusion. At 60, 120, and 240 minutes, 99.7% of detected cells were found in the liver, lungs, and spleen, with cells primarily retained in the liver. This is the first study using 3D cryo-imaging to track hMSCs in a rat lung injury model. hMSCs were retained primarily in the liver, with fewer detected in lungs and spleen. Effective bench-to-bedside clinical translation of cellular therapies requires careful understanding of cell fate through tracking. Tracking cells is important to measure cell retention so that delivery methods and cell dose can be optimized and so that biodistribution and clearance can be defined to better understand potential off-target toxicity and redosing strategies. This article demonstrates, for the first time, the use of three-dimensional cryo-imaging for single-cell quantitative tracking of intravenous infused clinical-grade mesenchymal stem cells in a clinically relevant model of lung injury. The important information learned in this study will help guide future clinical and translational stem cell therapies for lung injuries. ©AlphaMed Press.

Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes

PubMed Central

Datta, Rupsa; Heylman, Christopher; George, Steven C.; Gratton, Enrico

2016-01-01

In this work we demonstrate a label-free optical imaging technique to assess metabolic status and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes by two-photon fluorescence lifetime imaging of endogenous fluorophores. Our results show the sensitivity of this method to detect shifts in metabolism and oxidative stress in the cardiomyocytes upon pathological stimuli of hypoxia and cardiotoxic drugs. This non-invasive imaging technique could prove beneficial for drug development and screening, especially for in vitro cardiac models created from stem cell-derived cardiomyocytes and to study the pathogenesis of cardiac diseases and therapy. PMID:27231614

Cell type classifiers for breast cancer microscopic images based on fractal dimension texture analysis of image color layers.

PubMed

Jitaree, Sirinapa; Phinyomark, Angkoon; Boonyaphiphat, Pleumjit; Phukpattaranont, Pornchai

2015-01-01

Having a classifier of cell types in a breast cancer microscopic image (BCMI), obtained with immunohistochemical staining, is required as part of a computer-aided system that counts the cancer cells in such BCMI. Such quantitation by cell counting is very useful in supporting decisions and planning of the medical treatment of breast cancer. This study proposes and evaluates features based on texture analysis by fractal dimension (FD), for the classification of histological structures in a BCMI into either cancer cells or non-cancer cells. The cancer cells include positive cells (PC) and negative cells (NC), while the normal cells comprise stromal cells (SC) and lymphocyte cells (LC). The FD feature values were calculated with the box-counting method from binarized images, obtained by automatic thresholding with Otsu's method of the grayscale images for various color channels. A total of 12 color channels from four color spaces (RGB, CIE-L*a*b*, HSV, and YCbCr) were investigated, and the FD feature values from them were used with decision tree classifiers. The BCMI data consisted of 1,400, 1,200, and 800 images with pixel resolutions 128 × 128, 192 × 192, and 256 × 256, respectively. The best cross-validated classification accuracy was 93.87%, for distinguishing between cancer and non-cancer cells, obtained using the Cr color channel with window size 256. The results indicate that the proposed algorithm, based on fractal dimension features extracted from a color channel, performs well in the automatic classification of the histology in a BCMI. This might support accurate automatic cell counting in a computer-assisted system for breast cancer diagnosis. © Wiley Periodicals, Inc.

Synchrotron Time-Lapse Imaging of Lignocellulosic Biomass Hydrolysis: Tracking Enzyme Localization by Protein Autofluorescence and Biochemical Modification of Cell Walls by Microfluidic Infrared Microspectroscopy

PubMed Central

Devaux, Marie-Françoise; Jamme, Frédéric; André, William; Bouchet, Brigitte; Alvarado, Camille; Durand, Sylvie; Robert, Paul; Saulnier, Luc; Bonnin, Estelle; Guillon, Fabienne

2018-01-01

Tracking enzyme localization and following the local biochemical modification of the substrate should help explain the recalcitrance of lignocellulosic plant cell walls to enzymatic degradation. Time-lapse studies using conventional imaging require enzyme labeling and following the biochemical modifications of biopolymers found in plant cell walls, which cannot be easily achieved. In the present work, synchrotron facilities have been used to image the enzymatic degradation of lignocellulosic biomass without labeling the enzyme or the cell walls. Multichannel autofluorescence imaging of the protein and phenolic compounds after excitation at 275 nm highlighted the presence or absence of enzymes on cell walls and made it possible to track them during the reaction. Image analysis was used to quantify the fluorescence intensity variations. Consistent variations in the enzyme concentration were found locally for cell cavities and their surrounding cell walls. Microfluidic FT-IR microspectroscopy allowed for time-lapse tracking of local changes in the polysaccharides in cell walls during degradation. Hemicellulose degradation was found to occur prior to cellulose degradation using a Celluclast® preparation. Combining the fluorescence and FT-IR information yielded the conclusion that enzymes did not bind to lignified cell walls, which were consequently not degraded. Fluorescence multiscale imaging and FT-IR microspectroscopy showed an unexpected variability both in the initial biochemical composition and the degradation pattern, highlighting micro-domains in the cell wall of a given cell. Fluorescence intensity quantification showed that the enzymes were not evenly distributed, and their amount increased progressively on degradable cell walls. During degradation, adjacent cells were separated and the cell wall fragmented until complete degradation. PMID:29515611

Bioluminescent imaging demonstrates transplanted human embryonic stem cell-derived CD34+ cells preferentially develop into endothelial cells

PubMed Central

Tian, Xinghui; Hexum, Melinda K.; Penchev, Vesselin R.; Taylor, Russell J.; Shultz, Leonard D.; Kaufman, Dan S

2010-01-01

Human embryonic stem cells (hESCs) provide an important resource for novel regenerative medicine therapies and have been used to derive diverse cell populations, including hematopoietic and endothelial cells. However, it remains a challenge to achieve significant engraftment of hESC-derived blood cells when transplanted into animal models. To better understand mechanisms that enhance or limit the in vivo developmental potential of hESC-derived cells, we utilized hESCs that express firefly luciferase (luc) to allow non-invasive, real-time bioluminescent imaging of hESC-derived CD34+ cells transplanted into the liver of neonatal immunodeficient mice. Serial imaging demonstrated stable engraftment and expansion of the luc+ hESC-derived cells in vivo over several months. While we found that these hESC-derived CD34+ cells have bipotential ability to generate both hematopoietic and endothelial lineages in vitro, these studies demonstrate preferential differentiation into endothelial cells in vivo, with only low levels of hematopoietic cell engraftment. Therefore, these studies reveal key differences in the developmental potential of hESC-derived cells using in vitro and in vivo analyses. While transplanted hESC-derived CD34+ cells are well suited for revascularization therapies, additional measures are needed to provide higher levels of long-term hematopoietic engraftment. PMID:19711457

The application of digital image plane holography technology to identify Chinese herbal medicine

NASA Astrophysics Data System (ADS)

Wang, Huaying; Guo, Zhongjia; Liao, Wei; Zhang, Zhihui

2012-03-01

In this paper, the imaging technology of digital image plane holography to identify the Chinese herbal medicine is studied. The optical experiment system of digital image plane holography which is the special case of pre-magnification digital holography was built. In the record system, one is an object light by using plane waves which illuminates the object, and the other one is recording hologram by using spherical light wave as reference light. There is a Micro objective lens behind the object. The second phase factor which caus ed by the Micro objective lens can be eliminated by choosing the proper position of the reference point source when digital image plane holography is recorded by spherical light. In this experiment, we use the Lygodium cells and Onion cells as the object. The experiment results with Lygodium cells and Onion cells show that digital image plane holography avoid the process of finding recording distance by using auto-focusing approach, and the phase information of the object can be reconstructed more accurately. The digital image plane holography is applied to the microscopic imaging of cells more effectively, and it is suit to apply for the identify of Chinese Herbal Medicine. And it promotes the application of digital holographic in practice.

3D tomography of cells in micro-channels

NASA Astrophysics Data System (ADS)

Quint, S.; Christ, A. F.; Guckenberger, A.; Himbert, S.; Kaestner, L.; Gekle, S.; Wagner, C.

2017-09-01

We combine confocal imaging, microfluidics, and image analysis to record 3D-images of cells in flow. This enables us to recover the full 3D representation of several hundred living cells per minute. Whereas 3D confocal imaging has thus far been limited to steady specimens, we overcome this restriction and present a method to access the 3D shape of moving objects. The key of our principle is a tilted arrangement of the micro-channel with respect to the focal plane of the microscope. This forces cells to traverse the focal plane in an inclined manner. As a consequence, individual layers of passing cells are recorded, which can then be assembled to obtain the volumetric representation. The full 3D information allows for a detailed comparison with theoretical and numerical predictions unfeasible with, e.g., 2D imaging. Our technique is exemplified by studying flowing red blood cells in a micro-channel reflecting the conditions prevailing in the microvasculature. We observe two very different types of shapes: "croissants" and "slippers." Additionally, we perform 3D numerical simulations of our experiment to confirm the observations. Since 3D confocal imaging of cells in flow has not yet been realized, we see high potential in the field of flow cytometry where cell classification thus far mostly relies on 1D scattering and fluorescence signals.

Bioengineering anembryonic human trophoblast vesicles.

PubMed

Robins, Jared C; Morgan, Jeffrey R; Krueger, Paula; Carson, Sandra A

2011-02-01

Trophoblast cells in vivo form a 3-dimensional structure that promotes complex cell-to-cell interactions that cannot be studied with traditional monolayer culture. We describe a 3-dimensional trophoblast bioreactor to study cellular interactions. Nonadhesive agarose hydrogels were cast from molds using computer-assisted prototyping. Trophoblast cells were seeded into the gels for 10 days. Morphology, viability, and vesicle behavior were assessed. Trophoblast cells formed uniform spheroids. Serial sectioning on days 3, 7, and 10 revealed central vacuolization with a consistent outer rim 12.3-μ thick. The vesicle configuration has been confirmed with confocal imaging. Electron Microscopic (EM) imaging revealed its ultrastructure. The vesicles migrate across a fibronectin-coated surface and invaded basement membrane. Trophoblast cells cultured in a novel substrate-free 3-dimensional system form trophoblast vesicles. This new cell culture technique allows us to better study placental cell-to-cell interactions with the potential of forming microtissues.

Using wavelet denoising and mathematical morphology in the segmentation technique applied to blood cells images.

PubMed

Boix, Macarena; Cantó, Begoña

2013-04-01

Accurate image segmentation is used in medical diagnosis since this technique is a noninvasive pre-processing step for biomedical treatment. In this work we present an efficient segmentation method for medical image analysis. In particular, with this method blood cells can be segmented. For that, we combine the wavelet transform with morphological operations. Moreover, the wavelet thresholding technique is used to eliminate the noise and prepare the image for suitable segmentation. In wavelet denoising we determine the best wavelet that shows a segmentation with the largest area in the cell. We study different wavelet families and we conclude that the wavelet db1 is the best and it can serve for posterior works on blood pathologies. The proposed method generates goods results when it is applied on several images. Finally, the proposed algorithm made in MatLab environment is verified for a selected blood cells.

Computed tomography and magnetic resonance imaging of adult renal cell carcinoma associated with Xp11.2 translocation.

PubMed

Dang, Trien T; Ziv, Etay; Weinstein, Stefanie; Meng, Maxwell V; Wang, Zhen; Coakley, Fergus V

2012-01-01

This study aimed to report the computed tomography (CT) and magnetic resonance imaging (MRI) findings of renal cell carcinoma associated with Xp11.2 translocation in adults. We retrospectively identified 9 adults with renal cell carcinoma associated with Xp11.2 translocation who underwent baseline cross-sectional imaging with CT (n = 9) or MRI (n = 3). All available clinical, imaging, and histopathological records were reviewed. Mean patient age was 24 years (range, 18-45 years). Eight of 9 cancers demonstrated imaging findings of hemorrhage or necrosis (n = 3), advanced stage disease (n = 2), or both (n = 3) at CT or MRI. The possibility of renal cell carcinoma associated with Xp11.2 translocation should be considered for a renal mass seen in a patient 45 years or younger, which demonstrates hemorrhage or necrosis or advanced stage disease at CT or MRI.

Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein

NASA Astrophysics Data System (ADS)

Biteen, Julie S.; Thompson, Michael A.; Tselentis, Nicole K.; Shapiro, Lucy; Moerner, W. E.

2009-02-01

Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.

3D quantitative phase imaging of neural networks using WDT

NASA Astrophysics Data System (ADS)

Kim, Taewoo; Liu, S. C.; Iyer, Raj; Gillette, Martha U.; Popescu, Gabriel

2015-03-01

White-light diffraction tomography (WDT) is a recently developed 3D imaging technique based on a quantitative phase imaging system called spatial light interference microscopy (SLIM). The technique has achieved a sub-micron resolution in all three directions with high sensitivity granted by the low-coherence of a white-light source. Demonstrations of the technique on single cell imaging have been presented previously; however, imaging on any larger sample, including a cluster of cells, has not been demonstrated using the technique. Neurons in an animal body form a highly complex and spatially organized 3D structure, which can be characterized by neuronal networks or circuits. Currently, the most common method of studying the 3D structure of neuron networks is by using a confocal fluorescence microscope, which requires fluorescence tagging with either transient membrane dyes or after fixation of the cells. Therefore, studies on neurons are often limited to samples that are chemically treated and/or dead. WDT presents a solution for imaging live neuron networks with a high spatial and temporal resolution, because it is a 3D imaging method that is label-free and non-invasive. Using this method, a mouse or rat hippocampal neuron culture and a mouse dorsal root ganglion (DRG) neuron culture have been imaged in order to see the extension of processes between the cells in 3D. Furthermore, the tomogram is compared with a confocal fluorescence image in order to investigate the 3D structure at synapses.

Breast cancer cell targeted MR molecular imaging probe: Anti-MUC1 antibody-based magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Moradi Khaniabadi, P.; S. A Majid, A. M.; Asif, M.; Moradi Khaniabadi, B.; Shahbazi-Gahrouei, D.; Jaafar, M. S.

2017-05-01

Effective and specific diagnostic imaging techniques are important in early-stage breast cancer treatment. The objective of this study was to develop a specific breast cancer contrast agent for magnetic resonance imaging (MRI). In so doing, superparamagnetic iron oxide nanoparticles (SPIONs) were conjugated to C595 monoclonal antibody using EDC chemistry to produce nanoprobe with high relaxivity and narrow size (87.4±0.7 nm). To test the developed nanoprobe in vitro, assessments including Cell toxicity, targeting efficacy, cellular binding, and MR imaging were carried out. The results indicated that after 6 hrs incubation with MCF-7 cells at 200 to 25 µg Fe/ml doses, 76% to 16% T2 reduction was obtained. The presence of iron localised in MCF-7 cells measured by atomic absorption spectroscopy (AAS) was about 9.95±0.09 ppm iron/cell at higher doses of nanoprobe. Moreover, a linear relationship between iron concentration of nontoxic SPION-C595 and T2 relaxation times was observed. This study also revealed that developed nanoprobe might be used as a specific negative contrast agent for detecting breast cancer.

Imaging Transcriptional Regulation of Eukaryotic mRNA Genes: Advances and Outlook.

PubMed

Yao, Jie

2017-01-06

Regulation of eukaryotic transcription in vivo occurs at distinct stages. Previous research has identified many active or repressive transcription factors (TFs) and core transcription components and studied their functions in vitro and in vivo. Nonetheless, how individual TFs act in concert to regulate mRNA gene expression in a single cell remains poorly understood. Direct observation of TF assembly and disassembly and various biochemical reactions during transcription of a single-copy gene in vivo is the ideal approach to study this problem. Research in this area requires developing novel techniques for single-cell transcription imaging and integrating imaging studies into understanding the molecular biology of transcription. In the past decade, advanced cell imaging has enabled unprecedented capabilities to visualize individual TF molecules, to track single transcription sites, and to detect individual mRNA in fixed and living cells. These studies have raised several novel insights on transcriptional regulation such as the "hit-and-run" model and transcription bursting that could not be obtained by in vitro biochemistry analysis. At this point, the key question is how to achieve deeper understandings or discover novel mechanisms of eukaryotic transcriptional regulation by imaging transcription in single cells. Meanwhile, further technical advancements are likely required for visualizing distinct kinetic steps of transcription on a single-copy gene in vivo. This review article summarizes recent progress in the field and describes the challenges and opportunities ahead. Copyright © 2016 Elsevier Ltd. All rights reserved.

Neuronal Tracing with Magnetic Labels: NMR Imaging Methods, Preliminary Results, and New Optimized Coils.

NASA Astrophysics Data System (ADS)

Ghosh, Pratik

1992-01-01

The investigations focussed on in vivo NMR imaging studies of magnetic particles with and within neural cells. NMR imaging methods, both Fourier transform and projection reconstruction, were implemented and new protocols were developed to perform "Neuronal Tracing with Magnetic Labels" on small animal brains. Having performed the preliminary experiments with neuronal tracing, new optimized coils and experimental set-up were devised. A novel gradient coil technology along with new rf-coils were implemented, and optimized for future use with small animals in them. A new magnetic labelling procedure was developed that allowed labelling of billions of cells with ultra -small magnetite particles in a short time. The relationships among the viability of such cells, the amount of label and the contrast in the images were studied as quantitatively as possible. Intracerebral grafting of magnetite labelled fetal rat brain cells made it possible for the first time to attempt monitoring in vivo the survival, differentiation, and possible migration of both host and grafted cells in the host rat brain. This constituted the early steps toward future experiments that may lead to the monitoring of human brain grafts of fetal brain cells. Preliminary experiments with direct injection of horse radish peroxidase-conjugated magnetite particles into neurons, followed by NMR imaging, revealed a possible non-invasive alternative, allowing serial study of the dynamic transport pattern of tracers in single living animals. New gradient coils were built by using parallel solid-conductor ribbon cables that could be wrapped easily and quickly. Rapid rise times provided by these coils allowed implementation of fast imaging methods. Optimized rf-coil circuit development made it possible to understand better the sample-coil properties and the associated trade -offs in cases of small but conducting samples.

Controlled graphene encapsulation: a nanoscale shield for characterising single bacterial cells in liquid.

PubMed

Li, Jiayao; Zheng, Changxi; Liu, Boyin; Chou, Tsengming; Kim, Yeonuk; Qiu, Shi; Li, Jian; Yan, Wenyi; Fu, Jing

2018-06-11

High-resolution single-cell imaging in their native or near-native state has received considerable interest for decades. In this research, we present an innovative approach that can be employed to study both morphological and nano-mechanical properties of hydrated single bacterial cells. The proposed strategy is to encapsulate wet cells with monolayer graphene with a newly developed water membrane approach, followed by imaging with both electron microscopy (EM) and atomic force microscopy (AFM). A computational framework was developed to provide additional insights, with the detailed nanoindentation process on graphene modeled based on finite element method. The model was first validated by calibration with polymer materials of known properties, and the contribution of graphene was then studied and corrected to determine the actual moduli of the encapsulated hydrated sample. Aapplication of the proposed approach was performed on hydrated bacterial cells (Klebsiella pneumoniae) to correlate the structural and mechanical information. EM and EDS (energy-dispersive X-ray spectroscopy) imaging confirmed that the cells in their near-native stage can be studied inside the miniatured environment enabled with graphene encapsulation. The actual moduli of the encapsulated hydrated cells were determined based on the developed computational model in parallel, with results comparable with those acquired with Wet-AFM. It is expected that the successful establishment of controlled graphene encapsulation offers a new route for probing liquid/live cells with scanning probe microscopy, as well as correlative imaging of hydrated samples for both biological and material sciences. © 2018 IOP Publishing Ltd.

Revealing organization of cellulose in wood cell walls by Raman imaging

Treesearch

Umesh P. Agarwal; Sally A. Ralph

2007-01-01

Anisotropy of cellulose organization in mature black spruce wood cell wall was investigated by Raman imaging using a 1 [mu]m lateral-resolution capable confocal Raman microscope. In these studies, wood cross sections (CS) and radial longitudinal sections (LS) that were partially delignified by acid chlorite treatment were used. In the case of CS where latewood cells...

Automatic neuron segmentation and neural network analysis method for phase contrast microscopy images.

PubMed

Pang, Jincheng; Özkucur, Nurdan; Ren, Michael; Kaplan, David L; Levin, Michael; Miller, Eric L

2015-11-01

Phase Contrast Microscopy (PCM) is an important tool for the long term study of living cells. Unlike fluorescence methods which suffer from photobleaching of fluorophore or dye molecules, PCM image contrast is generated by the natural variations in optical index of refraction. Unfortunately, the same physical principles which allow for these studies give rise to complex artifacts in the raw PCM imagery. Of particular interest in this paper are neuron images where these image imperfections manifest in very different ways for the two structures of specific interest: cell bodies (somas) and dendrites. To address these challenges, we introduce a novel parametric image model using the level set framework and an associated variational approach which simultaneously restores and segments this class of images. Using this technique as the basis for an automated image analysis pipeline, results for both the synthetic and real images validate and demonstrate the advantages of our approach.

Imaging lysosomal highly reactive oxygen species and lighting up cancer cells and tumors enabled by a Si-rhodamine-based near-infrared fluorescent probe.

PubMed

Zhang, Hongxing; Liu, Jing; Liu, Chenlu; Yu, Pengcheng; Sun, Minjia; Yan, Xiaohan; Guo, Jian-Ping; Guo, Wei

2017-07-01

Lysosomes have recently been regarded as the attractive pharmacological targets for selectively killing of cancer cells via lysosomal cell death (LCD) pathway that is closely associated with reactive oxygen species (ROS). However, the details on the ROS-induced LCD of cancer cells are still poorly understood, partially due to the absence of a lysosome-targetable, robust, and biocompatible imaging tool for ROS. In this work, we brought forward a Si-rhodamine-based fluorescent probe, named PSiR, which could selectively and sensitively image the pathologically more relavent highly reactive oxygen species (hROS: HClO, HO, and ONOO - ) in lysosomes of cancer cells. Compared with many of the existing hROS fluorescent probes, its superiorities are mainly embodied in the high stability against autoxidation and photoxidation, near-infrared exitation and emission, fast fluorescence off-on response, and specific lysosomal localization. Its practicality has been demonstrated by the real-time imaging of hROS generation in lysosomes of human non-small-cell lung cancer cells stimulated by anticancer drug β-lapachone. Moreover, the probe was sensitive enough for basal hROS in cancer cells, allowing its further imaging applications to discriminate not only cancer cells from normal cells, but also tumors from healthy tissues. Overall, our results strongly indicated that PSiR is a very promising imaging tool for the studies of ROS-related LCD of cancer cells, screening of new anticancer drugs, and early diagnosis of cancers. Copyright © 2017 Elsevier Ltd. All rights reserved.

Gold-carbon dots for the intracellular imaging of cancer-derived exosomes.

PubMed

Jiang, Xiaoyue; Zong, Shenfei; Chen, Chen; Zhang, Yizhi; Wang, Zhuyuan; Cui, Yiping

2018-04-27

As a novel fluorescent nanomaterial, gold-carbon quantum dots (GCDs) possess high biocompatibility and can be easily synthesized by a microwave-assisted method. Owing to their small sizes and unique optical properties, GCDs can be applied to imaging of biological targets, such as cells, exosomes and other organelles. In this study, GCDs were used for fluorescence imaging of exosomes. Tumor-specific antibodies are attached to the GCDs, forming exosome specific nanoprobes. The nanoprobes can label exosomes via immuno-reactions and thus facilitate fluorescent imaging of exosomes. When incubated with live cells, exosomes labeled with the nanoprobes can be taken up by the cells. The intracellular experiments confirmed that the majority of exosomes were endocytosed by cells and transported to lysosomes. The manner by which exosomes were taken up and the intracellular distribution of exosomes are unaffected by the GCDs. The experimental results successfully demonstrated that the presented nanoprobe can be used to study the intrinsic intracellular behavior of tumor derived exosomes. We believe that the GCDs based nanoprobe holds a great promise in the study of exosome related cellular events, such as cancer metastasis.

Gold-carbon dots for the intracellular imaging of cancer-derived exosomes

NASA Astrophysics Data System (ADS)

Jiang, Xiaoyue; Zong, Shenfei; Chen, Chen; Zhang, Yizhi; Wang, Zhuyuan; Cui, Yiping

2018-04-01

As a novel fluorescent nanomaterial, gold-carbon quantum dots (GCDs) possess high biocompatibility and can be easily synthesized by a microwave-assisted method. Owing to their small sizes and unique optical properties, GCDs can be applied to imaging of biological targets, such as cells, exosomes and other organelles. In this study, GCDs were used for fluorescence imaging of exosomes. Tumor-specific antibodies are attached to the GCDs, forming exosome specific nanoprobes. The nanoprobes can label exosomes via immuno-reactions and thus facilitate fluorescent imaging of exosomes. When incubated with live cells, exosomes labeled with the nanoprobes can be taken up by the cells. The intracellular experiments confirmed that the majority of exosomes were endocytosed by cells and transported to lysosomes. The manner by which exosomes were taken up and the intracellular distribution of exosomes are unaffected by the GCDs. The experimental results successfully demonstrated that the presented nanoprobe can be used to study the intrinsic intracellular behavior of tumor derived exosomes. We believe that the GCDs based nanoprobe holds a great promise in the study of exosome related cellular events, such as cancer metastasis.

Morphological imaging and quantification of axial xylem tissue in Fraxinus excelsior L. through X-ray micro-computed tomography.

PubMed

Koddenberg, Tim; Militz, Holger

2018-05-05

The popularity of X-ray based imaging methods has continued to increase in research domains. In wood research, X-ray micro-computed tomography (XμCT) is useful for structural studies examining the three-dimensional and complex xylem tissue of trees qualitatively and quantitatively. In this study, XμCT made it possible to visualize and quantify the spatial xylem organization of the angiosperm species Fraxinus excelsior L. on the microscopic level. Through image analysis, it was possible to determine morphological characteristics of the cellular axial tissue (vessel elements, fibers, and axial parenchyma cells) three-dimensionally. X-ray imaging at high resolutions provides very distinct visual insight into the xylem structure. Numerical analyses performed through semi-automatic procedures made it possible to quickly quantify cell characteristics (length, diameter, and volume of cells). Use of various spatial resolutions (0.87-5 μm) revealed boundaries users should be aware of. Nevertheless, our findings, both qualitative and quantitative, demonstrate XμCT to be a valuable tool for studying the spatial cell morphology of F. excelsior. Copyright © 2018. Published by Elsevier Ltd.

Time-lapse microscopy and image processing for stem cell research: modeling cell migration

NASA Astrophysics Data System (ADS)

Gustavsson, Tomas; Althoff, Karin; Degerman, Johan; Olsson, Torsten; Thoreson, Ann-Catrin; Thorlin, Thorleif; Eriksson, Peter

2003-05-01

This paper presents hardware and software procedures for automated cell tracking and migration modeling. A time-lapse microscopy system equipped with a computer controllable motorized stage was developed. The performance of this stage was improved by incorporating software algorithms for stage motion displacement compensation and auto focus. The microscope is suitable for in-vitro stem cell studies and allows for multiple cell culture image sequence acquisition. This enables comparative studies concerning rate of cell splits, average cell motion velocity, cell motion as a function of cell sample density and many more. Several cell segmentation procedures are described as well as a cell tracking algorithm. Statistical methods for describing cell migration patterns are presented. In particular, the Hidden Markov Model (HMM) was investigated. Results indicate that if the cell motion can be described as a non-stationary stochastic process, then the HMM can adequately model aspects of its dynamic behavior.

Effect of microbubble ligation to cells on ultrasound signal enhancement: implications for targeted imaging.

PubMed

Lankford, Miles; Behm, Carolyn Z; Yeh, James; Klibanov, Alexander L; Robinson, Peter; Lindner, Jonathan R

2006-10-01

Molecular imaging with contrast-enhanced ultrasound (CEU) relies on the detection of microbubbles retained in regions of disease. The aim of this study was to determine whether microbubble attachment to cells influences their acoustic signal generation and stability. Biotinylated microbubbles were attached to streptavidin-coated plates to derive density versus intensity relations during low- and high-power imaging. To assess damping from microbubble attachment to solid or cell surfaces, in vitro imaging was performed for microbubbles charge-coupled to methacrylate spheres and for vascular cell adhesion molecule-1-targeted microbubbles attached to endothelial cells. Signal enhancement on plates increased according to acoustic power and microbubble site density up to 300 mm. Microbubble signal was reduced by attachment to solid spheres during high- and low-power imaging but was minimally reduced by attachment to endothelial cells and only at low power. Attachment of targeted microbubbles to rigid surfaces results in damping and a reduction of their acoustic signal, which is not seen when microbubbles are attached to cells. A reliable concentration versus intensity relationship can be expected from microbubble attachment to 2-dimensional surfaces until a very high site density is reached.

ExTzBox: A Glowing Cyclophane for Live-Cell Imaging.

PubMed

Roy, Indranil; Bobbala, Sharan; Zhou, Jiawang; Nguyen, Minh T; Nalluri, Siva Krishna Mohan; Wu, Yilei; Ferris, Daniel P; Scott, Evan Alexander; Wasielewski, Michael R; Stoddart, J Fraser

2018-06-13

The ideal fluorescent probe for live-cell imaging is bright and non-cytotoxic and can be delivered easily into the living cells in an efficient manner. The design of synthetic fluorophores having all three of these properties, however, has proved to be challenging. Here, we introduce a simple, yet effective, strategy based on well-established chemistry for designing a new class of fluorescent probes for live-cell imaging. A box-like hybrid cyclophane, namely ExTzBox·4X (6·4X, X = PF 6 - , Cl - ), has been synthesized by connecting an extended viologen (ExBIPY) and a dipyridyl thiazolothiazole (TzBIPY) unit in an end-to-end fashion with two p-xylylene linkers. Photophysical studies show that 6·4Cl has a quantum yield Φ F = 1.00. Furthermore, unlike its ExBIPY 2+ and TzBIPY 2+ building units, 6·4Cl is non-cytotoxic to RAW 264.7 macrophages, even with a loading concentration as high as 100 μM, presumably on account of its rigid box-like structure which prevents its intercalation into DNA and may inhibit other interactions with it. After gaining an understanding of the toxicity profile of 6·4Cl, we employed it in live-cell imaging. Confocal microscopy has demonstrated that 6 4+ is taken up by the RAW 264.7 macrophages, allowing the cells to glow brightly with blue laser excitation, without any hint of photobleaching or disruption of normal cell behavior under the imaging conditions. By contrast, the acyclic reference compound Me 2 TzBIPY·2Cl (4·2Cl) shows very little fluorescence inside the cells, which is quenched completely under the same imaging conditions. In vitro cell investigations underscore the significance of using highly fluorescent box-like rigid cyclophanes for live-cell imaging.

Culture of adult transgenic zebrafish retinal explants for live-cell imaging by multiphoton microscopy

PubMed Central

Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

2017-01-01

An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina i.e. they migrate between the basal inner nuclear layer (INL) and the outer nuclear layer (ONL), respectively, in a process described as interkinetic nuclear migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM. PMID:28287581

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy.

PubMed

Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

2017-02-24

An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP] mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments.

PubMed

Giarmarco, Michelle M; Cleghorn, Whitney M; Hurley, James B; Brockerhoff, Susan E

2018-05-09

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method's versatility. This protocol was developed for imaging Ca 2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca 2+ or metabolites in Müller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca 2+ , and energy homeostasis.

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

PubMed Central

Lee, Aih Cheun; Decourt, Boris; Suter, Daniel

2008-01-01

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones. PMID:19066568

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

PubMed Central

Gaylo, Alison; Overstreet, Michael G.; Fowell, Deborah J.

2016-01-01

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections. PMID:27078264

Advances in single-cell experimental design made possible by automated imaging platforms with feedback through segmentation.

PubMed

Crick, Alex J; Cammarota, Eugenia; Moulang, Katie; Kotar, Jurij; Cicuta, Pietro

2015-01-01

Live optical microscopy has become an essential tool for studying the dynamical behaviors and variability of single cells, and cell-cell interactions. However, experiments and data analysis in this area are often extremely labor intensive, and it has often not been achievable or practical to perform properly standardized experiments on a statistically viable scale. We have addressed this challenge by developing automated live imaging platforms, to help standardize experiments, increasing throughput, and unlocking previously impossible ones. Our real-time cell tracking programs communicate in feedback with microscope and camera control software, and they are highly customizable, flexible, and efficient. As examples of our current research which utilize these automated platforms, we describe two quite different applications: egress-invasion interactions of malaria parasites and red blood cells, and imaging of immune cells which possess high motility and internal dynamics. The automated imaging platforms are able to track a large number of motile cells simultaneously, over hours or even days at a time, greatly increasing data throughput and opening up new experimental possibilities. Copyright © 2015 Elsevier Inc. All rights reserved.

In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow

PubMed Central

Lo Celso, Cristina; Lin, Charles P; Scadden, David T

2011-01-01

In vivo imaging of transplanted hematopoietic stem and progenitor cells (HSPCs) was developed to investigate the relationship between HSPCs and components of their microenvironment in the bone marrow. In particular, it allows a direct observation of the behavior of hematopoietic cells during the first few days after transplantation, when the critical events in homing and early engraftment are occurring. By directly imaging these events in living animals, this method permits a detailed assessment of functions previously evaluated by crude assessments of cell counts (homing) or after prolonged periods (engraftment). This protocol offers a new means of investigating the role of cell-intrinsic and cell-extrinsic molecular regulators of hematopoiesis during the early stages of transplantation, and it is the first to allow the study of cell-cell interactions within the bone marrow in three dimensions and in real time. In this paper, we describe how to isolate, label and inject HSPCs, as well as how to perform calvarium intravital microscopy and analyze the resulting images. A typical experiment can be performed and analyzed in ~1 week. PMID:21212779

Thin-film optoacoustic transducers for subcellular Brillouin oscillation imaging of individual biological cells.

