Quantitative high-throughput population dynamics in continuous-culture by automated microscopy.

PubMed

Merritt, Jason; Kuehn, Seppe

2016-09-12

We present a high-throughput method to measure abundance dynamics in microbial communities sustained in continuous-culture. Our method uses custom epi-fluorescence microscopes to automatically image single cells drawn from a continuously-cultured population while precisely controlling culture conditions. For clonal populations of Escherichia coli our instrument reveals history-dependent resilience and growth rate dependent aggregation.

Hierarchical assembly of viral nanotemplates with encoded microparticles via nucleic acid hybridization.

PubMed

Tan, Wui Siew; Lewis, Christina L; Horelik, Nicholas E; Pregibon, Daniel C; Doyle, Patrick S; Yi, Hyunmin

2008-11-04

We demonstrate hierarchical assembly of tobacco mosaic virus (TMV)-based nanotemplates with hydrogel-based encoded microparticles via nucleic acid hybridization. TMV nanotemplates possess a highly defined structure and a genetically engineered high density thiol functionality. The encoded microparticles are produced in a high throughput microfluidic device via stop-flow lithography (SFL) and consist of spatially discrete regions containing encoded identity information, an internal control, and capture DNAs. For the hybridization-based assembly, partially disassembled TMVs were programmed with linker DNAs that contain sequences complementary to both the virus 5' end and a selected capture DNA. Fluorescence microscopy, atomic force microscopy (AFM), and confocal microscopy results clearly indicate facile assembly of TMV nanotemplates onto microparticles with high spatial and sequence selectivity. We anticipate that our hybridization-based assembly strategy could be employed to create multifunctional viral-synthetic hybrid materials in a rapid and high-throughput manner. Additionally, we believe that these viral-synthetic hybrid microparticles may find broad applications in high capacity, multiplexed target sensing.

High-Throughput Light Sheet Microscopy for the Automated Live Imaging of Larval Zebrafish

NASA Astrophysics Data System (ADS)

Baker, Ryan; Logan, Savannah; Dudley, Christopher; Parthasarathy, Raghuveer

The zebrafish is a model organism with a variety of useful properties; it is small and optically transparent, it reproduces quickly, it is a vertebrate, and there are a large variety of transgenic animals available. Because of these properties, the zebrafish is well suited to study using a variety of optical technologies including light sheet fluorescence microscopy (LSFM), which provides high-resolution three-dimensional imaging over large fields of view. Research progress, however, is often not limited by optical techniques but instead by the number of samples one can examine over the course of an experiment, which in the case of light sheet imaging has so far been severely limited. Here we present an integrated fluidic circuit and microscope which provides rapid, automated imaging of zebrafish using several imaging modes, including LSFM, Hyperspectral Imaging, and Differential Interference Contrast Microscopy. Using this system, we show that we can increase our imaging throughput by a factor of 10 compared to previous techniques. We also show preliminary results visualizing zebrafish immune response, which is sensitive to gut microbiota composition, and which shows a strong variability between individuals that highlights the utility of high throughput imaging. National Science Foundation, Award No. DBI-1427957.

Whole-animal imaging with high spatio-temporal resolution

NASA Astrophysics Data System (ADS)

Chhetri, Raghav; Amat, Fernando; Wan, Yinan; Höckendorf, Burkhard; Lemon, William C.; Keller, Philipp J.

2016-03-01

We developed isotropic multiview (IsoView) light-sheet microscopy in order to image fast cellular dynamics, such as cell movements in an entire developing embryo or neuronal activity throughput an entire brain or nervous system, with high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To achieve high temporal resolution and high spatial resolution at the same time, IsoView microscopy rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. In a post-processing step, these four views are then combined by means of high-throughput multiview deconvolution to yield images with a system resolution of ≤ 450 nm in all three dimensions. Using IsoView microscopy, we performed whole-animal functional imaging of Drosophila embryos and larvae at a spatial resolution of 1.1-2.5 μm and at a temporal resolution of 2 Hz for up to 9 hours. We also performed whole-brain functional imaging in larval zebrafish and multicolor imaging of fast cellular dynamics across entire, gastrulating Drosophila embryos with isotropic, sub-cellular resolution. Compared with conventional (spatially anisotropic) light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, such as lattice lightsheet microscopy or diSPIM, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.

Piezo-thermal Probe Array for High Throughput Applications

PubMed Central

Gaitas, Angelo; French, Paddy

2012-01-01

Microcantilevers are used in a number of applications including atomic-force microscopy (AFM). In this work, deflection-sensing elements along with heating elements are integrated onto micromachined cantilever arrays to increase sensitivity, and reduce complexity and cost. An array of probes with 5–10 nm gold ultrathin film sensors on silicon substrates for high throughput scanning probe microscopy is developed. The deflection sensitivity is 0.2 ppm/nm. Plots of the change in resistance of the sensing element with displacement are used to calibrate the probes and determine probe contact with the substrate. Topographical scans demonstrate high throughput and nanometer resolution. The heating elements are calibrated and the thermal coefficient of resistance (TCR) is 655 ppm/K. The melting temperature of a material is measured by locally heating the material with the heating element of the cantilever while monitoring the bending with the deflection sensing element. The melting point value measured with this method is in close agreement with the reported value in literature. PMID:23641125

High-throughput ultraviolet photoacoustic microscopy with multifocal excitation

NASA Astrophysics Data System (ADS)

Imai, Toru; Shi, Junhui; Wong, Terence T. W.; Li, Lei; Zhu, Liren; Wang, Lihong V.

2018-03-01

Ultraviolet photoacoustic microscopy (UV-PAM) is a promising intraoperative tool for surgical margin assessment (SMA), one that can provide label-free histology-like images with high resolution. In this study, using a microlens array and a one-dimensional (1-D) array ultrasonic transducer, we developed a high-throughput multifocal UV-PAM (MF-UV-PAM). Our new system achieved a 1.6 ± 0.2 μm lateral resolution and produced images 40 times faster than the previously developed point-by-point scanning UV-PAM. MF-UV-PAM provided a readily comprehensible photoacoustic image of a mouse brain slice with specific absorption contrast in ˜16 min, highlighting cell nuclei. Individual cell nuclei could be clearly resolved, showing its practical potential for intraoperative SMA.

Automated Analysis of siRNA Screens of Virus Infected Cells Based on Immunofluorescence Microscopy

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

We present an image analysis approach as part of a high-throughput microscopy screening system based on cell arrays for the identification of genes involved in Hepatitis C and Dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in cells, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behavior of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

SPIM-fluid: open source light-sheet based platform for high-throughput imaging

PubMed Central

Gualda, Emilio J.; Pereira, Hugo; Vale, Tiago; Estrada, Marta Falcão; Brito, Catarina; Moreno, Nuno

2015-01-01

Light sheet fluorescence microscopy has recently emerged as the technique of choice for obtaining high quality 3D images of whole organisms/embryos with low photodamage and fast acquisition rates. Here we present an open source unified implementation based on Arduino and Micromanager, which is capable of operating Light Sheet Microscopes for automatized 3D high-throughput imaging on three-dimensional cell cultures and model organisms like zebrafish, oriented to massive drug screening. PMID:26601007

A probabilistic approach to joint cell tracking and segmentation in high-throughput microscopy videos.

PubMed

Arbelle, Assaf; Reyes, Jose; Chen, Jia-Yun; Lahav, Galit; Riklin Raviv, Tammy

2018-04-22

We present a novel computational framework for the analysis of high-throughput microscopy videos of living cells. The proposed framework is generally useful and can be applied to different datasets acquired in a variety of laboratory settings. This is accomplished by tying together two fundamental aspects of cell lineage construction, namely cell segmentation and tracking, via a Bayesian inference of dynamic models. In contrast to most existing approaches, which aim to be general, no assumption of cell shape is made. Spatial, temporal, and cross-sectional variation of the analysed data are accommodated by two key contributions. First, time series analysis is exploited to estimate the temporal cell shape uncertainty in addition to cell trajectory. Second, a fast marching (FM) algorithm is used to integrate the inferred cell properties with the observed image measurements in order to obtain image likelihood for cell segmentation, and association. The proposed approach has been tested on eight different time-lapse microscopy data sets, some of which are high-throughput, demonstrating promising results for the detection, segmentation and association of planar cells. Our results surpass the state of the art for the Fluo-C2DL-MSC data set of the Cell Tracking Challenge (Maška et al., 2014). Copyright © 2018 Elsevier B.V. All rights reserved.

Oufti: An integrated software package for high-accuracy, high-throughput quantitative microscopy analysis

PubMed Central

Paintdakhi, Ahmad; Parry, Bradley; Campos, Manuel; Irnov, Irnov; Elf, Johan; Surovtsev, Ivan; Jacobs-Wagner, Christine

2016-01-01

Summary With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re-emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today’s single-cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand-alone, open-source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non-diffraction-limited fluorescence signals, and is scalable for high-throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis, and post-processing analysis, makes the software broadly accessible to users irrespective of their computational skills. PMID:26538279

High-throughput analysis of yeast replicative aging using a microfluidic system

PubMed Central

Jo, Myeong Chan; Liu, Wei; Gu, Liang; Dang, Weiwei; Qin, Lidong

2015-01-01

Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction. PMID:26170317

Micro-patterned agarose gel devices for single-cell high-throughput microscopy of E. coli cells.

PubMed

Priest, David G; Tanaka, Nobuyuki; Tanaka, Yo; Taniguchi, Yuichi

2017-12-21

High-throughput microscopy of bacterial cells elucidated fundamental cellular processes including cellular heterogeneity and cell division homeostasis. Polydimethylsiloxane (PDMS)-based microfluidic devices provide advantages including precise positioning of cells and throughput, however device fabrication is time-consuming and requires specialised skills. Agarose pads are a popular alternative, however cells often clump together, which hinders single cell quantitation. Here, we imprint agarose pads with micro-patterned 'capsules', to trap individual cells and 'lines', to direct cellular growth outwards in a straight line. We implement this micro-patterning into multi-pad devices called CapsuleHotel and LineHotel for high-throughput imaging. CapsuleHotel provides ~65,000 capsule structures per mm 2 that isolate individual Escherichia coli cells. In contrast, LineHotel provides ~300 line structures per mm that direct growth of micro-colonies. With CapsuleHotel, a quantitative single cell dataset of ~10,000 cells across 24 samples can be acquired and analysed in under 1 hour. LineHotel allows tracking growth of > 10 micro-colonies across 24 samples simultaneously for up to 4 generations. These easy-to-use devices can be provided in kit format, and will accelerate discoveries in diverse fields ranging from microbiology to systems and synthetic biology.

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

DOE PAGES

Rames, Matthew; Yu, Yadong; Ren, Gang

2014-08-15

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electronmore » microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high-resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography. Moreover, OpNS can be a high-throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.« less

Sequencing the Connectome

PubMed Central

Zador, Anthony M.; Dubnau, Joshua; Oyibo, Hassana K.; Zhan, Huiqing; Cao, Gang; Peikon, Ian D.

2012-01-01

Connectivity determines the function of neural circuits. Historically, circuit mapping has usually been viewed as a problem of microscopy, but no current method can achieve high-throughput mapping of entire circuits with single neuron precision. Here we describe a novel approach to determining connectivity. We propose BOINC (“barcoding of individual neuronal connections”), a method for converting the problem of connectivity into a form that can be read out by high-throughput DNA sequencing. The appeal of using sequencing is that its scale—sequencing billions of nucleotides per day is now routine—is a natural match to the complexity of neural circuits. An inexpensive high-throughput technique for establishing circuit connectivity at single neuron resolution could transform neuroscience research. PMID:23109909

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

PubMed

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

2017-09-14

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

Quantifying collagen orientation in breast tissue biopsies using SLIM (Conference Presentation)

NASA Astrophysics Data System (ADS)

Majeed, Hassaan; Okoro, Chukwuemeka; Balla, Andre; Toussaint, Kimani C.; Popescu, Gabriel

2017-02-01

Breast cancer is a major public health problem worldwide, being the most common type of cancer among women according to the World Health Organization (WHO). The WHO has further stressed the importance of an early determination of the disease course through prognostic markers. Recent studies have shown that the alignment of collagen fibers in tumor adjacent stroma correlate with poorer health outcomes in patients. Such studies have typically been carried out using Second-Harmonic Generation (SHG) microscopy. SHG images are very useful for quantifying collagen fiber orientation due their specificity to non-centrosymmetric structures in tissue, leading to high contrast in collagen rich areas. However, the imaging throughput in SHG microscopy is limited by its point scanning geometry. In this work, we show that SLIM, a wide-field high-throughput QPI technique, can be used to obtain the same information on collagen fiber orientation as is obtainable through SHG microscopy. We imaged a tissue microarray containing both benign and malignant cores using both SHG microscopy and SLIM. The cellular (non-collagenous) structures in the SLIM images were next segmented out using an algorithm developed in-house. Using the previously published Fourier Transform Second Harmonic Generation (FT-SHG) tool, the fiber orientations in SHG and segmented SLIM images were then quantified. The resulting histograms of fiber orientation angles showed that both SHG and SLIM generate similar measurements of collagen fiber orientation. The SLIM modality, however, can generate these results at much higher throughput due to its wide-field, whole-slide scanning capabilities.

Automated sample area definition for high-throughput microscopy.

PubMed

Zeder, M; Ellrott, A; Amann, R

2011-04-01

High-throughput screening platforms based on epifluorescence microscopy are powerful tools in a variety of scientific fields. Although some applications are based on imaging geometrically defined samples such as microtiter plates, multiwell slides, or spotted gene arrays, others need to cope with inhomogeneously located samples on glass slides. The analysis of microbial communities in aquatic systems by sample filtration on membrane filters followed by multiple fluorescent staining, or the investigation of tissue sections are examples. Therefore, we developed a strategy for flexible and fast definition of sample locations by the acquisition of whole slide overview images and automated sample recognition by image analysis. Our approach was tested on different microscopes and the computer programs are freely available (http://www.technobiology.ch). Copyright © 2011 International Society for Advancement of Cytometry.

Wide-field optical detection of nanoparticles using on-chip microscopy and self-assembled nanolenses

NASA Astrophysics Data System (ADS)

Mudanyali, Onur; McLeod, Euan; Luo, Wei; Greenbaum, Alon; Coskun, Ahmet F.; Hennequin, Yves; Allier, Cédric P.; Ozcan, Aydogan

2013-03-01

The direct observation of nanoscale objects is a challenging task for optical microscopy because the scattering from an individual nanoparticle is typically weak at optical wavelengths. Electron microscopy therefore remains one of the gold standard visualization methods for nanoparticles, despite its high cost, limited throughput and restricted field-of-view. Here, we describe a high-throughput, on-chip detection scheme that uses biocompatible wetting films to self-assemble aspheric liquid nanolenses around individual nanoparticles to enhance the contrast between the scattered and background light. We model the effect of the nanolens as a spatial phase mask centred on the particle and show that the holographic diffraction pattern of this effective phase mask allows detection of sub-100 nm particles across a large field-of-view of >20 mm2. As a proof-of-concept demonstration, we report on-chip detection of individual polystyrene nanoparticles, adenoviruses and influenza A (H1N1) viral particles.

Wide-field optical detection of nanoparticles using on-chip microscopy and self-assembled nanolenses

PubMed Central

Mudanyali, Onur; McLeod, Euan; Luo, Wei; Greenbaum, Alon; Coskun, Ahmet F.; Hennequin, Yves; Allier, Cédric P.; Ozcan, Aydogan

2013-01-01

The direct observation of nanoscale objects is a challenging task for optical microscopy because the scattering from an individual nanoparticle is typically weak at optical wavelengths. Electron microscopy therefore remains one of the gold standard visualization methods for nanoparticles, despite its high cost, limited throughput and restricted field-of-view. Here, we describe a high-throughput, on-chip detection scheme that uses biocompatible wetting films to self-assemble aspheric liquid nanolenses around individual nanoparticles to enhance the contrast between the scattered and background light. We model the effect of the nanolens as a spatial phase mask centred on the particle and show that the holographic diffraction pattern of this effective phase mask allows detection of sub-100 nm particles across a large field-of-view of >20 mm2. As a proof-of-concept demonstration, we report on-chip detection of individual polystyrene nanoparticles, adenoviruses and influenza A (H1N1) viral particles. PMID:24358054

Correlation of microstructure and thermo-mechanical properties of a novel hydrogen transport membrane

NASA Astrophysics Data System (ADS)

Zhang, Yongjun

A key part of the FutureGen concept is to support the production of hydrogen to fuel a "hydrogen economy," with the use of clean burning hydrogen in power-producing fuel cells, as well as for use as a transportation fuel. One of the key technical barriers to FutureGen deployment is reliable and efficient hydrogen separation technology. Most Hydrogen Transport Membrane (HTM) research currently focuses on separation technology and hydrogen flux characterization. No significant work has been performed on thermo-mechanical properties of HTMs. The objective of the thesis is to understand the structure-property correlation of HTM and to characterize (1) thermo mechanical properties under different reducing environments and thermal cycles (thermal shock), and (2) evaluate the stability of the novel HTM material. A novel HTM cermet bulk sample was characterized for its physical and mechanical properties at both room temperature and at elevated temperature up to 1000°C. Micro-structural properties and residual stresses were evaluated in order to understand the changing mechanism of the microstructure and its effects on the mechanical properties of materials. A correlation of the microstructural and thermo mechanical properties of the HTM system was established for both HTM and the substrate material. Mechanical properties of both selected structural ceramics and the novel HTM cermet bulk sample are affected mainly by porosity and microstructural features, such as grain size and pore size-distribution. The Young's Modulus (E-value) is positively correlated to the flexural strength for materials with similar crystallographic structure. However, for different crystallographic materials, physical properties are independent of mechanical properties. Microstructural properties, particularly, grain size and crystallographic structure, and thermodynamic properties are the main factors affecting the mechanical properties at both room and high temperatures. The HTM cermet behaves more like an elastic material at room temperature and as a ductile material at temperature above 850°C. The oxidation and the plasticity of Pd phase mainly affected the mechanical properties of HTM cermet at high temperature, also as a result of thermal cycling. Residual stress induced in the HTM by thermo cycles also plays a very critical role in defining the thermo-mechanical properties.

New Insight of Common Regulatory Pathways in Human Trabecular Meshwork Cells in Response to Dexamethasone and Prednisolone Using an Integrated Quantitative Proteomics: SWATH and MRM-HR Mass Spectrometry.

PubMed

Shan, Sze Wan; Do, Chi Wai; Lam, Thomas Chuen; Kong, Ricky Pak Wing; Li, King Kit; Chun, Ka Man; Stamer, William Daniel; To, Chi Ho

2017-10-06

The molecular pathophysiology of corticosteroid-induced ocular hypertension (CIH) is not well understood. To determine the biological mechanisms of CIH, this study investigated protein expression profiles of human trabecular meshwork (hTM) cells in response to dexamethasone and prednisolone treatment. Both discovery-based sequential windowed data independent acquisition of the total high-resolution mass spectra (SWATH-MS) and targeted based high resolution multiple reaction monitoring (MRM-HR) confirmation were applied using a hybrid quadrupole-time-of-flight mass spectrometer. A comprehensive list of 1759 proteins (1% FDR) was generated from the hTM. Quantitative proteomics revealed 20 differentially expressed proteins (p-value ≤ 0.05 and fold-change ≥ 1.5 or ≤ 0.67) commonly induced by prednisolone and dexamethasone, both at 300 nM. These included connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1), two proteins previously implicated in ocular hypertension, glaucoma, and the transforming growth factor-β pathway. Their gene expressions in response to corticosteroids were further confirmed using reverse-transcription polymerase chain reaction. Together with other novel proteins identified in the data sets, additional pathways implicated by these regulated proteins were the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, integrin cell surface interaction, extracellular matrix (ECM) proteoglycans, and ECM-receptor interaction. Our results indicated that an integrated platform of SWATH-MS and MRM-HR allows high throughput identification and confirmation of novel and known corticosteroid-regulated proteins in trabecular meshwork cells, demonstrating the power of this technique in extending the current understanding of the pathogenesis of CIH.

Unsupervised Clustering of Subcellular Protein Expression Patterns in High-Throughput Microscopy Images Reveals Protein Complexes and Functional Relationships between Proteins

PubMed Central

Handfield, Louis-François; Chong, Yolanda T.; Simmons, Jibril; Andrews, Brenda J.; Moses, Alan M.

2013-01-01

Protein subcellular localization has been systematically characterized in budding yeast using fluorescently tagged proteins. Based on the fluorescence microscopy images, subcellular localization of many proteins can be classified automatically using supervised machine learning approaches that have been trained to recognize predefined image classes based on statistical features. Here, we present an unsupervised analysis of protein expression patterns in a set of high-resolution, high-throughput microscope images. Our analysis is based on 7 biologically interpretable features which are evaluated on automatically identified cells, and whose cell-stage dependency is captured by a continuous model for cell growth. We show that it is possible to identify most previously identified localization patterns in a cluster analysis based on these features and that similarities between the inferred expression patterns contain more information about protein function than can be explained by a previous manual categorization of subcellular localization. Furthermore, the inferred cell-stage associated to each fluorescence measurement allows us to visualize large groups of proteins entering the bud at specific stages of bud growth. These correspond to proteins localized to organelles, revealing that the organelles must be entering the bud in a stereotypical order. We also identify and organize a smaller group of proteins that show subtle differences in the way they move around the bud during growth. Our results suggest that biologically interpretable features based on explicit models of cell morphology will yield unprecedented power for pattern discovery in high-resolution, high-throughput microscopy images. PMID:23785265

Pipeline for illumination correction of images for high-throughput microscopy.

PubMed

Singh, S; Bray, M-A; Jones, T R; Carpenter, A E

2014-12-01

The presence of systematic noise in images in high-throughput microscopy experiments can significantly impact the accuracy of downstream results. Among the most common sources of systematic noise is non-homogeneous illumination across the image field. This often adds an unacceptable level of noise, obscures true quantitative differences and precludes biological experiments that rely on accurate fluorescence intensity measurements. In this paper, we seek to quantify the improvement in the quality of high-content screen readouts due to software-based illumination correction. We present a straightforward illumination correction pipeline that has been used by our group across many experiments. We test the pipeline on real-world high-throughput image sets and evaluate the performance of the pipeline at two levels: (a) Z'-factor to evaluate the effect of the image correction on a univariate readout, representative of a typical high-content screen, and (b) classification accuracy on phenotypic signatures derived from the images, representative of an experiment involving more complex data mining. We find that applying the proposed post-hoc correction method improves performance in both experiments, even when illumination correction has already been applied using software associated with the instrument. To facilitate the ready application and future development of illumination correction methods, we have made our complete test data sets as well as open-source image analysis pipelines publicly available. This software-based solution has the potential to improve outcomes for a wide-variety of image-based HTS experiments. © 2014 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.

Custom Super-Resolution Microscope for the Structural Analysis of Nanostructures

DTIC Science & Technology

2018-05-29

research community. As part of our validation of the new design approach, we performed two - color imaging of pairs of adjacent oligo probes hybridized...nanostructures and biological targets. Our microscope features a large field of view and custom optics that facilitate 3D imaging and enhanced contrast in...our imaging throughput by creating two microscopy platforms for high-throughput, super-resolution materials characterization, with the AO set-up being

High-speed Fourier ptychographic microscopy based on programmable annular illuminations.

PubMed

Sun, Jiasong; Zuo, Chao; Zhang, Jialin; Fan, Yao; Chen, Qian

2018-05-16

High-throughput quantitative phase imaging (QPI) is essential to cellular phenotypes characterization as it allows high-content cell analysis and avoids adverse effects of staining reagents on cellular viability and cell signaling. Among different approaches, Fourier ptychographic microscopy (FPM) is probably the most promising technique to realize high-throughput QPI by synthesizing a wide-field, high-resolution complex image from multiple angle-variably illuminated, low-resolution images. However, the large dataset requirement in conventional FPM significantly limits its imaging speed, resulting in low temporal throughput. Moreover, the underlying theoretical mechanism as well as optimum illumination scheme for high-accuracy phase imaging in FPM remains unclear. Herein, we report a high-speed FPM technique based on programmable annular illuminations (AIFPM). The optical-transfer-function (OTF) analysis of FPM reveals that the low-frequency phase information can only be correctly recovered if the LEDs are precisely located at the edge of the objective numerical aperture (NA) in the frequency space. By using only 4 low-resolution images corresponding to 4 tilted illuminations matching a 10×, 0.4 NA objective, we present the high-speed imaging results of in vitro Hela cells mitosis and apoptosis at a frame rate of 25 Hz with a full-pitch resolution of 655 nm at a wavelength of 525 nm (effective NA = 0.8) across a wide field-of-view (FOV) of 1.77 mm 2 , corresponding to a space-bandwidth-time product of 411 megapixels per second. Our work reveals an important capability of FPM towards high-speed high-throughput imaging of in vitro live cells, achieving video-rate QPI performance across a wide range of scales, both spatial and temporal.

Molecular and Cellular Quantitative Microscopy: theoretical investigations, technological developments and applications to neurobiology

NASA Astrophysics Data System (ADS)

Esposito, Alessandro

2006-05-01

This PhD project aims at the development and evaluation of microscopy techniques for the quantitative detection of molecular interactions and cellular features. The primarily investigated techniques are Fαrster Resonance Energy Transfer imaging and Fluorescence Lifetime Imaging Microscopy. These techniques have the capability to quantitatively probe the biochemical environment of fluorophores. An automated microscope capable of unsupervised operation has been developed that enables the investigation of molecular and cellular properties at high throughput levels and the analysis of cellular heterogeneity. State-of-the-art Förster Resonance Energy Transfer imaging, Fluorescence Lifetime Imaging Microscopy, Confocal Laser Scanning Microscopy and the newly developed tools have been combined with cellular and molecular biology techniques for the investigation of protein-protein interactions, oligomerization and post-translational modifications of α-Synuclein and Tau, two proteins involved in Parkinson’s and Alzheimer’s disease, respectively. The high inter-disciplinarity of this project required the merging of the expertise of both the Molecular Biophysics Group at the Debye Institute - Utrecht University and the Cell Biophysics Group at the European Neuroscience Institute - Gαttingen University. This project was conducted also with the support and the collaboration of the Center for the Molecular Physiology of the Brain (Göttingen), particularly with the groups associated with the Molecular Quantitative Microscopy and Parkinson’s Disease and Aggregopathies areas. This work demonstrates that molecular and cellular quantitative microscopy can be used in combination with high-throughput screening as a powerful tool for the investigation of the molecular mechanisms of complex biological phenomena like those occurring in neurodegenerative diseases.

Cytopathological image analysis using deep-learning networks in microfluidic microscopy.

PubMed

Gopakumar, G; Hari Babu, K; Mishra, Deepak; Gorthi, Sai Siva; Sai Subrahmanyam, Gorthi R K

2017-01-01

Cytopathologic testing is one of the most critical steps in the diagnosis of diseases, including cancer. However, the task is laborious and demands skill. Associated high cost and low throughput drew considerable interest in automating the testing process. Several neural network architectures were designed to provide human expertise to machines. In this paper, we explore and propose the feasibility of using deep-learning networks for cytopathologic analysis by performing the classification of three important unlabeled, unstained leukemia cell lines (K562, MOLT, and HL60). The cell images used in the classification are captured using a low-cost, high-throughput cell imaging technique: microfluidics-based imaging flow cytometry. We demonstrate that without any conventional fine segmentation followed by explicit feature extraction, the proposed deep-learning algorithms effectively classify the coarsely localized cell lines. We show that the designed deep belief network as well as the deeply pretrained convolutional neural network outperform the conventionally used decision systems and are important in the medical domain, where the availability of labeled data is limited for training. We hope that our work enables the development of a clinically significant high-throughput microfluidic microscopy-based tool for disease screening/triaging, especially in resource-limited settings.

Breast cancer diagnosis using spatial light interference microscopy

NASA Astrophysics Data System (ADS)

Majeed, Hassaan; Kandel, Mikhail E.; Han, Kevin; Luo, Zelun; Macias, Virgilia; Tangella, Krishnarao; Balla, Andre; Popescu, Gabriel

2015-11-01

The standard practice in histopathology of breast cancers is to examine a hematoxylin and eosin (H&E) stained tissue biopsy under a microscope to diagnose whether a lesion is benign or malignant. This determination is made based on a manual, qualitative inspection, making it subject to investigator bias and resulting in low throughput. Hence, a quantitative, label-free, and high-throughput diagnosis method is highly desirable. We present here preliminary results showing the potential of quantitative phase imaging for breast cancer screening and help with differential diagnosis. We generated phase maps of unstained breast tissue biopsies using spatial light interference microscopy (SLIM). As a first step toward quantitative diagnosis based on SLIM, we carried out a qualitative evaluation of our label-free images. These images were shown to two pathologists who classified each case as either benign or malignant. This diagnosis was then compared against the diagnosis of the two pathologists on corresponding H&E stained tissue images and the number of agreements were counted. The agreement between SLIM and H&E based diagnosis was 88% for the first pathologist and 87% for the second. Our results demonstrate the potential and promise of SLIM for quantitative, label-free, and high-throughput diagnosis.

Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains.

PubMed

Müllenbroich, M Caroline; Silvestri, Ludovico; Onofri, Leonardo; Costantini, Irene; Hoff, Marcel Van't; Sacconi, Leonardo; Iannello, Giulio; Pavone, Francesco S

2015-10-01

Comprehensive mapping and quantification of neuronal projections in the central nervous system requires high-throughput imaging of large volumes with microscopic resolution. To this end, we have developed a confocal light-sheet microscope that has been optimized for three-dimensional (3-D) imaging of structurally intact clarified whole-mount mouse brains. We describe the optical and electromechanical arrangement of the microscope and give details on the organization of the microscope management software. The software orchestrates all components of the microscope, coordinates critical timing and synchronization, and has been written in a versatile and modular structure using the LabVIEW language. It can easily be adapted and integrated to other microscope systems and has been made freely available to the light-sheet community. The tremendous amount of data routinely generated by light-sheet microscopy further requires novel strategies for data handling and storage. To complete the full imaging pipeline of our high-throughput microscope, we further elaborate on big data management from streaming of raw images up to stitching of 3-D datasets. The mesoscale neuroanatomy imaged at micron-scale resolution in those datasets allows characterization and quantification of neuronal projections in unsectioned mouse brains.

High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Ito, Takuro; Jiang, Yiyue; Ozeki, Yasuyuki; Goda, Keisuke

2016-01-01

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary) and nitrogen-deficient (lipid-accumulated) E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.

Lack of P4H-TM in mice results in age-related retinal and renal alterations.

PubMed

Leinonen, Henri; Rossi, Maarit; Salo, Antti M; Tiainen, Päivi; Hyvärinen, Jaana; Pitkänen, Marja; Sormunen, Raija; Miinalainen, Ilkka; Zhang, Chi; Soininen, Raija; Kivirikko, Kari I; Koskelainen, Ari; Tanila, Heikki; Myllyharju, Johanna; Koivunen, Peppi

2016-09-01

Age-related macular degeneration (AMD), affecting the retinal pigment epithelium (RPE), is the leading cause of blindness in middle-aged and older people in developed countries. Genetic and environmental risk factors have been identified, but no effective cure exists. Using a mouse model we show that a transmembrane prolyl 4-hydroxylase (P4H-TM), which participates in the oxygen-dependent regulation of the hypoxia-inducible factor (HIF), is a potential novel candidate gene for AMD. We show that P4h-tm had its highest expression levels in the mouse RPE and brain, heart, lung, skeletal muscle and kidney. P4h-tm -/- mice were fertile and had a normal life span. Lack of P4h-tm stabilized HIF-1α in cortical neurons under normoxia, while in hypoxia it increased the expression of certain HIF target genes in tissues with high endogenous P4h-tm expression levels more than in wild-type mice. Renal erythropoietin levels increased in P4h-tm -/- mice with aging, but the resulting ∼2-fold increase in erythropoietin serum levels did not lead to erythrocytosis. Instead, accumulation of lipid-containing lamellar bodies in renal tubuli was detected in P4h-tm -/- mice with aging, resulting in inflammation and fibrosis, and later glomerular sclerosis and albuminuria. Lack of P4h-tm was associated with retinal thinning, rosette-like infoldings and drusen-like structure accumulation in RPE with aging, as is characteristic of AMD. Photoreceptor recycling was compromised, and electroretinograms revealed functional impairment of the cone pathway in adult P4h-tm -/- mice and cone and rod deficiency in middle-aged mice. P4H-TM is therefore imperative for normal vision, and potentially a novel candidate for age-induced diseases, such as AMD. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A comprehensive device modelling of perovskite solar cell with inorganic copper iodide as hole transport material

NASA Astrophysics Data System (ADS)

Zulqarnain Haider, Syed; Anwar, Hafeez; Wang, Mingqing

2018-03-01

Hole transport material (HTM) plays an important role in the efficiency and stability of perovskite solar cells (PSCs). Spiro-MeOTAD, the commonly used HTM, is costly and can be easily degraded by heat and moisture, thus offering hindrance to commercialize PSCs. There is dire need to find an alternate inorganic and stable HTM to exploit PSCs with their maximum capability. In this paper, a comprehensive device simulation is used to study various possible parameters that can influence the performance of perovskite solar cell with CuI as HTM. These include the effect of doping density, defect density and thickness of absorber layer, along with the influence of diffusion length of carriers as well as electron affinity of electron transport layer (ETM) and HTM on the performance of PSCs. In addition, hole mobility and doping density of HTM is also investigated. CuI is a p-type inorganic material with low cost and relatively high stability. It is found that concentration of dopant in absorber layer and HTM, the electron affinity of HTM and ETM affect the performance of solar cell minutely, while cell performance improves greatly with the reduction of defect density. Upon optimization of parameters, power conversion efficiency for this device is found to be 21.32%. The result shows that lead-based PSC with CuI as HTM is an efficient system. Enhancing the stability and reduction of defect density are critical factors for future research. These factors can be improved by better fabrication process and proper encapsulation of solar cell.

Near-common-path interferometer for imaging Fourier-transform spectroscopy in wide-field microscopy

PubMed Central

Wadduwage, Dushan N.; Singh, Vijay Raj; Choi, Heejin; Yaqoob, Zahid; Heemskerk, Hans; Matsudaira, Paul; So, Peter T. C.

2017-01-01

Imaging Fourier-transform spectroscopy (IFTS) is a powerful method for biological hyperspectral analysis based on various imaging modalities, such as fluorescence or Raman. Since the measurements are taken in the Fourier space of the spectrum, it can also take advantage of compressed sensing strategies. IFTS has been readily implemented in high-throughput, high-content microscope systems based on wide-field imaging modalities. However, there are limitations in existing wide-field IFTS designs. Non-common-path approaches are less phase-stable. Alternatively, designs based on the common-path Sagnac interferometer are stable, but incompatible with high-throughput imaging. They require exhaustive sequential scanning over large interferometric path delays, making compressive strategic data acquisition impossible. In this paper, we present a novel phase-stable, near-common-path interferometer enabling high-throughput hyperspectral imaging based on strategic data acquisition. Our results suggest that this approach can improve throughput over those of many other wide-field spectral techniques by more than an order of magnitude without compromising phase stability. PMID:29392168

Simple technique for high-throughput marking of distinguishable micro-areas for microscopy.

PubMed

Henrichs, Leonard F; Chen, L I; Bell, Andrew J

2016-04-01

Today's (nano)-functional materials, usually exhibiting complex physical properties require local investigation with different microscopy techniques covering different physical aspects such as dipolar and magnetic structure. However, often these must be employed on the very same sample position to be able to truly correlate those different information and corresponding properties. This can be very challenging if not impossible especially when samples lack prominent features for orientation. Here, we present a simple but effective method to mark hundreds of approximately 15×15 μm sample areas at one time by using a commercial transmission electron microscopy grid as shadow mask in combination with thin-film deposition. Areas can be easily distinguished when using a reference or finder grid structure as shadow mask. We show that the method is suitable to combine many techniques such as light microscopy, scanning probe microscopy and scanning electron microscopy. Furthermore, we find that best results are achieved when depositing aluminium on a flat sample surface using electron-beam evaporation which ensures good line-of-sight deposition. This inexpensive high-throughput method has several advantageous over other marking techniques such as focused ion-beam processing especially when batch processing or marking of many areas is required. Nevertheless, the technique could be particularly valuable, when used in junction with, for example focused ion-beam sectioning to obtain a thin lamellar of a particular pre-selected area. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy.

PubMed

Chen, Robert; Rishi, Harneet S; Potapov, Vladimir; Yamada, Masaki R; Yeh, Vincent J; Chow, Thomas; Cheung, Celia L; Jones, Austin T; Johnson, Terry D; Keating, Amy E; DeLoache, William C; Dueber, John E

2015-11-20

Dramatic progress has been made in the design and build phases of the design-build-test cycle for engineering cells. However, the test phase usually limits throughput, as many outputs of interest are not amenable to rapid analytical measurements. For example, phenotypes such as motility, morphology, and subcellular localization can be readily measured by microscopy, but analysis of these phenotypes is notoriously slow. To increase throughput, we developed microscopy-readable barcodes (MiCodes) composed of fluorescent proteins targeted to discernible organelles. In this system, a unique barcode can be genetically linked to each library member, making possible the parallel analysis of phenotypes of interest via microscopy. As a first demonstration, we MiCoded a set of synthetic coiled-coil leucine zipper proteins to allow an 8 × 8 matrix to be tested for specific interactions in micrographs consisting of mixed populations of cells. A novel microscopy-readable two-hybrid fluorescence localization assay for probing candidate interactions in the cytosol was also developed using a bait protein targeted to the peroxisome and a prey protein tagged with a fluorescent protein. This work introduces a generalizable, scalable platform for making microscopy amenable to higher-throughput library screening experiments, thereby coupling the power of imaging with the utility of combinatorial search paradigms.

Hole-Transporting Materials for Printable Perovskite Solar Cells

PubMed Central

Salunke, Jagadish K.; Priimagi, Arri

2017-01-01

Perovskite solar cells (PSCs) represent undoubtedly the most significant breakthrough in photovoltaic technology since the 1970s, with an increase in their power conversion efficiency from less than 5% to over 22% in just a few years. Hole-transporting materials (HTMs) are an essential building block of PSC architectures. Currently, 2,2’,7,7’-tetrakis-(N,N’-di-p-methoxyphenylamine)-9,9’-spirobifluorene), better known as spiro-OMeTAD, is the most widely-used HTM to obtain high-efficiency devices. However, it is a tremendously expensive material with mediocre hole carrier mobility. To ensure wide-scale application of PSC-based technologies, alternative HTMs are being proposed. Solution-processable HTMs are crucial to develop inexpensive, high-throughput and printable large-area PSCs. In this review, we present the most recent advances in the design and development of different types of HTMs, with a particular focus on mesoscopic PSCs. Finally, we outline possible future research directions for further optimization of the HTMs to achieve low-cost, stable and large-area PSCs. PMID:28914823

Massively Parallel Rogue Cell Detection Using Serial Time-Encoded Amplified Microscopy of Inertially Ordered Cells in High-Throughput Flow

DTIC Science & Technology

2012-08-01

techniques and STEAM imager. It couples the high-speed capability of the STEAM imager and differential phase contrast imaging of DIC / Nomarski microscopy...On 10 TPE chips, we obtained 9 homogenous and strong bonds, the failed bond being due to operator error and presence of air bubbles in the TPE...instruments, structural dynamics, and microelectromechanical systems (MEMS) via laser-scanning surface vibrometry , and observation of biomechanical motility

Massively Parallel Rogue Cell Detection Using Serial Time-Encoded Amplified Microscopy of Inertially Ordered Cells in High Throughput Flow

DTIC Science & Technology

2011-08-01

further chemical analysis of the cells. While in our proof-of-concept demonstration, we showed high- throughput screening of budding yeast and...of 8.0 mW/cm2 through the transparency mask for 90 seconds. The wafer was baked again at 95°C for 4 minutes then developed in SU-8 developer...sonicated in isopropanol for 5 minutes, sonicated in deionized H2O for 5 minutes, and baked at 65°C for at least 30 minutes. Holes were punched

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

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

2018-06-01

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

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

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

2018-04-01

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

Lensless digital holographic microscopy and its applications in biomedicine and environmental monitoring.

PubMed

Wu, Yichen; Ozcan, Aydogan

2018-03-01

Optical compound microscope has been a major tool in biomedical imaging for centuries. Its performance relies on relatively complicated, bulky and expensive lenses and alignment mechanics. In contrast, the lensless microscope digitally reconstructs microscopic images of specimens without using any lenses, as a result of which it can be made much smaller, lighter and lower-cost. Furthermore, the limited space-bandwidth product of objective lenses in a conventional microscope can be significantly surpassed by a lensless microscope. Such lensless imaging designs have enabled high-resolution and high-throughput imaging of specimens using compact, portable and cost-effective devices to potentially address various point-of-care, global-health and telemedicine related challenges. In this review, we discuss the operation principles and the methods behind lensless digital holographic on-chip microscopy. We also go over various applications that are enabled by cost-effective and compact implementations of lensless microscopy, including some recent work on air quality monitoring, which utilized machine learning for high-throughput and accurate quantification of particulate matter in air. Finally, we conclude with a brief future outlook of this computational imaging technology. Copyright © 2017 Elsevier Inc. All rights reserved.

The intrinsic stiffness of human trabecular meshwork cells increases with senescence

PubMed Central

Chang, Yow-Ren; Murphy, Christopher J.; Russell, Paul

2015-01-01

Dysfunction of the human trabecular meshwork (HTM) plays a central role in the age-associated disease glaucoma, a leading cause of irreversible blindness. The etiology remains poorly understood but cellular senescence, increased stiffness of the tissue, and the expression of Wnt antagonists such as secreted frizzled related protein-1 (SFRP1) have been implicated. However, it is not known if senescence is causally linked to either stiffness or SFRP1 expression. In this study, we utilized in vitro HTM senescence to determine the effect on cellular stiffening and SFRP1 expression. Stiffness of cultured cells was measured using atomic force microscopy and the morphology of the cytoskeleton was determined using immunofluorescent analysis. SFRP1 expression was measured using qPCR and immunofluorescent analysis. Senescent cell stiffness increased 1.88±0.14 or 2.57±0.14 fold in the presence or absence of serum, respectively. This was accompanied by increased vimentin expression, stress fiber formation, and SFRP1 expression. In aggregate, these data demonstrate that senescence may be a causal factor in HTM stiffening and elevated SFRP1 expression, and contribute towards disease progression. These findings provide insight into the etiology of glaucoma and, more broadly, suggest a causal link between senescence and altered tissue biomechanics in aging-associated diseases. PMID:25915531

Toward reliable and repeatable automated STEM-EDS metrology with high throughput

NASA Astrophysics Data System (ADS)

Zhong, Zhenxin; Donald, Jason; Dutrow, Gavin; Roller, Justin; Ugurlu, Ozan; Verheijen, Martin; Bidiuk, Oleksii

2018-03-01

New materials and designs in complex 3D architectures in logic and memory devices have raised complexity in S/TEM metrology. In this paper, we report about a newly developed, automated, scanning transmission electron microscopy (STEM) based, energy dispersive X-ray spectroscopy (STEM-EDS) metrology method that addresses these challenges. Different methodologies toward repeatable and efficient, automated STEM-EDS metrology with high throughput are presented: we introduce the best known auto-EDS acquisition and quantification methods for robust and reliable metrology and present how electron exposure dose impacts the EDS metrology reproducibility, either due to poor signalto-noise ratio (SNR) at low dose or due to sample modifications at high dose conditions. Finally, we discuss the limitations of the STEM-EDS metrology technique and propose strategies to optimize the process both in terms of throughput and metrology reliability.

High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy.

PubMed

Bravo-San Pedro, J M; Pietrocola, F; Sica, V; Izzo, V; Sauvat, A; Kepp, O; Maiuri, M C; Kroemer, G; Galluzzi, L

2017-01-01

Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned organelle commonly known as autophagosome. Various methods have been developed to quantify the size of the autophagosomal compartment, which is an indirect indicator of macroautophagic responses, based on the peculiar ability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; best known as LC3) to accumulate in forming autophagosomes upon maturation. One particularly convenient method to monitor the accumulation of mature LC3 within autophagosomes relies on a green fluorescent protein (GFP)-tagged variant of this protein and fluorescence microscopy. In physiological conditions, cells transfected temporarily or stably with a GFP-LC3-encoding construct exhibit a diffuse green fluorescence over the cytoplasm and nucleus. Conversely, in response to macroautophagy-promoting stimuli, the GFP-LC3 signal becomes punctate and often (but not always) predominantly cytoplasmic. The accumulation of GFP-LC3 in cytoplasmic dots, however, also ensues the blockage of any of the steps that ensure the degradation of mature autophagosomes, calling for the implementation of strategies that accurately discriminate between an increase in autophagic flux and an arrest in autophagic degradation. Various cell lines have been engineered to stably express GFP-LC3, which-combined with the appropriate controls of flux, high-throughput imaging stations, and automated image analysis-offer a relatively straightforward tool to screen large chemical or biological libraries for inducers or inhibitors of autophagy. Here, we describe a simple and robust method for the high-throughput quantification of GFP-LC3 + dots by automated fluorescence microscopy. © 2017 Elsevier Inc. All rights reserved.

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Interface Play between Perovskite and Hole Selective Layer on the Performance and Stability of Perovskite Solar Cells.

PubMed

Salado, Manuel; Idigoras, Jesus; Calio, Laura; Kazim, Samrana; Nazeeruddin, Mohammad Khaja; Anta, Juan A; Ahmad, Shahzada

2016-12-21

Perovskite solar cells with variety of hole selective contacts such as 2,2',7,7'-tetrakis(N,N-di-p-methoxyphenylamine)-9,9'-spirobifluorene (Spiro-OMeTAD), poly(3-hexylthiophene-2,5-diyl), poly[bis(4-phenyl)(2,5,6-trimentlyphenyl)amine], 5,10,15-trihexyl-3,8,13-tris(4-methoxyphenyl)-10,15-dihydro-5H-diindolo[3,2-a:3',2'-c]carbazole (HMPDI), and 2',7'-bis(bis(4-methoxyphenyl)amino)spiro[cyclopenta[2,1-b:3,4-b']dithiophene-4,9'-fluorene] were employed to elucidate its role at the interface of perovskite and metallic cathode. Microscopy images revealed Spiro-OMeTAD and HMPDI produce smoother and intimate contact between perovskite/hole transporting materials (HTM) interfaces among others evaluated here. This morphological feature appears to be connected with three fundamental facts: (1) hole injection to the HTM is much more efficient as evidenced by photoluminescence measurements, (2) recombination losses are less important as evidenced by intensity-modulated photovoltage spectroscopy and impedance spectroscopy measurements, and (3) fabricated solar cells are much more robust against degradation by moisture. Devices with higher open-circuit photovoltages are characterized by higher values of the recombination resistance extracted from the impedance data. The variation in device hysteresis behavior can be ascribed mainly due to the molecular interaction and the core of HTM employed. In all cases, this fact is related with a larger value of the low-frequency capacitance, which indicates that the HTM can induce specific slow processes of ion accumulation at the interface. Notably, these processes tend to slowly relax in time, as hysteresis is substantially reduced for aged devices.

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

Heusinkveld, Harm J.; Westerink, Remco H.S., E-mail: R.Westerink@uu.nl

Calcium plays a crucial role in virtually all cellular processes, including neurotransmission. The intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) is therefore an important readout in neurotoxicological and neuropharmacological studies. Consequently, there is an increasing demand for high-throughput measurements of [Ca{sup 2+}]{sub i}, e.g. using multi-well microplate readers, in hazard characterization, human risk assessment and drug development. However, changes in [Ca{sup 2+}]{sub i} are highly dynamic, thereby creating challenges for high-throughput measurements. Nonetheless, several protocols are now available for real-time kinetic measurement of [Ca{sup 2+}]{sub i} in plate reader systems, though the results of such plate reader-based measurements have beenmore » questioned. In view of the increasing use of plate reader systems for measurements of [Ca{sup 2+}]{sub i} a careful evaluation of current technologies is warranted. We therefore performed an extensive set of experiments, using two cell lines (PC12 and B35) and two fluorescent calcium-sensitive dyes (Fluo-4 and Fura-2), for comparison of a linear plate reader system with single cell fluorescence microscopy. Our data demonstrate that the use of plate reader systems for high-throughput real-time kinetic measurements of [Ca{sup 2+}]{sub i} is associated with many pitfalls and limitations, including erroneous sustained increases in fluorescence, limited sensitivity and lack of single cell resolution. Additionally, our data demonstrate that probenecid, which is often used to prevent dye leakage, effectively inhibits the depolarization-evoked increase in [Ca{sup 2+}]{sub i}. Overall, the data indicate that the use of current plate reader-based strategies for high-throughput real-time kinetic measurements of [Ca{sup 2+}]{sub i} is associated with caveats and limitations that require further investigation. - Research Highlights: > The use of plate readers for high-throughput screening of intracellular Ca{sup 2+} is associated with many pitfalls and limitations. > Single cell fluorescent microscopy is recommended for measurements of intracellular Ca{sup 2+}. > Dual-wavelength dyes (Fura-2) are preferred over single-wavelength dyes (Fluo-4) for measurements of intracellular Ca{sup 2+}. > Probenecid prevents dye leakage but abolishes depolarization-evoked Ca{sup 2+} influx, severely hampering measurements of Ca{sup 2+}. > In general, care should be taken when interpreting data from high-throughput kinetic measurements.« less

High Throughput Biological Analysis Using Multi-bit Magnetic Digital Planar Tags

NASA Astrophysics Data System (ADS)

Hong, B.; Jeong, J.-R.; Llandro, J.; Hayward, T. J.; Ionescu, A.; Trypiniotis, T.; Mitrelias, T.; Kopper, K. P.; Steinmuller, S. J.; Bland, J. A. C.

2008-06-01

We report a new magnetic labelling technology for high-throughput biomolecular identification and DNA sequencing. Planar multi-bit magnetic tags have been designed and fabricated, which comprise a magnetic barcode formed by an ensemble of micron-sized thin film Ni80Fe20 bars encapsulated in SU8. We show that by using a globally applied magnetic field and magneto-optical Kerr microscopy the magnetic elements in the multi-bit magnetic tags can be addressed individually and encoded/decoded remotely. The critical steps needed to show the feasibility of this technology are demonstrated, including fabrication, flow transport, remote writing and reading, and successful functionalization of the tags as verified by fluorescence detection. This approach is ideal for encoding information on tags in microfluidic flow or suspension, for such applications as labelling of chemical precursors during drug synthesis and combinatorial library-based high-throughput multiplexed bioassays.

Screening for Chemical Effects on Neuronal Proliferation and Neurite Outgrowth Using High-Content/High-Throughput Microscopy

EPA Science Inventory

The need to develop novel screening methods for developmental neurotoxicity in order to alleviate the demands of cost, time, and animals required for in vivo toxicity studies is well recognized. Accordingly, the U.S. EPA launched the ToxCast research program in 2007 to develop c...

High definition infrared chemical imaging of colorectal tissue using a Spero QCL microscope.

PubMed

Bird, B; Rowlette, J

2017-04-10

Mid-infrared microscopy has become a key technique in the field of biomedical science and spectroscopy. This label-free, non-destructive technique permits the visualisation of a wide range of intrinsic biochemical markers in tissues, cells and biofluids by detection of the vibrational modes of the constituent molecules. Together, infrared microscopy and chemometrics is a widely accepted method that can distinguish healthy and diseased states with high accuracy. However, despite the exponential growth of the field and its research world-wide, several barriers currently exist for its full translation into the clinical sphere, namely sample throughput and data management. The advent and incorporation of quantum cascade lasers (QCLs) into infrared microscopes could help propel the field over these remaining hurdles. Such systems offer several advantages over their FT-IR counterparts, a simpler instrument architecture, improved photon flux, use of room temperature camera systems, and the flexibility of a tunable illumination source. In this current study we explore the use of a QCL infrared microscope to produce high definition, high throughput chemical images useful for the screening of biopsied colorectal tissue.

Invited review article: high-speed flexure-guided nanopositioning: mechanical design and control issues.

PubMed

Yong, Y K; Moheimani, S O R; Kenton, B J; Leang, K K

2012-12-01

Recent interest in high-speed scanning probe microscopy for high-throughput applications including video-rate atomic force microscopy and probe-based nanofabrication has sparked attention on the development of high-bandwidth flexure-guided nanopositioning systems (nanopositioners). Such nanopositioners are designed to move samples with sub-nanometer resolution with positioning bandwidth in the kilohertz range. State-of-the-art designs incorporate uniquely designed flexure mechanisms driven by compact and stiff piezoelectric actuators. This paper surveys key advances in mechanical design and control of dynamic effects and nonlinearities, in the context of high-speed nanopositioning. Future challenges and research topics are also discussed.

High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis.

PubMed

Zheng, Xianlin; Lu, Yiqing; Zhao, Jiangbo; Zhang, Yuhai; Ren, Wei; Liu, Deming; Lu, Jie; Piper, James A; Leif, Robert C; Liu, Xiaogang; Jin, Dayong

2016-01-19

Compared with routine microscopy imaging of a few analytes at a time, rapid scanning through the whole sample area of a microscope slide to locate every single target object offers many advantages in terms of simplicity, speed, throughput, and potential for robust quantitative analysis. Existing techniques that accommodate solid-phase samples incorporating individual micrometer-sized targets generally rely on digital microscopy and image analysis, with intrinsically low throughput and reliability. Here, we report an advanced on-the-fly stage scanning method to achieve high-precision target location across the whole slide. By integrating X- and Y-axis linear encoders to a motorized stage as the virtual "grids" that provide real-time positional references, we demonstrate an orthogonal scanning automated microscopy (OSAM) technique which can search a coverslip area of 50 × 24 mm(2) in just 5.3 min and locate individual 15 μm lanthanide luminescent microspheres with standard deviations of 1.38 and 1.75 μm in X and Y directions. Alongside implementation of an autofocus unit that compensates the tilt of a slide in the Z-axis in real time, we increase the luminescence detection efficiency by 35% with an improved coefficient of variation. We demonstrate the capability of advanced OSAM for robust quantification of luminescence intensities and lifetimes for a variety of micrometer-scale luminescent targets, specifically single down-shifting and upconversion microspheres, crystalline microplates, and color-barcoded microrods, as well as quantitative suspension array assays of biotinylated-DNA functionalized upconversion nanoparticles.

High-throughput label-free screening of euglena gracilis with optofluidic time-stretch quantitative phase microscopy

NASA Astrophysics Data System (ADS)

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

2017-02-01

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, microalgal biofuel is expected to play a key role in reducing the detrimental effects of global warming since microalgae absorb atmospheric CO2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid contents and fail to characterize a diverse population of microalgal cells with single-cell resolution in a noninvasive and interference-free manner. Here we demonstrate high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy. In particular, we use Euglena gracilis - an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement) within lipid droplets. Our optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch phase-contrast microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase contents of every single cell at a high throughput of 10,000 cells/s. We characterize heterogeneous populations of E. gracilis cells under two different culture conditions to evaluate their lipid production efficiency. Our method holds promise as an effective analytical tool for microalgaebased biofuel production.

High-throughput microscopy must re-invent the microscope rather than speed up its functions

PubMed Central

Oheim, M

2007-01-01

Knowledge gained from the revolutions in genomics and proteomics has helped to identify many of the key molecules involved in cellular signalling. Researchers, both in academia and in the pharmaceutical industry, now screen, at a sub-cellular level, where and when these proteins interact. Fluorescence imaging and molecular labelling combine to provide a powerful tool for real-time functional biochemistry with molecular resolution. However, they traditionally have been work-intensive, required trained personnel, and suffered from low through-put due to sample preparation, loading and handling. The need for speeding up microscopy is apparent from the tremendous complexity of cellular signalling pathways, the inherent biological variability, as well as the possibility that the same molecule plays different roles in different sub-cellular compartments. Research institutes and companies have teamed up to develop imaging cytometers of ever-increasing complexity. However, to truly go high-speed, sub-cellular imaging must free itself from the rigid framework of current microscopes. PMID:17603553

Comparison of Nerve Growth Factor Receptor Binding Models Using Heterodimeric Muteins

PubMed Central

Mehta, Hrishikesh M.; Woo, Sang B.; Neet, Kenneth E.

2013-01-01

Nerve growth factor (NGF) is a homodimer that binds to two distinct receptor types, TrkA and p75, to support survival and differentiation of neurons. The high-affinity binding on the cell surface is believed to involve a heteroreceptor complex, but its exact nature is unclear. We developed a heterodimer (heteromutein) of two NGF muteins that can bind p75 and TrkA on opposite sides of the heterodimer, but not two TrkA receptors. Previously described muteins are Δ9/13 that is TrkA negative and 7-84-103 that is signal selective through TrkA. The heteromutein (Htm1) was used to study the heteroreceptor complex formation and function, in the putative absence of NGF-induced TrkA dimerization. Cellular binding assays indicated that Htm1 does not bind TrkA as efficiently as wild-type (wt) NGF but has better affinity than either homodimeric mutein. Htm1, 7-84-103, and Δ9/13 were each able to compete for cold-temperature, cold-chase stable binding on PC12 cells, indicating that binding to p75 was required for a portion of this high-affinity binding. Survival, neurite outgrowth, and MAPK signaling in PC12 cells also showed a reduced response for Htm1, compared with wtNGF, but was better than the parent muteins in the order wtNGF > Htm1 > 7-84-103 >> Δ9/13. Htm1 and 7-84-103 demonstrated similar levels of survival on cells expressing only TrkA. In the longstanding debate on the NGF receptor binding mechanism, our data support the ligand passing of NGF from p75 to TrkA involving a transient heteroreceptor complex of p75-NGF-TrkA. PMID:22903500

Ultrathin efficient perovskite solar cells employing a periodic structure of a composite hole conductor for elevated plasmonic light harvesting and hole collection

NASA Astrophysics Data System (ADS)

Long, Mingzhu; Chen, Zefeng; Zhang, Tiankai; Xiao, Yubin; Zeng, Xiaoliang; Chen, Jian; Yan, Keyou; Xu, Jianbin

2016-03-01

We developed a molecule/polymer composite hole transporting material (HTM) with a periodic microstructure for morphology replication of a corrugated Au electrode, which in combination plays a dual role in the optical and electronic enhancement of high performance perovskite solar cells (PSCs). The electro-optics revealed that perovskite couldn't readily extinct the red light even though the thickness increased to 370 nm, but we found that the quasi periodic microstructure composite (PMC) HTM in combination with the conformal Au electrode could promote the absorption through the enhanced cavity effects, leading to comparable absorption even using much thinner perovskite (240 nm). We identified that the cavity was the combination of Fabry-Pérot interferometer and surface plasmonic resonance, with light harvesting enhancement through surface plasmon polariton or waveguide modes that propagate in the plane of the perovskite layer. On the other hand, the PMC HTM increased hole conductivity by one order of magnitude with respect to standard spiro-OMeTAD HTM due to molecular packing and self-assembly, embodying traceable hole mobility and density elevation up to 3 times, and thus the hysteresis was greatly avoided. Owing to dual optical and electronic enhancement, the PMC PSC afforded high efficiency PSC using as thin as 240 nm perovskite layer, delivering a Voc of 1.05 V, Jsc of 22.9 mA cm-2, FF of 0.736, and efficiency amounting to 17.7% PCE, the highest efficiency with ultrathin perovskite layer.We developed a molecule/polymer composite hole transporting material (HTM) with a periodic microstructure for morphology replication of a corrugated Au electrode, which in combination plays a dual role in the optical and electronic enhancement of high performance perovskite solar cells (PSCs). The electro-optics revealed that perovskite couldn't readily extinct the red light even though the thickness increased to 370 nm, but we found that the quasi periodic microstructure composite (PMC) HTM in combination with the conformal Au electrode could promote the absorption through the enhanced cavity effects, leading to comparable absorption even using much thinner perovskite (240 nm). We identified that the cavity was the combination of Fabry-Pérot interferometer and surface plasmonic resonance, with light harvesting enhancement through surface plasmon polariton or waveguide modes that propagate in the plane of the perovskite layer. On the other hand, the PMC HTM increased hole conductivity by one order of magnitude with respect to standard spiro-OMeTAD HTM due to molecular packing and self-assembly, embodying traceable hole mobility and density elevation up to 3 times, and thus the hysteresis was greatly avoided. Owing to dual optical and electronic enhancement, the PMC PSC afforded high efficiency PSC using as thin as 240 nm perovskite layer, delivering a Voc of 1.05 V, Jsc of 22.9 mA cm-2, FF of 0.736, and efficiency amounting to 17.7% PCE, the highest efficiency with ultrathin perovskite layer. Electronic supplementary information (ESI) available: XRD patterns corresponding to the perovskite; AFM images of 3D PMC HTM perovskite solar cells; performance statistics for 3D PMC HTM; ultraviolet photoelectron spectra (UPS) of HTMs on FTO. See DOI: 10.1039/c5nr05042a

A simple and sensitive high-throughput GFP screening in woody and herbaceous plants.

