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.

Visual-servoing optical microscopy

DOEpatents

Callahan, Daniel E.; Parvin, Bahram

2009-06-09

The present invention provides methods and devices for the knowledge-based discovery and optimization of differences between cell types. In particular, the present invention provides visual servoing optical microscopy, as well as analysis methods. The present invention provides means for the close monitoring of hundreds of individual, living cells over time: quantification of dynamic physiological responses in multiple channels; real-time digital image segmentation and analysis; intelligent, repetitive computer-applied cell stress and cell stimulation; and the ability to return to the same field of cells for long-term studies and observation. The present invention further provides means to optimize culture conditions for specific subpopulations of cells.

Visual-servoing optical microscopy

DOEpatents

Callahan, Daniel E [Martinez, CA; Parvin, Bahram [Mill Valley, CA

2011-05-24

The present invention provides methods and devices for the knowledge-based discovery and optimization of differences between cell types. In particular, the present invention provides visual servoing optical microscopy, as well as analysis methods. The present invention provides means for the close monitoring of hundreds of individual, living cells over time; quantification of dynamic physiological responses in multiple channels; real-time digital image segmentation and analysis; intelligent, repetitive computer-applied cell stress and cell stimulation; and the ability to return to the same field of cells for long-term studies and observation. The present invention further provides means to optimize culture conditions for specific subpopulations of cells.

Visual-servoing optical microscopy

DOEpatents

Callahan, Daniel E; Parvin, Bahram

2013-10-01

The present invention provides methods and devices for the knowledge-based discovery and optimization of differences between cell types. In particular, the present invention provides visual servoing optical microscopy, as well as analysis methods. The present invention provides means for the close monitoring of hundreds of individual, living cells over time; quantification of dynamic physiological responses in multiple channels; real-time digital image segmentation and analysis; intelligent, repetitive computer-applied cell stress and cell stimulation; and the ability to return to the same field of cells for long-term studies and observation. The present invention further provides means to optimize culture conditions for specific subpopulations of cells.

Mathematical imaging methods for mitosis analysis in live-cell phase contrast microscopy.

PubMed

Grah, Joana Sarah; Harrington, Jennifer Alison; Koh, Siang Boon; Pike, Jeremy Andrew; Schreiner, Alexander; Burger, Martin; Schönlieb, Carola-Bibiane; Reichelt, Stefanie

2017-02-15

In this paper we propose a workflow to detect and track mitotic cells in time-lapse microscopy image sequences. In order to avoid the requirement for cell lines expressing fluorescent markers and the associated phototoxicity, phase contrast microscopy is often preferred over fluorescence microscopy in live-cell imaging. However, common specific image characteristics complicate image processing and impede use of standard methods. Nevertheless, automated analysis is desirable due to manual analysis being subjective, biased and extremely time-consuming for large data sets. Here, we present the following workflow based on mathematical imaging methods. In the first step, mitosis detection is performed by means of the circular Hough transform. The obtained circular contour subsequently serves as an initialisation for the tracking algorithm based on variational methods. It is sub-divided into two parts: in order to determine the beginning of the whole mitosis cycle, a backwards tracking procedure is performed. After that, the cell is tracked forwards in time until the end of mitosis. As a result, the average of mitosis duration and ratios of different cell fates (cell death, no division, division into two or more daughter cells) can be measured and statistics on cell morphologies can be obtained. All of the tools are featured in the user-friendly MATLAB®Graphical User Interface MitosisAnalyser. Copyright © 2017. Published by Elsevier Inc.

Extent of agreement between the body fluid model of Sysmex XN-20 and the manual microscopy method.

PubMed

Huang, Wei-Hua; Lu, Lin-Peng; Wu, Kang; Guo, Fang-Yu; Guo, Jie; Yu, Jing-Long; Zhou, Dao-Yin; Sun, Yi; Deng, An-Mei

2017-09-01

Although the correlations concerning cellular component analysis between the Sysmex XN-20 body fluid (BF) model and manual microscopy have been investigated by several studies, the extent of agreement between these two methods has not been investigated. A total of 90 BF samples were prospectively collected and analyzed using the Sysmex XN-20 BF model and microscopy. The extent of agreement between these two methods was evaluated using the Bland-Altman approach. Receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic accuracy of high-fluorescence (HF) BF cells for malignant diseases. The agreements of white blood cell (WBC), red blood cell (RBC), and percentages of neutrophils, lymphocytes, and monocytes between the Sysmex XN-20 BF model and manual microscopy were imperfect. The areas under the ROC curves for absolute and relative HF cells were 0.67 (95% confidence interval [CI]: 0.56-0.78) and 0.60 (95% CI: 0.48-0.72), respectively. Due to the Sysmex XN-20 BF model's imperfect agreement with manual microscopy and its weak diagnostic accuracy for malignant diseases, the current evidence does not support replacing manual microscopy with this model in clinical practice. © 2016 Wiley Periodicals, Inc.

New approaches for the analysis of confluent cell layers with quantitative phase digital holographic microscopy

NASA Astrophysics Data System (ADS)

Pohl, L.; Kaiser, M.; Ketelhut, S.; Pereira, S.; Goycoolea, F.; Kemper, Björn

2016-03-01

Digital holographic microscopy (DHM) enables high resolution non-destructive inspection of technical surfaces and minimally-invasive label-free live cell imaging. However, the analysis of confluent cell layers represents a challenge as quantitative DHM phase images in this case do not provide sufficient information for image segmentation, determination of the cellular dry mass or calculation of the cell thickness. We present novel strategies for the analysis of confluent cell layers with quantitative DHM phase contrast utilizing a histogram based-evaluation procedure. The applicability of our approach is illustrated by quantification of drug induced cell morphology changes and it is shown that the method is capable to quantify reliable global morphology changes of confluent cell layers.

Peculiarities of studying an isolated neuron by the method of laser interference microscopy

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

Yusipovich, Alexander I; Kazakova, Tatiana A; Erokhova, Liudmila A

2006-09-30

Actual aspects of using a new method of laser interference microscopy (LIM) for studying nerve cells are discussed. The peculiarities of the LIM display of neurons are demonstrated by the example of isolated neurons of a pond snail Lymnaea stagnalis. A comparative analysis of the images of the cell and subcellular structures of a neuron obtained by the methods of interference microscopy, optical transmission microscopy, and confocal microscopy is performed. Various aspects of the application of LIM for studying the lateral dimensions and internal structure of the cytoplasm and organelles of a neuron in cytology and cell physiology are discussed.more » (laser biology)« less

A Study of the Effects of High Power Pulsed 2450 MHz Microwaves, ELF modulated Microwaves, and ELF Fields on Human Lymphocytes and Selected Cell Lines

DTIC Science & Technology

1993-01-27

Considerable effect was expended in investigating shifts in intercellular calcium of one particular cell line, Jurket, using flow cytometry methods. No...culture. The following analysis were used to characterize the immortalized cell lines: flow cytometry , electron microscopy, two-dimensional protein gel...further characterized by flow cytometry , electron microscopy, two dimensional protein electrophoresis and nuclear run-off assay. Flow cytometric analysis of

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

Non-rigid multi-frame registration of cell nuclei in live cell fluorescence microscopy image data.

PubMed

Tektonidis, Marco; Kim, Il-Han; Chen, Yi-Chun M; Eils, Roland; Spector, David L; Rohr, Karl

2015-01-01

The analysis of the motion of subcellular particles in live cell microscopy images is essential for understanding biological processes within cells. For accurate quantification of the particle motion, compensation of the motion and deformation of the cell nucleus is required. We introduce a non-rigid multi-frame registration approach for live cell fluorescence microscopy image data. Compared to existing approaches using pairwise registration, our approach exploits information from multiple consecutive images simultaneously to improve the registration accuracy. We present three intensity-based variants of the multi-frame registration approach and we investigate two different temporal weighting schemes. The approach has been successfully applied to synthetic and live cell microscopy image sequences, and an experimental comparison with non-rigid pairwise registration has been carried out. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid enumeration of viable bacteria by image analysis

NASA Technical Reports Server (NTRS)

Singh, A.; Pyle, B. H.; McFeters, G. A.

1989-01-01

A direct viable counting method for enumerating viable bacteria was modified and made compatible with image analysis. A comparison was made between viable cell counts determined by the spread plate method and direct viable counts obtained using epifluorescence microscopy either manually or by automatic image analysis. Cultures of Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Yersinia enterocolitica and Pseudomonas aeruginosa were incubated at 35 degrees C in a dilute nutrient medium containing nalidixic acid. Filtered samples were stained for epifluorescence microscopy and analysed manually as well as by image analysis. Cells enlarged after incubation were considered viable. The viable cell counts determined using image analysis were higher than those obtained by either the direct manual count of viable cells or spread plate methods. The volume of sample filtered or the number of cells in the original sample did not influence the efficiency of the method. However, the optimal concentration of nalidixic acid (2.5-20 micrograms ml-1) and length of incubation (4-8 h) varied with the culture tested. The results of this study showed that under optimal conditions, the modification of the direct viable count method in combination with image analysis microscopy provided an efficient and quantitative technique for counting viable bacteria in a short time.

Live cell refractometry using Hilbert phase microscopy and confocal reflectance microscopy.

PubMed

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

2009-11-26

Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ.