PubMed

Pérez-Cota, Fernando; Smith, Richard J; Moradi, Emilia; Marques, Leonel; Webb, Kevin F; Clark, Matt

2015-10-01

At low frequencies ultrasound is a valuable tool to mechanically characterize and image biological tissues. There is much interest in using high-frequency ultrasound to investigate single cells. Mechanical characterization of vegetal and biological cells by measurement of Brillouin oscillations has been demonstrated using ultrasound in the GHz range. This paper presents a method to extend this technique from the previously reported single-point measurements and line scans into a high-resolution acoustic imaging tool. Our technique uses a three-layered metal-dielectric-metal film as a transducer to launch acoustic waves into the cell we want to study. The design of this transducer and measuring system is optimized to overcome the vulnerability of a cell to the exposure of laser light and heat without sacrificing the signal-to-noise ratio. The transducer substrate shields the cell from the laser radiation, efficiently generates acoustic waves, facilitates optical detection in transmission, and aids with heat dissipation away from the cell. This paper discusses the design of the transducers and instrumentation and presents Brillouin frequency images on phantom, fixed, and living cells.

Aptamer-conjugated Magnetic Nanoparticles as Targeted Magnetic Resonance Imaging Contrast Agent for Breast Cancer.

PubMed

Keshtkar, Mohammad; Shahbazi-Gahrouei, Daryoush; Khoshfetrat, Seyyed Mehdi; Mehrgardi, Masoud A; Aghaei, Mahmoud

2016-01-01

Early detection of breast cancer is the most effective way to improve the survival rate in women. Magnetic resonance imaging (MRI) offers high spatial resolution and good anatomic details, and its lower sensitivity can be improved by using targeted molecular imaging. In this study, AS1411 aptamer was conjugated to Fe 3 O 4 @Au nanoparticles for specific targeting of mouse mammary carcinoma (4T1) cells that overexpress nucleolin. In vitro cytotoxicity of aptamer-conjugated nanoparticles was assessed on 4T1 and HFFF-PI6 (control) cells. The ability of the synthesized nanoprobe to target specifically the nucleolin overexpressed cells was assessed with the MRI technique. Results show that the synthesized nanoprobe produced strongly darkened T 2 -weighted magnetic resonance (MR) images with 4T1 cells, whereas the MR images of HFFF-PI6 cells incubated with the nanoprobe are brighter, showing small changes compared to water. The results demonstrate that in a Fe concentration of 45 μg/mL, the nanoprobe reduced by 90% MR image intensity in 4T1 cells compared with the 27% reduction in HFFF-PI6 cells. Analysis of MR signal intensity showed statistically significant signal intensity difference between 4T1 and HFFF-PI6 cells treated with the nanoprobe. MRI experiments demonstrate the high potential of the synthesized nanoprobe as a specific MRI contrast agent for detection of nucleolin-expressing breast cancer cells.

Hyperspectral imaging for simultaneous measurements of two FRET biosensors in pancreatic β-cells.

PubMed

Elliott, Amicia D; Bedard, Noah; Ustione, Alessandro; Baird, Michelle A; Davidson, Michael W; Tkaczyk, Tomasz; Piston, David W

2017-01-01

Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges. We have previously demonstrated the IMS's capabilities for simultaneously imaging GFP and CFP/YFP-based biosensors in pancreatic β-cells. Here, we demonstrate a further capability of the IMS to image simultaneously two FRET biosensors with a single excitation band, one for cAMP and the other for Caspase-3. We use these measurements to measure simultaneously cAMP signaling and Caspase-3 activation in pancreatic β-cells during oxidative stress and hyperglycemia, which are essential components in the pathology of diabetes.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction

PubMed Central

Parashurama, Natesh; Ahn, Byeong-Cheol; Ziv, Keren; Ito, Ken; Paulmurugan, Ramasamy; Willmann, Jürgen K.; Chung, Jaehoon; Ikeno, Fumiaki; Swanson, Julia C.; Merk, Denis R.; Lyons, Jennifer K.; Yerushalmi, David; Teramoto, Tomohiko; Kosuge, Hisanori; Dao, Catherine N.; Ray, Pritha; Patel, Manishkumar; Chang, Ya-fang; Mahmoudi, Morteza; Cohen, Jeff Eric; Goldstone, Andrew Brooks; Habte, Frezghi; Bhaumik, Srabani; Yaghoubi, Shahriar; Robbins, Robert C.; Dash, Rajesh; Yang, Phillip C.; Brinton, Todd J.; Yock, Paul G.; McConnell, Michael V.

2016-01-01

Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow–derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. © RSNA, 2016 Online supplemental material is available for this article. PMID:27308957

Redox-responsive manganese dioxide nanoparticles for enhanced MR imaging and radiotherapy of lung cancer

NASA Astrophysics Data System (ADS)

Cho, Mi Hyeon; Choi, Eun-Seok; Kim, Sehee; Goh, Sung-Ho; Choi, Yongdoo

2017-12-01

In this study, we synthesized manganese dioxide nanoparticles (MnO2 NPs) stabilized with biocompatible polymers (polyvinylpyrrolidone and polyacrylic acid) and analyzed their effect on non-small cell lung cancer (NSCLC) cells with or without gefitinib resistance in vitro. MnO2 NPs showed glutathione (GSH)-responsive dissolution and subsequent enhancement in magnetic resonance (MR) imaging. Of note, treatment with MnO2 NPs induced significant cytotoxic effects on NSCLC cells, and additional dose-dependent therapeutic effects were obtained upon X-ray irradiation. Normal cells treated with MnO2 NPs were viable at the tested concentrations. In addition, increased therapeutic efficacy could be achieved when the cells were treated with MnO2 NPs in hypoxic conditions. Therefore, we conclude that the use of MnO2 NPs in MR imaging and combination radiotherapy may be an efficient strategy for the imaging and therapy of NSCLC.

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.

Functional imaging of the brain

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

Ell, P.J.; Jarritt, P.H.; Costa, D.C.

1987-07-01

The radionuclide tracer method is unique among all other imaging methodologies in its ability to trace organ or tissue function and metabolism. Physical processes such as electron or proton density assessment or resonance, edge identification, electrical or ultrasonic impedence, do not pertain to the image generation process in nuclear medicine, and if so, only in a rather secondary manner. The nuclear medicine imaging study is primarily a study of the chemical nature, distribution and interaction of the tracer/radiopharmaceutical utilized with the cellular system which requires investigation: the thyroid cells with sodium iodide, the recticular endothelial cells with colloidal particles, themore » adrenal medulla cells with metaiodobenzylguanidine, and so on. In the two most recent areas of nuclear medicine expansion, oncology (with labelled monoclonal antibodies) and neurology and psychiatry (with a whole new series of lipid soluble radiopharmaceuticals), specific cell systems can also be targeted and hence imaged and investigated. The study of structure as masterly performed by Virchow and all his successors over more than a century, is now definitely the prerogative of such imaging systems which excel with spatial and contrast resolution However the investigation of function and metabolism, has clearly passed from the laboratory animal protocol and experiment to the direct investigation in man, this being the achievement of the radionuclide tracer methodology. In this article, we review present interest and developments in that part of nuclear medicine activity which is aimed at the study of the neurological or psychiatric patient.« less

Study of the cell activity in three-dimensional cell culture by using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Arunngam, Pakajiraporn; Mahardika, Anggara; Hiroko, Matsuyoshi; Andriana, Bibin Bintang; Tabata, Yasuhiko; Sato, Hidetoshi

2018-02-01

The purpose of this study is to develop a estimation technique of local cell activity in cultured 3D cell aggregate with gelatin hydrogel microspheres by using Raman spectroscopy. It is an invaluable technique allowing real-time, nondestructive, and invasive measurement. Cells in body generally exist in 3D structure, which physiological cell-cell interaction enhances cell survival and biological functions. Although a 3D cell aggregate is a good model of the cells in living tissues, it was difficult to estimate their physiological conditions because there is no effective technique to make observation of intact cells in the 3D structure. In this study, cell aggregates were formed by MC3T-E1 (pre-osteoblast) cells and gelatin hydrogel microspheres. In appropriate condition MC3T-E1 cells can differentiate into osteoblast. We assume that the activity of the cell would be different according to the location in the aggregate because the cells near the surface of the aggregate have more access to oxygen and nutrient. Raman imaging technique was applied to measure 3D image of the aggregate. The concentration of the hydroxyapatite (HA) is generated by osteoblast was estimated with a strong band at 950-970 cm-1 which assigned to PO43- in HA. It reflects an activity of the specific site in the cell aggregate. The cell density in this specific site was analyzed by multivariate analysis of the 3D Raman image. Hence, the ratio between intensity and cell density in the site represents the cell activity.

Compact Wireless Microscope for In-Situ Time Course Study of Large Scale Cell Dynamics within an Incubator

NASA Astrophysics Data System (ADS)

Jin, Di; Wong, Dennis; Li, Junxiang; Luo, Zhang; Guo, Yiran; Liu, Bifeng; Wu, Qiong; Ho, Chih-Ming; Fei, Peng

2015-12-01

Imaging of live cells in a region of interest is essential to life science research. Unlike the traditional way that mounts CO2 incubator onto a bulky microscope for observation, here we propose a wireless microscope (termed w-SCOPE) that is based on the “microscope-in-incubator” concept and can be easily housed into a standard CO2 incubator for prolonged on-site observation of the cells. The w-SCOPE is capable of tunable magnification, remote control and wireless image transmission. At the same time, it is compact, measuring only ~10 cm in each dimension, and cost-effective. With the enhancement of compressive sensing computation, the acquired images can achieve a wide field of view (FOV) of ~113 mm2 as well as a cellular resolution of ~3 μm, which enables various forms of follow-up image-based cell analysis. We performed 12 hours time-lapse study on paclitaxel-treated MCF-7 and HEK293T cell lines using w-SCOPE. The analytic results, such as the calculated viability and therapeutic window, from our device were validated by standard cell detection assays and imaging-based cytometer. In addition to those end-point detection methods, w-SCOPE further uncovered the time course of the cell’s response to the drug treatment over the whole period of drug exposure.

Compact Wireless Microscope for In-Situ Time Course Study of Large Scale Cell Dynamics within an Incubator

PubMed Central

Jin, Di; Wong, Dennis; Li, Junxiang; Luo, Zhang; Guo, Yiran; Liu, Bifeng; Wu, Qiong; Ho, Chih-Ming; Fei, Peng

2015-01-01

Imaging of live cells in a region of interest is essential to life science research. Unlike the traditional way that mounts CO2 incubator onto a bulky microscope for observation, here we propose a wireless microscope (termed w-SCOPE) that is based on the “microscope-in-incubator” concept and can be easily housed into a standard CO2 incubator for prolonged on-site observation of the cells. The w-SCOPE is capable of tunable magnification, remote control and wireless image transmission. At the same time, it is compact, measuring only ~10 cm in each dimension, and cost-effective. With the enhancement of compressive sensing computation, the acquired images can achieve a wide field of view (FOV) of ~113 mm2 as well as a cellular resolution of ~3 μm, which enables various forms of follow-up image-based cell analysis. We performed 12 hours time-lapse study on paclitaxel-treated MCF-7 and HEK293T cell lines using w-SCOPE. The analytic results, such as the calculated viability and therapeutic window, from our device were validated by standard cell detection assays and imaging-based cytometer. In addition to those end-point detection methods, w-SCOPE further uncovered the time course of the cell’s response to the drug treatment over the whole period of drug exposure. PMID:26681552

Chimeric animal models in human stem cell biology.

PubMed

Glover, Joel C; Boulland, Jean-Luc; Halasi, Gabor; Kasumacic, Nedim

2009-01-01

The clinical use of stem cells for regenerative medicine is critically dependent on preclinical studies in animal models. In this review we examine some of the key issues and challenges in the use of animal models to study human stem cell biology-experimental standardization, body size, immunological barriers, cell survival factors, fusion of host and donor cells, and in vivo imaging and tracking. We focus particular attention on the various imaging modalities that can be used to track cells in living animals, comparing their strengths and weaknesses and describing technical developments that are likely to lead to new opportunities for the dynamic assessment of stem cell behavior in vivo. We then provide an overview of some of the most commonly used animal models, their advantages and disadvantages, and examples of their use for xenotypic transplantation of human stem cells, with separate reviews of models involving rodents, ungulates, nonhuman primates, and the chicken embryo. As the use of human somatic, embryonic, and induced pluripotent stem cells increases, so too will the range of applications for these animal models. It is likely that increasingly sophisticated uses of human/animal chimeric models will be developed through advances in genetic manipulation, cell delivery, and in vivo imaging.

Water-mediated green synthesis of PbS quantum dot and its glutathione and biotin conjugates for non-invasive live cell imaging

NASA Astrophysics Data System (ADS)

Vijaya Bharathi, M.; Maiti, Santanu; Sarkar, Bidisha; Ghosh, Kaustab; Paira, Priyankar

2018-03-01

This study addresses the cellular uptake of nanomaterials in the field of bio-applications. In the present study, we have synthesized water-soluble lead sulfide quantum dot (PbS QD) with glutathione and 3-MPA (mercaptopropionic acid) as the stabilizing ligand using a green approach. 3-MPA-capped QDs were further modified with streptavidin and then bound to biotin because of its high conjugation efficiency. Labelling and bio-imaging of cells with these bio-conjugated QDs were evaluated. The bright red fluorescence from these types of QDs in HeLa cells makes these materials suitable for deep tissue imaging.

Evanescent field microscopy techniques for studying dynamics at the surface of living cells

NASA Astrophysics Data System (ADS)

Sund, Susan E.

This thesis presents two distinct optical microscopy techniques for applications in cell biophysics: (a)the extension to living cells of an established technique, total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) for the first time in imaging mode; and (b)the novel development of polarized total internal reflection fluorescence (p- TIRF) to study membrane orientation in living cells. Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about the relevant chemical kinetic rates in vivo. TIR/FRAP, an established technique which can measure reversible biomolecular kinetic rates at surfaces, is extended here to measure kinetic parameters of microinjected rhodamine actin at the cytofacial surface of the plasma membrane of living cultured smooth muscle cells. For the first time, spatial imaging (with a CCD camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging allows production of spatially resolved images of kinetic data, and calculation of correlation distances, cell-wide gradients, and kinetic parameter dependence on initial fluorescence intensity. In living cells, membrane curvature occurs both in easily imaged large scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method, p-TIRF, is introduced here to visualize such regions. The method is based on fluorescence of the oriented membrane probe diI- C18-(3) (diI) excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane. A theoretical background of the technique and experimental verifications are presented in samples of protein solutions, model lipid bilayers, and living cells. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time- course maps of membrane orientations on diI labeled macrophages from which low visibility membrane structures can be identified and quantified. The TIR images are sharpened and contrast-enhanced by deconvoluting them with an experimentally-measured point spread function.

Development of a stained cell nuclei counting system

NASA Astrophysics Data System (ADS)

Timilsina, Niranjan; Moffatt, Christopher; Okada, Kazunori

2011-03-01

This paper presents a novel cell counting system which exploits the Fast Radial Symmetry Transformation (FRST) algorithm [1]. The driving force behind our system is a research on neurogenesis in the intact nervous system of Manduca Sexta or the Tobacco Hornworm, which was being studied to assess the impact of age, food and environment on neurogenesis. The varying thickness of the intact nervous system in this species often yields images with inhomogeneous background and inconsistencies such as varying illumination, variable contrast, and irregular cell size. For automated counting, such inhomogeneity and inconsistencies must be addressed, which no existing work has done successfully. Thus, our goal is to devise a new cell counting algorithm for the images with non-uniform background. Our solution adapts FRST: a computer vision algorithm which is designed to detect points of interest on circular regions such as human eyes. This algorithm enhances the occurrences of the stained-cell nuclei in 2D digital images and negates the problems caused by their inhomogeneity. Besides FRST, our algorithm employs standard image processing methods, such as mathematical morphology and connected component analysis. We have evaluated the developed cell counting system with fourteen digital images of Tobacco Hornworm's nervous system collected for this study with ground-truth cell counts by biology experts. Experimental results show that our system has a minimum error of 1.41% and mean error of 16.68% which is at least forty-four percent better than the algorithm without FRST.

Longitudinal confocal microscopy imaging of solid tumor destruction following adoptive T cell transfer

PubMed Central

Schietinger, Andrea; Arina, Ainhoa; Liu, Rebecca B; Wells, Sam; Huang, Jianhua; Engels, Boris; Bindokas, Vytas; Bartkowiak, Todd; Lee, David; Herrmann, Andreas; Piston, David W; Pittet, Mikael J; Lin, P Charles; Zal, Tomasz; Schreiber, Hans

2013-01-01

A fluorescence-based, high-resolution imaging approach was used to visualize longitudinally the cellular events unfolding during T cell-mediated tumor destruction. The dynamic interplay of T cells, cancer cells, cancer antigen loss variants, and stromal cells—all color-coded in vivo—was analyzed in established, solid tumors that had developed behind windows implanted on the backs of mice. Events could be followed repeatedly within precisely the same tumor region—before, during and after adoptive T cell therapy—thereby enabling for the first time a longitudinal in vivo evaluation of protracted events, an analysis not possible with terminal imaging of surgically exposed tumors. T cell infiltration, stromal interactions, and vessel destruction, as well as the functional consequences thereof, including the elimination of cancer cells and cancer cell variants were studied. Minimal perivascular T cell infiltrates initiated vascular destruction inside the tumor mass eventually leading to macroscopic central tumor necrosis. Prolonged engagement of T cells with tumor antigen-crosspresenting stromal cells correlated with high IFNγ cytokine release and bystander elimination of antigen-negative cancer cells. The high-resolution, longitudinal, in vivo imaging approach described here will help to further a better mechanistic understanding of tumor eradication by T cells and other anti-cancer therapies. PMID:24482750

Genetic Recombination Between Stromal and Cancer Cells Results in Highly Malignant Cells Identified by Color-Coded Imaging in a Mouse Lymphoma Model.

PubMed

Nakamura, Miki; Suetsugu, Atsushi; Hasegawa, Kousuke; Matsumoto, Takuro; Aoki, Hitomi; Kunisada, Takahiro; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Hoffman, Robert M

2017-12-01

The tumor microenvironment (TME) promotes tumor growth and metastasis. We previously established the color-coded EL4 lymphoma TME model with red fluorescent protein (RFP) expressing EL4 implanted in transgenic C57BL/6 green fluorescent protein (GFP) mice. Color-coded imaging of the lymphoma TME suggested an important role of stromal cells in lymphoma progression and metastasis. In the present study, we used color-coded imaging of RFP-lymphoma cells and GFP stromal cells to identify yellow-fluorescent genetically recombinant cells appearing only during metastasis. The EL4-RFP lymphoma cells were injected subcutaneously in C57BL/6-GFP transgenic mice and formed subcutaneous tumors 14 days after cell transplantation. The subcutaneous tumors were harvested and transplanted to the abdominal cavity of nude mice. Metastases to the liver, perigastric lymph node, ascites, bone marrow, and primary tumor were imaged. In addition to EL4-RFP cells and GFP-host cells, genetically recombinant yellow-fluorescent cells, were observed only in the ascites and bone marrow. These results indicate genetic exchange between the stromal and cancer cells. Possible mechanisms of genetic exchange are discussed as well as its ramifications for metastasis. J. Cell. Biochem. 118: 4216-4221, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

DALMATIAN: An Algorithm for Automatic Cell Detection and Counting in 3D.

PubMed

Shuvaev, Sergey A; Lazutkin, Alexander A; Kedrov, Alexander V; Anokhin, Konstantin V; Enikolopov, Grigori N; Koulakov, Alexei A

2017-01-01

Current 3D imaging methods, including optical projection tomography, light-sheet microscopy, block-face imaging, and serial two photon tomography enable visualization of large samples of biological tissue. Large volumes of data obtained at high resolution require development of automatic image processing techniques, such as algorithms for automatic cell detection or, more generally, point-like object detection. Current approaches to automated cell detection suffer from difficulties originating from detection of particular cell types, cell populations of different brightness, non-uniformly stained, and overlapping cells. In this study, we present a set of algorithms for robust automatic cell detection in 3D. Our algorithms are suitable for, but not limited to, whole brain regions and individual brain sections. We used watershed procedure to split regional maxima representing overlapping cells. We developed a bootstrap Gaussian fit procedure to evaluate the statistical significance of detected cells. We compared cell detection quality of our algorithm and other software using 42 samples, representing 6 staining and imaging techniques. The results provided by our algorithm matched manual expert quantification with signal-to-noise dependent confidence, including samples with cells of different brightness, non-uniformly stained, and overlapping cells for whole brain regions and individual tissue sections. Our algorithm provided the best cell detection quality among tested free and commercial software.

Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy.

PubMed

Wang, Quanli; Niemi, Jarad; Tan, Chee-Meng; You, Lingchong; West, Mike

2010-01-01

An increasingly common component of studies in synthetic and systems biology is analysis of dynamics of gene expression at the single-cell level, a context that is heavily dependent on the use of time-lapse movies. Extracting quantitative data on the single-cell temporal dynamics from such movies remains a major challenge. Here, we describe novel methods for automating key steps in the analysis of single-cell, fluorescent images-segmentation and lineage reconstruction-to recognize and track individual cells over time. The automated analysis iteratively combines a set of extended morphological methods for segmentation, and uses a neighborhood-based scoring method for frame-to-frame lineage linking. Our studies with bacteria, budding yeast and human cells, demonstrate the portability and usability of these methods, whether using phase, bright field or fluorescent images. These examples also demonstrate the utility of our integrated approach in facilitating analyses of engineered and natural cellular networks in diverse settings. The automated methods are implemented in freely available, open-source software.

AuNP-DG: deoxyglucose-labeled gold nanoparticles as X-ray computed tomography contrast agents for cancer imaging.

PubMed

Aydogan, Bulent; Li, Ji; Rajh, Tijana; Chaudhary, Ahmed; Chmura, Steven J; Pelizzari, Charles; Wietholt, Christian; Kurtoglu, Metin; Redmond, Peter

2010-10-01

To study the feasibility of using 2-deoxy-D-glucose (2-DG)-labeled gold nanoparticle (AuNP-DG) as a computed tomography (CT) contrast agent with tumor targeting capability through in vitro experiments. Gold nanoparticles (AuNP) were fabricated and were conjugated with 2-deoxy-D-glucose. The human alveolar epithelial cancer cell line, A-549, was chosen for the in vitro cellular uptake assay. Two groups of cell samples were incubated with the AuNP-DG and the unlabeled AuNP, respectively. Following the incubation, the cells were washed with sterile PBS to remove the excess gold nanoparticles and spun to cell pellets using a centrifuge. The cell pellets were imaged using a microCT scanner immediately after the centrifugation. The reconstructed CT images were analyzed using a commercial software package. Significant contrast enhancement in the cell samples incubated with the AuNP-DG with respect to the cell samples incubated with the unlabeled AuNP was observed in multiple CT slices. Results from this study demonstrate enhanced uptake of 2-DG-labeled gold nanoparticle by cancer cells in vitro and warrant further experiments to study the exact molecular mechanism by which the AuNP-DG is internalized and retained in the tumor cells.

Noncontact Viscoelastic Imaging of Living Cells Using a Long-Needle Atomic Force Microscope with Dual-Frequency Modulation

NASA Astrophysics Data System (ADS)

Guan, Dongshi; Charlaix, Elisabeth; Qi, Robert Z.; Tong, Penger

2017-10-01

Imaging of surface topography and elasticity of living cells can provide insight into the roles played by the cells' volumetric and mechanical properties and their response to external forces in regulating the essential cellular events and functions. Here, we report a unique technique of noncontact viscoelastic imaging of live cells using atomic force microscopy (AFM) with a long-needle glass probe. Because only the probe tip is placed in a liquid medium near the cell surface, the AFM cantilever in air functions well under dual-frequency modulation, retaining its high-quality resonant modes. The probe tip interacts with the cell surface through a minute hydrodynamic flow in the nanometer-thin gap region between them without physical contact. Quantitative measurements of the cell height, volume, and Young's modulus are conducted simultaneously. The experiment demonstrates that the long-needle AFM has a wide range of applications in the study of cell mechanics.

CellTracker (not only) for dummies.

PubMed

Piccinini, Filippo; Kiss, Alexa; Horvath, Peter

2016-03-15

Time-lapse experiments play a key role in studying the dynamic behavior of cells. Single-cell tracking is one of the fundamental tools for such analyses. The vast majority of the recently introduced cell tracking methods are limited to fluorescently labeled cells. An equally important limitation is that most software cannot be effectively used by biologists without reasonable expertise in image processing. Here we present CellTracker, a user-friendly open-source software tool for tracking cells imaged with various imaging modalities, including fluorescent, phase contrast and differential interference contrast (DIC) techniques. CellTracker is written in MATLAB (The MathWorks, Inc., USA). It works with Windows, Macintosh and UNIX-based systems. Source code and graphical user interface (GUI) are freely available at: http://celltracker.website/ horvath.peter@brc.mta.hu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Segmentation and classification of cell cycle phases in fluorescence imaging.

PubMed

Ersoy, Ilker; Bunyak, Filiz; Chagin, Vadim; Cardoso, M Christina; Palaniappan, Kannappan

2009-01-01

Current chemical biology methods for studying spatiotemporal correlation between biochemical networks and cell cycle phase progression in live-cells typically use fluorescence-based imaging of fusion proteins. Stable cell lines expressing fluorescently tagged protein GFP-PCNA produce rich, dynamically varying sub-cellular foci patterns characterizing the cell cycle phases, including the progress during the S-phase. Variable fluorescence patterns, drastic changes in SNR, shape and position changes and abundance of touching cells require sophisticated algorithms for reliable automatic segmentation and cell cycle classification. We extend the recently proposed graph partitioning active contours (GPAC) for fluorescence-based nucleus segmentation using regional density functions and dramatically improve its efficiency, making it scalable for high content microscopy imaging. We utilize surface shape properties of GFP-PCNA intensity field to obtain descriptors of foci patterns and perform automated cell cycle phase classification, and give quantitative performance by comparing our results to manually labeled data.

Bright fluorescent Streptococcus pneumoniae for live-cell imaging of host-pathogen interactions.

PubMed

Kjos, Morten; Aprianto, Rieza; Fernandes, Vitor E; Andrew, Peter W; van Strijp, Jos A G; Nijland, Reindert; Veening, Jan-Willem

2015-03-01

Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people but, at the same time, one of the major causes of infectious diseases such as pneumonia, meningitis, and sepsis. The shift from commensal to pathogen and its interaction with host cells are poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live-cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than those of the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent, and the GFP reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live-cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells than encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live-cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live-cell imaging of pneumococcal infection and help further understanding of the mechanisms of pneumococcal pathogenesis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Narrow band imaging versus autofluorescence imaging for head and neck squamous cell carcinoma detection: a prospective study.

PubMed

Ni, X-G; Zhang, Q-Q; Wang, G-Q

2016-11-01

This study aimed to compare the diagnostic effectiveness of narrow band imaging and autofluorescence imaging for malignant laryngopharyngeal tumours. Between May 2010 and October 2010, 50 consecutive patients with suspected laryngopharyngeal tumour underwent endoscopic laryngopharynx examination. The morphological characteristics of laryngopharyngeal lesions were analysed using high performance endoscopic systems equipped with narrow band imaging and autofluorescence imaging modes. The diagnostic effectiveness of white light image, narrow band imaging and autofluorescence imaging endoscopy for benign and malignant laryngopharyngeal lesions was evaluated. Under narrow band imaging endoscopy, the superficial microvessels of squamous cell carcinomas appeared as dark brown spots or twisted cords. Under autofluorescence imaging endoscopy, malignant lesions appeared as bright purple. The sensitivity of malignant lesion diagnosis was not significantly different between narrow band imaging and autofluorescence imaging modes, but was better than for white light image endoscopy (χ2 = 12.676, p = 0.002). The diagnostic specificity was significantly better in narrow band imaging mode than in both autofluorescence imaging and white light imaging mode (χ2 = 8.333, p = 0.016). Narrow band imaging endoscopy is the best option for the diagnosis and differential diagnosis of laryngopharyngeal tumours.

Imaging Cell Shape Change in Living Drosophila Embryos

PubMed Central

Figard, Lauren; Sokac, Anna Marie

2011-01-01

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells. The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle. Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope. PMID:21490577

Where in the Cell Are You? Probing HIV-1 Host Interactions through Advanced Imaging Techniques

PubMed Central

Dirk, Brennan S.; Van Nynatten, Logan R.; Dikeakos, Jimmy D.

2016-01-01

Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence. The type-1 human immunodeficiency virus (HIV-1) is a prime example of viral persistence within the host, having plagued the human population for decades. In recent years, advances in cellular imaging and molecular biology have aided the elucidation of key steps mediating the HIV-1 lifecycle and viral pathogenesis. Super-resolution imaging techniques such as stimulated emission depletion (STED) and photoactivation and localization microscopy (PALM) have been instrumental in studying viral assembly and release through both cell–cell transmission and cell–free viral transmission. Moreover, powerful methods such as Forster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) have shed light on the protein-protein interactions HIV-1 engages within the host to hijack the cellular machinery. Specific advancements in live cell imaging in combination with the use of multicolor viral particles have become indispensable to unravelling the dynamic nature of these virus-host interactions. In the current review, we outline novel imaging methods that have been used to study the HIV-1 lifecycle and highlight advancements in the cell culture models developed to enhance our understanding of the HIV-1 lifecycle. PMID:27775563

Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy.

PubMed

Rappaz, Benjamin; Cano, Elena; Colomb, Tristan; Kühn, Jonas; Depeursinge, Christian; Simanis, Viesturs; Magistretti, Pierre J; Marquet, Pierre

2009-01-01

Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.

Classification of Acute Myelogenous Leukemia (AML M2 and AML M3) using Momentum Back Propagation from Watershed Distance Transform Segmented Images

NASA Astrophysics Data System (ADS)

Suryani, Esti; Wiharto; Palgunadi, Sarngadi; Nurcahya Pradana, TP

2017-01-01

This study uses image processing to analyze white blood cell with leukemia indicated that includes the identification, analysis of shapes and sizes, as well as white blood cell count indicated the symptoms of leukemia. A case study in this research was blood cells, from the type of leukemia Acute Myelogenous Leukemia (AML), M2 and M3 in particular. Image processing operations used for segmentation by utilizing the color conversion from RGB (Red, Green dab Blue) to obtain white blood cell candidates. Furthermore, the white blood cells candidates are separated by other cells with active contour without edge. WBC (White Blood Cell) results still have intersected or overlap condition. Watershed distance transform method can separate overlap of WBC. Furthermore, the separation of the nucleus from the cytoplasm using the HSI (Hue Saturation Intensity). The further characteristic extraction process is done by calculating the area WBC, WBC edge, roundness, the ratio of the nucleus, the mean and standard deviation of pixel intensities. The feature extraction results are used for training and testing in determining the classification of AML: M2 and M3 by using the momentum backpropagation algorithm. The classification process is done by testing the numeric data input from the feature extraction results that have been entered in the database. K-Fold validation is used to divide the amount of training data and to test the classification of AML M2 and M3. The experiment results of eight images trials, the result, was 94.285% per cell accuracy and 75% per image accuracy

Computerized microscopic image analysis of follicular lymphoma

NASA Astrophysics Data System (ADS)

Sertel, Olcay; Kong, Jun; Lozanski, Gerard; Catalyurek, Umit; Saltz, Joel H.; Gurcan, Metin N.

2008-03-01

Follicular Lymphoma (FL) is a cancer arising from the lymphatic system. Originating from follicle center B cells, FL is mainly comprised of centrocytes (usually middle-to-small sized cells) and centroblasts (relatively large malignant cells). According to the World Health Organization's recommendations, there are three histological grades of FL characterized by the number of centroblasts per high-power field (hpf) of area 0.159 mm2. In current practice, these cells are manually counted from ten representative fields of follicles after visual examination of hematoxylin and eosin (H&E) stained slides by pathologists. Several studies clearly demonstrate the poor reproducibility of this grading system with very low inter-reader agreement. In this study, we are developing a computerized system to assist pathologists with this process. A hybrid approach that combines information from several slides with different stains has been developed. Thus, follicles are first detected from digitized microscopy images with immunohistochemistry (IHC) stains, (i.e., CD10 and CD20). The average sensitivity and specificity of the follicle detection tested on 30 images at 2×, 4× and 8× magnifications are 85.5+/-9.8% and 92.5+/-4.0%, respectively. Since the centroblasts detection is carried out in the H&E-stained slides, the follicles in the IHC-stained images are mapped to H&E-stained counterparts. To evaluate the centroblast differentiation capabilities of the system, 11 hpf images have been marked by an experienced pathologist who identified 41 centroblast cells and 53 non-centroblast cells. A non-supervised clustering process differentiates the centroblast cells from noncentroblast cells, resulting in 92.68% sensitivity and 90.57% specificity.