PubMed

Hily, Jean-Michel; Liu, Zongrang

2009-03-01

Green fluorescent protein (GFP) has been used widely as a powerful bioluminescent reporter, but its visualization by existing methods in tissues or whole plants and its utilization for high-throughput screening remains challenging in many species. Here, we report a fluorescence image analyzer-based method for GFP detection and its utility for high-throughput screening of transformed plants. Of three detection methods tested, the Typhoon fluorescence scanner was able to detect GFP fluorescence in all Arabidopsis thaliana tissues and apple leaves, while regular fluorescence microscopy detected it only in Arabidopsis flowers and siliques but barely in the leaves of either Arabidopsis or apple. The hand-held UV illumination method failed in all tissues of both species. Additionally, the Typhoon imager was able to detect GFP fluorescence in both green and non-green tissues of Arabidopsis seedlings as well as in imbibed seeds, qualifying it as a high-throughput screening tool, which was further demonstrated by screening the seedlings of primary transformed T(0) seeds. Of the 30,000 germinating Arabidopsis seedlings screened, at least 69 GFP-positive lines were identified, accounting for an approximately 0.23% transformation efficiency. About 14,000 seedlings grown in 16 Petri plates could be screened within an hour, making the screening process significantly more efficient and robust than any other existing high-throughput screening method for transgenic plants.

An Efficient Amphiphilic-Type Triphenylamine-Based Organic Hole Transport Material for High-Performance and Ambient-Stable Dopant-Free Perovskite and Organic Solar Cells.

PubMed

Reddy, Saripally Sudhaker; Park, Ho-Yeol; Kwon, Haeun; Shin, Jongmoon; Kim, Chang-Su; Song, Myungkwan; Jin, Sung-Ho

2018-04-25

A new set of simply structured triphenylamine-based small molecules are synthesized and evaluated as dopant-free hole transporting materials (HTMs) for high-performance perovskite solar cells (PSCs) and bulk heterojunction inverted organic solar cells (BHJ IOSCs). Surprisingly, the new amphiphilic-type HTM-1 (with internal hydrophilic groups and peripheral hydrophobic alkyl tails) showed better compatibility and performance than the actual target molecule, that is, HTM-2 in PSCs and BHJ IOSCs. Importantly, the HTM-1-based dopant-free PSCs and BHJ IOSCs exhibited high power conversion efficiencies (PCEs) of 11.45 % and 8.34 %, respectively. These performances are superior and comparable to those of standard HTMs Spiro-OMeTAD (2,2',7,7'-tetrakis(N,N-di-p-methoxyphenylamine)-9,9'-spirobifluorene) and PEDOT:PSS (poly(3,4-ethylenedioxythiophene)/polystyrene sulfonate) in PSCs and BHJ IOSCs, respectively. The enhanced device performance of the HTM-1-based PSCs is ascribed to its strong affinity towards the perovskite, properly aligned energy levels with respect to the perovskite valence band, and excellent hole transporting behavior. In addition, the well-organized energy levels of the HTMs showed excellent compatibility in BHJ IOSCs. The new amphiphilic-type HTM-based photovoltaic devices also showed long-term air stability over 700 h. These promising results offer new and unexpected prospects for engineering the interface between the photoactive material and HTMs in PSCs and BHJ IOSCs. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novel molecular diagnostic tools for malaria elimination: a review of options from the point of view of high-throughput and applicability in resource limited settings.

PubMed

Britton, Sumudu; Cheng, Qin; McCarthy, James S

2016-02-16

As malaria transmission continues to decrease, an increasing number of countries will enter pre-elimination and elimination. To interrupt transmission, changes in control strategies are likely to require more accurate identification of all carriers of Plasmodium parasites, both symptomatic and asymptomatic, using diagnostic tools that are highly sensitive, high throughput and with fast turnaround times preferably performed in local health service settings. Currently available immunochromatographic lateral flow rapid diagnostic tests and field microscopy are unlikely to consistently detect infections at parasite densities less than 100 parasites/µL making them insufficiently sensitive for detecting all carriers. Molecular diagnostic platforms, such as PCR and LAMP, are currently available in reference laboratories, but at a cost both financially and in turnaround time. This review describes the recent progress in developing molecular diagnostic tools in terms of their capacity for high throughput and potential for performance in non-reference laboratories for malaria elimination.

High throughput ion-channel pharmacology: planar-array-based voltage clamp.

PubMed

Kiss, Laszlo; Bennett, Paul B; Uebele, Victor N; Koblan, Kenneth S; Kane, Stefanie A; Neagle, Brad; Schroeder, Kirk

2003-02-01

Technological advances often drive major breakthroughs in biology. Examples include PCR, automated DNA sequencing, confocal/single photon microscopy, AFM, and voltage/patch-clamp methods. The patch-clamp method, first described nearly 30 years ago, was a major technical achievement that permitted voltage-clamp analysis (membrane potential control) of ion channels in most cells and revealed a role for channels in unimagined areas. Because of the high information content, voltage clamp is the best way to study ion-channel function; however, throughput is too low for drug screening. Here we describe a novel breakthrough planar-array-based HT patch-clamp technology developed by Essen Instruments capable of voltage-clamping thousands of cells per day. This technology provides greater than two orders of magnitude increase in throughput compared with the traditional voltage-clamp techniques. We have applied this method to study the hERG K(+) channel and to determine the pharmacological profile of QT prolonging drugs.

The Open Connectome Project Data Cluster: Scalable Analysis and Vision for High-Throughput Neuroscience.

PubMed

Burns, Randal; Roncal, William Gray; Kleissas, Dean; Lillaney, Kunal; Manavalan, Priya; Perlman, Eric; Berger, Daniel R; Bock, Davi D; Chung, Kwanghun; Grosenick, Logan; Kasthuri, Narayanan; Weiler, Nicholas C; Deisseroth, Karl; Kazhdan, Michael; Lichtman, Jeff; Reid, R Clay; Smith, Stephen J; Szalay, Alexander S; Vogelstein, Joshua T; Vogelstein, R Jacob

2013-01-01

We describe a scalable database cluster for the spatial analysis and annotation of high-throughput brain imaging data, initially for 3-d electron microscopy image stacks, but for time-series and multi-channel data as well. The system was designed primarily for workloads that build connectomes - neural connectivity maps of the brain-using the parallel execution of computer vision algorithms on high-performance compute clusters. These services and open-science data sets are publicly available at openconnecto.me. The system design inherits much from NoSQL scale-out and data-intensive computing architectures. We distribute data to cluster nodes by partitioning a spatial index. We direct I/O to different systems-reads to parallel disk arrays and writes to solid-state storage-to avoid I/O interference and maximize throughput. All programming interfaces are RESTful Web services, which are simple and stateless, improving scalability and usability. We include a performance evaluation of the production system, highlighting the effec-tiveness of spatial data organization.

An Automated High-throughput Array Microscope for Cancer Cell Mechanics

NASA Astrophysics Data System (ADS)

Cribb, Jeremy A.; Osborne, Lukas D.; Beicker, Kellie; Psioda, Matthew; Chen, Jian; O'Brien, E. Timothy; Taylor, Russell M., II; Vicci, Leandra; Hsiao, Joe Ping-Lin; Shao, Chong; Falvo, Michael; Ibrahim, Joseph G.; Wood, Kris C.; Blobe, Gerard C.; Superfine, Richard

2016-06-01

Changes in cellular mechanical properties correlate with the progression of metastatic cancer along the epithelial-to-mesenchymal transition (EMT). Few high-throughput methodologies exist that measure cell compliance, which can be used to understand the impact of genetic alterations or to screen the efficacy of chemotherapeutic agents. We have developed a novel array high-throughput microscope (AHTM) system that combines the convenience of the standard 96-well plate with the ability to image cultured cells and membrane-bound microbeads in twelve independently-focusing channels simultaneously, visiting all wells in eight steps. We use the AHTM and passive bead rheology techniques to determine the relative compliance of human pancreatic ductal epithelial (HPDE) cells, h-TERT transformed HPDE cells (HPNE), and four gain-of-function constructs related to EMT. The AHTM found HPNE, H-ras, Myr-AKT, and Bcl2 transfected cells more compliant relative to controls, consistent with parallel tests using atomic force microscopy and invasion assays, proving the AHTM capable of screening for changes in mechanical phenotype.

The Open Connectome Project Data Cluster: Scalable Analysis and Vision for High-Throughput Neuroscience

PubMed Central

Burns, Randal; Roncal, William Gray; Kleissas, Dean; Lillaney, Kunal; Manavalan, Priya; Perlman, Eric; Berger, Daniel R.; Bock, Davi D.; Chung, Kwanghun; Grosenick, Logan; Kasthuri, Narayanan; Weiler, Nicholas C.; Deisseroth, Karl; Kazhdan, Michael; Lichtman, Jeff; Reid, R. Clay; Smith, Stephen J.; Szalay, Alexander S.; Vogelstein, Joshua T.; Vogelstein, R. Jacob

2013-01-01

We describe a scalable database cluster for the spatial analysis and annotation of high-throughput brain imaging data, initially for 3-d electron microscopy image stacks, but for time-series and multi-channel data as well. The system was designed primarily for workloads that build connectomes— neural connectivity maps of the brain—using the parallel execution of computer vision algorithms on high-performance compute clusters. These services and open-science data sets are publicly available at openconnecto.me. The system design inherits much from NoSQL scale-out and data-intensive computing architectures. We distribute data to cluster nodes by partitioning a spatial index. We direct I/O to different systems—reads to parallel disk arrays and writes to solid-state storage—to avoid I/O interference and maximize throughput. All programming interfaces are RESTful Web services, which are simple and stateless, improving scalability and usability. We include a performance evaluation of the production system, highlighting the effec-tiveness of spatial data organization. PMID:24401992

Characterization and complete genome sequence of a panicovirus from Bermuda grass by high-throughput sequencing.

PubMed

Tahir, Muhammad N; Lockhart, Ben; Grinstead, Samuel; Mollov, Dimitre

2017-04-01

Bermuda grass samples were examined by transmission electron microscopy and 28-30 nm spherical virus particles were observed. Total RNA from these plants was subjected to high-throughput sequencing (HTS). The nearly full genome sequence of a panicovirus was identified from one HTS scaffold. Sanger sequencing was used to confirm the HTS results and complete the genome sequence of 4404 nt. This virus was provisionally named Bermuda grass latent virus (BGLV). Its predicted open reading frames follow the typical arrangement of the genus Panicovirus. Based on sequence comparisons and phylogenetic analyses BGLV differs from other viruses and therefore taxonomically it is a new member of the genus Panicovirus, family Tombusviridae.

High-throughput quantum cascade laser (QCL) spectral histopathology: a practical approach towards clinical translation.

PubMed

Pilling, Michael J; Henderson, Alex; Bird, Benjamin; Brown, Mick D; Clarke, Noel W; Gardner, Peter

2016-06-23

Infrared microscopy has become one of the key techniques in the biomedical research field for interrogating tissue. In partnership with multivariate analysis and machine learning techniques, it has become widely accepted as a method that can distinguish between normal and cancerous tissue with both high sensitivity and high specificity. While spectral histopathology (SHP) is highly promising for improved clinical diagnosis, several practical barriers currently exist, which need to be addressed before successful implementation in the clinic. Sample throughput and speed of acquisition are key barriers and have been driven by the high volume of samples awaiting histopathological examination. FTIR chemical imaging utilising FPA technology is currently state-of-the-art for infrared chemical imaging, and recent advances in its technology have dramatically reduced acquisition times. Despite this, infrared microscopy measurements on a tissue microarray (TMA), often encompassing several million spectra, takes several hours to acquire. The problem lies with the vast quantities of data that FTIR collects; each pixel in a chemical image is derived from a full infrared spectrum, itself composed of thousands of individual data points. Furthermore, data management is quickly becoming a barrier to clinical translation and poses the question of how to store these incessantly growing data sets. Recently, doubts have been raised as to whether the full spectral range is actually required for accurate disease diagnosis using SHP. These studies suggest that once spectral biomarkers have been predetermined it may be possible to diagnose disease based on a limited number of discrete spectral features. In this current study, we explore the possibility of utilising discrete frequency chemical imaging for acquiring high-throughput, high-resolution chemical images. Utilising a quantum cascade laser imaging microscope with discrete frequency collection at key diagnostic wavelengths, we demonstrate that we can diagnose prostate cancer with high sensitivity and specificity. Finally we extend the study to a large patient dataset utilising tissue microarrays, and show that high sensitivity and specificity can be achieved using high-throughput, rapid data collection, thereby paving the way for practical implementation in the clinic.

Making the Nanoworld Accessible: Nanoscience Education Using Scanning Probe Methods

NASA Astrophysics Data System (ADS)

Knorr, Daniel; Killgore, Jason; Gray, Tomoko; Ginger, David; Wei, Joseph; Chen, Yeechi; Sarikaya, Mehmet; Fong, Hanson; Griffith, Tom; Overney, Rene

2008-03-01

A partnership between researchers and educators at the University of Washington, North Seattle Community College and two companies, Nanosurf, AG and nanoScience Instruments has been forged to develop a nationally replicable model of a sustainable and up-to-date undergraduate teaching laboratory of scanning probe microscopy (SPM) methods applied to nanoscience and nanotechnology. Within this partnership a new paradigm of operating and maintaining a SPM laboratory has been developed that provides a truly hands-on experience in a classroom laboratory setting with a small student to instrument ratio involving a variety of SPM techniques and topics. To date, we have run a first successful undergraduate laboratory workshop, where students were able to have extensive hands-on experience on five SPM modes of operation including: electrostatic force microscopy involving photovoltaic polymeric materials, tunneling microscopy and the determination of the workfunction, and nanolithography using the dip-pen method. http://depts.washington.edu/nanolab/NUE/UNIQUE/NUE/UNIQUE.htm

High-speed laser microsurgery of alert fruit flies for fluorescence imaging of neural activity

PubMed Central

Sinha, Supriyo; Liang, Liang; Ho, Eric T. W.; Urbanek, Karel E.; Luo, Liqun; Baer, Thomas M.; Schnitzer, Mark J.

2013-01-01

Intravital microscopy is a key means of monitoring cellular function in live organisms, but surgical preparation of a live animal for microscopy often is time-consuming, requires considerable skill, and limits experimental throughput. Here we introduce a spatially precise (<1-µm edge precision), high-speed (<1 s), largely automated, and economical protocol for microsurgical preparation of live animals for optical imaging. Using a 193-nm pulsed excimer laser and the fruit fly as a model, we created observation windows (12- to 350-µm diameters) in the exoskeleton. Through these windows we used two-photon microscopy to image odor-evoked Ca2+ signaling in projection neuron dendrites of the antennal lobe and Kenyon cells of the mushroom body. The impact of a laser-cut window on fly health appears to be substantially less than that of conventional manual dissection, for our imaging durations of up to 18 h were ∼5–20 times longer than prior in vivo microscopy studies of hand-dissected flies. This improvement will facilitate studies of numerous questions in neuroscience, such as those regarding neuronal plasticity or learning and memory. As a control, we used phototaxis as an exemplary complex behavior in flies and found that laser microsurgery is sufficiently gentle to leave it intact. To demonstrate that our techniques are applicable to other species, we created microsurgical openings in nematodes, ants, and the mouse cranium. In conjunction with emerging robotic methods for handling and mounting flies or other small organisms, our rapid, precisely controllable, and highly repeatable microsurgical techniques should enable automated, high-throughput preparation of live animals for optical experimentation. PMID:24167298

Human trabecular meshwork cells exhibit several characteristics of, but are distinct from, adipose-derived mesenchymal stem cells.

PubMed

Morgan, Joshua T; Wood, Joshua A; Walker, Naomi J; Raghunathan, Vijay Krishna; Borjesson, Dori L; Murphy, Christopher J; Russell, Paul

2014-01-01

To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities.

Human Trabecular Meshwork Cells Exhibit Several Characteristics of, but Are Distinct from, Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Morgan, Joshua T.; Wood, Joshua A.; Walker, Naomi J.; Raghunathan, Vijay Krishna; Borjesson, Dori L.; Murphy, Christopher J.

2014-01-01

Abstract Purpose: To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). Methods: HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Results: Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. Conclusions: HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities. PMID:24456002

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

PubMed

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

2017-06-28

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

RAN Translation as a Therapeutic in ALS

DTIC Science & Technology

2017-05-01

allow for HTS via CRISPR or drug screens to complement the in vitro screens using high-throughput microscopy or FACS. Figure 5: Mammalian G4C2...poly-GP in yeast (Figure 6A). [filler about RPS25 here?] This effect was further investigated in mammalian Hap1 cell lines with a CRISPR -mediated

High performance computing environment for multidimensional image analysis

PubMed Central

Rao, A Ravishankar; Cecchi, Guillermo A; Magnasco, Marcelo

2007-01-01

Background The processing of images acquired through microscopy is a challenging task due to the large size of datasets (several gigabytes) and the fast turnaround time required. If the throughput of the image processing stage is significantly increased, it can have a major impact in microscopy applications. Results We present a high performance computing (HPC) solution to this problem. This involves decomposing the spatial 3D image into segments that are assigned to unique processors, and matched to the 3D torus architecture of the IBM Blue Gene/L machine. Communication between segments is restricted to the nearest neighbors. When running on a 2 Ghz Intel CPU, the task of 3D median filtering on a typical 256 megabyte dataset takes two and a half hours, whereas by using 1024 nodes of Blue Gene, this task can be performed in 18.8 seconds, a 478× speedup. Conclusion Our parallel solution dramatically improves the performance of image processing, feature extraction and 3D reconstruction tasks. This increased throughput permits biologists to conduct unprecedented large scale experiments with massive datasets. PMID:17634099

High performance computing environment for multidimensional image analysis.

PubMed

Rao, A Ravishankar; Cecchi, Guillermo A; Magnasco, Marcelo

2007-07-10

The processing of images acquired through microscopy is a challenging task due to the large size of datasets (several gigabytes) and the fast turnaround time required. If the throughput of the image processing stage is significantly increased, it can have a major impact in microscopy applications. We present a high performance computing (HPC) solution to this problem. This involves decomposing the spatial 3D image into segments that are assigned to unique processors, and matched to the 3D torus architecture of the IBM Blue Gene/L machine. Communication between segments is restricted to the nearest neighbors. When running on a 2 Ghz Intel CPU, the task of 3D median filtering on a typical 256 megabyte dataset takes two and a half hours, whereas by using 1024 nodes of Blue Gene, this task can be performed in 18.8 seconds, a 478x speedup. Our parallel solution dramatically improves the performance of image processing, feature extraction and 3D reconstruction tasks. This increased throughput permits biologists to conduct unprecedented large scale experiments with massive datasets.

Automated analysis of siRNA screens of cells infected by hepatitis C and dengue viruses based on immunofluorescence microscopy images

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

2008-03-01

We present an image analysis approach as part of a high-throughput microscopy siRNA-based screening system using cell arrays for the identification of cellular genes involved in hepatitis C and dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in the neighborhood of segmented cell nuclei, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment and single images. In particular, we propose a novel approach for the localization of regions of transfected cells within cell array images, which combines model-based circle fitting and grid fitting. By this scheme we integrate information from single cell array images and knowledge from the complete cell arrays. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behaviour of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

The effect of trace mineral source and concentration on ruminal digestion and mineral solubility.

PubMed

Genther, O N; Hansen, S L

2015-01-01

The objective of this experiment was to compare the effect of sources of sulfate trace mineral (STM) and hydroxy trace mineral (HTM) at different inclusions on digestibility of dry matter (DM) and neutral detergent fiber and solubility of Cu, Mn, and Zn in the rumen and abomasum of cattle. Five ruminally cannulated steers were used in a 5×5 Latin square design and individually fed a corn silage-based diet on an ad libitum basis. The 5 dietary treatments were as follows: control: no supplemental Cu, Mn, or Zn, analyzed to contain 7.4mg of Cu, 30.8mg of Mn, and 32.1mg of Zn per kilogram of diet DM (CON); low sulfate: 5mg of Cu/kg of DM supplemented from CuSO4, 15mg of Mn/kg of DM from MnSO4, and 30mg of Zn/kg of DM from ZnSO4; low HTM: 5mg of Cu/kg of DM supplemented from basic copper chloride (IntelliBond C; Micronutrients Inc., Indianapolis, IN), 15mg of Mn/kg of DM from manganese hydroxychloride (IntelliBond M; Micronutrients Inc.), and 30mg of Zn/kg of DM from zinc hydroxychloride (IntelliBond Z; Micronutrients Inc.); high sulfate: 25mg of Cu/kg of DM supplemented from CuSO4, 60mg of Mn/kg of DM from MnSO4, and 120mg of Zn/kg of DM from ZnSO4; and high HTM: 25mg of Cu/kg of DM supplemented from basic copper chloride, 60mg of Mn/kg of DM from manganese hydroxychloride, and 120mg of Zn/kg of DM from zinc hydroxychloride. Periods lasted for 12d, with 10d of diet adaptation. Dacron bags containing the CON total mixed ration were inserted on d 11 at 0h and were removed at 6, 12, 24, and 36h after insertion. Dry matter and neutral detergent fiber disappearances and rumen and simulated abomasal trace mineral solubilities were evaluated. Dietary treatment did not affect DM intake. Dry matter disappearance was lesser in supplemental TM treatments and greater in CON than the STM treatments, although the CON and HTM treatments did not differ. Neutral detergent fiber disappearance was not affected by treatment. Ruminally soluble Cu and Mn concentrations were least in CON and were lesser in HTM-containing treatments compared with STM treatments. However, in the abomasum, solubilities of Cu and Mn were similar across trace mineral sources. Ruminal and simulated abomasal soluble Zn was greater in the HTM treatments than in CON and STM, driven by the greater solubility of the high HTM treatment. Under the conditions of this study, supplementing trace minerals as STM decreased DM digestibility, whereas HTM did not affect DM digestibility. Additionally, Cu and Mn from HTM sources were relatively insoluble in the rumen but had similar solubility as STM at the pH found in the abomasum, suggesting that these minerals should be available for absorption in the intestine. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Integrated Device for Circulating Tumor Cell Capture, Characterization and Lens-Free Microscopy

DTIC Science & Technology

2012-08-01

peripheral blood of breast cancer patients indicates high metastatic potential and increased morbidity. Development of a cost - effective CTC detection and...microfilter platform captures CTC from the cancer patients’ blood cost effectively , where the larger CTC are preferentially retained on the membrane...development of a cost - effective and high-throughput CTC analysis system would revolutionize the field of CTC detection, prognosis, and therapeutic

Zinc Absorption from Milk Is Affected by Dilution but Not by Thermal Processing, and Milk Enhances Absorption of Zinc from High-Phytate Rice in Young Dutch Women.

PubMed

Talsma, Elise F; Moretti, Diego; Ly, Sou Chheng; Dekkers, Renske; van den Heuvel, Ellen Ghm; Fitri, Aditia; Boelsma, Esther; Stomph, Tjeerd Jan; Zeder, Christophe; Melse-Boonstra, Alida

2017-06-01

Background: Milk has been suggested to increase zinc absorption. The effect of processing and the ability of milk to enhance zinc absorption from other foods has not been measured directly in humans. Objective: We aimed to assess zinc absorption from 1 ) milk undergoing various processing and preparatory steps and 2 ) from intrinsically labeled high-phytate rice consumed with milk or water. Methods: Two randomized crossover studies were conducted in healthy young women [age:18-25 y; body mass index (in kg/m 2 ): 20-25]: 1 ) a milk study ( n = 19) comparing the consumption of 800 mL full-fat ultra-high temperature (UHT) milk [heat-treated milk (HTM)], full-fat UHT milk diluted 1:1 with water [heat-treated milk and water (MW)], water, or unprocessed (raw) milk (UM), each extrinsically labeled with 67 Zn, and 2 ) a rice study ( n = 18) comparing the consumption of 90 g intrinsically 67 Zn-labeled rice with 600 mL of water [rice and water (RW)] or full-fat UHT milk [rice and milk (RM)]. The fractional absorption of zinc (FAZ) was measured with the double-isotope tracer ratio method. In vitro, we assessed zinc extraction from rice blended into water, UM, or HTM with or without phytate. Results: FAZ from HTM was 25.5% (95% CI: 21.6%, 29.4%) and was not different from UM (27.8%; 95% CI: 24.2%, 31.4%). FAZ from water was higher (72.3%; 95% CI: 68.7%, 75.9%), whereas FAZ from MW was lower (19.7%; 95% CI: 17.5%, 21.9%) than HTM and UM (both P < 0.01). FAZ from RM (20.7%; 95% CI: 18.8%, 22.7%) was significantly higher than from RW (12.8%; 95% CI: 10.8%, 14.6%; P < 0.01). In vitro, HTM and UM showed several orders of magnitude higher extraction of zinc from rice with HTM than from rice with water at various phytate concentrations. Conclusions: Milk enhanced human FAZ from high-phytate rice by 62% compared with water. Diluting milk with water decreases its absorption-enhancing proprieties, whereas UHT processing does not. This trial was registered at the Dutch trial registry as NTR4267 (http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=4267). © 2017 American Society for Nutrition.

FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons.

PubMed

Bosch, Carles; Martínez, Albert; Masachs, Nuria; Teixeira, Cátia M; Fernaud, Isabel; Ulloa, Fausto; Pérez-Martínez, Esther; Lois, Carlos; Comella, Joan X; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

2015-01-01

The fine analysis of synaptic contacts is usually performed using transmission electron microscopy (TEM) and its combination with neuronal labeling techniques. However, the complex 3D architecture of neuronal samples calls for their reconstruction from serial sections. Here we show that focused ion beam/scanning electron microscopy (FIB/SEM) allows efficient, complete, and automatic 3D reconstruction of identified dendrites, including their spines and synapses, from GFP/DAB-labeled neurons, with a resolution comparable to that of TEM. We applied this technology to analyze the synaptogenesis of labeled adult-generated granule cells (GCs) in mice. 3D reconstruction of dendritic spines in GCs aged 3-4 and 8-9 weeks revealed two different stages of dendritic spine development and unexpected features of synapse formation, including vacant and branched dendritic spines and presynaptic terminals establishing synapses with up to 10 dendritic spines. Given the reliability, efficiency, and high resolution of FIB/SEM technology and the wide use of DAB in conventional EM, we consider FIB/SEM fundamental for the detailed characterization of identified synaptic contacts in neurons in a high-throughput manner.

FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons

PubMed Central

Bosch, Carles; Martínez, Albert; Masachs, Nuria; Teixeira, Cátia M.; Fernaud, Isabel; Ulloa, Fausto; Pérez-Martínez, Esther; Lois, Carlos; Comella, Joan X.; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

2015-01-01

The fine analysis of synaptic contacts is usually performed using transmission electron microscopy (TEM) and its combination with neuronal labeling techniques. However, the complex 3D architecture of neuronal samples calls for their reconstruction from serial sections. Here we show that focused ion beam/scanning electron microscopy (FIB/SEM) allows efficient, complete, and automatic 3D reconstruction of identified dendrites, including their spines and synapses, from GFP/DAB-labeled neurons, with a resolution comparable to that of TEM. We applied this technology to analyze the synaptogenesis of labeled adult-generated granule cells (GCs) in mice. 3D reconstruction of dendritic spines in GCs aged 3–4 and 8–9 weeks revealed two different stages of dendritic spine development and unexpected features of synapse formation, including vacant and branched dendritic spines and presynaptic terminals establishing synapses with up to 10 dendritic spines. Given the reliability, efficiency, and high resolution of FIB/SEM technology and the wide use of DAB in conventional EM, we consider FIB/SEM fundamental for the detailed characterization of identified synaptic contacts in neurons in a high-throughput manner. PMID:26052271

Near-field microscopy with a microfabricated solid immersion lens

NASA Astrophysics Data System (ADS)

Fletcher, Daniel Alden

2001-07-01

Diffraction of focused light prevents optical microscopes from resolving features in air smaller than half the wavelength, λ Spatial resolution can be improved by passing light through a sub-wavelength metal aperture scanned close to a sample, but aperture-based probes suffer from low optical throughput, typically below 10-4. An alternate and more efficient technique is solid immersion microscopy in which light is focused through a high refractive index Solid Immersion Lens (SIL). This work describes the fabrication, modeling, and use of a microfabricated SIL to obtain spatial resolution better than the diffraction limit in air with high optical throughput for infrared applications. SILs on the order of 10 μm in diameter are fabricated from single-crystal silicon and integrated onto silicon cantilevers with tips for scanning. We measure a focused spot size of λ/5 with optical throughput better than 10-1 at a wavelength of λ = 9.3 μm. Spatial resolution is improved to λ/10 with metal apertures fabricated directly on the tip of the silicon SIL. Microlenses have reduced spherical aberration and better transparency than large lenses but cannot be made arbitrarily small and still focus. We model the advantages and limitations of focusing in lenses close to the wavelength in diameter using an extension of Mie theory. We also investigate a new contrast mechanism unique to microlenses resulting from the decrease in field-of-view with lens diameter. This technique is shown to achieve λ/4 spatial resolution. We explore applications of the microfabricated silicon SIL for high spatial resolution thermal microscopy and biological spectroscopy. Thermal radiation is collected through the SIL from a heated surface with spatial resolution four times better than that of a diffraction- limited infrared microscope. Using a Fourier-transform infrared spectrometer, we observe absorption peaks in bacteria cells positioned at the focus of the silicon SIL.

Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy.

PubMed

Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

2017-08-01

To target bacterial pathogens that invade and proliferate inside host cells, it is necessary to design intervention strategies directed against bacterial attachment, cellular invasion and intracellular proliferation. We present an automated microscopy-based, fast, high-throughput method for analyzing size and number of intracellular bacterial colonies in infected tissue culture cells. Cells are seeded in 48-well plates and infected with a GFP-expressing bacterial pathogen. Following gentamicin treatment to remove extracellular pathogens, cells are fixed and cell nuclei stained. This is followed by automated microscopy and subsequent semi-automated spot detection to determine the number of intracellular bacterial colonies, their size distribution, and the average number per host cell. Multiple 48-well plates can be processed sequentially and the procedure can be completed in one working day. As a model we quantified intracellular bacterial colonies formed by uropathogenic Escherichia coli (UPEC) during infection of human kidney cells (HKC-8). Urinary tract infections caused by UPEC are among the most common bacterial infectious diseases in humans. UPEC can colonize tissues of the urinary tract and is responsible for acute, chronic, and recurrent infections. In the bladder, UPEC can form intracellular quiescent reservoirs, thought to be responsible for recurrent infections. In the kidney, UPEC can colonize renal epithelial cells and pass to the blood stream, either via epithelial cell disruption or transcellular passage, to cause sepsis. Intracellular colonies are known to be clonal, originating from single invading UPEC. In our experimental setup, we found UPEC CFT073 intracellular bacterial colonies to be heterogeneous in size and present in nearly one third of the HKC-8 cells. This high-throughput experimental format substantially reduces experimental time and enables fast screening of the intracellular bacterial load and cellular distribution of multiple bacterial isolates. This will be a powerful experimental tool facilitating the study of bacterial invasion, drug resistance, and the development of new therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

Maximizing the quantitative accuracy and reproducibility of Förster resonance energy transfer measurement for screening by high throughput widefield microscopy

PubMed Central

Schaufele, Fred

2013-01-01

Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) provides insights into the proximities and orientations of FPs as surrogates of the biochemical interactions and structures of the factors to which the FPs are genetically fused. As powerful as FRET methods are, technical issues have impeded their broad adoption in the biologic sciences. One hurdle to accurate and reproducible FRET microscopy measurement stems from variable fluorescence backgrounds both within a field and between different fields. Those variations introduce errors into the precise quantification of fluorescence levels on which the quantitative accuracy of FRET measurement is highly dependent. This measurement error is particularly problematic for screening campaigns since minimal well-to-well variation is necessary to faithfully identify wells with altered values. High content screening depends also upon maximizing the numbers of cells imaged, which is best achieved by low magnification high throughput microscopy. But, low magnification introduces flat-field correction issues that degrade the accuracy of background correction to cause poor reproducibility in FRET measurement. For live cell imaging, fluorescence of cell culture media in the fluorescence collection channels for the FPs commonly used for FRET analysis is a high source of background error. These signal-to-noise problems are compounded by the desire to express proteins at biologically meaningful levels that may only be marginally above the strong fluorescence background. Here, techniques are presented that correct for background fluctuations. Accurate calculation of FRET is realized even from images in which a non-flat background is 10-fold higher than the signal. PMID:23927839

Green-Solvent-Processable, Dopant-Free Hole-Transporting Materials for Robust and Efficient Perovskite Solar Cells.

PubMed

Lee, Junwoo; Malekshahi Byranvand, Mahdi; Kang, Gyeongho; Son, Sung Y; Song, Seulki; Kim, Guan-Woo; Park, Taiho

2017-09-06

In addition to having proper energy levels and high hole mobility (μ h ) without the use of dopants, hole-transporting materials (HTMs) used in n-i-p-type perovskite solar cells (PSCs) should be processed using green solvents to enable environmentally friendly device fabrication. Although many HTMs have been assessed, due to the limited solubility of HTMs in green solvents, no green-solvent-processable HTM has been reported to date. Here, we report on a green-solvent-processable HTM, an asymmetric D-A polymer (asy-PBTBDT) that exhibits superior solubility even in the green solvent, 2-methylanisole, which is a known food additive. The new HTM is well matched with perovskites in terms of energy levels and attains a high μ h (1.13 × 10 -3 cm 2 /(V s)) even without the use of dopants. Using the HTM, we produced robust PSCs with 18.3% efficiency (91% retention after 30 days without encapsulation under 50%-75% relative humidity) without dopants; with dopants (bis(trifluoromethanesulfonyl) imide and tert-butylpyridine, a 20.0% efficiency was achieved. Therefore, it is a first report for a green-solvent-processable hole-transporting polymer, exhibiting the highest efficiencies reported so far for n-i-p devices with and without the dopants.

High-throughput optofluidic system for the laser microsurgery of oocytes

NASA Astrophysics Data System (ADS)

Chandsawangbhuwana, Charlie; Shi, Linda Z.; Zhu, Qingyuan; Alliegro, Mark C.; Berns, Michael W.

2012-01-01

This study combines microfluidics with optical microablation in a microscopy system that allows for high-throughput manipulation of oocytes, automated media exchange, and long-term oocyte observation. The microfluidic component of the system transports oocytes from an inlet port into multiple flow channels. Within each channel, oocytes are confined against a microfluidic barrier using a steady fluid flow provided by an external computer-controlled syringe pump. This allows for easy media replacement without disturbing the oocyte location. The microfluidic and optical-laser microbeam ablation capabilities of the system were validated using surf clam (Spisula solidissima) oocytes that were immobilized in order to permit ablation of the 5 μm diameter nucleolinus within the oocyte nucleolus. Oocytes were the followed and assayed for polar body ejection.

High throughput on-chip analysis of high-energy charged particle tracks using lensfree imaging

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

Luo, Wei; Shabbir, Faizan; Gong, Chao

2015-04-13

We demonstrate a high-throughput charged particle analysis platform, which is based on lensfree on-chip microscopy for rapid ion track analysis using allyl diglycol carbonate, i.e., CR-39 plastic polymer as the sensing medium. By adopting a wide-area opto-electronic image sensor together with a source-shifting based pixel super-resolution technique, a large CR-39 sample volume (i.e., 4 cm × 4 cm × 0.1 cm) can be imaged in less than 1 min using a compact lensfree on-chip microscope, which detects partially coherent in-line holograms of the ion tracks recorded within the CR-39 detector. After the image capture, using highly parallelized reconstruction and ion track analysis algorithms running on graphics processingmore » units, we reconstruct and analyze the entire volume of a CR-39 detector within ∼1.5 min. This significant reduction in the entire imaging and ion track analysis time not only increases our throughput but also allows us to perform time-resolved analysis of the etching process to monitor and optimize the growth of ion tracks during etching. This computational lensfree imaging platform can provide a much higher throughput and more cost-effective alternative to traditional lens-based scanning optical microscopes for ion track analysis using CR-39 and other passive high energy particle detectors.« less

The Next Linear Collider Program

Science.gov Websites

/graphics.htm Snowmass 2001 http://snowmass2001.org/ Electrical Systems Modulators http://www -project.slac.stanford.edu/lc/local/electrical/e_home.htm DC Magnet Power http://www-project.slac.stanford.edu/lc/local /electrical/e_home.htm Global Systems http://www-project.slac.stanford.edu/lc/local/electrical/e_home.htm

Fast globally optimal segmentation of cells in fluorescence microscopy images.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2011-01-01

Accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression in high-throughput screening applications. We propose a new approach for segmenting cell nuclei which is based on active contours and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images of different cell types. We have also performed a quantitative comparison with previous segmentation approaches.

High-throughput label-free detection of aggregate platelets with optofluidic time-stretch microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Jiang, Yiyue; Lei, Cheng; Yasumoto, Atsushi; Ito, Takuro; Guo, Baoshan; Kobayashi, Hirofumi; Ozeki, Yasuyuki; Yatomi, Yutaka; Goda, Keisuke

2017-02-01

According to WHO, approximately 10 million new cases of thrombotic disorders are diagnosed worldwide every year. In the U.S. and Europe, their related diseases kill more people than those from AIDS, prostate cancer, breast cancer and motor vehicle accidents combined. Although thrombotic disorders, especially arterial ones, mainly result from enhanced platelet aggregability in the vascular system, visual detection of platelet aggregates in vivo is not employed in clinical settings. Here we present a high-throughput label-free platelet aggregate detection method, aiming at the diagnosis and monitoring of thrombotic disorders in clinical settings. With optofluidic time-stretch microscopy with a spatial resolution of 780 nm and an ultrahigh linear scanning rate of 75 MHz, it is capable of detecting aggregated platelets in lysed blood which flows through a hydrodynamic-focusing microfluidic device at a high throughput of 10,000 particles/s. With digital image processing and statistical analysis, we are able to distinguish them from single platelets and other blood cells via morphological features. The detection results are compared with results of fluorescence-based detection (which is slow and inaccurate, but established). Our results indicate that the method holds promise for real-time, low-cost, label-free, and minimally invasive detection of platelet aggregates, which is potentially applicable to detection of platelet aggregates in vivo and to the diagnosis and monitoring of thrombotic disorders in clinical settings. This technique, if introduced clinically, may provide important clinical information in addition to that obtained by conventional techniques for thrombotic disorder diagnosis, including ex vivo platelet aggregation tests.

Neural network control of focal position during time-lapse microscopy of cells.

PubMed

Wei, Ling; Roberts, Elijah

2018-05-09

Live-cell microscopy is quickly becoming an indispensable technique for studying the dynamics of cellular processes. Maintaining the specimen in focus during image acquisition is crucial for high-throughput applications, especially for long experiments or when a large sample is being continuously scanned. Automated focus control methods are often expensive, imperfect, or ill-adapted to a specific application and are a bottleneck for widespread adoption of high-throughput, live-cell imaging. Here, we demonstrate a neural network approach for automatically maintaining focus during bright-field microscopy. Z-stacks of yeast cells growing in a microfluidic device were collected and used to train a convolutional neural network to classify images according to their z-position. We studied the effect on prediction accuracy of the various hyperparameters of the neural network, including downsampling, batch size, and z-bin resolution. The network was able to predict the z-position of an image with ±1 μm accuracy, outperforming human annotators. Finally, we used our neural network to control microscope focus in real-time during a 24 hour growth experiment. The method robustly maintained the correct focal position compensating for 40 μm of focal drift and was insensitive to changes in the field of view. About ~100 annotated z-stacks were required to train the network making our method quite practical for custom autofocus applications.

High-throughput electrical characterization for robust overlay lithography control

NASA Astrophysics Data System (ADS)

Devender, Devender; Shen, Xumin; Duggan, Mark; Singh, Sunil; Rullan, Jonathan; Choo, Jae; Mehta, Sohan; Tang, Teck Jung; Reidy, Sean; Holt, Jonathan; Kim, Hyung Woo; Fox, Robert; Sohn, D. K.

2017-03-01

Realizing sensitive, high throughput and robust overlay measurement is a challenge in current 14nm and advanced upcoming nodes with transition to 300mm and upcoming 450mm semiconductor manufacturing, where slight deviation in overlay has significant impact on reliability and yield1). Exponentially increasing number of critical masks in multi-patterning lithoetch, litho-etch (LELE) and subsequent LELELE semiconductor processes require even tighter overlay specification2). Here, we discuss limitations of current image- and diffraction- based overlay measurement techniques to meet these stringent processing requirements due to sensitivity, throughput and low contrast3). We demonstrate a new electrical measurement based technique where resistance is measured for a macro with intentional misalignment between two layers. Overlay is quantified by a parabolic fitting model to resistance where minima and inflection points are extracted to characterize overlay control and process window, respectively. Analyses using transmission electron microscopy show good correlation between actual overlay performance and overlay obtained from fitting. Additionally, excellent correlation of overlay from electrical measurements to existing image- and diffraction- based techniques is found. We also discuss challenges of integrating electrical measurement based approach in semiconductor manufacturing from Back End of Line (BEOL) perspective. Our findings open up a new pathway for accessing simultaneous overlay as well as process window and margins from a robust, high throughput and electrical measurement approach.

Impact of Trace Minerals on Wound Healing of Footpad Dermatitis in Broilers.

PubMed

Chen, Juxing; Tellez, Guillermo; Escobar, Jeffery; Vazquez-Anon, Mercedes

2017-05-15

Footpad dermatitis (FPD) is used in the poultry industry as an animal welfare criterion to determine stocking density. Trace minerals (TM) play a role in skin integrity and wound healing. This study evaluated the impact of TM on FPD and consisted of 3 treatments supplemented with 0 (NTM), low (LTM) and high (HTM) TM levels in the same basal diet. On d21, 71% birds in all treatments developed mild FPD and pens were top-dressed with dry litter to promote FPD healing. Compared to NTM, LTM reduced area under the curve (AUC) of FPD lesion scores during d21-42, HTM reduced the AUC of FPD lesion scores during d7-21 and d21-42. LTM improved growth performance on d14, HTM improved growth performance on d14 and d28. LTM and/or HTM increased gene expression of VEGF, TIMP3, TIMP4, MMP13, ITGA2, ITGA3 and CD40, which promoted collagen synthesis, deposition and organization; cell migration, matrix remodeling, and angiogenesis. LTM and/or HTM increased inflammation by upregulating TNFα and IL-1β during the early wound healing phase and reduced inflammation by downregulating IL-1β during the late wound healing phase. Our findings showed that TM not only improved growth performance but also reduced FPD development by promoting FPD wound healing.

Mechanical phenotyping of tumor cells using a microfluidic cell squeezer device

NASA Astrophysics Data System (ADS)

Khan, Zeina S.; Kamyabi, Nabiollah; Vanapalli, Siva A.

2013-03-01

Studies have indicated that cancer cells have distinct mechanical properties compared to healthy cells. We are investigating the potential of cell mechanics as a biophysical marker for diagnostics and prognosis of cancer. To establish the significance of mechanical properties for cancer diagnostics, a high throughput method is desired. Although techniques such as atomic force microscopy are very precise, they are limited in throughput for cellular mechanical property measurements. To develop a device for high throughput mechanical characterization of tumor cells, we have fabricated a microfludic cell squeezer device that contains narrow micrometer-scale pores. Fluid flow is used to drive cells into these pores mimicking the flow-induced passage of circulating tumor cells through microvasculature. By integrating high speed imaging, the device allows for the simultaneous characterization of five different parameters including the blockage pressure, cell velocity, cell size, elongation and the entry time into squeezer. We have tested a variety of in vitro cell lines, including brain and prostate cancer cell lines, and have found that the entry time is the most sensitive measurement capable of differentiating between cell lines with differing invasiveness.

Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

PubMed Central

Pinzon, Neissa M.; Aukema, Kelly G.; Gralnick, Jeffrey A.; Wackett, Lawrence P.

2011-01-01

ABSTRACT A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone production as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. PMID:21712420

High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification.

PubMed

Kroll, Torsten; Schmidt, David; Schwanitz, Georg; Ahmad, Mubashir; Hamann, Jana; Schlosser, Corinne; Lin, Yu-Chieh; Böhm, Konrad J; Tuckermann, Jan; Ploubidou, Aspasia

2016-07-01

High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging. Here, the step-by-step development of an HCA assay protocol and an HCS bioinformatics analysis pipeline are described. The protocol's power is demonstrated by application to focal adhesion (FA) detection, quantitative analysis of multiple FA features, and functional annotation of signaling pathways regulating FA size, using primary data of a published RNAi screen. The assay and the underlying strategy are aimed at researchers performing microscopy-based quantitative analysis of subcellular features, on a small scale or in large HCS experiments. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

High-throughput microfluidics to control and measure signaling dynamics in single yeast cells

PubMed Central

Hansen, Anders S.; Hao, Nan; O'Shea, Erin K.

2015-01-01

Microfluidics coupled to quantitative time-lapse fluorescence microscopy is transforming our ability to control, measure, and understand signaling dynamics in single living cells. Here we describe a pipeline that incorporates multiplexed microfluidic cell culture, automated programmable fluid handling for cell perturbation, quantitative time-lapse microscopy, and computational analysis of time-lapse movies. We illustrate how this setup can be used to control the nuclear localization of the budding yeast transcription factor Msn2. Using this protocol, we generate oscillations of Msn2 localization and measure the dynamic gene expression response of individual genes in single cells. The protocol allows a single researcher to perform up to 20 different experiments in a single day, whilst collecting data for thousands of single cells. Compared to other protocols, the present protocol is relatively easy to adopt and higher-throughput. The protocol can be widely used to control and monitor single-cell signaling dynamics in other signal transduction systems in microorganisms. PMID:26158443

Contradictory cooling in a warmer world? the climate of the Mediterranean region during the ';Holocene Thermal Maximum'

NASA Astrophysics Data System (ADS)

Davis, B.

2013-12-01

Extensive evidence from high latitudes of the Northern Hemisphere indicates that temperatures were warmer than present during the early-mid Holocene, a period known as the Holocene thermal maximum (HTM). The existence of the HTM over lower mid-latitudes and the sub-tropics however is less clear, with pollen-based reconstructions in particular actually indicating a contrary cooling at this time in these regions. This apparent cooling is controversial because it is not shown in climate model simulations, which indicate that the HTM occurred across all extra-tropical latitudes of the Northern Hemisphere. This is also supported by alkenone based SST reconstructions, which also show a much more widespread HTM than indicated by the pollen data. Here this problem is investigated by reviewing the evidence both for, and against, the HTM in the Mediterranean region, which represents one of the most intensively studied regions of sub-tropical climate in the Northern Hemisphere. This evidence includes a large number of both marine and terrestrial records that can be directly compared due to their close proximity around the Mediterranean Sea. The results highlight the potential for bias in both marine and terrestrial climate proxies, but despite many criticisms of the pollen-based record, it is shown that the existence of more extensive temperate vegetation in the early-mid Holocene in the Mediterranean is difficult to explain by anything other than a cooler climate. For instance, vegetation models driven by climate model output show that the warmer climate suggested by the models produces a HTM vegetation even more arid than today. The results have important implications in the interpretation of proxy records, but perhaps most importantly, the potential for climate models to underestimate cooling processes in a warmer world needs further investigation.

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

NASA Astrophysics Data System (ADS)

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

2014-09-01

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

Bacterial cell identification in differential interference contrast microscopy images.

PubMed

Obara, Boguslaw; Roberts, Mark A J; Armitage, Judith P; Grau, Vicente

2013-04-23

Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins.

Peroxisystem: harnessing systems cell biology to study peroxisomes.

PubMed

Schuldiner, Maya; Zalckvar, Einat

2015-04-01

In recent years, high-throughput experimentation with quantitative analysis and modelling of cells, recently dubbed systems cell biology, has been harnessed to study the organisation and dynamics of simple biological systems. Here, we suggest that the peroxisome, a fascinating dynamic organelle, can be used as a good candidate for studying a complete biological system. We discuss several aspects of peroxisomes that can be studied using high-throughput systematic approaches and be integrated into a predictive model. Such approaches can be used in the future to study and understand how a more complex biological system, like a cell and maybe even ultimately a whole organism, works. © 2015 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

High-resolution, high-throughput imaging with a multibeam scanning electron microscope.

PubMed

Eberle, A L; Mikula, S; Schalek, R; Lichtman, J; Knothe Tate, M L; Zeidler, D

2015-08-01

Electron-electron interactions and detector bandwidth limit the maximal imaging speed of single-beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

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.

High-Throughput Synthesis and Structure of Zeolite ZSM-43 with Two-Directional 8-Ring Channels.

PubMed

Willhammar, Tom; Su, Jie; Yun, Yifeng; Zou, Xiaodong; Afeworki, Mobae; Weston, Simon C; Vroman, Hilda B; Lonergan, William W; Strohmaier, Karl G

2017-08-07

The aluminosilicate zeolite ZSM-43 (where ZSM = Zeolite Socony Mobil) was first synthesized more than 3 decades ago, but its chemical structure remained unsolved because of its poor crystallinity and small crystal size. Here we present optimization of the ZSM-43 synthesis using a high-throughput approach and subsequent structure determination by the combination of electron crystallographic methods and powder X-ray diffraction. The synthesis required the use of a combination of both inorganic (Cs + and K + ) and organic (choline) structure-directing agents. High-throughput synthesis enabled a screening of the synthesis conditions, which made it possible to optimize the synthesis, despite its complexity, in order to obtain a material with significantly improved crystallinity. When both rotation electron diffraction and high-resolution transmission electron microscopy imaging techniques are applied, the structure of ZSM-43 could be determined. The structure of ZSM-43 is a new zeolite framework type and possesses a unique two-dimensional channel system limited by 8-ring channels. ZSM-43 is stable upon calcination, and sorption measurements show that the material is suitable for adsorption of carbon dioxide as well as methane.

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

49 CFR 24.103 - Criteria for appraisals.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Appraisal Foundation at the following URL: http://www.appraisalfoundation.org/htm/USPAP2004/toc.htm. 2 The..., regulations and practices. It is available at http://www.usdoj.gov/enrd/land-ack/toc.htm or in soft cover...

Label-free detection of aggregated platelets in blood by machine-learning-aided optofluidic time-stretch microscopy.

PubMed

Jiang, Yiyue; Lei, Cheng; Yasumoto, Atsushi; Kobayashi, Hirofumi; Aisaka, Yuri; Ito, Takuro; Guo, Baoshan; Nitta, Nao; Kutsuna, Natsumaro; Ozeki, Yasuyuki; Nakagawa, Atsuhiro; Yatomi, Yutaka; Goda, Keisuke

2017-07-11

According to WHO, about 10 million new cases of thrombotic disorders are diagnosed worldwide every year. Thrombotic disorders, including atherothrombosis (the leading cause of death in the US and Europe), are induced by occlusion of blood vessels, due to the formation of blood clots in which aggregated platelets play an important role. The presence of aggregated platelets in blood may be related to atherothrombosis (especially acute myocardial infarction) and is, hence, useful as a potential biomarker for the disease. However, conventional high-throughput blood analysers fail to accurately identify aggregated platelets in blood. Here we present an in vitro on-chip assay for label-free, single-cell image-based detection of aggregated platelets in human blood. This assay builds on a combination of optofluidic time-stretch microscopy on a microfluidic chip operating at a high throughput of 10 000 blood cells per second with machine learning, enabling morphology-based identification and enumeration of aggregated platelets in a short period of time. By performing cell classification with machine learning, we differentiate aggregated platelets from single platelets and white blood cells with a high specificity and sensitivity of 96.6% for both. Our results indicate that the assay is potentially promising as predictive diagnosis and therapeutic monitoring of thrombotic disorders in clinical settings.

High-throughput monitoring of major cell functions by means of lensfree video microscopy

PubMed Central

Kesavan, S. Vinjimore; Momey, F.; Cioni, O.; David-Watine, B.; Dubrulle, N.; Shorte, S.; Sulpice, E.; Freida, D.; Chalmond, B.; Dinten, J. M.; Gidrol, X.; Allier, C.

2014-01-01

Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 – 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells. PMID:25096726

Automated imaging of cellular spheroids with selective plane illumination microscopy on a chip (Conference Presentation)

NASA Astrophysics Data System (ADS)

Paiè, Petra; Bassi, Andrea; Bragheri, Francesca; Osellame, Roberto

2017-02-01

Selective plane illumination microscopy (SPIM) is an optical sectioning technique that allows imaging of biological samples at high spatio-temporal resolution. Standard SPIM devices require dedicated set-ups, complex sample preparation and accurate system alignment, thus limiting the automation of the technique, its accessibility and throughput. We present a millimeter-scaled optofluidic device that incorporates selective plane illumination and fully automatic sample delivery and scanning. To this end an integrated cylindrical lens and a three-dimensional fluidic network were fabricated by femtosecond laser micromachining into a single glass chip. This device can upgrade any standard fluorescence microscope to a SPIM system. We used SPIM on a CHIP to automatically scan biological samples under a conventional microscope, without the need of any motorized stage: tissue spheroids expressing fluorescent proteins were flowed in the microchannel at constant speed and their sections were acquired while passing through the light sheet. We demonstrate high-throughput imaging of the entire sample volume (with a rate of 30 samples/min), segmentation and quantification in thick (100-300 μm diameter) cellular spheroids. This optofluidic device gives access to SPIM analyses to non-expert end-users, opening the way to automatic and fast screening of a high number of samples at subcellular resolution.

Architecture, component, and microbiome of biofilm involved in the fouling of membrane bioreactors.

PubMed

Inaba, Tomohiro; Hori, Tomoyuki; Aizawa, Hidenobu; Ogata, Atsushi; Habe, Hiroshi

2017-01-01

Biofilm formation on the filtration membrane and the subsequent clogging of membrane pores (called biofouling) is one of the most persistent problems in membrane bioreactors for wastewater treatment and reclamation. Here, we investigated the structure and microbiome of fouling-related biofilms in the membrane bioreactor using non-destructive confocal reflection microscopy and high-throughput Illumina sequencing of 16S rRNA genes. Direct confocal reflection microscopy indicated that the thin biofilms were formed and maintained regardless of the increasing transmembrane pressure, which is a common indicator of membrane fouling, at low organic-loading rates. Their solid components were primarily extracellular polysaccharides and microbial cells. In contrast, high organic-loading rates resulted in a rapid increase in the transmembrane pressure and the development of the thick biofilms mainly composed of extracellular lipids. High-throughput sequencing revealed that the biofilm microbiomes, including major and minor microorganisms, substantially changed in response to the organic-loading rates and biofilm development. These results demonstrated for the first time that the architectures, chemical components, and microbiomes of the biofilms on fouled membranes were tightly associated with one another and differed considerably depending on the organic-loading conditions in the membrane bioreactor, emphasizing the significance of alternative indicators other than the transmembrane pressure for membrane biofouling.

Recent advances in high-throughput QCL-based infrared microspectral imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Rowlette, Jeremy A.; Fotheringham, Edeline; Nichols, David; Weida, Miles J.; Kane, Justin; Priest, Allen; Arnone, David B.; Bird, Benjamin; Chapman, William B.; Caffey, David B.; Larson, Paul; Day, Timothy

2017-02-01

The field of infrared spectral imaging and microscopy is advancing rapidly due in large measure to the recent commercialization of the first high-throughput, high-spatial-definition quantum cascade laser (QCL) microscope. Having speed, resolution and noise performance advantages while also eliminating the need for cryogenic cooling, its introduction has established a clear path to translating the well-established diagnostic capability of infrared spectroscopy into clinical and pre-clinical histology, cytology and hematology workflows. Demand for even higher throughput while maintaining high-spectral fidelity and low-noise performance continues to drive innovation in QCL-based spectral imaging instrumentation. In this talk, we will present for the first time, recent technological advances in tunable QCL photonics which have led to an additional 10X enhancement in spectral image data collection speed while preserving the high spectral fidelity and SNR exhibited by the first generation of QCL microscopes. This new approach continues to leverage the benefits of uncooled microbolometer focal plane array cameras, which we find to be essential for ensuring both reproducibility of data across instruments and achieving the high-reliability needed in clinical applications. We will discuss the physics underlying these technological advancements as well as the new biomedical applications these advancements are enabling, including automated whole-slide infrared chemical imaging on clinically relevant timescales.

Enhanced photocurrent density of HTM-free perovskite solar cells by carbon quantum dots

NASA Astrophysics Data System (ADS)

Zou, Haiyuan; Guo, Daipeng; He, Bowen; Yu, Jiaguo; Fan, Ke

2018-02-01

Full-printable and hole transport material (HTM)-free perovskite solar cells (PSCs) with carbon counter electrodes feature high stability and low cost. However, the perovskite film prepared by conventional one-step solution-coating method always shows a relatively poor coverage on the substrate, leading to the limit of the photocurrent density. In this study, we incorporated carbon quantum dots (CQDs) in the perovskite films, and investigated their effects on the performance of TiO2 nanosheet-based and HTM-free PSCs. It was found that the addition of CQDs to the perovskite film can enhance the photocurrent density of PSCs, and the optimal PSC with 0.1% CQDs evolved 60% higher photocurrent density than the pristine one. The improved photocurrent density was attributed to the heterogeneous nuclei derived from CQDs during perovskite crystallization, which would increase amount of perovskite nuclei and form a fine perovskite grain, leading to a better coverage on the substrate. Moreover, due to the excellent conductivity, CQDs in perovskite films could efficiently transport the photo-excited electrons, accelerating the separation and mobilization of charge carriers. This study presents the incorporation of CQDs in perovskite as an efficient approach to promote the performance of HTM-free PSCs.

Light microscopy applications in systems biology: opportunities and challenges

PubMed Central

2013-01-01

Biological systems present multiple scales of complexity, ranging from molecules to entire populations. Light microscopy is one of the least invasive techniques used to access information from various biological scales in living cells. The combination of molecular biology and imaging provides a bottom-up tool for direct insight into how molecular processes work on a cellular scale. However, imaging can also be used as a top-down approach to study the behavior of a system without detailed prior knowledge about its underlying molecular mechanisms. In this review, we highlight the recent developments on microscopy-based systems analyses and discuss the complementary opportunities and different challenges with high-content screening and high-throughput imaging. Furthermore, we provide a comprehensive overview of the available platforms that can be used for image analysis, which enable community-driven efforts in the development of image-based systems biology. PMID:23578051

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

PubMed

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

2017-05-01

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

3D imaging of optically cleared tissue using a simplified CLARITY method and on-chip microscopy

PubMed Central

Zhang, Yibo; Shin, Yoonjung; Sung, Kevin; Yang, Sam; Chen, Harrison; Wang, Hongda; Teng, Da; Rivenson, Yair; Kulkarni, Rajan P.; Ozcan, Aydogan

2017-01-01

High-throughput sectioning and optical imaging of tissue samples using traditional immunohistochemical techniques can be costly and inaccessible in resource-limited areas. We demonstrate three-dimensional (3D) imaging and phenotyping in optically transparent tissue using lens-free holographic on-chip microscopy as a low-cost, simple, and high-throughput alternative to conventional approaches. The tissue sample is passively cleared using a simplified CLARITY method and stained using 3,3′-diaminobenzidine to target cells of interest, enabling bright-field optical imaging and 3D sectioning of thick samples. The lens-free computational microscope uses pixel super-resolution and multi-height phase recovery algorithms to digitally refocus throughout the cleared tissue and obtain a 3D stack of complex-valued images of the sample, containing both phase and amplitude information. We optimized the tissue-clearing and imaging system by finding the optimal illumination wavelength, tissue thickness, sample preparation parameters, and the number of heights of the lens-free image acquisition and implemented a sparsity-based denoising algorithm to maximize the imaging volume and minimize the amount of the acquired data while also preserving the contrast-to-noise ratio of the reconstructed images. As a proof of concept, we achieved 3D imaging of neurons in a 200-μm-thick cleared mouse brain tissue over a wide field of view of 20.5 mm2. The lens-free microscope also achieved more than an order-of-magnitude reduction in raw data compared to a conventional scanning optical microscope imaging the same sample volume. Being low cost, simple, high-throughput, and data-efficient, we believe that this CLARITY-enabled computational tissue imaging technique could find numerous applications in biomedical diagnosis and research in low-resource settings. PMID:28819645

High-throughput on-chip in vivo neural regeneration studies using femtosecond laser nano-surgery and microfluidics

NASA Astrophysics Data System (ADS)

Rohde, Christopher B.; Zeng, Fei; Gilleland, Cody; Samara, Chrysanthi; Yanik, Mehmet F.