Chemical imaging of molecular changes in a hydrated single cell by dynamic secondary ion mass spectrometry and super-resolution microscopy

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

Hua, Xin; Szymanski, Craig; Wang, Zhaoying

2016-01-01

Chemical imaging of single cells is important in capturing biological dynamics. Single cell correlative imaging is realized between structured illumination microscopy (SIM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) using System for Analysis at the Liquid Vacuum Interface (SALVI), a multimodal microreactor. SIM characterized cells and guided subsequent ToF-SIMS analysis. Dynamic ToF-SIMS provided time- and space-resolved cell molecular mapping. Lipid fragments were identified in the hydrated cell membrane. Principal component analysis was used to elucidate chemical component differences among mouse lung cells that uptake zinc oxide nanoparticles. Our results provided submicron chemical spatial mapping for investigations of cell dynamics atmore » the molecular level.« less

Nanoscale quantification of intracellular element concentration by X-ray fluorescence microscopy combined with X-ray phase contrast nanotomography

NASA Astrophysics Data System (ADS)

Gramaccioni, Chiara; Yang, Yang; Procopio, Alessandra; Pacureanu, Alexandra; Bohic, Sylvain; Malucelli, Emil; Iotti, Stefano; Farruggia, Giovanna; Bukreeva, Inna; Notargiacomo, Andrea; Fratini, Michela; Valenti, Piera; Rosa, Luigi; Berlutti, Francesca; Cloetens, Peter; Lagomarsino, Stefano

2018-01-01

We present here a correlative X-ray microscopy approach for quantitative single cell imaging of molar concentrations. By combining the elemental content provided by X-ray fluorescence microscopy and the morphology information extracted from X-ray phase nanotomography, we determine the intracellular molarity distributions. This correlative method was demonstrated on a freeze-dried human phagocytic cell to obtain the absolute elemental concentration maps of K, P, and Fe. The cell morphology results showed a very good agreement with atomic-force microscopy measurements. This work opens the way for non-destructive single cell chemical analysis down to the sub-cellular level using exclusively synchrotron radiation techniques. It will be of high interest in the case where it is difficult to access the morphology using atomic-force microscopy, for example, on frozen-hydrated cells or tissues.

Transmission electron microscopy, fluorescence microscopy, and confocal raman microscopic analysis of ultrastructural and compositional heterogeneity of Cornus alba L. wood cell wall.

PubMed

Ma, Jianfeng; Ji, Zhe; Zhou, Xia; Zhang, Zhiheng; Xu, Feng

2013-02-01

Transmission electron microscopy (TEM), fluorescence microscopy, and confocal Raman microscopy can be used to characterize ultrastructural and compositional heterogeneity of plant cell walls. In this study, TEM observations revealed the ultrastructural characterization of Cornus alba L. fiber, vessel, axial parenchyma, ray parenchyma, and pit membrane between cells, notably with the ray parenchyma consisting of two well-defined layers. Fluorescence microscopy evidenced that cell corner middle lamella was more lignified than adjacent compound middle lamella and secondary wall with variation in lignification level from cell to cell. In situ Raman images showed that the inhomogeneity in cell wall components (cellulose and lignin) among different cells and within morphologically distinct cell wall layers. As the significant precursors of lignin biosynthesis, the pattern of coniferyl alcohol and aldehyde (joint abbreviation Lignin-CAA for both structures) distribution in fiber cell wall was also identified by Raman images, with higher concentration occurring in the fiber secondary wall where there was the highest cellulose concentration. Moreover, noteworthy was the observation that higher concentration of lignin and very minor amounts of cellulose were visualized in the pit membrane areas. These complementary microanalytical methods provide more accurate and complete information with regard to ultrastructural and compositional characterization of plant cell walls.

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

PubMed

Kwak, In Hae; Son, Minjun; Hagen, Stephen J

2012-05-11

Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly. Copyright © 2012 Elsevier Inc. All rights reserved.

Single cell versus large population analysis: cell variability in elemental intracellular concentration and distribution.

PubMed

Malucelli, Emil; Procopio, Alessandra; Fratini, Michela; Gianoncelli, Alessandra; Notargiacomo, Andrea; Merolle, Lucia; Sargenti, Azzurra; Castiglioni, Sara; Cappadone, Concettina; Farruggia, Giovanna; Lombardo, Marco; Lagomarsino, Stefano; Maier, Jeanette A; Iotti, Stefano

2018-01-01

The quantification of elemental concentration in cells is usually performed by analytical assays on large populations missing peculiar but important rare cells. The present article aims at comparing the elemental quantification in single cells and cell population in three different cell types using a new approach for single cells elemental analysis performed at sub-micrometer scale combining X-ray fluorescence microscopy and atomic force microscopy. The attention is focused on the light element Mg, exploiting the opportunity to compare the single cell quantification to the cell population analysis carried out by a highly Mg-selective fluorescent chemosensor. The results show that the single cell analysis reveals the same Mg differences found in large population of the different cell strains studied. However, in one of the cell strains, single cell analysis reveals two cells with an exceptionally high intracellular Mg content compared with the other cells of the same strain. The single cell analysis allows mapping Mg and other light elements in whole cells at sub-micrometer scale. A detailed intensity correlation analysis on the two cells with the highest Mg content reveals that Mg subcellular localization correlates with oxygen in a different fashion with respect the other sister cells of the same strain. Graphical abstract Single cells or large population analysis this is the question!

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

PubMed

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

2004-07-01

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

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.

TLM-Tracker: software for cell segmentation, tracking and lineage analysis in time-lapse microscopy movies.

PubMed

Klein, Johannes; Leupold, Stefan; Biegler, Ilona; Biedendieck, Rebekka; Münch, Richard; Jahn, Dieter

2012-09-01

Time-lapse imaging in combination with fluorescence microscopy techniques enable the investigation of gene regulatory circuits and uncovered phenomena like culture heterogeneity. In this context, computational image processing for the analysis of single cell behaviour plays an increasing role in systems biology and mathematical modelling approaches. Consequently, we developed a software package with graphical user interface for the analysis of single bacterial cell behaviour. A new software called TLM-Tracker allows for the flexible and user-friendly interpretation for the segmentation, tracking and lineage analysis of microbial cells in time-lapse movies. The software package, including manual, tutorial video and examples, is available as Matlab code or executable binaries at http://www.tlmtracker.tu-bs.de.

Clinical laboratory urine analysis: comparison of the UriSed automated microscopic analyzer and the manual microscopy.

PubMed

Ma, Junlong; Wang, Chengbin; Yue, Jiaxin; Li, Mianyang; Zhang, Hongrui; Ma, Xiaojing; Li, Xincui; Xue, Dandan; Qing, Xiaoyan; Wang, Shengjiang; Xiang, Daijun; Cong, Yulong

2013-01-01

Several automated urine sediment analyzers have been introduced to clinical laboratories. Automated microscopic pattern recognition is a new technique for urine particle analysis. We evaluated the analytical and diagnostic performance of the UriSed automated microscopic analyzer and compared with manual microscopy for urine sediment analysis. Precision, linearity, carry-over, and method comparison were carried out. A total of 600 urine samples sent for urinalysis were assessed using the UriSed automated microscopic analyzer and manual microscopy. Within-run and between-run precision of the UriSed for red blood cells (RBC) and white blood cells (WBC) were acceptable at all levels (CV < 20%). Within-run and between-run imprecision of the UriSed testing for cast, squamous epithelial cells (EPI), and bacteria (BAC) were good at middle level and high level (CV < 20%). The linearity analysis revealed substantial agreement between the measured value and the theoretical value of the UriSed for RBC, WBC, cast, EPI, and BAC (r > 0.95). There was no carry-over. RBC, WBC, and squamous epithelial cells with sensitivities and specificities were more than 80% in this study. There is substantial agreement between the UriSed automated microscopic analyzer and the manual microscopy methods. The UriSed provides for a rapid turnaround time.

Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection.

PubMed

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

2017-10-01

Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacterial colonies in infected host cells (Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy, Ernstsen et al., 2017 [1]). The infected cells were imaged with a 10× objective and number of intracellular bacterial colonies, their size distribution and the number of cell nuclei were automatically quantified using a spot detection-tool. The spot detection-output was exported to Excel, where data analysis was performed. In this article, micrographs and spot detection data are made available to facilitate implementation of the method.

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells.

PubMed

Hofemeier, Arne D; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F W; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

2016-05-26

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO4(3-) symmetric stretch vibrations at 959 cm(-1) assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

NASA Astrophysics Data System (ADS)

Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

2016-05-01

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43- symmetric stretch vibrations at 959 cm-1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

PubMed Central

Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

2016-01-01

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43− symmetric stretch vibrations at 959 cm−1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis. PMID:27225821

Determination of the refractive index of dehydrated cells by means of digital holographic microscopy

NASA Astrophysics Data System (ADS)

Belashov, A. V.; Zhikhoreva, A. A.; Bespalov, V. G.; Vasyutinskii, O. S.; Zhilinskaya, N. T.; Novik, V. I.; Semenova, I. V.

2017-10-01

Spatial distributions of the integral refractive index in dehydrated cells of human oral cavity epithelium are obtained by means of digital holographic microscopy, and mean refractive index of the cells is determined. The statistical analysis of the data obtained is carried out, and absolute errors of the method are estimated for different experimental conditions.

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

PubMed

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

2017-09-01

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

Robust Nucleus/Cell Detection and Segmentation in Digital Pathology and Microscopy Images: A Comprehensive Review.

PubMed

Xing, Fuyong; Yang, Lin

2016-01-01

Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to interobserver variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literature. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast, fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation.

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

PubMed Central

Lerner, Thomas R.; Burden, Jemima J.; Nkwe, David O.; Pelchen-Matthews, Annegret; Domart, Marie-Charlotte; Durgan, Joanne; Weston, Anne; Jones, Martin L.; Peddie, Christopher J.; Carzaniga, Raffaella; Florey, Oliver; Marsh, Mark; Gutierrez, Maximiliano G.