Multiphoton fluorescence lifetime imaging of chemotherapy distribution in solid tumors

NASA Astrophysics Data System (ADS)

Carlson, Marjorie; Watson, Adrienne L.; Anderson, Leah; Largaespada, David A.; Provenzano, Paolo P.

2017-11-01

Doxorubicin is a commonly used chemotherapeutic employed to treat multiple human cancers, including numerous sarcomas and carcinomas. Furthermore, doxorubicin possesses strong fluorescent properties that make it an ideal reagent for modeling drug delivery by examining its distribution in cells and tissues. However, while doxorubicin fluorescence and lifetime have been imaged in live tissue, its behavior in archival samples that frequently result from drug and treatment studies in human and animal patients, and murine models of human cancer, has to date been largely unexplored. Here, we demonstrate imaging of doxorubicin intensity and lifetimes in archival formalin-fixed paraffin-embedded sections from mouse models of human cancer with multiphoton excitation and multiphoton fluorescence lifetime imaging microscopy (FLIM). Multiphoton excitation imaging reveals robust doxorubicin emission in tissue sections and captures spatial heterogeneity in cells and tissues. However, quantifying the amount of doxorubicin signal in distinct cell compartments, particularly the nucleus, often remains challenging due to strong signals in multiple compartments. The addition of FLIM analysis to display the spatial distribution of excited state lifetimes clearly distinguishes between signals in distinct compartments such as the cell nuclei versus cytoplasm and allows for quantification of doxorubicin signal in each compartment. Furthermore, we observed a shift in lifetime values in the nuclei of transformed cells versus nontransformed cells, suggesting a possible diagnostic role for doxorubicin lifetime imaging to distinguish normal versus transformed cells. Thus, data here demonstrate that multiphoton FLIM is a highly sensitive platform for imaging doxorubicin distribution in normal and diseased archival tissues.

A study on the cytotoxicity of carbon-based materials

DOE PAGES

Saha, Dipendu; Heldt, Caryn L.; Gencoglu, Maria F.; ...

2016-05-25

With an aim to understand the origin and key contributing factors towards carboninduced cytotoxicity, we have studied five different carbon samples with diverse surface area, pore width, shape and size, conductivity and surface functionality. All the carbon materials were characterized with surface area and pore size distribution, x-ray photoelectron spectroscopy (XPS) and electron microscopic imaging. We performed cytotoxicity study in Caco-2 cells by colorimetric assay, oxidative stress analysis by reactive oxygen species (ROX) detection, cellular metabolic activity measurement by adenosine triphosphate (ATP) depletion and visualization of cellular internalization by TEM imaging. The carbon materials demonstrated a varying degree of cytotoxicitymore » in contact with Caco-2 cells. The lowest cell survival rate was observed for nanographene, which possessed the minimal size amongst all the carbon samples under study. None of the carbons induced oxidative stress to the cells as indicated by the ROX generation results. Cellular metabolic activity study revealed that the carbon materials caused ATP depletion in cells and nanographene caused the highest depletion. Visual observation by TEM imaging indicated the cellular internalization of nanographene. This study confirmed that the size is the key cause of carbon-induced cytotoxicity and it is probably caused by the ATP depletion within the cell.« less

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.

In Vivo Multiphoton Microscopy for Investigating Biomechanical Properties of Human Skin.

PubMed

Liang, Xing; Graf, Benedikt W; Boppart, Stephen A

2011-06-01

The biomechanical properties of living cells depend on their molecular building blocks, and are important for maintaining structure and function in cells, the extracellular matrix, and tissues. These biomechanical properties and forces also shape and modify the cellular and extracellular structures under stress. While many studies have investigated the biomechanics of single cells or small populations of cells in culture, or the properties of organs and tissues, few studies have investigated the biomechanics of complex cell populations in vivo. With the use of advanced multiphoton microscopy to visualize in vivo cell populations in human skin, the biomechanical properties are investigated in a depth-dependent manner in the stratum corneum and epidermis using quasi-static mechanical deformations. A 2D elastic registration algorithm was used to analyze the images before and after deformation to determine displacements in different skin layers. In this feasibility study, the images and results from one human subject demonstrate the potential of the technique for revealing differences in elastic properties between the stratum corneum and the rest of the epidermis. This interrogational imaging methodology has the potential to enable a wide range of investigations for understanding how the biomechanical properties of in vivo cell populations influence function in health and disease.

Microscopic optical path length difference and polarization measurement system for cell analysis

NASA Astrophysics Data System (ADS)

Satake, H.; Ikeda, K.; Kowa, H.; Hoshiba, T.; Watanabe, E.

2018-03-01

In recent years, noninvasive, nonstaining, and nondestructive quantitative cell measurement techniques have become increasingly important in the medical field. These cell measurement techniques enable the quantitative analysis of living cells, and are therefore applied to various cell identification processes, such as those determining the passage number limit during cell culturing in regenerative medicine. To enable cell measurement, we developed a quantitative microscopic phase imaging system based on a Mach-Zehnder interferometer that measures the optical path length difference distribution without phase unwrapping using optical phase locking. The applicability of our phase imaging system was demonstrated by successful identification of breast cancer cells amongst normal cells. However, the cell identification method using this phase imaging system exhibited a false identification rate of approximately 7%. In this study, we implemented a polarimetric imaging system by introducing a polarimetric module to one arm of the Mach-Zehnder interferometer of our conventional phase imaging system. This module was comprised of a quarter wave plate and a rotational polarizer on the illumination side of the sample, and a linear polarizer on the optical detector side. In addition, we developed correction methods for the measurement errors of the optical path length and birefringence phase differences that arose through the influence of elements other than cells, such as the Petri dish. As the Petri dish holding the fluid specimens was transparent, it did not affect the amplitude information; however, the optical path length and birefringence phase differences were affected. Therefore, we proposed correction of the optical path length and birefringence phase for the influence of elements other than cells, as a prerequisite for obtaining highly precise phase and polarimetric images.

Quantitative image analysis for investigating cell-matrix interactions

NASA Astrophysics Data System (ADS)

Burkel, Brian; Notbohm, Jacob

2017-07-01

The extracellular matrix provides both chemical and physical cues that control cellular processes such as migration, division, differentiation, and cancer progression. Cells can mechanically alter the matrix by applying forces that result in matrix displacements, which in turn may localize to form dense bands along which cells may migrate. To quantify the displacements, we use confocal microscopy and fluorescent labeling to acquire high-contrast images of the fibrous material. Using a technique for quantitative image analysis called digital volume correlation, we then compute the matrix displacements. Our experimental technology offers a means to quantify matrix mechanics and cell-matrix interactions. We are now using these experimental tools to modulate mechanical properties of the matrix to study cell contraction and migration.

Genetic modification of embryonic stem cells with VEGF enhances cell survival and improves cardiac function.

PubMed

Xie, Xiaoyan; Cao, Feng; Sheikh, Ahmad Y; Li, Zongjin; Connolly, Andrew J; Pei, Xuetao; Li, Ren-Ke; Robbins, Robert C; Wu, Joseph C

2007-01-01

Cardiac stem cell therapy remains hampered by acute donor cell death posttransplantation and the lack of reliable methods for tracking cell survival in vivo. We hypothesize that cells transfected with inducible vascular endothelial growth factor 165 (VEGF(165)) can improve their survival as monitored by novel molecular imaging techniques. Mouse embryonic stem (ES) cells were transfected with an inducible, bidirectional tetracycline (Bi-Tet) promoter driving VEGF(165) and renilla luciferase (Rluc). Addition of doxycycline induced Bi-Tet expression of VEGF(165) and Rluc significantly compared to baseline (p<0.05). Expression of VEGF(165) enhanced ES cell proliferation and inhibited apoptosis as determined by Annexin-V staining. For noninvasive imaging, ES cells were transduced with a double fusion (DF) reporter gene consisting of firefly luciferase and enhanced green fluorescence protein (Fluc-eGFP). There was a robust correlation between cell number and Fluc activity (R(2)=0.99). Analysis by immunostaining, histology, and RT-PCR confirmed that expression of Bi-Tet and DF systems did not affect ES cell self-renewal or pluripotency. ES cells were differentiated into beating embryoid bodies expressing cardiac markers such as troponin, Nkx2.5, and beta-MHC. Afterward, 5 x 10(5) cells obtained from these beating embryoid bodies or saline were injected into the myocardium of SV129 mice (n=36) following ligation of the left anterior descending (LAD) artery. Bioluminescence imaging (BLI) and echocardiography showed that VEGF(165) induction led to significant improvements in both transplanted cell survival and cardiac function (p<0.05). This is the first study to demonstrate imaging of embryonic stem cell-mediated gene therapy targeting cardiovascular disease. With further validation, this platform may have broad applications for current basic research and further clinical studies.

Visualization of gene expression in the live subject using the Na/I symporter as a reporter gene: applications in biotherapy.

PubMed

Baril, Patrick; Martin-Duque, Pilar; Vassaux, Georges

2010-02-01

Biotherapies involve the utilization of antibodies, genetically modified viruses, bacteria or cells for therapeutic purposes. Molecular imaging has the potential to provide unique information that will guarantee their biosafety in humans and provide a rationale for the future development of new generations of reagents. In this context, non-invasive imaging of gene expression is an attractive prospect, allowing precise, spacio-temporal measurements of gene expression in longitudinal studies involving gene transfer vectors. With the emergence of cell therapies in regenerative medicine, it is also possible to track cells injected into subjects. In this context, the Na/I symporter (NIS) has been used in preclinical studies. Associated with a relevant radiotracer ((123)I(-), (124)I(-), (99m)TcO4(-)), NIS can be used to monitor gene transfer and the spread of selectively replicative viruses in tumours as well as in cells with a therapeutic potential. In addition to its imaging potential, NIS can be used as a therapeutic transgene through its ability to concentrate therapeutic doses of radionuclides in target cells. This dual property has applications in cancer treatment and could also be used to eradicate cells with therapeutic potential in the case of adverse events. Through experience acquired in preclinical studies, we can expect that non-invasive molecular imaging using NIS as a transgene will be pivotal for monitoring in vivo the exact distribution and pharmacodynamics of gene expression in a precise and quantitative way. This review highlights the applications of NIS in biotherapy, with a particular emphasis on image-guided radiotherapy, monitoring of gene and vector biodistribution and trafficking of stem cells.

Molecular Imaging of Vasa Vasorum Neovascularization via DEspR-targeted Contrast-enhanced Ultrasound Micro-imaging in Transgenic Atherosclerosis Rat Model

PubMed Central

Decano, Julius L.; Moran, Anne Marie; Ruiz-Opazo, Nelson; Herrera, Victoria L. M.

2011-01-01

Purpose Given that carotid vasa vasorum neovascularization is associated with increased risk for stroke and cardiac events, the present in vivo study was designed to investigate molecular imaging of carotid artery vasa vasorum neovascularization via target-specific contrast-enhanced ultrasound (CEU) micro-imaging. Procedures Molecular imaging was performed in male transgenic rats with carotid artery disease and non-transgenic controls using dual endothelin1/VEGFsp receptor (DEspR)-targeted microbubbles (MBD) and the Vevo770 micro-imaging system and CEU imaging software. Results DEspR-targeted CEU-positive imaging exhibited significantly higher contrast intensity signal (CIS)-levels and pre-/post-destruction CIS-differences in seven of 13 transgenic rats, in contrast to significantly lower CIS-levels and differences in control isotype-targeted microbubble (MBC)-CEU imaging (n =8) and in MBD CEU-imaging of five non-transgenic control rats (P<0.0001). Ex vivo immunofluorescence analysis demonstrated binding of MBD to DEspR-positive endothelial cells; and association of DEspR-targeted increased contrast intensity signals with DEspR expression in vasa vasorum neovessel and intimal lesions. In vitro analysis demonstrated dose-dependent binding of MBD to DEspR-positive human endothelial cells with increasing %cells bound and number of MBD per cell, in contrast to MBC or non-labeled microbubbles (P<0.0001). Conclusion In vivo DEspR-targeted molecular imaging detected increased DEspR-expression in carotid artery lesions and in expanded vasa vasorum neovessels in transgenic rats with carotid artery disease. Future studies are needed to determine predictive value for stroke or heart disease in this transgenic atherosclerosis rat model and translational applications. PMID:20972637

Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells.

PubMed

Akita, Kae; Higaki, Takumi; Kutsuna, Natsumaro; Hasezawa, Seiichiro

2015-01-01

Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells.

Far-field photostable optical nanoscopy (PHOTON) for real-time super-resolution single-molecular imaging of signaling pathways of single live cells

NASA Astrophysics Data System (ADS)

Huang, Tao; Browning, Lauren M.; Xu, Xiao-Hong Nancy

2012-04-01

Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions.Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr11739h

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

PubMed Central

Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

2014-01-01

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria. PMID:25358460

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

DOE PAGES

Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; ...

2014-10-31

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip windowmore » surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.« less

Clonal expansion under the microscope: studying lymphocyte activation and differentiation using live-cell imaging.

PubMed

Polonsky, Michal; Chain, Benjamin; Friedman, Nir

2016-03-01

Clonal expansion of lymphocytes is a hallmark of vertebrate adaptive immunity. A small number of precursor cells that recognize a specific antigen proliferate into expanded clones, differentiate and acquire various effector and memory phenotypes, which promote effective immune responses. Recent studies establish a large degree of heterogeneity in the level of expansion and in cell state between and within expanding clones. Studying these processes in vivo, while providing insightful information on the level of heterogeneity, is challenging due to the complex microenvironment and the inability to continuously track individual cells over extended periods of time. Live cell imaging of ex vivo cultures within micro fabricated arrays provides an attractive methodology for studying clonal expansion. These experiments facilitate continuous acquisition of a large number of parameters on cell number, proliferation, death and differentiation state, with single-cell resolution on thousands of expanding clones that grow within controlled environments. Such data can reveal stochastic and instructive mechanisms that contribute to observed heterogeneity and elucidate the sequential order of differentiation events. Intercellular interactions can also be studied within these arrays by following responses of a controlled number of interacting cells, all trapped within the same microwell. Here we describe implementations of live-cell imaging within microwell arrays for studies of lymphocyte clonal expansion, portray insights already gained from these experiments and outline directions for future research. These tools, together with in vivo experiments tracking single-cell responses, will expand our understanding of adaptive immunity and the ways by which it can be manipulated.

Imaging Single Cells in the Living Retina

PubMed Central

Williams, David R.

2011-01-01

A quarter century ago, we were limited to a macroscopic view of the retina inside the living eye. Since then, new imaging technologies, including confocal scanning laser ophthalmoscopy, optical coherence tomography, and adaptive optics fundus imaging, transformed the eye into a microscope in which individual cells can now be resolved noninvasively. These technologies have enabled a wide range of studies of the retina that were previously impossible. PMID:21596053

Comparison of wheat classification accuracy using different classifiers of the image-100 system

NASA Technical Reports Server (NTRS)

Dejesusparada, N. (Principal Investigator); Chen, S. C.; Moreira, M. A.; Delima, A. M.

1981-01-01

Classification results using single-cell and multi-cell signature acquisition options, a point-by-point Gaussian maximum-likelihood classifier, and K-means clustering of the Image-100 system are presented. Conclusions reached are that: a better indication of correct classification can be provided by using a test area which contains various cover types of the study area; classification accuracy should be evaluated considering both the percentages of correct classification and error of commission; supervised classification approaches are better than K-means clustering; Gaussian distribution maximum likelihood classifier is better than Single-cell and Multi-cell Signature Acquisition Options of the Image-100 system; and in order to obtain a high classification accuracy in a large and heterogeneous crop area, using Gaussian maximum-likelihood classifier, homogeneous spectral subclasses of the study crop should be created to derive training statistics.

Common fluorescent proteins for single-molecule localization microscopy

NASA Astrophysics Data System (ADS)

Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

2015-07-01

Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

Visualization of gene expression in the live subject using the Na/I symporter as a reporter gene: applications in biotherapy

PubMed Central

Baril, Patrick; Martin-Duque, Pilar; Vassaux, Georges

2010-01-01

Biotherapies involve the utilization of antibodies, genetically modified viruses, bacteria or cells for therapeutic purposes. Molecular imaging has the potential to provide unique information that will guarantee their biosafety in humans and provide a rationale for the future development of new generations of reagents. In this context, non-invasive imaging of gene expression is an attractive prospect, allowing precise, spacio-temporal measurements of gene expression in longitudinal studies involving gene transfer vectors. With the emergence of cell therapies in regenerative medicine, it is also possible to track cells injected into subjects. In this context, the Na/I symporter (NIS) has been used in preclinical studies. Associated with a relevant radiotracer (123I-, 124I-, 99mTcO4-), NIS can be used to monitor gene transfer and the spread of selectively replicative viruses in tumours as well as in cells with a therapeutic potential. In addition to its imaging potential, NIS can be used as a therapeutic transgene through its ability to concentrate therapeutic doses of radionuclides in target cells. This dual property has applications in cancer treatment and could also be used to eradicate cells with therapeutic potential in the case of adverse events. Through experience acquired in preclinical studies, we can expect that non-invasive molecular imaging using NIS as a transgene will be pivotal for monitoring in vivo the exact distribution and pharmacodynamics of gene expression in a precise and quantitative way. This review highlights the applications of NIS in biotherapy, with a particular emphasis on image-guided radiotherapy, monitoring of gene and vector biodistribution and trafficking of stem cells. This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x PMID:19814733

Nanobubbles as ultrasound contrast agent for facilitating small cell lung cancer imaging

PubMed Central

Wang, Jin-Ping; Zhou, Xiao-Lin; Yan, Ji-Ping; Zheng, Rong-Qin; Wang, Wei

2017-01-01

Background This study is to investigate whether liposome-loaded nanobubbles (NBs) have the potentials to carry anti-pro-gastrin releasing peptide (proGRP) antibody and enhance ultrasound imaging of small cell lung cancer (SCLC). Methods NBs were loaded with an antibody against SCLC (H446 cell line). A nude mouse model of SCLC tumor was established by a subcutaneous injection of tumor cell suspension in the dorsal skin. Images for contrast-enhanced ultrasound (CEUS) of xenograft tumors in the model were obtained through an intravenous injection of blank and targeting NBs. Results The targeted NBs showed a high binding affinity (90.2 ± 3.24%) of the H446 cells in vitro as compared to the blank NBs that have no affinity of the cells. In process of tumor imaging, no mice died of the NB application. CEUS imaging of the targeted NBs manifested significant increases in half-peak time, area under the curve and peak intensity as compared to the blank NBs. In the model of SCLC, treatment with targeting NBs resulted in a large amount of fluorescent dye accumulated in the tumor tissue but not the liver tissue. Conclusion Our results indicate that NBs can carry antibody traveling to the SCLC cells, whereas application of NBs is safe and reliable in serving as ultrasound contrast agents for improving SCLC imaging. PMID:29100457

Distribution of macrophages and plasma cells in apical periodontitis and their relationship with clinical and image data

PubMed Central

Azeredo, Stéphane V.; Brasil, Sabrina C.; Antunes, Henrique; Marques, Fábio V.; Pires, Fábio R.

2017-01-01

Background Macrophages and plasma cells play a key role in the regulation of innate and adaptive immunity. The aim of this study was to assess the presence of these cells in apical periodontitis and their distribution comparing with clinical and image data. Material and Methods Thirty-three lesions were selected and divided in two groups (17 periapical cysts and 16 periapical granulomas). Immunoreactions using anti-CD68 and anti-CD138 antibodies were carried out; image analysis was performed with an optical microscope and 5 high-power fields from each slide were evaluated leading to an average score of immunoexpression. This mean score was compared between the two groups and correlated with the clinical and image data. Results There was no statistically significant difference (p >0.05) for the mean average score of CD68+ macrophages and CD138+ plasma cells when comparing the two groups (cysts x granulomas) and the specimens included in each specific group. No statistically significant differences (p >0.05) were also observed when comparing the average scores with clinical and image data. Conclusions The presence of CD68+ macrophages and CD138+ plasma cells was similar in periapical cysts and granulomas and the presence of these cells did not correlate with clinical and image data from both groups. Key words:Macrophages, plasma cells, apical periodontitis, periapical granuloma, periapical cyst. PMID:29075406

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

Characterization of human breast cancer tissues by infrared imaging.

PubMed

Verdonck, M; Denayer, A; Delvaux, B; Garaud, S; De Wind, R; Desmedt, C; Sotiriou, C; Willard-Gallo, K; Goormaghtigh, E

2016-01-21

Fourier Transform InfraRed (FTIR) spectroscopy coupled to microscopy (IR imaging) has shown unique advantages in detecting morphological and molecular pathologic alterations in biological tissues. The aim of this study was to evaluate the potential of IR imaging as a diagnostic tool to identify characteristics of breast epithelial cells and the stroma. In this study a total of 19 breast tissue samples were obtained from 13 patients. For 6 of the patients, we also obtained Non-Adjacent Non-Tumor tissue samples. Infrared images were recorded on the main cell/tissue types identified in all breast tissue samples. Unsupervised Principal Component Analyses and supervised Partial Least Square Discriminant Analyses (PLS-DA) were used to discriminate spectra. Leave-one-out cross-validation was used to evaluate the performance of PLS-DA models. Our results show that IR imaging coupled with PLS-DA can efficiently identify the main cell types present in FFPE breast tissue sections, i.e. epithelial cells, lymphocytes, connective tissue, vascular tissue and erythrocytes. A second PLS-DA model could distinguish normal and tumor breast epithelial cells in the breast tissue sections. A patient-specific model reached particularly high sensitivity, specificity and MCC rates. Finally, we showed that the stroma located close or at distance from the tumor exhibits distinct spectral characteristics. In conclusion FTIR imaging combined with computational algorithms could be an accurate, rapid and objective tool to identify/quantify breast epithelial cells and differentiate tumor from normal breast tissue as well as normal from tumor-associated stroma, paving the way to the establishment of a potential complementary tool to ensure safe tumor margins.

Norcyanine dyes with benzo[c,d]indolium moiety: Spectral sensitivity with pH change for fluorescence pH imaging in living cells.

PubMed

Guan, Li; Liu, Qi; Zhang, Borui; Wang, Lanying

2017-01-01

Fluorescence pH imaging in living cells is a rapidly expanding research direction, however, it relies on the development of pH-sensitive fluorescent imaging agents. Here four norcyanine dyes with benzo[c,d]indolium moiety, exhibiting high spectral sensitivity with pH changes, were synthesized for fluorescence pH imaging in living cells, and characterized by 1 H NMR, 13 C NMR, IR, UV-Vis and HRMS. The investigation of their spectral properties in methanol and water showed that the absorption and emission maxima were in the region 488-618nm and 583-651nm, respectively, and four dyes exhibited high photostability. The pH spectral titrations showed that selective dye D1 had pH-dependent absorption spectral changes within the pH range of 2.4 to 9.4, and high fluorescent spectral sensitivity at pH5.0-8.0, with a pK a of 5.0. A cell association study indicated that dye D1 exhibited no or mild cytotoxicity at the application dose and duration, and could be accumulated in cells and mainly distributed in the cytoplasm, giving red fluorescence imaging. In particular, dye D1 could achieve pH-dependent fluorescence imaging in living cells with the increase of pH from 3.0 to 8.0, at excitation wavelength of 543nm and receiving wavelength of 655-755nm, which was valuable for studying the weak acidic, neutral and weak alkaline biological tissue compartments. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Ono, Kenji; Fuma, Kazuya; Tabata, Kaori

Magnetic resonance imaging (MRI) is a minimally invasive way to provide high spatial resolution tomograms. However, MRI has been considered to be useless for gene expression imaging compared to optical imaging. In this study, we used a ferritin reporter, binding with biogenic iron, to make it a powerful tool for gene expression imaging in MRI studies. GL261 mouse glioma cells were over-expressed with dual-reporter ferritin-DsRed under {beta}-actin promoter, then gene expression was observed by optical imaging and MRI in a brain tumor model. GL261 cells expressing ferritin-DsRed fusion protein showed enhanced visualizing effect by reducing T2-weighted signal intensity for inmore » vitro and in vivo MRI studies, as well as DsRed fluorescence for optical imaging. Furthermore, a higher contrast was achieved on T2-weighted images when permeating the plasma membrane of ferritin-DsRed-expressing GL261. Thus, a ferritin expression vector can be used as an MRI reporter to monitor in vivo gene expression.« less

Novel single-cell mega-size chambers for electrochemical etching of panorama position-sensitive polycarbonate ion image detectors

NASA Astrophysics Data System (ADS)

Sohrabi, Mehdi

2017-11-01

A novel development is made here by inventing panorama single-cell mega-size electrochemical etching (MS-ECE) chamber systems for processing panorama position-sensitive mega-size polycarbonate ion image detectors (MS-PCIDs) of potential for many neutron and ion detection applications in particular hydrogen ions or proton tracks and images detected for the first time in polycarbonates in this study. The MS-PCID is simply a large polycarbonate sheet of a desired size. The single-cell MS-ECE invented consists of two large equally sized transparent Plexiglas sheets as chamber walls holding a MS-PCID and the ECE chamber components tightly together. One wall has a large flat stainless steel electrode (dry cell) attached to it which is directly in contact with the MS-PCID and the other wall has a rod electrode with two holes to facilitate feeding and draining out the etching solution from the wet cell. A silicon rubber washer plays the role of the wet cell to hold the etchant and the electrical insulator to isolate the dry cell from the wet cell. A simple 50 Hz-HV home-made generator provides an adequate field strength through the two electrodes across the MS-ECE chamber. Two panorama single-cell MS-ECE chamber systems (circular and rectangular shapes) constructed were efficiently applied to processing the MS-PCIDs for 4π ion emission image detection of different gases in particular hydrogen ions or protons in a 3.5 kJ plasma focus device (PFD as uniquely observed by the unaided eyes). The panorama MS-PCID/MS-ECE image detection systems invented are novel with high potential for many applications in particular as applied to 4π panorama ion emission angular distribution image detection studies in PFD space, some results of which are presented and discussed.

Novel single-cell mega-size chambers for electrochemical etching of panorama position-sensitive polycarbonate ion image detectors.

PubMed

Sohrabi, Mehdi

2017-11-01

A novel development is made here by inventing panorama single-cell mega-size electrochemical etching (MS-ECE) chamber systems for processing panorama position-sensitive mega-size polycarbonate ion image detectors (MS-PCIDs) of potential for many neutron and ion detection applications in particular hydrogen ions or proton tracks and images detected for the first time in polycarbonates in this study. The MS-PCID is simply a large polycarbonate sheet of a desired size. The single-cell MS-ECE invented consists of two large equally sized transparent Plexiglas sheets as chamber walls holding a MS-PCID and the ECE chamber components tightly together. One wall has a large flat stainless steel electrode (dry cell) attached to it which is directly in contact with the MS-PCID and the other wall has a rod electrode with two holes to facilitate feeding and draining out the etching solution from the wet cell. A silicon rubber washer plays the role of the wet cell to hold the etchant and the electrical insulator to isolate the dry cell from the wet cell. A simple 50 Hz-HV home-made generator provides an adequate field strength through the two electrodes across the MS-ECE chamber. Two panorama single-cell MS-ECE chamber systems (circular and rectangular shapes) constructed were efficiently applied to processing the MS-PCIDs for 4π ion emission image detection of different gases in particular hydrogen ions or protons in a 3.5 kJ plasma focus device (PFD as uniquely observed by the unaided eyes). The panorama MS-PCID/MS-ECE image detection systems invented are novel with high potential for many applications in particular as applied to 4π panorama ion emission angular distribution image detection studies in PFD space, some results of which are presented and discussed.

Intravital live cell triggered imaging system reveals monocyte patrolling and macrophage migration in atherosclerotic arteries

NASA Astrophysics Data System (ADS)

McArdle, Sara; Chodaczek, Grzegorz; Ray, Nilanjan; Ley, Klaus

2015-02-01

Intravital multiphoton imaging of arteries is technically challenging because the artery expands with every heartbeat, causing severe motion artifacts. To study leukocyte activity in atherosclerosis, we developed the intravital live cell triggered imaging system (ILTIS). This system implements cardiac triggered acquisition as well as frame selection and image registration algorithms to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice. To minimize tissue damage, no mechanical stabilization is used and the artery is allowed to expand freely. ILTIS performs multicolor high frame-rate two-dimensional imaging and full-thickness three-dimensional imaging of beating arteries in live mice. The external carotid artery and its branches (superior thyroid and ascending pharyngeal arteries) were developed as a surgically accessible and reliable model of atherosclerosis. We use ILTIS to demonstrate Cx3cr1GFP monocytes patrolling the lumen of atherosclerotic arteries. Additionally, we developed a new reporter mouse (Apoe-/-Cx3cr1GFP/+Cd11cYFP) to image GFP+ and GFP+YFP+ macrophages "dancing on the spot" and YFP+ macrophages migrating within intimal plaque. ILTIS will be helpful to answer pertinent open questions in the field, including monocyte recruitment and transmigration, macrophage and dendritic cell activity, and motion of other immune cells.

Noninvasive Optical Tracking of Red Fluorescent Protein-Expressing Cancer Cells in a Model of Metastatic Breast Cancer 1*

PubMed Central

Winnard, Paul T; Kluth, Jessica B; Raman, Venu

2006-01-01

Abstract We have evaluated the use of the Xenogen IVIS 200 imaging system for real-time fluorescence protein-based optical imaging of metastatic progression in live animals. We found that green fluorescent protein-expressing cells (100 x 106) were not detectable in a mouse cadaver phantom experiment. However, a 10-fold lower number of tdTomato-expressing cells were easily detected. Mammary fat pad xenografts of stable MDA-MB-231-tdTomato cells were generated for the imaging of metastatic progression. At 2 weeks postinjection, barely palpable tumor burdens were easily detected at the sites of injection. At 8 weeks, a small contralateral mammary fat pad metastasis was imaged and, by 13 weeks, metastases to lymph nodes were detectable. Metastases with nodular composition were detectable within the rib cage region at 15 weeks. 3-D image reconstructions indicated that the detection of fluorescence extended to approximately 1 cm below the surface. A combination of intense tdTomato fluorescence, imaging at ≥ 620 nm (where autofluorescence is minimized), and the sensitivity of the Xenogen imager made this possible. This study demonstrates the utility of the noninvasive optical tracking of cancer cells during metastatic progression with endogenously expressed fluorescence protein reporters using detection wavelengths of ≥ 620 nm. PMID:17032496

Time-lapse cinematography in living Drosophila tissues: preparation of material.

PubMed

Davis, Ilan; Parton, Richard M

2006-11-01

The fruit fly, Drosophila melanogaster, has been an extraordinarily successful model organism for studying the genetic basis of development and evolution. It is arguably the best-understood complex multicellular model system, owing its success to many factors. Recent developments in imaging techniques, in particular sophisticated fluorescence microscopy methods and equipment, now allow cellular events to be studied at high resolution in living material. This ability has enabled the study of features that tend to be lost or damaged by fixation, such as transient or dynamic events. Although many of the techniques of live cell imaging in Drosophila are shared with the greater community of cell biologists working on other model systems, studying living fly tissues presents unique difficulties in keeping the cells alive, introducing fluorescent probes, and imaging through thick hazy cytoplasm. This protocol outlines the preparation of major tissue types amenable to study by time-lapse cinematography and different methods for keeping them alive.

Rapid enumeration of viable bacteria by image analysis

NASA Technical Reports Server (NTRS)

Singh, A.; Pyle, B. H.; McFeters, G. A.

1989-01-01

A direct viable counting method for enumerating viable bacteria was modified and made compatible with image analysis. A comparison was made between viable cell counts determined by the spread plate method and direct viable counts obtained using epifluorescence microscopy either manually or by automatic image analysis. Cultures of Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Yersinia enterocolitica and Pseudomonas aeruginosa were incubated at 35 degrees C in a dilute nutrient medium containing nalidixic acid. Filtered samples were stained for epifluorescence microscopy and analysed manually as well as by image analysis. Cells enlarged after incubation were considered viable. The viable cell counts determined using image analysis were higher than those obtained by either the direct manual count of viable cells or spread plate methods. The volume of sample filtered or the number of cells in the original sample did not influence the efficiency of the method. However, the optimal concentration of nalidixic acid (2.5-20 micrograms ml-1) and length of incubation (4-8 h) varied with the culture tested. The results of this study showed that under optimal conditions, the modification of the direct viable count method in combination with image analysis microscopy provided an efficient and quantitative technique for counting viable bacteria in a short time.

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

PubMed Central

Almassalha, Luay M.; Bauer, Greta M.; Chandler, John E.; Gladstein, Scott; Cherkezyan, Lusik; Stypula-Cyrus, Yolanda; Weinberg, Samuel; Zhang, Di; Thusgaard Ruhoff, Peder; Roy, Hemant K.; Subramanian, Hariharan; Chandel, Navdeep S.; Szleifer, Igal; Backman, Vadim

2016-01-01

The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure–function relationship in live cells. PMID:27702891

Imaging live cells at high spatiotemporal resolution for lab-on-a-chip applications.