2009-02-01

In recent years, the advantages of using small invertebrate animals as model systems for human disease have become increasingly apparent and have resulted in three Nobel Prizes in medicine or chemistry during the last six years for studies conducted on the nematode Caenorhabditis elegans (C. elegans). The availability of a wide array of species-specific genetic techniques, along with the transparency of the worm and its ability to grow in minute volumes make C. elegans an extremely powerful model organism. We present a suite of technologies for complex high-throughput whole-animal genetic and drug screens. We demonstrate a high-speed microfluidic sorter that can isolate and immobilize C. elegans in a well-defined geometry, an integrated chip containing individually addressable screening chambers for incubation and exposure of individual animals to biochemical compounds, and a device for delivery of compound libraries in standard multiwell plates to microfluidic devices. The immobilization stability obtained by these devices is comparable to that of chemical anesthesia and the immobilization process does not affect lifespan, progeny production, or other aspects of animal health. The high-stability enables the use of a variety of key optical techniques. We use this to demonstrate femtosecond-laser nanosurgery and three-dimensional multiphoton microscopy. Used alone or in various combinations these devices facilitate a variety of high-throughput assays using whole animals, including mutagenesis and RNAi and drug screens at subcellular resolution, as well as high-throughput high-precision manipulations such as femtosecond-laser nanosurgery for large-scale in vivo neural degeneration and regeneration studies.

Recovering the dynamics of root growth and development using novel image acquisition and analysis methods

PubMed Central

Wells, Darren M.; French, Andrew P.; Naeem, Asad; Ishaq, Omer; Traini, Richard; Hijazi, Hussein; Bennett, Malcolm J.; Pridmore, Tony P.

2012-01-01

Roots are highly responsive to environmental signals encountered in the rhizosphere, such as nutrients, mechanical resistance and gravity. As a result, root growth and development is very plastic. If this complex and vital process is to be understood, methods and tools are required to capture the dynamics of root responses. Tools are needed which are high-throughput, supporting large-scale experimental work, and provide accurate, high-resolution, quantitative data. We describe and demonstrate the efficacy of the high-throughput and high-resolution root imaging systems recently developed within the Centre for Plant Integrative Biology (CPIB). This toolset includes (i) robotic imaging hardware to generate time-lapse datasets from standard cameras under infrared illumination and (ii) automated image analysis methods and software to extract quantitative information about root growth and development both from these images and via high-resolution light microscopy. These methods are demonstrated using data gathered during an experimental study of the gravitropic response of Arabidopsis thaliana. PMID:22527394

Recovering the dynamics of root growth and development using novel image acquisition and analysis methods.

PubMed

Wells, Darren M; French, Andrew P; Naeem, Asad; Ishaq, Omer; Traini, Richard; Hijazi, Hussein I; Hijazi, Hussein; Bennett, Malcolm J; Pridmore, Tony P

2012-06-05

Roots are highly responsive to environmental signals encountered in the rhizosphere, such as nutrients, mechanical resistance and gravity. As a result, root growth and development is very plastic. If this complex and vital process is to be understood, methods and tools are required to capture the dynamics of root responses. Tools are needed which are high-throughput, supporting large-scale experimental work, and provide accurate, high-resolution, quantitative data. We describe and demonstrate the efficacy of the high-throughput and high-resolution root imaging systems recently developed within the Centre for Plant Integrative Biology (CPIB). This toolset includes (i) robotic imaging hardware to generate time-lapse datasets from standard cameras under infrared illumination and (ii) automated image analysis methods and software to extract quantitative information about root growth and development both from these images and via high-resolution light microscopy. These methods are demonstrated using data gathered during an experimental study of the gravitropic response of Arabidopsis thaliana.

Rapid analysis and exploration of fluorescence microscopy images.

PubMed

Pavie, Benjamin; Rajaram, Satwik; Ouyang, Austin; Altschuler, Jason M; Steininger, Robert J; Wu, Lani F; Altschuler, Steven J

2014-03-19

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard. Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Predicting the Oxygen-Binding Properties of Platinum Nanoparticle Ensembles by Combining High-Precision Electron Microscopy and Density Functional Theory.

PubMed

Aarons, Jolyon; Jones, Lewys; Varambhia, Aakash; MacArthur, Katherine E; Ozkaya, Dogan; Sarwar, Misbah; Skylaris, Chris-Kriton; Nellist, Peter D

2017-07-12

Many studies of heterogeneous catalysis, both experimental and computational, make use of idealized structures such as extended surfaces or regular polyhedral nanoparticles. This simplification neglects the morphological diversity in real commercial oxygen reduction reaction (ORR) catalysts used in fuel-cell cathodes. Here we introduce an approach that combines 3D nanoparticle structures obtained from high-throughput high-precision electron microscopy with density functional theory. Discrepancies between experimental observations and cuboctahedral/truncated-octahedral particles are revealed and discussed using a range of widely used descriptors, such as electron-density, d-band centers, and generalized coordination numbers. We use this new approach to determine the optimum particle size for which both detrimental surface roughness and particle shape effects are minimized.

Raman microspectrometer combined with scattering microscopy and lensless imaging for bacteria identification

NASA Astrophysics Data System (ADS)

Strola, S. A.; Schultz, E.; Allier, C. P.; DesRoches, B.; Lemmonier, J.; Dinten, J.-M.

2013-03-01

In this paper, we report on a compact prototype capable both of lensfree imaging, Raman spectrometry and scattering microscopy from bacteria samples. This instrument allows high-throughput real-time characterization without the need of markers, making it potentially suitable to field label-free biomedical and environmental applications. Samples are illuminated from above with a focused-collimated 532nm laser beam and can be x-y-z scanned. The bacteria detection is based on emerging lensfree imaging technology able to localize cells of interest over a large field-of-view of 24mm2. Raman signal and scattered light are then collected by separate measurement arms simultaneously. In the first arm the emission light is fed by a fiber into a prototype spectrometer, developed by Tornado Spectral System based on Tornado's High Throughput Virtual Slit (HTVS) novel technology. The enhanced light throughput in the spectral region of interest (500-1800 cm-1) reduces Raman acquisition time down to few seconds, thus facilitating experimental protocols and avoiding the bacteria deterioration induced by laser thermal heating. Scattered light impinging in the second arm is collected onto a charge-coupled-device. The reconstructed image allows studying the single bacteria diffraction pattern and their specific structural features. The characterization and identification of different bacteria have been performed to validate and optimize the acquisition system and the component setup. The results obtained demonstrate the benefits of these three techniques combination by providing the precise bacteria localization, their chemical composition and a morphology description. The procedure for a rapid identification of particular pathogen bacteria in a sample is illustrated.

Enhancement of the blood compatibility of dialyzer membranes by the physical adsorption of human thrombomodulin (ART-123).

PubMed

Omichi, Masaaki; Matsusaki, Michiya; Kato, Shinya; Maruyama, Ikuro; Akashi, Mitsuru

2010-11-01

ART-123 is a recombinant soluble human thrombomodulin (hTM) with excellent anticoagulant activity. We focused on improving the blood compatibility of the polysulfone-polyvinylpyrrolidone dialyzer surface by the physical adsorption of ART-123 onto the surface. The blood compatibility of the dialyzer with the hTM adsorbed membrane was evaluated by measuring the differential pressure between the arterial and the venous pressures and by blood parameters during blood circulation. The hTM adsorbed dialyzer membrane inhibited blood clot formation without heparin administration due to the anticoagulant activity of hTM for over 4 h. The physically adsorbed hTM was stable during blood circulation, and it did not affect activated clotting time, which is significant drawback of heparin administration, and blood cell counts of RBC, WBC, or platelets. The physical adsorption of hTM onto the dialyzer membrane will be a simple and safe method to prevent blood coagulation during dialysis instead of heparin administration. © 2010 Wiley Periodicals, Inc.

High-throughput screening of PLGA thin films utilizing hydrophobic fluorescent dyes for hydrophobic drug compounds.

PubMed

Steele, Terry W J; Huang, Charlotte L; Kumar, Saranya; Widjaja, Effendi; Chiang Boey, Freddy Yin; Loo, Joachim S C; Venkatraman, Subbu S

2011-10-01

Hydrophobic, antirestenotic drugs such as paclitaxel (PCTX) and rapamycin are often incorporated into thin film coatings for local delivery using implantable medical devices and polymers such as drug-eluting stents and balloons. Selecting the optimum coating formulation through screening the release profile of these drugs in thin films is time consuming and labor intensive. We describe here a high-throughput assay utilizing three model hydrophobic fluorescent compounds: fluorescein diacetate (FDAc), coumarin-6, and rhodamine 6G that were incorporated into poly(d,l-lactide-co-glycolide) (PLGA) and PLGA-polyethylene glycol films. Raman microscopy determined the hydrophobic fluorescent dye distribution within the PLGA thin films in comparison with that of PCTX. Their subsequent release was screened in a high-throughput assay and directly compared with HPLC quantification of PCTX release. It was observed that PCTX controlled-release kinetics could be mimicked by a hydrophobic dye that had similar octanol-water partition coefficient values and homogeneous dissolution in a PLGA matrix as the drug. In particular, FDAc was found to be the optimal hydrophobic dye at modeling the burst release as well as the total amount of PCTX released over a period of 30 days. Copyright © 2011 Wiley-Liss, Inc.

Medium-throughput processing of whole mount in situ hybridisation experiments into gene expression domains.

PubMed

Crombach, Anton; Cicin-Sain, Damjan; Wotton, Karl R; Jaeger, Johannes

2012-01-01

Understanding the function and evolution of developmental regulatory networks requires the characterisation and quantification of spatio-temporal gene expression patterns across a range of systems and species. However, most high-throughput methods to measure the dynamics of gene expression do not preserve the detailed spatial information needed in this context. For this reason, quantification methods based on image bioinformatics have become increasingly important over the past few years. Most available approaches in this field either focus on the detailed and accurate quantification of a small set of gene expression patterns, or attempt high-throughput analysis of spatial expression through binary pattern extraction and large-scale analysis of the resulting datasets. Here we present a robust, "medium-throughput" pipeline to process in situ hybridisation patterns from embryos of different species of flies. It bridges the gap between high-resolution, and high-throughput image processing methods, enabling us to quantify graded expression patterns along the antero-posterior axis of the embryo in an efficient and straightforward manner. Our method is based on a robust enzymatic (colorimetric) in situ hybridisation protocol and rapid data acquisition through wide-field microscopy. Data processing consists of image segmentation, profile extraction, and determination of expression domain boundary positions using a spline approximation. It results in sets of measured boundaries sorted by gene and developmental time point, which are analysed in terms of expression variability or spatio-temporal dynamics. Our method yields integrated time series of spatial gene expression, which can be used to reverse-engineer developmental gene regulatory networks across species. It is easily adaptable to other processes and species, enabling the in silico reconstitution of gene regulatory networks in a wide range of developmental contexts.

Biases in the Experimental Annotations of Protein Function and Their Effect on Our Understanding of Protein Function Space

PubMed Central

Schnoes, Alexandra M.; Ream, David C.; Thorman, Alexander W.; Babbitt, Patricia C.; Friedberg, Iddo

2013-01-01

The ongoing functional annotation of proteins relies upon the work of curators to capture experimental findings from scientific literature and apply them to protein sequence and structure data. However, with the increasing use of high-throughput experimental assays, a small number of experimental studies dominate the functional protein annotations collected in databases. Here, we investigate just how prevalent is the “few articles - many proteins” phenomenon. We examine the experimentally validated annotation of proteins provided by several groups in the GO Consortium, and show that the distribution of proteins per published study is exponential, with 0.14% of articles providing the source of annotations for 25% of the proteins in the UniProt-GOA compilation. Since each of the dominant articles describes the use of an assay that can find only one function or a small group of functions, this leads to substantial biases in what we know about the function of many proteins. Mass-spectrometry, microscopy and RNAi experiments dominate high throughput experiments. Consequently, the functional information derived from these experiments is mostly of the subcellular location of proteins, and of the participation of proteins in embryonic developmental pathways. For some organisms, the information provided by different studies overlap by a large amount. We also show that the information provided by high throughput experiments is less specific than those provided by low throughput experiments. Given the experimental techniques available, certain biases in protein function annotation due to high-throughput experiments are unavoidable. Knowing that these biases exist and understanding their characteristics and extent is important for database curators, developers of function annotation programs, and anyone who uses protein function annotation data to plan experiments. PMID:23737737

Validating a two-high-threshold measurement model for confidence rating data in recognition.

PubMed

Bröder, Arndt; Kellen, David; Schütz, Julia; Rohrmeier, Constanze

2013-01-01

Signal Detection models as well as the Two-High-Threshold model (2HTM) have been used successfully as measurement models in recognition tasks to disentangle memory performance and response biases. A popular method in recognition memory is to elicit confidence judgements about the presumed old/new status of an item, allowing for the easy construction of ROCs. Since the 2HTM assumes fewer latent memory states than response options are available in confidence ratings, the 2HTM has to be extended by a mapping function which models individual rating scale usage. Unpublished data from 2 experiments in Bröder and Schütz (2009) validate the core memory parameters of the model, and 3 new experiments show that the response mapping parameters are selectively affected by manipulations intended to affect rating scale use, and this is independent of overall old/new bias. Comparisons with SDT show that both models behave similarly, a case that highlights the notion that both modelling approaches can be valuable (and complementary) elements in a researcher's toolbox.

High blood pressure - children

MedlinePlus

... this page: //medlineplus.gov/ency/article/007696.htm High blood pressure - children To use the sharing features on this page, please enable JavaScript. High blood pressure (hypertension) is an increase in the force of ...

A space- and time-resolved single photon counting detector for fluorescence microscopy and spectroscopy

PubMed Central

Michalet, X.; Siegmund, O.H.W.; Vallerga, J.V.; Jelinsky, P.; Millaud, J.E.; Weiss, S.

2017-01-01

We have recently developed a wide-field photon-counting detector having high-temporal and high-spatial resolutions and capable of high-throughput (the H33D detector). Its design is based on a 25 mm diameter multi-alkali photocathode producing one photo electron per detected photon, which are then multiplied up to 107 times by a 3-microchannel plate stack. The resulting electron cloud is proximity focused on a cross delay line anode, which allows determining the incident photon position with high accuracy. The imaging and fluorescence lifetime measurement performances of the H33D detector installed on a standard epifluorescence microscope will be presented. We compare them to those of standard single-molecule detectors such as single-photon avalanche photodiode (SPAD) or electron-multiplying camera using model samples (fluorescent beads, quantum dots and live cells). Finally, we discuss the design and applications of future generation of H33D detectors for single-molecule imaging and high-throughput study of biomolecular interactions. PMID:29479130

Observation of the immune response of cells and tissue through multimodal label-free microscopy

NASA Astrophysics Data System (ADS)

Pavillon, Nicolas; Smith, Nicholas I.

2017-02-01

We present applications of a label-free approach to assess the immune response based on the combination of interferometric microscopy and Raman spectroscopy, which makes it possible to simultaneously acquire morphological and molecular information of live cells. We employ this approach to derive statistical models for predicting the activation state of macrophage cells based both on morphological parameters extracted from the high-throughput full-field quantitative phase imaging, and on the molecular content information acquired through Raman spectroscopy. We also employ a system for 3D imaging based on coherence gating, enabling specific targeting of the Raman channel to structures of interest within tissue.

78 FR 45939 - Changes in Flood Hazard Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-30

... Whiteland (12-03- Virginia Township team.com/lomrs.htm. 2075P). McMichael, Building, 209 Chairman, East... Tredyffrin (12-03- Michelle H. Municipal team.com/lomrs.htm. 2075P). Kichline, Building, 1100 Chairman... Scott K. York, Building and team.com/lomrs.htm. County (12-03- Chairman-at- Development 1164P). Large...

7 CFR 1755.900 - Abbreviations and Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... http://www.usda.gov/rus/telecom/listing_procedures/index_listing_procedures.htm, as well as additional....usda.gov/rus/telecom/publications/bulletins.htm. (2) Agency means the Rural Utilities Service, an... available on the Internet at http://www.usda.gov/rus/telecom/materials/lstomat.htm. (16) Loose tube buffer...

7 CFR 1755.900 - Abbreviations and Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... http://www.usda.gov/rus/telecom/listing_procedures/index_listing_procedures.htm, as well as additional....usda.gov/rus/telecom/publications/bulletins.htm. (2) Agency means the Rural Utilities Service, an... available on the Internet at http://www.usda.gov/rus/telecom/materials/lstomat.htm. (16) Loose tube buffer...

7 CFR 1755.900 - Abbreviations and Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... http://www.usda.gov/rus/telecom/listing_procedures/index_listing_procedures.htm, as well as additional....usda.gov/rus/telecom/publications/bulletins.htm. (2) Agency means the Rural Utilities Service, an... available on the Internet at http://www.usda.gov/rus/telecom/materials/lstomat.htm. (16) Loose tube buffer...

Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

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

Pinzon, NM; Aukema, KG; Gralnick, JA

A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone productionmore » as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. IMPORTANCE In recent years, there has been renewed interest in advanced biofuel sources such as bacterial hydrocarbon production. Previous studies used solvent extraction of bacterial cultures followed by gas chromatography-mass spectrometry (GC-MS) to detect and quantify ketones and hydrocarbons (Beller HR, Goh EB, Keasling JD, Appl. Environ. Microbiol. 76: 1212-1223, 2010; Sukovich DJ, Seffernick JL, Richman JE, Gralnick JA, Wackett LP, Appl. Environ. Microbiol. 76: 3850-3862, 2010). While these analyses are powerful and accurate, their labor-intensive nature makes them intractable to high-throughput screening; therefore, methods for rapid identification of bacterial strains that are overproducing hydrocarbons are needed. The use of high-throughput evaluation of bacterial and algal hydrophobic molecule production via Nile red fluorescence from lipids and esters was extended in this study to include hydrocarbons and ketones. This work demonstrated accurate, high-throughput detection of high-level bacterial long-chain ketone and hydrocarbon production by screening for increased fluorescence of the hydrophobic dye Nile red.« less

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

PubMed Central

Gaber, Rok; Majerle, Andreja; Jerala, Roman; Benčina, Mojca

2013-01-01

To effectively fight against the human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity. PMID:24287545

Live imaging of muscles in Drosophila metamorphosis: Towards high-throughput gene identification and function analysis.

PubMed

Puah, Wee Choo; Wasser, Martin

2016-03-01

Time-lapse microscopy in developmental biology is an emerging tool for functional genomics. Phenotypic effects of gene perturbations can be studied non-invasively at multiple time points in chronological order. During metamorphosis of Drosophila melanogaster, time-lapse microscopy using fluorescent reporters allows visualization of alternative fates of larval muscles, which are a model for the study of genes related to muscle wasting. While doomed muscles enter hormone-induced programmed cell death, a smaller population of persistent muscles survives to adulthood and undergoes morphological remodeling that involves atrophy in early, and hypertrophy in late pupation. We developed a method that combines in vivo imaging, targeted gene perturbation and image analysis to identify and characterize genes involved in muscle development. Macrozoom microscopy helps to screen for interesting muscle phenotypes, while confocal microscopy in multiple locations over 4-5 days produces time-lapse images that are used to quantify changes in cell morphology. Performing a similar investigation using fixed pupal tissues would be too time-consuming and therefore impractical. We describe three applications of our pipeline. First, we show how quantitative microscopy can track and measure morphological changes of muscle throughout metamorphosis and analyze genes involved in atrophy. Second, our assay can help to identify genes that either promote or prevent histolysis of abdominal muscles. Third, we apply our approach to test new fluorescent proteins as live markers for muscle development. We describe mKO2 tagged Cysteine proteinase 1 (Cp1) and Troponin-I (TnI) as examples of proteins showing developmental changes in subcellular localization. Finally, we discuss strategies to improve throughput of our pipeline to permit genome-wide screens in the future. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

High blood pressure and eye disease

MedlinePlus

... this page: //medlineplus.gov/ency/article/000999.htm High blood pressure and eye disease To use the sharing features on this page, please enable JavaScript. High blood pressure can damage blood vessels in the retina . The ...

The HTM Spatial Pooler-A Neocortical Algorithm for Online Sparse Distributed Coding.

PubMed

Cui, Yuwei; Ahmad, Subutai; Hawkins, Jeff

2017-01-01

Hierarchical temporal memory (HTM) provides a theoretical framework that models several key computational principles of the neocortex. In this paper, we analyze an important component of HTM, the HTM spatial pooler (SP). The SP models how neurons learn feedforward connections and form efficient representations of the input. It converts arbitrary binary input patterns into sparse distributed representations (SDRs) using a combination of competitive Hebbian learning rules and homeostatic excitability control. We describe a number of key properties of the SP, including fast adaptation to changing input statistics, improved noise robustness through learning, efficient use of cells, and robustness to cell death. In order to quantify these properties we develop a set of metrics that can be directly computed from the SP outputs. We show how the properties are met using these metrics and targeted artificial simulations. We then demonstrate the value of the SP in a complete end-to-end real-world HTM system. We discuss the relationship with neuroscience and previous studies of sparse coding. The HTM spatial pooler represents a neurally inspired algorithm for learning sparse representations from noisy data streams in an online fashion.

HTS for SMFS, organohalide respiration, new epigenetic mark, and a decoy receptor.

PubMed

2014-10-23

Each month, Chemistry & Biology Select highlights a selection of research reports from the recent literature. These highlights are a snapshot of interesting research done across the field of chemical biology. This month's Select highlights an on-chip platform for high-throughput force microscopy, a structural view of organohalide respiration, evidence that 5-hydroxymethylcytosine is an epigenetic mark, and use of a decoy receptor to thwart oncogene signaling.

Characterization of Pleurotus ostreatus Biofilms by Using the Calgary Biofilm Device

PubMed Central

Pesciaroli, Lorena; Petruccioli, Maurizio; Fedi, Stefano; Firrincieli, Andrea; Federici, Federico

2013-01-01

The adequacy of the Calgary biofilm device, often referred to as the MBEC system, as a high-throughput approach to the production and subsequent characterization of Pleurotus ostreatus biofilms was assessed. The hydroxyapatite-coating of pegs was necessary to enable biofilm attachment, and the standardization of vegetative inocula ensured a uniform distribution of P. ostreatus biofilms, which is necessary for high-throughput evaluations of several antimicrobials and exposure conditions. Scanning electron microscopy showed surface-associated growth, the occurrence of a complex aggregated growth organized in multilayers or hyphal bundles, and the encasement of hyphae within an extracellular matrix (ECM), the extent of which increased with time. Chemical analyses showed that biofilms differed from free-floating cultures for their higher contents of total sugars (TS) and ECM, with the latter being mainly composed of TS and, to a lesser extent, protein. Confocal laser scanning microscopy analysis of 4-day-old biofilms showed the presence of interspersed interstitial voids and water channels in the mycelial network, the density and compactness of which increased after a 7-day incubation, with the novel occurrence of ECM aggregates with an α-glucan moiety. In 4- and 7-day-old biofilms, tolerance to cadmium was increased by factors of 3.2 and 11.1, respectively, compared to coeval free-floating counterparts. PMID:23892744

Characterization of Pleurotus ostreatus biofilms by using the calgary biofilm device.

PubMed

Pesciaroli, Lorena; Petruccioli, Maurizio; Fedi, Stefano; Firrincieli, Andrea; Federici, Federico; D'Annibale, Alessandro

2013-10-01

The adequacy of the Calgary biofilm device, often referred to as the MBEC system, as a high-throughput approach to the production and subsequent characterization of Pleurotus ostreatus biofilms was assessed. The hydroxyapatite-coating of pegs was necessary to enable biofilm attachment, and the standardization of vegetative inocula ensured a uniform distribution of P. ostreatus biofilms, which is necessary for high-throughput evaluations of several antimicrobials and exposure conditions. Scanning electron microscopy showed surface-associated growth, the occurrence of a complex aggregated growth organized in multilayers or hyphal bundles, and the encasement of hyphae within an extracellular matrix (ECM), the extent of which increased with time. Chemical analyses showed that biofilms differed from free-floating cultures for their higher contents of total sugars (TS) and ECM, with the latter being mainly composed of TS and, to a lesser extent, protein. Confocal laser scanning microscopy analysis of 4-day-old biofilms showed the presence of interspersed interstitial voids and water channels in the mycelial network, the density and compactness of which increased after a 7-day incubation, with the novel occurrence of ECM aggregates with an α-glucan moiety. In 4- and 7-day-old biofilms, tolerance to cadmium was increased by factors of 3.2 and 11.1, respectively, compared to coeval free-floating counterparts.

Multifocal multiphoton microscopy with adaptive optical correction

NASA Astrophysics Data System (ADS)

Coelho, Simao; Poland, Simon; Krstajic, Nikola; Li, David; Monypenny, James; Walker, Richard; Tyndall, David; Ng, Tony; Henderson, Robert; Ameer-Beg, Simon

2013-02-01

Fluorescence lifetime imaging microscopy (FLIM) is a well established approach for measuring dynamic signalling events inside living cells, including detection of protein-protein interactions. The improvement in optical penetration of infrared light compared with linear excitation due to Rayleigh scattering and low absorption have provided imaging depths of up to 1mm in brain tissue but significant image degradation occurs as samples distort (aberrate) the infrared excitation beam. Multiphoton time-correlated single photon counting (TCSPC) FLIM is a method for obtaining functional, high resolution images of biological structures. In order to achieve good statistical accuracy TCSPC typically requires long acquisition times. We report the development of a multifocal multiphoton microscope (MMM), titled MegaFLI. Beam parallelization performed via a 3D Gerchberg-Saxton (GS) algorithm using a Spatial Light Modulator (SLM), increases TCSPC count rate proportional to the number of beamlets produced. A weighted 3D GS algorithm is employed to improve homogeneity. An added benefit is the implementation of flexible and adaptive optical correction. Adaptive optics performed by means of Zernike polynomials are used to correct for system induced aberrations. Here we present results with significant improvement in throughput obtained using a novel complementary metal-oxide-semiconductor (CMOS) 1024 pixel single-photon avalanche diode (SPAD) array, opening the way to truly high-throughput FLIM.

Functionalized CdS quantum dots-based luminescence probe for detection of heavy and transition metal ions in aqueous solution.

PubMed

Chen, Jinlong; Zheng, Aifang; Gao, Yingchun; He, Chiyang; Wu, Genhua; Chen, Youcun; Kai, Xiaoming; Zhu, Changqing

2008-03-01

Strong luminescence CdS quantum dots (QDs) have been prepared and modified with l-cysteine by a facile seeds-assistant technique in water. They are water-soluble and highly stable in aqueous solution. CdS QDs evaluated as a luminescence probe for heavy and transition metal (HTM) ions in aqueous solution was systematically studied. Five HTM ions such as silver(I) ion, copper(II) ion, mercury(II) ion, cobalt(II) ion, and nickel(II) ion significantly influence the photophysics of the emission from the functionalized CdS QDs. Experiment results showed that the fluorescence emission from CdS QDs was enhanced significantly by silver ion without any spectral shift, while several other bivalent HTM ions, such as Hg(2+), Cu(2+), Co(2+), and Ni(2+), exhibited effective optical quenching effect on QDs. Moreover, an obvious red-shift of emission band was observed in the quenching of CdS QDs for Hg(2+) and Cu(2+) ions. Under the optimal conditions, the response was linearly proportional to the concentration of Ag(+) ion ranging from 1.25 x 10(-7) to 5.0 x 10(-6)molL(-1) with a detection limit of 2.0 x 10(-8)molL(-1). The concentration dependence of the quenching effect on functionalized QDs for the other four HTM ions could be well described by typical Stern-Volmer equation, with the linear response of CdS QDs emission proportional to the concentration ranging from 1.50 x 10(-8) to 7.50 x 10(-7)molL(-1) for Hg(2+) ion, 3.0 x 10(-7) to 1.0 x 10(-5)molL(-1) for Ni(2+) ion, 4.59 x 10(-8) to 2.295 x 10(-6)molL(-1) for Cu(2+) ion, and 1.20 x 10(-7) to 6.0 x 10(-6)molL(-1) Co(2+) ion, respectively. Based on the distinct optical properties of CdS QDs system with the five HTM ions, and the relatively wide linear range and rapid response to HTM ions, CdS QDs can be developed as a potential identified luminescence probe for familiar HTM ions detection in aqueous solution.

A high-throughput comparative characterization of laser-induced soft tissue damage using 3D digital microscopy.

PubMed

Das, Debobrato; Reed, Stephanie; Klokkevold, Perry R; Wu, Benjamin M

2013-02-01

3D digital microscopy was used to develop a rapid alternative approach to quantify the effects of specific laser parameters on soft tissue ablation and charring in vitro without the use of conventional tissue processing techniques. Two diode lasers operating at 810 and 980 nm wavelengths were used to ablate three tissue types (bovine liver, turkey breast, and bovine muscle) at varying laser power (0.3, 1.0, and 2.0 W) and velocities (1-50 mm/s). Spectrophotometric analyses were performed on each tissue to determine tissue-specific absorption coefficients and were considered in creating wavelength-dependent energy attenuation models to evaluate minimum heat of tissue ablations. 3D surface contour profiles characterizing tissue damage revealed that ablation depth and tissue charring increased with laser power and decreased with lateral velocity independent of wavelength and tissue type. While bovine liver ablation and charring were statistically higher at 810 than 980 nm (p < 0.05), turkey breast and bovine muscle ablated and charred more at 980 than 810 nm (p < 0.05). Spectrophotometric analysis revealed that bovine liver tissue had a greater tissue-specific absorption coefficient at 810 than 980 nm, while turkey breast and bovine muscle had a larger absorption coefficient at 980 nm (p < 0.05). This rapid 3D microscopic analysis of robot-driven laser ablation yielded highly reproducible data that supported well-defined trends related to laser-tissue interactions and enabled high throughput characterization of many laser-tissue permutations. Since 3D microscopy quantifies entire lesions without altering the tissue specimens, conventional and immunohistologic techniques can be used, if desired, to further interrogate specific sections of the digitized lesions.

What impact do anxiety, depression, perceived control and technology capability have on whether patients with chronic heart failure take-up or continue to use home tele-monitoring services? Study design of ADaPT-HF.

PubMed

Crundall-Goode, Amanda; Goode, Kevin M; Clark, Andrew L

2017-04-01

Home tele-monitoring (HTM) is used to monitor the clinical signs and symptoms of patients with chronic heart failure (CHF) in order to reduce unplanned hospital admissions. However, not all patients who are referred will agree to use HTM, and some patients choose to withdraw early from its use. ADaPT-HF will investigate whether depression, anxiety, low perceived control, reduced technology capability, level of education, age or the severity or complexity of a patient's illness can predict refusal of, or early withdrawal from, HTM in patients with CHF. The study will recruit 288 patients who have been recently admitted to hospital with heart failure who have been referred for HTM. At the time of referral, patients will complete depression (nine-item Patient Health Questionnaire), anxiety (seven-item Generalised Anxiety Disorder questionnaire), perceived control (eight-item revised Controlled Attitudes Scale) and technology capability (ten-item Technology Readiness Index 2.0) screening questionnaires. In addition, data on demographics, diagnosis, clinical examination, socio-economic status, history of comorbidities, medication, biochemistry and haematology will be recorded. The primary outcome will be a composite of refusal of or early withdrawal from HTM. The principle analysis will be made using logistic regression. By establishing which factors influence a patient's decision to refuse or withdraw early from HTM, it may be possible to redesign HTM referral processes. It may be that patients with CHF who also have depression, anxiety, low control and poor technology skills should not be referred until they receive appropriate support or that they should be managed differently when they do receive HTM. The results of ADAPT-HF may provide a way of making more efficient and cost-effective use of HTM services.

High-throughput analysis of sub-visible mAb aggregate particles using automated fluorescence microscopy imaging.

PubMed

Paul, Albert Jesuran; Bickel, Fabian; Röhm, Martina; Hospach, Lisa; Halder, Bettina; Rettich, Nina; Handrick, René; Herold, Eva Maria; Kiefer, Hans; Hesse, Friedemann

2017-07-01

Aggregation of therapeutic proteins is a major concern as aggregates lower the yield and can impact the efficacy of the drug as well as the patient's safety. It can occur in all production stages; thus, it is essential to perform a detailed analysis for protein aggregates. Several methods such as size exclusion high-performance liquid chromatography (SE-HPLC), light scattering, turbidity, light obscuration, and microscopy-based approaches are used to analyze aggregates. None of these methods allows determination of all types of higher molecular weight (HMW) species due to a limited size range. Furthermore, quantification and specification of different HMW species are often not possible. Moreover, automation is a perspective challenge coming up with automated robotic laboratory systems. Hence, there is a need for a fast, high-throughput-compatible method, which can detect a broad size range and enable quantification and classification. We describe a novel approach for the detection of aggregates in the size range 1 to 1000 μm combining fluorescent dyes for protein aggregate labelling and automated fluorescence microscope imaging (aFMI). After appropriate selection of the dye and method optimization, our method enabled us to detect various types of HMW species of monoclonal antibodies (mAbs). Using 10 μmol L -1 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (Bis-ANS) in combination with aFMI allowed the analysis of mAb aggregates induced by different stresses occurring during downstream processing, storage, and administration. Validation of our results was performed by SE-HPLC, UV-Vis spectroscopy, and dynamic light scattering. With this new approach, we could not only reliably detect different HMW species but also quantify and classify them in an automated approach. Our method achieves high-throughput requirements and the selection of various fluorescent dyes enables a broad range of applications.

Integrated, multi-scale, spatial-temporal cell biology--A next step in the post genomic era.

PubMed

Horwitz, Rick

2016-03-01

New microscopic approaches, high-throughput imaging, and gene editing promise major new insights into cellular behaviors. When coupled with genomic and other 'omic information and "mined" for correlations and associations, a new breed of powerful and useful cellular models should emerge. These top down, coarse-grained, and statistical models, in turn, can be used to form hypotheses merging with fine-grained, bottom up mechanistic studies and models that are the back bone of cell biology. The goal of the Allen Institute for Cell Science is to develop the top down approach by developing a high throughput microscopy pipeline that is integrated with modeling, using gene edited hiPS cell lines in various physiological and pathological contexts. The output of these experiments and models will be an "animated" cell, capable of integrating and analyzing image data generated from experiments and models. Copyright © 2015 Elsevier Inc. All rights reserved.

High-Throughput Mapping of Single-Neuron Projections by Sequencing of Barcoded RNA.

PubMed

Kebschull, Justus M; Garcia da Silva, Pedro; Reid, Ashlan P; Peikon, Ian D; Albeanu, Dinu F; Zador, Anthony M

2016-09-07

Neurons transmit information to distant brain regions via long-range axonal projections. In the mouse, area-to-area connections have only been systematically mapped using bulk labeling techniques, which obscure the diverse projections of intermingled single neurons. Here we describe MAPseq (Multiplexed Analysis of Projections by Sequencing), a technique that can map the projections of thousands or even millions of single neurons by labeling large sets of neurons with random RNA sequences ("barcodes"). Axons are filled with barcode mRNA, each putative projection area is dissected, and the barcode mRNA is extracted and sequenced. Applying MAPseq to the locus coeruleus (LC), we find that individual LC neurons have preferred cortical targets. By recasting neuroanatomy, which is traditionally viewed as a problem of microscopy, as a problem of sequencing, MAPseq harnesses advances in sequencing technology to permit high-throughput interrogation of brain circuits. Copyright © 2016 Elsevier Inc. All rights reserved.

High-throughput autofluorescence flow cytometry of breast cancer metabolism (Conference Presentation)

NASA Astrophysics Data System (ADS)

Shah, Amy T.; Cannon, Taylor M.; Higginbotham, Jim N.; Skala, Melissa C.

2016-02-01

Tumor heterogeneity poses challenges for devising optimal treatment regimens for cancer patients. In particular, subpopulations of cells can escape treatment and cause relapse. There is a need for methods to characterize tumor heterogeneity of treatment response. Cell metabolism is altered in cancer (Warburg effect), and cells use the autofluorescent cofactor NADH in numerous metabolic reactions. Previous studies have shown that microscopy measurements of NADH autofluorescence are sensitive to treatment response in breast cancer, and these techniques typically assess hundreds of cells per group. An alternative approach is flow cytometry, which measures fluorescence on a single-cell level and is attractive for characterizing tumor heterogeneity because it achieves high-throughput analysis and cell sorting in millions of cells per group. Current applications for flow cytometry rely on staining with fluorophores. This study characterizes flow cytometry measurements of NADH autofluorescence in breast cancer cells. Preliminary results indicate flow cytometry of NADH is sensitive to cyanide perturbation, which inhibits oxidative phosphorylation, in nonmalignant MCF10A cells. Additionally, flow cytometry is sensitive to higher NADH intensity for HER2-positive SKBr3 cells compared with triple-negative MDA-MB-231 cells. These results agree with previous microscopy studies. Finally, a mixture of SKBr3 and MDA-MB-231 cells were sorted into each cell type using NADH intensity. Sorted cells were cultured, and microscopy validation showed the expected morphology for each cell type. Ultimately, flow cytometry could be applied to characterize tumor heterogeneity based on treatment response and sort cell subpopulations based on metabolic profile. These achievements could enable individualized treatment strategies and improved patient outcomes.

The Role of Healthcare Technology Management in Facilitating Medical Device Cybersecurity.

PubMed

Busdicker, Mike; Upendra, Priyanka

2017-09-02

This article discusses the role of healthcare technology management (HTM) in medical device cybersecurity and outlines concepts that are applicable to HTM professionals at a healthcare delivery organization or at an integrated delivery network, regardless of size. It provides direction for HTM professionals who are unfamiliar with the security aspects of managing healthcare technologies but are familiar with standards from The Joint Commission (TJC). It provides a useful set of recommendations, including relevant references for incorporating good security practices into HTM practice. Recommendations for policies, procedures, and processes referencing TJC standards are easily applicable to HTM departments with limited resources and to those with no resource concerns. The authors outline processes from their organization as well as best practices learned through information sharing at AAMI, National Health Information Sharing and Analysis Center (NH-ISAC), and Medical Device Innovation, Safety, and Security Consortium (MDISS) conferences and workshops.

Camera array based light field microscopy

PubMed Central

Lin, Xing; Wu, Jiamin; Zheng, Guoan; Dai, Qionghai

2015-01-01

This paper proposes a novel approach for high-resolution light field microscopy imaging by using a camera array. In this approach, we apply a two-stage relay system for expanding the aperture plane of the microscope into the size of an imaging lens array, and utilize a sensor array for acquiring different sub-apertures images formed by corresponding imaging lenses. By combining the rectified and synchronized images from 5 × 5 viewpoints with our prototype system, we successfully recovered color light field videos for various fast-moving microscopic specimens with a spatial resolution of 0.79 megapixels at 30 frames per second, corresponding to an unprecedented data throughput of 562.5 MB/s for light field microscopy. We also demonstrated the use of the reported platform for different applications, including post-capture refocusing, phase reconstruction, 3D imaging, and optical metrology. PMID:26417490

MoS2: a two-dimensional hole-transporting material for high-efficiency, low-cost perovskite solar cells

NASA Astrophysics Data System (ADS)

Kohnehpoushi, Saman; Nazari, Pariya; Abdollahi Nejand, Bahram; Eskandari, Mehdi

2018-05-01

In this work MoS2 thin film was studied as a potential two-dimensional (2D) hole-transporting material for fabrication of low-cost, durable and efficient perovskite solar cells. The thickness of MoS2 was studied as a potential factor in reaching high power conversion efficiency in perovskite solar cells. The thickness of the perovskite layer and the different metal back contacts gave distinct photovoltaic properties to the designed cells. The results show that a single sheet of MoS2 could considerably improve the power conversion efficacy of the device from 10.41% for a hole transport material (HTM)-free device to 20.43% for a device prepared with a 0.67 nm thick MoS2 layer as a HTM. On the back, Ag and Al collected the carriers more efficiently than Au due to the value of their metal contact work function with the TiO2 conduction band. The present work proposes a new architecture for the fabrication of low-cost, durable and efficient perovskite solar cells made from a low-cost and robust inorganic HTM and electron transport material.

MoS2: a two-dimensional hole-transporting material for high-efficiency, low-cost perovskite solar cells.

PubMed

Kohnehpoushi, Saman; Nazari, Pariya; Nejand, Bahram Abdollahi; Eskandari, Mehdi

2018-05-18

In this work MoS 2 thin film was studied as a potential two-dimensional (2D) hole-transporting material for fabrication of low-cost, durable and efficient perovskite solar cells. The thickness of MoS 2 was studied as a potential factor in reaching high power conversion efficiency in perovskite solar cells. The thickness of the perovskite layer and the different metal back contacts gave distinct photovoltaic properties to the designed cells. The results show that a single sheet of MoS 2 could considerably improve the power conversion efficacy of the device from 10.41% for a hole transport material (HTM)-free device to 20.43% for a device prepared with a 0.67 nm thick MoS 2 layer as a HTM. On the back, Ag and Al collected the carriers more efficiently than Au due to the value of their metal contact work function with the TiO 2 conduction band. The present work proposes a new architecture for the fabrication of low-cost, durable and efficient perovskite solar cells made from a low-cost and robust inorganic HTM and electron transport material.

High throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy

PubMed Central

Dumitriu, Dani; Rodriguez, Alfredo; Morrison, John H.

2012-01-01

Morphological features such as size, shape and density of dendritic spines have been shown to reflect important synaptic functional attributes and potential for plasticity. Here we describe in detail a protocol for obtaining detailed morphometric analysis of spines using microinjection of fluorescent dyes, high resolution confocal microscopy, deconvolution and image analysis using NeuronStudio. Recent technical advancements include better preservation of tissue resulting in prolonged ability to microinject, and algorithmic improvements that compensate for the residual Z-smear inherent in all optical imaging. Confocal imaging parameters were probed systematically for the identification of both optimal resolution as well as highest efficiency. When combined, our methods yield size and density measurements comparable to serial section transmission electron microscopy in a fraction of the time. An experiment containing 3 experimental groups with 8 subjects in each can take as little as one month if optimized for speed, or approximately 4 to 5 months if the highest resolution and morphometric detail is sought. PMID:21886104

Accurate virus quantitation using a Scanning Transmission Electron Microscopy (STEM) detector in a scanning electron microscope.

PubMed

Blancett, Candace D; Fetterer, David P; Koistinen, Keith A; Morazzani, Elaine M; Monninger, Mitchell K; Piper, Ashley E; Kuehl, Kathleen A; Kearney, Brian J; Norris, Sarah L; Rossi, Cynthia A; Glass, Pamela J; Sun, Mei G

2017-10-01

A method for accurate quantitation of virus particles has long been sought, but a perfect method still eludes the scientific community. Electron Microscopy (EM) quantitation is a valuable technique because it provides direct morphology information and counts of all viral particles, whether or not they are infectious. In the past, EM negative stain quantitation methods have been cited as inaccurate, non-reproducible, and with detection limits that were too high to be useful. To improve accuracy and reproducibility, we have developed a method termed Scanning Transmission Electron Microscopy - Virus Quantitation (STEM-VQ), which simplifies sample preparation and uses a high throughput STEM detector in a Scanning Electron Microscope (SEM) coupled with commercially available software. In this paper, we demonstrate STEM-VQ with an alphavirus stock preparation to present the method's accuracy and reproducibility, including a comparison of STEM-VQ to viral plaque assay and the ViroCyt Virus Counter. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Miniaturized video-microscopy system for near real-time water quality biomonitoring using microfluidic chip-based devices

NASA Astrophysics Data System (ADS)

Huang, Yushi; Nigam, Abhimanyu; Campana, Olivia; Nugegoda, Dayanthi; Wlodkowic, Donald

2016-12-01

Biomonitoring studies apply biological responses of sensitive biomonitor organisms to rapidly detect adverse environmental changes such as presence of physic-chemical stressors and toxins. Behavioral responses such as changes in swimming patterns of small aquatic invertebrates are emerging as sensitive endpoints to monitor aquatic pollution. Although behavioral responses do not deliver information on an exact type or the intensity of toxicants present in water samples, they could provide orders of magnitude higher sensitivity than lethal endpoints such as mortality. Despite the advantages of behavioral biotests performed on sentinel organisms, their wider application in real-time and near realtime biomonitoring of water quality is limited by the lack of dedicated and automated video-microscopy systems. Current behavioral analysis systems rely mostly on static test conditions and manual procedures that are time-consuming and labor intensive. Tracking and precise quantification of locomotory activities of multiple small aquatic organisms requires high-resolution optical data recording. This is often problematic due to small size of fast moving animals and limitations of culture vessels that are not specially designed for video data recording. In this work, we capitalized on recent advances in miniaturized CMOS cameras, high resolution optics and biomicrofluidic technologies to develop near real-time water quality sensing using locomotory activities of small marine invertebrates. We present proof-of-concept integration of high-resolution time-resolved video recording system and high-throughput miniaturized perfusion biomicrofluidic platform for optical tracking of nauplii of marine crustacean Artemia franciscana. Preliminary data demonstrate that Artemia sp. exhibits rapid alterations of swimming patterns in response to toxicant exposure. The combination of video-microscopy and biomicrofluidic platform facilitated straightforward recording of fast moving objects. We envisage that prospectively such system can be scaled up to perform high-throughput water quality sensing in a robotic biomonitoring facility.

Topography and refractometry of nanostructures using spatial light interference microscopy.

PubMed

Wang, Zhuo; Chun, Ik Su; Li, Xiuling; Ong, Zhun-Yong; Pop, Eric; Millet, Larry; Gillette, Martha; Popescu, Gabriel

2010-01-15

Spatial light interference microscopy (SLIM) is a novel method developed in our laboratory that provides quantitative phase images of transparent structures with a 0.3 nm spatial and 0.03 nm temporal accuracy owing to the white light illumination and its common path interferometric geometry. We exploit these features and demonstrate SLIM's ability to perform topography at a single atomic layer in graphene. Further, using a decoupling procedure that we developed for cylindrical structures, we extract the axially averaged refractive index of semiconductor nanotubes and a neurite of a live hippocampal neuron in culture. We believe that this study will set the basis for novel high-throughput topography and refractometry of man-made and biological nanostructures.

Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery.

PubMed

Simm, Jaak; Klambauer, Günter; Arany, Adam; Steijaert, Marvin; Wegner, Jörg Kurt; Gustin, Emmanuel; Chupakhin, Vladimir; Chong, Yolanda T; Vialard, Jorge; Buijnsters, Peter; Velter, Ingrid; Vapirev, Alexander; Singh, Shantanu; Carpenter, Anne E; Wuyts, Roel; Hochreiter, Sepp; Moreau, Yves; Ceulemans, Hugo

2018-05-17

In both academia and the pharmaceutical industry, large-scale assays for drug discovery are expensive and often impractical, particularly for the increasingly important physiologically relevant model systems that require primary cells, organoids, whole organisms, or expensive or rare reagents. We hypothesized that data from a single high-throughput imaging assay can be repurposed to predict the biological activity of compounds in other assays, even those targeting alternate pathways or biological processes. Indeed, quantitative information extracted from a three-channel microscopy-based screen for glucocorticoid receptor translocation was able to predict assay-specific biological activity in two ongoing drug discovery projects. In these projects, repurposing increased hit rates by 50- to 250-fold over that of the initial project assays while increasing the chemical structure diversity of the hits. Our results suggest that data from high-content screens are a rich source of information that can be used to predict and replace customized biological assays. Copyright © 2018 Elsevier Ltd. All rights reserved.

Improved throughput traction microscopy reveals pivotal role for matrix stiffness in fibroblast contractility and TGF-β responsiveness

PubMed Central

Marinković, Aleksandar; Mih, Justin D.; Park, Jin-Ah; Liu, Fei

2012-01-01

Lung fibroblast functions such as matrix remodeling and activation of latent transforming growth factor-β1 (TGF-β1) are associated with expression of the myofibroblast phenotype and are directly linked to fibroblast capacity to generate force and deform the extracellular matrix. However, the study of fibroblast force-generating capacities through methods such as traction force microscopy is hindered by low throughput and time-consuming procedures. In this study, we improved at the detail level methods for higher-throughput traction measurements on polyacrylamide hydrogels using gel-surface-bound fluorescent beads to permit autofocusing and automated displacement mapping, and transduction of fibroblasts with a fluorescent label to streamline cell boundary identification. Together these advances substantially improve the throughput of traction microscopy and allow us to efficiently compute the forces exerted by lung fibroblasts on substrates spanning the stiffness range present in normal and fibrotic lung tissue. Our results reveal that lung fibroblasts dramatically alter the forces they transmit to the extracellular matrix as its stiffness changes, with very low forces generated on matrices as compliant as normal lung tissue. Moreover, exogenous TGF-β1 selectively accentuates tractions on stiff matrices, mimicking fibrotic lung, but not on physiological stiffness matrices, despite equivalent changes in Smad2/3 activation. Taken together, these results demonstrate a pivotal role for matrix mechanical properties in regulating baseline and TGF-β1-stimulated contraction of lung fibroblasts and suggest that stiff fibrotic lung tissue may promote myofibroblast activation through contractility-driven events, whereas normal lung tissue compliance may protect against such feedback amplification of fibroblast activation. PMID:22659883

Graphene Microcapsule Arrays for Combinatorial Electron Microscopy and Spectroscopy in Liquids

DOE PAGES

Yulaev, Alexander; Guo, Hongxuan; Strelcov, Evgheni; ...

2017-04-27

Atomic-scale thickness, molecular impermeability, low atomic number, and mechanical strength make graphene an ideal electron-transparent membrane for material characterization in liquids and gases with scanning electron microscopy and spectroscopy. Here in this paper, we present a novel sample platform made of an array of thousands of identical isolated graphene-capped microchannels with high aspect ratio. A combination of a global wide field of view with high resolution local imaging of the array allows for high throughput in situ studies as well as for combinatorial screening of solutions, liquid interfaces, and immersed samples. We demonstrate the capabilities of this platform by studyingmore » a pure water sample in comparison with alkali halide solutions, a model electrochemical plating process, and beam-induced crystal growth in liquid electrolyte. Spectroscopic characterization of liquid interfaces and immersed objects with Auger and X-ray fluorescence analysis through the graphene membrane are also demonstrated.« less

Localization-based super-resolution imaging meets high-content screening.

PubMed

Beghin, Anne; Kechkar, Adel; Butler, Corey; Levet, Florian; Cabillic, Marine; Rossier, Olivier; Giannone, Gregory; Galland, Rémi; Choquet, Daniel; Sibarita, Jean-Baptiste

2017-12-01

Single-molecule localization microscopy techniques have proven to be essential tools for quantitatively monitoring biological processes at unprecedented spatial resolution. However, these techniques are very low throughput and are not yet compatible with fully automated, multiparametric cellular assays. This shortcoming is primarily due to the huge amount of data generated during imaging and the lack of software for automation and dedicated data mining. We describe an automated quantitative single-molecule-based super-resolution methodology that operates in standard multiwell plates and uses analysis based on high-content screening and data-mining software. The workflow is compatible with fixed- and live-cell imaging and allows extraction of quantitative data like fluorophore photophysics, protein clustering or dynamic behavior of biomolecules. We demonstrate that the method is compatible with high-content screening using 3D dSTORM and DNA-PAINT based super-resolution microscopy as well as single-particle tracking.

Measurement of replication structures at the nanometer scale using super-resolution light microscopy

PubMed Central

Baddeley, D.; Chagin, V. O.; Schermelleh, L.; Martin, S.; Pombo, A.; Carlton, P. M.; Gahl, A.; Domaing, P.; Birk, U.; Leonhardt, H.; Cremer, C.; Cardoso, M. C.

2010-01-01

DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses. PMID:19864256

Massively Parallel Rogue Cell Detection using Serial Time-Encoded Amplified Microscopy of Inertially Ordered Cells in High Throughput Flow

DTIC Science & Technology

2013-06-01

couples  the  high-­‐speed  capability  of  the   STEAM  imager  and  differential  phase... air  bubbles  in  the  TPE  mix.  Moreover,  TPE  chips  were  also  successfully  sealed  to  other  substrates...dynamics,  and  microelectromechanical  systems  (MEMS)  via  laser-­‐scanning  surface   vibrometry ,  and   observation

Wide-field two-photon microscopy with temporal focusing and HiLo background rejection

NASA Astrophysics Data System (ADS)

Yew, Elijah Y. S.; Choi, Heejin; Kim, Daekeun; So, Peter T. C.

2011-03-01

Scanningless depth-resolved microscopy is achieved through spatial-temporal focusing and has been demonstrated previously. The advantage of this method is that a large area may be imaged without scanning resulting in higher throughput of the imaging system. Because it is a widefield technique, the optical sectioning effect is considerably poorer than with conventional spatial focusing two-photon microscopy. Here we propose wide-field two-photon microscopy based on spatio-temporal focusing and employing background rejection based on the HiLo microscope principle. We demonstrate the effects of applying HiLo microscopy to widefield temporally focused two-photon microscopy.

Solution-Processed hybrid Sb2 S3 planar heterojunction solar cell

NASA Astrophysics Data System (ADS)

Huang, Wenxiao; Borazan, Ismail; Carroll, David

Thin-film solar cells based on inorganic absorbers permit a high efficiency and stability. Among or those absorber candidates, recently Sb2S3 has attracted extensive attention because of its suitable band gap (1.5eV ~1.7 eV) , strong optical absorption, low-cost and earth-abundant constituents. Currently high-efficiency Sb2S3 solar cells have absorber layer deposited on nanostructured TiO2 electrodes in combination with organic hole transport material (HTM) on top. However it's challenging to fill the nanostructured TiO2 layer with Sb2S3 and subsequently by HTM, this leads to uncovered surface permits charge recombination. And the existing of Sb2S3/TiO2/HTM triple interface will enhance the recombination due to the surface trap state. Therefore, a planar junction cell would not only have simpler structure with less steps to fabricate but also ideally also have a higher open circuit voltage because of less interface carrier recombination. By far there is limited research focusing on planar Sb2S3 solar cell, so the feasibility is still unclear. Here, we developed a low-toxic solution method to fabricate Sb2S3 thin film solar cell, then we studied the morphology of the Sb2S3 layer and its impact to the device performance. The best device with a structure of FTO/TiO2/Sb2S3/P3HT/Ag has PCE over 5% which is similar or higher than yet the best nanostructure devices with the same HTM. Furthermore, based on solution engineering and surface modification, we improved the Sb2S3 film quality and achieved a record PCE. .

High-throughput characterization of stresses in thin film materials libraries using Si cantilever array wafers and digital holographic microscopy.

PubMed

Lai, Y W; Hamann, S; Ehmann, M; Ludwig, A

2011-06-01

We report the development of an advanced high-throughput stress characterization method for thin film materials libraries sputter-deposited on micro-machined cantilever arrays consisting of around 1500 cantilevers on 4-inch silicon-on-insulator wafers. A low-cost custom-designed digital holographic microscope (DHM) is employed to simultaneously monitor the thin film thickness, the surface topography and the curvature of each of the cantilevers before and after deposition. The variation in stress state across the thin film materials library is then calculated by Stoney's equation based on the obtained radii of curvature of the cantilevers and film thicknesses. DHM with nanometer-scale out-of-plane resolution allows stress measurements in a wide range, at least from several MPa to several GPa. By using an automatic x-y translation stage, the local stresses within a 4-inch materials library are mapped with high accuracy within 10 min. The speed of measurement is greatly improved compared with the prior laser scanning approach that needs more than an hour of measuring time. A high-throughput stress measurement of an as-deposited Fe-Pd-W materials library was evaluated for demonstration. The fast characterization method is expected to accelerate the development of (functional) thin films, e.g., (magnetic) shape memory materials, whose functionality is greatly stress dependent. © 2011 American Institute of Physics

Structural Fingerprinting of Nanocrystals in the Transmission Electron Microscope

NASA Astrophysics Data System (ADS)

Rouvimov, Sergei; Plachinda, Pavel; Moeck, Peter

2010-03-01

Three novel strategies for the structurally identification of nanocrystals in a transmission electron microscope are presented. Either a single high-resolution transmission electron microscopy image [1] or a single precession electron diffractogram (PED) [2] may be employed. PEDs from fine-grained crystal powders may also be utilized. Automation of the former two strategies is in progress and shall lead to statistically significant results on ensembles of nanocrystals. Open-access databases such as the Crystallography Open Database which provides more than 81,500 crystal structure data sets [3] or its mainly inorganic and educational subsets [4] may be utilized. [1] http://www.scientificjournals.org/journals 2007/j/of/dissertation.htm [2] P. Moeck and S. Rouvimov, in: {Drugs and the Pharmaceutical Sciences}, Vol. 191, 2009, 270-313 [3] http://cod.ibt.lt, http://www.crystallography.net, http://cod.ensicaen.fr, http://nanocrystallography.org, http://nanocrystallography.net, http://journals.iucr.org/j/issues/2009/04/00/kk5039/kk5039.pdf [4] http://nanocrystallography.research.pdx.edu/CIF-searchable

Fusion of lens-free microscopy and mobile-phone microscopy images for high-color-accuracy and high-resolution pathology imaging

NASA Astrophysics Data System (ADS)

Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

2017-03-01

Digital pathology and telepathology require imaging tools with high-throughput, high-resolution and accurate color reproduction. Lens-free on-chip microscopy based on digital in-line holography is a promising technique towards these needs, as it offers a wide field of view (FOV >20 mm2) and high resolution with a compact, low-cost and portable setup. Color imaging has been previously demonstrated by combining reconstructed images at three discrete wavelengths in the red, green and blue parts of the visible spectrum, i.e., the RGB combination method. However, this RGB combination method is subject to color distortions. To improve the color performance of lens-free microscopy for pathology imaging, here we present a wavelet-based color fusion imaging framework, termed "digital color fusion microscopy" (DCFM), which digitally fuses together a grayscale lens-free microscope image taken at a single wavelength and a low-resolution and low-magnification color-calibrated image taken by a lens-based microscope, which can simply be a mobile phone based cost-effective microscope. We show that the imaging results of an H&E stained breast cancer tissue slide with the DCFM technique come very close to a color-calibrated microscope using a 40x objective lens with 0.75 NA. Quantitative comparison showed 2-fold reduction in the mean color distance using the DCFM method compared to the RGB combination method, while also preserving the high-resolution features of the lens-free microscope. Due to the cost-effective and field-portable nature of both lens-free and mobile-phone microscopy techniques, their combination through the DCFM framework could be useful for digital pathology and telepathology applications, in low-resource and point-of-care settings.

An Automatic Segmentation Method Combining an Active Contour Model and a Classification Technique for Detecting Polycomb-group Proteinsin High-Throughput Microscopy Images.