2017-01-01

ABSTRACT The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research. PMID:27445312

Correlative scanning-transmission electron microscopy reveals that a chimeric flavivirus is released as individual particles in secretory vesicles.

PubMed

Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

2014-01-01

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

Correlative Scanning-Transmission Electron Microscopy Reveals that a Chimeric Flavivirus Is Released as Individual Particles in Secretory Vesicles

PubMed Central

Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

2014-01-01

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations. PMID:24681578

Survival and Intra-Nuclear Trafficking of Burkholderia pseudomallei: Strategies of Evasion from Immune Surveillance?

PubMed

Vadivelu, Jamuna; Vellasamy, Kumutha Malar; Thimma, Jaikumar; Mariappan, Vanitha; Kang, Wen-Tyng; Choh, Leang-Chung; Shankar, Esaki M; Wong, Kum Thong

2017-01-01

During infection, successful bacterial clearance is achieved via the host immune system acting in conjunction with appropriate antibiotic therapy. However, it still remains a tip of the iceberg as to where persistent pathogens namely, Burkholderia pseudomallei (B. pseudomallei) reside/hide to escape from host immune sensors and antimicrobial pressure. We used transmission electron microscopy (TEM) to investigate post-mortem tissue sections of patients with clinical melioidosis to identify the localisation of a recently identified gut microbiome, B. pseudomallei within host cells. The intranuclear presence of B. pseudomallei was confirmed using transmission electron microscopy (TEM) of experimentally infected guinea pig spleen tissues and Live Z-stack, and ImageJ analysis of fluorescence microscopy analysis of in vitro infection of A549 human lung epithelial cells. TEM investigations revealed intranuclear localization of B. pseudomallei in cells of infected human lung and guinea pig spleen tissues. We also found that B. pseudomallei induced actin polymerization following infection of A549 human lung epithelial cells. Infected A549 lung epithelial cells using 3D-Laser scanning confocal microscopy (LSCM) and immunofluorescence microscopy confirmed the intranuclear localization of B. pseudomallei. B. pseudomallei was found within the nuclear compartment of host cells. The nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrrent episodes or recrudescence of infection.

Survival and Intra-Nuclear Trafficking of Burkholderia pseudomallei: Strategies of Evasion from Immune Surveillance?

PubMed Central

Vadivelu, Jamuna; Vellasamy, Kumutha Malar; Thimma, Jaikumar; Mariappan, Vanitha; Kang, Wen-Tyng; Choh, Leang-Chung; Wong, Kum Thong

2017-01-01

Background During infection, successful bacterial clearance is achieved via the host immune system acting in conjunction with appropriate antibiotic therapy. However, it still remains a tip of the iceberg as to where persistent pathogens namely, Burkholderia pseudomallei (B. pseudomallei) reside/hide to escape from host immune sensors and antimicrobial pressure. Methods We used transmission electron microscopy (TEM) to investigate post-mortem tissue sections of patients with clinical melioidosis to identify the localisation of a recently identified gut microbiome, B. pseudomallei within host cells. The intranuclear presence of B. pseudomallei was confirmed using transmission electron microscopy (TEM) of experimentally infected guinea pig spleen tissues and Live Z-stack, and ImageJ analysis of fluorescence microscopy analysis of in vitro infection of A549 human lung epithelial cells. Results TEM investigations revealed intranuclear localization of B. pseudomallei in cells of infected human lung and guinea pig spleen tissues. We also found that B. pseudomallei induced actin polymerization following infection of A549 human lung epithelial cells. Infected A549 lung epithelial cells using 3D-Laser scanning confocal microscopy (LSCM) and immunofluorescence microscopy confirmed the intranuclear localization of B. pseudomallei. Conclusion B. pseudomallei was found within the nuclear compartment of host cells. The nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrrent episodes or recrudescence of infection. PMID:28045926

Applications of microscopy in Salmonella research.

PubMed

Malt, Layla M; Perrett, Charlotte A; Humphrey, Suzanne; Jepson, Mark A

2015-01-01

Salmonella enterica is a Gram-negative enteropathogen that can cause localized infections, typically resulting in gastroenteritis, or systemic infection, e.g., typhoid fever, in humans and many other animals. Understanding the mechanisms by which Salmonella induces disease has been the focus of intensive research. This has revealed that Salmonella invasion requires dynamic cross-talk between the microbe and host cells, in which bacterial adherence rapidly leads to a complex sequence of cellular responses initiated by proteins translocated into the host cell by a type 3 secretion system. Once these Salmonella-induced responses have resulted in bacterial invasion, proteins translocated by a second type 3 secretion system initiate further modulation of cellular activities to enable survival and replication of the invading pathogen. Elucidation of the complex and highly dynamic pathogen-host interactions ultimately requires analysis at the level of single cells and single infection events. To achieve this goal, researchers have applied a diverse range of microscopy techniques to analyze Salmonella infection in models ranging from whole animal to isolated cells and simple eukaryotic organisms. For example, electron microscopy and high-resolution light microscopy techniques such as confocal microscopy can reveal the precise location of Salmonella and its relationship to cellular components. Widefield light microscopy is a simpler approach with which to study the interaction of bacteria with host cells and often has advantages for live cell imaging, enabling detailed analysis of the dynamics of infection and cellular responses. Here we review the use of imaging techniques in Salmonella research and compare the capabilities of different classes of microscope to address specific types of research question. We also provide protocols and notes on some microscopy techniques used routinely in our own research.

Evaluation of mobile digital light-emitting diode fluorescence microscopy in Hanoi, Viet Nam.

PubMed

Chaisson, L H; Reber, C; Phan, H; Switz, N; Nilsson, L M; Myers, F; Nhung, N V; Luu, L; Pham, T; Vu, C; Nguyen, H; Nguyen, A; Dinh, T; Nahid, P; Fletcher, D A; Cattamanchi, A

2015-09-01

Hanoi Lung Hospital, Hanoi, Viet Nam. To compare the accuracy of CellScopeTB, a manually operated mobile digital fluorescence microscope, with conventional microscopy techniques. Patients referred for sputum smear microscopy to the Hanoi Lung Hospital from May to September 2013 were included. Ziehl-Neelsen (ZN) smear microscopy, conventional light-emitting diode (LED) fluorescence microscopy (FM), CellScopeTB-based LED FM and Xpert(®) MTB/RIF were performed on sputum samples. The sensitivity and specificity of microscopy techniques were determined in reference to Xpert results, and differences were compared using McNemar's paired test of proportions. Of 326 patients enrolled, 93 (28.5%) were Xpert-positive for TB. The sensitivity of ZN microscopy, conventional LED FM, and CellScopeTB-based LED FM was respectively 37.6% (95%CI 27.8-48.3), 41.9% (95%CI 31.8-52.6), and 35.5% (95%CI 25.8-46.1). The sensitivity of CellScopeTB was similar to that of conventional LED FM (difference -6.5%, 95%CI -18.2 to 5.3, P = 0.33) and ZN microscopy (difference -2.2%, 95%CI -9.2 to 4.9, P = 0.73). The specificity was >99% for all three techniques. CellScopeTB performed similarly to conventional microscopy techniques in the hands of experienced TB microscopists. However, the sensitivity of all sputum microscopy techniques was low. Options enabled by digital microscopy, such as automated imaging with real-time computerized analysis, should be explored to increase sensitivity.

Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

PubMed Central

Young, Jonathan W; Locke, James C W; Altinok, Alphan; Rosenfeld, Nitzan; Bacarian, Tigran; Swain, Peter S; Mjolsness, Eric; Elowitz, Michael B

2014-01-01

Quantitative single-cell time-lapse microscopy is a powerful method for analyzing gene circuit dynamics and heterogeneous cell behavior. We describe the application of this method to imaging bacteria by using an automated microscopy system. This protocol has been used to analyze sporulation and competence differentiation in Bacillus subtilis, and to quantify gene regulation and its fluctuations in individual Escherichia coli cells. The protocol involves seeding and growing bacteria on small agarose pads and imaging the resulting microcolonies. Images are then reviewed and analyzed using our laboratory's custom MATLAB analysis code, which segments and tracks cells in a frame-to-frame method. This process yields quantitative expression data on cell lineages, which can illustrate dynamic expression profiles and facilitate mathematical models of gene circuits. With fast-growing bacteria, such as E. coli or B. subtilis, image acquisition can be completed in 1 d, with an additional 1–2 d for progressing through the analysis procedure. PMID:22179594

Computerized image analysis of cell-cell interactions in human renal tissue by using multi-channel immunoflourescent confocal microscopy

NASA Astrophysics Data System (ADS)

Peng, Yahui; Jiang, Yulei; Liarski, Vladimir M.; Kaverina, Natalya; Clark, Marcus R.; Giger, Maryellen L.

2012-03-01

Analysis of interactions between B and T cells in tubulointerstitial inflammation is important for understanding human lupus nephritis. We developed a computer technique to perform this analysis, and compared it with manual analysis. Multi-channel immunoflourescent-microscopy images were acquired from 207 regions of interest in 40 renal tissue sections of 19 patients diagnosed with lupus nephritis. Fresh-frozen renal tissue sections were stained with combinations of immunoflourescent antibodies to membrane proteins and counter-stained with a cell nuclear marker. Manual delineation of the antibodies was considered as the reference standard. We first segmented cell nuclei and cell membrane markers, and then determined corresponding cell types based on the distances between cell nuclei and specific cell-membrane marker combinations. Subsequently, the distribution of the shortest distance from T cell nuclei to B cell nuclei was obtained and used as a surrogate indicator of cell-cell interactions. The computer and manual analyses results were concordant. The average absolute difference was 1.1+/-1.2% between the computer and manual analysis results in the number of cell-cell distances of 3 μm or less as a percentage of the total number of cell-cell distances. Our computerized analysis of cell-cell distances could be used as a surrogate for quantifying cell-cell interactions as either an automated and quantitative analysis or for independent confirmation of manual analysis.