PubMed

Chin, Lip Ket; Lee, Chau-Hwang; Chen, Bi-Chang

2016-05-24

Conventional optical imaging techniques are limited by the diffraction limit and difficult-to-image biomolecular and sub-cellular processes in living specimens. Novel optical imaging techniques are constantly evolving with the desire to innovate an imaging tool that is capable of seeing sub-cellular processes in a biological system, especially in three dimensions (3D) over time, i.e. 4D imaging. For fluorescence imaging on live cells, the trade-offs among imaging depth, spatial resolution, temporal resolution and photo-damage are constrained based on the limited photons of the emitters. The fundamental solution to solve this dilemma is to enlarge the photon bank such as the development of photostable and bright fluorophores, leading to the innovation in optical imaging techniques such as super-resolution microscopy and light sheet microscopy. With the synergy of microfluidic technology that is capable of manipulating biological cells and controlling their microenvironments to mimic in vivo physiological environments, studies of sub-cellular processes in various biological systems can be simplified and investigated systematically. In this review, we provide an overview of current state-of-the-art super-resolution and 3D live cell imaging techniques and their lab-on-a-chip applications, and finally discuss future research trends in new and breakthrough research areas of live specimen 4D imaging in controlled 3D microenvironments.

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

PubMed Central

Lerner, Thomas R.; Burden, Jemima J.; Nkwe, David O.; Pelchen-Matthews, Annegret; Domart, Marie-Charlotte; Durgan, Joanne; Weston, Anne; Jones, Martin L.; Peddie, Christopher J.; Carzaniga, Raffaella; Florey, Oliver; Marsh, Mark; Gutierrez, Maximiliano G.

2017-01-01

ABSTRACT The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research. PMID:27445312

Reduced background autofluorescence for cell imaging using nanodiamonds and lanthanide chelates.

PubMed

Cordina, Nicole M; Sayyadi, Nima; Parker, Lindsay M; Everest-Dass, Arun; Brown, Louise J; Packer, Nicolle H

2018-03-14

Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence; and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules. The second method uses europium-chelating tags with long fluorescence lifetimes. These europium-chelating tags enhance background-free imaging due to the short fluorescent lifetimes of cellular autofluorescence. In this study, we used both methods to target E-selectin, a transmembrane glycoprotein that is activated by inflammation, to demonstrate background-free fluorescent staining in fixed endothelial cells. Our findings indicate that both FND and Europium based staining can improve fluorescent bio-imaging capabilities by reducing competition with cellular autofluorescence. 30 nm nanodiamonds coated with the E-selectin antibody was found to enable the most sensitive detective of E-selectin in inflamed cells, with a 40-fold increase in intensity detected.

A G protein-coupled receptor (GPCR) in red: live cell imaging of the kappa opioid receptor-tdTomato fusion protein (KOPR-tdT) in neuronal cells

PubMed Central

Huang, Peng; Chiu, Yi-Ting; Chen, Chongguang; Wang, Yujun; Liu-Chen, Lee-Yuan

2013-01-01

Introduction In contrast to green fluorescent protein and variants (GFPs), red fluorescent proteins (RFPs) have rarely been employed for generation of GPCR fusion proteins, likely because of formation of aggregates and cell toxicity of some RFPs. Among all the RFPs available, tdTomato (tdT), one of the non-aggregating RFP, has the highest brightness score (about 3 times that of eGFP) and unsurpassed photostability. Methods We fused tdT to the KOPR C-terminus. The KOPR-tdT cDNA construct was transfected into Neuro2A mouse neuroblastoma cell line (Neuro2A cells) and rat cortical primary neurons for characterization of pharmacological properties and imaging studies on KOPR trafficking. Results KOPR-tdT retained KOPR properties (cell surface expression, ligand binding, agonist-induced signaling and internalization) when expressed in Neuro2A cells and rat primary cortical neurons. Live cell imaging of KOPR-tdT enables visualization of time course of agonist-induced internalization of KOPR in real time for 60 min, without photobleaching and apparent cell toxicity. U50,488H-induced KOPR internalization occurred as early as 4 min and plateaued at about 30 min. A unique pattern of internalized KOPR in processes of primary neurons was induced by U50,488H. Discussion tdT is an alternative to, or even a better tool than, GFPs for fusing to GPCR for trafficking studies, because tdT has higher brightness and thus better resolution and less photobleaching problems due to reduced laser power used. It also has advantages associated with its longer-wavelength emission including spectral separation from autofluorescence and GFPs, reduced cell toxicity the laser may impose, and greater tissue penetration. These advantages of tdT over GPFs may be critical for live cell imaging studies of GPCRs in vitro and for studying GPCRs in vivo because of their low abundance. PMID:23856011

Regulating effect of epithalone on gastric endocrine cells in pinealectomized rats.

PubMed

Khavinson, V K; Popuchiev, V V; Kvetnoii, I M; Yuzhakov, V V; Kotlova, L N

2000-12-01

Endocrine cells in the stomach of pinealectomized rats after injection of epithalone (pineal gland peptide) were studied by immunohistochemical tests, morphometry, and image analysis microscopic images. A functional relationship was found between the pineal gland and stomach, which is regulated by peptides produced by the pineal gland.

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel.

PubMed

Bolgioni, Amanda F; Vittoria, Marc A; Ganem, Neil J

2018-05-14

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 °C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Spatiotemporal PET Imaging of Dynamic Metabolic Changes After Therapeutic Approaches of Induced Pluripotent Stem Cells, Neuronal Stem Cells, and a Chinese Patent Medicine in Stroke.

PubMed

Zhang, Hong; Song, Fahuan; Xu, Caiyun; Liu, Hao; Wang, Zefeng; Li, Jinhui; Wu, Shuang; YehuaShen; Chen, Yao; Zhu, Yunqi; Du, Ruili; Tian, Mei

2015-11-01

This study aimed to use spatiotemporal PET imaging to investigate the dynamic metabolic changes after a combined therapeutic approach of induced pluripotent stem cells (iPSCs), neuronal stem cells (NSCs), and Chinese patent medicine in a rat model of cerebral ischemia-reperfusion injury. Cerebral ischemia was established by the middle cerebral artery occlusion approach. Thirty-six male rats were randomly assigned to 1 of the 6 groups: control phosphate-buffered saline (PBS), Chinese patent medicine (Qing-kai-ling [QKL]), induced pluripotent stem cells (iPSCs), combination of iPSCs and QKL, neuronal stem cells (NSCs), and combination of NSCs and QKL. Serial (18)F-FDG small-animal PET imaging and neurofunctional tests were performed weekly. Autoradiographic imaging and immunohistochemical and immunofluorescent analyses were performed at 4 wk after stem cell transplantation. Compared with the PBS control group, significantly higher (18)F-FDG accumulations in the ipsilateral cerebral infarction were observed in 5 treatment groups from weeks 1-4. Interestingly, the most intensive (18)F-FDG accumulation was found in the NSCs + QKL group at week 1 but in the iPSCs + QKL group at week 4. The neurofunctional scores in the 5 treatment groups were significantly higher than that of the PBS group from week 3 to 4. In addition, there was a significant correlation between the PET imaging findings and neurofunctional recovery (P < 0.05) or glucose transporter-1 expression (P < 0.01). Immunohistochemical and immunofluorescence studies found that transplanted iPSCs survived and migrated to the ischemic region and expressed protein markers for cells of interest. Spatiotemporal PET imaging with (18)F-FDG demonstrated dynamic metabolic and functional recovery after iPSCs or NSCs combined with QKL in a rat model of cerebral ischemia-reperfusion injury. iPSCs or NSCs combined with Chinese medicine QKL seemed to be a better therapeutic approach than these stem cells used individually. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Label-Free, High Resolution, Multi-Modal Light Microscopy for Discrimination of Live Stem Cell Differentiation Status.

PubMed

Zhang, Jing; Moradi, Emilia; Somekh, Michael G; Mather, Melissa L

2018-01-15

A label-free microscopy method for assessing the differentiation status of stem cells is presented with potential application for characterization of therapeutic stem cell populations. The microscopy system is capable of characterizing live cells based on the use of evanescent wave microscopy and quantitative phase contrast (QPC) microscopy. The capability of the microscopy system is demonstrated by studying the differentiation of live immortalised neonatal mouse neural stem cells over a 15 day time course. Metrics extracted from microscope images are assessed and images compared with results from endpoint immuno-staining studies to illustrate the system's performance. Results demonstrate the potential of the microscopy system as a valuable tool for cell biologists to readily identify the differentiation status of unlabelled live cells.

The impact of functional imaging on radiation medicine.

PubMed

Sharma, Nidhi; Neumann, Donald; Macklis, Roger

2008-09-15

Radiation medicine has previously utilized planning methods based primarily on anatomic and volumetric imaging technologies such as CT (Computerized Tomography), ultrasound, and MRI (Magnetic Resonance Imaging). In recent years, it has become apparent that a new dimension of non-invasive imaging studies may hold great promise for expanding the utility and effectiveness of the treatment planning process. Functional imaging such as PET (Positron Emission Tomography) studies and other nuclear medicine based assays are beginning to occupy a larger place in the oncology imaging world. Unlike the previously mentioned anatomic imaging methodologies, functional imaging allows differentiation between metabolically dead and dying cells and those which are actively metabolizing. The ability of functional imaging to reproducibly select viable and active cell populations in a non-invasive manner is now undergoing validation for many types of tumor cells. Many histologic subtypes appear amenable to this approach, with impressive sensitivity and selectivity reported. For clinical radiation medicine, the ability to differentiate between different levels and types of metabolic activity allows the possibility of risk based focal treatments in which the radiation doses and fields are more tightly connected to the perceived risk of recurrence or progression at each location. This review will summarize many of the basic principles involved in the field of functional PET imaging for radiation oncology planning and describe some of the major relevant published data behind this expanding trend.

Imaging: Guiding the Clinical Translation of Cardiac Stem Cell Therapy

PubMed Central

Nguyen, Patricia K.; Lan, Feng; Wang, Yongming; Wu, Joseph C.

2011-01-01

Stem cells have been touted as the holy grail of medical therapy with promises to regenerate cardiac tissue, but it appears the jury is still out on this novel therapy. Using advanced imaging technology, scientists have discovered that these cells do not survive nor engraft long-term. In addition, only marginal benefit has been observed in large animal studies and human trials. However, all is not lost. Further application of advanced imaging technology will help scientists unravel the mysteries of stem cell therapy and address the clinical hurdles facing its routine implementation. In this review, we will discuss how advanced imaging technology will help investigators better define the optimal delivery method, improve survival and engraftment, and evaluate efficacy and safety. Insights gained from this review may direct the development of future preclinical investigations and clinical trials. PMID:21960727

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

Greenberg, S.D.; Smith, S.; Swank, P.R.

Visual cell profiles were used to analyze the distribution of atypical bronchial cells in sputum specimens from cigarette-smoking volunteers, cigarette-smoking asbestos workers and cigarette-smoking uranium miners. The preliminary results of these sputum visual cell profile studies have demonstrated distinctive distributions of bronchial cell atypias in progressive patterns of squamous metaplasia, mild, moderate and severe atypias and carcinoma, similar to those the authors have previously reported using cell image analysis techniques to determine an atypia status index (ASI). The information gained from this study will be helpful in further validating this ASI and subsequently achieving the ultimate goal of employing cellmore » image analysis for the rapid and precise identification of premalignant atypias in sputum.« less

Visualizing Viral Infection In Vivo by Multi-Photon Intravital Microscopy.

PubMed

Sewald, Xaver

2018-06-20

Viral pathogens have adapted to the host organism to exploit the cellular machinery for virus replication and to modulate the host cells for efficient systemic dissemination and immune evasion. Much of our knowledge of the effects that virus infections have on cells originates from in vitro imaging studies using experimental culture systems consisting of cell lines and primary cells. Recently, intravital microscopy using multi-photon excitation of fluorophores has been applied to observe virus dissemination and pathogenesis in real-time under physiological conditions in living organisms. Critical steps during viral infection and pathogenesis could be studied by direct visualization of fluorescent virus particles, virus-infected cells, and the immune response to viral infection. In this review, I summarize the latest research on in vivo studies of viral infections using multi-photon intravital microscopy (MP-IVM). Initially, the underlying principle of multi-photon microscopy is introduced and experimental challenges during microsurgical animal preparation and fluorescent labeling strategies for intravital imaging are discussed. I will further highlight recent studies that combine MP-IVM with optogenetic tools and transcriptional analysis as a powerful approach to extend the significance of in vivo imaging studies of viral pathogens.

Non-fused phospholes as fluorescent probes for imaging of lipid droplets in living cells

NASA Astrophysics Data System (ADS)

Öberg, Elisabet; Appelqvist, Hanna; Nilsson, K. Peter R.

2017-04-01

Molecular tools for fluorescent imaging of specific compartments in cells are essential for understanding the function and activity of cells. Here, we report the synthesis of a series of pyridyl- and thienyl-substituted phospholes and the evaluation of these dyes for fluorescent imaging of cells. The thienyl-substituted phospholes proved to be successful for staining of cultured normal and malignant cells due to their fluorescent properties and low toxicity. Co-staining experiments demonstrated that these probes target lipid droplets, which are, lipid-storage organelles found in the cytosol of nearly all cell types. Our findings confirm that thienyl-substituted phospholes can be utilized as fluorescent tools for vital staining of cells, and we foresee that these fluorescent dyes might be used in studies to unravel the roles that lipid droplets play in cellular physiology and their role in diseases.

An orientation-independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a Cnidarian model

PubMed Central

Malamy, Jocelyn; Shribak, Michael

2017-01-01

Epithelial cell dynamics can be difficult to study in intact animals or tissues. Here we use the medusa form of the hydrozoan Clytia hemisphaerica, which is covered with a monolayer of epithelial cells, to test the efficacy of an orientation-independent differential interference contrast (OI-DIC) microscope for in vivo imaging of wound healing. OI-DIC provides an unprecedented resolution phase image of epithelial cells closing a wound in a live, non-transgenic animal model. In particular, the OI-DIC microscope equipped with a 40×/0.75NA objective lens and using the illumination light with wavelength 546 nm demonstrated a resolution of 460 nm. The repair of individual cells, the adhesion of cells to close a gap, and the concomitant contraction of these cells during closure is clearly visualized. PMID:29345317

An orientation-independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a cnidarian model.

PubMed

Malamy, J E; Shribak, M

2018-06-01

Epithelial cell dynamics can be difficult to study in intact animals or tissues. Here we use the medusa form of the hydrozoan Clytia hemisphaerica, which is covered with a monolayer of epithelial cells, to test the efficacy of an orientation-independent differential interference contrast microscope for in vivo imaging of wound healing. Orientation-independent differential interference contrast provides an unprecedented resolution phase image of epithelial cells closing a wound in a live, nontransgenic animal model. In particular, the orientation-independent differential interference contrast microscope equipped with a 40x/0.75NA objective lens and using the illumination light with wavelength 546 nm demonstrated a resolution of 460 nm. The repair of individual cells, the adhesion of cells to close a gap, and the concomitant contraction of these cells during closure is clearly visualized. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Label free cell tracking in 3D tissue engineering constructs with high resolution imaging

NASA Astrophysics Data System (ADS)

Smith, W. A.; Lam, K.-P.; Dempsey, K. P.; Mazzocchi-Jones, D.; Richardson, J. B.; Yang, Y.

2014-02-01

Within the field of tissue engineering there is an emphasis on studying 3-D live tissue structures. Consequently, to investigate and identify cellular activities and phenotypes in a 3-D environment for all in vitro experiments, including shape, migration/proliferation and axon projection, it is necessary to adopt an optical imaging system that enables monitoring 3-D cellular activities and morphology through the thickness of the construct for an extended culture period without cell labeling. This paper describes a new 3-D tracking algorithm developed for Cell-IQ®, an automated cell imaging platform, which has been equipped with an environmental chamber optimized to enable capturing time-lapse sequences of live cell images over a long-term period without cell labeling. As an integral part of the algorithm, a novel auto-focusing procedure was developed for phase contrast microscopy equipped with 20x and 40x objectives, to provide a more accurate estimation of cell growth/trajectories by allowing 3-D voxels to be computed at high spatiotemporal resolution and cell density. A pilot study was carried out in a phantom system consisting of horizontally aligned nanofiber layers (with precise spacing between them), to mimic features well exemplified in cellular activities of neuronal growth in a 3-D environment. This was followed by detailed investigations concerning axonal projections and dendritic circuitry formation in a 3-D tissue engineering construct. Preliminary work on primary animal neuronal cells in response to chemoattractant and topographic cue within the scaffolds has produced encouraging results.

Sensitive dual color in vivo bioluminescence imaging using a new red codon optimized firefly luciferase and a green click beetle luciferase.

PubMed

Mezzanotte, Laura; Que, Ivo; Kaijzel, Eric; Branchini, Bruce; Roda, Aldo; Löwik, Clemens

2011-04-22

Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color-coupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99. Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate. We report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays, our study demonstrated enhanced sensitivity combined with spatially separate BL spectral emissions using a suitable spectral unmixing algorithm. This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future for more accurate quantitative gene expression studies in vivo by simultaneously monitoring two events in real time.

Sensitive Dual Color In Vivo Bioluminescence Imaging Using a New Red Codon Optimized Firefly Luciferase and a Green Click Beetle Luciferase

PubMed Central

Mezzanotte, Laura; Que, Ivo; Kaijzel, Eric; Branchini, Bruce; Roda, Aldo; Löwik, Clemens

2011-01-01

Background Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color-coupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99. Principal Findings Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate. Significance We report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays, our study demonstrated enhanced sensitivity combined with spatially separate BL spectral emissions using a suitable spectral unmixing algorithm. This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future for more accurate quantitative gene expression studies in vivo by simultaneously monitoring two events in real time. PMID:21544210

Imaging of the interaction of cancer cells and the lymphatic system.

PubMed

Tran Cao, Hop S; McElroy, Michele; Kaushal, Sharmeela; Hoffman, Robert M; Bouvet, Michael

2011-09-10

A thorough understanding of the lymphatic system and its interaction with cancer cells is crucial to our ability to fight cancer metastasis. Efforts to study the lymphatic system had previously been limited by the inability to visualize the lymphatic system in vivo in real time. Fluorescence imaging can address these limitations and allow for visualization of lymphatic delivery and trafficking of cancer cells and potentially therapeutic agents as well. Here, we review recent articles in which antibody-fluorophore conjugates are used to label the lymphatic network and fluorescent proteins to label cancer cells in the evaluation of lymphatic delivery and imaging. Copyright © 2011 Elsevier B.V. All rights reserved.

Long-term C. elegans immobilization enables high resolution developmental studies in vivo.

PubMed

Berger, Simon; Lattmann, Evelyn; Aegerter-Wilmsen, Tinri; Hengartner, Michael; Hajnal, Alex; deMello, Andrew; Casadevall I Solvas, Xavier

2018-05-01

Live-imaging of C. elegans is essential for the study of conserved cellular pathways (e.g. EGFR/Wnt signaling) and morphogenesis in vivo. However, the usefulness of live imaging as a research tool has been severely limited by the need to immobilize worms prior to and during imaging. Conventionally, immobilization is achieved by employing both physical and chemical interventions. These are known to significantly affect many physiological processes, and thus limit our understanding of dynamic developmental processes. Herein we present a novel, easy-to-use microfluidic platform for the long-term immobilization of viable, normally developing C. elegans, compatible with image acquisition at high resolution, thereby overcoming the limitations associated with conventional worm immobilization. The capabilities of the platform are demonstrated through the continuous assessment of anchor cell (AC) invasion and distal tip cell (DTC) migration in larval C. elegans and germ cell apoptosis in adult C. elegans in vivo for the first time.

Pulse splitter-based nonlinear microscopy for live-cardiomyocyte imaging

PubMed Central

Wang, Zhonghai; Qin, Wan; Shao, Yonghong; Ma, Siyu; Borg, Thomas K.; Gao, Bruce Z.

2015-01-01

Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al. in our two photon excitation fluorescence (TPEF) and SHG hybrid microscope. The system dramatically reduced photodamage to neonatal cardiomyocytes in early stages of culture, greatly increasing cell viability. Thus continuous imaging of live cardiomyocytes was achieved with a stronger laser and for a longer period than has been reported in the literature. The pulse splitter-based TPEF-SHG microscope constructed in this study was demonstrated to be an ideal imaging system for sarcomeric addition-related investigations of neonatal cardiomyocytes in early stages of culture. PMID:25767692

Analysis of Morphological Features of Benign and Malignant Breast Cell Extracted From FNAC Microscopic Image Using the Pearsonian System of Curves.

PubMed

Rajbongshi, Nijara; Bora, Kangkana; Nath, Dilip C; Das, Anup K; Mahanta, Lipi B

2018-01-01

Cytological changes in terms of shape and size of nuclei are some of the common morphometric features to study breast cancer, which can be observed by careful screening of fine needle aspiration cytology (FNAC) images. This study attempts to categorize a collection of FNAC microscopic images into benign and malignant classes based on family of probability distribution using some morphometric features of cell nuclei. For this study, features namely area, perimeter, eccentricity, compactness, and circularity of cell nuclei were extracted from FNAC images of both benign and malignant samples using an image processing technique. All experiments were performed on a generated FNAC image database containing 564 malignant (cancerous) and 693 benign (noncancerous) cell level images. The five-set extracted features were reduced to three-set (area, perimeter, and circularity) based on the mean statistic. Finally, the data were fitted to the generalized Pearsonian system of frequency curve, so that the resulting distribution can be used as a statistical model. Pearsonian system is a family of distributions where kappa (κ) is the selection criteria computed as functions of the first four central moments. For the benign group, kappa (κ) corresponding to area, perimeter, and circularity was -0.00004, 0.0000, and 0.04155 and for malignant group it was 1016942, 0.01464, and -0.3213, respectively. Thus, the family of distribution related to these features for the benign and malignant group were different, and therefore, characterization of their probability curve will also be different.

Observation of endoplasmic reticulum tubules via TOF-SIMS tandem mass spectrometry imaging of transfected cells.

PubMed

Chini, Corryn E; Fisher, Gregory L; Johnson, Ben; Tamkun, Michael M; Kraft, Mary L

2018-02-26

Advances in three-dimensional secondary ion mass spectrometry (SIMS) imaging have enabled visualizing the subcellular distributions of various lipid species within individual cells. However, the difficulty of locating organelles using SIMS limits efforts to study their lipid compositions. Here, the authors have assessed whether endoplasmic reticulum (ER)-Tracker Blue White DPX ® , which is a commercially available stain for visualizing the endoplasmic reticulum using fluorescence microscopy, produces distinctive ions that can be used to locate the endoplasmic reticulum using SIMS. Time-of-flight-SIMS tandem mass spectrometry (MS 2 ) imaging was used to identify positively and negatively charged ions produced by the ER-Tracker stain. Then, these ions were used to localize the stain and thus the endoplasmic reticulum, within individual human embryonic kidney cells that contained higher numbers of endoplasmic reticulum-plasma membrane junctions on their surfaces. By performing MS 2 imaging of selected ions in parallel with the precursor ion (MS 1 ) imaging, the authors detected a chemical interference native to the cell at the same nominal mass as the pentafluorophenyl fragment from the ER-Tracker stain. Nonetheless, the fluorine secondary ions produced by the ER-Tracker stain provided a distinctive signal that enabled locating the endoplasmic reticulum using SIMS. This simple strategy for visualizing the endoplasmic reticulum in individual cells using SIMS could be combined with existing SIMS methodologies for imaging intracellular lipid distribution and to study the lipid composition within the endoplasmic reticulum.

Bioinspired Nanocomplex for Spatiotemporal Imaging of Sequential mRNA Expression in Differentiating Neural Stem Cells

PubMed Central

2015-01-01

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions. PMID:25494492

Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

PubMed

Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

2014-12-23

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions.

Hallmarks in prostate cancer imaging with Ga68-PSMA-11-PET/CT with reference to detection limits and quantitative properties.

PubMed

Sanchez-Crespo, Alejandro; Jussing, Emma; Björklund, Ann-Charlotte; Pokrovskaja Tamm, Katja

2018-04-04

Gallium-68-labeled prostate-specific antigen positron emission tomography/computed tomography imaging (Ga68-PSMA-11-PET/CT) has emerged as a potential gold standard for prostate cancer (PCa) diagnosis. However, the imaging limitations of this technique at the early state of PCa recurrence/metastatic spread are still not well characterized. The aim of this study was to determine the quantitative properties and the fundamental imaging limits of Ga68-PSMA-11-PET/CT in localizing small PCa cell deposits. The human PCa LNCaP cells (PSMA expressing) were grown and collected as single cell suspension or as 3D-spheroids at different cell numbers and incubated with Ga68-PSMA-11. Thereafter, human HCT116 cells (PSMA negative) were added to a total cell number of 2 × 10 5 cells per tube. The tubes were then pelleted and the supernatant aspirated. A whole-body PET/CT scanner with a clinical routine protocol was used for imaging the pellets inside of a cylindrical water phantom with increasing amounts of background activity. The actual activity bound to the cells was also measured in an automatic gamma counter. Imaging detection limits and activity recovery coefficients as a function of LNCaP cell number were obtained. The effect of Ga68-PSMA-11 mass concentration on cell binding was also investigated in samples of LnCaP cells incubated with increasing concentrations of radioligand. A total of 1 × 10 4 LNCaP cells mixed in a pellet of 2 × 10 5 cells were required to reach a 50% detection probability with Ga68-PSMA-11-PET/CT without background. With a background level of 1 kBq/ml, between 4 × 10 5 and 1 × 10 6 cells are required. The radioligand equilibrium dissociation constant was 27.05 nM, indicating high binding affinity. Hence, the specific activity of the radioligand has a profound effect on image quantification. Ga68-PSMA-11-PET detects a small number of LNCaP cells even when they are mixed in a population of non-PSMA expressing cells and in the presence of background. The obtained image detection limits and characteristic quantification properties of Ga68-PSMA-11-PET/CT are essential hallmarks for the individualization of patient management. The use of the standardized uptake value for Ga68-PSMA-11-PET/CT image quantification should be precluded.

Molecule-Specific Imaging Analysis of Carcinogens in Breast Cancer Cells Using Time-of-Flight Secondary Ion Mass Spectrometry

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

Quong, J N; Knize, M G; Kulp, K S

2003-08-19

Imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) is used to study the localization of heterocyclic amines in MCF7 line of human breast cancer cells. The detection sensitivities of a model rodent mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were determined. Following an established criteria for the determination of status of freeze-fracture cells, the distribution of PhIP in the MCF7 cells are reported.

Cell labeling with magnetic nanoparticles: Opportunity for magnetic cell imaging and cell manipulation

PubMed Central

2013-01-01

This tutorial describes a method of controlled cell labeling with citrate-coated ultra small superparamagnetic iron oxide nanoparticles. This method may provide basically all kinds of cells with sufficient magnetization to allow cell detection by high-resolution magnetic resonance imaging (MRI) and to enable potential magnetic manipulation. In order to efficiently exploit labeled cells, quantify the magnetic load and deliver or follow-up magnetic cells, we herein describe the main requirements that should be applied during the labeling procedure. Moreover we present some recommendations for cell detection and quantification by MRI and detail magnetic guiding on some real-case studies in vitro and in vivo. PMID:24564857

Optical coherence microscopy for deep tissue imaging of the cerebral cortex with intrinsic contrast

PubMed Central

Srinivasan, Vivek J.; Radhakrishnan, Harsha; Jiang, James Y.; Barry, Scott; Cable, Alex E.

2012-01-01

In vivo optical microscopic imaging techniques have recently emerged as important tools for the study of neurobiological development and pathophysiology. In particular, two-photon microscopy has proved to be a robust and highly flexible method for in vivo imaging in highly scattering tissue. However, two-photon imaging typically requires extrinsic dyes or contrast agents, and imaging depths are limited to a few hundred microns. Here we demonstrate Optical Coherence Microscopy (OCM) for in vivo imaging of neuronal cell bodies and cortical myelination up to depths of ~1.3 mm in the rat neocortex. Imaging does not require the administration of exogenous dyes or contrast agents, and is achieved through intrinsic scattering contrast and image processing alone. Furthermore, using OCM we demonstrate in vivo, quantitative measurements of optical properties (index of refraction and attenuation coefficient) in the cortex, and correlate these properties with laminar cellular architecture determined from the images. Lastly, we show that OCM enables direct visualization of cellular changes during cell depolarization and may therefore provide novel optical markers of cell viability. PMID:22330462

Color-Coded Imaging of Syngeneic Orthotopic Malignant Lymphoma Interacting with Host Stromal Cells During Metastasis.

PubMed

Matsumoto, Takuro; Suetsugu, Atsushi; Hasegawa, Kosuke; Nakamura, Miki; Aoki, Hitomi; Kunisada, Takahiro; Tsurumi, Hisashi; Shimizu, Masahito; Hoffman, Robert M

2016-04-01

The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. In a previous study, EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were established and injected into the tail vein of C57/BL6 green fluorescent protein (GFP) transgenic mice. Metastasis was observed at multiple sites which were also enriched with host GFP-expressing stromal cells. In the present study, our aim was to establish an orthotopic model of EL4-RFP. In the present study, EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice as an orthotopic model of lymphoma. Resultant primary tumor and metastases were imaged with the Olympus FV1000 scanning laser confocal microscope. EL4-RFP metastasis was observed 21 days later. EL4-RFP tumors in the spleen (primary injection site), liver, supra-mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized by color-coded imaging. EL4-RFP metastases in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Small tumors were observed in the spleen, which were rich in host stromal cells. In the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture. Color-coded imaging of the lymphoma microenvironment suggests an important role of stromal cells in lymphoma progression and metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Magnetic resonance imaging tractography as a diagnostic tool in patients with spinal cord injury treated with human embryonic stem cells.

PubMed

Shroff, Geeta

2017-02-01

Introduction Spinal cord injury is a cause of severe disability and mortality. The pharmacological and non-pharmacological methods used, are unable to improve the quality of life in spinal cord injury. Spinal disorders have been treated with human embryonic stem cells. Magnetic resonance imaging and tractography were used as imaging modality to document the changes in the damaged cord, but the magnetic resonance imaging tractography was seen to be more sensitive in detecting the changes in the spinal cord. The present study was conducted to evaluate the diagnostic modality of magnetic resonance imaging tractography to determine the efficacy of human embryonic stem cells in chronic spinal cord injury. Materials and methods The study included the patients with spinal cord injury for whom magnetic resonance imaging tractography was performed before and after the therapy. Omniscan (gadodiamide) magnetic resonance imaging tractography was analyzed to assess the spinal defects and the improvement by human embryonic stem cell treatment. The patients were also scored by American Spinal Injury Association scale. Results Overall, 15 patients aged 15-44 years with clinical manifestations of spinal cord injury had magnetic resonance imaging tractography performed. The average treatment period was nine months. The majority of subjects ( n = 13) had American Spinal Injury Association score A, and two patients were at score C at the beginning of therapy. At the end of therapy, 10 patients were at score A, two patients were at score B and three patients were at score C. Improvements in patients were clearly understood through magnetic resonance imaging tractography as well as in clinical signs and symptoms. Conclusion Magnetic resonance imaging tractography can be a crucial diagnostic modality to assess the improvement in spinal cord injury patients.

Enhanced Bioactivity of Internally Functionalized Cationic Dendrimers with PEG Cores

DTIC Science & Technology

2012-11-09

Miltenyi) by counting 10 000 events. Cell Culture and Confocal Imaging. HeLa (CCL-2) were purchased from ATCC and cultured following manufacturer’s...concentration of PI before confocal imaging. Internalization Assay and Colocalization Studies. To monitor dendrimer internalization, cells were incubated...calcein. After 2 h of incubation at 37 °C, cells were washed three times with PBS and then analyzed by confocal microscopy. Ethidium Bromide Intercalation

Theranostic Gold Nanoantennas for Simultaneous Multiplexed Raman Imaging of Immunomarkers and Photothermal Therapy.

PubMed

Webb, Joseph A; Ou, Yu-Chuan; Faley, Shannon; Paul, Eden P; Hittinger, Joseph P; Cutright, Camden C; Lin, Eugene C; Bellan, Leon M; Bardhan, Rizia

2017-07-31

In this study, we demonstrate the theranostic capability of actively targeted, site-specific multibranched gold nanoantennas (MGNs) in triple-negative breast cancer (TNBC) cells in vitro. By utilizing multiplexed surface-enhanced Raman scattering (SERS) imaging, enabled by the narrow peak widths of Raman signatures, we simultaneously targeted immune checkpoint receptor programmed death ligand 1 (PDL1) and the epidermal growth factor receptor (EGFR) overexpressed in TNBC cells. A 1:1 mixture of MGNs functionalized with anti-PDL1 antibodies and Raman tag 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) and MGNs functionalized with anti-EGFR antibodies and Raman tag para -mercaptobenzoic acid ( p MBA) were incubated with the cells. SERS imaging revealed a cellular traffic map of MGN localization by surface binding and receptor-mediated endocytosis, enabling targeted diagnosis of both biomarkers. Furthermore, cells incubated with anti-EGFR- p MBA-MGNs and illuminated with an 808 nm laser for 15 min at 4.7 W/cm 2 exhibited photothermal cell death only within the laser spot (indicated by live/dead cell fluorescence assay). Therefore, this study not only provides an optical imaging platform that can track immunomarkers with spatiotemporal control but also demonstrates an externally controlled light-triggered therapeutic approach enabling receptor-specific treatment with biocompatible theranostic nanoprobes.