PubMed

Gregoretti, Francesco; Cesarini, Elisa; Lanzuolo, Chiara; Oliva, Gennaro; Antonelli, Laura

2016-01-01

The large amount of data generated in biological experiments that rely on advanced microscopy can be handled only with automated image analysis. Most analyses require a reliable cell image segmentation eventually capable of detecting subcellular structures.We present an automatic segmentation method to detect Polycomb group (PcG) proteins areas isolated from nuclei regions in high-resolution fluorescent cell image stacks. It combines two segmentation algorithms that use an active contour model and a classification technique serving as a tool to better understand the subcellular three-dimensional distribution of PcG proteins in live cell image sequences. We obtained accurate results throughout several cell image datasets, coming from different cell types and corresponding to different fluorescent labels, without requiring elaborate adjustments to each dataset.

New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.

PubMed

Schorb, Martin; Gaechter, Leander; Avinoam, Ori; Sieckmann, Frank; Clarke, Mairi; Bebeacua, Cecilia; Bykov, Yury S; Sonnen, Andreas F-P; Lihl, Reinhard; Briggs, John A G

2017-02-01

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Deep-branching Novel Lineages and High Diversity of Haptophytes in the Skagerrak (Norway) Uncovered by 454 Pyrosequencing

PubMed Central

Egge, Elianne S; Eikrem, Wenche; Edvardsen, Bente

2015-01-01

Microalgae in the division Haptophyta may be difficult to identify to species by microscopy because they are small and fragile. Here, we used high-throughput sequencing to explore the diversity of haptophytes in outer Oslofjorden, Skagerrak, and supplemented this with electron microscopy. Nano- and picoplanktonic subsurface samples were collected monthly for 2 yr, and the haptophytes were targeted by amplification of RNA/cDNA with Haptophyta-specific 18S ribosomal DNA V4 primers. Pyrosequencing revealed higher species richness of haptophytes than previously observed in the Skagerrak by microscopy. From ca. 400,000 reads we obtained 156 haptophyte operational taxonomic units (OTUs) after rigorous filtering and 99.5% clustering. The majority (84%) of the OTUs matched environmental sequences not linked to a morphological species, most of which were affiliated with the order Prymnesiales. Phylogenetic analyses including Oslofjorden OTUs and available cultured and environmental haptophyte sequences showed that several of the OTUs matched sequences forming deep-branching lineages, potentially representing novel haptophyte classes. Pyrosequencing also retrieved cultured species not previously reported by microscopy in the Skagerrak. Electron microscopy revealed species not yet genetically characterised and some potentially novel taxa. This study contributes to linking genotype to phenotype within this ubiquitous and ecologically important protist group, and reveals great, unknown diversity. PMID:25099994

Syphilis

MedlinePlus

... cdc.gov/std/syphilis/ stdfact-msm-syphilis.htm Congenital Syphilis - Fact Sheet https://www.cdc.gov/std/syphilis/ stdfact-congenital-syphilis.htm STDs during Pregnancy - Fact Sheet https://www. ...

A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells

PubMed Central

Forment, Josep V.; Jackson, Stephen P.

2016-01-01

Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, make it easier to quantify and allow a stream-lined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry1. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell-cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (no more than a working day from sample collection to quantification), requires less starting material compared to standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy. PMID:26226461

Efficient globally optimal segmentation of cells in fluorescence microscopy images using level sets and convex energy functionals.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2012-10-01

In high-throughput applications, accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression and the understanding of cell function. We propose an approach for segmenting cell nuclei which is based on active contours using level sets and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We consider three different well-known energy functionals for active contour-based segmentation and introduce convex formulations of these functionals. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images from different experiments comprising different cell types. We have also performed a quantitative comparison with previous segmentation approaches. Copyright © 2012 Elsevier B.V. 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.

High-throughput cocrystal slurry screening by use of in situ Raman microscopy and multi-well plate.

PubMed

Kojima, Takashi; Tsutsumi, Shunichirou; Yamamoto, Katsuhiko; Ikeda, Yukihiro; Moriwaki, Toshiya

2010-10-31

Cocrystal has attracted much attention in order to improve poor physicochemical properties, since cocrystal former crystallize with the ionic drugs as well as nonionic drugs. Cocrystal screening was usually conducted by crystallization, slurry and co-grinding techniques, however sensitivity, cost and time for screening were limited because of issues such as dissociation of cocrystal during crystallization and cost and time required for slurry and co-grinding methods. To overcome these issues, novel high-throughput cocrystal slurry screening was developed by using in situ Raman microscope and a multi-well plate. Cocrystal screening of indomethacin was conducted with 46 cocrystal formers and potential cocrystals were prepared on a large scale for the characterization with powder X-ray diffractometry, thermal analysis, and Raman microscopy and (1)H NMR spectroscopy. Compared with the characterization of scale-up cocrystals, the cocrystal screening indicated that indomethacin structured novel cocrystals with D/L-mandelic acid, nicotinamide, lactamide and benzamide which was not obtained in the screening with crystallization technique previously reported. In addition, the screening provided not only information of cocrystal formation within a day but also information of equilibrium of cocrystal formation and polymorphic transformation in one screening. Information obtained in this screening allows effective solid form selection by saving cost and time for the development. Copyright © 2010 Elsevier B.V. All rights reserved.

ElectroTaxis-on-a-Chip (ETC): an integrated quantitative high-throughput screening platform for electrical field-directed cell migration.

PubMed

Zhao, Siwei; Zhu, Kan; Zhang, Yan; Zhu, Zijie; Xu, Zhengping; Zhao, Min; Pan, Tingrui

2014-11-21

Both endogenous and externally applied electrical stimulation can affect a wide range of cellular functions, including growth, migration, differentiation and division. Among those effects, the electrical field (EF)-directed cell migration, also known as electrotaxis, has received broad attention because it holds great potential in facilitating clinical wound healing. Electrotaxis experiment is conventionally conducted in centimetre-sized flow chambers built in Petri dishes. Despite the recent efforts to adapt microfluidics for electrotaxis studies, the current electrotaxis experimental setup is still cumbersome due to the needs of an external power supply and EF controlling/monitoring systems. There is also a lack of parallel experimental systems for high-throughput electrotaxis studies. In this paper, we present a first independently operable microfluidic platform for high-throughput electrotaxis studies, integrating all functional components for cell migration under EF stimulation (except microscopy) on a compact footprint (the same as a credit card), referred to as ElectroTaxis-on-a-Chip (ETC). Inspired by the R-2R resistor ladder topology in digital signal processing, we develop a systematic approach to design an infinitely expandable microfluidic generator of EF gradients for high-throughput and quantitative studies of EF-directed cell migration. Furthermore, a vacuum-assisted assembly method is utilized to allow direct and reversible attachment of our device to existing cell culture media on biological surfaces, which separates the cell culture and device preparation/fabrication steps. We have demonstrated that our ETC platform is capable of screening human cornea epithelial cell migration under the stimulation of an EF gradient spanning over three orders of magnitude. The screening results lead to the identification of the EF-sensitive range of that cell type, which can provide valuable guidance to the clinical application of EF-facilitated wound healing.

WE-E-BRE-03: Biological Validation of a Novel High-Throughput Irradiator for Predictive Radiation Sensitivity Bioassays

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

Fowler, TL; Martin, JA; Shepard, AJ

2014-06-15

Purpose: The large dose-response variation in both tumor and normal cells between individual patients has led to the recent implementation of predictive bioassays of patient-specific radiation sensitivity in order to personalize radiation therapy. This exciting new clinical paradigm has led us to develop a novel high-throughput, variable dose-rate irradiator to accompany these efforts. Here we present the biological validation of this irradiator through the use of human cells as a relative dosimeter assessed by two metrics, DNA double-strand break repair pathway modulation and intercellular reactive oxygen species production. Methods: Immortalized human tonsilar epithelial cells were cultured in 96-well micro titermore » plates and irradiated in groups of eight wells to absorbed doses of 0, 0.5, 1, 2, 4, and 8 Gy. High-throughput immunofluorescent microscopy was used to detect γH2AX, a DNA double-strand break repair mechanism recruiter. The same analysis was performed with the cells stained with CM-H2DCFDA that produces a fluorescent adduct when exposed to reactive oxygen species during the irradiation cycle. Results: Irradiations of the immortalized human tonsilar epithelial cells at absorbed doses of 0, 0.5, 1, 2, 4, and 8 Gy produced excellent linearity in γH2AX and CM-H2DCFDA with R2 values of 0.9939 and 0.9595 respectively. Single cell gel electrophoresis experimentation for the detection of physical DNA double-strand breaks in ongoing. Conclusions: This work indicates significant potential for our high-throughput variable dose rate irradiator for patient-specific predictive radiation sensitivity bioassays. This irradiator provides a powerful tool by increasing the efficiency and number of assay techniques available to help personalize radiation therapy.« less

Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition.

PubMed

Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria

2014-08-30

Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.

Fluidized-bed technology enabling the integration of high temperature solar receiver CSP systems with steam and advanced power cycles

DOE PAGES

Sakadjian, B.; Hu, S.; Maryamchik, M.; ...

2015-06-05

Solar Particle Receivers (SPR) are under development to drive concentrating solar plants (CSP) towards higher operating temperatures to support higher efficiency power conversion cycles. The novel high temperature SPR-based CSP system uses solid particles as the heat transfer medium (HTM) in place of the more conventional fluids such as molten salt or steam used in current state-of-the-art CSP plants. The solar particle receiver (SPR) is designed to heat the HTM to temperatures of 800 °C or higher which is well above the operating temperatures of nitrate-based molten salt thermal energy storage (TES) systems. The solid particles also help overcome somemore » of the other challenges associated with molten salt-based systems such as freezing, instability and degradation. The higher operating temperatures and use of low cost HTM and higher efficiency power cycles are geared towards reducing costs associated with CSP systems. This paper describes the SPR-based CSP system with a focus on the fluidized-bed (FB) heat exchanger and its integration with various power cycles. Furthermore, the SPR technology provides a potential pathway to achieving the levelized cost of electricity (LCOE) target of $0.06/kWh that has been set by the U.S. Department of Energy's SunShot initiative.« less

Fluidized-bed technology enabling the integration of high temperature solar receiver CSP systems with steam and advanced power cycles

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

Sakadjian, B.; Hu, S.; Maryamchik, M.

Solar Particle Receivers (SPR) are under development to drive concentrating solar plants (CSP) towards higher operating temperatures to support higher efficiency power conversion cycles. The novel high temperature SPR-based CSP system uses solid particles as the heat transfer medium (HTM) in place of the more conventional fluids such as molten salt or steam used in current state-of-the-art CSP plants. The solar particle receiver (SPR) is designed to heat the HTM to temperatures of 800 °C or higher which is well above the operating temperatures of nitrate-based molten salt thermal energy storage (TES) systems. The solid particles also help overcome somemore » of the other challenges associated with molten salt-based systems such as freezing, instability and degradation. The higher operating temperatures and use of low cost HTM and higher efficiency power cycles are geared towards reducing costs associated with CSP systems. This paper describes the SPR-based CSP system with a focus on the fluidized-bed (FB) heat exchanger and its integration with various power cycles. Furthermore, the SPR technology provides a potential pathway to achieving the levelized cost of electricity (LCOE) target of $0.06/kWh that has been set by the U.S. Department of Energy's SunShot initiative.« less

Characterization of the NEXT Hollow Cathode Inserts After Long-Duration Testing

NASA Technical Reports Server (NTRS)

Mackey, J.; Shastry, R.; Soulas, G.

2017-01-01

Hollow dispenser cathode inserts are a critical element of electric propulsion systems, and should therefore be well understood during long term operation to ensure reliable system performance. This work destructively investigated cathode inserts from the NEXT long-duration test which demonstrated 51,184 hours of high-voltage operation, 918 kg of propellant throughput, and 35.5 MN-s of total impulse. The characterization methods used include scanning electron microscopy with energy dispersive spectroscopy and X-ray diffraction. Microscopy analysis has been performed on fractured surfaces, emission surfaces, and metallographically polished cross-sections of post-test inserts and unused inserts. Impregnate distribution, etch region thickness, impregnate chemical content, emission surface topography, and emission surface phase identification are the primary factors investigated.

Topography and refractometry of nanostructures using spatial light interference microscopy (SLIM)

PubMed Central

Wang, Zhuo; Chun, Ik Su; Li, Xiuling; Ong, Zhun-Yong; Pop, Eric; Millet, Larry; Gillette, Martha; Popescu, Gabriel

2010-01-01

Spatial Light Interference Microscopy (SLIM) is a novel method developed in our laboratory that provides quantitative phase images of transparent structures with 0.3 nm spatial and 0.03 nm temporal accuracy owing to the white light illumination and its common path interferometric geometry. We exploit these features and demonstrate SLIM's ability to perform topography at a single atomic layer in graphene. Further, using a decoupling procedure that we developed for cylindrical structures, we extract the axially-averaged refractive index of semiconductor nanotubes and a neurite of a live hippocampal neuron in culture. We believe that this study will set the basis for novel high-throughput topography and refractometry of man-made and biological nanostructures. PMID:20081970

heterogeneous mixture distributions for multi-source extreme rainfall

NASA Astrophysics Data System (ADS)

Ouarda, T.; Shin, J.; Lee, T. S.

2013-12-01

Mixture distributions have been used to model hydro-meteorological variables showing mixture distributional characteristics, e.g. bimodality. Homogeneous mixture (HOM) distributions (e.g. Normal-Normal and Gumbel-Gumbel) have been traditionally applied to hydro-meteorological variables. However, there is no reason to restrict the mixture distribution as the combination of one identical type. It might be beneficial to characterize the statistical behavior of hydro-meteorological variables from the application of heterogeneous mixture (HTM) distributions such as Normal-Gamma. In the present work, we focus on assessing the suitability of HTM distributions for the frequency analysis of hydro-meteorological variables. In the present work, in order to estimate the parameters of HTM distributions, the meta-heuristic algorithm (Genetic Algorithm) is employed to maximize the likelihood function. In the present study, a number of distributions are compared, including the Gamma-Extreme value type-one (EV1) HTM distribution, the EV1-EV1 HOM distribution, and EV1 distribution. The proposed distribution models are applied to the annual maximum precipitation data in South Korea. The Akaike Information Criterion (AIC), the root mean squared errors (RMSE) and the log-likelihood are used as measures of goodness-of-fit of the tested distributions. Results indicate that the HTM distribution (Gamma-EV1) presents the best fitness. The HTM distribution shows significant improvement in the estimation of quantiles corresponding to the 20-year return period. It is shown that extreme rainfall in the coastal region of South Korea presents strong heterogeneous mixture distributional characteristics. Results indicate that HTM distributions are a good alternative for the frequency analysis of hydro-meteorological variables when disparate statistical characteristics are presented.

On the Optimum Architecture of the Biologically Inspired Hierarchical Temporal Memory Model Applied to the Hand-Written Digit Recognition

NASA Astrophysics Data System (ADS)

Štolc, Svorad; Bajla, Ivan

2010-01-01

In the paper we describe basic functions of the Hierarchical Temporal Memory (HTM) network based on a novel biologically inspired model of the large-scale structure of the mammalian neocortex. The focus of this paper is in a systematic exploration of possibilities how to optimize important controlling parameters of the HTM model applied to the classification of hand-written digits from the USPS database. The statistical properties of this database are analyzed using the permutation test which employs a randomization distribution of the training and testing data. Based on a notion of the homogeneous usage of input image pixels, a methodology of the HTM parameter optimization is proposed. In order to study effects of two substantial parameters of the architecture: the patch size and the overlap in more details, we have restricted ourselves to the single-level HTM networks. A novel method for construction of the training sequences by ordering series of the static images is developed. A novel method for estimation of the parameter maxDist based on the box counting method is proposed. The parameter sigma of the inference Gaussian is optimized on the basis of the maximization of the belief distribution entropy. Both optimization algorithms can be equally applied to the multi-level HTM networks as well. The influences of the parameters transitionMemory and requestedGroupCount on the HTM network performance have been explored. Altogether, we have investigated 2736 different HTM network configurations. The obtained classification accuracy results have been benchmarked with the published results of several conventional classifiers.

Asthma and Hispanic Americans

MedlinePlus

... as bronchitis. Children living below or near the poverty level are more likely to have high levels ... www.cdc.gov/nchs/hdi.htm The proportional impact of asthma prevalence, health care use and mortality ...

Uninterrupted monitoring of drug effects in human-induced pluripotent stem cell-derived cardiomyocytes with bioluminescence Ca2+ microscopy.

PubMed

Suzuki, Kazushi; Onishi, Takahito; Nakada, Chieko; Takei, Shunsuke; Daniels, Matthew J; Nakano, Masahiro; Matsuda, Tomoki; Nagai, Takeharu

2018-05-18

Cardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for high-throughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca 2+ indicator GmNL(Ca 2+ ), and its application in a customized microscope for high-throughput drug screening. GmNL(Ca 2+ ) gives a 140% signal change with Ca 2+ , and can image drug-induced changes of Ca 2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca 2+ ) with this adaptation, we could image spontaneous Ca 2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.

Droplet Microarray Based on Superhydrophobic-Superhydrophilic Patterns for Single Cell Analysis.

PubMed

Jogia, Gabriella E; Tronser, Tina; Popova, Anna A; Levkin, Pavel A

2016-12-09

Single-cell analysis provides fundamental information on individual cell response to different environmental cues and is a growing interest in cancer and stem cell research. However, current existing methods are still facing challenges in performing such analysis in a high-throughput manner whilst being cost-effective. Here we established the Droplet Microarray (DMA) as a miniaturized screening platform for high-throughput single-cell analysis. Using the method of limited dilution and varying cell density and seeding time, we optimized the distribution of single cells on the DMA. We established culturing conditions for single cells in individual droplets on DMA obtaining the survival of nearly 100% of single cells and doubling time of single cells comparable with that of cells cultured in bulk cell population using conventional methods. Our results demonstrate that the DMA is a suitable platform for single-cell analysis, which carries a number of advantages compared with existing technologies allowing for treatment, staining and spot-to-spot analysis of single cells over time using conventional analysis methods such as microscopy.

Development of automated high throughput single molecular microfluidic detection platform for signal transduction analysis

NASA Astrophysics Data System (ADS)

Huang, Po-Jung; Baghbani Kordmahale, Sina; Chou, Chao-Kai; Yamaguchi, Hirohito; Hung, Mien-Chie; Kameoka, Jun

2016-03-01

Signal transductions including multiple protein post-translational modifications (PTM), protein-protein interactions (PPI), and protein-nucleic acid interaction (PNI) play critical roles for cell proliferation and differentiation that are directly related to the cancer biology. Traditional methods, like mass spectrometry, immunoprecipitation, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy require a large amount of sample and long processing time. "microchannel for multiple-parameter analysis of proteins in single-complex (mMAPS)"we proposed can reduce the process time and sample volume because this system is composed by microfluidic channels, fluorescence microscopy, and computerized data analysis. In this paper, we will present an automated mMAPS including integrated microfluidic device, automated stage and electrical relay for high-throughput clinical screening. Based on this result, we estimated that this automated detection system will be able to screen approximately 150 patient samples in a 24-hour period, providing a practical application to analyze tissue samples in a clinical setting.

Automated solid-phase subcloning based on beads brought into proximity by magnetic force.

PubMed

Hudson, Elton P; Nikoshkov, Andrej; Uhlen, Mathias; Rockberg, Johan

2012-01-01

In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.

Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force

PubMed Central

Hudson, Elton P.; Nikoshkov, Andrej; Uhlen, Mathias; Rockberg, Johan

2012-01-01

In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications. PMID:22624028

HTM Spatial Pooler With Memristor Crossbar Circuits for Sparse Biometric Recognition.

PubMed

James, Alex Pappachen; Fedorova, Irina; Ibrayev, Timur; Kudithipudi, Dhireesha

2017-06-01

Hierarchical Temporal Memory (HTM) is an online machine learning algorithm that emulates the neo-cortex. The development of a scalable on-chip HTM architecture is an open research area. The two core substructures of HTM are spatial pooler and temporal memory. In this work, we propose a new Spatial Pooler circuit design with parallel memristive crossbar arrays for the 2D columns. The proposed design was validated on two different benchmark datasets, face recognition, and speech recognition. The circuits are simulated and analyzed using a practical memristor device model and 0.18 μm IBM CMOS technology model. The databases AR, YALE, ORL, and UFI, are used to test the performance of the design in face recognition. TIMIT dataset is used for the speech recognition.

PIZZA: a phase-induced zonal Zernike apodization designed for stellar coronagraphy

NASA Astrophysics Data System (ADS)

Martinache, Frantz

2004-08-01

I explore here the possibilities offered by the general formalism of coronagraphy for the very special case of phase contrast. This technique, invented by Zernike, is commonly used in microscopy, to see phase objects such as micro-organisms, and in strioscopy, to control the quality of optics polishing. It may find application in telescope pupil apodization with significant advantages over classical pupil apodization techniques, including high throughput and no off-axis resolution loss, which is essential for exoplanet imaging.

High-Throughput and Label-Free Single Nanoparticle Sizing Based on Time-Resolved On-Chip Microscopy

DTIC Science & Technology

2015-02-17

12,13 soot ,6,14 ice crystals in clouds,15 and engineered nano- materials,16 among others. While there exist various nanoparticle detection and sizing...the sample of interest is placed on an optoelectronic sensor -array with typically less than 0.5 mm gap (z2) between the sample and sensor planes such...that, under unit mag- nification, the entire sensor active area serves as the imaging FOV, easily reaching >2030 mm2 with state-of-the-art CMOS

Snapshot Hyperspectral Volumetric Microscopy

NASA Astrophysics Data System (ADS)

Wu, Jiamin; Xiong, Bo; Lin, Xing; He, Jijun; Suo, Jinli; Dai, Qionghai

2016-04-01

The comprehensive analysis of biological specimens brings about the demand for capturing the spatial, temporal and spectral dimensions of visual information together. However, such high-dimensional video acquisition faces major challenges in developing large data throughput and effective multiplexing techniques. Here, we report the snapshot hyperspectral volumetric microscopy that computationally reconstructs hyperspectral profiles for high-resolution volumes of ~1000 μm × 1000 μm × 500 μm at video rate by a novel four-dimensional (4D) deconvolution algorithm. We validated the proposed approach with both numerical simulations for quantitative evaluation and various real experimental results on the prototype system. Different applications such as biological component analysis in bright field and spectral unmixing of multiple fluorescence are demonstrated. The experiments on moving fluorescent beads and GFP labelled drosophila larvae indicate the great potential of our method for observing multiple fluorescent markers in dynamic specimens.

Adverse outcome pathway (AOP) development and evaluation ...

EPA Pesticide Factsheets

The Adverse Outcome Pathway provides a construct for assembling mechanistic information at different levels of biological organization in a form designed to support regulatory decision making. In particular, it frames the link between molecular and cellular events that can be measured in high throughput toxicity testing and the organism or population-level events that are commonly relevant in defining risk. Recognizing the importance of this emerging framework, the Organisation for Economic Co-operation and Development (OECD) launched a program to support the development, documentation and consideration of AOPs by the international community in 2012 (http://www.oecd.org/chemicalsafety/testing/adverse-outcome-pathways-molecular-screening-and-toxicogenomics.htm). In 2014, a handbook (https://aopkb.org/common/AOP_Handbook.pdf) was developed to guide users in the documentation and evaluation of AOPs and their entry into an official knowledgebase. The handbook draws on longstanding experience in consideration of mechanistic data (e.g., mode of action analysis) to inform risk assessment. To further assist users, a training program was developed by members of the OECD Extended Advisory Group to teach users the basic principles of AOP development and the best practices as outlined in the OECD AOP handbook. Training sessions began in early 2015, and this course will provide training for interested SOT scientists. Following this course, all participants will be familiar w

Massively Parallel Rogue Cell Detection Using Serial Time-Encoded Amplified Microscopy of Inertially Ordered Cells in High-Throughput Flow

DTIC Science & Technology

2011-08-01

screening of budding yeast and detection of rare breast cancer cells in blood, our method should also be amenable to other applications in which high...to UV light with a power of 8.0 mW/cm2 through the transparency mask for 90 seconds. The wafer was baked again at 95°C for 4 minutes then developed...separated from the replica and sonicated in isopropanol for 5 minutes, sonicated in deionized H2O for 5 minutes, and baked at 65°C for at least 30

Active control of bright electron beams with RF optics for femtosecond microscopy

DOE PAGES

Williams, J.; Zhou, F.; Sun, T.; ...

2017-08-01

A frontier challenge in implementing femtosecond electron microscopy is to gain precise optical control of intense beams to mitigate collective space charge effects for significantly improving the throughput. In this paper, we explore the flexible uses of an RF cavity as a longitudinal lens in a high-intensity beam column for condensing the electron beams both temporally and spectrally, relevant to the design of ultrafast electron microscopy. Through the introduction of a novel atomic grating approach for characterization of electron bunch phase space and control optics, we elucidate the principles for predicting and controlling the phase space dynamics to reach optimalmore » compressions at various electron densities and generating conditions. We provide strategies to identify high-brightness modes, achieving ~100 fs and ~1 eV resolutions with 10 6 electrons per bunch, and establish the scaling of performance for different bunch charges. These results benchmark the sensitivity and resolution from the fundamental beam brightness perspective and also validate the adaptive optics concept to enable delicate control of the density-dependent phase space structures to optimize the performance, including delivering ultrashort, monochromatic, high-dose, or coherent electron bunches.« less

Active control of bright electron beams with RF optics for femtosecond microscopy

PubMed Central

Williams, J.; Zhou, F.; Sun, T.; Tao, Z.; Chang, K.; Makino, K.; Berz, M.; Duxbury, P. M.; Ruan, C.-Y.

2017-01-01

A frontier challenge in implementing femtosecond electron microscopy is to gain precise optical control of intense beams to mitigate collective space charge effects for significantly improving the throughput. Here, we explore the flexible uses of an RF cavity as a longitudinal lens in a high-intensity beam column for condensing the electron beams both temporally and spectrally, relevant to the design of ultrafast electron microscopy. Through the introduction of a novel atomic grating approach for characterization of electron bunch phase space and control optics, we elucidate the principles for predicting and controlling the phase space dynamics to reach optimal compressions at various electron densities and generating conditions. We provide strategies to identify high-brightness modes, achieving ∼100 fs and ∼1 eV resolutions with 106 electrons per bunch, and establish the scaling of performance for different bunch charges. These results benchmark the sensitivity and resolution from the fundamental beam brightness perspective and also validate the adaptive optics concept to enable delicate control of the density-dependent phase space structures to optimize the performance, including delivering ultrashort, monochromatic, high-dose, or coherent electron bunches. PMID:28868325

CellProfiler Tracer: exploring and validating high-throughput, time-lapse microscopy image data.

PubMed

Bray, Mark-Anthony; Carpenter, Anne E

2015-11-04

Time-lapse analysis of cellular images is an important and growing need in biology. Algorithms for cell tracking are widely available; what researchers have been missing is a single open-source software package to visualize standard tracking output (from software like CellProfiler) in a way that allows convenient assessment of track quality, especially for researchers tuning tracking parameters for high-content time-lapse experiments. This makes quality assessment and algorithm adjustment a substantial challenge, particularly when dealing with hundreds of time-lapse movies collected in a high-throughput manner. We present CellProfiler Tracer, a free and open-source tool that complements the object tracking functionality of the CellProfiler biological image analysis package. Tracer allows multi-parametric morphological data to be visualized on object tracks, providing visualizations that have already been validated within the scientific community for time-lapse experiments, and combining them with simple graph-based measures for highlighting possible tracking artifacts. CellProfiler Tracer is a useful, free tool for inspection and quality control of object tracking data, available from http://www.cellprofiler.org/tracer/.

High Throughput Screening for Anti–Trypanosoma cruzi Drug Discovery

PubMed Central

Alonso-Padilla, Julio; Rodríguez, Ana

2014-01-01

The discovery of new therapeutic options against Trypanosoma cruzi, the causative agent of Chagas disease, stands as a fundamental need. Currently, there are only two drugs available to treat this neglected disease, which represents a major public health problem in Latin America. Both available therapies, benznidazole and nifurtimox, have significant toxic side effects and their efficacy against the life-threatening symptomatic chronic stage of the disease is variable. Thus, there is an urgent need for new, improved anti–T. cruzi drugs. With the objective to reliably accelerate the drug discovery process against Chagas disease, several advances have been made in the last few years. Availability of engineered reporter gene expressing parasites triggered the development of phenotypic in vitro assays suitable for high throughput screening (HTS) as well as the establishment of new in vivo protocols that allow faster experimental outcomes. Recently, automated high content microscopy approaches have also been used to identify new parasitic inhibitors. These in vitro and in vivo early drug discovery approaches, which hopefully will contribute to bring better anti–T. cruzi drug entities in the near future, are reviewed here. PMID:25474364

High throughput screening for anti-Trypanosoma cruzi drug discovery.

PubMed

Alonso-Padilla, Julio; Rodríguez, Ana

2014-12-01

The discovery of new therapeutic options against Trypanosoma cruzi, the causative agent of Chagas disease, stands as a fundamental need. Currently, there are only two drugs available to treat this neglected disease, which represents a major public health problem in Latin America. Both available therapies, benznidazole and nifurtimox, have significant toxic side effects and their efficacy against the life-threatening symptomatic chronic stage of the disease is variable. Thus, there is an urgent need for new, improved anti-T. cruzi drugs. With the objective to reliably accelerate the drug discovery process against Chagas disease, several advances have been made in the last few years. Availability of engineered reporter gene expressing parasites triggered the development of phenotypic in vitro assays suitable for high throughput screening (HTS) as well as the establishment of new in vivo protocols that allow faster experimental outcomes. Recently, automated high content microscopy approaches have also been used to identify new parasitic inhibitors. These in vitro and in vivo early drug discovery approaches, which hopefully will contribute to bring better anti-T. cruzi drug entities in the near future, are reviewed here.

High resolution computational on-chip imaging of biological samples using sparsity constraint (Conference Presentation)

NASA Astrophysics Data System (ADS)

Rivenson, Yair; Wu, Chris; Wang, Hongda; Zhang, Yibo; Ozcan, Aydogan

2017-03-01

Microscopic imaging of biological samples such as pathology slides is one of the standard diagnostic methods for screening various diseases, including cancer. These biological samples are usually imaged using traditional optical microscopy tools; however, the high cost, bulkiness and limited imaging throughput of traditional microscopes partially restrict their deployment in resource-limited settings. In order to mitigate this, we previously demonstrated a cost-effective and compact lens-less on-chip microscopy platform with a wide field-of-view of >20-30 mm^2. The lens-less microscopy platform has shown its effectiveness for imaging of highly connected biological samples, such as pathology slides of various tissue samples and smears, among others. This computational holographic microscope requires a set of super-resolved holograms acquired at multiple sample-to-sensor distances, which are used as input to an iterative phase recovery algorithm and holographic reconstruction process, yielding high-resolution images of the samples in phase and amplitude channels. Here we demonstrate that in order to reconstruct clinically relevant images with high resolution and image contrast, we require less than 50% of the previously reported nominal number of holograms acquired at different sample-to-sensor distances. This is achieved by incorporating a loose sparsity constraint as part of the iterative holographic object reconstruction. We demonstrate the success of this sparsity-based computational lens-less microscopy platform by imaging pathology slides of breast cancer tissue and Papanicolaou (Pap) smears.

Transparent Nanopore Cavity Arrays Enable Highly Parallelized Optical Studies of Single Membrane Proteins on Chip.

PubMed

Diederichs, Tim; Nguyen, Quoc Hung; Urban, Michael; Tampé, Robert; Tornow, Marc

2018-06-13

Membrane proteins involved in transport processes are key targets for pharmaceutical research and industry. Despite continuous improvements and new developments in the field of electrical readouts for the analysis of transport kinetics, a well-suited methodology for high-throughput characterization of single transporters with nonionic substrates and slow turnover rates is still lacking. Here, we report on a novel architecture of silicon chips with embedded nanopore microcavities, based on a silicon-on-insulator technology for high-throughput optical readouts. Arrays containing more than 14 000 inverted-pyramidal cavities of 50 femtoliter volumes and 80 nm circular pore openings were constructed via high-resolution electron-beam lithography in combination with reactive ion etching and anisotropic wet etching. These cavities feature both, an optically transparent bottom and top cap. Atomic force microscopy analysis reveals an overall extremely smooth chip surface, particularly in the vicinity of the nanopores, which exhibits well-defined edges. Our unprecedented transparent chip design provides parallel and independent fluorescent readout of both cavities and buffer reservoir for unbiased single-transporter recordings. Spreading of large unilamellar vesicles with efficiencies up to 96% created nanopore-supported lipid bilayers, which are stable for more than 1 day. A high lipid mobility in the supported membrane was determined by fluorescent recovery after photobleaching. Flux kinetics of α-hemolysin were characterized at single-pore resolution with a rate constant of 0.96 ± 0.06 × 10 -3 s -1 . Here, we deliver an ideal chip platform for pharmaceutical research, which features high parallelism and throughput, synergistically combined with single-transporter resolution.

High-throughput methods for electron crystallography.

PubMed

Stokes, David L; Ubarretxena-Belandia, Iban; Gonen, Tamir; Engel, Andreas

2013-01-01

Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on titration of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described.

Highly Expandable Human iPS Cell-Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High-Throughput Screening.

PubMed

Cheng, Chialin; Fass, Daniel M; Folz-Donahue, Kat; MacDonald, Marcy E; Haggarty, Stephen J

2017-01-11

Reprogramming of human somatic cells into induced pluripotent stem (iPS) cells has greatly expanded the set of research tools available to investigate the molecular and cellular mechanisms underlying central nervous system (CNS) disorders. Realizing the promise of iPS cell technology for the identification of novel therapeutic targets and for high-throughput drug screening requires implementation of methods for the large-scale production of defined CNS cell types. Here we describe a protocol for generating stable, highly expandable, iPS cell-derived CNS neural progenitor cells (NPC) using multi-dimensional fluorescence activated cell sorting (FACS) to purify NPC defined by cell surface markers. In addition, we describe a rapid, efficient, and reproducible method for generating excitatory cortical-like neurons from these NPC through inducible expression of the pro-neural transcription factor Neurogenin 2 (iNgn2-NPC). Finally, we describe methodology for the use of iNgn2-NPC for probing human neuroplasticity and mechanisms underlying CNS disorders using high-content, single-cell-level automated microscopy assays. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Improvement of blood compatibility on polysulfone-polyvinylpyrrolidone blend films as a model membrane of dialyzer by physical adsorption of recombinant soluble human thrombomodulin (ART-123).

PubMed

Omichi, Masaaki; Matsusaki, Michiya; Maruyama, Ikuro; Akashi, Mitsuru

2012-01-01

ART-123 is a recombinant soluble human thrombomodulin (hTM) with potent anticoagulant activity, and is available for developing antithrombogenic surfaces by immobilization. We focused on improving blood compatibility on the dialyzer surface by the physical adsorption of ART-123 as a safe yet simple method without using chemical reagents. The physical adsorption mechanism and anticoagulant activities of adsorbed hTM on the surface of a polysulfone (PSF) membrane containing polyvinylpyrrolidone (PVP) as a model dialyzer were investigated in detail. The PVP content of the PSF-PVP films was saturated at 20 wt% after immersion in Tris-HCl buffer, even with the addition of over 20 wt% PVP. The surface morphology of the PSF-PVP films was strongly influenced by the PVP content, because PVP covered the outermost surface of the PSF-PVP films. The adsorption speed of hTM slowed dramatically with increasing PVP content up to 10 wt%, but the maximum adsorption amount of hTM onto the PSF-PVP film surface was almost the same, regardless of the PVP content. The PSF-PVP film with the physically adsorbed hTM showed higher protein C activity as compared to the PSF film, it showed excellent blood compatibility due to the protein C activity and the inhibition properties of platelet adhesion. The physical adsorption of hTM can be useful as a safe yet simple method to improve the blood compatibility of a dialyzer surface.

Three-dimensional nanoscale imaging by plasmonic Brownian microscopy

NASA Astrophysics Data System (ADS)

Labno, Anna; Gladden, Christopher; Kim, Jeongmin; Lu, Dylan; Yin, Xiaobo; Wang, Yuan; Liu, Zhaowei; Zhang, Xiang

2017-12-01

Three-dimensional (3D) imaging at the nanoscale is a key to understanding of nanomaterials and complex systems. While scanning probe microscopy (SPM) has been the workhorse of nanoscale metrology, its slow scanning speed by a single probe tip can limit the application of SPM to wide-field imaging of 3D complex nanostructures. Both electron microscopy and optical tomography allow 3D imaging, but are limited to the use in vacuum environment due to electron scattering and to optical resolution in micron scales, respectively. Here we demonstrate plasmonic Brownian microscopy (PBM) as a way to improve the imaging speed of SPM. Unlike photonic force microscopy where a single trapped particle is used for a serial scanning, PBM utilizes a massive number of plasmonic nanoparticles (NPs) under Brownian diffusion in solution to scan in parallel around the unlabeled sample object. The motion of NPs under an evanescent field is three-dimensionally localized to reconstruct the super-resolution topology of 3D dielectric objects. Our method allows high throughput imaging of complex 3D structures over a large field of view, even with internal structures such as cavities that cannot be accessed by conventional mechanical tips in SPM.

Lens-free computational imaging of capillary morphogenesis within three-dimensional substrates

NASA Astrophysics Data System (ADS)

Weidling, John; Isikman, Serhan O.; Greenbaum, Alon; Ozcan, Aydogan; Botvinick, Elliot

2012-12-01

Endothelial cells cultured in three-dimensional (3-D) extracellular matrices spontaneously form microvessels in response to soluble and matrix-bound factors. Such cultures are common for the study of angiogenesis and may find widespread use in drug discovery. Vascular networks are imaged over weeks to measure the distribution of vessel morphogenic parameters. Measurements require micron-scale spatial resolution, which for light microscopy comes at the cost of limited field-of-view (FOV) and shallow depth-of-focus (DOF). Small FOVs and DOFs necessitate lateral and axial mechanical scanning, thus limiting imaging throughput. We present a lens-free holographic on-chip microscopy technique to rapidly image microvessels within a Petri dish over a large volume without any mechanical scanning. This on-chip method uses partially coherent illumination and a CMOS sensor to record in-line holographic images of the sample. For digital reconstruction of the measured holograms, we implement a multiheight phase recovery method to obtain phase images of capillary morphogenesis over a large FOV (24 mm2) with ˜1.5 μm spatial resolution. On average, measured capillary length in our method was within approximately 2% of lengths measured using a 10× microscope objective. These results suggest lens-free on-chip imaging is a useful toolset for high-throughput monitoring and quantitative analysis of microvascular 3-D networks.

CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation

PubMed Central

2013-01-01

The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening. PMID:23938087

A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA.

PubMed

Diot, Alan; Hinks-Roberts, Alex; Lodge, Tiffany; Liao, Chunyan; Dombi, Eszter; Morten, Karl; Brady, Stefen; Fratter, Carl; Carver, Janet; Muir, Rebecca; Davis, Ryan; Green, Charlotte J; Johnston, Iain; Hilton-Jones, David; Sue, Carolyn; Mortiboys, Heather; Poulton, Joanna

2015-10-01

Mitophagy is a cellular mechanism for the recycling of mitochondrial fragments. This process is able to improve mitochondrial DNA (mtDNA) quality in heteroplasmic mtDNA disease, in which mutant mtDNA co-exists with normal mtDNA. In disorders where the load of mutant mtDNA determines disease severity it is likely to be an important determinant of disease progression. Measuring mitophagy is technically demanding. We used pharmacological modulators of autophagy to validate two techniques for quantifying mitophagy. First we used the IN Cell 1000 analyzer to quantify mitochondrial co-localisation with LC3-II positive autophagosomes. Unlike conventional fluorescence and electron microscopy, this high-throughput system is sufficiently sensitive to detect transient low frequency autophagosomes. Secondly, because mitophagy preferentially removes pathogenic heteroplasmic mtDNA mutants, we developed a heteroplasmy assay based on loss of m.3243A>G mtDNA, during culture conditions requiring oxidative metabolism ("energetic stress"). The effects of the pharmacological modulators on these two measures were consistent, confirming that the high throughput imaging output (autophagosomes co-localising with mitochondria) reflects mitochondrial quality control. To further validate these methods, we performed a more detailed study using metformin, the most commonly prescribed antidiabetic drug that is still sometimes used in Maternally Inherited Diabetes and Deafness (MIDD). This confirmed our initial findings and revealed that metformin inhibits mitophagy at clinically relevant concentrations, suggesting that it may have novel therapeutic uses. Copyright © 2015. Published by Elsevier Ltd.

CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation.

PubMed

Hodneland, Erlend; Kögel, Tanja; Frei, Dominik Michael; Gerdes, Hans-Hermann; Lundervold, Arvid

2013-08-09

: The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening.

High throughput microscopy identifies bisphenol AP, a bisphenol A analog, as a novel AR down-regulator.

PubMed

Stossi, Fabio; Dandekar, Radhika D; Bolt, Michael J; Newberg, Justin Y; Mancini, Maureen G; Kaushik, Akash K; Putluri, Vasanta; Sreekumar, Arun; Mancini, Michael A

2016-03-29

Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.

Mapping whole-brain activity with cellular resolution by light-sheet microscopy and high-throughput image analysis (Conference Presentation)

NASA Astrophysics Data System (ADS)

Silvestri, Ludovico; Rudinskiy, Nikita; Paciscopi, Marco; Müllenbroich, Marie Caroline; Costantini, Irene; Sacconi, Leonardo; Frasconi, Paolo; Hyman, Bradley T.; Pavone, Francesco S.

2016-03-01

Mapping neuronal activity patterns across the whole brain with cellular resolution is a challenging task for state-of-the-art imaging methods. Indeed, despite a number of technological efforts, quantitative cellular-resolution activation maps of the whole brain have not yet been obtained. Many techniques are limited by coarse resolution or by a narrow field of view. High-throughput imaging methods, such as light sheet microscopy, can be used to image large specimens with high resolution and in reasonable times. However, the bottleneck is then moved from image acquisition to image analysis, since many TeraBytes of data have to be processed to extract meaningful information. Here, we present a full experimental pipeline to quantify neuronal activity in the entire mouse brain with cellular resolution, based on a combination of genetics, optics and computer science. We used a transgenic mouse strain (Arc-dVenus mouse) in which neurons which have been active in the last hours before brain fixation are fluorescently labelled. Samples were cleared with CLARITY and imaged with a custom-made confocal light sheet microscope. To perform an automatic localization of fluorescent cells on the large images produced, we used a novel computational approach called semantic deconvolution. The combined approach presented here allows quantifying the amount of Arc-expressing neurons throughout the whole mouse brain. When applied to cohorts of mice subject to different stimuli and/or environmental conditions, this method helps finding correlations in activity between different neuronal populations, opening the possibility to infer a sort of brain-wide 'functional connectivity' with cellular resolution.

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

PubMed Central

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

2009-01-01

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

Layer-controllable graphene by plasma thinning and post-annealing

NASA Astrophysics Data System (ADS)

Zhang, Lufang; Feng, Shaopeng; Xiao, Shaoqing; Shen, Gang; Zhang, Xiumei; Nan, Haiyan; Gu, Xiaofeng; Ostrikov, Kostya (Ken)

2018-05-01

The electronic structure of graphene depends crucially on its layer number and therefore engineering the number of graphene's atomic stacking layers is of great importance for the preparation of graphene-based devices. In this paper, we demonstrated a relatively less invasive, high-throughput and uniform large-area plasma thinning of graphene based on direct bombardment effect of fast-moving ionic hydrogen or argon species. Any desired number of graphene layers including trilayer, bilayer and monolayer can be obtained. Structural changes of graphene layers are studied by optical microscopy, Raman spectroscopy and atomic force microscopy. Post annealing is adopted to self-heal the lattice defects induced by the ion bombardment effect. This plasma etching technique is efficient and compatible with semiconductor manufacturing processes, and may find important applications for graphene-based device fabrication.

76 FR 66311 - Draft Documents To Support Submission of an Electronic Common Technical Document; Availability

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-26

.../DevelopmentApprovalProcess/FormsSubmissionRequirements/ElectronicSubmissions/ucm253101.htm , http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm , or http...

Volume determination of irregularly-shaped quasi-spherical nanoparticles.

PubMed

Attota, Ravi Kiran; Liu, Eileen Cherry

2016-11-01

Nanoparticles (NPs) are widely used in diverse application areas, such as medicine, engineering, and cosmetics. The size (or volume) of NPs is one of the most important parameters for their successful application. It is relatively straightforward to determine the volume of regular NPs such as spheres and cubes from a one-dimensional or two-dimensional measurement. However, due to the three-dimensional nature of NPs, it is challenging to determine the proper physical size of many types of regularly and irregularly-shaped quasi-spherical NPs at high-throughput using a single tool. Here, we present a relatively simple method that determines a better volume estimate of NPs by combining measurements from their top-down projection areas and peak heights using two tools. The proposed method is significantly faster and more economical than the electron tomography method. We demonstrate the improved accuracy of the combined method over scanning electron microscopy (SEM) or atomic force microscopy (AFM) alone by using modeling, simulations, and measurements. This study also exposes the existence of inherent measurement biases for both SEM and AFM, which usually produce larger measured diameters with SEM than with AFM. However, in some cases SEM measured diameters appear to have less error compared to AFM measured diameters, especially for widely used IS-NPs such as of gold, and silver. The method provides a much needed, proper high-throughput volumetric measurement method useful for many applications. Graphical Abstract The combined method for volume determination of irregularly-shaped quasi-spherical nanoparticles.

Three-dimensional Imaging and Scanning: Current and Future Applications for Pathology

PubMed Central

Farahani, Navid; Braun, Alex; Jutt, Dylan; Huffman, Todd; Reder, Nick; Liu, Zheng; Yagi, Yukako; Pantanowitz, Liron

2017-01-01

Imaging is vital for the assessment of physiologic and phenotypic details. In the past, biomedical imaging was heavily reliant on analog, low-throughput methods, which would produce two-dimensional images. However, newer, digital, and high-throughput three-dimensional (3D) imaging methods, which rely on computer vision and computer graphics, are transforming the way biomedical professionals practice. 3D imaging has been useful in diagnostic, prognostic, and therapeutic decision-making for the medical and biomedical professions. Herein, we summarize current imaging methods that enable optimal 3D histopathologic reconstruction: Scanning, 3D scanning, and whole slide imaging. Briefly mentioned are emerging platforms, which combine robotics, sectioning, and imaging in their pursuit to digitize and automate the entire microscopy workflow. Finally, both current and emerging 3D imaging methods are discussed in relation to current and future applications within the context of pathology. PMID:28966836

All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide.

PubMed

Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

2017-02-10

While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.

All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

PubMed Central

Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

2017-01-01

While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab. PMID:28186188

All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

NASA Astrophysics Data System (ADS)

Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

2017-02-01

While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.

Cloning of the cDNA for a hematopoietic cell-specific protein related to CD20 and the {beta} subunit of the high-affinity IgE receptor: Evidence for a family of proteins with four membrane-spanning regions

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

Adra, C.N.; Morrison, P.; Lim, B.

1994-10-11

The authors report the cloning of the cDNA for a human gene whose mRNA is expressed specifically in hematopoietic cells. A long open reading frame in the 1.7-kb mRNA encodes a 214-aa protein of 25 kDa with four hydrophobic regions consistent with a protein that traverses the membrane four times. To reflect the structure and expression of this gene in diverse hematopoietic lineages of lymphoid and myeloid origin, the authors named the gene HTm{sub 4}. The protein is about 20% homologous to two other {open_quotes}four-transmembrane{close_quotes} proteins; the B-cell-specific antigen CD20 and the {beta} subunit of the high-affinity receptor for IgE,more » Fc{sub {epsilon}}RI{beta}. The highest homologies among the three proteins are found in the transmembrane domains, but conserved residues are also recognized in the inter-transmembrane domains and in the N and C termini. Using fluorescence in situ hybridization, they localized HTm{sub 4} to human chromosome 11q12-13.1, where the CD20 and Fc{sub {epsilon}}RI{beta} genes are also located. Both the murine homologue for CD20, Ly-44, and the murine Fc{sub {epsilon}}RI{beta} gene map to the same region in murine chromosome 19. The authors propose that the HTm{sub 4}, CD20, and Fc{sub {epsilon}}RI{beta} genes evolved from the same ancestral gene to form a family of four-transmembrane proteins. It is possible that other related members exist. Similar to CD20 and Fc{sub {epsilon}}RI{beta}, it is likely that Htm{sub 4} has a role in signal transduction and, like Fc{sub {epsilon}}RI{beta}, might be a subunit associated with receptor complexes.« less

High-throughput nanoparticle sizing using lensfree holographic microscopy and liquid nanolenses (Conference Presentation)

NASA Astrophysics Data System (ADS)

McLeod, Euan

2016-03-01

The sizing of individual nanoparticles and the recovery of the distributions of sizes from populations of nanoparticles provide valuable information in virology, exosome analysis, air and water quality monitoring, and nanomaterials synthesis. Conventional approaches for nanoparticle sizing include those based on costly or low-throughput laboratory-scale equipment such as transmission electron microscopy or nanoparticle tracking analysis, as well as those approaches that only provide population-averaged quantities, such as dynamic light scattering. Some of these limitations can be overcome using a new family of alternative approaches based on quantitative phase imaging that combines lensfree holographic on-chip microscopy with self-assembled liquid nanolenses. In these approaches, the particles of interest are deposited onto a glass coverslip and the sample is coated with either pure liquid polyethylene glycol (PEG) or aqueous solutions of PEG. Due to surface tension, the PEG self-assembles into nano-scale lenses around the particles of interest. These nanolenses enhance the scattering signatures of the embedded particles such that individual nanoparticles as small as 40 nm are clearly visible in phase images reconstructed from captured holograms. The magnitude of the phase quantitatively corresponds to particle size with an accuracy of +/-11 nm. This family of approaches can individually size more than 10^5 particles in parallel, can handle a large dynamic range of particle sizes (40 nm - 100s of microns), and can accurately size multi-modal distributions of particles. Furthermore, the entire approach has been implemented in a compact and cost-effective device suitable for use in the field or in low-resource settings.

Ultrafast Microfluidic Cellular Imaging by Optical Time-Stretch.

PubMed

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

2016-01-01

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

Holocene thermal maximum in the western Arctic (0-180°W)

USGS Publications Warehouse

Kaufman, D.S.; Ager, T.A.; Anderson, N.J.; Anderson, P.M.; Andrews, John T.; Bartlein, P.J.; Brubaker, L.B.; Coats, Larry L.; Cwynar, L.C.; Duvall, M.L.; Dyke, A.S.; Edwards, M.E.; Eisner, Wendy R.; Gajewski, K.; Geirsdottir, A.; Hu, F.-S.; Jennings, A.E.; Kaplan, M.R.; Kerwin, M.W.; Lozhkin, A.V.; MacDonald, G.M.; Miller, G.H.; Mock, Cary J.; Oswald, W.W.; Otto-Bliesner, B. L.; Porinchu, David F.; Ruhland, K.; Smol, J.P.; Steig, E.J.; Wolfe, B.B.

2004-01-01

The spatio-temporal pattern of peak Holocene warmth (Holocene thermal maximum, HTM) is traced over 140 sites across the Western Hemisphere of the Arctic (0-180??W; north of ???60??N). Paleoclimate inferences based on a wide variety of proxy indicators provide clear evidence for warmer-than-present conditions at 120 of these sites. At the 16 terrestrial sites where quantitative estimates have been obtained, local HTM temperatures (primarily summer estimates) were on average 1.6??0.8??C higher than present (approximate average of the 20th century), but the warming was time-transgressive across the western Arctic. As the precession-driven summer insolation anomaly peaked 12-10ka (thousands of calendar years ago), warming was concentrated in northwest North America, while cool conditions lingered in the northeast. Alaska and northwest Canada experienced the HTM between ca 11 and 9ka, about 4000yr prior to the HTM in northeast Canada. The delayed warming in Quebec and Labrador was linked to the residual Laurentide Ice Sheet, which chilled the region through its impact on surface energy balance and ocean circulation. The lingering ice also attests to the inherent asymmetry of atmospheric and oceanic circulation that predisposes the region to glaciation and modulates the pattern of climatic change. The spatial asymmetry of warming during the HTM resembles the pattern of warming observed in the Arctic over the last several decades. Although the two warmings are described at different temporal scales, and the HTM was additionally affected by the residual Laurentide ice, the similarities suggest there might be a preferred mode of variability in the atmospheric circulation that generates a recurrent pattern of warming under positive radiative forcing. Unlike the HTM, however, future warming will not be counterbalanced by the cooling effect of a residual North American ice sheet. ?? 2003 Elsevier Ltd. All rights reserved.

Holocene thermal maximum in the western Arctic (0-180°W)

NASA Astrophysics Data System (ADS)

Kaufman, D. S.; Ager, T. A.; Anderson, N. J.; Anderson, P. M.; Andrews, J. T.; Bartlein, P. J.; Brubaker, L. B.; Coats, L. L.; Cwynar, L. C.; Duvall, M. L.; Dyke, A. S.; Edwards, M. E.; Eisner, W. R.; Gajewski, K.; Geirsdóttir, A.; Hu, F. S.; Jennings, A. E.; Kaplan, M. R.; Kerwin, M. W.; Lozhkin, A. V.; MacDonald, G. M.; Miller, G. H.; Mock, C. J.; Oswald, W. W.; Otto-Bliesner, B. L.; Porinchu, D. F.; Rühland, K.; Smol, J. P.; Steig, E. J.; Wolfe, B. B.

2004-03-01

The spatio-temporal pattern of peak Holocene warmth (Holocene thermal maximum, HTM) is traced over 140 sites across the Western Hemisphere of the Arctic (0-180°W; north of ˜60°N). Paleoclimate inferences based on a wide variety of proxy indicators provide clear evidence for warmer-than-present conditions at 120 of these sites. At the 16 terrestrial sites where quantitative estimates have been obtained, local HTM temperatures (primarily summer estimates) were on average 1.6±0.8°C higher than present (approximate average of the 20th century), but the warming was time-transgressive across the western Arctic. As the precession-driven summer insolation anomaly peaked 12-10 ka (thousands of calendar years ago), warming was concentrated in northwest North America, while cool conditions lingered in the northeast. Alaska and northwest Canada experienced the HTM between ca 11 and 9 ka, about 4000 yr prior to the HTM in northeast Canada. The delayed warming in Quebec and Labrador was linked to the residual Laurentide Ice Sheet, which chilled the region through its impact on surface energy balance and ocean circulation. The lingering ice also attests to the inherent asymmetry of atmospheric and oceanic circulation that predisposes the region to glaciation and modulates the pattern of climatic change. The spatial asymmetry of warming during the HTM resembles the pattern of warming observed in the Arctic over the last several decades. Although the two warmings are described at different temporal scales, and the HTM was additionally affected by the residual Laurentide ice, the similarities suggest there might be a preferred mode of variability in the atmospheric circulation that generates a recurrent pattern of warming under positive radiative forcing. Unlike the HTM, however, future warming will not be counterbalanced by the cooling effect of a residual North American ice sheet.

Rigorous quantitative elemental microanalysis by scanning electron microscopy/energy dispersive x-ray spectrometry (SEM/EDS) with spectrum processing by NIST DTSA-II

NASA Astrophysics Data System (ADS)

Newbury, Dale E.; Ritchie, Nicholas W. M.

2014-09-01

Quantitative electron-excited x-ray microanalysis by scanning electron microscopy/silicon drift detector energy dispersive x-ray spectrometry (SEM/SDD-EDS) is capable of achieving high accuracy and high precision equivalent to that of the high spectral resolution wavelength dispersive x-ray spectrometer even when severe peak interference occurs. The throughput of the SDD-EDS enables high count spectra to be measured that are stable in calibration and resolution (peak shape) across the full deadtime range. With this high spectral stability, multiple linear least squares peak fitting is successful for separating overlapping peaks and spectral background. Careful specimen preparation is necessary to remove topography on unknowns and standards. The standards-based matrix correction procedure embedded in the NIST DTSA-II software engine returns quantitative results supported by a complete error budget, including estimates of the uncertainties from measurement statistics and from the physical basis of the matrix corrections. NIST DTSA-II is available free for Java-platforms at: http://www.cstl.nist.gov/div837/837.02/epq/dtsa2/index.html).

Solution-Processed Cu(In, Ga)(S, Se)2 Nanocrystal as Inorganic Hole-Transporting Material for Efficient and Stable Perovskite Solar Cells.

PubMed

Xu, Lu; Deng, Lin-Long; Cao, Jing; Wang, Xin; Chen, Wei-Yi; Jiang, Zhiyuan

2017-12-01

Perovskite solar cells are emerging as one of the most promising candidates for solar energy harvesting. To date, most of the high-performance perovskite solar cells have exclusively employed organic hole-transporting materials (HTMs) such as 2,2',7,7'-tetrakis-(N,N-di-p-methoxyphenylamine)-9,9'-spirobifluorene (spiro-OMeTAD) or polytriarylamine (PTAA) which are often expensive and have low hole mobility. Almost all these HTMs reported needed lithium salt, e.g., lithium bis(trifluoromethylsulfonyl)imide (Li-TFSI) doping, to improve hole mobility and performance. However, the use of Li-TFSI should be avoided because the hygroscopic nature of Li-TFSI could cause decomposition of perovskite and reduce device stability. Herein, we employed solution-processed CuIn 0.1 Ga 0.9 (S 0.9 Se 0.1 ) 2 (CIGSSe) nanocrystals as a novel inorganic HTM in perovskite solar cells. A power conversion efficiency of 9.15% was obtained for CIGSSe-based devices with improved stability, compared to devices using spiro-OMeTAD as HTM. This work offers a promising candidate of Cu-based inorganic HTM for efficient and stable perovskite solar cells.

Step-by-step guide to building an inexpensive 3D printed motorized positioning stage for automated high-content screening microscopy.

PubMed

Schneidereit, Dominik; Kraus, Larissa; Meier, Jochen C; Friedrich, Oliver; Gilbert, Daniel F

2017-06-15

High-content screening microscopy relies on automation infrastructure that is typically proprietary, non-customizable, costly and requires a high level of skill to use and maintain. The increasing availability of rapid prototyping technology makes it possible to quickly engineer alternatives to conventional automation infrastructure that are low-cost and user-friendly. Here, we describe a 3D printed inexpensive open source and scalable motorized positioning stage for automated high-content screening microscopy and provide detailed step-by-step instructions to re-building the device, including a comprehensive parts list, 3D design files in STEP (Standard for the Exchange of Product model data) and STL (Standard Tessellation Language) format, electronic circuits and wiring diagrams as well as software code. System assembly including 3D printing requires approx. 30h. The fully assembled device is light-weight (1.1kg), small (33×20×8cm) and extremely low-cost (approx. EUR 250). We describe positioning characteristics of the stage, including spatial resolution, accuracy and repeatability, compare imaging data generated with our device to data obtained using a commercially available microplate reader, demonstrate its suitability to high-content microscopy in 96-well high-throughput screening format and validate its applicability to automated functional Cl - - and Ca 2+ -imaging with recombinant HEK293 cells as a model system. A time-lapse video of the stage during operation and as part of a custom assembled screening robot can be found at https://vimeo.com/158813199. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography

PubMed Central

Beckwith, Marianne Sandvold; Beckwith, Kai Sandvold; Sikorski, Pawel; Skogaker, Nan Tostrup

2015-01-01

Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D. PMID:26406896

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

Moving an Expeditionary Force: Three Case Studies in Afghanistan

DTIC Science & Technology

2003-01-01

its individual parts.” 258 Stephen R. Covey, The 7 Habits of Highly Effective People (New York: Simon & Schuster, 1989), 186. 48 anywhere in the world...transcon.htm, accessed 26 January 2003]. Covey, Stephen R. The 7 Habits of Highly Effective People . New York: Simon & Schuster, 1989. Department of

Terrorism and Cybercrime

DTIC Science & Technology

2008-05-01

www.fbi.gov/congress/congress03/farnan051503.htm 63 Federal Bureau of Investigations. “Netting Cyber Criminals : Inside the Connecticut Computer Crimes Task...http://www.fbi.gov/cyberinvest/cyberhome.htm Federal Bureau of Investigations. “Netting Cyber Criminals : Inside the Connecticut Computer Crimes

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.