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.

Analysis and modification of defective surface aggregates on PCDTBT:PCBM solar cell blends using combined Kelvin probe, conductive and bimodal atomic force microscopy

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

Noh, Hanaul; Diaz, Alfredo J.; Solares, Santiago D.

Organic photovoltaic systems comprising donor polymers and acceptor fullerene derivatives are attractive for inexpensive energy harvesting. Extensive research on polymer solar cells has provided insight into the factors governing device-level efficiency and stability. However, the detailed investigation of nanoscale structures is still challenging. Here we demonstrate the analysis and modification of unidentified surface aggregates. The aggregates are characterized electrically by Kelvin probe force microscopy and conductive atomic force microscopy (C-AFM), whereby the correlation between local electrical potential and current confirms a defective charge transport. Bimodal AFM modification confirms that the aggregates exist on top of the solar cell structure, andmore » is used to remove them and to reveal the underlying active layer. The systematic analysis of the surface aggregates suggests that the structure consists of PCBM molecules.« less

Analysis and modification of defective surface aggregates on PCDTBT:PCBM solar cell blends using combined Kelvin probe, conductive and bimodal atomic force microscopy

DOE PAGES

Noh, Hanaul; Diaz, Alfredo J.; Solares, Santiago D.

2017-03-08

Organic photovoltaic systems comprising donor polymers and acceptor fullerene derivatives are attractive for inexpensive energy harvesting. Extensive research on polymer solar cells has provided insight into the factors governing device-level efficiency and stability. However, the detailed investigation of nanoscale structures is still challenging. Here we demonstrate the analysis and modification of unidentified surface aggregates. The aggregates are characterized electrically by Kelvin probe force microscopy and conductive atomic force microscopy (C-AFM), whereby the correlation between local electrical potential and current confirms a defective charge transport. Bimodal AFM modification confirms that the aggregates exist on top of the solar cell structure, andmore » is used to remove them and to reveal the underlying active layer. The systematic analysis of the surface aggregates suggests that the structure consists of PCBM molecules.« less

Analysis and modification of defective surface aggregates on PCDTBT:PCBM solar cell blends using combined Kelvin probe, conductive and bimodal atomic force microscopy

PubMed Central

Noh, Hanaul; Diaz, Alfredo J

2017-01-01

Organic photovoltaic systems comprising donor polymers and acceptor fullerene derivatives are attractive for inexpensive energy harvesting. Extensive research on polymer solar cells has provided insight into the factors governing device-level efficiency and stability. However, the detailed investigation of nanoscale structures is still challenging. Here we demonstrate the analysis and modification of unidentified surface aggregates. The aggregates are characterized electrically by Kelvin probe force microscopy and conductive atomic force microscopy (C-AFM), whereby the correlation between local electrical potential and current confirms a defective charge transport. Bimodal AFM modification confirms that the aggregates exist on top of the solar cell structure, and is used to remove them and to reveal the underlying active layer. The systematic analysis of the surface aggregates suggests that the structure consists of PCBM molecules. PMID:28382247

Automated analysis of high-content microscopy data with deep learning.

PubMed

Kraus, Oren Z; Grys, Ben T; Ba, Jimmy; Chong, Yolanda; Frey, Brendan J; Boone, Charles; Andrews, Brenda J

2017-04-18

Existing computational pipelines for quantitative analysis of high-content microscopy data rely on traditional machine learning approaches that fail to accurately classify more than a single dataset without substantial tuning and training, requiring extensive analysis. Here, we demonstrate that the application of deep learning to biological image data can overcome the pitfalls associated with conventional machine learning classifiers. Using a deep convolutional neural network (DeepLoc) to analyze yeast cell images, we show improved performance over traditional approaches in the automated classification of protein subcellular localization. We also demonstrate the ability of DeepLoc to classify highly divergent image sets, including images of pheromone-arrested cells with abnormal cellular morphology, as well as images generated in different genetic backgrounds and in different laboratories. We offer an open-source implementation that enables updating DeepLoc on new microscopy datasets. This study highlights deep learning as an important tool for the expedited analysis of high-content microscopy data. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Live CLEM imaging to analyze nuclear structures at high resolution.

PubMed

Haraguchi, Tokuko; Osakada, Hiroko; Koujin, Takako

2015-01-01

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

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

PubMed

Johnson, Heath E; Haugh, Jason M

2013-12-02

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

Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood.

PubMed Central

Oosterwijk, J C; Knepflé, C F; Mesker, W E; Vrolijk, H; Sloos, W C; Pattenier, H; Ravkin, I; van Ommen, G J; Kanhai, H H; Tanke, H J

1998-01-01

This article explores the feasibility of the use of automated microscopy and image analysis to detect the presence of rare fetal nucleated red blood cells (NRBCs) circulating in maternal blood. The rationales for enrichment and for automated image analysis for "rare-event" detection are reviewed. We also describe the application of automated image analysis to 42 maternal blood samples, using a protocol consisting of one-step enrichment followed by immunocytochemical staining for fetal hemoglobin (HbF) and FISH for X- and Y-chromosomal sequences. Automated image analysis consisted of multimode microscopy and subsequent visual evaluation of image memories containing the selected objects. The FISH results were compared with the results of conventional karyotyping of the chorionic villi. By use of manual screening, 43% of the slides were found to be positive (>=1 NRBC), with a mean number of 11 NRBCs (range 1-40). By automated microscopy, 52% were positive, with on average 17 NRBCs (range 1-111). There was a good correlation between both manual and automated screening, but the NRBC yield from automated image analysis was found to be superior to that from manual screening (P=.0443), particularly when the NRBC count was >15. Seven (64%) of 11 XY fetuses were correctly diagnosed by FISH analysis of automatically detected cells, and all discrepancies were restricted to the lower cell-count range. We believe that automated microscopy and image analysis reduce the screening workload, are more sensitive than manual evaluation, and can be used to detect rare HbF-containing NRBCs in maternal blood. PMID:9837832

Microscopy and microanalysis 1996

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

Bailey, G.W.; Corbett, J.M.; Dimlich, R.V.W.

1996-12-31

The Proceedings of this Annual Meeting contain paper of members from the three societies. These proceedings emphasizes the common research interests and attempts to eliminate some unwanted overlap. Topics covered are: microscopic analysis of animals with altered gene expression and in-situ gene and antibody localizations, high-resolution elemental mapping of nucleoprofein interactions, plant biology and pathology, quantitative HREM analysis of perfect and defected materials, computational methods for TEM image analysis, high-resolution FESM in materials research, frontiers in polymer microscopy and microanalysis, oxidation and corrosion, micro XRD and XRF, molecular microspectroscopy and spectral imaging, advances in confocal and multidimensional light microscopy, analyticalmore » electron microscopy in biology, correlative microscopy in biological sciences, grain-boundary microengineering, surfaces and interfaces, telepresence microscopy in education and research, MSA educational outreach, quantitative electron probe microanalysis, frontiers of analytical electron microscopy, critical issues in ceramic microstructures, dynamic organization of the cell, pathology, microbiology, high-resolution biological and cryo SEM, and scanning-probe microscopy.« less

The Role of IQGAP1 in Breast Carcinoma

DTIC Science & Technology

2011-10-01

study! of! the! pathogenesis! of! breast! cancer.! These! include! analysis ! of! intracellular! signaling!by!Western!blotting,! determination!of! cell...proliferation!by! sulforhodamine!B! staining,! fluorescence: activated!cell!sorting!(FACS)! analysis ,!stable!cell!line!generation,!production!of!and...transduction!using!retroviral! and!lentiviral!supernatants,! immunocytochemistry!and!confocal! laser!microscopy,! immunohistochemistry,!and! analysis

Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane

PubMed Central

Paparelli, Laura; Corthout, Nikky; Wakefield, Devin L.; Sannerud, Ragna; Jovanovic-Talisman, Tijana; Annaert, Wim; Munck, Sebastian

2016-01-01

Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH). We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided. PMID:27603951

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.

The three dimensionality of cell membranes: lamellar to cubic membrane transition as investigated by electron microscopy.

PubMed

Chong, Ketpin; Deng, Yuru

2012-01-01

Biological membranes are generally perceived as phospholipid bilayer structures that delineate in a lamellar form the cell surface and intracellular organelles. However, much more complex and highly convoluted membrane organizations are ubiquitously present in many cell types under certain types of stress, states of disease, or in the course of viral infections. Their occurrence under pathological conditions make such three-dimensionally (3D) folded and highly ordered membranes attractive biomarkers. They have also stimulated great biomedical interest in understanding the molecular basis of their formation. Currently, the analysis of such membrane arrangements, which include tubulo-reticular structures (TRS) or cubic membranes of various subtypes, is restricted to electron microscopic methods, including tomography. Preservation of membrane structures during sample preparation is the key to understand their true 3D nature. This chapter discusses methods for appropriate sample preparations to successfully examine and analyze well-preserved highly ordered membranes by electron microscopy. Processing methods and analysis conditions for green algae (Zygnema sp.) and amoeba (Chaos carolinense), mammalian cells in culture and primary tissue cells are described. We also discuss methods to identify cubic membranes by transmission electron microscopy (TEM) with the aid of a direct template matching method and by computer simulation. A 3D analysis of cubic cell membrane topology by electron tomography is described as well as scanning electron microscopy (SEM) to investigate surface contours of isolated mitochondria with cubic membrane arrangement. Copyright © 2012 Elsevier Inc. All rights reserved.