Theranostic Gold Nanoantennas for Simultaneous Multiplexed Raman Imaging of Immunomarkers and Photothermal Therapy

PubMed Central

2017-01-01

In this study, we demonstrate the theranostic capability of actively targeted, site-specific multibranched gold nanoantennas (MGNs) in triple-negative breast cancer (TNBC) cells in vitro. By utilizing multiplexed surface-enhanced Raman scattering (SERS) imaging, enabled by the narrow peak widths of Raman signatures, we simultaneously targeted immune checkpoint receptor programmed death ligand 1 (PDL1) and the epidermal growth factor receptor (EGFR) overexpressed in TNBC cells. A 1:1 mixture of MGNs functionalized with anti-PDL1 antibodies and Raman tag 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) and MGNs functionalized with anti-EGFR antibodies and Raman tag para-mercaptobenzoic acid (pMBA) were incubated with the cells. SERS imaging revealed a cellular traffic map of MGN localization by surface binding and receptor-mediated endocytosis, enabling targeted diagnosis of both biomarkers. Furthermore, cells incubated with anti-EGFR–pMBA–MGNs and illuminated with an 808 nm laser for 15 min at 4.7 W/cm2 exhibited photothermal cell death only within the laser spot (indicated by live/dead cell fluorescence assay). Therefore, this study not only provides an optical imaging platform that can track immunomarkers with spatiotemporal control but also demonstrates an externally controlled light-triggered therapeutic approach enabling receptor-specific treatment with biocompatible theranostic nanoprobes. PMID:28782050

Aptamer-conjugated and drug-loaded acoustic droplets for ultrasound theranosis.

PubMed

Wang, Chung-Hsin; Kang, Shih-Tsung; Lee, Ya-Hsuan; Luo, Yun-Ling; Huang, Yu-Fen; Yeh, Chih-Kuang

2012-02-01

Tumor therapy requires multi-functional treatment strategies with specific targeting of therapeutics to reduce general toxicity and increase efficacy. In this study we fabricated and functionally tested aptamer-conjugated and doxorubicin (DOX)-loaded acoustic droplets comprising cores of liquid perfluoropentane compound and lipid-based shell materials. Conjugation of sgc8c aptamers provided the ability to specifically target CCRF-CEM cells for both imaging and therapy. High-intensity focused ultrasound (HIFU) was introduced to trigger targeted acoustic droplet vaporization (ADV) which resulted in both mechanical cancer cell destruction by inertial cavitation and chemical treatment through localized drug release. HIFU insonation showed a 56.8% decrease in cell viability with aptamer-conjugated droplets, representing a 4.5-fold increase in comparison to non-conjugated droplets. In addition, the fully-vaporized droplets resulted in the highest DOX uptake by cancer cells, compared to non-vaporized or partially vaporized droplets. Optical studies clearly illustrated the transient changes that occurred upon ADV of droplet-targeted CEM cells, and B-mode ultrasound imaging revealed contrast enhancement by ADV in ultrasound images. In conclusion, our fabricated droplets functioned as a hybrid chemical and mechanical strategy for the specific destruction of cancer cells upon ultrasound-mediated ADV, while simultaneously providing ultrasound imaging capability. Copyright © 2011 Elsevier Ltd. All rights reserved.

Interaction of amino acid-functionalized silver nanoparticles and Candida albicans polymorphs: A deep-UV fluorescence imaging study.

PubMed

Dojčilović, Radovan; Pajović, Jelena D; Božanić, Dušan K; Bogdanović, Una; Vodnik, Vesna V; Dimitrijević-Branković, Suzana; Miljković, Miona G; Kaščaková, Slavka; Réfrégiers, Matthieu; Djoković, Vladimir

2017-07-01

The interaction of the tryptophan functionalized Ag nanoparticles and live Candida albicans cells was studied by synchrotron excitation deep-ultraviolet (DUV) fluorescence imaging at the DISCO beamline of Synchrotron SOLEIL. DUV imaging showed that incubation of the fungus with functionalized nanoparticles results in significant increase in the fluorescence signal. The analysis of the images revealed that the interaction of the nanoparticles with (pseudo)hyphae polymorphs of the diploid fungus was less pronounced than in the case of yeast cells or budding spores. The changes in the intensity of the fluorescence signals of the cells after incubation were followed in [327-353nm] and [370-410nm] spectral ranges that correspond to the fluorescence of tryptophan in non-polar and polar environment, respectively. As a consequence of the environmental sensitivity of the silver-tryptophan fluorescent nanoprobe, we were able to determine the possible accumulation sites of the nanoparticles. The analysis of the intensity decay kinetics showed that the photobleaching effects were more pronounced in the case of the functionalized nanoparticle treated cells. The results of time-integrated emission in the mentioned spectral ranges suggested that the nanoparticles penetrate the cells, but that the majority of the nanoparticles attach to the cells' surfaces. Copyright © 2017 Elsevier B.V. All rights reserved.

A study of the native cell wall structures of the marine alga Ventricaria ventricosa (Siphonocladales, Chlorophyceae) using atomic force microscopy.

PubMed

Eslick, Enid M; Beilby, Mary J; Moon, Anthony R

2014-04-01

A substantial proportion of the architecture of the plant cell wall remains unknown with a few cell wall models being proposed. Moreover, even less is known about the green algal cell wall. Techniques that allow direct visualization of the cell wall in as near to its native state are of importance in unravelling the spatial arrangement of cell wall structures and hence in the development of cell wall models. Atomic force microscopy (AFM) was used to image the native cell wall of living cells of Ventricaria ventricosa (V. ventricosa) at high resolution under physiological conditions. The cell wall polymers were identified mainly qualitatively via their structural appearance. The cellulose microfibrils (CMFs) were easily recognizable and the imaging results indicate that the V. ventricosa cell wall has a cross-fibrillar structure throughout. We found the native wall to be abundant in matrix polysaccharides existing in different curing states. The soft phase matrix polysaccharides susceptible by the AFM scanning tip existed as a glutinous fibrillar meshwork, possibly incorporating both the pectic- and hemicellulosic-type substances. The hard phase matrix producing clearer images, revealed coiled fibrillar structures associated with CMFs, sometimes being resolved as globular structures by the AFM tip. The coiling fibrillar structures were also seen in the images of isolated cell wall fragments. The mucilaginous component of the wall was discernible from the gelatinous cell wall matrix as it formed microstructural domains over the surface. AFM has been successful in imaging the native cell wall and revealing novel findings such as the 'coiling fibrillar structures' and cell wall components which have previously not been seen, that is, the gelatinous matrix phase.

Image analysis driven single-cell analytics for systems microbiology.

PubMed

Balomenos, Athanasios D; Tsakanikas, Panagiotis; Aspridou, Zafiro; Tampakaki, Anastasia P; Koutsoumanis, Konstantinos P; Manolakos, Elias S

2017-04-04

Time-lapse microscopy is an essential tool for capturing and correlating bacterial morphology and gene expression dynamics at single-cell resolution. However state-of-the-art computational methods are limited in terms of the complexity of cell movies that they can analyze and lack of automation. The proposed Bacterial image analysis driven Single Cell Analytics (BaSCA) computational pipeline addresses these limitations thus enabling high throughput systems microbiology. BaSCA can segment and track multiple bacterial colonies and single-cells, as they grow and divide over time (cell segmentation and lineage tree construction) to give rise to dense communities with thousands of interacting cells in the field of view. It combines advanced image processing and machine learning methods to deliver very accurate bacterial cell segmentation and tracking (F-measure over 95%) even when processing images of imperfect quality with several overcrowded colonies in the field of view. In addition, BaSCA extracts on the fly a plethora of single-cell properties, which get organized into a database summarizing the analysis of the cell movie. We present alternative ways to analyze and visually explore the spatiotemporal evolution of single-cell properties in order to understand trends and epigenetic effects across cell generations. The robustness of BaSCA is demonstrated across different imaging modalities and microscopy types. BaSCA can be used to analyze accurately and efficiently cell movies both at a high resolution (single-cell level) and at a large scale (communities with many dense colonies) as needed to shed light on e.g. how bacterial community effects and epigenetic information transfer play a role on important phenomena for human health, such as biofilm formation, persisters' emergence etc. Moreover, it enables studying the role of single-cell stochasticity without losing sight of community effects that may drive it.

Near-infrared fluorescent proteins for multicolor in vivo imaging

PubMed Central

Shcherbakova, Daria M.; Verkhusha, Vladislav V.

2013-01-01

Near-infrared fluorescent proteins are in high demand for in vivo imaging. We developed four spectrally distinct fluorescent proteins, iRFP670, iRFP682, iRFP702, and iRFP720, from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging in living cells and mice using standard approaches. Five iRFPs including previously engineered iRFP713 allow multicolor imaging in living mice with spectral unmixing. PMID:23770755

Adaptive optical imaging through complex living plant cells

NASA Astrophysics Data System (ADS)

Tamada, Yosuke; Hayano, Yutaka; Murata, Takashi; Oya, Shin; Honma, Yusuke; Kanazawa, Minoru; Miura, Noriaki; Hasebe, Mitsuyasu; Kamei, Yasuhiro; Hattori, Masayuki

2017-04-01

Live-cell imaging using fluorescent molecules is now essential for biological researches. However, images of living cells are accompanied with blur, which becomes stronger according to the depth inside the cells and tissues. This image blur is caused by the disturbance on light that goes through optically inhomogeneous living cells and tissues. Here, we show adaptive optics (AO) imaging of living plant cells. AO has been developed in astronomy to correct the disturbance on light caused by atmospheric turbulence. We developed AO microscope effective for the observation of living plant cells with strong disturbance by chloroplasts, and successfully obtained clear images inside plant cells.

Portable multispectral imaging system for oral cancer diagnosis

NASA Astrophysics Data System (ADS)

Hsieh, Yao-Fang; Ou-Yang, Mang; Lee, Cheng-Chung

2013-09-01

This study presents the portable multispectral imaging system that can acquire the image of specific spectrum in vivo for oral cancer diagnosis. According to the research literature, the autofluorescence of cells and tissue have been widely applied to diagnose oral cancer. The spectral distribution is difference for lesions of epithelial cells and normal cells after excited fluorescence. We have been developed the hyperspectral and multispectral techniques for oral cancer diagnosis in three generations. This research is the third generation. The excited and emission spectrum for the diagnosis are acquired from the research of first generation. The portable system for detection of oral cancer is modified for existing handheld microscope. The UV LED is used to illuminate the surface of oral cavity and excite the cells to produce fluorescent. The image passes through the central channel and filters out unwanted spectrum by the selection of filter, and focused by the focus lens on the image sensor. Therefore, we can achieve the specific wavelength image via fluorescence reaction. The specificity and sensitivity of the system are 85% and 90%, respectively.

Tc-99m Glu-Cys-Gly-His-Gly-Lys (ECG-HGK), a novel Tc-99m labeled hexapeptide for molecular tumor imaging.

PubMed

Kim, Dae-Weung; Kim, Myoung Hyoun; Kim, Chang Guhn

2016-03-01

Domain 5 of kinin-free high molecular weight kininogen inhibits the adhesion of many tumor cell lines, and it has been reported that the histidine-glycine-lysine (HGK)-rich region might be responsible for inhibition of cell adhesion. The authors developed HGK-containing hexapeptide, glutamic acid-cysteine-glycine (ECG)-HGK, and evaluated the utility of Tc-99m ECG-HGK for tumor imaging. Hexapeptide, ECG-HGK was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling efficiency was evaluated. The uptake of Tc-99m ECG-HGK within HT-1080 cells was evaluated in vitro. In HT-1080 tumor-bearing mice, gamma imaging and biodistribution studies were performed. The complexes Tc-99m ECG-HGK was prepared in high yield. The uptake of Tc-99m ECG-HGK within the HT-1080 tumor cells had been demonstrated by in vitro studies. The gamma camera imaging in the murine model showed that Tc-99m ECG-HGK was accumulated substantially in the HT-1080 tumor (tumor-to-muscle ratio = 5.7 ± 1.4 at 4 h), and the tumoral uptake was blocked by the co-injection of excess HGK (tumor-to-muscle ratio = 2.8 ± 0.6 at 4 h). In the present study, Tc-99m ECG-HGK was developed as a new tumor imaging agents. Our in vitro and in vivo studies revealed specific function of Tc-99m ECG-HGK for tumor imaging. Copyright © 2016 John Wiley & Sons, Ltd.

Folic acid-functionalized up-conversion nanoparticles: toxicity studies in vivo and in vitro and targeted imaging applications

NASA Astrophysics Data System (ADS)

Sun, Lining; Wei, Zuwu; Chen, Haige; Liu, Jinliang; Guo, Jianjian; Cao, Ming; Wen, Tieqiao; Shi, Liyi

2014-07-01

Folate receptors (FRs) are overexpressed on a variety of human cancer cells and tissues, including cancers of the breast, ovaries, endometrium, and brain. This over-expression of FRs can be used to target folate-linked imaging specifically to FR-expressing tumors. Fluorescence is emerging as a powerful new modality for molecular imaging in both the diagnosis and treatment of disease. Combining innovative molecular biology and chemistry, we prepared three kinds of folate-targeted up-conversion nanoparticles as imaging agents (UCNC-FA: UCNC-Er-FA, UCNC-Tm-FA, and UCNC-Er,Tm-FA). In vivo and in vitro toxicity studies showed that these nanoparticles have both good biocompatibility and low toxicity. Moreover, the up-conversion luminescence imaging indicated that they have good targeting to HeLa cells and can therefore serve as potential fluorescent contrast agents.Folate receptors (FRs) are overexpressed on a variety of human cancer cells and tissues, including cancers of the breast, ovaries, endometrium, and brain. This over-expression of FRs can be used to target folate-linked imaging specifically to FR-expressing tumors. Fluorescence is emerging as a powerful new modality for molecular imaging in both the diagnosis and treatment of disease. Combining innovative molecular biology and chemistry, we prepared three kinds of folate-targeted up-conversion nanoparticles as imaging agents (UCNC-FA: UCNC-Er-FA, UCNC-Tm-FA, and UCNC-Er,Tm-FA). In vivo and in vitro toxicity studies showed that these nanoparticles have both good biocompatibility and low toxicity. Moreover, the up-conversion luminescence imaging indicated that they have good targeting to HeLa cells and can therefore serve as potential fluorescent contrast agents. Electronic supplementary information (ESI) available: Up-conversion luminescence spectra of UCNC-Er and UCNC-Er-FA, UCNC-Tm and UCNC-Tm-FA. Confocal luminescence imaging data collected as a series along the Z optical axis. See DOI: 10.1039/c4nr02312a

Theoretical Analysis of Novel Quasi-3D Microscopy of Cell Deformation

PubMed Central

Qiu, Jun; Baik, Andrew D.; Lu, X. Lucas; Hillman, Elizabeth M. C.; Zhuang, Zhuo; Guo, X. Edward

2012-01-01

A novel quasi-three-dimensional (quasi-3D) microscopy technique has been developed to enable visualization of a cell under dynamic loading in two orthogonal planes simultaneously. The three-dimensional (3D) dynamics of the mechanical behavior of a cell under fluid flow can be examined at a high temporal resolution. In this study, a numerical model of a fluorescently dyed cell was created in 3D space, and the cell was subjected to uniaxial deformation or unidirectional fluid shear flow via finite element analysis (FEA). Therefore, the intracellular deformation in the simulated cells was exactly prescribed. Two-dimensional fluorescent images simulating the quasi-3D technique were created from the cell and its deformed states in 3D space using a point-spread function (PSF) and a convolution operation. These simulated original and deformed images were processed by a digital image correlation technique to calculate quasi-3D-based intracellular strains. The calculated strains were compared to the prescribed strains, thus providing a theoretical basis for the measurement of the accuracy of quasi-3D and wide-field microscopy-based intracellular strain measurements against the true 3D strains. The signal-to-noise ratio (SNR) of the simulated quasi-3D images was also modulated using additive Gaussian noise, and a minimum SNR of 12 was needed to recover the prescribed strains using digital image correlation. Our computational study demonstrated that quasi-3D strain measurements closely recovered the true 3D strains in uniform and fluid flow cellular strain states to within 5% strain error. PMID:22707985

Photoluminescence Imaging and LBIC Characterization of Defects in mc-Si Solar Cells

NASA Astrophysics Data System (ADS)

Sánchez, L. A.; Moretón, A.; Guada, M.; Rodríguez-Conde, S.; Martínez, O.; González, M. A.; Jiménez, J.

2018-05-01

Today's photovoltaic market is dominated by multicrystalline silicon (mc-Si) based solar cells with around 70% of worldwide production. In order to improve the quality of the Si material, a proper characterization of the electrical activity in mc-Si solar cells is essential. A full-wafer characterization technique such as photoluminescence imaging (PLi) provides a fast inspection of the wafer defects, though at the expense of the spatial resolution. On the other hand, a study of the defects at a microscopic scale can be achieved through the light-beam induced current technique. The combination of these macroscopic and microscopic resolution techniques allows a detailed study of the electrical activity of defects in mc-Si solar cells. In this work, upgraded metallurgical-grade Si solar cells are studied using these two techniques.

Gold nanorods for cell imaging with confocal reflectance microscopy and two-photon fluorescence microscopy

NASA Astrophysics Data System (ADS)

Chen, Ji-Yao; Wang, Pei-Nan

2010-02-01

Gold nanorods have unique optical properties as their two photon absorption cross sections are very high and their spectral positions of extinction bands can be controlled by their aspect ratio only, so that gold nanorods have been considered as agents for cell imaging. Two-photon photoluminescence imaging could be used to detect the cellular gold nanorods with the high power femto-second (fs) infrared laser, but may cause the photothermal effect melting the rods. The 3-D distribution of gold nanorods in living cells also can be measured by confocal reflectance microscopy with a very low laser power, and thus the cell damaging can be avoided. In this work, these two methods were comparatively studied in living rat basophilic leukemia (RBL-2H3) cells.

Using simulated fluorescence cell micrographs for the evaluation of cell image segmentation algorithms.

PubMed

Wiesmann, Veit; Bergler, Matthias; Palmisano, Ralf; Prinzen, Martin; Franz, Daniela; Wittenberg, Thomas

2017-03-18

Manual assessment and evaluation of fluorescent micrograph cell experiments is time-consuming and tedious. Automated segmentation pipelines can ensure efficient and reproducible evaluation and analysis with constant high quality for all images of an experiment. Such cell segmentation approaches are usually validated and rated in comparison to manually annotated micrographs. Nevertheless, manual annotations are prone to errors and display inter- and intra-observer variability which influence the validation results of automated cell segmentation pipelines. We present a new approach to simulate fluorescent cell micrographs that provides an objective ground truth for the validation of cell segmentation methods. The cell simulation was evaluated twofold: (1) An expert observer study shows that the proposed approach generates realistic fluorescent cell micrograph simulations. (2) An automated segmentation pipeline on the simulated fluorescent cell micrographs reproduces segmentation performances of that pipeline on real fluorescent cell micrographs. The proposed simulation approach produces realistic fluorescent cell micrographs with corresponding ground truth. The simulated data is suited to evaluate image segmentation pipelines more efficiently and reproducibly than it is possible on manually annotated real micrographs.

Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations

PubMed Central

Gustafsson, Nils; Culley, Siân; Ashdown, George; Owen, Dylan M.; Pereira, Pedro Matos; Henriques, Ricardo

2016-01-01

Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours. PMID:27514992

Raman hyperspectral imaging of iron transport across membranes in cells

NASA Astrophysics Data System (ADS)

Das, Anupam; Costa, Xavier Felipe; Khmaladze, Alexander; Barroso, Margarida; Sharikova, Anna

2016-09-01

Raman scattering microscopy is a powerful imaging technique used to identify chemical composition, structural and conformational state of molecules of complex samples in biology, biophysics, medicine and materials science. In this work, we have shown that Raman techniques allow the measurement of the iron content in protein mixtures and cells. Since the mechanisms of iron acquisition, storage, and excretion by cells are not completely understood, improved knowledge of iron metabolism can offer insight into many diseases in which iron plays a role in the pathogenic process, such as diabetes, neurodegenerative diseases, cancer, and metabolic syndrome. Understanding of the processes involved in cellular iron metabolism will improve our knowledge of cell functioning. It will also have a big impact on treatment of diseases caused by iron deficiency (anemias) and iron overload (hereditary hemochromatosis). Previously, Raman studies have shown substantial differences in spectra of transferrin with and without bound iron, thus proving that it is an appropriate technique to determine the levels of bound iron in the protein mixture. We have extended these studies to obtain hyperspectral images of transferrin in cells. By employing a Raman scanning microscope together with spectral detection by a highly sensitive back-illuminated cooled CCD camera, we were able to rapidly acquire and process images of fixed cells with chemical selectivity. We discuss and compare various methods of hyperspectral Raman image analysis and demonstrate the use of these methods to characterize cellular iron content without the need for dye labeling.

A single frame: imaging live cells twenty-five years ago.

PubMed

Fink, Rachel

2011-07-01

In the mid-1980s live-cell imaging was changed by the introduction of video techniques, allowing new ways to collect and store data. The increased resolution obtained by manipulating video signals, the ability to use time-lapse videocassette recorders to study events that happen over long time intervals, and the introduction of fluorescent probes and sensitive video cameras opened research avenues previously unavailable. The author gives a personal account of this evolution, focusing on cell migration studies at the Marine Biological Laboratory 25 years ago. Copyright © 2011 Wiley-Liss, Inc.

In vivo imaging of T cell lymphoma infiltration process at the colon.

PubMed

Ueda, Yoshibumi; Ishiwata, Toshiyuki; Shinji, Seiichi; Arai, Tomio; Matsuda, Yoko; Aida, Junko; Sugimoto, Naotoshi; Okazaki, Toshiro; Kikuta, Junichi; Ishii, Masaru; Sato, Moritoshi

2018-03-05

The infiltration and proliferation of cancer cells in the secondary organs are of great interest, since they contribute to cancer metastasis. However, cancer cell dynamics in the secondary organs have not been elucidated at single-cell resolution. In the present study, we established an in vivo model using two-photon microscopy to observe how infiltrating cancer cells form assemblages from single T-cell lymphomas, EL4 cells, in the secondary organs. Using this model, after inoculation of EL4 cells in mice, we discovered that single EL4 cells infiltrated into the colon. In the early stage, sporadic elongated EL4 cells became lodged in small blood vessels. Real-time imaging revealed that, whereas more than 70% of EL4 cells did not move during a 1-hour observation, other EL4 cells irregularly moved even in small vessels and dynamically changed shape upon interacting with other cells. In the late stages, EL4 cells formed small nodules composed of several EL4 cells in blood vessels as well as crypts, suggesting the existence of diverse mechanisms of nodule formation. The present in vivo imaging system is instrumental to dissect cancer cell dynamics during metastasis in other organs at the single-cell level.

Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells

PubMed Central

Akita, Kae; Higaki, Takumi; Kutsuna, Natsumaro; Hasezawa, Seiichiro

2015-01-01

Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells. PMID:26039484

Age estimation using exfoliative cytology and radiovisiography: A comparative study

PubMed Central

Nallamala, Shilpa; Guttikonda, Venkateswara Rao; Manchikatla, Praveen Kumar; Taneeru, Sravya

2017-01-01

Introduction: Age estimation is one of the essential factors in establishing the identity of an individual. Among various methods, exfoliative cytology (EC) is a unique, noninvasive technique, involving simple, and pain-free collection of intact cells from the oral cavity for microscopic examination. Objective: The study was undertaken with an aim to estimate the age of an individual from the average cell size of their buccal smears calculated using image analysis morphometric software and the pulp–tooth area ratio in mandibular canine of the same individual using radiovisiography (RVG). Materials and Methods: Buccal smears were collected from 100 apparently healthy individuals. After fixation in 95% alcohol, the smears were stained using Papanicolaou stain. The average cell size was measured using image analysis software (Image-Pro Insight 8.0). The RVG images of mandibular canines were obtained, pulp and tooth areas were traced using AutoCAD 2010 software, and area ratio was calculated. The estimated age was then calculated using regression analysis. Results: The paired t-test between chronological age and estimated age by cell size and pulp–tooth area ratio was statistically nonsignificant (P > 0.05). Conclusion: In the present study, age estimated by pulp–tooth area ratio and EC yielded good results. PMID:29657491

Age estimation using exfoliative cytology and radiovisiography: A comparative study.

PubMed

Nallamala, Shilpa; Guttikonda, Venkateswara Rao; Manchikatla, Praveen Kumar; Taneeru, Sravya

2017-01-01

Age estimation is one of the essential factors in establishing the identity of an individual. Among various methods, exfoliative cytology (EC) is a unique, noninvasive technique, involving simple, and pain-free collection of intact cells from the oral cavity for microscopic examination. The study was undertaken with an aim to estimate the age of an individual from the average cell size of their buccal smears calculated using image analysis morphometric software and the pulp-tooth area ratio in mandibular canine of the same individual using radiovisiography (RVG). Buccal smears were collected from 100 apparently healthy individuals. After fixation in 95% alcohol, the smears were stained using Papanicolaou stain. The average cell size was measured using image analysis software (Image-Pro Insight 8.0). The RVG images of mandibular canines were obtained, pulp and tooth areas were traced using AutoCAD 2010 software, and area ratio was calculated. The estimated age was then calculated using regression analysis. The paired t -test between chronological age and estimated age by cell size and pulp-tooth area ratio was statistically nonsignificant ( P > 0.05). In the present study, age estimated by pulp-tooth area ratio and EC yielded good results.

Extraction of the number of peroxisomes in yeast cells by automated image analysis.

PubMed

Niemistö, Antti; Selinummi, Jyrki; Saleem, Ramsey; Shmulevich, Ilya; Aitchison, John; Yli-Harja, Olli

2006-01-01

An automated image analysis method for extracting the number of peroxisomes in yeast cells is presented. Two images of the cell population are required for the method: a bright field microscope image from which the yeast cells are detected and the respective fluorescent image from which the number of peroxisomes in each cell is found. The segmentation of the cells is based on clustering the local mean-variance space. The watershed transformation is thereafter employed to separate cells that are clustered together. The peroxisomes are detected by thresholding the fluorescent image. The method is tested with several images of a budding yeast Saccharomyces cerevisiae population, and the results are compared with manually obtained results.

Supervised graph hashing for histopathology image retrieval and classification.

PubMed

Shi, Xiaoshuang; Xing, Fuyong; Xu, KaiDi; Xie, Yuanpu; Su, Hai; Yang, Lin

2017-12-01

In pathology image analysis, morphological characteristics of cells are critical to grade many diseases. With the development of cell detection and segmentation techniques, it is possible to extract cell-level information for further analysis in pathology images. However, it is challenging to conduct efficient analysis of cell-level information on a large-scale image dataset because each image usually contains hundreds or thousands of cells. In this paper, we propose a novel image retrieval based framework for large-scale pathology image analysis. For each image, we encode each cell into binary codes to generate image representation using a novel graph based hashing model and then conduct image retrieval by applying a group-to-group matching method to similarity measurement. In order to improve both computational efficiency and memory requirement, we further introduce matrix factorization into the hashing model for scalable image retrieval. The proposed framework is extensively validated with thousands of lung cancer images, and it achieves 97.98% classification accuracy and 97.50% retrieval precision with all cells of each query image used. Copyright © 2017 Elsevier B.V. All rights reserved.

Optical Imaging of Targeted β-Galactosidase in Brain Tumors to Detect EGFR Levels

PubMed Central

Broome, Ann-Marie; Ramamurthy, Gopal; Lavik, Kari; Liggett, Alexander; Kinstlinger, Ian; Basilion, James

2015-01-01

A current limitation in molecular imaging is that it often requires genetic manipulation of cancer cells for noninvasive imaging. Other methods to detect tumor cells in vivo using exogenously delivered and functionally active reporters, such as β-gal, are required. We report the development of a platform system for linking β-gal to any number of different ligands or antibodies for in vivo targeting to tissue or cells, without the requirement for genetic engineering of the target cells prior to imaging. Our studies demonstrate significant uptake in vitro and in vivo of an EGFR-targeted β-gal complex. We were then able to image orthotopic brain tumor accumulation and localization of the targeted enzyme when a fluorophore was added to the complex, as well as validate the internalization of the intravenously administered β-gal reporter complex ex vivo. After fluorescence imaging localized the β-gal complexes to the brain tumor, we topically applied a bioluminescent β-gal substrate to serial sections of the brain to evaluate the delivery and integrity of the enzyme. Finally, robust bioluminescence of the EGFR-targeted β-gal complex was captured within the tumor during noninvasive in vivo imaging. PMID:25775241

Optical imaging of targeted β-galactosidase in brain tumors to detect EGFR levels.

PubMed

Broome, Ann-Marie; Ramamurthy, Gopal; Lavik, Kari; Liggett, Alexander; Kinstlinger, Ian; Basilion, James

2015-04-15

A current limitation in molecular imaging is that it often requires genetic manipulation of cancer cells for noninvasive imaging. Other methods to detect tumor cells in vivo using exogenously delivered and functionally active reporters, such as β-gal, are required. We report the development of a platform system for linking β-gal to any number of different ligands or antibodies for in vivo targeting to tissue or cells, without the requirement for genetic engineering of the target cells prior to imaging. Our studies demonstrate significant uptake in vitro and in vivo of an EGFR-targeted β-gal complex. We were then able to image orthotopic brain tumor accumulation and localization of the targeted enzyme when a fluorophore was added to the complex, as well as validate the internalization of the intravenously administered β-gal reporter complex ex vivo. After fluorescence imaging localized the β-gal complexes to the brain tumor, we topically applied a bioluminescent β-gal substrate to serial sections of the brain to evaluate the delivery and integrity of the enzyme. Finally, robust bioluminescence of the EGFR-targeted β-gal complex was captured within the tumor during noninvasive in vivo imaging.

Functionalization of titanium surface with chitosan via silanation: 3D CLSM imaging of cell biocompatibility behaviour.

PubMed

Attik, G N; D'Almeida, M; Toury, B; Grosgogeat, B

2013-09-16

Biocompatibility ranks as one of the most important properties of dental materials. One of the criteria for biocompatibility is the absence of material toxicity to cells, according to the ISO 7405 and 10993 recommendations. Among numerous available methods for toxicity assessment; 3-dimensional Confocal Laser Scanning Microscopy (3D CLSM) imaging was chosen because it provides an accurate and sensitive index of living cell behavior in contact with chitosan coated tested implants. The purpose of this study was to investigate the in vitro biocompatibility of functionalized titanium with chitosan via a silanation using sensitive and innovative 3D CLSM imaging as an investigation method for cytotoxicity assessment. The biocompatibility of four samples (controls cells, TA6V, TA6V-TESBA and TA6V-TESBAChitosan) was compared in vitro after 24h of exposure. Confocal imaging was performed on cultured human gingival fibroblast (HGF1) like cells using Live/Dead® staining. Image series were obtained with a FV10i confocal biological inverted system and analyzed with FV10-ASW 3.1 Software (Olympus France). Image analysis showed no cytotoxicity in the presence of the three tested substrates after 24 h of contact. A slight decrease of cell viability was found in contact with TA6V-TESBA with and without chitosan compared to negative control cells. Our findings highlighted the use of 3D CLSM confocal imaging as a sensitive method to evaluate qualitatively and quantitatively the biocompatibility behavior of functionalized titanium with chitosan via a silanation. The biocompatibility of the new functionalized coating to HGF1 cells is as good as the reference in biomedical device implantation TA6V.

New water soluble Hg2 + selective fluorescent calix[4]arenes: Synthesis and application in living cells imaging

NASA Astrophysics Data System (ADS)

Oguz, Mehmet; Bhatti, Asif Ali; Karakurt, Serdar; Aktas, Mehmet; Yilmaz, Mustafa

2017-01-01

The present study demonstrates the synthesis of water-soluble fluorescent calix[4]arenes (6 and 7) and its application in living cell imaging for Hg2 + detection at a low level. The synthesized fluorescent ligands 6 and 7 were characterized by 1H NMR technique. The fluorescent study showed both water soluble ligands were Hg2 + selective and follow photo-induced electron transfer (PET) process. From the fluorimeter titration experiment detection limit was calculated as 1.14 × 10- 5 and 3.42 × 10- 5 for ligand 6 and 7, respectively. From the Benesi-Hildebrand plot binding constant values were evaluated as 666.7 and 733.3 M- 1 for 6 and 7, respectively. The interactions between ligands 6 and 7 and Hg2 + were also demonstrated in living cells, SW-620, using Fluorescent Cell Imager. While ligands 6 and 7 alone show fluorescent properties, they loss their action with the presence of Hg2 + in SW-620 cells.

Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

NASA Astrophysics Data System (ADS)

Winckler, Pascale; Lartigue, Lydia; Giannone, Gregory; de Giorgi, Francesca; Ichas, François; Sibarita, Jean-Baptiste; Lounis, Brahim; Cognet, Laurent

2013-08-01

Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.