Efficient purification and concentration of viruses from a large body of high turbidity seawater.

PubMed

Sun, Guowei; Xiao, Jinzhou; Wang, Hongming; Gong, Chaowen; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

2014-01-01

Marine viruses are the most abundant entities in the ocean and play crucial roles in the marine ecological system. However, understanding of viral diversity on large scale depends on efficient and reliable viral purification and concentration techniques. Here, we report on developing an efficient method to purify and concentrate viruses from large body of high turbidity seawater. The developed method characterizes with high viral recovery efficiency, high concentration factor, high viral particle densities and high-throughput, and is reliable for viral concentration from high turbidity seawater. Recovered viral particles were used directly for subsequent analysis by epifluorescence microscopy, transmission electron microscopy and metagenomic sequencing. Three points are essential for this method:•The sampled seawater (>150 L) was initially divided into two parts, water fraction and settled matter fraction, after natural sedimentation.•Both viruses in the water fraction concentrated by tangential flow filtration (TFF) and viruses isolated from the settled matter fraction were considered as the whole viral community in high turbidity seawater.•The viral concentrates were re-concentrated by using centrifugal filter device in order to obtain high density of viral particles.

Machine learning and computer vision approaches for phenotypic profiling.

PubMed

Grys, Ben T; Lo, Dara S; Sahin, Nil; Kraus, Oren Z; Morris, Quaid; Boone, Charles; Andrews, Brenda J

2017-01-02

With recent advances in high-throughput, automated microscopy, there has been an increased demand for effective computational strategies to analyze large-scale, image-based data. To this end, computer vision approaches have been applied to cell segmentation and feature extraction, whereas machine-learning approaches have been developed to aid in phenotypic classification and clustering of data acquired from biological images. Here, we provide an overview of the commonly used computer vision and machine-learning methods for generating and categorizing phenotypic profiles, highlighting the general biological utility of each approach. © 2017 Grys et al.

Machine learning and computer vision approaches for phenotypic profiling

PubMed Central

Morris, Quaid

2017-01-01

With recent advances in high-throughput, automated microscopy, there has been an increased demand for effective computational strategies to analyze large-scale, image-based data. To this end, computer vision approaches have been applied to cell segmentation and feature extraction, whereas machine-learning approaches have been developed to aid in phenotypic classification and clustering of data acquired from biological images. Here, we provide an overview of the commonly used computer vision and machine-learning methods for generating and categorizing phenotypic profiles, highlighting the general biological utility of each approach. PMID:27940887

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

PubMed Central

McCarthy, Jason R.; Weissleder, Ralph

2007-01-01

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

Digital micromirror device-based laser-illumination Fourier ptychographic microscopy

PubMed Central

Kuang, Cuifang; Ma, Ye; Zhou, Renjie; Lee, Justin; Barbastathis, George; Dasari, Ramachandra R.; Yaqoob, Zahid; So, Peter T. C.

2015-01-01

We report a novel approach to Fourier ptychographic microscopy (FPM) by using a digital micromirror device (DMD) and a coherent laser source (532 nm) for generating spatially modulated sample illumination. Previously demonstrated FPM systems are all based on partially-coherent illumination, which offers limited throughput due to insufficient brightness. Our FPM employs a high power coherent laser source to enable shot-noise limited high-speed imaging. For the first time, a digital micromirror device (DMD), imaged onto the back focal plane of the illumination objective, is used to generate spatially modulated sample illumination field for ptychography. By coding the on/off states of the micromirrors, the illumination plane wave angle can be varied at speeds more than 4 kHz. A set of intensity images, resulting from different oblique illuminations, are used to numerically reconstruct one high-resolution image without obvious laser speckle. Experiments were conducted using a USAF resolution target and a fiber sample, demonstrating high-resolution imaging capability of our system. We envision that our approach, if combined with a coded-aperture compressive-sensing algorithm, will further improve the imaging speed in DMD-based FPM systems. PMID:26480361

Digital micromirror device-based laser-illumination Fourier ptychographic microscopy.

PubMed

Kuang, Cuifang; Ma, Ye; Zhou, Renjie; Lee, Justin; Barbastathis, George; Dasari, Ramachandra R; Yaqoob, Zahid; So, Peter T C

2015-10-19

We report a novel approach to Fourier ptychographic microscopy (FPM) by using a digital micromirror device (DMD) and a coherent laser source (532 nm) for generating spatially modulated sample illumination. Previously demonstrated FPM systems are all based on partially-coherent illumination, which offers limited throughput due to insufficient brightness. Our FPM employs a high power coherent laser source to enable shot-noise limited high-speed imaging. For the first time, a digital micromirror device (DMD), imaged onto the back focal plane of the illumination objective, is used to generate spatially modulated sample illumination field for ptychography. By coding the on/off states of the micromirrors, the illumination plane wave angle can be varied at speeds more than 4 kHz. A set of intensity images, resulting from different oblique illuminations, are used to numerically reconstruct one high-resolution image without obvious laser speckle. Experiments were conducted using a USAF resolution target and a fiber sample, demonstrating high-resolution imaging capability of our system. We envision that our approach, if combined with a coded-aperture compressive-sensing algorithm, will further improve the imaging speed in DMD-based FPM systems.

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

PubMed Central

Chiaraviglio, Lucius; Kang, Yoon-Suk; Kirby, James E.

2016-01-01

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila. PMID:27911388

Secretory glands and microvascular systems imaged in aqueous solution by atmospheric scanning electron microscopy (ASEM).

PubMed

Yamazawa, Toshiko; Nakamura, Naotoshi; Sato, Mari; Sato, Chikara

2016-12-01

Exocrine glands, e.g., salivary and pancreatic glands, play an important role in digestive enzyme secretion, while endocrine glands, e.g., pancreatic islets, secrete hormones that regulate blood glucose levels. The dysfunction of these secretory organs immediately leads to various diseases, such as diabetes or Sjögren's syndrome, by poorly understood mechanisms. Gland-related diseases have been studied by optical microscopy (OM), and at higher resolution by transmission electron microscopy (TEM) of Epon embedded samples, which necessitates hydrophobic sample pretreatment. Here, we report the direct observation of tissue in aqueous solution by atmospheric scanning electron microscopy (ASEM). Salivary glands, lacrimal glands, and pancreas were fixed, sectioned into slabs, stained with phosphotungstic acid (PTA), and inspected in radical scavenger d-glucose solution from below by an inverted scanning electron microscopy (SEM), guided by optical microscopy from above to target the tissue substructures. A 2- to 3-µm specimen thickness was visualized by the SEM. In secretory cells, cytoplasmic vesicles and other organelles were clearly imaged at high resolution, and the former could be classified according to the degree of PTA staining. In islets of Langerhans, the microvascular system used as an outlet by the secretory cells was also clearly observed. Microvascular system is also critically involved in the onset of diabetic complications and was clearly visible in subcutaneous tissue imaged by ASEM. The results suggest the use of in-solution ASEM for histology and to study vesicle secretion systems. Further, the high-throughput of ASEM makes it a potential tool for the diagnosis of exocrine and endocrine-related diseases. © 2016 Wiley Periodicals, Inc.

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.

Partially coherent lensfree tomographic microscopy⋄

PubMed Central

Isikman, Serhan O.; Bishara, Waheb; Ozcan, Aydogan

2012-01-01

Optical sectioning of biological specimens provides detailed volumetric information regarding their internal structure. To provide a complementary approach to existing three-dimensional (3D) microscopy modalities, we have recently demonstrated lensfree optical tomography that offers high-throughput imaging within a compact and simple platform. In this approach, in-line holograms of objects at different angles of partially coherent illumination are recorded using a digital sensor-array, which enables computing pixel super-resolved tomographic images of the specimen. This imaging modality, which forms the focus of this review, offers micrometer-scale 3D resolution over large imaging volumes of, for example, 10–15 mm3, and can be assembled in light weight and compact architectures. Therefore, lensfree optical tomography might be particularly useful for lab-on-a-chip applications as well as for microscopy needs in resource-limited settings. PMID:22193016

Niacin to Boost Your HDL "Good" Cholesterol

MedlinePlus

... the AIM-HIGH trial. U.S. Food and Drug Administration. http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/ucm256841.htm. Accessed May 27, 2011. May 16, 2018 Original article: http://www.mayoclinic.org/diseases-conditions/ ...

Strategy to Boost the Efficiency of Mixed-Ion Perovskite Solar Cells: Changing Geometry of the Hole Transporting Material.

PubMed

Zhang, Jinbao; Xu, Bo; Johansson, Malin B; Vlachopoulos, Nick; Boschloo, Gerrit; Sun, Licheng; Johansson, Erik M J; Hagfeldt, Anders

2016-07-26

The hole transporting material (HTM) is an essential component in perovskite solar cells (PSCs) for efficient extraction and collection of the photoinduced charges. Triphenylamine- and carbazole-based derivatives have extensively been explored as alternative and economical HTMs for PSCs. However, the improvement of their power conversion efficiency (PCE), as well as further investigation of the relationship between the chemical structure of the HTMs and the photovoltaic performance, is imperatively needed. In this respect, a simple carbazole-based HTM X25 was designed on the basis of a reference HTM, triphenylamine-based X2, by simply linking two neighboring phenyl groups in a triphenylamine unit through a carbon-carbon single bond. It was found that a lowered highest occupied molecular orbital (HOMO) energy level was obtained for X25 compared to that of X2. Besides, the carbazole moiety in X25 improved the molecular planarity as well as conductivity property in comparison with the triphenylamine unit in X2. Utilizing the HTM X25 in a solar cell with mixed-ion perovskite [HC(NH2)2]0.85(CH3NH3)0.15Pb(I0.85Br0.15)3, a highest reported PCE of 17.4% at 1 sun (18.9% under 0.46 sun) for carbazole-based HTM in PSCs was achieved, in comparison of a PCE of 14.7% for triphenylamine-based HTM X2. From the steady-state photoluminescence and transient photocurrent/photovoltage measurements, we conclude that (1) the lowered HOMO level for X25 compared to X2 favored a higher open-circuit voltage (Voc) in PSCs; (2) a more uniform formation of X25 capping layer than X2 on the surface of perovskite resulted in more efficient hole transport and charge extraction in the devices. In addition, the long-term stability of PSCs with X25 is significantly enhanced compared to X2 due to its good uniformity of HTM layer and thus complete coverage on the perovskite. The results provide important information to further develop simple and efficient small molecular HTMs applied in solar cells.

Harnessing cell-to-cell variations to probe bacterial structure and biophysics

NASA Astrophysics Data System (ADS)

Cass, Julie A.

Advances in microscopy and biotechnology have given us novel insights into cellular biology and physics. While bacteria were long considered to be relatively unstructured, the development of fluorescence microscopy techniques, and spatially and temporally resolved high-throughput quantitative studies, have uncovered that the bacterial cell is highly organized, and its structure rigorously maintained. In this thesis I will describe our gateTool software, designed to harness cell-to-cell variations to probe bacterial structure, and discuss two exciting aspects of structure that we have employed gateTool to investigate: (i) chromosome organization and the cellular mechanisms for controlling DNA dynamics, and (ii) the study of cell wall synthesis, and how the genes in the synthesis pathway impact cellular shape. In the first project, we develop a spatial and temporal mapping of cell-cycle-dependent chromosomal organization, and use this quantitative map to discover that chromosomal loci segregate from midcell with universal dynamics. In the second project, I describe preliminary time- lapse and snapshot imaging analysis suggesting phentoypical coherence across peptidoglycan synthesis pathways.

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

NASA Astrophysics Data System (ADS)

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

2016-02-01

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

Automated profiling of individual cell-cell interactions from high-throughput time-lapse imaging microscopy in nanowell grids (TIMING).

PubMed

Merouane, Amine; Rey-Villamizar, Nicolas; Lu, Yanbin; Liadi, Ivan; Romain, Gabrielle; Lu, Jennifer; Singh, Harjeet; Cooper, Laurence J N; Varadarajan, Navin; Roysam, Badrinath

2015-10-01

There is a need for effective automated methods for profiling dynamic cell-cell interactions with single-cell resolution from high-throughput time-lapse imaging data, especially, the interactions between immune effector cells and tumor cells in adoptive immunotherapy. Fluorescently labeled human T cells, natural killer cells (NK), and various target cells (NALM6, K562, EL4) were co-incubated on polydimethylsiloxane arrays of sub-nanoliter wells (nanowells), and imaged using multi-channel time-lapse microscopy. The proposed cell segmentation and tracking algorithms account for cell variability and exploit the nanowell confinement property to increase the yield of correctly analyzed nanowells from 45% (existing algorithms) to 98% for wells containing one effector and a single target, enabling automated quantification of cell locations, morphologies, movements, interactions, and deaths without the need for manual proofreading. Automated analysis of recordings from 12 different experiments demonstrated automated nanowell delineation accuracy >99%, automated cell segmentation accuracy >95%, and automated cell tracking accuracy of 90%, with default parameters, despite variations in illumination, staining, imaging noise, cell morphology, and cell clustering. An example analysis revealed that NK cells efficiently discriminate between live and dead targets by altering the duration of conjugation. The data also demonstrated that cytotoxic cells display higher motility than non-killers, both before and during contact. broysam@central.uh.edu or nvaradar@central.uh.edu 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.

High-throughput multipesticides residue analysis in earthworms by the improvement of purification method: Development and application of magnetic Fe3 O4 -SiO2 nanoparticles based dispersive solid-phase extraction.

PubMed

Sun, Yuhan; Qi, Peipei; Cang, Tao; Wang, Zhiwei; Wang, Xiangyun; Yang, Xuewei; Wang, Lidong; Xu, Xiahong; Wang, Qiang; Wang, Xinquan; Zhao, Changshan

2018-06-01

As a key representative organism, earthworms can directly illustrate the influence of pesticides on environmental organisms in soil ecosystems. The present work aimed to develop a high-throughput multipesticides residue analytical method for earthworms using solid-liquid extraction with acetonitrile as the solvent and magnetic material-based dispersive solid-phase extraction for purification. Magnetic Fe 3 O 4 nanoparticles were modified with a thin silica layer to form Fe 3 O 4 -SiO 2 nanoparticles, which were fully characterized by field-emission scanning electron microscopy, transmission electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffractometry, and vibrating sample magnetometry. The Fe 3 O 4 -SiO 2 nanoparticles were used as the separation media in dispersive solid-phase extraction with primary secondary amine and ZrO 2 as the cleanup adsorbents to eliminate matrix interferences. The amounts of nanoparticles and adsorbents were optimized for the simultaneous determination of 44 pesticides and six metabolites in earthworms by liquid chromatography with tandem mass spectrometry. The method performance was systematically validated with satisfactory results. The limits of quantification were 20 μg/kg for all analytes studied, while the recoveries of the target analytes ranged from 65.1 to 127% with relative standard deviation values lower than 15.0%. The developed method was subsequently utilized to explore the bioaccumulation of bitertanol in earthworms exposed to contaminated soil, verifying its feasibility for real sample analysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

30 CFR 1210.56 - Where can I find more information on how to complete the royalty report?

Code of Federal Regulations, 2011 CFR

2011-07-01

... http://www.mrm.boemre.gov/ReportingServices/Handbooks/Handbks.htm or from ONRR at P.O. Box 5760, Denver... on our Internet Web site at http://www.mrm.boemre.gov/ReportingServices/Forms/AFSOil_Gas.htm, or you...

76 FR 16795 - The National Antimicrobial Resistance Monitoring System Strategic Plan 2011-2015; Request for...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-03-25

....fda.gov/AnimalVeterinary/SafetyHealth/AntimicrobialResistance/NationalAntimicrobialResistanceMonitoringSystem/default.htm , http://www.fda.gov/AnimalVeterinary/SafetyHealth/AntimicrobialResistance/NationalAntimicrobialResistanceMonitoringSystem/ucm062630.htm , http://www.fda.gov/AnimalVeterinary/Safety...

77 FR 58560 - Proposed Flood Hazard Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2012-09-21

... resolution process. SRPs are independent panels of experts in hydrology, hydraulics, and other pertinent.../dnr/water/6497.htm City of Tipton Tipton County Courthouse, 101 East Jefferson Street, Tipton, IN... Incorporated Areas Maps Available for Inspection Online at: http://www.in.gov/dnr/water/6648.htm City of...

Effects of hydroxy trace minerals on oxidative metabolism, cytological endometritis, and performance of transition dairy cows.

PubMed

Yasui, T; Ryan, C M; Gilbert, R O; Perryman, K R; Overton, T R

2014-01-01

Multiparous Holstein cows (n=60) were used to determine effects of supplementing hydroxy forms of Zn, Cu, and Mn compared with 2 other common supplementation strategies on oxidative metabolism, cytological endometritis, and performance of transition cows. After a 1-wk pretreatment period, cows were assigned randomly to 1 of 3 dietary treatments from 21 d before expected calving through 84 d postcalving. Dietary treatments administered by daily top-dressing included (1) inorganic sulfate forms of Zn, Cu, and Mn (ITM); (2) a blend (75:25) of sulfates and organic complexes of Zn, Cu, and Mn (ITM/OTM); and (3) hydroxy trace minerals (HTM) of Zn, Cu, and Mn. The resulting dietary concentrations of supplemental Zn, Cu, and Mn were similar among treatments and averaged 40, 10, and 27 mg/kg, respectively, before calving and 59, 15, and 40 mg/kg, respectively, after calving. Total concentrations of Zn, Cu, and Mn averaged 80, 16, and 62 mg/kg during the prepartum period and 102, 23, and 75 mg/kg, respectively, during the postpartum period. Overall, effects of treatment on milk yield and milk composition were not significant. Cows fed HTM during the prepartum period had higher body weight (BW) than those fed ITM during the prepartum period and had higher BW during the postpartum period than those fed the other treatments; however, BW change, body condition score, and body condition score change were not affected by treatment. Plasma total antioxidant capacity was lower in cows fed HTM than ITM but was not different from cows fed ITM/OTM. Cows fed HTM tended to have lower concentrations of plasma thiobarbituric acid reactive substances than those fed ITM during the whole study period, but plasma thiobarbituric acid reactive substances were not different between HTM and ITM/OTM. Plasma haptoglobin was lower in cows fed HTM than ITM/OTM at 1 wk postpartum. Endometrial cytology 7d postcalving and cytological endometritis as assessed on 1d between 40 and 60 d postcalving was not affected by treatment. In conclusion, supplementation with HTM sources of Zn, Cu, and Mn modulated plasma variables related to oxidative metabolism compared with supplementation with ITM; however, HTM and ITM/OTM resulted in similar responses. Furthermore, the source of trace minerals did not affect performance or uterine health in this experiment. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Association of Sleep Groups and Sleep Survey Results of High School Students

ERIC Educational Resources Information Center

Anderson, Stephen A.; MacDonald, Lawrence; Frost, Frederica C.

2006-01-01

In January 2003 the High School Late Start Committee of the Northville Public Schools sent surveys to all families, faculty, and middle and high school students. The descriptive results of this survey can be found at www.northville.k12.mi.us/hr/late_start_committee_surveysummary.htm. This study focuses on the responses of the high school students,…

Soy

MedlinePlus

... Soy URL of this page: //medlineplus.gov/ency/article/007204.htm Soy To use the sharing features on this page, please enable JavaScript. Humans have been eating soy beans for almost 5000 years. The soybean is high in protein. The quality of protein from soy equals that of protein ...

High-throughput isotropic mapping of whole mouse brain using multi-view light-sheet microscopy

NASA Astrophysics Data System (ADS)

Nie, Jun; Li, Yusha; Zhao, Fang; Ping, Junyu; Liu, Sa; Yu, Tingting; Zhu, Dan; Fei, Peng

2018-02-01

Light-sheet fluorescence microscopy (LSFM) uses an additional laser-sheet to illuminate selective planes of the sample, thereby enabling three-dimensional imaging at high spatial-temporal resolution. These advantages make LSFM a promising tool for high-quality brain visualization. However, even by the use of LSFM, the spatial resolution remains insufficient to resolve the neural structures across a mesoscale whole mouse brain in three dimensions. At the same time, the thick-tissue scattering prevents a clear observation from the deep of brain. Here we use multi-view LSFM strategy to solve this challenge, surpassing the resolution limit of standard light-sheet microscope under a large field-of-view (FOV). As demonstrated by the imaging of optically-cleared mouse brain labelled with thy1-GFP, we achieve a brain-wide, isotropic cellular resolution of 3μm. Besides the resolution enhancement, multi-view braining imaging can also recover complete signals from deep tissue scattering and attenuation. The identification of long distance neural projections across encephalic regions can be identified and annotated as a result.

Time-Gated Orthogonal Scanning Automated Microscopy (OSAM) for High-speed Cell Detection and Analysis

NASA Astrophysics Data System (ADS)

Lu, Yiqing; Xi, Peng; Piper, James A.; Huo, Yujing; Jin, Dayong

2012-11-01

We report a new development of orthogonal scanning automated microscopy (OSAM) incorporating time-gated detection to locate rare-event organisms regardless of autofluorescent background. The necessity of using long-lifetime (hundreds of microseconds) luminescent biolabels for time-gated detection implies long integration (dwell) time, resulting in slow scan speed. However, here we achieve high scan speed using a new 2-step orthogonal scanning strategy to realise on-the-fly time-gated detection and precise location of 1-μm lanthanide-doped microspheres with signal-to-background ratio of 8.9. This enables analysis of a 15 mm × 15 mm slide area in only 3.3 minutes. We demonstrate that detection of only a few hundred photoelectrons within 100 μs is sufficient to distinguish a target event in a prototype system using ultraviolet LED excitation. Cytometric analysis of lanthanide labelled Giardia cysts achieved a signal-to-background ratio of two orders of magnitude. Results suggest that time-gated OSAM represents a new opportunity for high-throughput background-free biosensing applications.

Horse Training and Management: Program of Excellence.

ERIC Educational Resources Information Center

Lane, Marvin

This report on Lamar Community College's Horse Training and Management (HTM) program assesses the quality of the educational experience provided by the program, the quality of the faculty and students, institutional financial commitment to the program, contribution of the HTM program to state and local economic development, and external funding…

78 FR 14565 - Changes in Flood Hazard Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-06

..., Building and team.com/lomrs.htm. County (12-03- Chairman at Development 1200P). Large, Loudoun Department... D. Public Works, team.com/lomrs.htm. 2078P). Sessoms, Jr., Municipal Center, Mayor, City of Building... flood insurance premium rates for new buildings and the contents of those buildings. For rating purposes...

78 FR 48885 - Changes in Flood Hazard Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-12

...- August 12, 2013...... 420713 Whitpain (12-03- Joseph J. Palmer, Building, 960 team.com/lomrs.htm. 1849P... team.com/lomrs.htm. City of Sachse, Department, 5560 3815 Sachse Road, Highway 78, Building B, Sachse... flood insurance premium rates for new buildings and the contents of those buildings. For rating purposes...

78 FR 18233 - Medical Devices; Technical Amendment

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-26

... the right column: Section Remove Add 814.20 http://www.fda.gov/ http://www.fda.gov/ cdrh/devadvice.../ PremarketApproval PMA/default.htm. 822.7 http://www.fda.gov/ http://www.fda.gov/ cdrh/ombudsman/ AboutFDA/ dispute.html. CentersOffices/ OfficeofMedicalPr oductsandTobacco/ CDRH/ CDRHOmbudsman/ default.htm. 822.15...

77 FR 74859 - Proposed Flood Hazard Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-18

... resolution process. SRPs are independent panels of experts in hydrology, hydraulics, and other pertinent...://www.in.gov/dnr/water/6401.htm City of Delphi Carroll County Area Plan Commission, Carroll County... Inspection Online at: http://www.in.gov/dnr/water/7339.htm Town of Cumberland Municipal Building, 11501 East...

78 FR 28888 - Proposed Flood Hazard Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2013-05-16

... resolution process. SRPs are independent panels of experts in hydrology, hydraulics, and other pertinent.../dnr/water/6670.htm City of Oakland City City Hall, 210 East Washington Street, Oakland City, IN 47660... Areas Maps Available for Inspection Online at: http://www.in.gov/dnr/water/6472.htm City of Martinsville...

78 FR 8181 - Proposed Flood Hazard Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-05

... panels of experts in hydrology, hydraulics, and other pertinent sciences established to review... Inspection Online at: http://www.in.gov/dnr/water/6476.htm City of Hartford City Blackford County Area... Online at: http://www.in.gov/dnr/water/5775.htm City of Carmel Department of Community Services, One...

77 FR 67016 - Proposed Flood Hazard Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-08

... resolution process. SRPs are independent panels of experts in hydrology, hydraulics, and other pertinent... at: http://www.in.gov/dnr/water/6644.htm Town of Brookville Franklin County Government Center, Area...://www.in.gov/dnr/water/6415.htm Town of Liberty Union County Area Plan Commission, 6 West South Street...

78 FR 310 - Draft Revision of Guidance for Industry on Providing Regulatory Submissions in Electronic Format...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-03

...ApprovalProcess/FormsSubmissionRequirements/ElectronicSubmissions/ucm253101.htm , http://www.regulations.../Drugs/DevelopmentApprovalProcess/FormsSubmissionRequirements/ElectronicSubmissions/ucm253101.htm , http...), in a format that FDA can process, review, and archive. Currently, the Agency can process, review, and...

Diagnosis of breast cancer biopsies using quantitative phase imaging

NASA Astrophysics Data System (ADS)

Majeed, Hassaan; Kandel, Mikhail E.; Han, Kevin; Luo, Zelun; Macias, Virgilia; Tangella, Krishnarao; Balla, Andre; Popescu, Gabriel

2015-03-01

The standard practice in the histopathology of breast cancers is to examine a hematoxylin and eosin (H&E) stained tissue biopsy under a microscope. The pathologist looks at certain morphological features, visible under the stain, to diagnose whether a tumor is benign or malignant. This determination is made based on qualitative inspection making it subject to investigator bias. Furthermore, since this method requires a microscopic examination by the pathologist it suffers from low throughput. A quantitative, label-free and high throughput method for detection of these morphological features from images of tissue biopsies is, hence, highly desirable as it would assist the pathologist in making a quicker and more accurate diagnosis of cancers. We present here preliminary results showing the potential of using quantitative phase imaging for breast cancer screening and help with differential diagnosis. We generated optical path length maps of unstained breast tissue biopsies using Spatial Light Interference Microscopy (SLIM). As a first step towards diagnosis based on quantitative phase imaging, we carried out a qualitative evaluation of the imaging resolution and contrast of our label-free phase images. These images were shown to two pathologists who marked the tumors present in tissue as either benign or malignant. This diagnosis was then compared against the diagnosis of the two pathologists on H&E stained tissue images and the number of agreements were counted. In our experiment, the agreement between SLIM and H&E based diagnosis was measured to be 88%. Our preliminary results demonstrate the potential and promise of SLIM for a push in the future towards quantitative, label-free and high throughput diagnosis.

Vision-based Nano Robotic System for High-throughput Non-embedded Cell Cutting

NASA Astrophysics Data System (ADS)

Shang, Wanfeng; Lu, Haojian; Wan, Wenfeng; Fukuda, Toshio; Shen, Yajing

2016-03-01

Cell cutting is a significant task in biology study, but the highly productive non-embedded cell cutting is still a big challenge for current techniques. This paper proposes a vision-based nano robotic system and then realizes automatic non-embedded cell cutting with this system. First, the nano robotic system is developed and integrated with a nanoknife inside an environmental scanning electron microscopy (ESEM). Then, the positions of the nanoknife and the single cell are recognized, and the distance between them is calculated dynamically based on image processing. To guarantee the positioning accuracy and the working efficiency, we propose a distance-regulated speed adapting strategy, in which the moving speed is adjusted intelligently based on the distance between the nanoknife and the target cell. The results indicate that the automatic non-embedded cutting is able to be achieved within 1-2 mins with low invasion benefiting from the high precise nanorobot system and the sharp edge of nanoknife. This research paves a way for the high-throughput cell cutting at cell’s natural condition, which is expected to make significant impact on the biology studies, especially for the in-situ analysis at cellular and subcellular scale, such as cell interaction investigation, neural signal transduction and low invasive cell surgery.

High-throughput, high-resolution X-ray phase contrast tomographic microscopy for visualisation of soft tissue

NASA Astrophysics Data System (ADS)

McDonald, S. A.; Marone, F.; Hintermüller, C.; Bensadoun, J.-C.; Aebischer, P.; Stampanoni, M.

2009-09-01

The use of conventional absorption based X-ray microtomography can become limited for samples showing only very weak absorption contrast. However, a wide range of samples studied in biology and materials science can produce significant phase shifts of the X-ray beam, and thus the use of the phase signal can provide substantially increased contrast and therefore new and otherwise inaccessible information. The application of two approaches for high-throughput, high-resolution X-ray phase contrast tomography, both available on the TOMCAT beamline of the SLS, is illustrated. Differential Phase Contrast (DPC) imaging uses a grating interferometer and a phase-stepping technique. It has been integrated into the beamline environment on TOMCAT in terms of the fast acquisition and reconstruction of data and the availability to scan samples within an aqueous environment. The second phase contrast approach is a modified transfer of intensity approach that can yield the 3D distribution of the phase (refractive index) of a weakly absorbing object from a single tomographic dataset. These methods are being used for the evaluation of cell integrity in 3D, with the specific aim of following and analyzing progressive cell degeneration to increase knowledge of the mechanistic events of neurodegenerative disorders such as Parkinson's disease.

Brain Connectivity as a DNA Sequencing Problem

NASA Astrophysics Data System (ADS)

Zador, Anthony

The mammalian cortex consists of millions or billions of neurons, each connected to thousands of other neurons. Traditional methods for determining the brain connectivity rely on microscopy to visualize neuronal connections, but such methods are slow, labor-intensive and often lack single neuron resolution. We have recently developed a new method, MAPseq, to recast the determination of brain wiring into a form that can exploit the tremendous recent advances in high-throughput DNA sequencing. DNA sequencing technology has outpaced even Moore's law, so that the cost of sequencing the human genome has dropped from a billion dollars in 2001 to below a thousand dollars today. MAPseq works by introducing random sequences of DNA-``barcodes''-to tag neurons uniquely. With MAPseq, we can determine the connectivity of over 50K single neurons in a single mouse cortex in about a week, an unprecedented throughput, ushering in the era of ``big data'' for brain wiring. We are now developing analytical tools and algorithms to make sense of these novel data sets.

Fluorescence lifetime microscopy with a time- and space-resolved single-photon counting detector

PubMed Central

Michalet, X.; Siegmund, O.H.W.; Vallerga, J.V.; Jelinsky, P.; Pinaud, F. F.; Millaud, J.E.; Weiss, S.

2017-01-01

We have recently developed a wide-field photon-counting detector (the H33D detector) having high-temporal and high-spatial resolutions and capable of recording up to 500,000 photons per sec. Its temporal performance has been previously characterized using solutions of fluorescent materials with different lifetimes, and its spatial resolution using sub-diffraction objects (beads and quantum dots). Here we show its application to fluorescence lifetime imaging of live cells and compare its performance to a scanning confocal TCSPC approach. With the expected improvements in photocathode sensitivity and increase in detector throughput, this technology appears as a promising alternative to the current lifetime imaging solutions. PMID:29449756

Out-of-focal plane imaging by leakage radiation microscopy

NASA Astrophysics Data System (ADS)

Zhu, Liangfu; Zhang, Douguo; Wang, Ruxue; Wen, Xiaolei; Wang, Pei; Ming, Hai; Badugu, Ramachandram; Lakowicz, Joseph R.

2017-09-01

Leakage radiation microscopy (LRM) is used to investigate the optical properties of surfaces. The front-focal plane (FFP) image with LRM reveals the structural features on the surfaces. A back-focal plane (BFP) image with LRM reveals the angular distribution of the radiation. Herein, we experimentally demonstrate that the out-of-focal plane (OFP) images present a link between the FFP and BFP images and provide optical information that cannot be resolved by either FFP or BFP images. The OFP image provides a link between the spatial location of the emission and the angular distribution from the same location, and thus information about the film’s discontinuity, nonuniformity or variable thickness can be uncovered. The use of OFP imaging will extend the scope and applications of the LRM and coupled emission imaging, which are powerful tools in nanophotonics and high throughput fluorescence screening.

Analyzing Carbohydrate-Protein Interaction Based on Single Plasmonic Nanoparticle by Conventional Dark Field Microscopy.

PubMed

Jin, Hong-Ying; Li, Da-Wei; Zhang, Na; Gu, Zhen; Long, Yi-Tao

2015-06-10

We demonstrated a practical method to analyze carbohydrate-protein interaction based on single plasmonic nanoparticles by conventional dark field microscopy (DFM). Protein concanavalin A (ConA) was modified on large sized gold nanoparticles (AuNPs), and dextran was conjugated on small sized AuNPs. As the interaction between ConA and dextran resulted in two kinds of gold nanoparticles coupled together, which caused coupling of plasmonic oscillations, apparent color changes (from green to yellow) of the single AuNPs were observed through DFM. Then, the color information was instantly transformed into a statistic peak wavelength distribution in less than 1 min by a self-developed statistical program (nanoparticleAnalysis). In addition, the interaction between ConA and dextran was proved with biospecific recognition. This approach is high-throughput and real-time, and is a convenient method to analyze carbohydrate-protein interaction at the single nanoparticle level efficiently.

Photon-counting-based diffraction phase microscopy combined with single-pixel imaging

NASA Astrophysics Data System (ADS)

Shibuya, Kyuki; Araki, Hiroyuki; Iwata, Tetsuo

2018-04-01

We propose a photon-counting (PC)-based quantitative-phase imaging (QPI) method for use in diffraction phase microscopy (DPM) that is combined with a single-pixel imaging (SPI) scheme (PC-SPI-DPM). This combination of DPM with the SPI scheme overcomes a low optical throughput problem that has occasionally prevented us from obtaining quantitative-phase images in DPM through use of a high-sensitivity single-channel photodetector such as a photomultiplier tube (PMT). The introduction of a PMT allowed us to perform PC with ease and thus solved a dynamic range problem that was inherent to SPI. As a proof-of-principle experiment, we performed a comparison study of analogue-based SPI-DPM and PC-SPI-DPM for a 125-nm-thick indium tin oxide (ITO) layer coated on a silica glass substrate. We discuss the basic performance of the method and potential future modifications of the proposed system.

Rapid, High-Throughput Tracking of Bacterial Motility in 3D via Phase-Contrast Holographic Video Microscopy

PubMed Central

Cheong, Fook Chiong; Wong, Chui Ching; Gao, YunFeng; Nai, Mui Hoon; Cui, Yidan; Park, Sungsu; Kenney, Linda J.; Lim, Chwee Teck

2015-01-01

Tracking fast-swimming bacteria in three dimensions can be extremely challenging with current optical techniques and a microscopic approach that can rapidly acquire volumetric information is required. Here, we introduce phase-contrast holographic video microscopy as a solution for the simultaneous tracking of multiple fast moving cells in three dimensions. This technique uses interference patterns formed between the scattered and the incident field to infer the three-dimensional (3D) position and size of bacteria. Using this optical approach, motility dynamics of multiple bacteria in three dimensions, such as speed and turn angles, can be obtained within minutes. We demonstrated the feasibility of this method by effectively tracking multiple bacteria species, including Escherichia coli, Agrobacterium tumefaciens, and Pseudomonas aeruginosa. In addition, we combined our fast 3D imaging technique with a microfluidic device to present an example of a drug/chemical assay to study effects on bacterial motility. PMID:25762336

Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

PubMed

Bunyak, Filiz; Palaniappan, Kannappan; Chagin, Vadim; Cardoso, M

2009-01-01

Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.

Quantitative Image Restoration in Bright Field Optical Microscopy.

PubMed

Gutiérrez-Medina, Braulio; Sánchez Miranda, Manuel de Jesús

2017-11-07

Bright field (BF) optical microscopy is regarded as a poor method to observe unstained biological samples due to intrinsic low image contrast. We introduce quantitative image restoration in bright field (QRBF), a digital image processing method that restores out-of-focus BF images of unstained cells. Our procedure is based on deconvolution, using a point spread function modeled from theory. By comparing with reference images of bacteria observed in fluorescence, we show that QRBF faithfully recovers shape and enables quantify size of individual cells, even from a single input image. We applied QRBF in a high-throughput image cytometer to assess shape changes in Escherichia coli during hyperosmotic shock, finding size heterogeneity. We demonstrate that QRBF is also applicable to eukaryotic cells (yeast). Altogether, digital restoration emerges as a straightforward alternative to methods designed to generate contrast in BF imaging for quantitative analysis. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Optimizing ultrafast wide field-of-view illumination for high-throughput multi-photon imaging and screening of mutant fluorescent proteins

NASA Astrophysics Data System (ADS)

Stoltzfus, Caleb; Mikhailov, Alexandr; Rebane, Aleksander

2017-02-01

Fluorescence induced by 1wo-photon absorption (2PA) and three-photon absorption (3PA) is becoming an increasingly important tool for deep-tissue microscopy, especially in conjunction with genetically-encoded functional probes such as fluorescent proteins (FPs). Unfortunately, the efficacy of the multi-photon excitation of FPs is notoriously low, and because relations between a biological fluorophore's nonlinear-optical properties and its molecular structure are inherently complex, there are no practical avenues available that would allow boosting the performance of current FPs. Here we describe a novel method, where we apply directed evolution to optimize the 2PA properties of EGFP. Key to the success of this approach consists in high-throughput screening of mutants that would allow selection of variants with promising 2PA and 3PA properties in a broad near-IR excitation range of wavelength. For this purpose, we construct and test a wide field-of-view (FOV), femtosecond imaging system that we then use to quantify the multi-photon excited fluorescence in the 550- 1600 nm range of tens of thousands of E. coli colonies expressing randomly mutated FPs in a standard 10 cm diameter Petri dish configuration. We present a quantitative analysis of different factors that are currently limiting the maximum throughput of the femtosecond multi-photon screening techniques and also report on quantitative measurement of absolute 2PA and 3PA cross sections spectra.

AI-augmented time stretch microscopy

NASA Astrophysics Data System (ADS)

Mahjoubfar, Ata; Chen, Claire L.; Lin, Jiahao; Jalali, Bahram

2017-02-01

Cell reagents used in biomedical analysis often change behavior of the cells that they are attached to, inhibiting their native signaling. On the other hand, label-free cell analysis techniques have long been viewed as challenging either due to insufficient accuracy by limited features, or because of low throughput as a sacrifice of improved precision. We present a recently developed artificial-intelligence augmented microscope, which builds upon high-throughput time stretch quantitative phase imaging (TS-QPI) and deep learning to perform label-free cell classification with record high-accuracy. Our system captures quantitative optical phase and intensity images simultaneously by frequency multiplexing, extracts multiple biophysical features of the individual cells from these images fused, and feeds these features into a supervised machine learning model for classification. The enhanced performance of our system compared to other label-free assays is demonstrated by classification of white blood T-cells versus colon cancer cells and lipid accumulating algal strains for biofuel production, which is as much as five-fold reduction in inaccuracy. This system obtains the accuracy required in practical applications such as personalized drug development, while the cells remain intact and the throughput is not sacrificed. Here, we introduce a data acquisition scheme based on quadrature phase demodulation that enables interruptionless storage of TS-QPI cell images. Our proof of principle demonstration is capable of saving 40 TB of cell images in about four hours, i.e. pictures of every single cell in 10 mL of a sample.

Attributed relational graphs for cell nucleus segmentation in fluorescence microscopy images.

PubMed

Arslan, Salim; Ersahin, Tulin; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem

2013-06-01

More rapid and accurate high-throughput screening in molecular cellular biology research has become possible with the development of automated microscopy imaging, for which cell nucleus segmentation commonly constitutes the core step. Although several promising methods exist for segmenting the nuclei of monolayer isolated and less-confluent cells, it still remains an open problem to segment the nuclei of more-confluent cells, which tend to grow in overlayers. To address this problem, we propose a new model-based nucleus segmentation algorithm. This algorithm models how a human locates a nucleus by identifying the nucleus boundaries and piecing them together. In this algorithm, we define four types of primitives to represent nucleus boundaries at different orientations and construct an attributed relational graph on the primitives to represent their spatial relations. Then, we reduce the nucleus identification problem to finding predefined structural patterns in the constructed graph and also use the primitives in region growing to delineate the nucleus borders. Working with fluorescence microscopy images, our experiments demonstrate that the proposed algorithm identifies nuclei better than previous nucleus segmentation algorithms.

Microbubble Enzyme-Linked Immunosorbent Assay for the Detection of Targeted Microbubbles in in Vitro Static Binding Assays.

PubMed

Wischhusen, Jennifer; Padilla, Frederic

2017-07-01

Targeted microbubbles (MBs) are ultrasound contrast agents that are functionalized with a ligand for ultrasound molecular imaging of endothelial markers. Novel targeted MBs are characterized in vitro by incubation in protein-coated wells, followed by binding quantification by microscopy or ultrasound imaging. Both methods provide operator-dependent results: Between 3 and 20 fields of view from a heterogeneous sample are typically selected for analysis by microscopy, and in ultrasound imaging, different acoustic settings affect signal intensities. This study proposes a new method to reproducibly quantify MB binding based on enzyme-linked immunosorbent assay (ELISA), in which bound MBs are revealed with an enzyme-linked antibody. MB-ELISA was adapted to in vitro static binding assays, incubating the MBs in inverted position or by agitation, and compared with microscopy. The specificity and sensitivity of MB-ELISA enable the reliable quantification of MB binding in a rapid, high-throughput and whole-well analysis, facilitating the characterization of new targeted contrast agents. Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

40 CFR 1037.810 - Incorporation by reference.

Code of Federal Regulations, 2013 CFR

2013-07-01

... must publish a notice of the change in the Federal Register and the material must be available to the... software is also available for download at http://www.epa.gov/otaq/climate/gem.htm. (2) [Reserved] (d... working version of this software is also available for download at http://www.epa.gov/otaq/climate/gem.htm. ...

76 FR 55390 - Guidance on Exculpatory Language in Informed Consent, Draft

Federal Register 2010, 2011, 2012, 2013, 2014

2011-09-07

...://www.fda.gov/ScienceResearch/SpecialTopics/RunningClinicalTrials/default.htm , is intended to assist... , http://www.hhs.gov/ohrp/newsroom/rfc/index.html , or http://www.fda.gov/ScienceResearch/SpecialTopics/RunningClinicalTrials/default.htm . III. Request for Comments OHRP and FDA are making their joint draft...

78 FR 32676 - Changes in Flood Hazard Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2013-05-31

... flood insurance premium rates for new buildings and the contents of those buildings. For rating purposes...://www.rampp-team.com/ September 23, 2013........ 050084 Springs (13-06- Ruth Carney, 111 Opera lomrs.htm... http://www.rampp-team.com/ July 25, 2013 340343 Hanover (12-02- Ronald F. Engineering lomrs.htm. 1077P...

77 FR 73490 - Proposed Flood Hazard Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-10

... resolution process. SRPs are independent panels of experts in hydrology, hydraulics, and other pertinent...://www.in.gov/dnr/water/6573.htm City of Rising Sun City Hall, 200 North Walnut Street, Rising Sun, IN....in.gov/dnr/water/7357.htm Town of Gosport Town Hall, 13 South 3rd Street, Gosport, IN 47433. Town of...

Addressing the Big-Earth-Data Variety Challenge with the Hierarchical Triangular Mesh

NASA Technical Reports Server (NTRS)

Rilee, Michael L.; Kuo, Kwo-Sen; Clune, Thomas; Oloso, Amidu; Brown, Paul G.; Yu, Honfeng

2016-01-01

We have implemented an updated Hierarchical Triangular Mesh (HTM) as the basis for a unified data model and an indexing scheme for geoscience data to address the variety challenge of Big Earth Data. We observe that, in the absence of variety, the volume challenge of Big Data is relatively easily addressable with parallel processing. The more important challenge in achieving optimal value with a Big Data solution for Earth Science (ES) data analysis, however, is being able to achieve good scalability with variety. With HTM unifying at least the three popular data models, i.e. Grid, Swath, and Point, used by current ES data products, data preparation time for integrative analysis of diverse datasets can be drastically reduced and better variety scaling can be achieved. In addition, since HTM is also an indexing scheme, when it is used to index all ES datasets, data placement alignment (or co-location) on the shared nothing architecture, which most Big Data systems are based on, is guaranteed and better performance is ensured. Moreover, our updated HTM encoding turns most geospatial set operations into integer interval operations, gaining further performance advantages.

Morphological changes and viability of primary cultured human ocular trabecular meshwork cells after exposure to air.

PubMed

Kopsachilis, Nikolaos; Tsaousis, Konstantinos T; Carifi, Gianluca; Welge-Luessen, Ulrich

2014-06-01

To investigate the possible toxic effect of air exposure for an in vitro model of primary human ocular trabecular meshwork cells (HTM). HTM were isolated from five donor eyes and cultivated at 37 °C. After reaching confluence the cells were seeded on two well chamber slides. The chamber slides were turned upside down in a Petri culture dish full of culture medium and filled with air using a 5 ml syringe, starting this way the exposure of the cells to the air. Subsequently they were placed in the incubation chamber at 37 °C. Six groups of HTM cultures were set up: group 1 consisted of samples in which HTM were exposed to air for 30 min, group 2 for 1 h, group 3 for 3 h, group 4 for 6 h, group 5 for 12 h and group 6 for 24 h. At 3 h after exposure, the morphology of the cells was still intact, at 6 h few cells appeared deformed and exhibited characteristics of more senescent cells. At 12 h after exposure to air the HTM cells started losing their typical morphology and appeared enlarged and compromised. Viability was superior to 94% in groups 1-3 while for groups 4, 5, 6 it was 82.7%, 39.5% and 12.7% respectively. The toxic effect of air exposure for the studied in vitro model of HTM is not significant for the time period of one to three hours. However it starts reducing viability and alternating morphology 6 h after exposure until the time period of 24 h, where the percentage of living cells is drastically decreased. Therefore, we suggest that the use of an air bubble especially in glaucomatous patients should be applied with caution. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of Microscopy, Nested-PCR, and Real-Time-PCR Assays Using High-Throughput Screening of Pooled Samples for Diagnosis of Malaria in Asymptomatic Carriers from Areas of Endemicity in Myanmar

PubMed Central

Wang, Bo; Han, Soe-Soe; Cho, Cho; Han, Jin-Hee; Cheng, Yang; Lee, Seong-Kyun; Galappaththy, Gawrie N. L; Thimasarn, Krongthong; Soe, Myat Thu; Oo, Htet Wai; Kyaw, Myat Phone

2014-01-01

Asymptomatic infection is an important obstacle for controlling disease in countries where malaria is endemic. Because asymptomatic carriers do not seek treatment for their infections, they can have high levels of gametocytes and constitute a reservoir available for new infection. We employed a sample pooling/PCR-based molecular detection strategy for screening malaria infection in residents from areas of Myanmar where malaria is endemic. Blood samples (n = 1,552) were collected from residents in three areas of malaria endemicity (Kayin State, Bago, and Tanintharyi regions) of Myanmar. Two nested PCR and real-time PCR assays showed that asymptomatic infection was detected in about 1.0% to 9.4% of residents from the surveyed areas. The sensitivities of the two nested PCR and real-time PCR techniques were higher than that of microscopy examination (sensitivity, 100% versus 26.4%; kappa values, 0.2 to 0.5). Among the three regions, parasite-positive samples were highly detected in subjects from the Bago and Tanintharyi regions. Active surveillance of residents from regions of intense malaria transmission would reduce the risk of morbidity and mitigate transmission to the population in these areas of endemicity. Our data demonstrate that PCR-based molecular techniques are more efficient than microscopy for nationwide surveillance of malaria in countries where malaria is endemic. PMID:24648557

Solvent influence upon structure & throughput of poly vinyledene fluoride thin film nano-patterns by imprint lithography

NASA Astrophysics Data System (ADS)

Sankar, M. S. Ravi; Gangineni, R. B.

2018-04-01

This work aims at understanding the solvent influence upon the throughput and structure of poly vinyledene fluoride (PVDF)nano-patterned films. The PVDF thin films are deposited by spin coating method using Dimethylsulfoxide (DMSO), Tetrahydrofuran (THF) and 2-butanone solvents. The nano-patterns are realized by imprinting SONY 700 MB CD aluminum constructions on PVDF thin filmsusing imprint lithography technique under ambient annealing temperature and pressure. Surface morphology &imprint pattern transfer quality is evaluated with Atomic force microscopy (AFM). Raman spectroscopy is used for evaluating the structural evolutions with respect to solvent & patterning.

Environmental High-content Fluorescence Microscopy (e-HCFM) of Tara Oceans Samples Provides a View of Global Ocean Protist Biodiversity

NASA Astrophysics Data System (ADS)

Coelho, L. P.; Colin, S.; Sunagawa, S.; Karsenti, E.; Bork, P.; Pepperkok, R.; de Vargas, C.

2016-02-01

Protists are responsible for much of the diversity in the eukaryotic kingdomand are crucial to several biogeochemical processes of global importance (e.g.,the carbon cycle). Recent global investigations of these organisms have reliedon sequence-based approaches. These methods do not, however, capture thecomplex functional morphology of these organisms nor can they typically capturephenomena such as interactions (except indirectly through statistical means).Direct imaging of these organisms, can therefore provide a valuable complementto sequencing and, when performed quantitatively, provide measures ofstructures and interaction patterns which can then be related back to sequencebased measurements. Towards this end, we developed a framework, environmentalhigh-content fluorescence microscopy (e-HCFM) which can be applied toenvironmental samples composed of mixed communities. This strategy is based ongeneral purposes dyes that stain major structures in eukaryotes. Samples areimaged using scanning confocal microscopy, resulting in a three-dimensionalimage-stack. High-throughput can be achieved using automated microscopy andcomputational analysis. Standard bioimage informatics segmentation methodscombined with feature computation and machine learning results in automatictaxonomic assignments to the objects that are imaged in addition to severalbiochemically relevant measurements (such as biovolumes, fluorescenceestimates) per organism. We provide results on 174 image acquisition from TaraOcean samples, which cover organisms from 5 to 180 microns (82 samples in the5-20 fraction, 96 in the 20-180 fraction). We show a validation of the approachboth on technical grounds (demonstrating the high accuracy of automatedclassification) and provide results obtain from image analysis and fromintegrating with other data, such as associated environmental parametersmeasured in situ as well as perspectives on integration with sequenceinformation.

75 FR 41121 - Hazardous and Solid Waste Management System; Identification and Listing of Special Wastes...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-15

.../fossil/ccr-rule/ccr-form.htm . If you would like to present oral testimony and are unable to preregister... SUPPLEMENTARY INFORMATION for information on how to register. ADDRESSES: The public hearings will be held at the....gov/epawaste/nonhaz/industrial/special/fossil/ccr-rule/ccr-hearing.htm . Those persons planning to...

Training a US Army Chemical Corps Company in the Current Operating Environment

DTIC Science & Technology

2009-02-20

Recon Vehicle. http://www.globalsecurity.org/military/systems/ground/ iav - nbc.htm (accessed December 14, 2008). 5    in addition to his or her duties...systems/ground/ iav - nbc.htm (accessed December 14, 2008). Meyer, Josh. "Al Qaeda is said to focus again on WMD." Los Angeles Times, February 3, 2008: A

78 FR 36176 - Commission Information Collection Activities (FERC-551); Comment Request; Extension

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-17

...: Reporting of Flow Volume and Capacity by Interstate Natural Gas Pipelines. OMB Control No.: 1902-0243. Type... Code of Federal Regulations 1320.3. Type of Respondents: Interstate Natural Gas Pipelines. Estimate of...) ( http://bls.gov/oes/current/naics2_22.htm and http://www.bls.gov/news.release/ecec.nr0.htm ). Comments...

78 FR 20331 - Changes in Flood Hazard Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-04

... flood insurance premium rates for new buildings and the contents of those buildings. For rating purposes............ 350070 (12-06-1488P). David Coss, Avenue, Santa Fe, team.com/lomrs.htm. Mayor, City of NM 87501. Santa Fe... Design http://www.rampp- May 28, 2013......... 405381 06-1019P). Dewey F. Office, 2317 team.com/lomrs.htm...

Air Force’s Combat Aircraft: A Future Holding into the Past

DTIC Science & Technology

2010-04-07

Global Security, “9M111 / SA-19 GRISON ,” Globalsecurity.org, http://www.globalsecurity.org/military/world/ russia/sa-19.htm (accessed March 13, 2010...world/russia/9k338.htm (assessed March 13, 2010). Global Security. “9M111 / SA-19 GRISON .” Globalsecurity.org. http://www.globalsecurity. org

50 CFR 622.14 - Area closures related to the Deepwater Horizon oil spill.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Web site: http://sero.nmfs.noaa.gov/deepwater_horizon_oil_spill.htm. (b) Gulf EEZ area closure related... the Gulf EEZ identified in the map shown on the NMFS Web site: http://sero.nmfs.noaa.gov/deepwater... shown on the NMFS Web site: http://sero.nmfs.noaa.gov/deepwater_horizon_oil_spill.htm. ...

A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination

PubMed Central

van der Gaast—de Jongh, Christa E.; Diavatopoulos, Dimitri A.; de Jonge, Marien I.

2017-01-01

The respiratory pathogen Streptococcus pneumoniae is a major cause of diseases such as otitis media, pneumonia, sepsis and meningitis. The first step towards infection is colonization of the nasopharynx. Recently, it was shown that agglutinating antibodies play an important role in the prevention of mucosal colonization with S. pneumoniae. Here, we present a novel method to quantify antibody-dependent pneumococcal agglutination in a high-throughput manner using flow cytometry. We found that the concentration of agglutinating antibodies against pneumococcal capsule are directly correlated with changes in the size and complexity of bacterial aggregates, as measured by flow cytometry and confirmed by light microscopy. Using the increase in size, we determined the agglutination index. The cutoff value was set by measuring a series of non-agglutinating antibodies. With this method, we show that not only anti-polysaccharide capsule antibodies are able to induce agglutination but that also anti-PspA protein antibodies have agglutinating capabilities. In conclusion, we have described and validated a novel method to quantify pneumococcal agglutination, which can be used to screen sera from murine or human vaccination studies, in a high-throughput manner. PMID:28288168

High-throughput fabrication of anti-counterfeiting colloid-based photoluminescent microtags using electrical nanoimprint lithography

NASA Astrophysics Data System (ADS)

Diaz, R.; Palleau, E.; Poirot, D.; Sangeetha, N. M.; Ressier, L.

2014-08-01

This work demonstrates the excellent capability of the recently developed electrical nanoimprint lithography (e-NIL) technique for quick, high-throughput production of well-defined colloid assemblies on surfaces. This is shown by fabricating micron-sized photoluminescent quick response (QR) codes based on the electrostatic directed trapping (so called nanoxerography process) of 28 nm colloidal lanthanide-doped upconverting NaYF4 nanocrystals. Influencing experimental parameters have been optimized and the contribution of triboelectrification in e-NIL was evidenced. Under the chosen conditions, more than 300 000 nanocrystal-based QR codes were fabricated on a 4 inch silicon wafer, in less than 15 min. These microtags were then transferred to transparent flexible films, to be easily integrated onto desired products. Invisible to the naked eye, they can be decoded and authenticated using an optical microscopy image of their specific photoluminescence mapping. Beyond this very promising application for product tracking and the anti-counterfeiting strategies, e-NIL nanoxerography, potentially applicable to any types of charged and/or polarizable colloids and pattern geometries opens up tremendous opportunities for industrial scale production of various other kinds of colloid-based devices and sensors.

High-throughput single-molecule force spectroscopy for membrane proteins

NASA Astrophysics Data System (ADS)

Bosshart, Patrick D.; Casagrande, Fabio; Frederix, Patrick L. T. M.; Ratera, Merce; Bippes, Christian A.; Müller, Daniel J.; Palacin, Manuel; Engel, Andreas; Fotiadis, Dimitrios

2008-09-01

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ~400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ~200 (AdiC) and ~400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

Magnetic high throughput screening system for the development of nano-sized molecularly imprinted polymers for controlled delivery of curcumin.

PubMed

Piletska, Elena V; Abd, Bashar H; Krakowiak, Agata S; Parmar, Anitha; Pink, Demi L; Wall, Katie S; Wharton, Luke; Moczko, Ewa; Whitcombe, Michael J; Karim, Kal; Piletsky, Sergey A

2015-05-07

Curcumin is a versatile anti-inflammatory and anti-cancer agent known for its low bioavailability, which could be improved by developing materials capable of binding and releasing drug in a controlled fashion. The present study describes the preparation of magnetic nano-sized Molecularly Imprinted Polymers (nanoMIPs) for the controlled delivery of curcumin and their high throughput characterisation using microtitre plates modified with magnetic inserts. NanoMIPs were synthesised using functional monomers chosen with the aid of molecular modelling. The rate of release of curcumin from five polymers was studied under aqueous conditions and was found to correlate well with the binding energies obtained computationally. The presence of specific monomers was shown to be significant in ensuring effective binding of curcumin and to the rate of release obtained. Characterisation of the polymer particles was carried out using dynamic light scattering (DLS) technique and scanning electron microscopy (SEM) in order to establish the relationship between irradiation time and particle size. The protocols optimised during this study could be used as a blueprint for the development of nanoMIPs capable of the controlled release of potentially any compound of interest.

High Throughput Multiplex PCR and Probe-based Detection with Luminex Beads for Seven Intestinal Parasites

PubMed Central

Taniuchi, Mami; Verweij, Jaco J.; Noor, Zannatun; Sobuz, Shihab U.; van Lieshout, Lisette; Petri, William A.; Haque, Rashidul; Houpt, Eric R.

2011-01-01

Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites—Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis—were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites. PMID:21292910

Microarrays for the evaluation of cell-biomaterial surface interactions

NASA Astrophysics Data System (ADS)

Thissen, H.; Johnson, G.; McFarland, G.; Verbiest, B. C. H.; Gengenbach, T.; Voelcker, N. H.

2007-01-01

The evaluation of cell-material surface interactions is important for the design of novel biomaterials which are used in a variety of biomedical applications. While traditional in vitro test methods have routinely used samples of relatively large size, microarrays representing different biomaterials offer many advantages, including high throughput and reduced sample handling. Here, we describe the simultaneous cell-based testing of matrices of polymeric biomaterials, arrayed on glass slides with a low cell-attachment background coating. Arrays were constructed using a microarray robot at 6 fold redundancy with solid pins having a diameter of 375 μm. Printed solutions contained at least one monomer, an initiator and a bifunctional crosslinker. After subsequent UV polymerisation, the arrays were washed and characterised by X-ray photoelectron spectroscopy. Cell culture experiments were carried out over 24 hours using HeLa cells. After labelling with CellTracker ® Green for the final hour of incubation and subsequent fixation, the arrays were scanned. In addition, individual spots were also viewed by fluorescence microscopy. The evaluation of cell-surface interactions in high-throughput assays as demonstrated here is a key enabling technology for the effective development of future biomaterials.

DM-BLD: differential methylation detection using a hierarchical Bayesian model exploiting local dependency.