A software solution for recording circadian oscillator features in time-lapse live cell microscopy.

PubMed

Sage, Daniel; Unser, Michael; Salmon, Patrick; Dibner, Charna

2010-07-06

Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/dedicated tracking software. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts. In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets. Our software solution, Circadian Gene Express (CGE), is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and efficient recording of large number of cell parameters, including level of reporter protein expression, velocity, direction of movement, and others. CGE proves to be useful for the analysis of widefield fluorescent microscopy datasets, as well as for bioluminescence imaging. Moreover, it might be easily adaptable for confocal image analysis by manually choosing one of the focal planes of each z-stack of the various time points of a time series. CGE is a Java plugin for ImageJ; it is freely available at: http://bigwww.epfl.ch/sage/soft/circadian/.

Time lapse microscopy observation of cellular structural changes and image analysis of drug treated cancer cells to characterize the cellular heterogeneity.

PubMed

Vaiyapuri, Periasamy S; Ali, Alshatwi A; Mohammad, Akbarsha A; Kandhavelu, Jeyalakshmi; Kandhavelu, Meenakshisundaram

2015-01-01

The effect of Calotropis gigantea latex (CGLX) on human mammary carcinoma cells is not well established. We present the results of this drug activity at total population and single cell level. CGLX inhibited the growth of MCF7 cancer cells at lower IC50 concentration (17 µL/mL). Microscopy of IC50 drug treated cells at 24 hr confirming the appearance of morphological characteristics of apoptotic and necrotic cells, associated with 70% of DNA damage. FACS analysis confirmed that, 10 and 20% of the disruption of cellular mitochondrial nature by at 24 and 48 h, respectively. Microscopic image analysis of total population level proved that MMP changes were statistically significant with P values. The cell to cell variation was confirmed by functional heterogeneity analysis which proves that CGLX was able to induce the apoptosis without the contribution of mitochondria. We conclude that CGLX inhibits cell proliferation, survival, and heterogeneity of pathways in human mammary carcinoma cells. © 2014 Wiley Periodicals, Inc.

Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

2014-09-01

We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

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

Lin, Jian; Zheng, Wei; Wang, Zi

2014-09-08

We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

Robust Nucleus/Cell Detection and Segmentation in Digital Pathology and Microscopy Images: A Comprehensive Review

PubMed Central

Xing, Fuyong; Yang, Lin

2016-01-01

Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to inter-observer variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literatures. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast (DIC), fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation. PMID:26742143

Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria.

PubMed

Wheeler, Richard; Mesnage, Stéphane; Boneca, Ivo G; Hobbs, Jamie K; Foster, Simon J

2011-12-01

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms. © 2011 Blackwell Publishing Ltd.

Revealing 3D Ultrastructure and Morphology of Stem Cell Spheroids by Electron Microscopy.

PubMed

Jaros, Josef; Petrov, Michal; Tesarova, Marketa; Hampl, Ales

2017-01-01

Cell culture methods have been developed in efforts to produce biologically relevant systems for developmental and disease modeling, and appropriate analytical tools are essential. Knowledge of ultrastructural characteristics represents the basis to reveal in situ the cellular morphology, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. The traditional method for 3D visualization of ultrastructural components, serial sectioning using transmission electron microscopy (TEM), is very labor-intensive due to contentious TEM slice preparation and subsequent image processing of the whole collection. In this chapter, we present serial block-face scanning electron microscopy, together with complex methodology for spheroid formation, contrasting of cellular compartments, image processing, and 3D visualization. The described technique is effective for detailed morphological analysis of stem cell spheroids, organoids, as well as organotypic cell cultures.

Unraveling Cell Processes: Interference Imaging Interwoven with Data Analysis

PubMed Central

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

2006-01-01

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

[Watching dance of the molecules - CARS microscopy].

PubMed

Korczyński, Jaroslaw; Kubiak, Katarzyna; Węgłowska, Edyta

2017-01-01

CARS (Coherent Anti-Stokes Raman Scattering) microscopy is an imaging method for living cells visualization as well as for food or cosmetics material analysis without the need for staining. The near infrared laser source generates the CARS signal - the characteristic intrinsic vibrational contrast of the molecules in a sample which is no longer caused by staining, but by the molecules themselves. It provides the benefit of a non-toxic, non-destructive and almost noninvasive method for sample imaging. CARS can easily be combined with fluorescence confocal microscopy so it is an excellent complementary imaging method. In this article we showed some of the applications for this technology: imaging of lipid droplets inside human HaCaT cells and analysis of the composition of cosmetic products. Moreover we believe, that soon new fields of application become accessible for this rapidly developing branch of microscopy.

[application of the analytical transmission electron microscopy techniques for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in mammalian cells].

PubMed

Shebanova, A S; Bogdanov, A G; Ismagulova, T T; Feofanov, A V; Semenyuk, P I; Muronets, V I; Erokhina, M V; Onishchenko, G E; Kirpichnikov, M P; Shaitan, K V

2014-01-01

This work represents the results of the study on applicability of the modern methods of analytical transmission electron microscopy for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in A549 cell, human lung adenocarcinoma cell line. A comparative analysis of images of the nanoparticles in the cells obtained in the bright field mode of transmission electron microscopy, under dark-field scanning transmission electron microscopy and high-angle annular dark field scanning transmission electron was performed. For identification of nanoparticles in the cells the analytical techniques, energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy, were compared when used in the mode of obtaining energy spectrum from different particles and element mapping. It was shown that the method for electron tomography is applicable to confirm that nanoparticles are localized in the sample but not coated by contamination. The possibilities and fields of utilizing different techniques for analytical transmission electron microscopy for detection, visualization and identification of nanoparticles in the biological samples are discussed.

Nuclear microscopy in biomedical analysis with special emphasis on clinical metal biology

NASA Astrophysics Data System (ADS)

Lindh, Ulf; Frisk, Peter; Nyström, Joakim; Danersund, Antero; Hudecek, Romuald; Lindvall, Anders; Thunell, Stig

1997-07-01

Nuclear microscopy based upon developments in high energy ion beam techniques is by now an accepted technique in many fields of research. The advancements into the biomedical field have, however, been slower than expected. A major factor explaining this tendency is the availability of nuclear microscopy. This paper reviews briefly the biomedical work using nuclear microscopy that has been carried out since the 4 th International Conference on Nuclear Microprobe Technology and Applications held in Shanghai. Nuclear microscopy of isolated individual blood cells from patients adversely affected by metal exposure from dental amalgam has been performed both before and after removal of the metallic fillings. The elemental profile of blood cells was more or less normalised after treatment. Some of these results will be presented to illustrate a medical application. Results from bulk analysis by ICP-MS of erythrocytes and plasma before and after treatment will also be presented to illustrate the difference in information content between these two approaches as well as the need for complementary information in solving biomedical problems. As part of a larger study of acute porphyria, nuclear microscopy of blood cells was included among the 78 laboratory tests. The approach in this study was unbiased in the sense that no hypothesis was formulated as to which laboratory parameters would be the most explanatory for health or disease. Multivariate discriminant analysis was applied to the large amounts of data acquired. This approach led to the hypothesis that oxidative stress increased the synthesis of manganese-dependent superoxide dismutase in the mitochondria of polymorphonuclear leukocytes, explaining the increase of manganese in these cells. Antioxidant therapy was therefore applied to a couple of patients with porphyria, however, without clinical success.

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

PubMed

Zhu, Hongying; Ozcan, Aydogan

2015-01-01

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

Learning a cost function for microscope image segmentation.

PubMed

Nilufar, Sharmin; Perkins, Theodore J

2014-01-01

Quantitative analysis of microscopy images is increasingly important in clinical researchers' efforts to unravel the cellular and molecular determinants of disease, and for pathological analysis of tissue samples. Yet, manual segmentation and measurement of cells or other features in images remains the norm in many fields. We report on a new system that aims for robust and accurate semi-automated analysis of microscope images. A user interactively outlines one or more examples of a target object in a training image. We then learn a cost function for detecting more objects of the same type, either in the same or different images. The cost function is incorporated into an active contour model, which can efficiently determine optimal boundaries by dynamic programming. We validate our approach and compare it to some standard alternatives on three different types of microscopic images: light microscopy of blood cells, light microscopy of muscle tissue sections, and electron microscopy cross-sections of axons and their myelin sheaths.

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.

The quest for four-dimensional imaging in plant cell biology: it's just a matter of time

PubMed Central

Domozych, David S.

2012-01-01

Background Analysis of plant cell dynamics over time, or four-dimensional imaging (4-DI), represents a major goal of plant science. The ability to resolve structures in the third dimension within the cell or tissue during developmental events or in response to environmental or experimental stresses (i.e. 4-DI) is critical to our understanding of gene expression, post-expression modulations of macromolecules and sub-cellular system interactions. Scope Microscopy-based technologies have been profoundly integral to this type of investigation, and new and refined microscopy technologies now allow for the visualization of cell dynamics with unprecedented resolution, contrast and experimental versatility. However, certain realities of light and electron microscopy, choice of specimen and specimen preparation techniques limit the scope of readily attaining 4-DI. Today, the plant microscopist must use a combinatorial strategy whereby multiple microscopy-based investigations are used. Modern fluorescence, confocal laser scanning, transmission electron and scanning electron microscopy provide effective conduits for synthesizing data detailing live cell dynamics and highly resolved snapshots of specific cell structures that will ultimately lead to 4-DI. This review provides a synopsis of such technologies available. PMID:22628381

Atomic force microscopy as a tool for the investigation of living cells.