Studying leukemia metastasis and therapy monitoring by in vivo imaging and flow cytometer

NASA Astrophysics Data System (ADS)

Wei, Xunbin; Li, Yan; Tan, Yuan; Zhang, Li; Chen, Yun; Liu, Guangda; Chen, Tong; Gu, Zhenqin; Wang, Guiying; Zhou, Zenghui; Wang, Li; Wang, Chen

2007-11-01

Cytotoxic chemotherapy agents are the foundation of current leukemia therapy. For a large number of adult and elderly patients, however, treatment options are poor. These patients may suffer from disease that is resistant to conventional chemotherapy or may not be candidates for curative therapies because of advanced age or poor medical conditions. To control disease in these patients, new therapies must be developed that are selectively targeted to unique characteristics of leukemic cell growth and metastasis. A large body of elegant work in the field of immunology has demonstrated the mechanisms whereby leukocytes traffic to specific sites within the body. Vascular cell adhesion molecules and chemicalattractants combine to direct white blood cells to appropriate environments. Although it has been hypothesized that leukemic white blood cells home to hematopoietic organs using mechanisms similar to those of their benign leukocyte counterparts, detailed study of leukemic cell transit through bone marrow has yet to be undertaken. We develop the "in vivo microscopy" to study the mechanisms that govern leukemic cell spread through the bone marrow microenvironment in vivo in real-time confocal infrared fluorescence imaging. A recently developed "in vivo flow cytometer" and optical imaging are used to assess leukemic cell spreading and the circulation kinetics of leukemic cells. A real- time quantitative monitoring of circulating leukemic cells by the in vivo flow cytometer will be useful to assess the effectiveness of the potential therapeutic interventions.

In-Vivo Imaging of Cell Migration Using Contrast Enhanced MRI and SVM Based Post-Processing.

PubMed

Weis, Christian; Hess, Andreas; Budinsky, Lubos; Fabry, Ben

2015-01-01

The migration of cells within a living organism can be observed with magnetic resonance imaging (MRI) in combination with iron oxide nanoparticles as an intracellular contrast agent. This method, however, suffers from low sensitivity and specificty. Here, we developed a quantitative non-invasive in-vivo cell localization method using contrast enhanced multiparametric MRI and support vector machines (SVM) based post-processing. Imaging phantoms consisting of agarose with compartments containing different concentrations of cancer cells labeled with iron oxide nanoparticles were used to train and evaluate the SVM for cell localization. From the magnitude and phase data acquired with a series of T2*-weighted gradient-echo scans at different echo-times, we extracted features that are characteristic for the presence of superparamagnetic nanoparticles, in particular hyper- and hypointensities, relaxation rates, short-range phase perturbations, and perturbation dynamics. High detection quality was achieved by SVM analysis of the multiparametric feature-space. The in-vivo applicability was validated in animal studies. The SVM detected the presence of iron oxide nanoparticles in the imaging phantoms with high specificity and sensitivity with a detection limit of 30 labeled cells per mm3, corresponding to 19 μM of iron oxide. As proof-of-concept, we applied the method to follow the migration of labeled cancer cells injected in rats. The combination of iron oxide labeled cells, multiparametric MRI and a SVM based post processing provides high spatial resolution, specificity, and sensitivity, and is therefore suitable for non-invasive in-vivo cell detection and cell migration studies over prolonged time periods.

Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

PubMed

Svitkina, Tatyana M

2017-05-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platinum Replica Electron Microscopy: Imaging the Cytoskeleton Globally and Locally

PubMed Central

SVITKINA, Tatyana M.

2017-01-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the “comfort zones” of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. PMID:28323208

Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

PubMed

Autour, Alexis; C Y Jeng, Sunny; D Cawte, Adam; Abdolahzadeh, Amir; Galli, Angela; Panchapakesan, Shanker S S; Rueda, David; Ryckelynck, Michael; Unrau, Peter J

2018-02-13

Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

Hetero-bivalent Imaging Agents for Simultaneous Targeting Prostate-Specific Membrane Antigen (PSMA) and Hepsin

DTIC Science & Technology

2013-09-01

Suzuki cross-coupling reaction. The effects of solvent and base on the synthesis of 3 were studied using Pd(PPh3)4 as catalyst . DMF, ethanol, and DMSO...PSMA/hepsin for in vitro cell uptake and in vivo imaging studies . Compound 13 showed a low but detectable increased cell uptake into the developed...have been comprehensive clinical studies whether PSA testing is an efficient biomarker in diagnosing PCa and reducing PCa deaths. Two European studies

Three-dimensional in vitro cancer spheroid models for Photodynamic Therapy: Strengths and Opportunities

NASA Astrophysics Data System (ADS)

Evans, Conor

2015-03-01

Three dimensional, in vitro spheroid cultures offer considerable utility for the development and testing of anticancer photodynamic therapy regimens. More complex than monolayer cultures, three-dimensional spheroid systems replicate many of the important cell-cell and cell-matrix interactions that modulate treatment response in vivo. Simple enough to be grown by the thousands and small enough to be optically interrogated, spheroid cultures lend themselves to high-content and high-throughput imaging approaches. These advantages have enabled studies investigating photosensitizer uptake, spatiotemporal patterns of therapeutic response, alterations in oxygen diffusion and consumption during therapy, and the exploration of mechanisms that underlie therapeutic synergy. The use of quantitative imaging methods, in particular, has accelerated the pace of three-dimensional in vitro photodynamic therapy studies, enabling the rapid compilation of multiple treatment response parameters in a single experiment. Improvements in model cultures, the creation of new molecular probes of cell state and function, and innovations in imaging toolkits will be important for the advancement of spheroid culture systems for future photodynamic therapy studies.

Novel snapshot hyperspectral imager for fluorescence imaging

NASA Astrophysics Data System (ADS)

Chandler, Lynn; Chandler, Andrea; Periasamy, Ammasi

2018-02-01

Hyperspectral imaging has emerged as a new technique for the identification and classification of biological tissue1. Benefitting recent developments in sensor technology, the new class of hyperspectral imagers can capture entire hypercubes with single shot operation and it shows great potential for real-time imaging in biomedical sciences. This paper explores the use of a SnapShot imager in fluorescence imaging via microscope for the very first time. Utilizing the latest imaging sensor, the Snapshot imager is both compact and attachable via C-mount to any commercially available light microscope. Using this setup, fluorescence hypercubes of several cells were generated, containing both spatial and spectral information. The fluorescence images were acquired with one shot operation for all the emission range from visible to near infrared (VIS-IR). The paper will present the hypercubes obtained images from example tissues (475-630nm). This study demonstrates the potential of application in cell biology or biomedical applications for real time monitoring.

Cryo-imaging of fluorescently labeled single cells in a mouse

NASA Astrophysics Data System (ADS)

Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

2009-02-01

We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron-scale, fluorescence, and bright field image data. Here we describe our image preprocessing, analysis, and visualization techniques. Processing improves axial resolution, reduces subsurface fluorescence by 97%, and enables single cell detection and counting. High quality 3D volume renderings enable us to evaluate cell distribution patterns. Applications include the myriad of biomedical experiments using fluorescent reporter gene and exogenous fluorophore labeling of cells in applications such as stem cell regenerative medicine, cancer, tissue engineering, etc.

In vivo nanoparticle imaging of innate immune cells can serve as a marker of disease severity in a model of multiple sclerosis.

PubMed

Kirschbaum, Klara; Sonner, Jana K; Zeller, Matthias W; Deumelandt, Katrin; Bode, Julia; Sharma, Rakesh; Krüwel, Thomas; Fischer, Manuel; Hoffmann, Angelika; Costa da Silva, Milene; Muckenthaler, Martina U; Wick, Wolfgang; Tews, Björn; Chen, John W; Heiland, Sabine; Bendszus, Martin; Platten, Michael; Breckwoldt, Michael O

2016-11-15

Innate immune cells play a key role in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Current clinical imaging is restricted to visualizing secondary effects of inflammation, such as gliosis and blood-brain barrier disruption. Advanced molecular imaging, such as iron oxide nanoparticle imaging, can allow direct imaging of cellular and molecular activity, but the exact cell types that phagocytose nanoparticles in vivo and how phagocytic activity relates to disease severity is not well understood. In this study we used MRI to map inflammatory infiltrates using high-field MRI and fluorescently labeled cross-linked iron oxide nanoparticles for cell tracking. We confirmed nanoparticle uptake and MR detectability ex vivo. Using in vivo MRI, we identified extensive nanoparticle signal in the cerebellar white matter and circumscribed cortical gray matter lesions that developed during the disease course (4.6-fold increase of nanoparticle accumulation in EAE compared with healthy controls, P < 0.001). Nanoparticles showed good cellular specificity for innate immune cells in vivo, labeling activated microglia, infiltrating macrophages, and neutrophils, whereas there was only sparse uptake by adaptive immune cells. Importantly, nanoparticle signal correlated better with clinical disease than conventional gadolinium (Gd) imaging (r, 0.83 for nanoparticles vs. 0.71 for Gd-imaging, P < 0.001). We validated our approach using the Food and Drug Administration-approved iron oxide nanoparticle ferumoxytol. Our results show that noninvasive molecular imaging of innate immune responses can serve as an imaging biomarker of disease activity in autoimmune-mediated neuroinflammation with potential clinical applications in a wide range of inflammatory diseases.

Imaging Neuroinflammation – from Bench to Bedside

PubMed Central

Pulli, Benjamin; Chen, John W

2014-01-01

Neuroinflammation plays a central role in a variety of neurological diseases, including stroke, multiple sclerosis, Alzheimer’s disease, and malignant CNS neoplasms, among many other. Different cell types and molecular mediators participate in a cascade of events in the brain that is ultimately aimed at control, regeneration and repair, but leads to damage of brain tissue under pathological conditions. Non-invasive molecular imaging of key players in the inflammation cascade holds promise for identification and quantification of the disease process before it is too late for effective therapeutic intervention. In this review, we focus on molecular imaging techniques that target inflammatory cells and molecules that are of interest in neuroinflammation, especially those with high translational potential. Over the past decade, a plethora of molecular imaging agents have been developed and tested in animal models of (neuro)inflammation, and a few have been translated from bench to bedside. The most promising imaging techniques to visualize neuroinflammation include MRI, positron emission tomography (PET), single photon emission computed tomography (SPECT), and optical imaging methods. These techniques enable us to image adhesion molecules to visualize endothelial cell activation, assess leukocyte functions such as oxidative stress, granule release, and phagocytosis, and label a variety of inflammatory cells for cell tracking experiments. In addition, several cell types and their activation can be specifically targeted in vivo, and consequences of neuroinflammation such as neuronal death and demyelination can be quantified. As we continue to make progress in utilizing molecular imaging technology to study and understand neuroinflammation, increasing efforts and investment should be made to bring more of these novel imaging agents from the “bench to bedside.” PMID:25525560

Differentiating intratumoral melanocytes from Langerhans cells in nonmelanocytic pigmented skin tumors in vivo by label-free third-harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Weng, Wei-Hung; Liao, Yi-Hua; Tsai, Ming-Rung; Wei, Ming-Liang; Huang, Hsin-Yi; Sun, Chi-Kuang

2016-07-01

Morphology and distribution of melanocytes are critical imaging information for the diagnosis of melanocytic lesions. However, how to image intratumoral melanocytes noninvasively in pigmented skin tumors is seldom investigated. Third-harmonic generation (THG) is shown to be enhanced by melanin, whereas high accuracy has been demonstrated using THG microscopy for in vivo differential diagnosis of nonmelanocytic pigmented skin tumors. It is thus desirable to investigate if label-free THG microscopy was capable to in vivo identify intratumoral melanocytes. In this study, histopathological correlations of label-free THG images with the immunohistochemical images stained with human melanoma black (HMB)-45 and cluster of differentiation 1a (CD1a) were made. The correlation results indicated that the intratumoral THG-bright dendritic-cell-like signals were endogenously derived from melanocytes rather than Langerhans cells (LCs). The consistency between THG-bright dendritic-cell-like signals and HMB-45 melanocyte staining showed a kappa coefficient of 0.807, 84.6% sensitivity, and 95% specificity. In contrast, a kappa coefficient of -0.37, 21.7% sensitivity, and 30% specificity were noted between the THG-bright dendritic-cell-like signals and CD1a staining for LCs. Our study indicates the capability of noninvasive label-free THG microscopy to differentiate intratumoral melanocytes from LCs, which is not feasible in previous in vivo label-free clinical-imaging modalities.

Fluorine-containing nanoemulsions for MRI cell tracking

PubMed Central

Janjic, Jelena M.; Ahrens, Eric T.

2009-01-01

In this article we review the chemistry and nanoemulsion formulation of perfluorocarbons used for in vivo 19F MRI cell tracking. In this application, cells of interest are labeled in culture using a perfluorocarbon nanoemulsion. Labeled cells are introduced into a subject and tracked using 19F MRI or NMR spectroscopy. In the same imaging session, a high-resolution, conventional (1H) image can be used to place the 19F-labeled cells into anatomical context. Perfluorocarbon-based 19F cell tracking is a useful technology because of the high specificity for labeled cells, ability to quantify cell accumulations, and biocompatibility. This technology can be widely applied to studies of inflammation, cellular regenerative medicine, and immunotherapy. PMID:19920872

Motion of single MreB bacterial actin proteins in Caulobacter show treadmilling in vivo

NASA Astrophysics Data System (ADS)

Moerner, W. E.; Kim, Soyeon; Gitai, Zemer; Kinkhabwala, Anika; McAdams, Harley; Shapiro, Lucy

2006-03-01

Ensemble imaging of a bacterial actin homologue, the MreB protein, suggests that the MreB proteins form a dynamic filamentous spiral along the long axis of the cell in Caulobacter crescentus. MreB contracts and expands along the cell axis and plays an important role in cell shape and polarity maintenance, as well as chromosome segregation and translocation of the origin of replication during cell division. In this study we investigated the real-time polymerization of MreB in Caulobacter crescentus using single-molecule fluorescence imaging. With time-lapse imaging, polymerized MreB could be distinguished from cytoplasmic MreB monomers, because single monomeric MreB showed fast motion characteristic of Brownian diffusion, while single polymerized MreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer implies that treadmilling is the predominant mechanism in MreB filament formation. These single-molecule imaging experiments provide the first available information on the velocity of bacterial actin polymerization in a living cell.

pHLIP-FIRE, a Cell Insertion-Triggered Fluorescent Probe for Imaging Tumors Demonstrates Targeted Cargo Delivery In Vivo

PubMed Central

2015-01-01

We have developed an improved tool for imaging acidic tumors by reporting the insertion of a transmembrane helix: the pHLIP-Fluorescence Insertion REporter (pHLIP-FIRE). In acidic tissues, such as tumors, peptides in the pHLIP family insert as α-helices across cell membranes. The cell-inserting end of the pHLIP-FIRE peptide has a fluorophore–fluorophore or fluorophore–quencher pair. A pair member is released by disulfide cleavage after insertion into the reducing environment inside a cell, resulting in dequenching of the probe. Thus, the fluorescence of the pHLIP-FIRE probe is enhanced upon cell-insertion in the targeted tissues but is suppressed elsewhere due to quenching. Targeting studies in mice bearing breast tumors show strong signaling by pHLIP-FIRE, with a contrast index of ∼17, demonstrating (i) direct imaging of pHLIP insertion and (ii) cargo translocation in vivo. Imaging and targeted cargo delivery should each have clinical applications. PMID:25184440

Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins.

PubMed

Venkataramani, Varun; Kardorff, Markus; Herrmannsdörfer, Frank; Wieneke, Ralph; Klein, Alina; Tampé, Robert; Heilemann, Mike; Kuner, Thomas

2018-04-03

With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the 'labeling barrier' and to bypass photobleaching in multi-plane, whole-cell 3D experiments.

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice.

PubMed

Feeks, James A; Hunter, Jennifer J

2017-05-01

In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina.

An entirely automated method to score DSS-induced colitis in mice by digital image analysis of pathology slides

PubMed Central

Kozlowski, Cleopatra; Jeet, Surinder; Beyer, Joseph; Guerrero, Steve; Lesch, Justin; Wang, Xiaoting; DeVoss, Jason; Diehl, Lauri

2013-01-01

SUMMARY The DSS (dextran sulfate sodium) model of colitis is a mouse model of inflammatory bowel disease. Microscopic symptoms include loss of crypt cells from the gut lining and infiltration of inflammatory cells into the colon. An experienced pathologist requires several hours per study to score histological changes in selected regions of the mouse gut. In order to increase the efficiency of scoring, Definiens Developer software was used to devise an entirely automated method to quantify histological changes in the whole H&E slide. When the algorithm was applied to slides from historical drug-discovery studies, automated scores classified 88% of drug candidates in the same way as pathologists’ scores. In addition, another automated image analysis method was developed to quantify colon-infiltrating macrophages, neutrophils, B cells and T cells in immunohistochemical stains of serial sections of the H&E slides. The timing of neutrophil and macrophage infiltration had the highest correlation to pathological changes, whereas T and B cell infiltration occurred later. Thus, automated image analysis enables quantitative comparisons between tissue morphology changes and cell-infiltration dynamics. PMID:23580198

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

PubMed Central

Malide, Daniela; Métais, Jean-Yves; Dunbar, Cynthia E.

2014-01-01

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner. PMID:25145579

Upright Imaging of Drosophila Egg Chambers

PubMed Central

Manning, Lathiena; Starz-Gaiano, Michelle

2015-01-01

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective. PMID:25867882

Investigations of (99m)Tc-labeled glucarate as a SPECT radiotracer for non-small cell lung cancer (NSCLC) and potential tumor uptake mechanism.

PubMed

Meng, Lanfang; Xiu, Yan; Li, Yanli; Xu, Xiaobo; Li, Shanqun; Li, Xiao; Pak, Koon Y; Shi, Hongcheng; Cheng, Dengfeng

2015-07-01

This study attempted to evaluate the feasibility of (99m)Tc-labeled glucarate ((99m)Tc-GLA) imaging in non-small cell lung cancer (NSCLC) and the potential tumor uptake mechanism. Cell lysates from two NSCLC cell lines, H292 and H1975, were immunoblotted with anti-glucose transporter 5 (GLUT5) antibody for Western blotting. Thereafter, the two cell lines were used to examine cellular uptake of (99m)Tc-GLA with or without fructose. SPECT/CT imaging studies were performed on small animals bearing H292 and H1975 tumors. Biodistribution studies were also conducted to achieve accurate tissue uptake of this tracer in two tumor models. Hematoxylin & eosin (H&E) staining and GLUT5, Ki67 and cytokeratin-7 (CK-7) immunohistochemistry (IHC) analysis were further investigated on tumor tissues. In Western blotting, H292 cells showed higher levels of GLUT5 compared to the H1975 cells. Meanwhile, the in vitro cell assays indicated GLUT5-dependent uptake of (99m)Tc-GLA in H292 and H1975 cells. The fructose competition assays showed a significant decrease in (99m)Tc-GLA uptake by H292 and H1975 cells when fructose was added. The (99m)Tc-GLA accumulation was as much as two-fold higher in H292 implanted tumors than in H1975 implanted tumors. (99m)Tc-GLA exhibited rapid clearance pharmacokinetics and reasonable uptake in human NSCLC H292 (1.69±0.37 ID%/g) and H1975 (0.89±0.06 ID%/g) implanted tumors at 30min post injection. Finally, the expression of GLUT5, Ki67 and CK-7 on tumor tissues also exhibited positive correlation with the in vitro cell test results and in vivo SPECT/CT imaging results in xenograft tumors. Both in vitro and ex vivo studies demonstrated that the uptake of (99m)Tc-GLA in NSCLC is highly related to GLUT5 expression. Imaging and further IHC results support that (99m)Tc-GLA could be a promising SPECT imaging agent for NSCLC diagnosis and prognosis evaluation. Copyright © 2015 Elsevier Inc. All rights reserved.

[Future perspectives for diagnostic imaging in urology: from anatomic and functional to molecular imaging].

PubMed

Macis, Giuseppe; Di Giovanni, Silvia; Di Franco, Davide; Bonomo, Lorenzo

2013-01-01

The future approach of diagnostic imaging in urology follows the technological progress, which made the visualization of in vivo molecular processes possible. From anatomo-morphological diagnostic imaging and through functional imaging molecular radiology is reached. Based on molecular probes, imaging is aimed at assessing the in vivo molecular processes, their physiology and function at cellular level. The future imaging will investigate the complex tumor functioning as metabolism, aerobic glycolysis in particular, angiogenesis, cell proliferation, metastatic potential, hypoxia, apoptosis and receptors expressed by neoplastic cells. Methods for performing molecular radiology are CT, MRI, PET-CT, PET-MRI, SPECT and optical imaging. Molecular ultrasound combines technological advancement with targeted contrast media based on microbubbles, this allowing the selective registration of microbubble signal while that of stationary tissues is suppressed. An experimental study was carried out where the ultrasound molecular probe BR55 strictly bound to prostate tumor results in strong enhancement in the early phase after contrast, this contrast being maintained in the late phase. This late enhancement is markedly significant for the detection of prostatic cancer foci and to guide the biopsy sampling. The 124I-cG250 molecular antibody which is strictly linked to cellular carbonic anhydrase IX of clear cell renal carcinoma, allows the acquisition of diagnostic PET images of clear cell renal carcinoma without biopsy. This WG-250 (RENCAREX) antibody was used as a therapy in metastatic clear cell renal carcinoma. Future advancements and applications will result in early cancer diagnosis, personalized therapy that will be specific according to the molecular features of cancer and leading to the development of catheter-based multichannel molecular imaging devices for cystoscopy-based molecular imaging diagnosis and intervention.

A spectral k-means approach to bright-field cell image segmentation.

PubMed

Bradbury, Laura; Wan, Justin W L

2010-01-01

Automatic segmentation of bright-field cell images is important to cell biologists, but difficult to complete due to the complex nature of the cells in bright-field images (poor contrast, broken halo, missing boundaries). Standard approaches such as level set segmentation and active contours work well for fluorescent images where cells appear as round shape, but become less effective when optical artifacts such as halo exist in bright-field images. In this paper, we present a robust segmentation method which combines the spectral and k-means clustering techniques to locate cells in bright-field images. This approach models an image as a matrix graph and segment different regions of the image by computing the appropriate eigenvectors of the matrix graph and using the k-means algorithm. We illustrate the effectiveness of the method by segmentation results of C2C12 (muscle) cells in bright-field images.

Tumor Targeting via Sialic Acid: [68Ga]DOTA-en-pba as a New Tool for Molecular Imaging of Cancer with PET.

PubMed

Tsoukalas, Charalambos; Geninatti-Crich, Simonetta; Gaitanis, Anastasios; Tsotakos, Theodoros; Paravatou-Petsotas, Maria; Aime, Silvio; Jiménez-Juárez, Rogelio; Anagnostopoulos, Constantinos D; Djanashvili, Kristina; Bouziotis, Penelope

2018-02-20

The aim of this study was to demonstrate the potential of Ga-68-labeled macrocycle (DOTA-en-pba) conjugated with phenylboronic vector for tumor recognition by positron emission tomography (PET), based on targeting of the overexpressed sialic acid (Sia). The imaging reporter DOTA-en-pba was synthesized and labeled with Ga-68 at high efficiency. Cell binding assay on Mel-C and B16-F10 melanoma cells was used to evaluate melanin production and Sia overexpression to determine the best model for demonstrating the capability of [ 68 Ga]DOTA-en-pba to recognize tumors. The in vivo PET imaging was done with B16-F10 tumor-bearing SCID mice injected with [ 68 Ga]DOTA-en-pba intravenously. Tumor, blood, and urine metabolites were assessed to evaluate the presence of a targeting agent. The affinity of [ 68 Ga]DOTA-en-pba to Sia was demonstrated on B16-F10 melanoma cells, after the production of melanin as well as Sia overexpression was proved to be up to four times higher in this cell line compared to that in Mel-C cells. Biodistribution studies in B16-F10 tumor-bearing SCID mice showed blood clearance at the time points studied, while uptake in the tumor peaked at 60 min post-injection (6.36 ± 2.41 % ID/g). The acquired PET images were in accordance with the ex vivo biodistribution results. Metabolite assessment on tumor, blood, and urine samples showed that [ 68 Ga]DOTA-en-pba remains unmetabolized up to at least 60 min post-injection. Our work is the first attempt for in vivo imaging of cancer by targeting overexpression of sialic acid on cancer cells with a radiotracer in PET.

Gold nanoparticles for non-invasive cell tracking with CT imaging

NASA Astrophysics Data System (ADS)

Meir, Rinat; Betzer, Oshra; Barnoy, Eran; Motiei, Menachem; Popovtzer, Rachela

2018-02-01

Cell-based therapies use living cells with therapeutic traits to treat various diseases. This is a beneficial alternative for diseases that existing medicine cannot cure efficiently. However, inconsistent results in clinical trials are preventing the advancement and implementation of cell-based therapy. In order to explain such results, there is a need to discover the fate of the transplanted cells. To answer this need, we developed a technique for noninvasive in vivo cell tracking, which uses gold nanoparticles as contrast agents for CT imaging. Herein, we investigate the design principles of this technique for intramuscular transplantation of therapeutic cells. Longitudinal studies were performed, demonstrating the ability to track cells over long periods of time. As few as 500 cells could be detected and a way to quantify the number of cells visualized by CT was demonstrated. This cell-tracking technology has the potential to become an essential tool in pre-clinical studies as well as in clinical trials and advance cell therapy.

Quantum dots-based double imaging combined with organic dye imaging to establish an automatic computerized method for cancer Ki67 measurement.

PubMed

Wang, Lin-Wei; Qu, Ai-Ping; Liu, Wen-Lou; Chen, Jia-Mei; Yuan, Jing-Ping; Wu, Han; Li, Yan; Liu, Juan

2016-02-03

As a widely used proliferative marker, Ki67 has important impacts on cancer prognosis, especially for breast cancer (BC). However, variations in analytical practice make it difficult for pathologists to manually measure Ki67 index. This study is to establish quantum dots (QDs)-based double imaging of nuclear Ki67 as red signal by QDs-655, cytoplasmic cytokeratin (CK) as yellow signal by QDs-585, and organic dye imaging of cell nucleus as blue signal by 4',6-diamidino-2-phenylindole (DAPI), and to develop a computer-aided automatic method for Ki67 index measurement. The newly developed automatic computerized Ki67 measurement could efficiently recognize and count Ki67-positive cancer cell nuclei with red signals and cancer cell nuclei with blue signals within cancer cell cytoplasmic with yellow signals. Comparisons of computerized Ki67 index, visual Ki67 index, and marked Ki67 index for 30 patients of 90 images with Ki67 ≤ 10% (low grade), 10% < Ki67 < 50% (moderate grade), and Ki67 ≥ 50% (high grade) showed computerized Ki67 counting is better than visual Ki67 counting, especially for Ki67 low and moderate grades. Based on QDs-based double imaging and organic dye imaging on BC tissues, this study successfully developed an automatic computerized Ki67 counting method to measure Ki67 index.

Comparing an FPGA to a Cell for an Image Processing Application

NASA Astrophysics Data System (ADS)

Rakvic, Ryan N.; Ngo, Hau; Broussard, Randy P.; Ives, Robert W.

2010-12-01

Modern advancements in configurable hardware, most notably Field-Programmable Gate Arrays (FPGAs), have provided an exciting opportunity to discover the parallel nature of modern image processing algorithms. On the other hand, PlayStation3 (PS3) game consoles contain a multicore heterogeneous processor known as the Cell, which is designed to perform complex image processing algorithms at a high performance. In this research project, our aim is to study the differences in performance of a modern image processing algorithm on these two hardware platforms. In particular, Iris Recognition Systems have recently become an attractive identification method because of their extremely high accuracy. Iris matching, a repeatedly executed portion of a modern iris recognition algorithm, is parallelized on an FPGA system and a Cell processor. We demonstrate a 2.5 times speedup of the parallelized algorithm on the FPGA system when compared to a Cell processor-based version.

Influence of sample preparation and identification of subcelluar structures in quantitative holographic phase contrast microscopy

NASA Astrophysics Data System (ADS)

Kemper, Björn; Schmidt, Lisa; Przibilla, Sabine; Rommel, Christina; Vollmer, Angelika; Ketelhut, Steffi; Schnekenburger, Jürgen; von Bally, Gert

2010-04-01

Digital holographic microscopy (DHM) provides label-free quantitative phase contrast with low demands on sample preparation. Nevertheless, for DHM measurements on fixed cells the mounting medium has to be considered while the phase contrast of living cells may be influenced by the used buffer solution. To quantify these effects, the maximum cell caused phase contrast and the visibility of the nucleoli were analyzed. A second aim of the study was to identify subcellular components in DHM phase contrast images. Therefore, comparative investigations using bright field imaging, DHM and fluorescence microscopy with 4',6- Diamidino-2-phenylindol (DAPI) staining were performed. DAPI-staining visualizes cell components containing DNA. The obtained results demonstrate exemplarily for two tumor cell lines that from DHM phase contrast images of fixed cells in phosphate buffer saline (PBS) cell thickness values are obtained which are comparable to living cells. Furthermore, it is shown that in many cases nucleus components can be identified only by DHM phase contrast.

Multicell migration tracking within angiogenic networks by deep learning-based segmentation and augmented Bayesian filtering.

PubMed

Wang, Mengmeng; Ong, Lee-Ling Sharon; Dauwels, Justin; Asada, H Harry

2018-04-01

Cell migration is a key feature for living organisms. Image analysis tools are useful in studying cell migration in three-dimensional (3-D) in vitro environments. We consider angiogenic vessels formed in 3-D microfluidic devices (MFDs) and develop an image analysis system to extract cell behaviors from experimental phase-contrast microscopy image sequences. The proposed system initializes tracks with the end-point confocal nuclei coordinates. We apply convolutional neural networks to detect cell candidates and combine backward Kalman filtering with multiple hypothesis tracking to link the cell candidates at each time step. These hypotheses incorporate prior knowledge on vessel formation and cell proliferation rates. The association accuracy reaches 86.4% for the proposed algorithm, indicating that the proposed system is able to associate cells more accurately than existing approaches. Cell culture experiments in 3-D MFDs have shown considerable promise for improving biology research. The proposed system is expected to be a useful quantitative tool for potential microscopy problems of MFDs.

Automatic digital image analysis for identification of mitotic cells in synchronous mammalian cell cultures.

PubMed

Eccles, B A; Klevecz, R R

1986-06-01

Mitotic frequency in a synchronous culture of mammalian cells was determined fully automatically and in real time using low-intensity phase-contrast microscopy and a newvicon video camera connected to an EyeCom III image processor. Image samples, at a frequency of one per minute for 50 hours, were analyzed by first extracting the high-frequency picture components, then thresholding and probing for annular objects indicative of putative mitotic cells. Both the extraction of high-frequency components and the recognition of rings of varying radii and discontinuities employed novel algorithms. Spatial and temporal relationships between annuli were examined to discern the occurrences of mitoses, and such events were recorded in a computer data file. At present, the automatic analysis is suited for random cell proliferation rate measurements or cell cycle studies. The automatic identification of mitotic cells as described here provides a measure of the average proliferative activity of the cell population as a whole and eliminates more than eight hours of manual review per time-lapse video recording.

Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer.

PubMed

De, Abhijit; Gambhir, Sanjiv Sam

2005-12-01

This study demonstrates a significant advancement of imaging of a distance-dependent physical process, known as the bioluminescent resonance energy transfer (BRET2) signal in living subjects, by using a cooled charge-coupled device (CCD) camera. A CCD camera-based spectral imaging strategy enables simultaneous visualization and quantitation of BRET signal from live cells and cells implanted in living mice. We used the BRET2 system, which utilizes Renilla luciferase (hRluc) protein and its substrate DeepBlueC (DBC) as an energy donor and a mutant green fluorescent protein (GFP2) as the acceptor. To accomplish this objective in this proof-of-principle study, the donor and acceptor proteins were fused to FKBP12 and FRB, respectively, which are known to interact only in the presence of the small molecule mediator rapamycin. Mammalian cells expressing these fusion constructs were imaged using a cooled-CCD camera either directly from culture dishes or by implanting them into mice. By comparing the emission photon yields in the presence and absence of rapamycin, the specific BRET signal was determined. The CCD imaging approach of BRET signal is particularly appealing due to its capacity to seamlessly bridge the gap between in vitro and in vivo studies. This work validates BRET as a powerful tool for interrogating and observing protein-protein interactions directly at limited depths in living mice.

Magnetic Resonance Imaging of Electrolysis.