PubMed

Wang, Xiao; Gu, Jinghua; Hilakivi-Clarke, Leena; Clarke, Robert; Xuan, Jianhua

2017-01-15

The advent of high-throughput DNA methylation profiling techniques has enabled the possibility of accurate identification of differentially methylated genes for cancer research. The large number of measured loci facilitates whole genome methylation study, yet posing great challenges for differential methylation detection due to the high variability in tumor samples. We have developed a novel probabilistic approach, D: ifferential M: ethylation detection using a hierarchical B: ayesian model exploiting L: ocal D: ependency (DM-BLD), to detect differentially methylated genes based on a Bayesian framework. The DM-BLD approach features a joint model to capture both the local dependency of measured loci and the dependency of methylation change in samples. Specifically, the local dependency is modeled by Leroux conditional autoregressive structure; the dependency of methylation changes is modeled by a discrete Markov random field. A hierarchical Bayesian model is developed to fully take into account the local dependency for differential analysis, in which differential states are embedded as hidden variables. Simulation studies demonstrate that DM-BLD outperforms existing methods for differential methylation detection, particularly when the methylation change is moderate and the variability of methylation in samples is high. DM-BLD has been applied to breast cancer data to identify important methylated genes (such as polycomb target genes and genes involved in transcription factor activity) associated with breast cancer recurrence. A Matlab package of DM-BLD is available at http://www.cbil.ece.vt.edu/software.htm CONTACT: Xuan@vt.eduSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Alginate Oligosaccharides Inhibit Fungal Cell Growth and Potentiate the Activity of Antifungals against Candida and Aspergillus spp

PubMed Central

Tøndervik, Anne; Sletta, Håvard; Klinkenberg, Geir; Emanuel, Charlotte; Powell, Lydia C.; Pritchard, Manon F.; Khan, Saira; Craine, Kieron M.; Onsøyen, Edvar; Rye, Phil D.; Wright, Chris; Thomas, David W.; Hill, Katja E.

2014-01-01

The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD600; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity. PMID:25409186

Metabolomic and high-throughput sequencing analysis-modern approach for the assessment of biodeterioration of materials from historic buildings.

PubMed

Gutarowska, Beata; Celikkol-Aydin, Sukriye; Bonifay, Vincent; Otlewska, Anna; Aydin, Egemen; Oldham, Athenia L; Brauer, Jonathan I; Duncan, Kathleen E; Adamiak, Justyna; Sunner, Jan A; Beech, Iwona B

2015-01-01

Preservation of cultural heritage is of paramount importance worldwide. Microbial colonization of construction materials, such as wood, brick, mortar, and stone in historic buildings can lead to severe deterioration. The aim of the present study was to give modern insight into the phylogenetic diversity and activated metabolic pathways of microbial communities colonized historic objects located in the former Auschwitz II-Birkenau concentration and extermination camp in Oświecim, Poland. For this purpose we combined molecular, microscopic and chemical methods. Selected specimens were examined using Field Emission Scanning Electron Microscopy (FESEM), metabolomic analysis and high-throughput Illumina sequencing. FESEM imaging revealed the presence of complex microbial communities comprising diatoms, fungi and bacteria, mainly cyanobacteria and actinobacteria, on sample surfaces. Microbial diversity of brick specimens appeared higher than that of the wood and was dominated by algae and cyanobacteria, while wood was mainly colonized by fungi. DNA sequences documented the presence of 15 bacterial phyla representing 99 genera including Halomonas, Halorhodospira, Salinisphaera, Salinibacterium, Rubrobacter, Streptomyces, Arthrobacter and nine fungal classes represented by 113 genera including Cladosporium, Acremonium, Alternaria, Engyodontium, Penicillium, Rhizopus, and Aureobasidium. Most of the identified sequences were characteristic of organisms implicated in deterioration of wood and brick. Metabolomic data indicated the activation of numerous metabolic pathways, including those regulating the production of primary and secondary metabolites, for example, metabolites associated with the production of antibiotics, organic acids and deterioration of organic compounds. The study demonstrated that a combination of electron microscopy imaging with metabolomic and genomic techniques allows to link the phylogenetic information and metabolic profiles of microbial communities and to shed new light on biodeterioration processes.

Metabolomic and high-throughput sequencing analysis—modern approach for the assessment of biodeterioration of materials from historic buildings

PubMed Central

Gutarowska, Beata; Celikkol-Aydin, Sukriye; Bonifay, Vincent; Otlewska, Anna; Aydin, Egemen; Oldham, Athenia L.; Brauer, Jonathan I.; Duncan, Kathleen E.; Adamiak, Justyna; Sunner, Jan A.; Beech, Iwona B.

2015-01-01

Preservation of cultural heritage is of paramount importance worldwide. Microbial colonization of construction materials, such as wood, brick, mortar, and stone in historic buildings can lead to severe deterioration. The aim of the present study was to give modern insight into the phylogenetic diversity and activated metabolic pathways of microbial communities colonized historic objects located in the former Auschwitz II–Birkenau concentration and extermination camp in Oświecim, Poland. For this purpose we combined molecular, microscopic and chemical methods. Selected specimens were examined using Field Emission Scanning Electron Microscopy (FESEM), metabolomic analysis and high-throughput Illumina sequencing. FESEM imaging revealed the presence of complex microbial communities comprising diatoms, fungi and bacteria, mainly cyanobacteria and actinobacteria, on sample surfaces. Microbial diversity of brick specimens appeared higher than that of the wood and was dominated by algae and cyanobacteria, while wood was mainly colonized by fungi. DNA sequences documented the presence of 15 bacterial phyla representing 99 genera including Halomonas, Halorhodospira, Salinisphaera, Salinibacterium, Rubrobacter, Streptomyces, Arthrobacter and nine fungal classes represented by 113 genera including Cladosporium, Acremonium, Alternaria, Engyodontium, Penicillium, Rhizopus, and Aureobasidium. Most of the identified sequences were characteristic of organisms implicated in deterioration of wood and brick. Metabolomic data indicated the activation of numerous metabolic pathways, including those regulating the production of primary and secondary metabolites, for example, metabolites associated with the production of antibiotics, organic acids and deterioration of organic compounds. The study demonstrated that a combination of electron microscopy imaging with metabolomic and genomic techniques allows to link the phylogenetic information and metabolic profiles of microbial communities and to shed new light on biodeterioration processes. PMID:26483760

A high-throughput quantitative expression analysis of cancer-related genes in human HepG2 cells in response to limonene, a potential anticancer agent.

PubMed

Hafidh, Rand R; Hussein, Saba Z; MalAllah, Mohammed Q; Abdulamir, Ahmed S; Abu Bakar, Fatimah

2017-11-14

Citrus bioactive compounds, as active anticancer agent, have been under focus by several studies worldwide. However, the underlying genes responsible for the anticancer potential have not been sufficiently highlighted. The current study investigated the gene expression profile of hepatocellular carcinoma, HepG2, cells after treatment with Limonene. The concentration that killed 50% of HepG2 cells was used to elucidate the genetic mechanisms of limonene anticancer activity. The apoptotic induction was detected by flow cytometry and confocal fluorescence microscope. Two of pro-apoptotic events, caspase-3 activation and phosphatidylserine translocation were manifested by confocal fluorescence microscopy. High-throughput real-time PCR was used to profile 1023 cancer-related genes in 16 different gene families related to the cancer development. In comparison to untreated cells, limonene increased the percentage of apoptotic cells up to 89.61%, by flow cytometry, and 48.2% by fluorescence microscopy. There was a significant limonene-driven differential gene expression of HepG2 cells in 15 different gene families. Limonene was shown to significantly (>2log) up-regulate and down-regulate 14 and 59 genes, respectively. The affected gene families, from most to least affected, were apoptosis induction, signal transduction, cancer genes augmentation, alteration in kinases expression, inflammation, DNA damage repair, and cell cycle proteins. The current study reveals that limonene could be a promising, cheap, and effective anticancer compound. The broad spectrum of limonene anticancer activity is interesting for anticancer drug development. Further research is needed to confirm the current findings and to examine the anticancer potential of limonene along with underlying mechanisms on different cell lines. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Quantifying Golgi structure using EM: combining volume-SEM and stereology for higher throughput.

PubMed

Ferguson, Sophie; Steyer, Anna M; Mayhew, Terry M; Schwab, Yannick; Lucocq, John Milton

2017-06-01

Investigating organelles such as the Golgi complex depends increasingly on high-throughput quantitative morphological analyses from multiple experimental or genetic conditions. Light microscopy (LM) has been an effective tool for screening but fails to reveal fine details of Golgi structures such as vesicles, tubules and cisternae. Electron microscopy (EM) has sufficient resolution but traditional transmission EM (TEM) methods are slow and inefficient. Newer volume scanning EM (volume-SEM) methods now have the potential to speed up 3D analysis by automated sectioning and imaging. However, they produce large arrays of sections and/or images, which require labour-intensive 3D reconstruction for quantitation on limited cell numbers. Here, we show that the information storage, digital waste and workload involved in using volume-SEM can be reduced substantially using sampling-based stereology. Using the Golgi as an example, we describe how Golgi populations can be sensed quantitatively using single random slices and how accurate quantitative structural data on Golgi organelles of individual cells can be obtained using only 5-10 sections/images taken from a volume-SEM series (thereby sensing population parameters and cell-cell variability). The approach will be useful in techniques such as correlative LM and EM (CLEM) where small samples of cells are treated and where there may be variable responses. For Golgi study, we outline a series of stereological estimators that are suited to these analyses and suggest workflows, which have the potential to enhance the speed and relevance of data acquisition in volume-SEM.

Microfluidic system for high throughput characterisation of echogenic particles.

PubMed

Rademeyer, Paul; Carugo, Dario; Lee, Jeong Yu; Stride, Eleanor

2015-01-21

Echogenic particles, such as microbubbles and volatile liquid micro/nano droplets, have shown considerable potential in a variety of clinical diagnostic and therapeutic applications. The accurate prediction of their response to ultrasound excitation is however extremely challenging, and this has hindered the optimisation of techniques such as quantitative ultrasound imaging and targeted drug delivery. Existing characterisation techniques, such as ultra-high speed microscopy provide important insights, but suffer from a number of limitations; most significantly difficulty in obtaining large data sets suitable for statistical analysis and the need to physically constrain the particles, thereby altering their dynamics. Here a microfluidic system is presented that overcomes these challenges to enable the measurement of single echogenic particle response to ultrasound excitation. A co-axial flow focusing device is used to direct a continuous stream of unconstrained particles through the combined focal region of an ultrasound transducer and a laser. Both the optical and acoustic scatter from individual particles are then simultaneously recorded. Calibration of the device and example results for different types of echogenic particle are presented, demonstrating a high throughput of up to 20 particles per second and the ability to resolve changes in particle radius down to 0.1 μm with an uncertainty of less than 3%.

Identification of Novel Pro-Migratory, Cancer-Associated Genes Using Quantitative, Microscopy-Based Screening

PubMed Central

Naffar-Abu-Amara, Suha; Shay, Tal; Galun, Meirav; Cohen, Naomi; Isakoff, Steven J.; Kam, Zvi; Geiger, Benjamin

2008-01-01

Background Cell migration is a highly complex process, regulated by multiple genes, signaling pathways and external stimuli. To discover genes or pharmacological agents that can modulate the migratory activity of cells, screening strategies that enable the monitoring of diverse migratory parameters in a large number of samples are necessary. Methodology In the present study, we describe the development of a quantitative, high-throughput cell migration assay, based on a modified phagokinetic tracks (PKT) procedure, and apply it for identifying novel pro-migratory genes in a cancer-related gene library. In brief, cells are seeded on fibronectin-coated 96-well plates, covered with a monolayer of carboxylated latex beads. Motile cells clear the beads, located along their migratory paths, forming tracks that are visualized using an automated, transmitted-light screening microscope. The tracks are then segmented and characterized by multi-parametric, morphometric analysis, resolving a variety of morphological and kinetic features. Conclusions In this screen we identified 4 novel genes derived from breast carcinoma related cDNA library, whose over-expression induces major alteration in the migration of the stationary MCF7 cells. This approach can serve for high throughput screening for novel ways to modulate cellular migration in pathological states such as tumor metastasis and invasion. PMID:18213366

Phenotype classification of single cells using SRS microscopy, RNA sequencing, and microfluidics (Conference Presentation)

NASA Astrophysics Data System (ADS)

Streets, Aaron M.; Cao, Chen; Zhang, Xiannian; Huang, Yanyi

2016-03-01

Phenotype classification of single cells reveals biological variation that is masked in ensemble measurement. This heterogeneity is found in gene and protein expression as well as in cell morphology. Many techniques are available to probe phenotypic heterogeneity at the single cell level, for example quantitative imaging and single-cell RNA sequencing, but it is difficult to perform multiple assays on the same single cell. In order to directly track correlation between morphology and gene expression at the single cell level, we developed a microfluidic platform for quantitative coherent Raman imaging and immediate RNA sequencing (RNA-Seq) of single cells. With this device we actively sort and trap cells for analysis with stimulated Raman scattering microscopy (SRS). The cells are then processed in parallel pipelines for lysis, and preparation of cDNA for high-throughput transcriptome sequencing. SRS microscopy offers three-dimensional imaging with chemical specificity for quantitative analysis of protein and lipid distribution in single cells. Meanwhile, the microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved RNA-Seq detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. By combining coherent Raman microscopy with RNA sequencing, we can better understand the relationship between cellular morphology and gene expression at the single-cell level.

High-throughput methods for characterizing the mechanical properties of coatings

NASA Astrophysics Data System (ADS)

Siripirom, Chavanin

The characterization of mechanical properties in a combinatorial and high-throughput workflow has been a bottleneck that reduced the speed of the materials development process. High-throughput characterization of the mechanical properties was applied in this research in order to reduce the amount of sample handling and to accelerate the output. A puncture tester was designed and built to evaluate the toughness of materials using an innovative template design coupled with automation. The test is in the form of a circular free-film indentation. A single template contains 12 samples which are tested in a rapid serial approach. Next, the operational principles of a novel parallel dynamic mechanical-thermal analysis instrument were analyzed in detail for potential sources of errors. The test uses a model of a circular bilayer fixed-edge plate deformation. A total of 96 samples can be analyzed simultaneously which provides a tremendous increase in efficiency compared with a conventional dynamic test. The modulus values determined by the system had considerable variation. The errors were observed and improvements to the system were made. A finite element analysis was used to analyze the accuracy given by the closed-form solution with respect to testing geometries, such as thicknesses of the samples. A good control of the thickness of the sample was proven to be crucial to the accuracy and precision of the output. Then, the attempt to correlate the high-throughput experiments and conventional coating testing methods was made. Automated nanoindentation in dynamic mode was found to provide information on the near-surface modulus and could potentially correlate with the pendulum hardness test using the loss tangent component. Lastly, surface characterization of stratified siloxane-polyurethane coatings was carried out with X-ray photoelectron spectroscopy, Rutherford backscattering spectroscopy, transmission electron microscopy, and nanoindentation. The siloxane component segregates to the surface during curing. The distribution of siloxane as a function of thickness into the sample showed differences depending on the formulation parameters. The coatings which had higher siloxane content near the surface were those coatings found to perform well in field tests.

Polymer surface functionalities that control human embryoid body cell adhesion revealed by high throughput surface characterization of combinatorial material microarrays

PubMed Central

Yang, Jing; Mei, Ying; Hook, Andrew L.; Taylor, Michael; Urquhart, Andrew J.; Bogatyrev, Said R.; Langer, Robert; Anderson, Daniel G.; Davies, Martyn C.; Alexander, Morgan R.

2010-01-01

High throughput materials discovery using combinatorial polymer microarrays to screen for new biomaterials with new and improved function is established as a powerful strategy. Here we combine this screening approach with high throughput surface characterisation (HT-SC) to identify surface structure-function relationships. We explore how this combination can help to identify surface chemical moieties that control protein adsorption and subsequent cellular response. The adhesion of human embryoid body (hEB) cells to a large number (496) of different acrylate polymers synthesized in a microarray format is screened using a high throughput procedure. To determine the role of the polymer surface properties on hEB cell adhesion, detailed HT-SC of these acrylate polymers is carried out using time of flight secondary ion mass spectrometry (ToF SIMS), x-ray photoelectron spectroscopy (XPS), pico litre drop sessile water contact angle (WCA) measurement and atomic force microscopy (AFM). A structure-function relationship is identified between the ToF SIMS analysis of the surface chemistry after a fibronectin (Fn) pre-conditioning step and the cell adhesion to each spot using the multivariate analysis technique partial least squares (PLS) regression. Secondary ions indicative of the adsorbed Fn correlate with increased cell adhesion whereas glycol and other functionalities from the polymers are identified that reduce cell adhesion. Furthermore, a strong relationship between the ToF SIMS spectra of bare polymers and the cell adhesion to each spot is identified using PLS regression. This identifies a role for both the surface chemistry of the bare polymer and the pre-adsorbed Fn, as-represented in the ToF SIMS spectra, in controlling cellular adhesion. In contrast, no relationship is found between cell adhesion and wettability, surface roughness, elemental or functional surface composition. The correlation between ToF SIMS data of the surfaces and the cell adhesion demonstrates the ability of identifying surface moieties that control protein adsorption and subsequent cell adhesion using ToF SIMS and multivariate analysis. PMID:20832108

Modification of CuI based Hole Transport Material for Solid State DSSC Application

NASA Astrophysics Data System (ADS)

Hanif, Q. A.; Ramelan, A. H.; Saputri, L. N. M. Z.; Wahyuningsih, S.

2018-03-01

Modification of Hole Transport Material (HTM) with addition of tetramethylethylenediamine (TMED) and ammonium thiocyanate (NH4SCN) have been conducted. Copper Iodide (CuI) were used as the main component of HTM. Several volume variations of TMED (0.1; 0.2; and 0.4 mL) was added into 0.05 M CuI solutions. While TMED: NH4SCN ratio were 1:1,1:2,2:1 also introduced to the dissolved CuI. Optical properties of these materials showed the band gap energy value ranging from 2.38 to 3.79 eV. The conductivity of HTM has been measured and showed the maximum value in CuI added with 0.4 mL TMED = 0.29 S m-1, and ratio of TMED: NH4SCN=2:1 added into CuI = 0.39 S m-1, these value were increased compared to the CuI conductivity itself (0.26 S m-1). The effect of this modification towards SS DSSC efficiency also has been monitored. The SS DSSC construction consists of TiO2 nanorods sensitized by N3, HTM, and platinum as a counter electrode. The performance of SS DSSC showed rising efficiency as follows TiO2|N3|CuI

A District Approach to Countering Afghanistan’s Insurgency

DTIC Science & Technology

2009-12-01

actualization. Abraham Maslow , A Theory of Human Motivation, 1943, http://psychclassics.yorku.ca/ Maslow /motivation.htm (accessed October 29, 2009...Lyon Press, 2002. Maslow , Abraham . A Theory of Human Motivation, 1943. http://psychclassics.yorku.ca/ Maslow /motivation.htm (accessed October 29...established village and district political hierarchies may effectively deny insurgents sanctuary, critical resources, and serve to isolate and separate the

Maritime Trade Defense: Establishing the Joint Force Maritime Trade Component Commander

DTIC Science & Technology

2012-05-04

GlobalSecurity.Org, "Sealift in World War II,” http://www.globalsecurity.org/military/systems/ship/sealift- ww2 .htm, (accessed 1 May 2012). 11 Ibid, 203. 12 David...military/systems/ship/sealift- ww2 .htm (accessed 1 May 2012). 19 Daniel Y. Coulter, "Globalization of Maritime Commerce: The Rise of Hub Ports," in

Array Formatting of the Heat-Transfer Method (HTM) for the Detection of Small Organic Molecules by Molecularly Imprinted Polymers

PubMed Central

Wackers, Gideon; Vandenryt, Thijs; Cornelis, Peter; Kellens, Evelien; Thoelen, Ronald; De Ceuninck, Ward; Losada-Pérez, Patricia; van Grinsven, Bart; Peeters, Marloes; Wagner, Patrick

2014-01-01

In this work we present the first steps towards a molecularly imprinted polymer (MIP)-based biomimetic sensor array for the detection of small organic molecules via the heat-transfer method (HTM). HTM relies on the change in thermal resistance upon binding of the target molecule to the MIP-type receptor. A flow-through sensor cell was developed, which is segmented into four quadrants with a volume of 2.5 μL each, allowing four measurements to be done simultaneously on a single substrate. Verification measurements were conducted, in which all quadrants received a uniform treatment and all four channels exhibited a similar response. Subsequently, measurements were performed in quadrants, which were functionalized with different MIP particles. Each of these quadrants was exposed to the same buffer solution, spiked with different molecules, according to the MIP under analysis. With the flow cell design we could discriminate between similar small organic molecules and observed no significant cross-selectivity. Therefore, the MIP array sensor platform with HTM as a readout technique, has the potential to become a low-cost analysis tool for bioanalytical applications. PMID:24955945

MPLM Donatello is offloaded at the SLF

NASA Technical Reports Server (NTRS)

2001-01-01

At the Shuttle Landing Facility, cranes are poised to help offload the Italian Space Agency's Multi- Purpose Logistics Module Donatello from the Airbus '''Beluga''' air cargo plane that brought it from the factory of Alenia Aerospazio in Turin, Italy. The third of three for the International Space Station, the module will be transported to the Space Station Processing Facility for processing. Among the activities for the payload test team are integrated electrical tests with other Station elements in the SSPF, leak tests, electrical and software compatibility tests with the Space Shuttle (using the Cargo Integrated Test equipment) and an Interface Verification Test once the module is installed in the Space Shuttle's payload bay at the launch pad. The most significant mechanical task to be performed on Donatello in the SSPF is the installation and outfitting of the racks for carrying the various experiments and cargo.

Hydrogel Droplet Microfluidics for High-Throughput Single Molecule/Cell Analysis.

PubMed

Zhu, Zhi; Yang, Chaoyong James

2017-01-17

Heterogeneity among individual molecules and cells has posed significant challenges to traditional bulk assays, due to the assumption of average behavior, which would lose important biological information in heterogeneity and result in a misleading interpretation. Single molecule/cell analysis has become an important and emerging field in biological and biomedical research for insights into heterogeneity between large populations at high resolution. Compared with the ensemble bulk method, single molecule/cell analysis explores the information on time trajectories, conformational states, and interactions of individual molecules/cells, all key factors in the study of chemical and biological reaction pathways. Various powerful techniques have been developed for single molecule/cell analysis, including flow cytometry, atomic force microscopy, optical and magnetic tweezers, single-molecule fluorescence spectroscopy, and so forth. However, some of them have the low-throughput issue that has to analyze single molecules/cells one by one. Flow cytometry is a widely used high-throughput technique for single cell analysis but lacks the ability for intercellular interaction study and local environment control. Droplet microfluidics becomes attractive for single molecule/cell manipulation because single molecules/cells can be individually encased in monodisperse microdroplets, allowing high-throughput analysis and manipulation with precise control of the local environment. Moreover, hydrogels, cross-linked polymer networks that swell in the presence of water, have been introduced into droplet microfluidic systems as hydrogel droplet microfluidics. By replacing an aqueous phase with a monomer or polymer solution, hydrogel droplets can be generated on microfluidic chips for encapsulation of single molecules/cells according to the Poisson distribution. The sol-gel transition property endows the hydrogel droplets with new functionalities and diversified applications in single molecule/cell analysis. The hydrogel can act as a 3D cell culture matrix to mimic the extracellular environment for long-term single cell culture, which allows further heterogeneity study in proliferation, drug screening, and metastasis at the single-cell level. The sol-gel transition allows reactions in solution to be performed rapidly and efficiently with product storage in the gel for flexible downstream manipulation and analysis. More importantly, controllable sol-gel regulation provides a new way to maintain phenotype-genotype linkages in the hydrogel matrix for high throughput molecular evolution. In this Account, we will review the hydrogel droplet generation on microfluidics, single molecule/cell encapsulation in hydrogel droplets, as well as the progress made by our group and others in the application of hydrogel droplet microfluidics for single molecule/cell analysis, including single cell culture, single molecule/cell detection, single cell sequencing, and molecular evolution.

Significantly improved precision of cell migration analysis in time-lapse video microscopy through use of a fully automated tracking system

PubMed Central

2010-01-01

Background Cell motility is a critical parameter in many physiological as well as pathophysiological processes. In time-lapse video microscopy, manual cell tracking remains the most common method of analyzing migratory behavior of cell populations. In addition to being labor-intensive, this method is susceptible to user-dependent errors regarding the selection of "representative" subsets of cells and manual determination of precise cell positions. Results We have quantitatively analyzed these error sources, demonstrating that manual cell tracking of pancreatic cancer cells lead to mis-calculation of migration rates of up to 410%. In order to provide for objective measurements of cell migration rates, we have employed multi-target tracking technologies commonly used in radar applications to develop fully automated cell identification and tracking system suitable for high throughput screening of video sequences of unstained living cells. Conclusion We demonstrate that our automatic multi target tracking system identifies cell objects, follows individual cells and computes migration rates with high precision, clearly outperforming manual procedures. PMID:20377897

All-printed capacitors with continuous solution dispensing technology

NASA Astrophysics Data System (ADS)

Ge, Yang; Plötner, Matthias; Berndt, Andreas; Kumar, Amit; Voit, Brigitte; Pospiech, Doris; Fischer, Wolf-Joachim

2017-09-01

Printed electronics have been introduced into the commercial markets in recent years. Various printing technologies have emerged aiming to process printed electronic devices with low cost, environmental friendliness, and compatibility with large areas and flexible substrates. The aim of this study is to propose a continuous solution dispensing technology for processing all-printed thin-film capacitors on glass substrates using a leading-edge printing instrument. Among all printing technologies, this study provides concrete proof of the following outstanding advantages of this technology: high tolerance to inks, high throughput, low cost, and precise pattern transfers. Ag nanoparticle ink based on glycol ethers was used to print the electrodes. To obtain dielectric ink, a copolymer powder of poly(methyl methacrylate-co-benzoylphenyl methacrylate) containing crosslinkable side groups was dissolved in anisole. Various layouts were designed to support multiple electronic applications. Scanning electron microscopy and atomic force microscopy were used to investigate the all-printed capacitor layers formed using the proposed process. Additionally, the printed capacitors were electrically characterized under direct current and alternating current. The measured electrical properties of the printed capacitors were consistent with the theoretical results.

sideSPIM - selective plane illumination based on a conventional inverted microscope.

PubMed

Hedde, Per Niklas; Malacrida, Leonel; Ahrar, Siavash; Siryaporn, Albert; Gratton, Enrico

2017-09-01

Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or vice versa . Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to 5 stacks/s. Additionally, sample handling is compatible with established methods and switching magnification to change the field of view from single cells to whole organisms does not require labor intensive adjustments of the system.

Deep learning massively accelerates super-resolution localization microscopy.

PubMed

Ouyang, Wei; Aristov, Andrey; Lelek, Mickaël; Hao, Xian; Zimmer, Christophe

2018-06-01

The speed of super-resolution microscopy methods based on single-molecule localization, for example, PALM and STORM, is limited by the need to record many thousands of frames with a small number of observed molecules in each. Here, we present ANNA-PALM, a computational strategy that uses artificial neural networks to reconstruct super-resolution views from sparse, rapidly acquired localization images and/or widefield images. Simulations and experimental imaging of microtubules, nuclear pores, and mitochondria show that high-quality, super-resolution images can be reconstructed from up to two orders of magnitude fewer frames than usually needed, without compromising spatial resolution. Super-resolution reconstructions are even possible from widefield images alone, though adding localization data improves image quality. We demonstrate super-resolution imaging of >1,000 fields of view containing >1,000 cells in ∼3 h, yielding an image spanning spatial scales from ∼20 nm to ∼2 mm. The drastic reduction in acquisition time and sample irradiation afforded by ANNA-PALM enables faster and gentler high-throughput and live-cell super-resolution imaging.

Molecular threading: mechanical extraction, stretching and placement of DNA molecules from a liquid-air interface.

PubMed

Payne, Andrew C; Andregg, Michael; Kemmish, Kent; Hamalainen, Mark; Bowell, Charlotte; Bleloch, Andrew; Klejwa, Nathan; Lehrach, Wolfgang; Schatz, Ken; Stark, Heather; Marblestone, Adam; Church, George; Own, Christopher S; Andregg, William

2013-01-01

We present "molecular threading", a surface independent tip-based method for stretching and depositing single and double-stranded DNA molecules. DNA is stretched into air at a liquid-air interface, and can be subsequently deposited onto a dry substrate isolated from solution. The design of an apparatus used for molecular threading is presented, and fluorescence and electron microscopies are used to characterize the angular distribution, straightness, and reproducibility of stretched DNA deposited in arrays onto elastomeric surfaces and thin membranes. Molecular threading demonstrates high straightness and uniformity over length scales from nanometers to micrometers, and represents an alternative to existing DNA deposition and linearization methods. These results point towards scalable and high-throughput precision manipulation of single-molecule polymers.

Strategic and Operational Plan for Integrating Transcriptomics ...

EPA Pesticide Factsheets

Plans for incorporating high throughput transcriptomics into the current high throughput screening activities at NCCT; the details are in the attached slide presentation presentation on plans for incorporating high throughput transcriptomics into the current high throughput screening activities at NCCT, given at the OECD meeting on June 23, 2016

High-Throughput Experimental Approach Capabilities | Materials Science |

Science.gov Websites

NREL High-Throughput Experimental Approach Capabilities High-Throughput Experimental Approach by yellow and is for materials in the upper right sector. NREL's high-throughput experimental ,Te) and oxysulfide sputtering Combi-5: Nitrides and oxynitride sputtering We also have several non

Ultra-High Efficiency / Low Hydrogen Embrittlement Nanostructured Zn-Based Electrodeposits as Environmentally Benign Cd-Replacement Coatings for High Strength Steel Fasteners

DTIC Science & Technology

2011-04-01

forma The prese corrosion coating) w conversio converted not develo before and a : Example un ted Zn-Ni ac : Example s Zn-Ni alkalin nt of scribing...Protection Agency, 1998. Retrieved from web July 30th 2009. Available online at www.epa.gov/ iris /subst/0141.htm. 3 M. Bielawski, Surface and

The RootScope: a simple high-throughput screening system for quantitating gene expression dynamics in plant roots

PubMed Central

2013-01-01

Background High temperature stress responses are vital for plant survival. The mechanisms that plants use to sense high temperatures are only partially understood and involve multiple sensing and signaling pathways. Here we describe the development of the RootScope, an automated microscopy system for quantitating heat shock responses in plant roots. Results The promoter of Hsp17.6 was used to build a Hsp17.6p:GFP transcriptional reporter that is induced by heat shock in Arabidopsis. An automated fluorescence microscopy system which enables multiple roots to be imaged in rapid succession was used to quantitate Hsp17.6p:GFP response dynamics. Hsp17.6p:GFP signal increased with temperature increases from 28°C to 37°C. At 40°C the kinetics and localization of the response are markedly different from those at 37°C. This suggests that different mechanisms mediate heat shock responses above and below 37°C. Finally, we demonstrate that Hsp17.6p:GFP expression exhibits wave like dynamics in growing roots. Conclusions The RootScope system is a simple and powerful platform for investigating the heat shock response in plants. PMID:24119322

Learning from Heterogeneous Data Sources: An Application in Spatial Proteomics

PubMed Central

Breckels, Lisa M.; Holden, Sean B.; Wojnar, David; Mulvey, Claire M.; Christoforou, Andy; Groen, Arnoud; Trotter, Matthew W. B.; Kohlbacher, Oliver; Lilley, Kathryn S.; Gatto, Laurent

2016-01-01

Sub-cellular localisation of proteins is an essential post-translational regulatory mechanism that can be assayed using high-throughput mass spectrometry (MS). These MS-based spatial proteomics experiments enable us to pinpoint the sub-cellular distribution of thousands of proteins in a specific system under controlled conditions. Recent advances in high-throughput MS methods have yielded a plethora of experimental spatial proteomics data for the cell biology community. Yet, there are many third-party data sources, such as immunofluorescence microscopy or protein annotations and sequences, which represent a rich and vast source of complementary information. We present a unique transfer learning classification framework that utilises a nearest-neighbour or support vector machine system, to integrate heterogeneous data sources to considerably improve on the quantity and quality of sub-cellular protein assignment. We demonstrate the utility of our algorithms through evaluation of five experimental datasets, from four different species in conjunction with four different auxiliary data sources to classify proteins to tens of sub-cellular compartments with high generalisation accuracy. We further apply the method to an experiment on pluripotent mouse embryonic stem cells to classify a set of previously unknown proteins, and validate our findings against a recent high resolution map of the mouse stem cell proteome. The methodology is distributed as part of the open-source Bioconductor pRoloc suite for spatial proteomics data analysis. PMID:27175778

Spatially Fourier-encoded photoacoustic microscopy using a digital micromirror device.

PubMed

Liang, Jinyang; Gao, Liang; Li, Chiye; Wang, Lihong V

2014-02-01

We have developed spatially Fourier-encoded photoacoustic (PA) microscopy using a digital micromirror device. The spatial intensity distribution of laser pulses is Fourier-encoded, and a series of such encoded PA measurements allows one to decode the spatial distribution of optical absorption. The throughput and Fellgett advantages were demonstrated by imaging a chromium target. By using 63 spatial elements, the signal-to-noise ratio in the recovered PA signal was enhanced by ∼4×. The system was used to image two biological targets, a monolayer of red blood cells and melanoma cells.

Spatially Fourier-encoded photoacoustic microscopy using a digital micromirror device

PubMed Central

Liang, Jinyang; Gao, Liang; Li, Chiye; Wang, Lihong V.

2014-01-01

We have developed spatially Fourier-encoded photoacoustic microscopy using a digital micromirror device. The spatial intensity distribution of laser pulses is Fourier-encoded, and a series of such encoded photoacoustic measurements allows one to decode the spatial distribution of optical absorption. The throughput and Fellgett advantages were demonstrated by imaging a chromium target. By using 63 spatial elements, the signal-to-noise ratio in the recovered photoacoustic signal was enhanced by ~4×. The system was used to image two biological targets, a monolayer of red blood cells and melanoma cells. PMID:24487832

The Role of Experiential Learning in Nurturing Management Competencies in Hospitality and Tourism Management Students: Perceptions from Students, Faculty, and Industry Professionals

ERIC Educational Resources Information Center

Jack, Kristen

2011-01-01

Previous research has revealed that industry professionals and educators in the field of Hospitality and Tourism Management (HTM) agree that classroom theory and experience in the field are essential components of undergraduate HTM education. Yet there is some disagreement on internship hour requirements, and limited data on actual outcomes. This…

77 FR 14972 - Discrimination on the Basis of Disability in Federally Assisted Programs and Activities

Federal Register 2010, 2011, 2012, 2013, 2014

2012-03-14

...'s ADA Web site contains links to HTML and PDF versions at www.ada.gov/regs2010/ADAregs2010.htm . \\15... Transportation Barriers Compliance Board) in 1991 (1991 ADAAG). DOJ's ADA Web site contains links to HTML and PDF.../2010ADAstandards_index.htm . There are links to HTML, PDF screen, and PDF print versions of the 2010 Standards and...

Adding Nonlinear Tools to the Strategist’s Toolbox

DTIC Science & Technology

2001-06-01

between interests. Idealists base their arguments on the pioneers like Hugo Grotius (1583-1645) and Immanuel Kant (1724-1804). The Idealist school based...12Blupete, [website]: available from http://www.blupete.com/Literature/ Biographies / Philosophy/Popper.htm; Internet. 13Glesne and Peshkin, 24. 39...www.blupete.com/Literature/ Biographies / Philosophy/Popper.htm. Internet. Dean Robbins, Brent. Mythos and Logos. Available from http://www.geocities.com/ Athens

Binary ROCs in Perception and Recognition Memory Are Curved

ERIC Educational Resources Information Center

Dube, Chad; Rotello, Caren M.

2012-01-01

In recognition memory, a classic finding is that receiver operating characteristics (ROCs) are curvilinear. This has been taken to support the fundamental assumptions of signal detection theory (SDT) over discrete-state models such as the double high-threshold model (2HTM), which predicts linear ROCs. Recently, however, Broder and Schutz (2009)…

CellAnimation: an open source MATLAB framework for microscopy assays.

PubMed

Georgescu, Walter; Wikswo, John P; Quaranta, Vito

2012-01-01

Advances in microscopy technology have led to the creation of high-throughput microscopes that are capable of generating several hundred gigabytes of images in a few days. Analyzing such wealth of data manually is nearly impossible and requires an automated approach. There are at present a number of open-source and commercial software packages that allow the user to apply algorithms of different degrees of sophistication to the images and extract desired metrics. However, the types of metrics that can be extracted are severely limited by the specific image processing algorithms that the application implements, and by the expertise of the user. In most commercial software, code unavailability prevents implementation by the end user of newly developed algorithms better suited for a particular type of imaging assay. While it is possible to implement new algorithms in open-source software, rewiring an image processing application requires a high degree of expertise. To obviate these limitations, we have developed an open-source high-throughput application that allows implementation of different biological assays such as cell tracking or ancestry recording, through the use of small, relatively simple image processing modules connected into sophisticated imaging pipelines. By connecting modules, non-expert users can apply the particular combination of well-established and novel algorithms developed by us and others that are best suited for each individual assay type. In addition, our data exploration and visualization modules make it easy to discover or select specific cell phenotypes from a heterogeneous population. CellAnimation is distributed under the Creative Commons Attribution-NonCommercial 3.0 Unported license (http://creativecommons.org/licenses/by-nc/3.0/). CellAnimationsource code and documentation may be downloaded from www.vanderbilt.edu/viibre/software/documents/CellAnimation.zip. Sample data are available at www.vanderbilt.edu/viibre/software/documents/movies.zip. walter.georgescu@vanderbilt.edu Supplementary data available at Bioinformatics online.

Cellular Fibronectin Expression in Human Trabecular Meshwork and Induction by Transforming Growth Factor-β2

PubMed Central

Medina-Ortiz, Wanda E.; Belmares, Ricardo; Neubauer, Sandra; Wordinger, Robert J.; Clark, Abbot F.

2013-01-01

Purpose. Levels of TGF-β2 are higher in POAG aqueous humor, causing deposition of extracellular matrix (ECM) proteins, including fibronectin (FN), in the glaucomatous human trabecular meshwork (HTM) that may be responsible for elevated IOP. The purpose of this study was to identify the expression of cellular FN (cFN) isoforms (EDA and EDB) in HTM cells and tissues, and to determine whether TGF-β2 can induce cFN expression and fibril formation in cultured HTM cells. Methods. Expression of cFN mRNA isoforms and induction by recombinant TGF-β2 (5 ng/mL) were determined by quantitative RT-PCR. The TGF-β2 induction of EDA isoform protein expression and FN fibril formation were analyzed using Western immunoblots and immunocytochemistry (ICC), respectively. Immunohistochemistry (IHC) analysis was used to examine total FN and EDA isoform expression in normal (NTM) and glaucomatous (GTM) trabecular meshwork (TM) tissues. Results. Both cFN mRNA isoforms were expressed in cultured HTM cells and were induced by TGF-β2 after 2, 4, and 7 days (P < 0.05). Similarly, EDA isoform protein and fibril formation were increased after 4 and 7 days of TGF-β2 treatment. Finally, GTM tissues had significantly greater EDA isoform protein levels (1.7-fold, P < 0.05) compared to NTM tissues. Conclusions. This study demonstrated that cFN isoforms are expressed and induced in HTM cells by TGF-β2. Also, increased EDA isoform protein levels were seen in GTM tissues. Our findings suggest that induction of cFN isoform expression in the TM ECM may be a novel pathologic mechanism involved in the TM changes associated with glaucoma. PMID:24030464

Testing of Streptococcus pneumoniae for resistance to penicillin.

PubMed Central

Marshall, K J; Musher, D M; Watson, D; Mason, E O

1993-01-01

The increasing prevalence of penicillin-resistant Streptococcus pneumoniae requires antibiotic susceptibility tests that can be done with greater ease and reliability. We measured the MIC of penicillin for pneumococci by the tube macrodilution method with Mueller-Hinton broth (MHB), Haemophilus Test Medium (HTM), Todd-Hewitt broth with 0.5% yeast extract (THY), and MHB with 3% lysed horse blood (LHB). Eight (19%) and 6 (14%) of 42 pneumococcal isolates failed to generate turbid growth in MHB and HTM, respectively, whereas all pneumococcal isolates did so in THY and LHB. For those strains that replicated to turbidity, the mean MICs of penicillin were lower in MHB and HTM than in THY and LHB, with differences being significant (P < 0.05) for comparisons with LHB. Four isolates appeared to be penicillin susceptible in HTM but were actually moderately resistant in THY and LHB, and two isolates appeared to be moderately resistant but were resistant. A similar failure to detect resistance was seen with MHB. S. pneumoniae ATCC 49619, a moderately penicillin-resistant strain that has been proposed for quality control testing, gave variable results in MHB or THM and appeared to be susceptible to penicillin in some assays, whereas the MICs for S. pneumoniae ATCC 49619 in THY or LHB fell within a twofold dilution range, with geometric means of 0.16 and 0.18 micrograms/ml, respectively. Pneumococcal isolates thus may appear falsely susceptible to penicillin when tested in MHB or HTM. LHB remains the standard medium; however, because THY is an easily prepared clear medium that can be used in automated systems and appears to yield results similar to those obtained with LHB, THY deserves consideration for routine use. PMID:8501225

A high brightness probe of polymer nanoparticles for biological imaging

NASA Astrophysics Data System (ADS)

Zhou, Sirong; Zhu, Jiarong; Li, Yaping; Feng, Liheng

2018-03-01

Conjugated polymer nanoparticles (CPNs) with high brightness in long wavelength region were prepared by the nano-precipitation method. Based on fluorescence resonance energy transfer (FRET) mechanism, the high brightness property of the CPNs was realized by four different emission polymers. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) displayed that the CPNs possessed a spherical structure and an average diameter of 75 nm. Analysis assays showed that the CPNs had excellent biocompatibility, good photostability and low cytotoxicity. The CPNs were bio-modified with a cell penetrating peptide (Tat, a targeted element) through covalent link. Based on the entire wave fluorescence emission, the functionalized CPNs1-4 can meet multichannel and high throughput assays in cell and organ imaging. The contribution of the work lies in not only providing a new way to obtain a high brightness imaging probe in long wavelength region, but also using targeted cell and organ imaging.

Precision platform for convex lens-induced confinement microscopy

NASA Astrophysics Data System (ADS)

Berard, Daniel; McFaul, Christopher M. J.; Leith, Jason S.; Arsenault, Adriel K. J.; Michaud, François; Leslie, Sabrina R.

2013-10-01

We present the conception, fabrication, and demonstration of a versatile, computer-controlled microscopy device which transforms a standard inverted fluorescence microscope into a precision single-molecule imaging station. The device uses the principle of convex lens-induced confinement [S. R. Leslie, A. P. Fields, and A. E. Cohen, Anal. Chem. 82, 6224 (2010)], which employs a tunable imaging chamber to enhance background rejection and extend diffusion-limited observation periods. Using nanopositioning stages, this device achieves repeatable and dynamic control over the geometry of the sample chamber on scales as small as the size of individual molecules, enabling regulation of their configurations and dynamics. Using microfluidics, this device enables serial insertion as well as sample recovery, facilitating temporally controlled, high-throughput measurements of multiple reagents. We report on the simulation and experimental characterization of this tunable chamber geometry, and its influence upon the diffusion and conformations of DNA molecules over extended observation periods. This new microscopy platform has the potential to capture, probe, and influence the configurations of single molecules, with dramatically improved imaging conditions in comparison to existing technologies. These capabilities are of immediate interest to a wide range of research and industry sectors in biotechnology, biophysics, materials, and chemistry.

Automated 100-Position Specimen Loader and Image Acquisition System for Transmission Electron Microscopy

PubMed Central

Lefman, Jonathan; Morrison, Robert; Subramaniam, Sriram

2007-01-01

We report the development of a novel, multi-specimen imaging system for high-throughput transmission electron microscopy. Our cartridge-based loading system, called the “Gatling”, permits the sequential examination of as many as 100 specimens in the microscope for room temperature electron microscopy using mechanisms for rapid and automated specimen exchange. The software for the operation of the Gatling and automated data acquisition has been implemented in an updated version of our in-house program AutoEM. In the current implementation of the system, the time required to deliver 95 specimens into the microscope and collect overview images from each is about 13 hours. Regions of interest are identified from a low magnification atlas generation from each specimen and an unlimited number of higher magnifications images can be subsequently acquired from these regions using fully automated data acquisition procedures that can be controlled from a remote interface. We anticipate that the availability of the Gatling will greatly accelerate the speed of data acquisition for a variety of applications in biology, materials science and nanotechnology that require rapid screening and image analysis of multiple specimens. PMID:17240161

Hole-Transport Materials for Perovskite Solar Cells.

PubMed

Calió, Laura; Kazim, Samrana; Grätzel, Michael; Ahmad, Shahzada

2016-11-14

The pressure to move towards renewable energy has inspired researchers to look for ideas in photovoltaics that may lead to a major breakthrough. Recently the use of perovskites as a light harvester has lead to stunning progress. The power conversion efficiency of perovskite solar cells is now approaching parity (>22 %) with that of the established technology which took decades to reach this level of performance. The use of a hole transport material (HTM) remains indispensable in perovskite solar cells. Perovskites can conduct holes, but they are present at low levels, and for efficient charge extraction a HTM layer is a prerequisite. Herein we provide an overview of the diverse types of HTM available, from organic to inorganic, in the hope of encouraging further research and the optimization of these materials. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kinetics of liquid-mediated crystallization of amorphous Ge from multi-frame dynamic transmission electron microscopy

DOE PAGES

Santala, M. K.; Raoux, S.; Campbell, G. H.

2015-12-24

The kinetics of laser-induced, liquid-mediated crystallization of amorphous Ge thin films were studied using multi-frame dynamic transmission electron microscopy (DTEM), a nanosecond-scale photo-emission transmission electron microscopy technique. In these experiments, high temperature gradients are established in thin amorphous Ge films with a 12-ns laser pulse with a Gaussian spatial profile. The hottest region at the center of the laser spot crystallizes in ~100 ns and becomes nano-crystalline. Over the next several hundred nanoseconds crystallization continues radially outward from the nano-crystalline region forming elongated grains, some many microns long. The growth rate during the formation of these radial grains is measuredmore » with time-resolved imaging experiments. Crystal growth rates exceed 10 m/s, which are consistent with crystallization mediated by a very thin, undercooled transient liquid layer, rather than a purely solid-state transformation mechanism. The kinetics of this growth mode have been studied in detail under steady-state conditions, but here we provide a detailed study of liquid-mediated growth in high temperature gradients. Unexpectedly, the propagation rate of the crystallization front was observed to remain constant during this growth mode even when passing through large local temperature gradients, in stark contrast to other similar studies that suggested the growth rate changed dramatically. As a result, the high throughput of multi-frame DTEM provides gives a more complete picture of the role of temperature and temperature gradient on laser crystallization than previous DTEM experiments.« less

Kinetics of liquid-mediated crystallization of amorphous Ge from multi-frame dynamic transmission electron microscopy

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

Santala, M. K., E-mail: melissa.santala@oregonstate.edu; Campbell, G. H.; Raoux, S.

2015-12-21

The kinetics of laser-induced, liquid-mediated crystallization of amorphous Ge thin films were studied using multi-frame dynamic transmission electron microscopy (DTEM), a nanosecond-scale photo-emission transmission electron microscopy technique. In these experiments, high temperature gradients are established in thin amorphous Ge films with a 12-ns laser pulse with a Gaussian spatial profile. The hottest region at the center of the laser spot crystallizes in ∼100 ns and becomes nano-crystalline. Over the next several hundred nanoseconds crystallization continues radially outward from the nano-crystalline region forming elongated grains, some many microns long. The growth rate during the formation of these radial grains is measured withmore » time-resolved imaging experiments. Crystal growth rates exceed 10 m/s, which are consistent with crystallization mediated by a very thin, undercooled transient liquid layer, rather than a purely solid-state transformation mechanism. The kinetics of this growth mode have been studied in detail under steady-state conditions, but here we provide a detailed study of liquid-mediated growth in high temperature gradients. Unexpectedly, the propagation rate of the crystallization front was observed to remain constant during this growth mode even when passing through large local temperature gradients, in stark contrast to other similar studies that suggested the growth rate changed dramatically. The high throughput of multi-frame DTEM provides gives a more complete picture of the role of temperature and temperature gradient on laser crystallization than previous DTEM experiments.« less

2008 U.S. Coast Guard Innovation Expo

DTIC Science & Technology

2008-11-20

for Capability (CG-7) Public Release Authorized DCO Outcomes • Mission Performance Plans, policies, strategic analysis and planning, assessments and...Untitled Document 2008 U.S.htm[5/26/ 2016 2:24:38 PM] 2008 U.S. Coast Guard Innovation Expo “Collaboration Fuels Innovation” Virginia Beach, Virginia...Untitled Document 2008 U.S.htm[5/26/ 2016 2:24:38 PM] Panelists: Mr. Tom Tomaiko, Maritime Security Program Manager, Borders and Maritime Security

Effects of various media on the activity of NXL103 (formerly XRP 2868), a new oral streptogramin, against Haemophilus influenzae.

PubMed

Pankuch, Glenn A; Hoellman, Dianne; Bryskier, André; Lowther, John; Appelbaum, Peter C

2006-11-01

The activity of NXL103 against 108 strains of Haemophilus influenzae was tested using Haemophilus test media (HTM) obtained from various sources. With the exception of those obtained with stored HTM, MICs did not differ significantly, with MIC(50) and MIC(90) values of 0.5 and 0.5 to 1 microg/ml, respectively, in each medium.

Peacekeeping/Stabilization and Conflict Transitions: Background and Congressional Action on the Civilian Response/Reserve Corps and Other Civilian Stabilization and Reconstruction Capabilities

DTIC Science & Technology

2009-02-05

Peace Operations. February 24, 2000 http://www.fas.org/irp/ offdocs/pdd/pdd-71-4.htm, hereafter referred to as PDD-71 White Paper; and U.S. Text ... Summary of Presidential Decision Directive 71, http://www.fas.org/irp/offdocs/pdd/pdd-71-1.htm. 8 See CRS Report RL32321, Policing in Peacekeeping and

Dynamic electronic institutions in agent oriented cloud robotic systems.

PubMed

Nagrath, Vineet; Morel, Olivier; Malik, Aamir; Saad, Naufal; Meriaudeau, Fabrice

2015-01-01

The dot-com bubble bursted in the year 2000 followed by a swift movement towards resource virtualization and cloud computing business model. Cloud computing emerged not as new form of computing or network technology but a mere remoulding of existing technologies to suit a new business model. Cloud robotics is understood as adaptation of cloud computing ideas for robotic applications. Current efforts in cloud robotics stress upon developing robots that utilize computing and service infrastructure of the cloud, without debating on the underlying business model. HTM5 is an OMG's MDA based Meta-model for agent oriented development of cloud robotic systems. The trade-view of HTM5 promotes peer-to-peer trade amongst software agents. HTM5 agents represent various cloud entities and implement their business logic on cloud interactions. Trade in a peer-to-peer cloud robotic system is based on relationships and contracts amongst several agent subsets. Electronic Institutions are associations of heterogeneous intelligent agents which interact with each other following predefined norms. In Dynamic Electronic Institutions, the process of formation, reformation and dissolution of institutions is automated leading to run time adaptations in groups of agents. DEIs in agent oriented cloud robotic ecosystems bring order and group intellect. This article presents DEI implementations through HTM5 methodology.

Fourier Ptychographic Microscopy for Rapid, High-Resolution Imaging of Circulating Tumor Cells Enriched by Microfiltration.

PubMed

Williams, Anthony; Chung, Jaebum; Yang, Changhuei; Cote, Richard J

2017-01-01

Examining the hematogenous compartment for evidence of metastasis has increased significantly within the oncology research community in recent years, due to the development of technologies aimed at the enrichment of circulating tumor cells (CTCs), the subpopulation of primary tumor cells that gain access to the circulatory system and are responsible for colonization at distant sites. In contrast to other technologies, filtration-based CTC enrichment, which exploits differences in size between larger tumor cells and surrounding smaller, non-tumor blood cells, has the potential to improve CTC characterization through isolation of tumor cell populations with greater molecular heterogeneity. However, microscopic analysis of uneven filtration surfaces containing CTCs is laborious, time-consuming, and inconsistent, preventing widespread use of filtration-based enrichment technologies. Here, integrated with a microfiltration-based CTC and rare cell enrichment device we have previously described, we present a protocol for Fourier Ptychographic Microscopy (FPM), a method that, unlike many automated imaging platforms, produces high-speed, high-resolution images that can be digitally refocused, allowing users to observe objects of interest present on multiple focal planes within the same image frame. The development of a cost-effective and high-throughput CTC analysis system for filtration-based enrichment technologies could have profound clinical implications for improved CTC detection and analysis.

Interlaced zone plate optics for hard X-ray imaging in the 10 nm range

DOE PAGES

Mohacsi, Istvan; Vartiainen, Ismo; Rosner, Benedikt; ...

2017-03-08

Multi-keV X-ray microscopy has been particularly successful in bridging the resolution gap between optical and electron microscopy. However, resolutions below 20 nm are still considered challenging, as high throughput direct imaging methods are limited by the availability of suitable optical elements. In order to bridge this gap, we present a new type of Fresnel zone plate lenses aimed at the sub-20 and the sub-10 nm resolution range. By extending the concept of double-sided zone plate stacking, we demonstrate the doubling of the effective line density and thus the resolution and provide large aperture, single- chip optical devices with 15 andmore » 7 nm smallest zone widths. The detailed characterization of these lenses shows excellent optical properties with focal spots down to 7.8 nm. Furthermore, beyond wave front characterization, the zone plates also excel in typical imaging scenarios, verifying their resolution close to their diffraction limited optical performance.« less

High-throughput growth temperature optimization of ferroelectric SrxBa1-xNb2O6 epitaxial thin films using a temperature gradient method

NASA Astrophysics Data System (ADS)

Ohkubo, I.; Christen, H. M.; Kalinin, Sergei V.; Jellison, G. E.; Rouleau, C. M.; Lowndes, D. H.

2004-02-01

We have developed a multisample film growth method on a temperature-gradient substrate holder to quickly optimize the film growth temperature in pulsed-laser deposition. A smooth temperature gradient is achieved, covering a range of temperatures from 200 to 830 °C. In a single growth run, the optimal growth temperature for SrxBa1-xNb2O6 thin films on MgO(001) substrates was determined to be 750 °C, based on results from ellipsometry and piezoresponse force microscopy. Variations in optical properties and ferroelectric domains structures were clearly observed as function of growth temperature, and these physical properties can be related to their different crystalline quality. Piezoresponse force microscopy indicated the formation of uniform ferroelectric film for deposition temperatures above 750 °C. At 660 °C, isolated micron-sized ferroelectric islands were observed, while samples deposited below 550 °C did not exhibit clear piezoelectric contrast.

Interlaced zone plate optics for hard X-ray imaging in the 10 nm range

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

Mohacsi, Istvan; Vartiainen, Ismo; Rosner, Benedikt

Multi-keV X-ray microscopy has been particularly successful in bridging the resolution gap between optical and electron microscopy. However, resolutions below 20 nm are still considered challenging, as high throughput direct imaging methods are limited by the availability of suitable optical elements. In order to bridge this gap, we present a new type of Fresnel zone plate lenses aimed at the sub-20 and the sub-10 nm resolution range. By extending the concept of double-sided zone plate stacking, we demonstrate the doubling of the effective line density and thus the resolution and provide large aperture, single- chip optical devices with 15 andmore » 7 nm smallest zone widths. The detailed characterization of these lenses shows excellent optical properties with focal spots down to 7.8 nm. Furthermore, beyond wave front characterization, the zone plates also excel in typical imaging scenarios, verifying their resolution close to their diffraction limited optical performance.« less

Interlaced zone plate optics for hard X-ray imaging in the 10 nm range

PubMed Central

Mohacsi, Istvan; Vartiainen, Ismo; Rösner, Benedikt; Guizar-Sicairos, Manuel; Guzenko, Vitaliy A.; McNulty, Ian; Winarski, Robert; Holt, Martin V.; David, Christian

2017-01-01

Multi-keV X-ray microscopy has been particularly successful in bridging the resolution gap between optical and electron microscopy. However, resolutions below 20 nm are still considered challenging, as high throughput direct imaging methods are limited by the availability of suitable optical elements. In order to bridge this gap, we present a new type of Fresnel zone plate lenses aimed at the sub-20 and the sub-10 nm resolution range. By extending the concept of double-sided zone plate stacking, we demonstrate the doubling of the effective line density and thus the resolution and provide large aperture, singlechip optical devices with 15 and 7 nm smallest zone widths. The detailed characterization of these lenses shows excellent optical properties with focal spots down to 7.8 nm. Beyond wave front characterization, the zone plates also excel in typical imaging scenarios, verifying their resolution close to their diffraction limited optical performance.

Interlaced zone plate optics for hard X-ray imaging in the 10 nm range

NASA Astrophysics Data System (ADS)

Mohacsi, Istvan; Vartiainen, Ismo; Rösner, Benedikt; Guizar-Sicairos, Manuel; Guzenko, Vitaliy A.; McNulty, Ian; Winarski, Robert; Holt, Martin V.; David, Christian

2017-03-01

Multi-keV X-ray microscopy has been particularly successful in bridging the resolution gap between optical and electron microscopy. However, resolutions below 20 nm are still considered challenging, as high throughput direct imaging methods are limited by the availability of suitable optical elements. In order to bridge this gap, we present a new type of Fresnel zone plate lenses aimed at the sub-20 and the sub-10 nm resolution range. By extending the concept of double-sided zone plate stacking, we demonstrate the doubling of the effective line density and thus the resolution and provide large aperture, singlechip optical devices with 15 and 7 nm smallest zone widths. The detailed characterization of these lenses shows excellent optical properties with focal spots down to 7.8 nm. Beyond wave front characterization, the zone plates also excel in typical imaging scenarios, verifying their resolution close to their diffraction limited optical performance.

Development of high-productivity, strong cation-exchange adsorbers for protein capture by graft polymerization from membranes with different pore sizes

PubMed Central

Chenette, Heather C.S.; Robinson, Julie R.; Hobley, Eboni; Husson, Scott M.

2012-01-01

This paper describes the surface modification of macroporous membranes using ATRP (atom transfer radical polymerization) to create cation-exchange adsorbers with high protein binding capacity at high product throughput. The work is motivated by the need for a more economical and rapid capture step in downstream processing of protein therapeutics. Membranes with three reported nominal pore sizes (0.2, 0.45, 1.0 μm) were modified with poly(3-sulfopropyl methacrylate, potassium salt) tentacles, to create a high density of protein binding sites. A special formulation was used in which the monomer was protected by a crown ether to enable surface-initiated ATRP of this cationic polyelectrolyte. Success with modification was supported by chemical analysis using Fourier-transform infrared spectroscopy and indirectly by measurement of pure water flux as a function of polymerization time. Uniformity of modification within the membranes was visualized with confocal laser scanning microscopy. Static and dynamic binding capacities were measured using lysozyme protein to allow comparisons with reported performance data for commercial cation-exchange materials. Dynamic binding capacities were measured for flow rates ranging from 13 to 109 column volumes (CV)/min. Results show that this unique ATRP formulation can be used to fabricate cation-exchange membrane adsorbers with dynamic binding capacities as high as 70 mg/mL at a throughput of 100 CV/min and unprecedented productivity of 300 mg/mL/min. PMID:23175597

Evaluating multiplexed next-generation sequencing as a method in palynology for mixed pollen samples.

PubMed

Keller, A; Danner, N; Grimmer, G; Ankenbrand, M; von der Ohe, K; von der Ohe, W; Rost, S; Härtel, S; Steffan-Dewenter, I

2015-03-01

The identification of pollen plays an important role in ecology, palaeo-climatology, honey quality control and other areas. Currently, expert knowledge and reference collections are essential to identify pollen origin through light microscopy. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach, but the assessment of mixed pollen samples originating from multiple plant species is still a tedious and error-prone task. Next-generation sequencing has been proposed to avoid this hindrance. In this study we assessed mixed pollen probes through next-generation sequencing of amplicons from the highly variable, species-specific internal transcribed spacer 2 region of nuclear ribosomal DNA. Further, we developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. We assessed in total 16 mixed pollen samples, 14 originated from honeybee colonies and two from solitary bee nests. The sequencing technique resulted in higher taxon richness (deeper assignments and more identified taxa) compared to light microscopy. Abundance estimations from sequencing data were significantly correlated with counted abundances through light microscopy. Simulation analyses of taxon specificity and sensitivity indicate that 96% of taxa present in the database are correctly identifiable at the genus level and 70% at the species level. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialised palynological expert knowledge. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

77 FR 39959 - Draft Guidance To Implement Requirements for the Treatment of Air Quality Monitoring Data...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-06

... of the revised draft High Winds Guidance document, the EPA identifies example technical analyses that... identified analyses and any additional technical analyses that air agencies could use to demonstrate that the... Web site at http://www.epa.gov/ttn/analysis/exevents.htm for additional details on the draft non...