PubMed

Morkvėnaitė-Vilkončienė, Inga; Ramanavičienė, Almira; Ramanavičius, Arūnas

2013-01-01

Atomic force microscopy is a valuable and useful tool for the imaging and investigation of living cells in their natural environment at high resolution. Procedures applied to living cell preparation before measurements should be adapted individually for different kinds of cells and for the desired measurement technique. Different ways of cell immobilization, such as chemical fixation on the surface, entrapment in the pores of a membrane, or growing them directly on glass cover slips or on plastic substrates, result in the distortion or appearance of artifacts in atomic force microscopy images. Cell fixation allows the multiple use of samples and storage for a prolonged period; it also increases the resolution of imaging. Different atomic force microscopy modes are used for the imaging and analysis of living cells. The contact mode is the best for cell imaging because of high resolution, but it is usually based on the following: (i) image formation at low interaction force, (ii) low scanning speed, and (iii) usage of "soft," low resolution cantilevers. The tapping mode allows a cell to behave like a very solid material, and destructive shear forces are minimized, but imaging in liquid is difficult. The force spectroscopy mode is used for measuring the mechanical properties of cells; however, obtained results strongly depend on the cell fixation method. In this paper, the application of 3 atomic force microscopy modes including (i) contact, (ii) tapping, and (iii) force spectroscopy for the investigation of cells is described. The possibilities of cell preparation for the measurements, imaging, and determination of mechanical properties of cells are provided. The applicability of atomic force microscopy to diagnostics and other biomedical purposes is discussed.

Super-resolution binding activated localization microscopy through reversible change of DNA conformation.

PubMed

Szczurek, Aleksander; Birk, Udo; Knecht, Hans; Dobrucki, Jurek; Mai, Sabine; Cremer, Christoph

2018-01-01

Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine.

Super-resolution binding activated localization microscopy through reversible change of DNA conformation

PubMed Central

Knecht, Hans; Dobrucki, Jurek; Mai, Sabine

2018-01-01

ABSTRACT Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine. PMID:29297245

Quantitative phase-digital holographic microscopy: a new imaging modality to identify original cellular biomarkers of diseases

NASA Astrophysics Data System (ADS)

Marquet, P.; Rothenfusser, K.; Rappaz, B.; Depeursinge, C.; Jourdain, P.; Magistretti, P. J.

2016-03-01

Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.

Insights into the prominent effect of mahanimbine on Acanthamoeba castellanii: Cell profiling analysis based on microscopy techniques

NASA Astrophysics Data System (ADS)

Hashim, Fatimah; Amin, Nakisah Mat

2017-02-01

Mahanimbine (MH), has been shown to have antiamoeba properties. Therefore, the aim of this study was to assess the growth inhibitory mechanisms of MH on Acanthamoeba castellanii, a causative agents for Acanthamoeba keratitis. The IC50 value obtained for MH against A. castellanii was 1.18 µg/ml. Light and scanning electron microscopy observation showed that most cells were in cystic appearance. While transmission electron microscopy observation revealed changes at the ultrastructural level and fluorescence microscopy observation indicated the induction of apoptosis and autophagic activity in the amoeba cytoplasms. In conclusion, MH has very potent anti-amoebic properties on A. castellanii as is shown by cytotoxicity analyses based on microscopy techniques.

Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy

NASA Astrophysics Data System (ADS)

Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

2015-03-01

Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy.

PubMed

Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

2015-03-09

Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

Automated analysis of time-lapse fluorescence microscopy images: from live cell images to intracellular foci.

PubMed

Dzyubachyk, Oleh; Essers, Jeroen; van Cappellen, Wiggert A; Baldeyron, Céline; Inagaki, Akiko; Niessen, Wiro J; Meijering, Erik

2010-10-01

Complete, accurate and reproducible analysis of intracellular foci from fluorescence microscopy image sequences of live cells requires full automation of all processing steps involved: cell segmentation and tracking followed by foci segmentation and pattern analysis. Integrated systems for this purpose are lacking. Extending our previous work in cell segmentation and tracking, we developed a new system for performing fully automated analysis of fluorescent foci in single cells. The system was validated by applying it to two common tasks: intracellular foci counting (in DNA damage repair experiments) and cell-phase identification based on foci pattern analysis (in DNA replication experiments). Experimental results show that the system performs comparably to expert human observers. Thus, it may replace tedious manual analyses for the considered tasks, and enables high-content screening. The described system was implemented in MATLAB (The MathWorks, Inc., USA) and compiled to run within the MATLAB environment. The routines together with four sample datasets are available at http://celmia.bigr.nl/. The software is planned for public release, free of charge for non-commercial use, after publication of this article.

Method for semi-automated microscopy of filtration-enriched circulating tumor cells.

PubMed

Pailler, Emma; Oulhen, Marianne; Billiot, Fanny; Galland, Alexandre; Auger, Nathalie; Faugeroux, Vincent; Laplace-Builhé, Corinne; Besse, Benjamin; Loriot, Yohann; Ngo-Camus, Maud; Hemanda, Merouan; Lindsay, Colin R; Soria, Jean-Charles; Vielh, Philippe; Farace, Françoise

2016-07-14

Circulating tumor cell (CTC)-filtration methods capture high numbers of CTCs in non-small-cell lung cancer (NSCLC) and metastatic prostate cancer (mPCa) patients, and hold promise as a non-invasive technique for treatment selection and disease monitoring. However filters have drawbacks that make the automation of microscopy challenging. We report the semi-automated microscopy method we developed to analyze filtration-enriched CTCs from NSCLC and mPCa patients. Spiked cell lines in normal blood and CTCs were enriched by ISET (isolation by size of epithelial tumor cells). Fluorescent staining was carried out using epithelial (pan-cytokeratins, EpCAM), mesenchymal (vimentin, N-cadherin), leukocyte (CD45) markers and DAPI. Cytomorphological staining was carried out with Mayer-Hemalun or Diff-Quik. ALK-, ROS1-, ERG-rearrangement were detected by filter-adapted-FISH (FA-FISH). Microscopy was carried out using an Ariol scanner. Two combined assays were developed. The first assay sequentially combined four-color fluorescent staining, scanning, automated selection of CD45(-) cells, cytomorphological staining, then scanning and analysis of CD45(-) cell phenotypical and cytomorphological characteristics. CD45(-) cell selection was based on DAPI and CD45 intensity, and a nuclear area >55 μm(2). The second assay sequentially combined fluorescent staining, automated selection of CD45(-) cells, FISH scanning on CD45(-) cells, then analysis of CD45(-) cell FISH signals. Specific scanning parameters were developed to deal with the uneven surface of filters and CTC characteristics. Thirty z-stacks spaced 0.6 μm apart were defined as the optimal setting, scanning 82 %, 91 %, and 95 % of CTCs in ALK-, ROS1-, and ERG-rearranged patients respectively. A multi-exposure protocol consisting of three separate exposure times for green and red fluorochromes was optimized to analyze the intensity, size and thickness of FISH signals. The semi-automated microscopy method reported here increases the feasibility and reliability of filtration-enriched CTC assays and can help progress towards their validation and translation to the clinic.

Novel microscopy-based screening method reveals regulators of contact-dependent intercellular transfer

PubMed Central

Michael Frei, Dominik; Hodneland, Erlend; Rios-Mondragon, Ivan; Burtey, Anne; Neumann, Beate; Bulkescher, Jutta; Schölermann, Julia; Pepperkok, Rainer; Gerdes, Hans-Hermann; Kögel, Tanja

2015-01-01

Contact-dependent intercellular transfer (codeIT) of cellular constituents can have functional consequences for recipient cells, such as enhanced survival and drug resistance. Pathogenic viruses, prions and bacteria can also utilize this mechanism to spread to adjacent cells and potentially evade immune detection. However, little is known about the molecular mechanism underlying this intercellular transfer process. Here, we present a novel microscopy-based screening method to identify regulators and cargo of codeIT. Single donor cells, carrying fluorescently labelled endocytic organelles or proteins, are co-cultured with excess acceptor cells. CodeIT is quantified by confocal microscopy and image analysis in 3D, preserving spatial information. An siRNA-based screening using this method revealed the involvement of several myosins and small GTPases as codeIT regulators. Our data indicates that cellular protrusions and tubular recycling endosomes are important for codeIT. We automated image acquisition and analysis to facilitate large-scale chemical and genetic screening efforts to identify key regulators of codeIT. PMID:26271723

Multimodal biophotonic workstation for live cell analysis.

PubMed

Esseling, Michael; Kemper, Björn; Antkowiak, Maciej; Stevenson, David J; Chaudet, Lionel; Neil, Mark A A; French, Paul W; von Bally, Gert; Dholakia, Kishan; Denz, Cornelia

2012-01-01

A reliable description and quantification of the complex physiology and reactions of living cells requires a multimodal analysis with various measurement techniques. We have investigated the integration of different techniques into a biophotonic workstation that can provide biological researchers with these capabilities. The combination of a micromanipulation tool with three different imaging principles is accomplished in a single inverted microscope which makes the results from all the techniques directly comparable. Chinese Hamster Ovary (CHO) cells were manipulated by optical tweezers while the feedback was directly analyzed by fluorescence lifetime imaging, digital holographic microscopy and dynamic phase-contrast microscopy. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The effects of viscoelastic polymer substrates on adult stem cell differentiation

NASA Astrophysics Data System (ADS)

Chang, Chungchueh; Fields, Adam; Ramek, Alex; Jurukovski, Vladimir; Simon, Marcia; Rafailovich, Miriam

2009-03-01

Dental Pulp Stem Cells (DPSCs) are known to differentiate in either bone, dentine, or nerve tissue by different environment signals. In this study, we have determined whether differentiation could only through modification of the substrate mechanics. Atomic Force Microscopy (AFM) on Shear Modulation Force Microscopy (SMFM) mode indicated that the spun-cast polybutadiene (PB) thin films could be used to provide different stiffness substrates by changing the thicknesses of thin films. DPSCs were then plated on these substrates and cultured in standard media. After 28 days incubation, Lasar Scanning Confocal Microscopy (LSCM) with mercury lamp indicated that the crystals were observed only on hard surfaces. The Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray analysis (EDX analysis) indicated that the crystals are calcium phosphates. The Glancing Incidence Diffraction (GID) was also used to determine the structure of crystals. These results indicate that DPSCs could be differentiated into osteoblasts by mechanical stimuli from substrate mechanics.