PubMed Central

Meir, Arie; Hjouj, Mohammad; Rubinsky, Liel; Rubinsky, Boris

2015-01-01

This study explores the hypothesis that Magnetic Resonance Imaging (MRI) can image the process of electrolysis by detecting pH fronts. The study has relevance to real time control of cell ablation with electrolysis. To investigate the hypothesis we compare the following MR imaging sequences: T1 weighted, T2 weighted and Proton Density (PD), with optical images acquired using pH-sensitive dyes embedded in a physiological saline agar solution phantom treated with electrolysis and discrete measurements with a pH microprobe. We further demonstrate the biological relevance of our work using a bacterial E. Coli model, grown on the phantom. The results demonstrate the ability of MRI to image electrolysis produced pH changes in a physiological saline phantom and show that these changes correlate with cell death in the E. Coli model grown on the phantom. The results are promising and invite further experimental research. PMID:25659942

Advanced cardiac chemical exchange saturation transfer (cardioCEST) MRI for in vivo cell tracking and metabolic imaging

PubMed Central

Pumphrey, Ashley; Yang, Zhengshi; Ye, Shaojing; Powell, David K.; Thalman, Scott; Watt, David S.; Abdel-Latif, Ahmed; Unrine, Jason; Thompson, Katherine; Fornwalt, Brandon; Ferrauto, Giuseppe; Vandsburger, Moriel

2016-01-01

An improved pre-clinical cardiac chemical exchange saturation transfer (CEST) pulse sequence (cardioCEST) was used to selectively visualize paramagnetic CEST (paraCEST)-labeled cells following intramyocardial implantation. In addition, cardioCEST was used to examine the effect of diet-induced obesity upon myocardial creatine CEST contrast. CEST pulse sequences were designed from standard turbo-spin-echo and gradient-echo sequences, and a cardiorespiratory-gated steady-state cine gradient-echo sequence. In vitro validation studies performed in phantoms composed of 20mM Eu-HPDO3A, 20mM Yb-HPDO3A, or saline demonstrated similar CEST contrast by spin-echo and gradient-echo pulse sequences. Skeletal myoblast cells (C2C12) were labeled with either Eu-HPDO3A or saline using a hypotonic swelling procedure and implanted into the myocardium of C57B6/J mice. Inductively coupled plasma mass spectrometry confirmed cellular levels of Eu of 2.1 × 10−3 ng/cell in Eu-HPDO3A-labeled cells and 2.3 × 10−5 ng/cell in saline-labeled cells. In vivo cardioCEST imaging of labeled cells at ±15ppm was performed 24 h after implantation and revealed significantly elevated asymmetric magnetization transfer ratio values in regions of Eu-HPDO3A-labeled cells when compared with surrounding myocardium or saline-labeled cells. We further utilized the cardioCEST pulse sequence to examine changes in myocardial creatine in response to diet-induced obesity by acquiring pairs of cardioCEST images at ±1.8 ppm. While ventricular geometry and function were unchanged between mice fed either a high-fat diet or a corresponding control low-fat diet for 14 weeks, myocardial creatine CEST contrast was significantly reduced in mice fed the high-fat diet. The selective visualization of paraCEST-labeled cells using cardioCEST imaging can enable investigation of cell fate processes in cardioregenerative medicine, or multiplex imaging of cell survival with imaging of cardiac structure and function and additional imaging of myocardial creatine. PMID:26684053

A Scalable Perfusion Culture System with Miniature Peristaltic Pumps for Live-Cell Imaging Assays with Provision for Microfabricated Scaffolds

PubMed Central

Balakrishnan, Sreenath; Suma, M.S.; Raju, Shilpa R.; Bhargav, Santosh D.B.; Arunima, S.; Das, Saumitra

2015-01-01

Abstract We present a perfusion culture system with miniature bioreactors and peristaltic pumps. The bioreactors are designed for perfusion, live-cell imaging studies, easy incorporation of microfabricated scaffolds, and convenience of operation in standard cell culture techniques. By combining with miniature peristaltic pumps—one for each bioreactor to avoid cross-contamination and to maintain desired flow rate in each—we have made a culture system that facilitates perfusion culture inside standard incubators. This scalable system can support multiple parallel perfusion experiments. The major components are fabricated by three-dimensional printing using VeroWhite, which we show to be amenable to ex vivo cell culture. Furthermore, the components of the system can be reused, thus making it economical. We validate the system and illustrate its versatility by culturing primary rat hepatocytes, live imaging the growth of mouse fibroblasts (NIH 3T3) on microfabricated ring-scaffolds inserted into the bioreactor, performing perfusion culture of breast cancer cells (MCF7), and high-magnification imaging of hepatocarcinoma cells (HuH7). PMID:26309810

Three-dimensional micro-scale strain mapping in living biological soft tissues.

PubMed

Moo, Eng Kuan; Sibole, Scott C; Han, Sang Kuy; Herzog, Walter

2018-04-01

Non-invasive characterization of the mechanical micro-environment surrounding cells in biological tissues at multiple length scales is important for the understanding of the role of mechanics in regulating the biosynthesis and phenotype of cells. However, there is a lack of imaging methods that allow for characterization of the cell micro-environment in three-dimensional (3D) space. The aims of this study were (i) to develop a multi-photon laser microscopy protocol capable of imprinting 3D grid lines onto living tissue at a high spatial resolution, and (ii) to develop image processing software capable of analyzing the resulting microscopic images and performing high resolution 3D strain analyses. Using articular cartilage as the biological tissue of interest, we present a novel two-photon excitation imaging technique for measuring the internal 3D kinematics in intact cartilage at sub-micrometer resolution, spanning length scales from the tissue to the cell level. Using custom image processing software, we provide accurate and robust 3D micro-strain analysis that allows for detailed qualitative and quantitative assessment of the 3D tissue kinematics. This novel technique preserves tissue structural integrity post-scanning, therefore allowing for multiple strain measurements at different time points in the same specimen. The proposed technique is versatile and opens doors for experimental and theoretical investigations on the relationship between tissue deformation and cell biosynthesis. Studies of this nature may enhance our understanding of the mechanisms underlying cell mechano-transduction, and thus, adaptation and degeneration of soft connective tissues. We presented a novel two-photon excitation imaging technique for measuring the internal 3D kinematics in intact cartilage at sub-micrometer resolution, spanning from tissue length scale to cellular length scale. Using a custom image processing software (lsmgridtrack), we provide accurate and robust micro-strain analysis that allowed for detailed qualitative and quantitative assessment of the 3D tissue kinematics. The approach presented here can also be applied to other biological tissues such as meniscus and annulus fibrosus, as well as tissue-engineered tissues for the characterization of their mechanical properties. This imaging technique opens doors for experimental and theoretical investigation on the relationship between tissue deformation and cell biosynthesis. Studies of this nature may enhance our understanding of the mechanisms underlying cell mechano-transduction, and thus, adaptation and degeneration of soft connective tissues. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Automated detection of analyzable metaphase chromosome cells depicted on scanned digital microscopic images

NASA Astrophysics Data System (ADS)

Qiu, Yuchen; Wang, Xingwei; Chen, Xiaodong; Li, Yuhua; Liu, Hong; Li, Shibo; Zheng, Bin

2010-02-01

Visually searching for analyzable metaphase chromosome cells under microscopes is quite time-consuming and difficult. To improve detection efficiency, consistency, and diagnostic accuracy, an automated microscopic image scanning system was developed and tested to directly acquire digital images with sufficient spatial resolution for clinical diagnosis. A computer-aided detection (CAD) scheme was also developed and integrated into the image scanning system to search for and detect the regions of interest (ROI) that contain analyzable metaphase chromosome cells in the large volume of scanned images acquired from one specimen. Thus, the cytogeneticists only need to observe and interpret the limited number of ROIs. In this study, the high-resolution microscopic image scanning and CAD performance was investigated and evaluated using nine sets of images scanned from either bone marrow (three) or blood (six) specimens for diagnosis of leukemia. The automated CAD-selection results were compared with the visual selection. In the experiment, the cytogeneticists first visually searched for the analyzable metaphase chromosome cells from specimens under microscopes. The specimens were also automated scanned and followed by applying the CAD scheme to detect and save ROIs containing analyzable cells while deleting the others. The automated selected ROIs were then examined by a panel of three cytogeneticists. From the scanned images, CAD selected more analyzable cells than initially visual examinations of the cytogeneticists in both blood and bone marrow specimens. In general, CAD had higher performance in analyzing blood specimens. Even in three bone marrow specimens, CAD selected 50, 22, 9 ROIs, respectively. Except matching with the initially visual selection of 9, 7, and 5 analyzable cells in these three specimens, the cytogeneticists also selected 41, 15 and 4 new analyzable cells, which were missed in initially visual searching. This experiment showed the feasibility of applying this CAD-guided high-resolution microscopic image scanning system to prescreen and select ROIs that may contain analyzable metaphase chromosome cells. The success and the further improvement of this automated scanning system may have great impact on the future clinical practice in genetic laboratories to detect and diagnose diseases.

Four-Dimensional Imaging of T Cells in Kidney Transplant Rejection.

PubMed

Hughes, Andrew D; Lakkis, Fadi G; Oberbarnscheidt, Martin H

2018-06-01

Kidney transplantation is the treatment of choice for ESRD but is complicated by the response of the recipient's immune system to nonself histocompatibility antigens on the graft, resulting in rejection. Multiphoton intravital microscopy, referred to as four-dimensional imaging because it records dynamic events in three-dimensional tissue volumes, has emerged as a powerful tool to study immunologic processes in living animals. Here, we will review advances in understanding the complex mechanisms of T cell-mediated rejection made possible by four-dimensional imaging of mouse renal allografts. We will summarize recent data showing that activated (effector) T cell migration to the graft is driven by cognate antigen presented by dendritic cells that surround and penetrate peritubular capillaries, and that T cell-dendritic cell interactions persist in the graft over time, maintaining the immune response in the tissue. Copyright © 2018 by the American Society of Nephrology.

Lung Cancer Pathological Image Analysis Using a Hidden Potts Model

PubMed Central

Li, Qianyun; Yi, Faliu; Wang, Tao; Xiao, Guanghua; Liang, Faming

2017-01-01

Nowadays, many biological data are acquired via images. In this article, we study the pathological images scanned from 205 patients with lung cancer with the goal to find out the relationship between the survival time and the spatial distribution of different types of cells, including lymphocyte, stroma, and tumor cells. Toward this goal, we model the spatial distribution of different types of cells using a modified Potts model for which the parameters represent interactions between different types of cells and estimate the parameters of the Potts model using the double Metropolis-Hastings algorithm. The double Metropolis-Hastings algorithm allows us to simulate samples approximately from a distribution with an intractable normalizing constant. Our numerical results indicate that the spatial interaction between the lymphocyte and tumor cells is significantly associated with the patient’s survival time, and it can be used together with the cell count information to predict the survival of the patients. PMID:28615918

Robust Nucleus/Cell Detection and Segmentation in Digital Pathology and Microscopy Images: A Comprehensive Review.

PubMed

Xing, Fuyong; Yang, Lin

2016-01-01

Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to interobserver variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literature. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast, fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation.

Cellular Imaging With MRI.

PubMed

Makela, Ashley V; Murrell, Donna H; Parkins, Katie M; Kara, Jenna; Gaudet, Jeffrey M; Foster, Paula J

2016-10-01

Cellular magnetic resonance imaging (MRI) is an evolving field of imaging with strong translational and research potential. The ability to detect, track, and quantify cells in vivo and over time allows for studying cellular events related to disease processes and may be used as a biomarker for decisions about treatments and for monitoring responses to treatments. In this review, we discuss methods for labeling cells, various applications for cellular MRI, the existing limitations, strategies to address these shortcomings, and clinical cellular MRI.

Automated processing of label-free Raman microscope images of macrophage cells with standardized regression for high-throughput analysis.

PubMed

Milewski, Robert J; Kumagai, Yutaro; Fujita, Katsumasa; Standley, Daron M; Smith, Nicholas I

2010-11-19

Macrophages represent the front lines of our immune system; they recognize and engulf pathogens or foreign particles thus initiating the immune response. Imaging macrophages presents unique challenges, as most optical techniques require labeling or staining of the cellular compartments in order to resolve organelles, and such stains or labels have the potential to perturb the cell, particularly in cases where incomplete information exists regarding the precise cellular reaction under observation. Label-free imaging techniques such as Raman microscopy are thus valuable tools for studying the transformations that occur in immune cells upon activation, both on the molecular and organelle levels. Due to extremely low signal levels, however, Raman microscopy requires sophisticated image processing techniques for noise reduction and signal extraction. To date, efficient, automated algorithms for resolving sub-cellular features in noisy, multi-dimensional image sets have not been explored extensively. We show that hybrid z-score normalization and standard regression (Z-LSR) can highlight the spectral differences within the cell and provide image contrast dependent on spectral content. In contrast to typical Raman imaging processing methods using multivariate analysis, such as single value decomposition (SVD), our implementation of the Z-LSR method can operate nearly in real-time. In spite of its computational simplicity, Z-LSR can automatically remove background and bias in the signal, improve the resolution of spatially distributed spectral differences and enable sub-cellular features to be resolved in Raman microscopy images of mouse macrophage cells. Significantly, the Z-LSR processed images automatically exhibited subcellular architectures whereas SVD, in general, requires human assistance in selecting the components of interest. The computational efficiency of Z-LSR enables automated resolution of sub-cellular features in large Raman microscopy data sets without compromise in image quality or information loss in associated spectra. These results motivate further use of label free microscopy techniques in real-time imaging of live immune cells.

Live-cell CRISPR imaging in plants reveals dynamic telomere movements.

PubMed

Dreissig, Steven; Schiml, Simon; Schindele, Patrick; Weiss, Oda; Rutten, Twan; Schubert, Veit; Gladilin, Evgeny; Mette, Michael F; Puchta, Holger; Houben, Andreas

2017-08-01

Elucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non-coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long-standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR-dCas9, we reveal dynamic telomere movements of up to 2 μm over 30 min during interphase. Furthermore, we show that CRISPR-dCas9 can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Imaging Complex Protein Metabolism in Live Organisms by Stimulated Raman Scattering Microscopy with Isotope Labeling

PubMed Central

2016-01-01

Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse–chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial–temporal resolution. Second, by tracking the methyl group (CH3) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse–chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice in vivo. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans. PMID:25560305

Multicolor Fluorescence Imaging of Traumatic Brain Injury in a Cryolesion Mouse Model

PubMed Central

2012-01-01

Traumatic brain injury is characterized by initial tissue damage, which then can lead to secondary processes such as cell death and blood-brain-barrier disruption. Clinical and preclinical studies of traumatic brain injury typically employ anatomical imaging techniques and there is a need for new molecular imaging methods that provide complementary biochemical information. Here, we assess the ability of a targeted, near-infrared fluorescent probe, named PSS-794, to detect cell death in a brain cryolesion mouse model that replicates certain features of traumatic brain injury. In short, the model involves brief contact of a cold rod to the head of a living, anesthetized mouse. Using noninvasive whole-body fluorescence imaging, PSS-794 permitted visualization of the cryolesion in the living animal. Ex vivo imaging and histological analysis confirmed PSS-794 localization to site of brain cell death. The nontargeted, deep-red Tracer-653 was validated as a tracer dye for monitoring blood-brain-barrier disruption, and a binary mixture of PSS-794 and Tracer-653 was employed for multicolor imaging of cell death and blood-brain-barrier permeability in a single animal. The imaging data indicates that at 3 days after brain cryoinjury the amount of cell death had decreased significantly, but the integrity of the blood-brain-barrier was still impaired; at 7 days, the blood-brain-barrier was still three times more permeable than before cryoinjury. PMID:22860222

Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

PubMed

Mandracchia, Biagio; Gennari, Oriella; Marchesano, Valentina; Paturzo, Melania; Ferraro, Pietro

2017-09-01

The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cationic Organochalcogen with Monomer/Excimer Emissions for Dual-Color Live Cell Imaging and Cell Damage Diagnosis.

PubMed

Chao, Xi-Juan; Wang, Kang-Nan; Sun, Li-Li; Cao, Qian; Ke, Zhuo-Feng; Cao, Du-Xia; Mao, Zong-Wan

2018-04-25

Studies on the development of fluorescent organic molecules with different emission colors for imaging of organelles and their biomedical application are gaining lots of focus recently. Here, we report two cationic organochalcogens 1 and 2, both of which exhibit very weak green emission (Φ 1 = 0.12%; Φ 2 = 0.09%) in dilute solution as monomers, but remarkably enhanced green emission upon interaction with nucleic acids and large red-shifted emission in aggregate state by the formation of excimers at high concentration. More interestingly, the monomer emission and excimer-like emission can be used for dual color imaging of different organelles. Upon passively diffusing into cells, both probes selectively stain nucleoli with strong green emission upon 488 nm excitation, whereas upon 405 nm excitation, a completely different stain pattern by staining lysosomes (for 1) or mitochondria (for 2) with distinct red emission is observed because of the highly concentrated accumulation in these organelles. Studies on the mechanism of the accumulation in lysosomes (for 1) or mitochondria (for 2) found that the accumulations of the probes are dependent on the membrane permeabilization, which make the probes have great potential in diagnosing cell damage by sensing lysosomal or mitochondrial membrane permeabilization. The study is demonstrative, for the first time, of two cationic molecules for dual-color imaging nucleoli and lysosomes (1)/mitochondria (2) simultaneously in live cell based on monomer and excimer-like emission, respectively, and more importantly, for diagnosing cell damage.

Incubator-independent cell-culture perfusion platform for continuous long-term microelectrode array electrophysiology and time-lapse imaging

PubMed Central

Saalfrank, Dirk; Konduri, Anil Krishna; Latifi, Shahrzad; Habibey, Rouhollah; Golabchi, Asiyeh; Martiniuc, Aurel Vasile; Knoll, Alois; Ingebrandt, Sven; Blau, Axel

2015-01-01

Most in vitro electrophysiology studies extract information and draw conclusions from representative, temporally limited snapshot experiments. This approach bears the risk of missing decisive moments that may make a difference in our understanding of physiological events. This feasibility study presents a simple benchtop cell-culture perfusion system adapted to commercial microelectrode arrays (MEAs), multichannel electrophysiology equipment and common inverted microscopy stages for simultaneous and uninterrupted extracellular electrophysiology and time-lapse imaging at ambient CO2 levels. The concept relies on a transparent, replica-casted polydimethylsiloxane perfusion cap, gravity- or syringe-pump-driven perfusion and preconditioning of pH-buffered serum-free cell-culture medium to ambient CO2 levels at physiological temperatures. The low-cost microfluidic in vitro enabling platform, which allows us to image cultures immediately after cell plating, is easy to reproduce and is adaptable to the geometries of different cell-culture containers. It permits the continuous and simultaneous multimodal long-term acquisition or manipulation of optical and electrophysiological parameter sets, thereby considerably widening the range of experimental possibilities. Two exemplary proof-of-concept long-term MEA studies on hippocampal networks illustrate system performance. Continuous extracellular recordings over a period of up to 70 days revealed details on both sudden and gradual neural activity changes in maturing cell ensembles with large intra-day fluctuations. Correlated time-lapse imaging unveiled rather static macroscopic network architectures with previously unreported local morphological oscillations on the timescale of minutes. PMID:26543581

Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice.

PubMed

Bhaumik, S; Lewis, X Z; Gambhir, S S

2004-01-01

We have recently demonstrated that Renilla luciferase (Rluc) is a promising bioluminescence reporter gene that can be used for noninvasive optical imaging of reporter gene expression in living mice, with the aid of a cooled charged couple device (CCD) camera. In the current study, we explore the expression of a novel synthetic Renilla luciferase reporter gene (hRluc) in living mice, which has previously been reported to be a more sensitive reporter than native Rluc in mammalian cells. We explore the strategies of simultaneous imaging of both Renilla luciferase enzyme (RL) and synthetic Renilla luciferase enzyme (hRL):coelenterazine (substrate for RL/hRL) in the same living mouse. We also demonstrate that hRL:coelenterazine can yield a higher signal when compared to Firefly luciferase enzyme (FL): D-Luciferin, both in cell culture studies and when imaged from cells at the surface and from lungs of living mice. These studies demonstrate that hRluc should be a useful primary reporter gene with high sensitivity when used alone or in conjunction with other bioluminescence reporter genes for imaging in living rodents. (c) 2004 Society of Photo-Optical Instrumentation Engineers.

Visualizing inducible nitric-oxide synthase in living cells with a heme-binding fluorescent inhibitor.

PubMed

Panda, Koustubh; Chawla-Sarkar, Mamta; Santos, Cecile; Koeck, Thomas; Erzurum, Serpil C; Parkinson, John F; Stuehr, Dennis J

2005-07-19

The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme specificity, and was essentially irreversible. PIF was used to successfully image iNOS expressed in RAW264.7 cells, HEK293T cells, human A549 epithelial cells, and freshly obtained human lung epithelium. PIF was used to estimate a half-life for iNOS of 1.8 h in HEK293T cells. Our work reveals that fluorescent probes like PIF will be valuable for studying iNOS cell biology and in understanding the pathophysiology of diseases that involve dysfunctional iNOS expression.

Live cell imaging combined with high-energy single-ion microbeam

NASA Astrophysics Data System (ADS)

Guo, Na; Du, Guanghua; Liu, Wenjing; Guo, Jinlong; Wu, Ruqun; Chen, Hao; Wei, Junzhe

2016-03-01

DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10-3 s-1 and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10-2 s-1.

Hyperspectral imaging flow cytometer

DOEpatents

Sinclair, Michael B.; Jones, Howland D. T.

2017-10-25

A hyperspectral imaging flow cytometer can acquire high-resolution hyperspectral images of particles, such as biological cells, flowing through a microfluidic system. The hyperspectral imaging flow cytometer can provide detailed spatial maps of multiple emitting species, cell morphology information, and state of health. An optimized system can image about 20 cells per second. The hyperspectral imaging flow cytometer enables many thousands of cells to be characterized in a single session.

Functionalized iron oxide nanoparticles for controlling the movement of immune cells

NASA Astrophysics Data System (ADS)

White, Ethan E.; Pai, Alex; Weng, Yiming; Suresh, Anil K.; van Haute, Desiree; Pailevanian, Torkom; Alizadeh, Darya; Hajimiri, Ali; Badie, Behnam; Berlin, Jacob M.

2015-04-01

Immunotherapy is currently being investigated for the treatment of many diseases, including cancer. The ability to control the location of immune cells during or following activation would represent a powerful new technique for this field. Targeted magnetic delivery is emerging as a technique for controlling cell movement and localization. Here we show that this technique can be extended to microglia, the primary phagocytic immune cells in the central nervous system. The magnetized microglia were generated by loading the cells with iron oxide nanoparticles functionalized with CpG oligonucleotides, serving as a proof of principle that nanoparticles can be used to both deliver an immunostimulatory cargo to cells and to control the movement of the cells. The nanoparticle-oligonucleotide conjugates are efficiently internalized, non-toxic, and immunostimulatory. We demonstrate that the in vitro migration of the adherent, loaded microglia can be controlled by an external magnetic field and that magnetically-induced migration is non-cytotoxic. In order to capture video of this magnetically-induced migration of loaded cells, a novel 3D-printed ``cell box'' was designed to facilitate our imaging application. Analysis of cell movement velocities clearly demonstrate increased cell velocities toward the magnet. These studies represent the initial step towards our final goal of using nanoparticles to both activate immune cells and to control their trafficking within the diseased brain.Immunotherapy is currently being investigated for the treatment of many diseases, including cancer. The ability to control the location of immune cells during or following activation would represent a powerful new technique for this field. Targeted magnetic delivery is emerging as a technique for controlling cell movement and localization. Here we show that this technique can be extended to microglia, the primary phagocytic immune cells in the central nervous system. The magnetized microglia were generated by loading the cells with iron oxide nanoparticles functionalized with CpG oligonucleotides, serving as a proof of principle that nanoparticles can be used to both deliver an immunostimulatory cargo to cells and to control the movement of the cells. The nanoparticle-oligonucleotide conjugates are efficiently internalized, non-toxic, and immunostimulatory. We demonstrate that the in vitro migration of the adherent, loaded microglia can be controlled by an external magnetic field and that magnetically-induced migration is non-cytotoxic. In order to capture video of this magnetically-induced migration of loaded cells, a novel 3D-printed ``cell box'' was designed to facilitate our imaging application. Analysis of cell movement velocities clearly demonstrate increased cell velocities toward the magnet. These studies represent the initial step towards our final goal of using nanoparticles to both activate immune cells and to control their trafficking within the diseased brain. Electronic supplementary information (ESI) available: Transmission electron microscopy images of the particles, additional independent experiments for the NFκB activity and exocytosis assays, TEM images for the SPION untreated cells, bright field microscopy images of the cells alone in the presence and absence of magnet, images of the magnetic movement experiments at higher doses of SPION, full uncropped images of the post-migration LIVE/DEAD assay, and a video file of cell movement. See DOI: 10.1039/c3nr04421a

High MRI performance fluorescent mesoporous silica-coated magnetic nanoparticles for tracking neural progenitor cells in an ischemic mouse model.

PubMed

Zhang, Lu; Wang, Yao; Tang, Yaohui; Jiao, Zheng; Xie, Chengying; Zhang, Haijiao; Gu, Ping; Wei, Xunbin; Yang, Guo-Yuan; Gu, Hongchen; Zhang, Chunfu

2013-05-21

Multifunctional probes with high MRI sensitivity and high efficiency for cell labeling are desirable for MR cell imaging. Herein, we have fabricated fluorescent mesoporous silica-coated superparamagnetic iron oxide nanoparticles (fmSiO4@SPIONs) for neural progenitor cell (C17.2) MR imaging. FmSiO4@SPIONs were discrete and uniform in size, and had a clear core-shell structure. The magnetic core size was about 10 nm and the fluorescent mesoporous silica coating layer was around 20 nm. Compared with fluorescent dense silica-coated SPIONs (fdSiO4@SPIONs) with a similar size, fmSiO4@SPIONs demonstrated higher MR sensitivity and cell labeling efficiency. When implanted into the right hemisphere of stroke mice, contralateral to the ischemic territory, a small amount of labeled cells were able to be tracked migrating to the lesion sites using a clinical MRI scanner (3 T). More impressively, even when administered intravenously, the labeled cells could also be monitored homing to the ischemic area. MRI observations were corroborated by histological studies of the brain tissues. Our study demonstrated that fmSiO4@SPIONs are highly effective for cell imaging and hold great promise for MRI cell tracking in future.

Rotation is the primary motion of paired human epidermal keratinocytes.

PubMed

Tate, Sota; Imai, Matome; Matsushita, Natsuki; Nishimura, Emi K; Higashiyama, Shigeki; Nanba, Daisuke

2015-09-01

Collective motion of keratinocytes is involved in morphogenesis, homeostasis, and wound healing of the epidermis. Yet how the collective motion of keratinocytes emerges from the behavior of individual cells is still largely unknown. The aim of this study was to find the cellular behavior that links single and collective motion of keratinocytes. We investigated the behavior of two-cell colonies of HaCaT keratinocytes by a combination of time-lapse imaging and image processing. The two-cell colonies of HaCaT cells were formed as a contacted pair of keratinocyte clones. Image analysis and cell culture experiments revealed that the rotational speed of two-cell colonies was positively associated with their proliferative capacity. α6 integrin was required for the rotational motion of two-cell keratinocyte colonies. We also confirmed that two-cell colonies of keratinocytes predominantly exhibited the rotational, but not translational, motion, two modes of motion in a contact pair of rotating objects. The rotational motion is the primary motion of two-cell keratinocyte colonies and its speed is positively associated with their proliferative capacity. This study suggests that the assembly of rotating keratinocytes generates the collective motion of proliferative keratinocytes during morphogenesis and wound healing of the epidermis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

High Resolution Helium Ion Scanning Microscopy of the Rat Kidney

PubMed Central

Rice, William L.; Van Hoek, Alfred N.; Păunescu, Teodor G.; Huynh, Chuong; Goetze, Bernhard; Singh, Bipin; Scipioni, Larry; Stern, Lewis A.; Brown, Dennis

2013-01-01

Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide significant advances in our understanding of cell surface structures and membrane organization. PMID:23505418

Time-lapse total internal reflection fluorescence video of acetylcholine receptor cluster formation on myotubes.

PubMed

Wang, M D; Axelrod, D

1994-09-01

To study when and where acetylcholine receptor (AChR) clusters appear on developing rat myotubes in primary culture, we have made time-lapse movies of total internal reflection fluorescence (TIRF) overlaid with schlieren transmitted light images. The receptors, including the ones newly incorporated into the membrane, were labeled with rhodamine alpha-bungarotoxin (R-BT) continuously present in the medium. Since TIRF illuminates only cell-substrate contact regions where almost all of the AChR clusters are located, background fluorescence from fluorophores either in the bulk solution or inside the cells can be suppressed. Also, because TIRF minimizes the exposure of the cell interior to light, the healthy survival of the culture during imaging procedures is much enhanced relative to standard epi- (or trans-) illumination. During the experiment, cells were kept alive on the microscope stage at 37 degrees C in an atmosphere of 10% CO2. Two digital images were recorded by a CCD camera every 20 min: the schlieren image of the cells and the TIRF image of the clusters. After background subtraction, the cluster image was displayed in pseudocolors, overlaid onto the cell images, and recorded as 3 frames on a videotape. The final movies are thus able to summarize a week-long experiment in less than a minute. These movies and images show that clusters form often shortly after the myoblast fusion but sometimes much later, and the formation takes place very rapidly (a few hours). The clusters have an average lifetime of around a day, much shorter than the lifetime of a typical myotube. The brightest and largest clusters tend to be the longest-lived. The cluster formation seems to be associated with the contacts of myotubes at the glass substrate, but not with cell-cell contacts or myoblast fusion into myotubes. New AChR continuously appear in preexisting clusters: after photobleaching, the fluorescence of some clusters recovers within an hour.

Imaging as characterization techniques for thin-film cadmium telluride photovoltaics

NASA Astrophysics Data System (ADS)

Zaunbrecher, Katherine

The goal of increasing the efficiency of solar cell devices is a universal one. Increased photovoltaic (PV) performance means an increase in competition with other energy technologies. One way to improve PV technologies is to develop rapid, accurate characterization tools for quality control. Imaging techniques developed over the past decade are beginning to fill that role. Electroluminescence (EL), photoluminescence (PL), and lock-in thermography are three types of imaging implemented in this study to provide a multifaceted approach to studying imaging as applied to thin-film CdTe solar cells. Images provide spatial information about cell operation, which in turn can be used to identify defects that limit performance. This study began with developing EL, PL, and dark lock-in thermography (DLIT) for CdTe. Once imaging data were acquired, luminescence and thermography signatures of non-uniformities that disrupt the generation and collection of carriers were identified and cataloged. Additional data acquisition and analysis were used to determine luminescence response to varying operating conditions. This includes acquiring spectral data, varying excitation conditions, and correlating luminescence to device performance. EL measurements show variations in a cell's local voltage, which include inhomogeneities in the transparent-conductive oxide (TCO) front contact, CdS window layer, and CdTe absorber layer. EL signatures include large gradients, local reduction of luminescence, and local increases in luminescence on the interior of the device as well as bright spots located on the cell edges. The voltage bias and spectral response were analyzed to determine the response of these non-uniformities and surrounding areas. PL images of CdTe have not shown the same level of detail and features compared to their EL counterparts. Many of the signatures arise from reflections and severe inhomogeneities, but the technique is limited by the external illumination source used to excite carriers. Measurements on unfinished CdS and CdTe films reveal changes in signal after post-deposition processing treatments. DLIT images contained heat signatures arising from defect-related current crowding. Forward- and reverse-bias measurements revealed hot spots related to shunt and weak-diode defects. Modeling and previous studies done on Cu(In,Ga)Se 2 thin-film solar cells aided in identifying the physical causes of these thermographic and luminescence signatures. Imaging data were also coupled with other characterization techniques to provide a more comprehensive examination of nonuniform features and their origins and effects on device performance. These techniques included light-beam-induced-current (LBIC) measurements, which provide spatial quantum efficiency maps of the cell at varying resolutions, as well as time-resolved photoluminescence and spectral PL mapping. Local drops in quantum efficiency seen in LBIC typically corresponded with reductions in EL signal while minority-carrier lifetime values acquired by time-resolved PL measurements correlate with PL intensity.

Autofluorescence-based diagnostic UV imaging of tissues and cells

NASA Astrophysics Data System (ADS)

Renkoski, Timothy E.

Cancer is the second leading cause of death in the United States, and its early diagnosis is critical to improving treatment options and patient outcomes. In autofluorescence (AF) imaging, light of controlled wavelengths is projected onto tissue, absorbed by specific molecules, and re-emitted at longer wavelengths. Images of re-emitted light are used together with spectral information to infer tissue functional information and diagnosis. This dissertation describes AF imaging studies of three different organs using data collected from fresh human surgical specimens. In the ovary study, illumination was at 365 nm, and images were captured at 8 emission wavelengths. Measurements from a multispectral imaging system and fiber optic probe were used to map tissue diagnosis at every image pixel. For the colon and pancreas studies, instrumentation was developed extending AF imaging capability to sub-300 nm excitation. Images excited in the deep UV revealed tryptophan and protein content which are believed to change with disease state. Several excitation wavelength bands from 280 nm to 440 nm were investigated. Microscopic AF images collected in the pancreas study included both cultured and primary cells. Several findings are reported. A method of transforming fiber optic probe spectra for direct comparison with imager spectra was devised. Normalization of AF data by green reflectance data was found useful in correcting hemoglobin absorption. Ratio images, both AF and reflectance, were formulated to highlight growths in the colon. Novel tryptophan AF images were found less useful for colon diagnostics than the new ratio techniques. Microscopic tryptophan AF images produce useful visualization of cellular protein content, but their diagnostic value requires further study.

Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy

PubMed Central

Vélez-Ortega, A. Catalina; Frolenkov, Gregory I.

2016-01-01

The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3 to 4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette –which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier– is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface. Here we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations. PMID:27259929

Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy.

PubMed

Vélez-Ortega, A Catalina; Frolenkov, Gregory I

2016-01-01

The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3-4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette-which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier-is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface.Here, we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations.

Imaging Stem Cells Implanted in Infarcted Myocardium

PubMed Central

Zhou, Rong; Acton, Paul D.; Ferrari, Victor A.

2008-01-01

Stem cell–based cellular cardiomyoplasty represents a promising therapy for myocardial infarction. Noninvasive imaging techniques would allow the evaluation of survival, migration, and differentiation status of implanted stem cells in the same subject over time. This review describes methods for cell visualization using several corresponding noninvasive imaging modalities, including magnetic resonance imaging, positron emission tomography, single-photon emission computed tomography, and bioluminescent imaging. Reporter-based cell visualization is compared with direct cell labeling for short- and long-term cell tracking. PMID:17112999

In Vivo Detection of Hyperoxia-Induced Pulmonary Endothelial Cell Death Using 99mTc-Duramycin

PubMed Central

Audi, Said H.; Jacobs, Elizabeth R.; Zhao, Ming; Roerig, David L.; Haworth, Steven T.; Clough, Anne V.