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy

PubMed Central

Eum, Juneyong; Kwak, Jina; Kim, Hee Joung; Ki, Seoyoung; Lee, Kooyeon; Raslan, Ahmed A.; Park, Ok Kyu; Chowdhury, Md Ashraf Uddin; Her, Song; Kee, Yun; Kwon, Seung-Hae; Hwang, Byung Joon

2016-01-01

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis. PMID:27869673

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

PubMed Central

Wang, Li; Carnegie, Graeme K.

2013-01-01

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction. PMID:23979513

Flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction.

PubMed

Wang, Li; Carnegie, Graeme K

2013-08-15

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

An Unsupervised kNN Method to Systematically Detect Changes in Protein Localization in High-Throughput Microscopy Images.

PubMed

Lu, Alex Xijie; Moses, Alan M

2016-01-01

Despite the importance of characterizing genes that exhibit subcellular localization changes between conditions in proteome-wide imaging experiments, many recent studies still rely upon manual evaluation to assess the results of high-throughput imaging experiments. We describe and demonstrate an unsupervised k-nearest neighbours method for the detection of localization changes. Compared to previous classification-based supervised change detection methods, our method is much simpler and faster, and operates directly on the feature space to overcome limitations in needing to manually curate training sets that may not generalize well between screens. In addition, the output of our method is flexible in its utility, generating both a quantitatively ranked list of localization changes that permit user-defined cut-offs, and a vector for each gene describing feature-wise direction and magnitude of localization changes. We demonstrate that our method is effective at the detection of localization changes using the Δrpd3 perturbation in Saccharomyces cerevisiae, where we capture 71.4% of previously known changes within the top 10% of ranked genes, and find at least four new localization changes within the top 1% of ranked genes. The results of our analysis indicate that simple unsupervised methods may be able to identify localization changes in images without laborious manual image labelling steps.

Exploring the diversity of Listeria monocytogenes biofilm architecture by high-throughput confocal laser scanning microscopy and the predominance of the honeycomb-like morphotype.

PubMed

Guilbaud, Morgan; Piveteau, Pascal; Desvaux, Mickaël; Brisse, Sylvain; Briandet, Romain

2015-03-01

Listeria monocytogenes is involved in food-borne illness with a high mortality rate. The persistence of the pathogen along the food chain can be associated with its ability to form biofilms on inert surfaces. While most of the phenotypes associated with biofilms are related to their spatial organization, most published data comparing biofilm formation by L. monocytogenes isolates are based on the quantitative crystal violet assay, which does not give access to structural information. Using a high-throughput confocal-imaging approach, the aim of this work was to decipher the structural diversity of biofilms formed by 96 L. monocytogenes strains isolated from various environments. Prior to large-scale analysis, an experimental design was created to improve L. monocytogenes biofilm formation in microscopic-grade microplates, with special emphasis on the growth medium composition. Microscopic analysis of biofilms formed under the selected conditions by the 96 isolates revealed only weak correlation between the genetic lineages of the isolates and the structural properties of the biofilms. However, a gradient in their geometric descriptors (biovolume, mean thickness, and roughness), ranging from flat multilayers to complex honeycomb-like structures, was shown. The dominant honeycomb-like morphotype was characterized by hollow voids hosting free-swimming cells and localized pockets containing mixtures of dead cells and extracellular DNA (eDNA). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Characterization and Fate of Gun and Rocket Propellant Residues on Testing and Training Ranges

DTIC Science & Technology

2008-01-01

ext/reg_e.html. 36. United States Environmental Protection Agency (2007) Integrated Risk Information System: Hydrogen cyanide, http://www.epa.gov/ iris ...Environmental Protection Agency (2007) Integrated Risk Information System: Benzene, http://www.epa.gov/ iris /subst/0276.htm 42. United States Environmental...Protection Agency (2007) Integrated Risk Information System: Toluene, http://www.epa.gov/ iris /subst/0118.htm. 43. National Library of Medicine’s TOXNET

Effects of Various Media on the Activity of NXL103 (Formerly XRP 2868), a New Oral Streptogramin, against Haemophilus influenzae

PubMed Central

Pankuch, Glenn A.; Hoellman, Dianne; Bryskier, André; Lowther, John; Appelbaum, Peter C.

2006-01-01

The activity of NXL103 against 108 strains of Haemophilus influenzae was tested using Haemophilus test media (HTM) obtained from various sources. With the exception of those obtained with stored HTM, MICs did not differ significantly, with MIC50 and MIC90 values of 0.5 and 0.5 to 1 μg/ml, respectively, in each medium. PMID:17065630

Rational design of dipolar chromophore as an efficient dopant-free hole-transporting material for perovskite solar cells

DOE PAGES

Li, Zhong’an; Zhu, Zonglong; Chueh, Chu -Chen; ...

2016-08-23

In this paper, an electron donor-acceptor (D-A) substituted dipolar chromophore ( BTPA-TCNE) is developed to serve as an efficient dopant-free hole-transporting material (HTM) for perovskite solar cells (PVSCs). BTPA-TCNE is synthesized via a simple reaction between a triphenylamine-based Michler’s base and tetracyanoethylene. This chromophore possesses a zwitterionic resonance structure in the ground state, as evidenced by X-ray crystallography and transient absorption spectroscopies. Moreover, BTPA-TCNE shows an antiparallel molecular packing (i.e., centrosymmetric dimers) in its crystalline state, which cancels out its overall molecular dipole moment to facilitate charge transport. Finally, BTPA-TCNE can be employed as an effective dopant-free HTM to realizemore » an efficient (PCE ≈ 17.0%) PVSC in the conventional n-i-p configuration, outperforming the control device with doped spiro-OMeTAD HTM.« less

Ratiometric and turn-on monitoring for heavy and transition metal ions in aqueous solution with a fluorescent peptide sensor.

PubMed

Joshi, Bishnu Prasad; Park, Junwon; Lee, Wan In; Lee, Keun-Hyeung

2009-05-15

A novel fluorescent peptide sensor containing tryptophan (donor) and dansyl fluorophore (acceptor) was synthesized for monitoring heavy and transition metal (HTM) ions on the basis of metal ion binding motif (Cys-X-X-X-Cys). The peptide probe successfully exhibited a turn on and ratiometric response for several heavy metal ions such as Hg(2+), Cd(2+), Pb(2+), Zn(2+), and Ag(+) in aqueous solution. The enhancements of emission intensity were achieved in the presence of the HTM ions by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The detection limits of the sensor for Cd(2+), Pb(2+), Zn(2+), and Ag(+) were lower than the EPA's drinking water maximum contaminant levels (MCL). We described the fluorescent enhancement, binding affinity, and detection limit of the peptide probe for HTM ions.

Analyzing Patents Generated by SBIR Firms

DTIC Science & Technology

2014-04-30

ac/ido/oeip/ taf /us_gov.htm   ^Åèìáëáíáçå=oÉëÉ~êÅÜ=mêçÖê~ãW= `êÉ~íáåÖ=póåÉêÖó=Ñçê=fåÑçêãÉÇ=`Ü~åÖÉ= = - 392 - A 2006 RAND report on the DoD SBIR...ac/ido/oeip/ taf /reports.htm#misc_info ^Åèìáëáíáçå=oÉëÉ~êÅÜ=mêçÖê~ãW= `êÉ~íáåÖ=póåÉêÖó=Ñçê=fåÑçêãÉÇ=`Ü~åÖÉ= = - 400 - patent protection and are...view of the many variations which may occur in corporate identifications. http://www.uspto.gov/web/offices/ac/ido/oeip/ taf /topo_04.htm#Desc 16

Accurate assessment of liver steatosis in animal models using a high throughput Raman fiber optic probe.

PubMed

Hewitt, Kevin C; Ghassemi Rad, Javad; McGregor, Hanna C; Brouwers, Erin; Sapp, Heidi; Short, Michael A; Fashir, Samia B; Zeng, Haishan; Alwayn, Ian P

2015-10-07

Due to the shortage of healthy donor organs, steatotic livers are commonly used for transplantation, placing patients at higher risk for graft dysfunction and lower survival rates. Raman Spectroscopy is a technique which has shown the ability to rapidly detect the vibration state of C-H bonds in triglycerides. The aim of this study is to determine whether conventional Raman spectroscopy can reliably detect and quantify fat in an animal model of liver steatosis. Mice and rats fed a methionine and choline-deficient (MCD) and control diets were sacrificed on one, two, three and four weeks' time points. A confocal Raman microscope, a commercial Raman (iRaman) fiber optic probe and a highly sensitive Raman fiber optic probe system, the latter utilizing a 785 nm excitation laser, were used to detect changes in the Raman spectra of steatotic mouse livers. Thin layer chromatography was used to assess the triglyceride content of liver specimens, and sections were scored blindly for fat content using histological examination. Principal component analysis (PCA) of Raman spectra was used to extract the principal components responsible for spectroscopic differences with MCD week (time on MCD diet). Confocal Raman microscopy revealed the presence of saturated fats in mice liver sections. A commercially available handheld Raman spectroscopy probe could not distinguish the presence of fat in the liver whereas our specially designed, high throughput Raman system could clearly distinguish lobe-specific changes in fat content. In the left lobe in particular, the Raman PC scores exhibited a significant correlation (R(2) = 0.96) with the gold standard, blinded scoring by histological examination. The specially designed, high throughput Raman system can be used for clinical purposes. Its application to the field of transplantation would enable surgeons to determine the hepatic fat content of the donor's liver in the field prior to proceeding with organ retrieval. Next steps include validating these results in a prospective analysis of human liver transplantation implant biopsies.

High Throughput PBTK: Open-Source Data and Tools for ...

EPA Pesticide Factsheets

Presentation on High Throughput PBTK at the PBK Modelling in Risk Assessment meeting in Ispra, Italy Presentation on High Throughput PBTK at the PBK Modelling in Risk Assessment meeting in Ispra, Italy

Characterization of a fluorescent hydrogel synthesized using chitosan, polyvinyl alcohol and 9-anthraldehyde for the selective detection and discrimination of trace Fe3+ and Fe2+ in water for live-cell imaging.

PubMed

Maity, Santu; Parshi, Nira; Prodhan, Chandraday; Chaudhuri, Keya; Ganguly, Jhuma

2018-08-01

A three-dimensional fluorescent hydrogel based on chitosan, polyvinyl alcohol and 9-anthraldehyde (ChPA) has been successfully designed and synthesized for the selective detection and discrimination of Fe 3+ and Fe 2+ in aqueous environment. The unique characteristics of ChPA has been confirmed by the Fourier-transform infrared spectroscopy (FTIR), rheological measurement, scanning electron microscopy (SEM), thermogravimetry and differential thermogravimetry (TG-DTG), ultraviolet-visible spectroscopy (UV-vis), fluorescence studies, transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDX), x-ray diffraction (XRD) and dynamic light scattering (DLS). The emission intensity at 516 nm of the hydrogel has been enhanced remarkably with the addition of Fe 3+ due to the inhibition of the photoinduced electron transfer (PET) process. However, it gets strongly quenched in the case of Fe 2+ owing to chelation enhanced quenching (CHEQ). The probe (ChPA) causes no significant change in the fluorescence and becomes highly specific and sensitive towards Fe 3+ and Fe 2+ compared to other interfering heavy and transition metal ions (HTM). The detection limits of the sensor for the Fe 3+ and Fe 2+ are 0.124 nM and 0.138 nM, respectively. The probe is also promising as a selective sensor for the Fe 3+ and Fe 2+ in the fluorescence imaging of living cells. Thus, such a probe opens up new opportunities to improve the chitosan based fluorescent chemosensor having biocompatibility, biodegradability, sufficient thermal stability and stability in a wide pH range. Copyright © 2018 Elsevier Ltd. All rights reserved.

Forward Masking in Cochlear Implant Users: Electrophysiological and Psychophysical Data Using Pulse Train Maskers.

PubMed

Adel, Youssef; Hilkhuysen, Gaston; Noreña, Arnaud; Cazals, Yves; Roman, Stéphane; Macherey, Olivier

2017-06-01

Electrical stimulation of auditory nerve fibers using cochlear implants (CI) shows psychophysical forward masking (pFM) up to several hundreds of milliseconds. By contrast, recovery of electrically evoked compound action potentials (eCAPs) from forward masking (eFM) was shown to be more rapid, with time constants no greater than a few milliseconds. These discrepancies suggested two main contributors to pFM: a rapid-recovery process due to refractory properties of the auditory nerve and a slow-recovery process arising from more central structures. In the present study, we investigate whether the use of different maskers between eCAP and psychophysical measures, specifically single-pulse versus pulse train maskers, may have been a source of confound.In experiment 1, we measured eFM using the following: a single-pulse masker, a 300-ms low-rate pulse train masker (LTM, 250 pps), and a 300-ms high-rate pulse train masker (HTM, 5000 pps). The maskers were presented either at same physical current (Φ) or at same perceptual (Ψ) level corresponding to comfortable loudness. Responses to a single-pulse probe were measured for masker-probe intervals ranging from 1 to 512 ms. Recovery from masking was much slower for pulse trains than for the single-pulse masker. When presented at Φ level, HTM produced more and longer-lasting masking than LTM. However, results were inconsistent when LTM and HTM were compared at Ψ level. In experiment 2, masked detection thresholds of single-pulse probes were measured using the same pulse train masker conditions. In line with our eFM findings, masked thresholds for HTM were higher than those for LTM at Φ level. However, the opposite result was found when the pulse trains were presented at Ψ level.Our results confirm the presence of slow-recovery phenomena at the level of the auditory nerve in CI users, as previously shown in animal studies. Inconsistencies between eFM and pFM results, despite using the same masking conditions, further underline the importance of comparing electrophysiological and psychophysical measures with identical stimulation paradigms.

Mexico-U.S. Relations: Issues for Congress

DTIC Science & Technology

2010-06-03

tuna to the U.S. market. A longstanding dispute involving sugar and high fructose corn syrup was resolved in 2006.95 Trucking96 Since 1995, the...Hillary Clinton led a cabinet-level delegation to Mexico for a meeting of the U.S.-Mexico Mérida Initiative High Level Group during which she announced a...http://www.state.gov/p/inl/rls/nrcrpt/2010/index.htm). The report praises the “ambitious pace” of the Mexican government’s efforts to arrest high -level

Isolation of Biologically Active Exosomes from Plasma of Patients with Cancer.

PubMed

Hong, Chang-Sook; Funk, Sonja; Whiteside, Theresa L

2017-01-01

A method for exosome isolation from human plasma was developed for rapid, high-throughput processing of plasma specimens obtained from patients with cancer. This method removes the bulk of plasma proteins associated with exosomes and can be used for comparative examinations of exosomes and their content in serial specimens of patients' plasma, allowing for monitoring changes in exosome numbers, profiles, and functions in the course of cancer progression or during therapy. The plasma-derived exosomes can be recovered in quantities sufficient for the characterization of their morphology by transmission electron microscopy (TEM), size and concentration by qNano, protein/lipid ratios, nucleic acid extraction, molecular profiling by Western blots or immune arrays, and functional assays.

Compartmental genomics in living cells revealed by single-cell nanobiopsy.

PubMed

Actis, Paolo; Maalouf, Michelle M; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R Adam; Pourmand, Nader

2014-01-28

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.

Application of ToxCast High-Throughput Screening and ...

EPA Pesticide Factsheets

Slide presentation at the SETAC annual meeting on High-Throughput Screening and Modeling Approaches to Identify Steroidogenesis Distruptors Slide presentation at the SETAC annual meeting on High-Throughput Screening and Modeling Approaches to Identify Steroidogenssis Distruptors

High permeable microporous structured carbon counter electrode assisted by polystyrene sphere for fully printable perovskite solar cells

NASA Astrophysics Data System (ADS)

Tao, Haijun; Li, Yongtao; Zhang, Chuanxiang; Wang, Kang; Wang, Jiayue; Tan, Bin; Han, Linxuan; Tao, Jie

2018-03-01

The permeability of the carbon counter electrode (CCE) is critical to the HTM-free fully printable perovskite solar cells. In this work, we report a CCE assisted by polystyrene spheres (PS-spheres) as pore-forming agent to gain microporous structure for a better permeability. Due to its decomposition temperature at 400 °C, the porous structure is obtained in the carbon layer easily. By optimization towards the contents of PS-spheres in CCE, the filling rate of perovskite solution and the photovoltaic performance of the device have been significantly improved. Using this method, an average efficiency enhancement of 22% has been obtained for HTM-free fully printable perovskite solar cells, resulting in a better fill rate of CH3NH3PbI3 and an efficiency of 4.49%. This kind of CCE with the advantages of simple, easy preparation process and well performance, show excellent potential application in perovskite solar cells.

What Are We Missing A Call for Red Teaming Within the Domestic Maritime Domain for Anti-Terrorism Programs

DTIC Science & Technology

2015-12-01

Sherman Kent Center, Central Intelligence Agency 3, no. 2 (October 2004), https://www.cia.gov/ library /kent-center-occasional-papers/vol3no2.htm...Naval Intelligence (Norfolk, VA: Naval Warfare Development Command, 2008), http://www.nwdc.navy.mil/content/ Library /Documents/NDPs/ ndp2/ndp20007.htm...Fathali M. Moghaddam, “ Multiculturalism and Intergroup Relations,” American Psychological Association, November 2011, 95. 114 Matherly, The Red Teaming

Measuring Cultural Change - A Management Focused Approach To Improving The Safety Culture

DTIC Science & Technology

2002-05-01

draws, and areas of potential harm for the patient Improved communication for the entire health care team. Cost saving with a heighten awareness of all...www.ama-assn.org/sci- pubs/amnews/pick_01/prsb0312.htm. Agency for Health Care Research and Quality, (2002). Patient fact sheet: five steps to...safer health care. Retrieved 25 March 2002 from the World Wide Web at: http://www.ahrq.gov/ consumer /5steps.htm. American Hospital Association

A pilot study: the effect of healing touch on anxiety, stress, pain, pain medication usage, and physiological measures in hospitalized sickle cell disease adults experiencing a vaso-occlusive pain episode.

PubMed

Thomas, Linda S; Stephenson, Nancy; Swanson, Mel; Jesse, D Elizabeth; Brown, Sylvia

2013-12-01

This pilot study was conducted to determine the effectiveness of Healing Touch on anxiety, stress, pain, pain medication usage, and selected physiological measures of hospitalized adults with sickle cell disease experiencing a vaso-occlusive pain episode. Healing Touch sessions were administered for 30 minutes on four consecutive days, and the self-reported data on anxiety, stress, pain, and the selected physiological data were collected while controlling for music and presence. A parallel-group randomized control trial comparing the effects of Healing Touch with Music (HTM) to Attention Control with Music (ACM). Due to the small sample size, there were no statistically significant changes in any between-group comparisons, except for present pain on Day 4 for the ACM group. For both groups, the within-group comparison showed a nonsignificant reduction in physiological parameters, a statistically significant reduction in anxiety and stress for the ACM group after Day 4, and a statistically significant reduction in stress in the HTM group after Days 2 and 4. The pre- to postintervention reductions in present pain were greater in the HTM group across all 4 days, but the only statistically significant within groups findings were in the HTM group (p < .01) on Day 1. Further research is needed.

Health technopole: innovation applied to clinical engineering & health technology management education.

PubMed

Vilcahuaman, L; Rivas, R

2010-01-01

In the Peruvian Health System, Clinical Engineering does not exist as a topic of intervention. 59% of biomedical equipment is officially classified as operational, however next to apply the correct classification methodology and include security issues, only 10% of the equipment are suitable for use in patients. The serious consequences for patients, is opposite to the increased public investment in the health sector. Reversing this context leads to structural changes at all levels of the organization and they will be achievable only through an appropriate educational program. A strategy focused on joint of capacities called Health Technopole has managed to implement an innovative Model of Education in Healthcare Technology Management HTM and Clinical Engineering CE aimed at solving this problem. The proposal focused on strategies to strengthen the educational goals such as creating HTM & CE Units in hospitals, the implementation of the methodology: Problem Based Learning and Project Management in HTM & CE in classroom and on line courses. The process includes an effective interaction with global organizations through teleconferences, Internships, Workshops and Seminars. A key component was the sustained multidisciplinary approach. Health Technopole CENGETS is an expert adviser for the Ministry of Health and is called for trainings, design training programs for regional governments and also supports global organizations such as PAHO / WHO and ORAS / CONHU. The proposal of innovation applied to HTM & CE Education is effective and is a benchmark for similar countries.

Enhancing Perovskite Solar Cell Performance by Interface Engineering Using CH3NH3PbBr0.9I2.1 Quantum Dots.

PubMed

Cha, Mingyang; Da, Peimei; Wang, Jun; Wang, Weiyi; Chen, Zhanghai; Xiu, Faxian; Zheng, Gengfeng; Wang, Zhong-Sheng

2016-07-13

To improve the interfacial charge transfer that is crucial to the performance of perovskite solar cells, the interface engineering in a device should be rationally designed. Here we have developed an interface engineering method to tune the photovoltaic performance of planar-heterojunction perovskite solar cells by incorporating MAPbBr3-xIx (MA = CH3NH3) quantum dots (QDs) between the MAPbI3 perovskite film and the hole-transporting material (HTM) layer. By adjustment of the Br:I ratio, the as-synthesized MAPbBr3-xIx QDs show tunable fluorescence and band edge positions. When the valence band (VB) edge of MAPbBr3-xIx QDs is located below that of the MAPbI3 perovskite, the hole transfer from the MAPbI3 perovskite film to the HTM layer is hindered, and hence, the power conversion efficiency decreases. In contrast, when the VB edge of MAPbBr3-xIx QDs is located between the VB edge of the MAPbI3 perovskite film and the highest occupied molecular orbital of the HTM layer, the hole transfer from the MAPbI3 perovskite film to the HTM layer is well-facilitated, resulting in significant improvements in the fill factor, short-circuit photocurrent, and power conversion efficiency.

Germanium photodetectors fabricated on 300 mm silicon wafers for near-infrared focal plane arrays

NASA Astrophysics Data System (ADS)

Zeller, John W.; Rouse, Caitlin; Efstathiadis, Harry; Dhar, Nibir K.; Wijewarnasuriya, Priyalal; Sood, Ashok K.

2017-09-01

SiGe p-i-n photodetectors have been fabricated on 300 mm (12") diameter silicon (Si) wafers utilizing high throughput, large-area complementary metal-oxide semiconductor (CMOS) technologies. These Ge photodetectors are designed to operate in room temperature environments without cooling, and thus have potential size and cost advantages over conventional cooled infrared detectors. The two-step fabrication process for the p-i-n photodetector devices, designed to minimize the formation of defects and threading dislocations, involves low temperature epitaxial growth of a thin p+ (boron) Ge seed/buffer layer, followed by higher temperature deposition of a thicker Ge intrinsic layer. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) demonstrated uniform layer compositions with well defined layer interfaces and reduced dislocation density. Time-of-flight secondary ion mass spectroscopy (TOF-SIMS) was likewise employed to analyze the doping levels of the p+ and n+ layers. Current-voltage (I-V) measurements demonstrated that these SiGe photodetectors, when exposed to incident visible-NIR radiation, exhibited dark currents down below 1 μA and significant enhancement in photocurrent at -1 V. The zero-bias photocurrent was also relatively high, showing a minimal drop compared to that at -1 V bias.

sideSPIM – selective plane illumination based on a conventional inverted microscope

PubMed Central

Hedde, Per Niklas; Malacrida, Leonel; Ahrar, Siavash; Siryaporn, Albert; Gratton, Enrico

2017-01-01

Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or vice versa. Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to 5 stacks/s. Additionally, sample handling is compatible with established methods and switching magnification to change the field of view from single cells to whole organisms does not require labor intensive adjustments of the system. PMID:29026679

Metabolic microscopy of head and neck cancer organoids

NASA Astrophysics Data System (ADS)

Shah, Amy T.; Skala, Melissa C.

2016-03-01

Studies for head and neck cancer have primarily relied on cell lines or in vivo animal studies. However, a technique that combines the benefits of high-throughput in vitro studies with a complex, physiologically relevant microenvironment would be advantageous for understanding drug effects. Organoids provide a unique platform that fulfills these goals. Organoids are generated from excised and digested tumor tissue and are grown in culture. Fluorescence microscopy provides high-resolution images on a similar spatial scale as organoids. In particular, autofluorescence imaging of the metabolic cofactors NAD(P)H and FAD can provide insight into response to anti-cancer treatment. The optical redox ratio reflects relative amounts of NAD(P)H and FAD, and the fluorescence lifetime reflects enzyme activity of NAD(P)H and FAD. This study optimizes and characterizes the generation and culture of organoids grown from head and neck cancer tissue. Additionally, organoids were treated for 24 hours with a standard chemotherapy, and metabolic response in the organoids was measured using optical metabolic imaging. Ultimately, combining head and neck cancer organoids with optical metabolic imaging could be applied to test drug sensitivity for drug development studies as well as treatment planning for cancer patients.

The high throughput biomedicine unit at the institute for molecular medicine Finland: high throughput screening meets precision medicine.

PubMed

Pietiainen, Vilja; Saarela, Jani; von Schantz, Carina; Turunen, Laura; Ostling, Paivi; Wennerberg, Krister

2014-05-01

The High Throughput Biomedicine (HTB) unit at the Institute for Molecular Medicine Finland FIMM was established in 2010 to serve as a national and international academic screening unit providing access to state of the art instrumentation for chemical and RNAi-based high throughput screening. The initial focus of the unit was multiwell plate based chemical screening and high content microarray-based siRNA screening. However, over the first four years of operation, the unit has moved to a more flexible service platform where both chemical and siRNA screening is performed at different scales primarily in multiwell plate-based assays with a wide range of readout possibilities with a focus on ultraminiaturization to allow for affordable screening for the academic users. In addition to high throughput screening, the equipment of the unit is also used to support miniaturized, multiplexed and high throughput applications for other types of research such as genomics, sequencing and biobanking operations. Importantly, with the translational research goals at FIMM, an increasing part of the operations at the HTB unit is being focused on high throughput systems biological platforms for functional profiling of patient cells in personalized and precision medicine projects.

High Throughput Screening For Hazard and Risk of Environmental Contaminants

EPA Science Inventory

High throughput toxicity testing provides detailed mechanistic information on the concentration response of environmental contaminants in numerous potential toxicity pathways. High throughput screening (HTS) has several key advantages: (1) expense orders of magnitude less than an...

Parallelized multi–graphics processing unit framework for high-speed Gabor-domain optical coherence microscopy

PubMed Central

Tankam, Patrice; Santhanam, Anand P.; Lee, Kye-Sung; Won, Jungeun; Canavesi, Cristina; Rolland, Jannick P.

2014-01-01

Abstract. Gabor-domain optical coherence microscopy (GD-OCM) is a volumetric high-resolution technique capable of acquiring three-dimensional (3-D) skin images with histological resolution. Real-time image processing is needed to enable GD-OCM imaging in a clinical setting. We present a parallelized and scalable multi-graphics processing unit (GPU) computing framework for real-time GD-OCM image processing. A parallelized control mechanism was developed to individually assign computation tasks to each of the GPUs. For each GPU, the optimal number of amplitude-scans (A-scans) to be processed in parallel was selected to maximize GPU memory usage and core throughput. We investigated five computing architectures for computational speed-up in processing 1000×1000 A-scans. The proposed parallelized multi-GPU computing framework enables processing at a computational speed faster than the GD-OCM image acquisition, thereby facilitating high-speed GD-OCM imaging in a clinical setting. Using two parallelized GPUs, the image processing of a 1×1×0.6  mm3 skin sample was performed in about 13 s, and the performance was benchmarked at 6.5 s with four GPUs. This work thus demonstrates that 3-D GD-OCM data may be displayed in real-time to the examiner using parallelized GPU processing. PMID:24695868

Parallelized multi-graphics processing unit framework for high-speed Gabor-domain optical coherence microscopy.

PubMed

Tankam, Patrice; Santhanam, Anand P; Lee, Kye-Sung; Won, Jungeun; Canavesi, Cristina; Rolland, Jannick P

2014-07-01

Gabor-domain optical coherence microscopy (GD-OCM) is a volumetric high-resolution technique capable of acquiring three-dimensional (3-D) skin images with histological resolution. Real-time image processing is needed to enable GD-OCM imaging in a clinical setting. We present a parallelized and scalable multi-graphics processing unit (GPU) computing framework for real-time GD-OCM image processing. A parallelized control mechanism was developed to individually assign computation tasks to each of the GPUs. For each GPU, the optimal number of amplitude-scans (A-scans) to be processed in parallel was selected to maximize GPU memory usage and core throughput. We investigated five computing architectures for computational speed-up in processing 1000×1000 A-scans. The proposed parallelized multi-GPU computing framework enables processing at a computational speed faster than the GD-OCM image acquisition, thereby facilitating high-speed GD-OCM imaging in a clinical setting. Using two parallelized GPUs, the image processing of a 1×1×0.6  mm3 skin sample was performed in about 13 s, and the performance was benchmarked at 6.5 s with four GPUs. This work thus demonstrates that 3-D GD-OCM data may be displayed in real-time to the examiner using parallelized GPU processing.

78 FR 7427 - National and Governmental Advisory Committees to the U.S. Representative to the Commission for...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-01

... Rapparteur on the rights of Indigenous peoples in the United Nations Office of the High Commissioner for Human Rights. A copy of the agenda will be posted at http://www.epa.gov/ofacmo/nacgac-page.htm . DATES... February 7, 2013. Meeting Access: For information on access or services for individuals with disabilities...

Interfacial Engineering of Perovskite Solar Cells by Employing a Hydrophobic Copper Phthalocyanine Derivative as Hole-Transporting Material with Improved Performance and Stability.

PubMed

Jiang, Xiaoqing; Yu, Ze; Lai, Jianbo; Zhang, Yuchen; Hu, Maowei; Lei, Ning; Wang, Dongping; Yang, Xichuan; Sun, Licheng

2017-04-22

In high-performance perovskite solar cells (PSCs), hole-transporting materials (HTMs) play an important role in extracting and transporting the photo-generated holes from the perovskite absorber to the cathode, thus reducing unwanted recombination losses and enhancing the photovoltaic performance. Herein, solution-processable tetra-4-(bis(4-tert-butylphenyl)amino)phenoxy-substituted copper phthalocyanine (CuPc-OTPAtBu) was synthesized and explored as a HTM in PSCs. The optical, electrochemical, and thermal properties were fully characterized for this organic metal complex. The photovoltaic performance of PSCs employing this CuPc derivative as a HTM was further investigated, in combination with a mixed-ion perovskite as a light absorber and a low-cost vacuum-free carbon as cathode. The optimized devices [doped with 6 % (w/w) tetrafluoro-tetracyano-quinodimethane (F4TCNQ)] showed a decent power conversion efficiency of 15.0 %, with an open-circuit voltage of 1.01 V, a short-circuit current density of 21.9 mA cm -2 , and a fill factor of 0.68. Notably, the PSC devices studied also exhibited excellent long-term durability under ambient condition for 720 h, mainly owing to the introduction of the hydrophobic HTM interlayer, which prevents moisture penetration into the perovskite film. The present work emphasizes that solution-processable CuPc holds a great promise as a class of alternative HTMs that can be further explored for efficient and stable PSCs in the future. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Planar Perovskite Solar Cells with High Open-Circuit Voltage Containing a Supramolecular Iron Complex as Hole Transport Material Dopant.

PubMed

Saygili, Yasemin; Turren-Cruz, Silver-Hamill; Olthof, Selina; Saes, Bartholomeus Wilhelmus Henricus; Pehlivan, Ilknur Bayrak; Saliba, Michael; Meerholz, Klaus; Edvinsson, Tomas; Zakeeruddin, Shaik M; Grätzel, Michael; Correa-Baena, Juan-Pablo; Hagfeldt, Anders; Freitag, Marina; Tress, Wolfgang

2018-04-26

In perovskite solar cells (PSCs), the most commonly used hole transport material (HTM) is spiro-OMeTAD, which is typically doped by metalorganic complexes, for example, based on Co, to improve charge transport properties and thereby enhance the photovoltaic performance of the device. In this study, we report a new hemicage-structured iron complex, 1,3,5-tris(5'-methyl-2,2'-bipyridin-5-yl)ethylbenzene Fe(III)-tris(bis(trifluoromethylsulfonyl)imide), as a p-type dopant for spiro-OMeTAD. The formal redox potential of this compound was measured as 1.29 V vs. the standard hydrogen electrode, which is slightly (20 mV) more positive than that of the commercial cobalt dopant FK209. Photoelectron spectroscopy measurements confirm that the iron complex acts as an efficient p-dopant, as evidenced in an increase of the spiro-OMeTAD work function. When fabricating planar PSCs with the HTM spiro-OMeTAD doped by 5 mol % of the iron complex, a power conversion efficiency of 19.5 % (AM 1.5G, 100 mW cm -2 ) is achieved, compared to 19.3 % for reference devices with FK209. Open circuit voltages exceeding 1.2 V at 1 sun and reaching 1.27 V at 3 suns indicate that recombination at the perovskite/HTM interface is low when employing this iron complex. This work contributes to recent endeavors to reduce recombination losses in perovskite solar cells. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

High Throughput Transcriptomics: From screening to pathways

EPA Science Inventory

The EPA ToxCast effort has screened thousands of chemicals across hundreds of high-throughput in vitro screening assays. The project is now leveraging high-throughput transcriptomic (HTTr) technologies to substantially expand its coverage of biological pathways. The first HTTr sc...

Holocene temperature shifts around Greenland: Paleolimnological approaches to quantifying past warmth and documenting its consequences

NASA Astrophysics Data System (ADS)

Axford, Y.; Lasher, G. E.; McFarlin, J. M.; Francis, D. R.; Kelly, M. A.; Langdon, P. G.; Levy, L.; Osburn, M. R.; Osterberg, E. C.

2015-12-01

Insolation-driven warmth across the Arctic during the early to middle Holocene (the Holocene Thermal Maximum, or HTM) represents a geologically accessible analog for future warming and its impacts. Improved constraints on the magnitude and seasonality of HTM warmth around Greenland's margins can advance the use of paleoclimate data to test and improve climate and ice sheet models. Here we present an overview of our recent efforts to reconstruct climate through the Holocene around the margins of the Greenland Ice Sheet using multiple proxies in lake sediments. We use insect (chironomid) assemblages to derive quantitative estimates of Holocene temperatures at sites with minimal soil and vegetation development near the eastern, northwestern and western margins of the ice sheet. Our chironomid-based temperature reconstructions consistently imply HTM July air temperatures 3 to 4.5 °C warmer than the pre-industrial late Holocene in these sectors of Greenland. The timing of reconstructed peak warmth differs between sites, with onset varying from ~10 ka to ~6.5 ka, but in good agreement with glacial geology and other evidence from each region. Our reconstructed temperature anomalies are larger than those typically inferred from annually-integrated indicators from the ice sheet itself, but comparable to the few other quantitative summer temperature estimates available from beyond the ice sheet on Greenland. Additional records are needed to confirm the magnitude of HTM warmth and to better define its seasonality and spatial pattern. To provide independent constraints on paleotemperatures and to elucidate additional aspects of Holocene paleoclimate, we are also employing oxygen isotopes of chironomid remains and other aquatic organic materials, and molecular organic proxies, in parallel (see Lasher et al. and McFarlin et al., this meeting). Combined with glacial geologic evidence, these multi-proxy records elucidate diverse aspects of HTM climate around Greenland - including temperature, hydroclimate, and the response of Greenland's glaciers to past climate change.

Reducing hole transporter use and increasing perovskite solar cell stability with dual-role polystyrene microgel particles.

PubMed

Chen, Mu; Mokhtar, Muhamad Z; Whittaker, Eric; Lian, Qing; Hamilton, Bruce; O'Brien, Paul; Zhu, Mingning; Cui, Zhengxing; Haque, Saif A; Saunders, Brian R

2017-07-20

Perovskite solar cells (PSCs) are a disruptive technology that continues to attract considerable attention due to their remarkable and sustained power conversion efficiency increase. Improving PSC stability and reducing expensive hole transport material (HTM) usage are two aspects that are gaining increased attention. In a new approach, we investigate the ability of insulating polystyrene microgel particles (MGs) to increase PSC stability and replace the majority of the HTM phase. MGs are sub-micrometre crosslinked polymer particles that swell in a good solvent. The MGs were prepared using a scalable emulsion polymerisation method. Mixed HTM/MG dispersions were subsequently spin-coated onto PSCs and formed composite HTM-MG layers. The HTMs employed were poly(triaryl amine) (PTAA), poly(3-hexylthiophene) (P3HT) and Spiro-MeOTAD (Spiro). The MGs formed mechanically robust composite HTMs with PTAA and P3HT. In contrast, Spiro-MG composites contained micro-cracks due the inability of the relatively small Spiro molecules to interdigitate. The efficiencies for the PSCs containing PTAA-MG and P3HT-MG decreased by only ∼20% compared to control PSCs despite PTAA and P3HT being the minority phases. They occupied only ∼35 vol% of the composite HTMs. An unexpected finding from the study was that the MGs dispersed well within the PTAA matrix. This morphology aided strong quenching of the CH 3 NH 3 PbI 3-x Cl x fluorescence. In addition, the open circuit voltages for the PSCs prepared using P3HT-MG increased by ∼170 mV compared to control PSCs. To demonstrate their versatility the MGs were also used to encapsulate P3HT-based PSCs. Solar cell stability data for the latter as well as those for PSCs containing composite HTM-MG were both far superior compared to data measured for a control PSC. Since MGs can reduce conjugated polymer use and increase stability they have good potential as dual-role PSC additives.

Quantitative description on structure-property relationships of Li-ion battery materials for high-throughput computations

NASA Astrophysics Data System (ADS)

Wang, Youwei; Zhang, Wenqing; Chen, Lidong; Shi, Siqi; Liu, Jianjun

2017-12-01

Li-ion batteries are a key technology for addressing the global challenge of clean renewable energy and environment pollution. Their contemporary applications, for portable electronic devices, electric vehicles, and large-scale power grids, stimulate the development of high-performance battery materials with high energy density, high power, good safety, and long lifetime. High-throughput calculations provide a practical strategy to discover new battery materials and optimize currently known material performances. Most cathode materials screened by the previous high-throughput calculations cannot meet the requirement of practical applications because only capacity, voltage and volume change of bulk were considered. It is important to include more structure-property relationships, such as point defects, surface and interface, doping and metal-mixture and nanosize effects, in high-throughput calculations. In this review, we established quantitative description of structure-property relationships in Li-ion battery materials by the intrinsic bulk parameters, which can be applied in future high-throughput calculations to screen Li-ion battery materials. Based on these parameterized structure-property relationships, a possible high-throughput computational screening flow path is proposed to obtain high-performance battery materials.

Quantitative description on structure-property relationships of Li-ion battery materials for high-throughput computations.

PubMed

Wang, Youwei; Zhang, Wenqing; Chen, Lidong; Shi, Siqi; Liu, Jianjun

2017-01-01

Li-ion batteries are a key technology for addressing the global challenge of clean renewable energy and environment pollution. Their contemporary applications, for portable electronic devices, electric vehicles, and large-scale power grids, stimulate the development of high-performance battery materials with high energy density, high power, good safety, and long lifetime. High-throughput calculations provide a practical strategy to discover new battery materials and optimize currently known material performances. Most cathode materials screened by the previous high-throughput calculations cannot meet the requirement of practical applications because only capacity, voltage and volume change of bulk were considered. It is important to include more structure-property relationships, such as point defects, surface and interface, doping and metal-mixture and nanosize effects, in high-throughput calculations. In this review, we established quantitative description of structure-property relationships in Li-ion battery materials by the intrinsic bulk parameters, which can be applied in future high-throughput calculations to screen Li-ion battery materials. Based on these parameterized structure-property relationships, a possible high-throughput computational screening flow path is proposed to obtain high-performance battery materials.

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

Yulaev, Alexander; Guo, Hongxuan; Strelcov, Evgheni

Atomic-scale thickness, molecular impermeability, low atomic number, and mechanical strength make graphene an ideal electron-transparent membrane for material characterization in liquids and gases with scanning electron microscopy and spectroscopy. Here in this paper, we present a novel sample platform made of an array of thousands of identical isolated graphene-capped microchannels with high aspect ratio. A combination of a global wide field of view with high resolution local imaging of the array allows for high throughput in situ studies as well as for combinatorial screening of solutions, liquid interfaces, and immersed samples. We demonstrate the capabilities of this platform by studyingmore » a pure water sample in comparison with alkali halide solutions, a model electrochemical plating process, and beam-induced crystal growth in liquid electrolyte. Spectroscopic characterization of liquid interfaces and immersed objects with Auger and X-ray fluorescence analysis through the graphene membrane are also demonstrated.« less

High Throughput Experimental Materials Database

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

Zakutayev, Andriy; Perkins, John; Schwarting, Marcus

The mission of the High Throughput Experimental Materials Database (HTEM DB) is to enable discovery of new materials with useful properties by releasing large amounts of high-quality experimental data to public. The HTEM DB contains information about materials obtained from high-throughput experiments at the National Renewable Energy Laboratory (NREL).

20180311 - High Throughput Transcriptomics: From screening to pathways (SOT 2018)

EPA Science Inventory

The EPA ToxCast effort has screened thousands of chemicals across hundreds of high-throughput in vitro screening assays. The project is now leveraging high-throughput transcriptomic (HTTr) technologies to substantially expand its coverage of biological pathways. The first HTTr sc...

Evaluation of Sequencing Approaches for High-Throughput Transcriptomics - (BOSC)

EPA Science Inventory

Whole-genome in vitro transcriptomics has shown the capability to identify mechanisms of action and estimates of potency for chemical-mediated effects in a toxicological framework, but with limited throughput and high cost. The generation of high-throughput global gene expression...

The Strategic Importance of Defeating Underground Facilities

DTIC Science & Technology

2012-03-20

German Submarine Pens: October 1942-April 1945,‖ December 7, 2010. http://www.usaaf.net/ ww2 /uboats/uboatspg6.htm (accessed 10 December 2011) 48...1945,‖ December 7, 2010. http://www.usaaf.net/ ww2 /uboats/uboatspg6.htm (accessed 10 December 2011). 51 Bradham, Hitler’s U-Boat Fortresses, 55. 52...The U.S. Army Air Forces in World War II, ―Bombing German Submarine Pens: October 1942-April 1945,‖ December 7, 2010. http://www.usaaf.net/ ww2

Ethnic Conflict in Indonesia: Causes and Recommended Measures

DTIC Science & Technology

2005-12-01

prepared to celebrate Hari Raya Idul Fitri, a holy day for Moslems throughout the world. Before that, at the end of 1998 certain scattered occurrences of...op. cit., p. 34. 132 Haris Azhar and Syamsul Alam Agus. “Poso: An Area Put to Conflict.” <file://F:\\Poso An Area Put to Conflict.htm> (accessed...Paper-04/0. Jakarta, 2004. Azhar, Haris , and Alam Agus Syamsul. “Poso: An Area Put to Conflict.” <file://F:\\Poso AnAreaPuttoConflict.htm> (accessed

Refractive Conditions of Amazon Environment and Its Effects on Ground and Airborne Radar and ESM Systems

DTIC Science & Technology

2003-09-01

superrefraction and trapping layers (the last one forming ducts) were investigated , as well as multiple layers. The multiple layers studied were made by 18...and Surveillance for the Amazon. Avionics Magazine of June 2002, from: http://www.aviationtoday.com/reports/avionics/previous/0602/0602sivam.htm...2003. Fisch, G., Marengo, J. A., & Nobre, C. A. Climate in Amazonia . From: http://www.mct.gov.br/clima/ingles/comunic_old/cinpe03.htm accessed in

Extracting Fluorescent Reporter Time Courses of Cell Lineages from High-Throughput Microscopy at Low Temporal Resolution

PubMed Central

Downey, Mike J.; Jeziorska, Danuta M.; Ott, Sascha; Tamai, T. Katherine; Koentges, Georgy; Vance, Keith W.; Bretschneider, Till

2011-01-01

The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted to different cell types and fluorescent markers. PMID:22194797

Quantitative imaging for discovery and assembly of the metabo-regulome

PubMed Central

Okumoto, Sakiko; Takanaga, Hitomi; Frommer, Wolf B.

2009-01-01

Summary Little is known about regulatory networks that control metabolic flux in plant cells. Detailed understanding of regulation is crucial for synthetic biology. The difficulty of measuring metabolites with cellular and subcellular precision is a major roadblock. New tools have been developed for monitoring extracellular, cytosolic, organellar and vacuolar ion and metabolite concentrations with a time resolution of milliseconds to hours. Genetically encoded sensors allow quantitative measurement of steady-state concentrations of ions, signaling molecules and metabolites and their respective changes over time. Fluorescence resonance energy transfer (FRET) sensors exploit conformational changes in polypeptides as a proxy for analyte concentrations. Subtle effects of analyte binding on the conformation of the recognition element are translated into a FRET change between two fused green fluorescent protein (GFP) variants, enabling simple monitoring of analyte concentrations using fluorimetry or fluorescence microscopy. Fluorimetry provides information averaged over cell populations, while microscopy detects differences between cells or populations of cells. The genetically encoded sensors can be targeted to subcellular compartments or the cell surface. Confocal microscopy ultimately permits observation of gradients or local differences within a compartment. The FRET assays can be adapted to high-throughput analysis to screen mutant populations in order to systematically identify signaling networks that control individual steps in metabolic flux. PMID:19138219

Wide-field lensless fluorescent microscopy using a tapered fiber-optic faceplate on a chip.

PubMed

Coskun, Ahmet F; Sencan, Ikbal; Su, Ting-Wei; Ozcan, Aydogan

2011-09-07

We demonstrate lensless fluorescent microscopy over a large field-of-view of ~60 mm(2) with a spatial resolution of <4 µm. In this on-chip fluorescent imaging modality, the samples are placed on a fiber-optic faceplate that is tapered such that the density of the fiber-optic waveguides on the top facet is >5 fold larger than the bottom one. Placed on this tapered faceplate, the fluorescent samples are pumped from the side through a glass hemisphere interface. After excitation of the samples, the pump light is rejected through total internal reflection that occurs at the bottom facet of the sample substrate. The fluorescent emission from the sample is then collected by the smaller end of the tapered faceplate and is delivered to an opto-electronic sensor-array to be digitally sampled. Using a compressive sampling algorithm, we decode these raw lensfree images to validate the resolution (<4 µm) of this on-chip fluorescent imaging platform using microparticles as well as labeled Giardia muris cysts. This wide-field lensfree fluorescent microscopy platform, being compact and high-throughput, might provide a valuable tool especially for cytometry, rare cell analysis (involving large area microfluidic systems) as well as for microarray imaging applications.

Protein 3D Structure and Electron Microscopy Map Retrieval Using 3D-SURFER2.0 and EM-SURFER.

PubMed

Han, Xusi; Wei, Qing; Kihara, Daisuke

2017-12-08

With the rapid growth in the number of solved protein structures stored in the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB), it is essential to develop tools to perform real-time structure similarity searches against the entire structure database. Since conventional structure alignment methods need to sample different orientations of proteins in the three-dimensional space, they are time consuming and unsuitable for rapid, real-time database searches. To this end, we have developed 3D-SURFER and EM-SURFER, which utilize 3D Zernike descriptors (3DZD) to conduct high-throughput protein structure comparison, visualization, and analysis. Taking an atomic structure or an electron microscopy map of a protein or a protein complex as input, the 3DZD of a query protein is computed and compared with the 3DZD of all other proteins in PDB or EMDB. In addition, local geometrical characteristics of a query protein can be analyzed using VisGrid and LIGSITE CSC in 3D-SURFER. This article describes how to use 3D-SURFER and EM-SURFER to carry out protein surface shape similarity searches, local geometric feature analysis, and interpretation of the search results. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

High Throughput Determination of Critical Human Dosing Parameters (SOT)

EPA Science Inventory

High throughput toxicokinetics (HTTK) is a rapid approach that uses in vitro data to estimate TK for hundreds of environmental chemicals. Reverse dosimetry (i.e., reverse toxicokinetics or RTK) based on HTTK data converts high throughput in vitro toxicity screening (HTS) data int...

High Throughput Determinations of Critical Dosing Parameters (IVIVE workshop)

EPA Science Inventory

High throughput toxicokinetics (HTTK) is an approach that allows for rapid estimations of TK for hundreds of environmental chemicals. HTTK-based reverse dosimetry (i.e, reverse toxicokinetics or RTK) is used in order to convert high throughput in vitro toxicity screening (HTS) da...

Optimization of high-throughput nanomaterial developmental toxicity testing in zebrafish embryos

EPA Science Inventory

Nanomaterial (NM) developmental toxicities are largely unknown. With an extensive variety of NMs available, high-throughput screening methods may be of value for initial characterization of potential hazard. We optimized a zebrafish embryo test as an in vivo high-throughput assay...

Multiplexed multi-scale imaging: novel roles for the scaffold protein IQGAP1 in epithelial cell development (Conference Presentation)

NASA Astrophysics Data System (ADS)

Schweikhard, Volker

2016-02-01

The precise sub-cellular spatial localization of multi-protein complexes is increasingly recognized as a key mechanism governing the organization of mammalian cells. Consequently, there is a need for novel microscopy techniques capable of investigating such sub-cellular architectures in comprehensive detail. Here, we applied a novel multiplexed STORM super-resolution microscopy technique, in combination with high-throughput immunofluorescence microscopy and live-cell imaging, to investigate the roles of the scaffold protein IQGAP1 in epithelial cells. IQGAP1 is known to orchestrate a wide range of biological processes, including intracellular signaling, cytoskeletal regulation, cell-cell adhesion, and protein trafficking, by forming distinct complexes with a number of known interaction partners, and recruiting these complexes to specific subcellular locations. Our results demonstrate that, in addition to supporting epithelial adherens junctions by associating with specialized cortical actin structures, IQGAP1 plays a second role in which it controls the confinement of a unique, previously undocumented class of membranous compartments to the basal actin cortex. These largely immotile yet highly dynamic structures appear transiently as cells merge into clusters and establish of apical-basolateral (epithelial) polarity, and are identified as an intermediate compartment in the endocytic recycling pathways for cell junction complexes and cell surface receptors. Although these two functions of IQGAP1 occur in parallel and largely independently of each other, they both support the maturation and maintenance of polarized epithelial cell architectures.

Optical tracking of embryonic vertebrates behavioural responses using automated time-resolved video-microscopy system

NASA Astrophysics Data System (ADS)

Walpitagama, Milanga; Kaslin, Jan; Nugegoda, Dayanthi; Wlodkowic, Donald

2016-12-01

The fish embryo toxicity (FET) biotest performed on embryos of zebrafish (Danio rerio) has gained significant popularity as a rapid and inexpensive alternative approach in chemical hazard and risk assessment. The FET was designed to evaluate acute toxicity on embryonic stages of fish exposed to the test chemical. The current standard, similar to most traditional methods for evaluating aquatic toxicity provides, however, little understanding of effects of environmentally relevant concentrations of chemical stressors. We postulate that significant environmental effects such as altered motor functions, physiological alterations reflected in heart rate, effects on development and reproduction can occur at sub-lethal concentrations well below than LC10. Behavioral studies can, therefore, provide a valuable integrative link between physiological and ecological effects. Despite the advantages of behavioral analysis development of behavioral toxicity, biotests is greatly hampered by the lack of dedicated laboratory automation, in particular, user-friendly and automated video microscopy systems. In this work we present a proof-of-concept development of an optical system capable of tracking embryonic vertebrates behavioral responses using automated and vastly miniaturized time-resolved video-microscopy. We have employed miniaturized CMOS cameras to perform high definition video recording and analysis of earliest vertebrate behavioral responses. The main objective was to develop a biocompatible embryo positioning structures that were suitable for high-throughput imaging as well as video capture and video analysis algorithms. This system should support the development of sub-lethal and behavioral markers for accelerated environmental monitoring.

Microfluidic device for a rapid immobilization of zebrafish larvae in environmental scanning electron microscopy.

PubMed

Akagi, Jin; Zhu, Feng; Skommer, Joanna; Hall, Chris J; Crosier, Philip S; Cialkowski, Michal; Wlodkowic, Donald

2015-03-01

Small vertebrate model organisms have recently gained popularity as attractive experimental models that enhance our understanding of human tissue and organ development. Despite a large body of evidence using optical spectroscopy for the characterization of small model organism on chip-based devices, no attempts have been so far made to interface microfabricated technologies with environmental scanning electron microscopy (ESEM). Conventional scanning electron microscopy requires high vacuum environments and biological samples must be, therefore, submitted to many preparative procedures to dehydrate, fix, and subsequently stain the sample with gold-palladium deposition. This process is inherently low-throughput and can introduce many analytical artifacts. This work describes a proof-of-concept microfluidic chip-based system for immobilizing zebrafish larvae for ESEM imaging that is performed in a gaseous atmosphere, under low vacuum mode and without any need for sample staining protocols. The microfabricated technology provides a user-friendly and simple interface to perform ESEM imaging on zebrafish larvae. Presented lab-on-a-chip device was fabricated using a high-speed infrared laser micromachining in a biocompatible poly(methyl methacrylate) thermoplastic. It consisted of a reservoir with multiple semispherical microwells designed to hold the yolk of dechorionated zebrafish larvae. Immobilization of the larvae was achieved by a gentle suction generated during blotting of the medium. Trapping region allowed for multiple specimens to be conveniently positioned on the chip-based device within few minutes for ESEM imaging. © 2014 International Society for Advancement of Cytometry.

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

PubMed

De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

2011-10-01

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay

PubMed Central

De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M.

2011-01-01

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin. PMID:22006542

Imaging and identification of waterborne parasites using a chip-scale microscope.

PubMed

Lee, Seung Ah; Erath, Jessey; Zheng, Guoan; Ou, Xiaoze; Willems, Phil; Eichinger, Daniel; Rodriguez, Ana; Yang, Changhuei

2014-01-01

We demonstrate a compact portable imaging system for the detection of waterborne parasites in resource-limited settings. The previously demonstrated sub-pixel sweeping microscopy (SPSM) technique is a lens-less imaging scheme that can achieve high-resolution (<1 µm) bright-field imaging over a large field-of-view (5.7 mm×4.3 mm). A chip-scale microscope system, based on the SPSM technique, can be used for automated and high-throughput imaging of protozoan parasite cysts for the effective diagnosis of waterborne enteric parasite infection. We successfully imaged and identified three major types of enteric parasite cysts, Giardia, Cryptosporidium, and Entamoeba, which can be found in fecal samples from infected patients. We believe that this compact imaging system can serve well as a diagnostic device in challenging environments, such as rural settings or emergency outbreaks.

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

Enhancing the reliability and throughput of neurosphere culture on hydrogel microwell arrays.

PubMed

Cordey, Myriam; Limacher, Monika; Kobel, Stefan; Taylor, Verdon; Lutolf, Matthias P

2008-10-01

The neurosphere assay is the standard retrospective assay to test the self-renewal capability and multipotency of neural stem cells (NSCs) in vitro. However, it has recently become clear that not all neurospheres are derived from a NSC and that on conventional cell culture substrates, neurosphere motility may cause frequent neurosphere "merging" [Nat Methods 2006;3:801-806; Stem Cells 2007;25:871-874]. Combining biomimetic hydrogel matrix technology with microengineering, we developed a microwell array platform on which NSC fate and neurosphere formation can be unequivocally attributed to a single founding cell. Using time-lapse microscopy and retrospective immunostaining, the fate of several hundred single NSCs was quantified. Compared with conventional neurosphere culture methods on plastic dishes, we detected a more than 100% increase in single NSC viability on soft hydrogels. Effective confinement of single proliferating cells to microwells led to neurosphere formation of vastly different sizes, a high percentage of which showed stem cell phenotypes after one week in culture. The reliability and increased throughput of this platform should help to better elucidate the function of sphere-forming stem/progenitor cells independent of their proliferation dynamics. Disclosure of potential conflicts of interest is found at the end of this article.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR).

PubMed

Crosby, Priya; Hoyle, Nathaniel P; O'Neill, John S

2017-12-13

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Microfluidic Flow Injection Analysis with Thermal Lens Microscopic Detection for Determination of NGAL

NASA Astrophysics Data System (ADS)

Radovanović, Tatjana; Liu, Mingqiang; Likar, Polona; Klemenc, Matjaž; Franko, Mladen

2015-06-01

A combined microfluidic flow injection analysis-thermal lens microscopy (FIA-TLM) system was applied for determination of neutrophil gelatinase-associated lipocalin (NGAL)—a biomarker of acute kidney injury. NGAL was determined following a commercial ELISA assay and transfer of the resulting solution into the FIA-TLM system with a 100 m deep microchannel. At an excitation power of 100 mW, the FIA-TLM provided about seven times lower limits of detection (1.5 pg as compared to a conventional ELISA test, and a sample throughput of six samples per minute, which compares favorably with sample throughput of the microtiter plate reader, which reads 96 wells in about 30 min. Comparison of results for NGAL in plasma samples from healthy individuals and for NGAL dynamics in patients undergoing coronary angiography measured with transmission mode spectrometry on a microtiter plate reader and with FIA-TLM showed good agreement. In addition to improved LOD, the high sensitivity of FIA-TLM offers possibilities of a further reduction of the total reaction time of the NGAL ELISA test by sacrificing some of the sensitivity while reducing the duration of individual incubation steps.

Artifact mitigation of ptychography integrated with on-the-fly scanning probe microscopy

DOE PAGES

Huang, Xiaojing; Yan, Hanfei; Ge, Mingyuan; ...

2017-07-11

In this paper, we report our experiences with conducting ptychography simultaneously with the X-ray fluorescence measurement using the on-the-fly mode for efficient multi-modality imaging. We demonstrate that the periodic artifact inherent to the raster scan pattern can be mitigated using a sufficiently fine scan step size to provide an overlap ratio of >70%. This allows us to obtain transmitted phase contrast images with enhanced spatial resolution from ptychography while maintaining the fluorescence imaging with continuous-motion scans on pixelated grids. Lastly, this capability will greatly improve the competence and throughput of scanning probe X-ray microscopy.

Towards Extending Forward Kinematic Models on Hyper-Redundant Manipulator to Cooperative Bionic Arms

NASA Astrophysics Data System (ADS)

Singh, Inderjeet; Lakhal, Othman; Merzouki, Rochdi

2017-01-01

Forward Kinematics is a stepping stone towards finding an inverse solution and subsequently a dynamic model of a robot. Hence a study and comparison of various Forward Kinematic Models (FKMs) is necessary for robot design. This paper deals with comparison of three FKMs on the same hyper-redundant Compact Bionic Handling Assistant (CBHA) manipulator under same conditions. The aim of this study is to project on modeling cooperative bionic manipulators. Two of these methods are quantitative methods, Arc Geometry HTM (Homogeneous Transformation Matrix) Method and Dual Quaternion Method, while the other one is Hybrid Method which uses both quantitative as well as qualitative approach. The methods are compared theoretically and experimental results are discussed to add further insight to the comparison. HTM is the widely used and accepted technique, is taken as reference and trajectory deviation in other techniques are compared with respect to HTM. Which method allows obtaining an accurate kinematic behavior of the CBHA, controlled in the real-time.

Spatial tuning of acoustofluidic pressure nodes by altering net sonic velocity enables high-throughput, efficient cell sorting

DOE PAGES

Jung, Seung-Yong; Notton, Timothy; Fong, Erika; ...