Gold nanoparticle uptake in whole cells in liquid examined by environmental scanning electron microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2014-02-01

The size of gold nanoparticles (AuNPs) can influence various aspects of their cellular uptake. Light microscopy is not capable of resolving most AuNPs, while electron microscopy (EM) is not practically capable of acquiring the necessary statistical data from many cells and the results may suffer from various artifacts. Here, we demonstrate the use of a fast EM method for obtaining high-resolution data from a much larger population of cells than is usually feasible with conventional EM. A549 (human lung carcinoma) cells were subjected to uptake protocols with 10, 15, or 30 nm diameter AuNPs with adsorbed serum proteins. After 20 min, 24 h, or 45 h, the cells were fixed and imaged in whole in a thin layer of liquid water with environmental scanning electron microscopy equipped with a scanning transmission electron microscopy detector. The fast preparation and imaging of 145 whole cells in liquid allowed collection of nanoscale data within an exceptionally small amount of time of ~80 h. Analysis of 1,041 AuNP-filled vesicles showed that the long-term AuNP storing lysosomes increased their average size by 80 nm when AuNPs with 30 nm diameter were uptaken, compared to lysosomes of cells incubated with AuNPs of 10 and 15 nm diameter.

Technician Consistency in Specular Microscopy Measurements: A "Real-World" Retrospective Analysis of a United States Eye Bank.

PubMed

Rand, Gabriel M; Kwon, Ji Won; Gore, Patrick K; McCartney, Mitchell D; Chuck, Roy S

2017-10-01

To quantify consistency of endothelial cell density (ECD) measurements among technicians in a single US eye bank operating under typical operating conditions. In this retrospective analysis of 51 microscopy technicians using a semiautomated counting method on 35,067 eyes from July 2007 to May 2015, technician- and date-related marginal ECD effects were calculated using linear regression models. ECD variance was correlated with the number of specular microscopy technicians. Technician mean ECDs ranged from 2386 ± 431 to 3005 ± 560 cells/mm. Nine technicians had statistically and clinically significant marginal effects. Annual mean ECDs adjusted for changes in technicians ranged from 2422 ± 433 to 2644 ± 430 cells/mm. The period of 2007 to 2009 had statistically and clinically significant marginal effects. There was a nonstatistically significant association between the number of technicians and ECD standard deviation. There was significant ECD variability associated with specular microscopy technicians and with the date of measurement. We recommend that eye banks collect data related to laboratory factors that have been shown to influence ECD variability.

Biomimetic Coating on Porous Alumina for Tissue Engineering: Characterisation by Cell Culture and Confocal Microscopy

PubMed Central

Kolos, Elizabeth; Ruys, Andrew J

2015-01-01

In this study porous alumina samples were prepared and then coated using the biomimetic coating technique using a five times Simulated Body Fluid (5.0SBF) as the growth solution. A coating was achieved after pre-treatment with concentrated acid. From elemental analysis, the coating contained calcium and phosphorous, but also sodium and chlorine. Halite was identified by XRD, a sodium chloride phase. Sintering was done to remove the halite phase. Once halite was burnt off, the calcium phosphate crystals were not covered with halite and, therefore, the apatite phases can be clearly observed. Cell culturing showed sufficient cell attachment to the less porous alumina, Sample B, that has more calcium phosphate growth, while the porous alumina, Sample A, with minimal calcium phosphate growth attained very little cell attachment. This is likely due to the contribution that calcium phosphate plays in the attachment of bone-like cells to a bioinert ceramic such as alumina. These results were repeated on both SEM and confocal microscopy analysis. Confocal microscopy was a novel characterisation approach which gave useful information and was a visual aid.

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection.

PubMed

Pucci, Angela; Mattioli, Claudia; Matteucci, Marco; Lorenzini, Daniele; Panvini, Francesca; Pacini, Simone; Ippolito, Chiara; Celiento, Michele; De Martino, Andrea; Dolfi, Amelio; Belgio, Beatrice; Bortolotti, Uberto; Basolo, Fulvio; Bartoloni, Giovanni

2018-05-22

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.

HeLa cells response to photodynamic treatment with Radachlorin at various irradiation parameters

NASA Astrophysics Data System (ADS)

Belashov, A. V.; Zhikhoreva, A. A.; Belyaeva, T. N.; Kornilova, E. S.; Petrov, N. V.; Salova, A. V.; Semenova, I. V.; Vasyutinskii, O. S.

2017-07-01

Measurements of average phase shifts introduced by living HeLa cells to probe wave front were carried out. Variations of this value were monitored in the course of morphological changes caused by photodynamic treatment at various irradiation doses. Observations of changes in living cells were also performed by means of far field optical microscopy and confocal fluorescent microscopy. Quantitative analysis of the data obtained shows that average phase shift introduced by the cells may either increase or decrease depending upon major parameters of the treatment.

Quantitative study of mammalian cells by scanning transmission soft X-ray microscopy

NASA Astrophysics Data System (ADS)

Shinohara, K.; Ohigashi, T.; Toné, S.; Kado, M.; Ito, A.

2017-06-01

Molecular distribution in mammalian cells was studied by soft X-ray scanning transmission microscopy with respect to the quantitative aspect of analysis. NEXAFS profiles at the C, N and O K-absorption edges were combined and used for the analysis. For the estimation of quantity for nucleic acids and proteins, NEXAFS profiles of DNA and bovine serum albumin (BSA) at the N K-absorption edge were applied assuming that those were their representatives. The method has a potential to explore the other molecular components than nucleic acids and proteins.

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

PubMed Central

Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony M.

2014-01-01

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds. PMID:25106872

Nuclear microscopy in trace-element biology — from cellular studies to the clinic

NASA Astrophysics Data System (ADS)

Lindh, Ulf

1993-05-01

The concentration and distribution of trace and major elements in cells are of great interest in cell biology. PIXE can provide elemental concentrations in the bulk of cells or organelles as other bulk techniques such as atomic absorption spectrophotometry and nuclear activation analysis. Supplementary information, perhaps more exciting, on the intracellular distributions of trace elements can be provided using nuclear microscopy. Intracellular distributions of trace elements in normal and malignant cells are presented. The toxicity of mercury and cadmium can be prevented by supplementation of the essential trace element selenium. Some results from an experimental animal model are discussed. The intercellular distribution of major and trace elements in isolated blood cells, as revealed by nuclear microscopy, provides useful clinical information. Examples are given concerning inflammatory connective-tissue diseases and the chronic fatigue syndrome.

Analyzing the dynamics of cell cycle processes from fixed samples through ergodic principles

PubMed Central

Wheeler, Richard John

2015-01-01

Tools to analyze cyclical cellular processes, particularly the cell cycle, are of broad value for cell biology. Cell cycle synchronization and live-cell time-lapse observation are widely used to analyze these processes but are not available for many systems. Simple mathematical methods built on the ergodic principle are a well-established, widely applicable, and powerful alternative analysis approach, although they are less widely used. These methods extract data about the dynamics of a cyclical process from a single time-point “snapshot” of a population of cells progressing through the cycle asynchronously. Here, I demonstrate application of these simple mathematical methods to analysis of basic cyclical processes—cycles including a division event, cell populations undergoing unicellular aging, and cell cycles with multiple fission (schizogony)—as well as recent advances that allow detailed mapping of the cell cycle from continuously changing properties of the cell such as size and DNA content. This includes examples using existing data from mammalian, yeast, and unicellular eukaryotic parasite cell biology. Through the ongoing advances in high-throughput cell analysis by light microscopy, electron microscopy, and flow cytometry, these mathematical methods are becoming ever more important and are a powerful complementary method to traditional synchronization and time-lapse cell cycle analysis methods. PMID:26543196

Scanning transmission ion microscopy mass measurements for quantitative trace element analysis within biological samples and validation using atomic force microscopy thickness measurements

NASA Astrophysics Data System (ADS)

Devès, Guillaume; Cohen-Bouhacina, Touria; Ortega, Richard

2004-10-01

We used the nuclear microprobe techniques, micro-PIXE (particle-induced X-ray emission), micro-RBS (Rutherford backscattering spectrometry) and scanning transmission ion microscopy (STIM) in order to perform the characterization of trace element content and spatial distribution within biological samples (dehydrated cultured cells, tissues). The normalization of PIXE results was usually expressed in terms of sample dry mass as determined by micro-RBS recorded simultaneously to micro-PIXE. However, the main limit of RBS mass measurement is the sample mass loss occurring during irradiation and which could be up to 30% of the initial sample mass. We present here a new methodology for PIXE normalization and quantitative analysis of trace element within biological samples based on dry mass measurement performed by mean of STIM. The validation of STIM cell mass measurements was obtained in comparison with AFM sample thickness measurements. Results indicated the reliability of STIM mass measurement performed on biological samples and suggested that STIM should be performed for PIXE normalization. Further information deriving from direct confrontation of AFM and STIM analysis could as well be obtained, like in situ measurements of cell specific gravity within cells compartment (nucleolus and cytoplasm).