2014-01-01

Introduction: 99mTc-duramycin, DU, is a SPECT biomarker of tissue injury identifying cell death. The objective of this study is to investigate the potential of DU imaging to quantify capillary endothelial cell death in rat lung injury resulting from hyperoxia exposure as a model of acute lung injury. Methods: Rats were exposed to room air (normoxic) or >98% O2 for 48 or 60 hours. DU was injected i.v. in anesthetized rats, scintigraphy images were acquired at steady-state, and lung DU uptake was quantified from the images. Post-mortem, the lungs were removed for histological studies. Sequential lung sections were immunostained for caspase activation and endothelial and epithelial cells. Results: Lung DU uptake increased significantly (p < 0.001) by 39% and 146% in 48-hr and 60-hr exposed rats, respectively, compared to normoxic rats. There was strong correlation (r2 = 0.82, p = 0.005) between lung DU uptake and the number of cleaved caspase 3 (CC3) positive cells, and endothelial cells accounted for more than 50% of CC3 positive cells in the hyperoxic lungs. Histology revealed preserved lung morphology through 48 hours. By 60 hours there was evidence of edema, and modest neutrophilic infiltrate. Conclusions: Rat lung DU uptake in vivo increased after just 48 hours of >98% O2 exposure, prior to the onset of any substantial evidence of lung injury. These results suggest that apoptotic endothelial cells are the primary contributors to the enhanced DU lung uptake, and support the utility of DU imaging for detecting early endothelial cell death in vivo. PMID:25218023

Gd2O3-doped silica @ Au nanoparticles for in vitro imaging cancer biomarkers using surface-enhanced Raman scattering.

PubMed

Xiao, Lifu; Tian, Xiumei; Harihar, Sitaram; Li, Qifei; Li, Li; Welch, Danny R; Zhou, Anhong

2017-06-15

There has been an interest in developing multimodal approaches to combine the advantages of individual imaging modalities, as well as to compensate for respective weaknesses. We previously reported a composite nano-system composed of gadolinium-doped mesoporous silica nanoparticle and gold nanoparticle (Gd-Au NPs) as an efficient MRI contrast agent for in vivo cancer imaging. However, MRI lacks sensitivity and is unsuitable for in vitro cancer detection. Thus, here we performed a study to use the Gd-Au NPs for detection and imaging of a widely recognized human cancer biomarker, epidermal growth factor receptor (EGFR), in individual human cancer cells with surface-enhanced Raman scattering (SERS). The Gd-Au NPs were sequentially conjugated with a monoclonal antibody recognizing EGFR and a Raman reporter molecule, 4-meraptobenzoic acid (MBA), to generate a characteristic SERS signal at 1075cm -1 . By spatially mapping the SERS intensity at 1075cm -1 , cellular distribution of EGFR and its relocalization on the plasma membrane were measured in situ. In addition, the EGFR expression levels in three human cancer cell lines (S18, A431 and A549) were measured using this SERS probe, which were consistent with the comparable measurements using immunoblotting and immunofluorescence. Our SERS results show that functionalized Gd-Au NPs successfully targeted EGFR molecules in three human cancer cell lines and monitored changes in single cell EGFR distribution in situ, demonstrating its potential to study cell activity under physiological conditions. This SERS study, combined with our previous MRI study, suggests the Gd-Au nanocomposite is a promising candidate contrast agent for multimodal cancer imaging. Copyright © 2017 Elsevier B.V. All rights reserved.

Gd2O3-doped silica @ Au nanoparticles for in vitro imaging cancer biomarkers using surface-enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Xiao, Lifu; Tian, Xiumei; Harihar, Sitaram; Li, Qifei; Li, Li; Welch, Danny R.; Zhou, Anhong

2017-06-01

There has been an interest in developing multimodal approaches to combine the advantages of individual imaging modalities, as well as to compensate for respective weaknesses. We previously reported a composite nano-system composed of gadolinium-doped mesoporous silica nanoparticle and gold nanoparticle (Gd-Au NPs) as an efficient MRI contrast agent for in vivo cancer imaging. However, MRI lacks sensitivity and is unsuitable for in vitro cancer detection. Thus, here we performed a study to use the Gd-Au NPs for detection and imaging of a widely recognized human cancer biomarker, epidermal growth factor receptor (EGFR), in individual human cancer cells with surface-enhanced Raman scattering (SERS). The Gd-Au NPs were sequentially conjugated with a monoclonal antibody recognizing EGFR and a Raman reporter molecule, 4-meraptobenzoic acid (MBA), to generate a characteristic SERS signal at 1075 cm- 1. By spatially mapping the SERS intensity at 1075 cm- 1, cellular distribution of EGFR and its relocalization on the plasma membrane were measured in situ. In addition, the EGFR expression levels in three human cancer cell lines (S18, A431 and A549) were measured using this SERS probe, which were consistent with the comparable measurements using immunoblotting and immunofluorescence. Our SERS results show that functionalized Gd-Au NPs successfully targeted EGFR molecules in three human cancer cell lines and monitored changes in single cell EGFR distribution in situ, demonstrating its potential to study cell activity under physiological conditions. This SERS study, combined with our previous MRI study, suggests the Gd-Au nanocomposite is a promising candidate contrast agent for multimodal cancer imaging.

NanoTopoChip: High-throughput nanotopographical cell instruction.

PubMed

Hulshof, Frits F B; Zhao, Yiping; Vasilevich, Aliaksei; Beijer, Nick R M; de Boer, Meint; Papenburg, Bernke J; van Blitterswijk, Clemens; Stamatialis, Dimitrios; de Boer, Jan

2017-10-15

Surface topography is able to influence cell phenotype in numerous ways and offers opportunities to manipulate cells and tissues. In this work, we develop the Nano-TopoChip and study the cell instructive effects of nanoscale topographies. A combination of deep UV projection lithography and conventional lithography was used to fabricate a library of more than 1200 different defined nanotopographies. To illustrate the cell instructive effects of nanotopography, actin-RFP labeled U2OS osteosarcoma cells were cultured and imaged on the Nano-TopoChip. Automated image analysis shows that of many cell morphological parameters, cell spreading, cell orientation and actin morphology are mostly affected by the nanotopographies. Additionally, by using modeling, the changes of cell morphological parameters could by predicted by several feature shape parameters such as lateral size and spacing. This work overcomes the technological challenges of fabricating high quality defined nanoscale features on unprecedented large surface areas of a material relevant for tissue culture such as PS and the screening system is able to infer nanotopography - cell morphological parameter relationships. Our screening platform provides opportunities to identify and study the effect of nanotopography with beneficial properties for the culture of various cell types. The nanotopography of biomaterial surfaces can be modified to influence adhering cells with the aim to improve the performance of medical implants and tissue culture substrates. However, the necessary knowledge of the underlying mechanisms remains incomplete. One reason for this is the limited availability of high-resolution nanotopographies on relevant biomaterials, suitable to conduct systematic biological studies. The present study shows the fabrication of a library of nano-sized surface topographies with high fidelity. The potential of this library, called the 'NanoTopoChip' is shown in a proof of principle HTS study which demonstrates how cells are affected by nanotopographies. The large dataset, acquired by quantitative high-content imaging, allowed us to use predictive modeling to describe how feature dimensions affect cell morphology. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Effects of cholesterol on plasma membrane lipid order in MCF-7 cells by two-photon microscopy

NASA Astrophysics Data System (ADS)

Zeng, Yixiu; Chen, Jianling; Yang, Hongqin; Wang, Yuhua; Li, Hui; Xie, Shusen

2014-09-01

Lipid rafts are cholesterol- and glycosphingolipids- enriched microdomains on plasma membrane surface of mammal cells, involved in a variety of cellular processes. Depleting cholesterol from the plasma membrane by drugs influences the trafficking of lipid raft markers. Optical imaging techniques are powerful tools to study lipid rafts in live cells due to its noninvasive feature. In this study, breast cancer cells MCF-7 were treated with different concentrations of MβCD to deplete cholesterol and an environmentally sensitive fluorescence probe, Laurdan was loaded to image lipid order by two-photon microscopy. The generalized polarization (GP) values were calculated to distinguish the lipid order and disorder phase. GP images and GP distributions of native and cholesterol-depleted MCF-7 cells were obtained. Our results suggest that even at low concentration (0.5 mM) of MβCD, the morphology of the MCF-7 cells changes. Small high GP areas (lipid order phase) decrease more rapidly than low GP areas (lipid disorder phase), indicating that lipid raft structure was altered more severely than nonraft domains. The data demonstrates that cholesterol dramatically affect raft coverage and plasma membrane fluidity in living cells.

High-resolution imaging of cellular processes across textured surfaces using an indexed-matched elastomer.

PubMed

Ravasio, Andrea; Vaishnavi, Sree; Ladoux, Benoit; Viasnoff, Virgile

2015-03-01

Understanding and controlling how cells interact with the microenvironment has emerged as a prominent field in bioengineering, stem cell research and in the development of the next generation of in vitro assays as well as organs on a chip. Changing the local rheology or the nanotextured surface of substrates has proved an efficient approach to improve cell lineage differentiation, to control cell migration properties and to understand environmental sensing processes. However, introducing substrate surface textures often alters the ability to image cells with high precision, compromising our understanding of molecular mechanisms at stake in environmental sensing. In this paper, we demonstrate how nano/microstructured surfaces can be molded from an elastomeric material with a refractive index matched to the cell culture medium. Once made biocompatible, contrast imaging (differential interference contrast, phase contrast) and high-resolution fluorescence imaging of subcellular structures can be implemented through the textured surface using an inverted microscope. Simultaneous traction force measurements by micropost deflection were also performed, demonstrating the potential of our approach to study cell-environment interactions, sensing processes and cellular force generation with unprecedented resolution. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Review of free software tools for image analysis of fluorescence cell micrographs.

PubMed

Wiesmann, V; Franz, D; Held, C; Münzenmayer, C; Palmisano, R; Wittenberg, T

2015-01-01

An increasing number of free software tools have been made available for the evaluation of fluorescence cell micrographs. The main users are biologists and related life scientists with no or little knowledge of image processing. In this review, we give an overview of available tools and guidelines about which tools the users should use to segment fluorescence micrographs. We selected 15 free tools and divided them into stand-alone, Matlab-based, ImageJ-based, free demo versions of commercial tools and data sharing tools. The review consists of two parts: First, we developed a criteria catalogue and rated the tools regarding structural requirements, functionality (flexibility, segmentation and image processing filters) and usability (documentation, data management, usability and visualization). Second, we performed an image processing case study with four representative fluorescence micrograph segmentation tasks with figure-ground and cell separation. The tools display a wide range of functionality and usability. In the image processing case study, we were able to perform figure-ground separation in all micrographs using mainly thresholding. Cell separation was not possible with most of the tools, because cell separation methods are provided only by a subset of the tools and are difficult to parametrize and to use. Most important is that the usability matches the functionality of a tool. To be usable, specialized tools with less functionality need to fulfill less usability criteria, whereas multipurpose tools need a well-structured menu and intuitive graphical user interface. © 2014 Fraunhofer-Institute for Integrated Circuits IIS Journal of Microscopy © 2014 Royal Microscopical Society.

FITC labeled silica nanoparticles as efficient cell tags: uptake and photostability study in endothelial cells.

PubMed

Veeranarayanan, Srivani; Poulose, Aby Cheruvathoor; Mohamed, Sheikh; Aravind, Athulya; Nagaoka, Yutaka; Yoshida, Yasuhiko; Maekawa, Toru; Kumar, D Sakthi

2012-03-01

The use of fluorescent nanomaterials has gained great importance in the field of medical imaging. Many traditional imaging technologies have been reported utilizing dyes in the past. These methods face drawbacks due to non-specific accumulation and photobleaching of dyes. We studied the uptake and internalization of two different sized (30 nm and 100 nm) FITC labeled silica nanoparticles in Human umbilical vein endothelial cell line. These nanomaterials show high biocompatability and are highly photostable inside live cells for increased period of time in comparison to the dye alone. To our knowledge, we report for the first time the use of 30 nm fluorescent silica nanoparticles as efficient endothelial tags along with the well studied 100 nm particles. We also have emphasized the good photostability of these materials in live cells.

Imaging windows for long-term intravital imaging

PubMed Central

Alieva, Maria; Ritsma, Laila; Giedt, Randy J; Weissleder, Ralph; van Rheenen, Jacco

2014-01-01

Intravital microscopy is increasingly used to visualize and quantitate dynamic biological processes at the (sub)cellular level in live animals. By visualizing tissues through imaging windows, individual cells (e.g., cancer, host, or stem cells) can be tracked and studied over a time-span of days to months. Several imaging windows have been developed to access tissues including the brain, superficial fascia, mammary glands, liver, kidney, pancreas, and small intestine among others. Here, we review the development of imaging windows and compare the most commonly used long-term imaging windows for cancer biology: the cranial imaging window, the dorsal skin fold chamber, the mammary imaging window, and the abdominal imaging window. Moreover, we provide technical details, considerations, and trouble-shooting tips on the surgical procedures and microscopy setups for each imaging window and explain different strategies to assure imaging of the same area over multiple imaging sessions. This review aims to be a useful resource for establishing the long-term intravital imaging procedure. PMID:28243510

Imaging windows for long-term intravital imaging: General overview and technical insights.

PubMed

Alieva, Maria; Ritsma, Laila; Giedt, Randy J; Weissleder, Ralph; van Rheenen, Jacco

2014-01-01

Intravital microscopy is increasingly used to visualize and quantitate dynamic biological processes at the (sub)cellular level in live animals. By visualizing tissues through imaging windows, individual cells (e.g., cancer, host, or stem cells) can be tracked and studied over a time-span of days to months. Several imaging windows have been developed to access tissues including the brain, superficial fascia, mammary glands, liver, kidney, pancreas, and small intestine among others. Here, we review the development of imaging windows and compare the most commonly used long-term imaging windows for cancer biology: the cranial imaging window, the dorsal skin fold chamber, the mammary imaging window, and the abdominal imaging window. Moreover, we provide technical details, considerations, and trouble-shooting tips on the surgical procedures and microscopy setups for each imaging window and explain different strategies to assure imaging of the same area over multiple imaging sessions. This review aims to be a useful resource for establishing the long-term intravital imaging procedure.

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

PubMed Central

Burke, Russell T.; Orth, James D.

2016-01-01

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls. PMID:27213923

Six-color intravital two-photon imaging of brain tumors and their dynamic microenvironment.

PubMed

Ricard, Clément; Debarbieux, Franck Christian

2014-01-01

The majority of intravital studies on brain tumor in living animal so far rely on dual color imaging. We describe here a multiphoton imaging protocol to dynamically characterize the interactions between six cellular components in a living mouse. We applied this methodology to a clinically relevant glioblastoma multiforme (GBM) model designed in reporter mice with targeted cell populations labeled by fluorescent proteins of different colors. This model permitted us to make non-invasive longitudinal and multi-scale observations of cell-to-cell interactions. We provide examples of such 5D (x,y,z,t,color) images acquired on a daily basis from volumes of interest, covering most of the mouse parietal cortex at subcellular resolution. Spectral deconvolution allowed us to accurately separate each cell population as well as some components of the extracellular matrix. The technique represents a powerful tool for investigating how tumor progression is influenced by the interactions of tumor cells with host cells and the extracellular matrix micro-environment. It will be especially valuable for evaluating neuro-oncological drug efficacy and target specificity. The imaging protocol provided here can be easily translated to other mouse models of neuropathologies, and should also be of fundamental interest for investigations in other areas of systems biology.

Image Guidance in Stem Cell Therapeutics: Unfolding the Blindfold.

PubMed

Bukhari, Amirali B; Dutta, Shruti; De, Abhijit

2015-01-01

Stem cell therapeutics is the future of regenerative medicine in the modern world. Many studies have been instigated with the hope of translating the outcome for the treatment of several disease conditions ranging from heart and neuronal disease to malignancies as grave as cancers. Stem cell therapeutics undoubtedly holds great promise on the front of regenerative medicine, however, the correct distribution and homing of these stem cells to the host site remained blinded until the recent advances in the discipline of molecular imaging. Herein, we discuss the various imaging guidance applied for determination of the proper delivery of various types of stem cell used as therapeutics for various maladies. Additionally, we scrutinize the use of several indirect labeling mechanisms for efficient tagging of the reporter entity for image guidance. Further, the promise of improving patient healthcare has led to the initiation of several clinical trials worldwide. However, in number of the cases, the benefits arrive with a price heavy enough to pose a serious health risk, one such being formation of teratomas. Thus numerous challenges and methodological obstacles must be overcome before their eloquent clinical impact can be realized. Therefore, we also discuss several clinical trials that have taken into consideration the various imaging guided protocols to monitor correct delivery and understand the distribution of therapeutic stem cells in real time.

Ex-vivo imaging of excised tissue using vital dyes and confocal microscopy

PubMed Central

Johnson, Simon; Rabinovitch, Peter

2012-01-01

Vital dyes routinely used for staining cultured cells can also be used to stain and image live tissue slices ex-vivo. Staining tissue with vital dyes allows researchers to collect structural and functional data simultaneously and can be used for qualitative or quantitative fluorescent image collection. The protocols presented here are useful for structural and functional analysis of viable properties of cells in intact tissue slices, allowing for the collection of data in a structurally relevant environment. With these protocols, vital dyes can be applied as a research tool to disease processes and properties of tissue not amenable to cell culture based studies. PMID:22752953

Live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix.

PubMed

Shih, Wenting; Yamada, Soichiro

2011-12-22

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network. By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

In Vivo Bioluminescence Tomography for Monitoring Breast Tumor Growth and Metastatic Spreading: Comparative Study and Mathematical Modeling

PubMed Central

Mollard, Séverine; Fanciullino, Raphaelle; Giacometti, Sarah; Serdjebi, Cindy; Benzekry, Sebastien; Ciccolini, Joseph

2016-01-01

This study aimed at evaluating the reliability and precision of Diffuse Luminescent Imaging Tomography (DLIT) for monitoring primary tumor and metastatic spreading in breast cancer mice, and to develop a biomathematical model to describe the collected data. Using orthotopic mammary fat pad model of breast cancer (MDAMB231-Luc) in mice, we monitored tumor and metastatic spreading by three-dimensional (3D) bioluminescence and cross-validated it with standard bioluminescence imaging, caliper measurement and necropsy examination. DLIT imaging proved to be reproducible and reliable throughout time. It was possible to discriminate secondary lesions from the main breast cancer, without removing the primary tumor. Preferential metastatic sites were lungs, peritoneum and lymph nodes. Necropsy examinations confirmed DLIT measurements. Marked differences in growth profiles were observed, with an overestimation of the exponential phase when using a caliper as compared with bioluminescence. Our mathematical model taking into account the balance between living and necrotic cells proved to be able to reproduce the experimental data obtained with a caliper or DLIT imaging, because it could discriminate proliferative living cells from a more composite mass consisting of tumor cells, necrotic cell, or inflammatory tissues. DLIT imaging combined with mathematical modeling could be a powerful and informative tool in experimental oncology. PMID:27812027

Quantitative characterization of the imaging limits of diffuse low-grade oligodendrogliomas.

PubMed

Gerin, Chloé; Pallud, Johan; Deroulers, Christophe; Varlet, Pascale; Oppenheim, Catherine; Roux, Francois-Xavier; Chrétien, Fabrice; Thomas, Stephen R; Grammaticos, Basile; Badoual, Mathilde

2013-10-01

Supratentorial diffuse low-grade gliomas in adults extend beyond maximal visible MRI-defined abnormalities, and a gap exists between the imaging signal changes and the actual tumor margins. Direct quantitative comparisons between imaging and histological analyses are lacking to date. However, they are of the utmost importance if one wishes to develop realistic models for diffuse glioma growth. In this study, we quantitatively compared the cell concentration and the edema fraction from human histological biopsy samples (BSs) performed inside and outside imaging abnormalities during serial imaging-based stereotactic biopsy of diffuse low-grade gliomas. The cell concentration was significantly higher in BSs located inside (1189 ± 378 cell/mm(2)) than outside (740 ± 124 cell/mm(2)) MRI-defined abnormalities (P = .0003). The edema fraction was significantly higher in BSs located inside (mean, 45% ± 23%) than outside (mean, 5 %± 9%) MRI-defined abnormalities (P < .0001). At borders of the MRI-defined abnormalities, 20% of the tissue surface area was occupied by edema and only 3% by tumor cells. The cycling cell concentration was significantly higher in BSs located inside (10 ± 12 cell/mm(2)), compared with outside (0.5 ± 0.9 cell/mm(2)), MRI-defined abnormalities (P = .0001). We showed that the margins of T2-weighted signal changes are mainly correlated with the edema fraction. In 62.5% of patients, the cycling tumor cell fraction (defined as the ratio of the cycling tumor cell concentration to the total number of tumor cells) was higher at the limits of the MRI-defined abnormalities than closer to the center of the tumor. In the remaining patients, the cycling tumor cell fraction increased towards the center of the tumor.

Fluoridated hydroxyapatite: Eu3+ nanorods-loaded folate-conjugated D-α-tocopheryl polyethylene glycol succinate (vitamin E TPGS) micelles for targeted imaging of cancer cells

NASA Astrophysics Data System (ADS)

Wan, Dong; Liu, Weijiao; Wang, Lei; Wang, Hao; Pan, Jie

2016-03-01

In this study, fluoridated hydroxyapatite: Eu3+ nanorod-loaded folate-conjugated TPGS micelles were prepared by thin-film hydration. The findings in this study demonstrate that micelles show improved dispersion, high stability, and excellent fluorescent property in aqueous solutions, suitable for targeted imaging of cancer cells with over-expressing folate receptors on their surface. The micelles designed in this study will be a promising tool for early detection of cancer.

Investigation of diseases through red blood cells' shape using photoacoustic response technique

NASA Astrophysics Data System (ADS)

Biswas, Deblina; Gorey, Abhijeet; Chen, Goerge C. K.; Sharma, Norman; Vasudevan, Srivathsan

2015-03-01

Photoacoustic (PA) imaging is a non-invasive real-time technique, widely applied to many biomedical imaging studies in the recent years. While most of these studies have been focussed on obtaining an image after reconstruction, various features of time domain signal (e.g. amplitude, width, rise and relaxation time) would provide very high sensitivity in detecting morphological changes in cells during a biological study. Different haematological disorders (e.g., sickle cell anaemia, thalassemia) exhibit significant morphological cellular changes. In this context, this study explores the possibility of utilizing the developed photoacoustic response technique to apply onto blood samples. Results of our preliminary study demonstrate that there is a significant change in signal amplitude due to change in concentration of the blood. Thus it shows the sensitivity of the developed photoacoustic technique towards red blood cell count (related to haematological disease like anaemia). Subsequently, morphological changes in RBC (i.e. swollen and shrunk compared to normal RBC) induced by hypotonic and hypertonic solutions respectively were also experimented. The result shows a distinct change in PA signal amplitude. This would serve as a diagnostic signature for many future studies on cellular morphological disorders.

Synthesis and evaluation of a novel urea-based 68Ga-complex for imaging PSMA binding in tumor.

PubMed

Zha, Zhihao; Ploessl, Karl; Choi, Seok Rye; Wu, Zehui; Zhu, Lin; Kung, Hank F

2018-04-01

Prostate specific membrane antigen (PSMA) is a well-established target for diagnostic and therapeutic applications for prostate cancer. It is know that [ 68 Ga]PSMA 11 ([ 68 Ga]Glu-NH-CO-NH-Lys(Ahx)-HBED-CC) is the most well studied PET imaging agent for detecting over expressed PSMA binding sites of tumors in humans. In an effort to provide new agents with improved characteristics for PET imaging, we report a novel [ 68 Ga]-Glu-NH-CO-NH-Lys(Ahx)-linker-HBED-CC conjugate with a novel O-(carboxymethyl)-L-tyrosine, as the linker group. Radiosynthesis was performed by a direct method. In vitro binding and cell internalization of [ 68 Ga]10 was investigated in PSMA positive LNCaP cell lines. Biodistribution and MicroPET imaging studies were performed in LNCaP tumor bearing mice. In vitro binding to LNCaP cells showed that nat Ga labeled O-(carboxymethyl)-L-tyrosine conjugate, [ nat Ga]10, displayed excellent affinity and specificity (IC 50  = 16.5 nM) a value comparable to that of PSMA 11. In vitro cell binding and internalization showed excellent uptake and retention; [ 68 Ga]10 displayed significantly higher cellular internalization than [ 68 Ga]PSMA 11 (12.5 vs 7.4% ID/10 6 cells at 1 h). Biodistribution studies in LNCaP tumor-bearing mice exhibited a high specific uptake in PSMA expressing tumors and fast clearance in normal organs (19.7 tumor/blood; 20.7 tumor/muscle at 1 h after iv injection). MicroPET imaging studies in mice confirmed that [ 68 Ga]10 displayed excellent uptake and distinctive tumor localization, which was blocked by iv injection of a competing drug, 2-PMPA. The preliminary results strongly suggest that [ 68 Ga]10 may be promising candidates as a PET imaging radiotracer for detecting PSMA expression in prostate cancer. Copyright © 2018. Published by Elsevier Inc.

A Deeper Look into Type 1 Diabetes – Imaging Immune Responses during Onset of Disease

PubMed Central

Christoffersson, Gustaf; von Herrath, Matthias G.

2016-01-01

Cytotoxic T lymphocytes execute the killing of insulin-producing beta cells during onset of type 1 diabetes mellitus (T1D). The research community has come far in dissecting the major events in the development of this disease, but still the trigger and high-resolved information of the immunological events leading up to beta cell loss are missing. During the past decades, intravital imaging of immune responses has led to significant scientific breakthroughs in diverse models of disease, including T1D. Dynamic imaging of immune cells at the pancreatic islets during T1D onset has been made possible through the development of both advanced microscopes, and animal models that allow long-term immobilization of the pancreas. The use of these modalities has revealed a milling microenvironment at the pancreatic islets during disease onset with a plethora of active players. Clues to answering the remaining questions in this disease may lie in intravital imaging, including how key immune cells traffic to and from the pancreas, and how cells interact at this target tissue. This review highlights and discusses recent studies, models, and techniques focused to understand the immune responses during T1D onset through intravital imaging. PMID:27574523

Activatable Fluorescence Probe via Self-Immolative Intramolecular Cyclization for Histone Deacetylase Imaging in Live Cells and Tissues.

PubMed

Liu, Xianjun; Xiang, Meihao; Tong, Zongxuan; Luo, Fengyan; Chen, Wen; Liu, Feng; Wang, Fenglin; Yu, Ru-Qin; Jiang, Jian-Hui

2018-05-01

Histone deacetylases (HDACs) play essential roles in transcription regulation and are valuable theranostic targets. However, there are no activatable fluorescent probes for imaging of HDAC activity in live cells. Here, we develop for the first time a novel activatable two-photon fluorescence probe that enables in situ imaging of HDAC activity in living cells and tissues. The probe is designed by conjugating an acetyl-lysine mimic substrate to a masked aldehyde-containing fluorophore via a cyanoester linker. Upon deacetylation by HDAC, the probe undergoes a rapid self-immolative intramolecular cyclization reaction, producing a cyanohydrin intermediate that is spontaneously rapidly decomposed into the highly fluorescent aldehyde-containing two-photon fluorophore. The probe is shown to exhibit high sensitivity, high specificity, and fast response for HDAC detection in vitro. Imaging studies reveal that the probe is able to directly visualize and monitor HDAC activity in living cells. Moreover, the probe is demonstrated to have the capability of two-photon imaging of HDAC activity in deep tissue slices up to 130 μm. This activatable fluorescent probe affords a useful tool for evaluating HDAC activity and screening HDAC-targeting drugs in both live cell and tissue assays.

Multi-color localization microscopy of fixed cells as a promising tool to study organization of bacterial cytoskeleton

NASA Astrophysics Data System (ADS)

Vedyaykin, A. D.; Gorbunov, V. V.; Sabantsev, A. V.; Polinovskaya, V. S.; Vishnyakov, I. E.; Melnikov, A. S.; Serdobintsev, P. Yu; Khodorkovskii, M. A.

2015-11-01

Localization microscopy allows visualization of biological structures with resolution well below the diffraction limit. Localization microscopy was used to study FtsZ organization in Escherichia coli previously in combination with fluorescent protein labeling, but the fact that fluorescent chimeric protein was unable to rescue temperature-sensitive ftsZ mutants suggests that obtained images may not represent native FtsZ structures faithfully. Indirect immunolabeling of FtsZ not only overcomes this problem, but also allows the use of the powerful visualization methods arsenal available for different structures in fixed cells. In this work we simultaneously obtained super-resolution images of FtsZ structures and diffraction-limited or super-resolution images of DNA and cell surface in E. coli, which allows for the study of the spatial arrangement of FtsZ structures with respect to the nucleoid positions and septum formation.

Fluorescence Imaging of Two-Photon Linear Dichroism: Cholesterol Depletion Disrupts Molecular Orientation in Cell Membranes

PubMed Central

Benninger, Richard K. P.; Önfelt, Björn; Neil, Mark A. A.; Davis, Daniel M.; French, Paul M. W.

2005-01-01

The plasma membrane of cells is an ordered environment, giving rise to anisotropic orientation and restricted motion of molecules and proteins residing in the membrane. At the same time as being an organized matrix of defined structure, the cell membrane is heterogeneous and dynamic. Here we present a method where we use fluorescence imaging of linear dichroism to measure the orientation of molecules relative to the cell membrane. By detecting linear dichroism as well as fluorescence anisotropy, the orientation parameters are separated from dynamic properties such as rotational diffusion and homo energy transfer (energy migration). The sensitivity of the technique is enhanced by using two-photon excitation for higher photo-selection compared to single photon excitation. We show here that we can accurately image lipid organization in whole cell membranes and in delicate structures such as membrane nanotubes connecting two cells. The speed of our wide-field imaging system makes it possible to image changes in orientation and anisotropy occurring on a subsecond timescale. This is demonstrated by time-lapse studies showing that cholesterol depletion rapidly disrupts the orientation of a fluorophore located within the hydrophobic region of the cell membrane but not of a surface bound probe. This is consistent with cholesterol having an important role in stabilizing and ordering the lipid tails within the plasma membrane. PMID:15520272

Statistical organelle dissection of Arabidopsis guard cells using image database LIPS.

PubMed

Higaki, Takumi; Kutsuna, Natsumaro; Hosokawa, Yoichiroh; Akita, Kae; Ebine, Kazuo; Ueda, Takashi; Kondo, Noriaki; Hasezawa, Seiichiro

2012-01-01

To comprehensively grasp cell biological events in plant stomatal movement, we have captured microscopic images of guard cells with various organelles markers. The 28,530 serial optical sections of 930 pairs of Arabidopsis guard cells have been released as a new image database, named Live Images of Plant Stomata (LIPS). We visualized the average organellar distributions in guard cells using probabilistic mapping and image clustering techniques. The results indicated that actin microfilaments and endoplasmic reticulum (ER) are mainly localized to the dorsal side and connection regions of guard cells. Subtractive images of open and closed stomata showed distribution changes in intracellular structures, including the ER, during stomatal movement. Time-lapse imaging showed that similar ER distribution changes occurred during stomatal opening induced by light irradiation or femtosecond laser shots on neighboring epidermal cells, indicating that our image analysis approach has identified a novel ER relocation in stomatal opening.

Does flat epithelial atypia have rounder nuclei than columnar cell change/hyperplasia? A morphometric approach to columnar cell lesions of the breast.

PubMed

Yamashita, Yoshiko; Ichihara, Shu; Moritani, Suzuko; Yoon, Han-Seung; Yamaguchi, Masahiro

2016-06-01

Columnar cell lesions of the breast encompass columnar cell change/hyperplasia (CCC/CCH) and flat epithelial atypia (FEA). These have attracted researchers because emerging data suggest that FEA may represent the earliest histologically detectable non-obligate precursor of breast cancer. However, it is occasionally difficult to distinguish FEA from CCC/CCH because of similar histology. Although the nuclei of FEA are frequently described as relatively round compared with those of CCC/CCH, there are few morphometric studies to support this statement. The aim of this study was to provide objective data as to the nuclear shape in columnar cell lesions. As a shape descriptor, we adopted ellipticity that is defined by the formula 2b/2a, where a is the length of the long axis of the ellipse and b is the length of the short axis. Contrary to circularity, ellipticity reflects the overall configuration of an ellipse irrespective of surface irregularity. Our image analysis included generating whole slide images, extracting glandular cell nuclei, measuring nuclear ellipticity, and superimposing graded colors based on execution of results on the captured images. A total of 7917 nuclei extracted from 22 FEA images and 5010 nuclei extracted from 13 CCC/CCH images were analyzed. There was a significant difference in nuclear roundness between FEA and CCC/CCH with mean ellipticity values of 0.723 and 0.679, respectively (p < 0.001, Welch's t test). Furthermore, FEA with malignancy had significantly rounder nuclei than FEA without malignancy (p < 0.001). Our preliminary results suggest that nuclear ellipticity is a key parameter in reproducibly classifying columnar cell lesions of the breast.