2015-01-07

Particle sorting using acoustofluidics has enormous potential but widespread adoption has been limited by complex device designs and low throughput. Here, we report high-throughput separation of particles and T lymphocytes (600 μL min -1) by altering the net sonic velocity to reposition acoustic pressure nodes in a simple two-channel device. Finally, the approach is generalizable to other microfluidic platforms for rapid, high-throughput analysis.

Quantitative description on structure–property relationships of Li-ion battery materials for high-throughput computations

PubMed Central

Wang, Youwei; Zhang, Wenqing; Chen, Lidong; Shi, Siqi; Liu, Jianjun

2017-01-01

Abstract Li-ion batteries are a key technology for addressing the global challenge of clean renewable energy and environment pollution. Their contemporary applications, for portable electronic devices, electric vehicles, and large-scale power grids, stimulate the development of high-performance battery materials with high energy density, high power, good safety, and long lifetime. High-throughput calculations provide a practical strategy to discover new battery materials and optimize currently known material performances. Most cathode materials screened by the previous high-throughput calculations cannot meet the requirement of practical applications because only capacity, voltage and volume change of bulk were considered. It is important to include more structure–property relationships, such as point defects, surface and interface, doping and metal-mixture and nanosize effects, in high-throughput calculations. In this review, we established quantitative description of structure–property relationships in Li-ion battery materials by the intrinsic bulk parameters, which can be applied in future high-throughput calculations to screen Li-ion battery materials. Based on these parameterized structure–property relationships, a possible high-throughput computational screening flow path is proposed to obtain high-performance battery materials. PMID:28458737

Rational Design of Molecular Hole-Transporting Materials for Perovskite Solar Cells: Direct versus Inverted Device Configurations.

PubMed

Grisorio, Roberto; Iacobellis, Rosabianca; Listorti, Andrea; De Marco, Luisa; Cipolla, Maria Pia; Manca, Michele; Rizzo, Aurora; Abate, Antonio; Gigli, Giuseppe; Suranna, Gian Paolo

2017-07-26

Due to a still limited understanding of the reasons making 2,2',7,7'-tetrakis(N,N-di-p-methoxyphenylamine)-9,9'-spirobifluorene (Spiro-OMeTAD) the state-of-the-art hole-transporting material (HTM) for emerging photovoltaic applications, the molecular tailoring of organic components for perovskite solar cells (PSCs) lacks in solid design criteria. Charge delocalization in radical cationic states can undoubtedly be considered as one of the essential prerequisites for an HTM, but this aspect has been investigated to a relatively minor extent. In marked contrast with the 3-D structure of Spiro-OMeTAD, truxene-based HTMs Trux1 and Trux2 have been employed for the first time in PSCs fabricated with a direct (n-i-p) or inverted (p-i-n) architecture, exhibiting a peculiar behavior with respect to the referential HTM. Notwithstanding the efficient hole extraction from the perovskite layer exhibited by Trux1 and Trux2 in direct configuration devices, their photovoltaic performances were detrimentally affected by their poor hole transport. Conversely, an outstanding improvement of the photovoltaic performances in dopant-free inverted configuration devices compared to Spiro-OMeTAD was recorded, ascribable to the use of thinner HTM layers. The rationalization of the photovoltaic performances exhibited by different configuration devices discussed in this paper can provide new and unexpected prospects for engineering the interface between the active layer of perovskite-based solar cells and the hole transporters.

Breakthrough Air Force Capabilities Spawned By Basic Research

DTIC Science & Technology

2007-04-01

AboutAFOSR/about_history.htm>. 15 Ibid. 16 Ibid. 4 1990s • Titanium Aluminides . AFOSR-sponsored research looked at titanium aluminides , which are...the 1970s, AFOSR sponsored research on ways to increase the strength and fatigue resistance of titanium , aluminum, and nickel alloys used for...High-Efficiency Swept Shock Compressor Blades . AFOSR-sponsored research looked at making rotor blades in axial flow compressors more efficient

Trustworthiness as a Limitation on Network Neutrality

DTIC Science & Technology

2007-01-01

June 24, 2005) (Civil No. 1:05CV01272), available at http://www.usdoj.gov/atr/cases/f209700/209728.htm (alleging that actuarial consulting firms moved...43 David D. Clark, Requirements for a Future Internet: Security as a Case Study , ver. 2.0...general solutions; and several high-profile cases had data re-indentified after release. See Bruce Schneier, Anonymity and the Netflix Dataset

An Assessment of the 2006 Lebanon-Israeli War

DTIC Science & Technology

2009-05-29

globe .1 Although the invasion of Iraq and some of the fighting in Afghanistan is representative of traditional high intensity conflict, the enduring...members, but also other terror organizations including the Irish Republican Army, Japanese Red Army, and Spanish Basque separatists.13...Swidey, “Tipping Point,” Boston Globe (27 April, 2003), http://home.comcast.net/~neilswidey/tipping.htm (accessed April 3, 2009). 29 Collelo

High-throughput screening (HTS) and modeling of the retinoid ...

EPA Pesticide Factsheets

Presentation at the Retinoids Review 2nd workshop in Brussels, Belgium on the application of high throughput screening and model to the retinoid system Presentation at the Retinoids Review 2nd workshop in Brussels, Belgium on the application of high throughput screening and model to the retinoid system

Evaluating High Throughput Toxicokinetics and Toxicodynamics for IVIVE (WC10)

EPA Science Inventory

High-throughput screening (HTS) generates in vitro data for characterizing potential chemical hazard. TK models are needed to allow in vitro to in vivo extrapolation (IVIVE) to real world situations. The U.S. EPA has created a public tool (R package “httk” for high throughput tox...

High-throughput RAD-SNP genotyping for characterization of sugar beet genotypes

USDA-ARS?s Scientific Manuscript database

High-throughput SNP genotyping provides a rapid way of developing resourceful set of markers for delineating the genetic architecture and for effective species discrimination. In the presented research, we demonstrate a set of 192 SNPs for effective genotyping in sugar beet using high-throughput mar...

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays (SOT)

EPA Science Inventory

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput s...

A quantitative literature-curated gold standard for kinase-substrate pairs

PubMed Central

2011-01-01

We describe the Yeast Kinase Interaction Database (KID, http://www.moseslab.csb.utoronto.ca/KID/), which contains high- and low-throughput data relevant to phosphorylation events. KID includes 6,225 low-throughput and 21,990 high-throughput interactions, from greater than 35,000 experiments. By quantitatively integrating these data, we identified 517 high-confidence kinase-substrate pairs that we consider a gold standard. We show that this gold standard can be used to assess published high-throughput datasets, suggesting that it will enable similar rigorous assessments in the future. PMID:21492431

High-Throughput Industrial Coatings Research at The Dow Chemical Company.

PubMed

Kuo, Tzu-Chi; Malvadkar, Niranjan A; Drumright, Ray; Cesaretti, Richard; Bishop, Matthew T

2016-09-12

At The Dow Chemical Company, high-throughput research is an active area for developing new industrial coatings products. Using the principles of automation (i.e., using robotic instruments), parallel processing (i.e., prepare, process, and evaluate samples in parallel), and miniaturization (i.e., reduce sample size), high-throughput tools for synthesizing, formulating, and applying coating compositions have been developed at Dow. In addition, high-throughput workflows for measuring various coating properties, such as cure speed, hardness development, scratch resistance, impact toughness, resin compatibility, pot-life, surface defects, among others have also been developed in-house. These workflows correlate well with the traditional coatings tests, but they do not necessarily mimic those tests. The use of such high-throughput workflows in combination with smart experimental designs allows accelerated discovery and commercialization.

Outlook for Development of High-throughput Cryopreservation for Small-bodied Biomedical Model Fishes★

PubMed Central

Tiersch, Terrence R.; Yang, Huiping; Hu, E.

2011-01-01

With the development of genomic research technologies, comparative genome studies among vertebrate species are becoming commonplace for human biomedical research. Fish offer unlimited versatility for biomedical research. Extensive studies are done using these fish models, yielding tens of thousands of specific strains and lines, and the number is increasing every day. Thus, high-throughput sperm cryopreservation is urgently needed to preserve these genetic resources. Although high-throughput processing has been widely applied for sperm cryopreservation in livestock for decades, application in biomedical model fishes is still in the concept-development stage because of the limited sample volumes and the biological characteristics of fish sperm. High-throughput processing in livestock was developed based on advances made in the laboratory and was scaled up for increased processing speed, capability for mass production, and uniformity and quality assurance. Cryopreserved germplasm combined with high-throughput processing constitutes an independent industry encompassing animal breeding, preservation of genetic diversity, and medical research. Currently, there is no specifically engineered system available for high-throughput of cryopreserved germplasm for aquatic species. This review is to discuss the concepts and needs for high-throughput technology for model fishes, propose approaches for technical development, and overview future directions of this approach. PMID:21440666

A Solution-Processed Transparent NiO Hole-Extraction Layer for High-Performance Inverted Perovskite Solar Cells.

PubMed

Tang, Li Juan; Chen, Xiao; Wen, Tian Yu; Yang, Shuang; Zhao, Jun Jie; Qiao, Hong Wei; Hou, Yu; Yang, Hua Gui

2018-02-26

A highly transparent NiO layer was prepared by a solution processing method with nickel(II) 2-ethylhexanoate in non-polar solvent and utilized as HTM in perovskite solar cells. Excellent optical transmittance and the matched energy level lead to the enhanced power conversion efficiency (PCE, 18.15 %) than that of conventional sol-gel-processed NiO-based device (12.98 %). © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-throughput measurements of biochemical responses using the plate::vision multimode 96 minilens array reader.

PubMed

Huang, Kuo-Sen; Mark, David; Gandenberger, Frank Ulrich

2006-01-01

The plate::vision is a high-throughput multimode reader capable of reading absorbance, fluorescence, fluorescence polarization, time-resolved fluorescence, and luminescence. Its performance has been shown to be quite comparable with other readers. When the reader is integrated into the plate::explorer, an ultrahigh-throughput screening system with event-driven software and parallel plate-handling devices, it becomes possible to run complicated assays with kinetic readouts in high-density microtiter plate formats for high-throughput screening. For the past 5 years, we have used the plate::vision and the plate::explorer to run screens and have generated more than 30 million data points. Their throughput, performance, and robustness have speeded up our drug discovery process greatly.

Compartmental Genomics in Living Cells Revealed by Single-Cell Nanobiopsy

PubMed Central

Actis, Paolo; Maalouf, Michelle; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R. Adam; Pourmand, Nader

2014-01-01

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis. PMID:24279711

Automated imaging system for single molecules

DOEpatents

Schwartz, David Charles; Runnheim, Rodney; Forrest, Daniel

2012-09-18

There is provided a high throughput automated single molecule image collection and processing system that requires minimal initial user input. The unique features embodied in the present disclosure allow automated collection and initial processing of optical images of single molecules and their assemblies. Correct focus may be automatically maintained while images are collected. Uneven illumination in fluorescence microscopy is accounted for, and an overall robust imaging operation is provided yielding individual images prepared for further processing in external systems. Embodiments described herein are useful in studies of any macromolecules such as DNA, RNA, peptides and proteins. The automated image collection and processing system and method of same may be implemented and deployed over a computer network, and may be ergonomically optimized to facilitate user interaction.

Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)

PubMed Central

Sergé, Arnauld; Bernard, Anne-Marie; Phélipot, Marie-Claire; Bertaux, Nicolas; Fallet, Mathieu; Grenot, Pierre; Marguet, Didier; He, Hai-Tao; Hamon, Yannick

2013-01-01

We introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells. PMID:24086124

Visualizing Oxidative Cellular Stress Induced by Nanoparticles in the Subcytotoxic Range Using Fluorescence Lifetime Imaging.

PubMed

Balke, Jens; Volz, Pierre; Neumann, Falko; Brodwolf, Robert; Wolf, Alexander; Pischon, Hannah; Radbruch, Moritz; Mundhenk, Lars; Gruber, Achim D; Ma, Nan; Alexiev, Ulrike

2018-06-01

Nanoparticles hold a great promise in biomedical science. However, due to their unique physical and chemical properties they can lead to overproduction of intracellular reactive oxygen species (ROS). As an important mechanism of nanotoxicity, there is a great need for sensitive and high-throughput adaptable single-cell ROS detection methods. Here, fluorescence lifetime imaging microscopy (FLIM) is employed for single-cell ROS detection (FLIM-ROX) providing increased sensitivity and enabling high-throughput analysis in fixed and live cells. FLIM-ROX owes its sensitivity to the discrimination of autofluorescence from the unique fluorescence lifetime of the ROS reporter dye. The effect of subcytotoxic amounts of cationic gold nanoparticles in J774A.1 cells and primary human macrophages on ROS generation is investigated. FLIM-ROX measures very low ROS levels upon gold nanoparticle exposure, which is undetectable by the conventional method. It is demonstrated that cellular morphology changes, elevated senescence, and DNA damage link the resulting low-level oxidative stress to cellular adverse effects and thus nanotoxicity. Multiphoton FLIM-ROX enables the quantification of spatial ROS distribution in vivo, which is shown for skin tissue as a target for nanoparticle exposure. Thus, this innovative method allows identifying of low-level ROS in vitro and in vivo and, subsequently, promotes understanding of ROS-associated nanotoxicity. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Time to "go large" on biofilm research: advantages of an omics approach.

PubMed

Azevedo, Nuno F; Lopes, Susana P; Keevil, Charles W; Pereira, Maria O; Vieira, Maria J

2009-04-01

In nature, the biofilm mode of life is of great importance in the cell cycle for many microorganisms. Perhaps because of biofilm complexity and variability, the characterization of a given microbial system, in terms of biofilm formation potential, structure and associated physiological activity, in a large-scale, standardized and systematic manner has been hindered by the absence of high-throughput methods. This outlook is now starting to change as new methods involving the utilization of microtiter-plates and automated spectrophotometry and microscopy systems are being developed to perform large-scale testing of microbial biofilms. Here, we evaluate if the time is ripe to start an integrated omics approach, i.e., the generation and interrogation of large datasets, to biofilms--"biofomics". This omics approach would bring much needed insight into how biofilm formation ability is affected by a number of environmental, physiological and mutational factors and how these factors interplay between themselves in a standardized manner. This could then lead to the creation of a database where biofilm signatures are identified and interrogated. Nevertheless, and before embarking on such an enterprise, the selection of a versatile, robust, high-throughput biofilm growing device and of appropriate methods for biofilm analysis will have to be performed. Whether such device and analytical methods are already available, particularly for complex heterotrophic biofilms is, however, very debatable.

iLAP: a workflow-driven software for experimental protocol development, data acquisition and analysis

PubMed Central

2009-01-01

Background In recent years, the genome biology community has expended considerable effort to confront the challenges of managing heterogeneous data in a structured and organized way and developed laboratory information management systems (LIMS) for both raw and processed data. On the other hand, electronic notebooks were developed to record and manage scientific data, and facilitate data-sharing. Software which enables both, management of large datasets and digital recording of laboratory procedures would serve a real need in laboratories using medium and high-throughput techniques. Results We have developed iLAP (Laboratory data management, Analysis, and Protocol development), a workflow-driven information management system specifically designed to create and manage experimental protocols, and to analyze and share laboratory data. The system combines experimental protocol development, wizard-based data acquisition, and high-throughput data analysis into a single, integrated system. We demonstrate the power and the flexibility of the platform using a microscopy case study based on a combinatorial multiple fluorescence in situ hybridization (m-FISH) protocol and 3D-image reconstruction. iLAP is freely available under the open source license AGPL from http://genome.tugraz.at/iLAP/. Conclusion iLAP is a flexible and versatile information management system, which has the potential to close the gap between electronic notebooks and LIMS and can therefore be of great value for a broad scientific community. PMID:19941647

Isolation of Exosome-Like Nanoparticles and Analysis of MicroRNAs Derived from Coconut Water Based on Small RNA High-Throughput Sequencing.

PubMed

Zhao, Zhehao; Yu, Siran; Li, Min; Gui, Xin; Li, Ping

2018-03-21

In this study, the presence of microRNAs in coconut water was identified by real-time polymerase chain reaction (PCR) based on the results of high-throughput small RNA sequencing. In addition, the differences in microRNA content between immature and mature coconut water were compared. A total of 47 known microRNAs belonging to 25 families and 14 new microRNAs were identified in coconut endosperm. Through analysis using a target gene prediction software, potential microRNA target genes were identified in the human genome. Real-time PCR showed that the level of most microRNAs was higher in mature coconut water than in immature coconut water. Then, exosome-like nanoparticles were isolated from coconut water. After ultracentrifugation, some particle structures were seen in coconut water samples using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate fluorescence staining. Subsequent scanning electron microscopy observation and dynamic light scattering analysis also revealed some exosome-like nanoparticles in coconut water, and the mean diameters of the particles detected by the two methods were 13.16 and 59.72 nm, respectively. In conclusion, there are extracellular microRNAs in coconut water, and their levels are higher in mature coconut water than in immature coconut water. Some exosome-like nanoparticles were isolated from coconut water, and the diameter of these particles was smaller than that of animal-derived exosomes.

Endophyte Microbiome Diversity in Micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

PubMed Central

Lucero, Mary E.; Unc, Adrian; Cooke, Peter; Dowd, Scot; Sun, Shulei

2011-01-01

Microbial diversity associated with micropropagated Atriplex species was assessed using microscopy, isolate culturing, and sequencing. Light, electron, and confocal microscopy revealed microbial cells in aseptically regenerated leaves and roots. Clone libraries and tag-encoded FLX amplicon pyrosequencing (TEFAP) analysis amplified sequences from callus homologous to diverse fungal and bacterial taxa. Culturing isolated some seed borne endophyte taxa which could be readily propagated apart from the host. Microbial cells were observed within biofilm-like residues associated with plant cell surfaces and intercellular spaces. Various universal primers amplified both plant and microbial sequences, with different primers revealing different patterns of fungal diversity. Bacterial and fungal TEFAP followed by alignment with sequences from curated databases revealed 7 bacterial and 17 ascomycete taxa in A. canescens, and 5 bacterial taxa in A. torreyi. Additional diversity was observed among isolates and clone libraries. Micropropagated Atriplex retains a complex, intimately associated microbiome which includes diverse strains well poised to interact in manners that influence host physiology. Microbiome analysis was facilitated by high throughput sequencing methods, but primer biases continue to limit recovery of diverse sequences from even moderately complex communities. PMID:21437280

CoPt/TiN films nanopatterned by RF plasma etching towards dot-patterned magnetic media

NASA Astrophysics Data System (ADS)

Szívós, János; Pothorszky, Szilárd; Soltys, Jan; Serényi, Miklós; An, Hongyu; Gao, Tenghua; Deák, András; Shi, Ji; Sáfrán, György

2018-03-01

CoPt thin films as possible candidates for Bit Patterned magnetic Media (BPM) were prepared and investigated by electron microscopy techniques and magnetic measurements. The structure and morphology of the Direct Current (DC) sputtered films with N incorporation were revealed in both as-prepared and annealed state. Nanopatterning of the samples was carried out by means of Radio Frequency (RF) plasma etching through a Langmuir-Blodgett film of silica nanospheres that is a fast and high throughput technique. As a result, the samples with hexagonally arranged 100 nm size separated dots of fct-phase CoPt were obtained. The influence of the order of nanopatterning and anneling on the nanostructure formation was revealed. The magnetic properties of the nanopatterned fct CoPt films were investigated by Vibrating Sample Magnetometer (VSM) and Magnetic Force Microscopy (MFM). The results show that CoPt thin film nanopatterned by means of the RF plasma etching technique is promising candidate to a possible realization of BPM. Furthermore, this technique is versatile and suitable for scaling up to technological and industrial applications.

Complete Prevention of Dendrite Formation in Zn Metal Anodes by Means of Pulsed Charging Protocols.

PubMed

Garcia, Grecia; Ventosa, Edgar; Schuhmann, Wolfgang

2017-06-07

Zn metal as anode in rechargeable batteries, such as Zn/air or Zn/Ni, suffers from poor cyclability. The formation of Zn dendrites upon cycling is the key limiting step. We report a systematic study of the influence of pulsed electroplating protocols on the formation of Zn dendrites and in turn on strategies to completely prevent Zn dendrite formation. Because of the large number of variables in electroplating protocols, a scanning droplet cell technique was adapted as a high-throughput methodology in which a descriptor of the surface roughness can be in situ derived by means of electrochemical impedance spectroscopy. Upon optimizing the electroplating protocol by controlling nucleation, zincate ion depletion, and zincate ion diffusion, scanning electron microscopy and atomic force microscopy confirmed the growth of uniform and homogenous Zn deposits with a complete prevention of dendrite growth. The implementation of pulsed electroplating as the charging protocol for commercially available Ni-Zn batteries leads to substantially prolonged cyclability demonstrating the benefits of pulsed charging in Zn metal-based batteries.

A system architecture for online data interpretation and reduction in fluorescence microscopy

NASA Astrophysics Data System (ADS)

Röder, Thorsten; Geisbauer, Matthias; Chen, Yang; Knoll, Alois; Uhl, Rainer

2010-01-01

In this paper we present a high-throughput sample screening system that enables real-time data analysis and reduction for live cell analysis using fluorescence microscopy. We propose a novel system architecture capable of analyzing a large amount of samples during the experiment and thus greatly minimizing the post-analysis phase that is the common practice today. By utilizing data reduction algorithms, relevant information of the target cells is extracted from the online collected data stream, and then used to adjust the experiment parameters in real-time, allowing the system to dynamically react on changing sample properties and to control the microscope setup accordingly. The proposed system consists of an integrated DSP-FPGA hybrid solution to ensure the required real-time constraints, to execute efficiently the underlying computer vision algorithms and to close the perception-action loop. We demonstrate our approach by addressing the selective imaging of cells with a particular combination of markers. With this novel closed-loop system the amount of superfluous collected data is minimized, while at the same time the information entropy increases.

Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication

PubMed Central

Degtyarev, Michael; Reichelt, Mike; Lin, Kui

2014-01-01

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

TCP Throughput Profiles Using Measurements over Dedicated Connections

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

Rao, Nageswara S.; Liu, Qiang; Sen, Satyabrata

Wide-area data transfers in high-performance computing infrastructures are increasingly being carried over dynamically provisioned dedicated network connections that provide high capacities with no competing traffic. We present extensive TCP throughput measurements and time traces over a suite of physical and emulated 10 Gbps connections with 0-366 ms round-trip times (RTTs). Contrary to the general expectation, they show significant statistical and temporal variations, in addition to the overall dependencies on the congestion control mechanism, buffer size, and the number of parallel streams. We analyze several throughput profiles that have highly desirable concave regions wherein the throughput decreases slowly with RTTs, inmore » stark contrast to the convex profiles predicted by various TCP analytical models. We present a generic throughput model that abstracts the ramp-up and sustainment phases of TCP flows, which provides insights into qualitative trends observed in measurements across TCP variants: (i) slow-start followed by well-sustained throughput leads to concave regions; (ii) large buffers and multiple parallel streams expand the concave regions in addition to improving the throughput; and (iii) stable throughput dynamics, indicated by a smoother Poincare map and smaller Lyapunov exponents, lead to wider concave regions. These measurements and analytical results together enable us to select a TCP variant and its parameters for a given connection to achieve high throughput with statistical guarantees.« less

Enhancing high throughput toxicology - development of putative adverse outcome pathways linking US EPA ToxCast screening targets to relevant apical hazards.

EPA Science Inventory

High throughput toxicology programs, such as ToxCast and Tox21, have provided biological effects data for thousands of chemicals at multiple concentrations. Compared to traditional, whole-organism approaches, high throughput assays are rapid and cost-effective, yet they generall...

Evaluation of High-Throughput Chemical Exposure Models via Analysis of Matched Environmental and Biological Media Measurements

EPA Science Inventory

The U.S. EPA, under its ExpoCast program, is developing high-throughput near-field modeling methods to estimate human chemical exposure and to provide real-world context to high-throughput screening (HTS) hazard data. These novel modeling methods include reverse methods to infer ...

The development of a general purpose ARM-based processing unit for the ATLAS TileCal sROD

NASA Astrophysics Data System (ADS)

Cox, M. A.; Reed, R.; Mellado, B.

2015-01-01

After Phase-II upgrades in 2022, the data output from the LHC ATLAS Tile Calorimeter will increase significantly. ARM processors are common in mobile devices due to their low cost, low energy consumption and high performance. It is proposed that a cost-effective, high data throughput Processing Unit (PU) can be developed by using several consumer ARM processors in a cluster configuration to allow aggregated processing performance and data throughput while maintaining minimal software design difficulty for the end-user. This PU could be used for a variety of high-level functions on the high-throughput raw data such as spectral analysis and histograms to detect possible issues in the detector at a low level. High-throughput I/O interfaces are not typical in consumer ARM System on Chips but high data throughput capabilities are feasible via the novel use of PCI-Express as the I/O interface to the ARM processors. An overview of the PU is given and the results for performance and throughput testing of four different ARM Cortex System on Chips are presented.

Ultra-high throughput real-time instruments for capturing fast signals and rare events

NASA Astrophysics Data System (ADS)

Buckley, Brandon Walter

Wide-band signals play important roles in the most exciting areas of science, engineering, and medicine. To keep up with the demands of exploding internet traffic, modern data centers and communication networks are employing increasingly faster data rates. Wide-band techniques such as pulsed radar jamming and spread spectrum frequency hopping are used on the battlefield to wrestle control of the electromagnetic spectrum. Neurons communicate with each other using transient action potentials that last for only milliseconds at a time. And in the search for rare cells, biologists flow large populations of cells single file down microfluidic channels, interrogating them one-by-one, tens of thousands of times per second. Studying and enabling such high-speed phenomena pose enormous technical challenges. For one, parasitic capacitance inherent in analog electrical components limits their response time. Additionally, converting these fast analog signals to the digital domain requires enormous sampling speeds, which can lead to significant jitter and distortion. State-of-the-art imaging technologies, essential for studying biological dynamics and cells in flow, are limited in speed and sensitivity by finite charge transfer and read rates, and by the small numbers of photo-electrons accumulated in short integration times. And finally, ultra-high throughput real-time digital processing is required at the backend to analyze the streaming data. In this thesis, I discuss my work in developing real-time instruments, employing ultrafast optical techniques, which overcome some of these obstacles. In particular, I use broadband dispersive optics to slow down fast signals to speeds accessible to high-bit depth digitizers and signal processors. I also apply telecommunication multiplexing techniques to boost the speeds of confocal fluorescence microscopy. The photonic time stretcher (TiSER) uses dispersive Fourier transformation to slow down analog signals before digitization and processing. The act of time-stretching effectively boosts the performance of the back-end electronics and digital signal processors. The slowed down signals reach the back-end electronics with reduced bandwidth, and are therefore less affected by high-frequency roll-off and distortion. Time-stretching also increases the effective sampling rate of analog-to-digital converters and reduces aperture jitter, thereby improving resolution. Finally, the instantaneous throughputs of digital signal processors are enhanced by the stretch factor to otherwise unattainable speeds. Leveraging these unique capabilities, TiSER becomes the ideal tool for capturing high-speed signals and characterizing rare phenomena. For this thesis, I have developed techniques to improve the spectral efficiency, bandwidth, and resolution of TiSER using polarization multiplexing, all-optical modulation, and coherent dispersive Fourier transformation. To reduce the latency and improve the data handling capacity, I have also designed and implemented a real-time digital signal processing electronic backend, achieving 1.5 tera-bit per second instantaneous processing throughput. Finally, I will present results from experiments highlighting TiSER's impact in real-world applications. Confocal fluorescence microscopy is the most widely used method for unveiling the molecular composition of biological specimens. However, the weak optical emission of fluorescent probes and the tradeoff between imaging speed and sensitivity is problematic for acquiring blur-free images of fast phenomena and cells flowing at high speed. Here I introduce a new fluorescence imaging modality, which leverages techniques from wireless communication to reach record pixel and frame rates. Termed Fluorescence Imaging using Radio-frequency tagged Emission (FIRE), this new imaging modality is capable of resolving never before seen dynamics in living cells - such as action potentials in neurons and metabolic waves in astrocytes - as well as performing high-content image assays of cells and particles in high-speed flow.

[Current applications of high-throughput DNA sequencing technology in antibody drug research].

PubMed

Yu, Xin; Liu, Qi-Gang; Wang, Ming-Rong

2012-03-01

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.

Two-Step Physical Deposition of a Compact CuI Hole-Transport Layer and the Formation of an Interfacial Species in Perovskite Solar Cells.

PubMed

Gharibzadeh, Saba; Nejand, Bahram Abdollahi; Moshaii, Ahmad; Mohammadian, Nasim; Alizadeh, Amir Hossein; Mohammadpour, Rahele; Ahmadi, Vahid; Alizadeh, Abdolali

2016-08-09

A simple and practical approach is introduced for the deposition of CuI as an inexpensive inorganic hole-transport material (HTM) for the fabrication of low cost perovskite solar cells (PSCs) by gas-solid phase transformation of Cu to CuI. The method provides a uniform and well-controlled CuI layer with large grains and good compactness that prevents the direct connection between the contact electrodes. Solar cells prepared with CuI as the HTM with Au electrodes displays an exceptionally high short-circuit current density of 32 mA cm(-2) , owing to an interfacial species formed between the perovskite and the Cu resulting in a long wavelength contribution to the incident photon-to-electron conversion efficiency (IPCE), and an overall power conversion efficiency (PCE) of 7.4 %. The growth of crystalline and uniform CuI on a low roughness perovskite layer leads to remarkably high charge extraction in the cells, which originates from the high hole mobility of CuI in addition to a large number of contact points between CuI and the perovskite layer. In addition, the solvent-free method has no damaging side effect on the perovskite layer, which makes it an appropriate method for large scale applications of CuI in perovskite solar cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lessons from high-throughput protein crystallization screening: 10 years of practical experience

PubMed Central

JR, Luft; EH, Snell; GT, DeTitta

2011-01-01

Introduction X-ray crystallography provides the majority of our structural biological knowledge at a molecular level and in terms of pharmaceutical design is a valuable tool to accelerate discovery. It is the premier technique in the field, but its usefulness is significantly limited by the need to grow well-diffracting crystals. It is for this reason that high-throughput crystallization has become a key technology that has matured over the past 10 years through the field of structural genomics. Areas covered The authors describe their experiences in high-throughput crystallization screening in the context of structural genomics and the general biomedical community. They focus on the lessons learnt from the operation of a high-throughput crystallization screening laboratory, which to date has screened over 12,500 biological macromolecules. They also describe the approaches taken to maximize the success while minimizing the effort. Through this, the authors hope that the reader will gain an insight into the efficient design of a laboratory and protocols to accomplish high-throughput crystallization on a single-, multiuser-laboratory or industrial scale. Expert Opinion High-throughput crystallization screening is readily available but, despite the power of the crystallographic technique, getting crystals is still not a solved problem. High-throughput approaches can help when used skillfully; however, they still require human input in the detailed analysis and interpretation of results to be more successful. PMID:22646073

High-throughput screening based on label-free detection of small molecule microarrays

NASA Astrophysics Data System (ADS)

Zhu, Chenggang; Fei, Yiyan; Zhu, Xiangdong

2017-02-01

Based on small-molecule microarrays (SMMs) and oblique-incidence reflectivity difference (OI-RD) scanner, we have developed a novel high-throughput drug preliminary screening platform based on label-free monitoring of direct interactions between target proteins and immobilized small molecules. The screening platform is especially attractive for screening compounds against targets of unknown function and/or structure that are not compatible with functional assay development. In this screening platform, OI-RD scanner serves as a label-free detection instrument which is able to monitor about 15,000 biomolecular interactions in a single experiment without the need to label any biomolecule. Besides, SMMs serves as a novel format for high-throughput screening by immobilization of tens of thousands of different compounds on a single phenyl-isocyanate functionalized glass slide. Based on the high-throughput screening platform, we sequentially screened five target proteins (purified target proteins or cell lysate containing target protein) in high-throughput and label-free mode. We found hits for respective target protein and the inhibition effects for some hits were confirmed by following functional assays. Compared to traditional high-throughput screening assay, the novel high-throughput screening platform has many advantages, including minimal sample consumption, minimal distortion of interactions through label-free detection, multi-target screening analysis, which has a great potential to be a complementary screening platform in the field of drug discovery.

High-performance inverted planar heterojunction perovskite solar cells based on a solution-processed CuOx hole transport layer.

PubMed

Sun, Weihai; Li, Yunlong; Ye, Senyun; Rao, Haixia; Yan, Weibo; Peng, Haitao; Li, Yu; Liu, Zhiwei; Wang, Shufeng; Chen, Zhijian; Xiao, Lixin; Bian, Zuqiang; Huang, Chunhui

2016-05-19

During the past several years, methylammonium lead halide perovskites have been widely investigated as light absorbers for thin-film photovoltaic cells. Among the various device architectures, the inverted planar heterojunction perovskite solar cells have attracted special attention for their relatively simple fabrication and high efficiencies. Although promising efficiencies have been obtained in the inverted planar geometry based on poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) sulfonic acid ( PSS) as the hole transport material (HTM), the hydrophilicity of the PSS is a critical factor for long-term stability. In this paper, a CuOx hole transport layer from a facile solution-processed method was introduced into the inverted planar heterojunction perovskite solar cells. After the optimization of the devices, a champion PCE of 17.1% was obtained with an open circuit voltage (Voc) of 0.99 V, a short-circuit current (Jsc) of 23.2 mA cm(-2) and a fill factor (FF) of 74.4%. Furthermore, the unencapsulated device cooperating with the CuOx film exhibited superior performance in the stability test, compared to the device involving the PSS layer, indicating that CuOx could be a promising HTM for replacing PSS in inverted planar heterojunction perovskite solar cells.

Efficient Carbon-Based CsPbBr3 Inorganic Perovskite Solar Cells by Using Cu-Phthalocyanine as Hole Transport Material

NASA Astrophysics Data System (ADS)

Liu, Zhiyong; Sun, Bo; Liu, Xingyue; Han, Jinghui; Ye, Haibo; Shi, Tielin; Tang, Zirong; Liao, Guanglan

2018-06-01

Metal halide perovskite solar cells (PSCs) have attracted extensive research interest for next-generation solution-processed photovoltaic devices because of their high solar-to-electric power conversion efficiency (PCE) and low fabrication cost. Although the world's best PSC successfully achieves a considerable PCE of over 20% within a very limited timeframe after intensive efforts, the stability, high cost, and up-scaling of PSCs still remain issues. Recently, inorganic perovskite material, CsPbBr3, is emerging as a promising photo-sensitizer with excellent durability and thermal stability, but the efficiency is still embarrassing. In this work, we intend to address these issues by exploiting CsPbBr3 as light absorber, accompanied by using Cu-phthalocyanine (CuPc) as hole transport material (HTM) and carbon as counter electrode. The optimal device acquires a decent PCE of 6.21%, over 60% higher than those of the HTM-free devices. The systematic characterization and analysis reveal a more effective charge transfer process and a suppressed charge recombination in PSCs after introducing CuPc as hole transfer layer. More importantly, our devices exhibit an outstanding durability and a promising thermal stability, making it rather meaningful in future fabrication and application of PSCs.[Figure not available: see fulltext.

Studying post-etching silicon crystal defects on 300mm wafer by automatic defect review AFM

NASA Astrophysics Data System (ADS)

Zandiatashbar, Ardavan; Taylor, Patrick A.; Kim, Byong; Yoo, Young-kook; Lee, Keibock; Jo, Ahjin; Lee, Ju Suk; Cho, Sang-Joon; Park, Sang-il

2016-03-01

Single crystal silicon wafers are the fundamental elements of semiconductor manufacturing industry. The wafers produced by Czochralski (CZ) process are very high quality single crystalline materials with known defects that are formed during the crystal growth or modified by further processing. While defects can be unfavorable for yield for some manufactured electrical devices, a group of defects like oxide precipitates can have both positive and negative impacts on the final device. The spatial distribution of these defects may be found by scattering techniques. However, due to limitations of scattering (i.e. light wavelength), many crystal defects are either poorly classified or not detected. Therefore a high throughput and accurate characterization of their shape and dimension is essential for reviewing the defects and proper classification. While scanning electron microscopy (SEM) can provide high resolution twodimensional images, atomic force microscopy (AFM) is essential for obtaining three-dimensional information of the defects of interest (DOI) as it is known to provide the highest vertical resolution among all techniques [1]. However AFM's low throughput, limited tip life, and laborious efforts for locating the DOI have been the limitations of this technique for defect review for 300 mm wafers. To address these limitations of AFM, automatic defect review AFM has been introduced recently [2], and is utilized in this work for studying DOI on 300 mm silicon wafer. In this work, we carefully etched a 300 mm silicon wafer with a gaseous acid in a reducing atmosphere at a temperature and for a sufficient duration to decorate and grow the crystal defects to a size capable of being detected as light scattering defects [3]. The etched defects form a shallow structure and their distribution and relative size are inspected by laser light scattering (LLS). However, several groups of defects couldn't be properly sized by the LLS due to the very shallow depth and low light scattering. Likewise, SEM cannot be used effectively for post-inspection defect review and classification of these very shallow types of defects. To verify and obtain accurate shape and three-dimensional information of those defects, automatic defect review AFM (ADR AFM) is utilized for accurate locating and imaging of DOI. In ADR AFM, non-contact mode imaging is used for non-destructive characterization and preserving tip sharpness for data repeatability and reproducibility. Locating DOI and imaging are performed automatically with a throughput of many defects per hour. Topography images of DOI has been collected and compared with SEM images. The ADR AFM has been shown as a non-destructive metrology tool for defect review and obtaining three-dimensional topography information.

Telescopes and recording systems used by amateurs for studying planets in our solar system - an overview

NASA Astrophysics Data System (ADS)

Kowollik, S.; Gaehrken, B.; Fiedler, M.; Gerstheimer, R.; Sohl, F.; Koschny, D.

2008-09-01

During the last couple of years, engaged amateur astronomers have benefited by the rapid development in the field of commercial CCD cameras, video techniques, and the availability of mirror telescopes with high quality. Until recently, such technical equipment and the related handling experience had been reserved to research institutes. This contribution presents the potential capabilities of amateur astronomers and describes the approach to the production of data. The quality of the used telescopes is described with respect to aperture and resolving power; as well as the quantum efficiency of the used sensitive b/w CCD cameras with respect to the detectable wavelength. Beyond these facts the necessary exposure times for CCD images using special filters are discussed. Today's amateur astronomers are able to image the bodies of the solar system in the wavelength range between 340 and 1050 nm [1], [2], [3], [4]. This covers a wide range of the spectrum which is investigated with cameras on board of space telescopes or planetary probes. While space probes usually obtain high-resolution images of individual Surface or atmospheric features of the planets, the images of amateur astronomers show the entire surface of the observed planet. Both datasets together permit a more comprehensive analysis of the data aquired in each case. The "Venus Amateur Observing Project" of the European Space Agency [5] is a first step into a successful co-operation between amateur astronomers and planetary scientists. Individual CCD images captured through the turbulent atmosphere of the Earth usually show characteristic distortions of the arriving wave fronts. If one captures hundreds or thousands of images on a video stream in very short time, there will be always also undistorted images within the data. Computer programmes are available to identify and retrieve these undistorted images and store them for further processing [7]. This method is called "Lucky Imaging" and it allows to achieve nearly the theoretical limit of telescopic resolution. By stacking the undistorted images, the signal-to-noise ratio of the data can be increased significantly. "Lucky Imaging" has become a standard in the amateur community since several years. Contrary to space based observations the data rate is not limited by the capacity of any radio transmission, but only limited by the scanning rate and capacity of a modern computer hard disk. An individual video with the uncompreesed raw data can be as large as 4 to 5 GB. EPSC Abstracts, Vol. 3, EPSC2008-A-00191, 2008 European Planetary Science Congress, Author(s) 2008 In addition to the video data, so-called meta data such as the observing location, the recording time, the used filter, environmental conditions (air temperature, wind velocity, air humidity and Seeing) are also documented. From these meta data, the central meridian (CM) of the observed planet during the time of image acqusition can be determined. After data reduction the resulting images can be used to produce map projections or position measurements of albedo structures on the planetary surface or of details within atmospheric features. Amateur astronomers can observe objects in the solar system for large continuous time periods due to the large number of the existing observers e. g. the members of the Association of Lunar & Planetary Observers [6] and their telescopes. They can and react very fast to special events, since they do not have to submit requests for telescope time to a national or international organization. References: [1] Venusimages in uv-light: B. Gährken: http://www.astrode.de/venus07.htm R. Gerstheimer: http://www.astromanie.de/astromania/galerie/venus/venus.html S. Kowollik: http://www.sternwarte-zollern-alb.de/mitarbeiterseiten/kowollik/venus M. Weigand: http://www.skytrip.de/venus2007.htm [2] Images of planets in visible light: M. Fiedler: http://bilder.astroclub-radebeul.de/kategorien.php?action=showukats&kat=0 R. Gerstheimer: http://www.astromanie.de/ S. Kowollik: http://www.sternwarte-zollern-alb.de/mitarbeiterseiten/kowollik [3] Images of planets in methane band light: S. Kowollik: http://www.sternwarte-zollern-alb.de/beobachtungen/methanband/index-gb.htm [4] Images of planets in ir-light: S. Kowollik: http://www.sternwarte-zollern-alb.de/beobachtungen/ir/index-gb.htm [5] ESA amateur astronomer observing campaign: http://sci.esa.int/science-e/www/object/index.cfm?fobjectid=38833 http://www.rssd.esa.int/index.php?project=VENUS [6] Association of Lunar & Planetary Observation (ALPO): http://alpo-astronomy.org/ [7] Software: Cor Berrevoets (Registax): http://www.astronomie.be/registax/ Christian Buil (IRIS): http://www.astrosurf.com/buil/us/iris/iris.htm Georg Dittié (Giotto): http://www.videoastronomy.org/giotto.htm Grischa Hahn (WinJupos): http://www.grischa-hahn.homepage.t-online.de/astro/winjupos/index.htm

Electrodeposition and Screening of Photoelectrochemical Activity in Conjugated Polymers Using Scanning Electrochemical Cell Microscopy.

PubMed

Aaronson, Barak D B; Garoz-Ruiz, Jesus; Byers, Joshua C; Colina, Alvaro; Unwin, Patrick R

2015-11-24

A number of renewable energy systems require an understanding and correlation of material properties and photoelectrochemical activity on the micro to nanoscale. Among these, conducting polymer electrodes continue to be important materials. In this contribution, an ultrasensitive scanning electrochemical cell microscopy (SECCM) platform is used to electrodeposit microscale thin films of poly(3-hexylthiophene) (P3HT) on an optically transparent gold electrode and to correlate the morphology (film thickness and structural order) with photoactivity. The electrochemical growth of P3HT begins with a thin ordered film up to 10 nm thick, after which a second more disordered film is deposited, as revealed by micro-Raman spectroscopy. A decrease in photoactivity for the thicker films, measured in situ immediately following film deposition, is attributed to an increase in bulk film disorder that limits charge transport. Higher resolution ex situ SECCM phototransient measurements, using a smaller diameter probe, show local variations in photoactivity within a given deposit. Even after aging, thinner, more ordered regions within a deposit exhibit sustained enhanced photocurrent densities compared to areas where the film is thicker and more disordered. The platform opens up new possibilities for high-throughput combinatorial correlation studies, by allowing materials fabrication and high spatial resolution probing of processes in photoelectrochemical materials.

Specimen preparation, imaging, and analysis protocols for knife-edge scanning microscopy.

PubMed

Choe, Yoonsuck; Mayerich, David; Kwon, Jaerock; Miller, Daniel E; Sung, Chul; Chung, Ji Ryang; Huffman, Todd; Keyser, John; Abbott, Louise C

2011-12-09

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM), developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi), vascular networks (India ink), and cell body distribution (Nissl). The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

Measuring molecular biomarkers in epidemiologic studies: laboratory techniques and biospecimen considerations.

PubMed

Erickson, Heidi S

2012-09-28

The future of personalized medicine depends on the ability to efficiently and rapidly elucidate a reliable set of disease-specific molecular biomarkers. High-throughput molecular biomarker analysis methods have been developed to identify disease risk, diagnostic, prognostic, and therapeutic targets in human clinical samples. Currently, high throughput screening allows us to analyze thousands of markers from one sample or one marker from thousands of samples and will eventually allow us to analyze thousands of markers from thousands of samples. Unfortunately, the inherent nature of current high throughput methodologies, clinical specimens, and cost of analysis is often prohibitive for extensive high throughput biomarker analysis. This review summarizes the current state of high throughput biomarker screening of clinical specimens applicable to genetic epidemiology and longitudinal population-based studies with a focus on considerations related to biospecimens, laboratory techniques, and sample pooling. Copyright © 2012 John Wiley & Sons, Ltd.

Malic Acid Carbon Dots: From Super-resolution Live-Cell Imaging to Highly Efficient Separation.

PubMed

Zhi, Bo; Cui, Yi; Wang, Shengyang; Frank, Benjamin P; Williams, Denise N; Brown, Richard P; Melby, Eric S; Hamers, Robert J; Rosenzweig, Zeev; Fairbrother, D Howard; Orr, Galya; Haynes, Christy L

2018-06-15

As-synthesized malic acid carbon dots are found to possess photoblinking properties that are outstanding and superior compared to those of conventional dyes. Considering their excellent biocompatibility, malic acid carbon dots are suitable for super-resolution fluorescence localization microscopy under a variety of conditions, as we demonstrate in fixed and live trout gill epithelial cells. In addition, during imaging experiments, the so-called "excitation wavelength-dependent" emission was not observed for individual as-made malic acid carbon dots, which motivated us to develop a time-saving and high-throughput separation technique to isolate malic acid carbon dots into fractions of different particle size distributions using C 18 reversed-phase silica gel column chromatography. This post-treatment allowed us to determine how particle size distribution influences the optical properties of malic acid carbon dot fractions, that is, optical band gap energies and photoluminescence behaviors.

Remotely Controlled Mixers for Light Microscopy Module (LMM) Colloid Samples

NASA Technical Reports Server (NTRS)

Kurk, Michael A. (Andy)

2015-01-01

Developed by NASA Glenn Research Center, the LMM aboard the International Space Station (ISS) is enabling multiple biomedical science experiments. Techshot, Inc., has developed a series of colloid specialty cell systems (C-SPECS) for use in the colloid science experiment module on the LMM. These low-volume mixing devices will enable uniform particle density and remotely controlled repetition of LMM colloid experiments. By automating the experiment process, C-SPECS allow colloid samples to be processed more quickly. In addition, C-SPECS will minimize the time the crew will need to spend on colloid experiments as well as eliminate the need for multiple and costly colloid samples, which are expended after a single examination. This high-throughput capability will lead to more efficient and productive use of the LMM. As commercial launch vehicles begin routine visits to the ISS, C-SPECS could become a significant means to process larger quantities of high-value materials for commercial customers.

MPLM Donatello is offloaded at the SLF

NASA Technical Reports Server (NTRS)

2001-01-01

At the KSC Shuttle Landing Facility, the Italian Space Agency's Multi- Purpose Logistics Module Donatello begins rolling out of the Airbus '''Beluga''' air cargo plane that brought it from the factory of Alenia Aerospazio in Turin, Italy. The third of three for the International Space Station, the module will be transported to the Space Station Processing Facility for processing. Among the activities for the payload test team are integrated electrical tests with other Station elements in the SSPF, leak tests, electrical and software compatibility tests with the Space Shuttle (using the Cargo Integrated Test equipment) and an Interface Verification Test once the module is installed in the Space Shuttle's payload bay at the launch pad. The most significant mechanical task to be performed on Donatello in the SSPF is the installation and outfitting of the racks for carrying the various experiments and cargo.

MPLM Donatello is offloaded at the SLF

NASA Technical Reports Server (NTRS)

2001-01-01

At the KSC Shuttle Landing Facility, an Airbus '''Beluga''' air cargo plane opens to reveal its cargo, the Italian Space Agency's Multi- Purpose Logistics Module Donatello, from the factory of Alenia Aerospazio in Turin, Italy. The third of three for the International Space Station, the module will be transported to the Space Station Processing Facility for processing. Among the activities for the payload test team are integrated electrical tests with other Station elements in the SSPF, leak tests, electrical and software compatibility tests with the Space Shuttle (using the Cargo Integrated Test equipment) and an Interface Verification Test once the module is installed in the Space Shuttle's payload bay at the launch pad. The most significant mechanical task to be performed on Donatello in the SSPF is the installation and outfitting of the racks for carrying the various experiments and cargo.

MPLM Donatello is offloaded at the SLF

NASA Technical Reports Server (NTRS)

2001-01-01

At the KSC Shuttle Landing Facility, the Italian Space Agency's Multi- Purpose Logistics Module Donatello rolls out of the Airbus '''Beluga''' air cargo plane that brought it from the factory of Alenia Aerospazio in Turin, Italy. The third of three for the International Space Station, the module will be transported to the Space Station Processing Facility for processing. Among the activities for the payload test team are integrated electrical tests with other Station elements in the SSPF, leak tests, electrical and software compatibility tests with the Space Shuttle (using the Cargo Integrated Test equipment) and an Interface Verification Test once the module is installed in the Space Shuttle's payload bay at the launch pad. The most significant mechanical task to be performed on Donatello in the SSPF is the installation and outfitting of the racks for carrying the various experiments and cargo.

40 CFR Table 9 to Subpart Eeee of... - Continuous Compliance With Operating Limits-High Throughput Transfer Racks

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 12 2010-07-01 2010-07-01 true Continuous Compliance With Operating Limits-High Throughput Transfer Racks 9 Table 9 to Subpart EEEE of Part 63 Protection of Environment...—Continuous Compliance With Operating Limits—High Throughput Transfer Racks As stated in §§ 63.2378(a) and (b...

Accelerating the design of solar thermal fuel materials through high throughput simulations.

PubMed

Liu, Yun; Grossman, Jeffrey C

2014-12-10

Solar thermal fuels (STF) store the energy of sunlight, which can then be released later in the form of heat, offering an emission-free and renewable solution for both solar energy conversion and storage. However, this approach is currently limited by the lack of low-cost materials with high energy density and high stability. In this Letter, we present an ab initio high-throughput computational approach to accelerate the design process and allow for searches over a broad class of materials. The high-throughput screening platform we have developed can run through large numbers of molecules composed of earth-abundant elements and identifies possible metastable structures of a given material. Corresponding isomerization enthalpies associated with the metastable structures are then computed. Using this high-throughput simulation approach, we have discovered molecular structures with high isomerization enthalpies that have the potential to be new candidates for high-energy density STF. We have also discovered physical principles to guide further STF materials design through structural analysis. More broadly, our results illustrate the potential of using high-throughput ab initio simulations to design materials that undergo targeted structural transitions.

High-speed atomic force microscopy and peak force tapping control

NASA Astrophysics Data System (ADS)

Hu, Shuiqing; Mininni, Lars; Hu, Yan; Erina, Natalia; Kindt, Johannes; Su, Chanmin

2012-03-01

ITRS Roadmap requires defect size measurement below 10 nanometers and challenging classifications for both blank and patterned wafers and masks. Atomic force microscope (AFM) is capable of providing metrology measurement in 3D at sub-nanometer accuracy but has long suffered from drawbacks in throughput and limitation of slow topography imaging without chemical information. This presentation focus on two disruptive technology developments, namely high speed AFM and quantitative nanomechanical mapping, which enables high throughput measurement with capability of identifying components through concurrent physical property imaging. The high speed AFM technology has allowed the imaging speed increase by 10-100 times without loss of the data quality. Such improvement enables the speed of defect review on a wafer to increase from a few defects per hour to nearly 100 defects an hour, approaching the requirements of ITRS Roadmap. Another technology development, Peak Force Tapping, substantially simplified the close loop system response, leading to self-optimization of most challenging samples groups to generate expert quality data. More importantly, AFM also simultaneously provides a series of mechanical property maps with a nanometer spatial resolution during defect review. These nanomechanical maps (including elastic modulus, hardness, and surface adhesion) provide complementary information for elemental analysis, differentiate defect materials by their physical properties, and assist defect classification beyond topographic measurements. This paper will explain the key enabling technologies, namely high speed tip-scanning AFM using innovative flexure design and control algorithm. Another critical element is AFM control using Peak Force Tapping, in which the instantaneous tip-sample interaction force is measured and used to derive a full suite of physical properties at each imaging pixel. We will provide examples of defect review data on different wafers and media disks. The similar AFM-based defect review capacity was also applied to EUV masks.

The combination of gas-phase fluorophore technology and automation to enable high-throughput analysis of plant respiration.

PubMed

Scafaro, Andrew P; Negrini, A Clarissa A; O'Leary, Brendan; Rashid, F Azzahra Ahmad; Hayes, Lucy; Fan, Yuzhen; Zhang, You; Chochois, Vincent; Badger, Murray R; Millar, A Harvey; Atkin, Owen K

2017-01-01

Mitochondrial respiration in the dark ( R dark ) is a critical plant physiological process, and hence a reliable, efficient and high-throughput method of measuring variation in rates of R dark is essential for agronomic and ecological studies. However, currently methods used to measure R dark in plant tissues are typically low throughput. We assessed a high-throughput automated fluorophore system of detecting multiple O 2 consumption rates. The fluorophore technique was compared with O 2 -electrodes, infrared gas analysers (IRGA), and membrane inlet mass spectrometry, to determine accuracy and speed of detecting respiratory fluxes. The high-throughput fluorophore system provided stable measurements of R dark in detached leaf and root tissues over many hours. High-throughput potential was evident in that the fluorophore system was 10 to 26-fold faster per sample measurement than other conventional methods. The versatility of the technique was evident in its enabling: (1) rapid screening of R dark in 138 genotypes of wheat; and, (2) quantification of rarely-assessed whole-plant R dark through dissection and simultaneous measurements of above- and below-ground organs. Variation in absolute R dark was observed between techniques, likely due to variation in sample conditions (i.e. liquid vs. gas-phase, open vs. closed systems), indicating that comparisons between studies using different measuring apparatus may not be feasible. However, the high-throughput protocol we present provided similar values of R dark to the most commonly used IRGA instrument currently employed by plant scientists. Together with the greater than tenfold increase in sample processing speed, we conclude that the high-throughput protocol enables reliable, stable and reproducible measurements of R dark on multiple samples simultaneously, irrespective of plant or tissue type.

Simultaneous Measurements of Auto-Immune and Infectious Disease Specific Antibodies Using a High Throughput Multiplexing Tool

PubMed Central

Asati, Atul; Kachurina, Olga; Kachurin, Anatoly

2012-01-01

Considering importance of ganglioside antibodies as biomarkers in various immune-mediated neuropathies and neurological disorders, we developed a high throughput multiplexing tool for the assessment of gangliosides-specific antibodies based on Biolpex/Luminex platform. In this report, we demonstrate that the ganglioside high throughput multiplexing tool is robust, highly specific and demonstrating ∼100-fold higher concentration sensitivity for IgG detection than ELISA. In addition to the ganglioside-coated array, the high throughput multiplexing tool contains beads coated with influenza hemagglutinins derived from H1N1 A/Brisbane/59/07 and H1N1 A/California/07/09 strains. Influenza beads provided an added advantage of simultaneous detection of ganglioside- and influenza-specific antibodies, a capacity important for the assay of both infectious antigen-specific and autoimmune antibodies following vaccination or disease. Taken together, these results support the potential adoption of the ganglioside high throughput multiplexing tool for measuring ganglioside antibodies in various neuropathic and neurological disorders. PMID:22952605

High-throughput sample adaptive offset hardware architecture for high-efficiency video coding

NASA Astrophysics Data System (ADS)

Zhou, Wei; Yan, Chang; Zhang, Jingzhi; Zhou, Xin

2018-03-01

A high-throughput hardware architecture for a sample adaptive offset (SAO) filter in the high-efficiency video coding video coding standard is presented. First, an implementation-friendly and simplified bitrate estimation method of rate-distortion cost calculation is proposed to reduce the computational complexity in the mode decision of SAO. Then, a high-throughput VLSI architecture for SAO is presented based on the proposed bitrate estimation method. Furthermore, multiparallel VLSI architecture for in-loop filters, which integrates both deblocking filter and SAO filter, is proposed. Six parallel strategies are applied in the proposed in-loop filters architecture to improve the system throughput and filtering speed. Experimental results show that the proposed in-loop filters architecture can achieve up to 48% higher throughput in comparison with prior work. The proposed architecture can reach a high-operating clock frequency of 297 MHz with TSMC 65-nm library and meet the real-time requirement of the in-loop filters for 8 K × 4 K video format at 132 fps.

An integrated laser trap/flow control video microscope for the study of single biomolecules.

PubMed Central

Wuite, G J; Davenport, R J; Rappaport, A; Bustamante, C

2000-01-01

We have developed an integrated laser trap/flow control video microscope for mechanical manipulation of single biopolymers. The instrument is automated to maximize experimental throughput. A single-beam optical trap capable of trapping micron-scale polystyrene beads in the middle of a 200-microm-deep microchamber is used, making it possible to insert a micropipette inside this chamber to hold a second bead by suction. Together, these beads function as easily exchangeable surfaces between which macromolecules of interest can be attached. A computer-controlled flow system is used to exchange the liquid in the chamber and to establish a flow rate with high precision. The flow and the optical trap can be used to exert forces on the beads, the displacements of which can be measured either by video microscopy or by laser deflection. To test the performance of this instrument, individual biotinylated DNA molecules were assembled between two streptavidin beads, and the DNA elasticity was characterized using both laser trap and flow forces. DNA extension under varying forces was measured by video microscopy. The combination of the flow system and video microscopy is a versatile design that is particularly useful for the study of systems susceptible to laser-induced damage. This capability was demonstrated by following the translocation of transcribing RNA polymerase up to 650 s. PMID:10920045

Stochastic optical reconstruction microscopy-based relative localization analysis (STORM-RLA) for quantitative nanoscale assessment of spatial protein organization.

PubMed

Veeraraghavan, Rengasayee; Gourdie, Robert G

2016-11-07

The spatial association between proteins is crucial to understanding how they function in biological systems. Colocalization analysis of fluorescence microscopy images is widely used to assess this. However, colocalization analysis performed on two-dimensional images with diffraction-limited resolution merely indicates that the proteins are within 200-300 nm of each other in the xy-plane and within 500-700 nm of each other along the z-axis. Here we demonstrate a novel three-dimensional quantitative analysis applicable to single-molecule positional data: stochastic optical reconstruction microscopy-based relative localization analysis (STORM-RLA). This method offers significant advantages: 1) STORM imaging affords 20-nm resolution in the xy-plane and <50 nm along the z-axis; 2) STORM-RLA provides a quantitative assessment of the frequency and degree of overlap between clusters of colabeled proteins; and 3) STORM-RLA also calculates the precise distances between both overlapping and nonoverlapping clusters in three dimensions. Thus STORM-RLA represents a significant advance in the high-throughput quantitative assessment of the spatial organization of proteins. © 2016 Veeraraghavan and Gourdie. 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).

Development of new photon-counting detectors for single-molecule fluorescence microscopy.

PubMed

Michalet, X; Colyer, R A; Scalia, G; Ingargiola, A; Lin, R; Millaud, J E; Weiss, S; Siegmund, Oswald H W; Tremsin, Anton S; Vallerga, John V; Cheng, A; Levi, M; Aharoni, D; Arisaka, K; Villa, F; Guerrieri, F; Panzeri, F; Rech, I; Gulinatti, A; Zappa, F; Ghioni, M; Cova, S

2013-02-05

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

Development of new photon-counting detectors for single-molecule fluorescence microscopy

PubMed Central

Michalet, X.; Colyer, R. A.; Scalia, G.; Ingargiola, A.; Lin, R.; Millaud, J. E.; Weiss, S.; Siegmund, Oswald H. W.; Tremsin, Anton S.; Vallerga, John V.; Cheng, A.; Levi, M.; Aharoni, D.; Arisaka, K.; Villa, F.; Guerrieri, F.; Panzeri, F.; Rech, I.; Gulinatti, A.; Zappa, F.; Ghioni, M.; Cova, S.

2013-01-01

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level. PMID:23267185