Automated segmentation and tracking of non-rigid objects in time-lapse microscopy videos of polymorphonuclear neutrophils.

PubMed

Brandes, Susanne; Mokhtari, Zeinab; Essig, Fabian; Hünniger, Kerstin; Kurzai, Oliver; Figge, Marc Thilo

2015-02-01

Time-lapse microscopy is an important technique to study the dynamics of various biological processes. The labor-intensive manual analysis of microscopy videos is increasingly replaced by automated segmentation and tracking methods. These methods are often limited to certain cell morphologies and/or cell stainings. In this paper, we present an automated segmentation and tracking framework that does not have these restrictions. In particular, our framework handles highly variable cell shapes and does not rely on any cell stainings. Our segmentation approach is based on a combination of spatial and temporal image variations to detect moving cells in microscopy videos. This method yields a sensitivity of 99% and a precision of 95% in object detection. The tracking of cells consists of different steps, starting from single-cell tracking based on a nearest-neighbor-approach, detection of cell-cell interactions and splitting of cell clusters, and finally combining tracklets using methods from graph theory. The segmentation and tracking framework was applied to synthetic as well as experimental datasets with varying cell densities implying different numbers of cell-cell interactions. We established a validation framework to measure the performance of our tracking technique. The cell tracking accuracy was found to be >99% for all datasets indicating a high accuracy for connecting the detected cells between different time points. Copyright © 2014 Elsevier B.V. All rights reserved.

Architecture of dermatophyte cell Walls: Electron microscopic and biochemical analysis

NASA Technical Reports Server (NTRS)

Nozawa, Y.; Kitajima, Y.

1984-01-01

A review with 83 references on the cell wall structure of dermatophytes is presented. Topics discussed include separation and preparation of cell walls; microstructure of cell walls by electron microscopy; chemical composition of cell walls; structural model of cell walls; and morphological structure of cell walls.

Microgravity effects during fertilization, cell division, development, and calcium metabolism in sea urchins

NASA Technical Reports Server (NTRS)

Schatten, Heide

1996-01-01

The overall objectives of this project are to explore the role of microgravity during fertilization, early development, cytoskeletal organization, and skeletal calcium deposition in a model development system: the sea urchin eggs and embryos. While pursuing these objectives, we have also helped to develop, test, and fly the Aquatic Research Facility (ARF) system. Cells were fixed at preselected time points to preserve the structures and organelles of interest with regards to cell biology events during development. The protocols used for the analysis of the results had been developed during the earlier part of this research and were applied for post-flight analysis using light and (immuno)fluorescence microscopy, scanning electron microscopy, and transmission electron microscopy. The structures of interest are: microtubules during fertilization, cell division, and cilia movement; microfilaments during cell surface restructuring and cell division; centrosomes and centrioles during cell division, cell differentiation, and cilia formation and movement; membranes, Golgi, endoplasmic reticulum, mitochondria, and chromosomes at all stages of development; and calcium deposits during spicule formation in late-stage embryos. In addition to further explore aspects important or living in space, several aspects of this research are also aimed at understanding diseases that affect humans on Earth which may be accelerated in space.

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

PubMed Central

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

2013-01-01

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

Polychromatic flow cytometry is more sensitive than microscopy in detecting small monoclonal plasma cell populations.

PubMed

Tran, Daniel N; Smith, Sandy A B C; Brown, David A; Parker, Andrew J C; Joseph, Joanne E; Armstrong, Nicola; Sewell, William A

2017-03-01

There is an emerging role for flow cytometry (FC) in the assessment of small populations of plasma cells (PC). However, FC's utility has been questioned due to consistent underestimation of the percentage of PC compared to microscopy. A retrospective study was performed on bone marrow samples analysed by 8-colour FC. Plasma cell populations were classified as polyclonal or monoclonal based on FC analysis. FC findings were compared with microscopy of aspirates, histology and immunohistochemistry of trephine biopsies, and immunofixation (IFX) of serum and/or urine. FC underestimated PC compared to aspirate and trephine microscopy. The 10% diagnostic cutoff for MM on aspirate microscopy corresponded to a 3.5% cutoff on FC. Abnormal plasma cell morphology by aspirate microscopy and clonality by FC correlated in 229 of 294 cases (78%). However, in 50 cases, FC demonstrated a monoclonal population but microscopy reported no abnormality. In 15 cases, abnormalities were reported by microscopy but not by FC. Clonality assessment by trephine microscopy and FC agreed in 251/280 cases (90%), but all 29 discordant cases were monoclonal by FC and not monoclonal by microscopy. These cases had fewer PC and proportionally more polyclonal PC, and when IFX detected a paraprotein, it had the same light chain as in the PC determined by FC. FC was more sensitive in detecting monoclonal populations that were small or accompanied by polyclonal PC. This study supports the inclusion of FC in the evaluation of PC, especially in the assessment of small populations. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

Investigating Antibacterial Effects of Garlic (Allium sativum) Concentrate and Garlic-Derived Organosulfur Compounds on Campylobacter jejuni by Using Fourier Transform Infrared Spectroscopy, Raman Spectroscopy, and Electron Microscopy ▿ †

PubMed Central

Lu, Xiaonan; Rasco, Barbara A.; Jabal, Jamie M. F.; Aston, D. Eric; Lin, Mengshi; Konkel, Michael E.

2011-01-01

Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy were used to study the cell injury and inactivation of Campylobacter jejuni from exposure to antioxidants from garlic. C. jejuni was treated with various concentrations of garlic concentrate and garlic-derived organosulfur compounds in growth media and saline at 4, 22, and 35°C. The antimicrobial activities of the diallyl sulfides increased with the number of sulfur atoms (diallyl sulfide < diallyl disulfide < diallyl trisulfide). FT-IR spectroscopy confirmed that organosulfur compounds are responsible for the substantial antimicrobial activity of garlic, much greater than those of garlic phenolic compounds, as indicated by changes in the spectral features of proteins, lipids, and polysaccharides in the bacterial cell membranes. Confocal Raman microscopy (532-nm-gold-particle substrate) and Raman mapping of a single bacterium confirmed the intracellular uptake of sulfur and phenolic components. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed to verify cell damage. Principal-component analysis (PCA), discriminant function analysis (DFA), and soft independent modeling of class analogs (SIMCA) were performed, and results were cross validated to differentiate bacteria based upon the degree of cell injury. Partial least-squares regression (PLSR) was employed to quantify and predict actual numbers of healthy and injured bacterial cells remaining following treatment. PLSR-based loading plots were investigated to further verify the changes in the cell membrane of C. jejuni treated with organosulfur compounds. We demonstrated that bacterial injury and inactivation could be accurately investigated by complementary infrared and Raman spectroscopies using a chemical-based, “whole-organism fingerprint” with the aid of chemometrics and electron microscopy. PMID:21642409

Quantitative phase imaging of human red blood cells using phase-shifting white light interference microscopy with colour fringe analysis

NASA Astrophysics Data System (ADS)

Singh Mehta, Dalip; Srivastava, Vishal

2012-11-01

We report quantitative phase imaging of human red blood cells (RBCs) using phase-shifting interference microscopy. Five phase-shifted white light interferograms are recorded using colour charge coupled device camera. White light interferograms were decomposed into red, green, and blue colour components. The phase-shifted interferograms of each colour were then processed by phase-shifting analysis and phase maps for red, green, and blue colours were reconstructed. Wavelength dependent refractive index profiles of RBCs were computed from the single set of white light interferogram. The present technique has great potential for non-invasive determination of refractive index variation and morphological features of cells and tissues.

FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.

PubMed

Kuipers, Jeroen; van Ham, Tjakko J; Kalicharan, Ruby D; Veenstra-Algra, Anneke; Sjollema, Klaas A; Dijk, Freark; Schnell, Ulrike; Giepmans, Ben N G

2015-04-01

Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named "FLIPPER", which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM.

Spectro-microscopy of living plant cells.

PubMed

Harter, Klaus; Meixner, Alfred J; Schleifenbaum, Frank

2012-01-01

Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techniques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent developments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassinosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime (FLT). Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy (FIDSAM), has been developed. FIDSAM is capable of imaging low-expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of Förster resonance energy transfer (FRET) for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quantitative analysis of the photosystem I and II (PSI/PSII) ratio in the chloroplasts of living Arabidopsis plants at room temperature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiological and molecular topics can be addressed and valuable insights into molecular and subcellular processes can be obtained in living plants.

Contributed review: Review of integrated correlative light and electron microscopy.

PubMed

Timmermans, F J; Otto, C

2015-01-01

New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemically or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.

Live Cell Refractometry Using Hilbert Phase Microscopy and Confocal Reflectance Microscopy†

PubMed Central

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

2010-01-01

Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ. PMID:19803506

Quantitative evaluation method for differentiation of C2C12 myoblasts by ultrasonic microscopy

NASA Astrophysics Data System (ADS)

Takanashi, Kyoichi; Washiya, Mamoru; Ota, Kazuki; Yoshida, Sachiko; Hozumi, Naohiro; Kobayashi, Kazuto

2017-07-01

Cell differentiation was evaluated by ultrasonic microscopy. However, there were some regions that showed a lower acoustic impedance than the culture liquid. It was considered that, in such regions, the cells were not perfectly in contact with the film substrate. Hence, a waveform analysis was performed, and compensated acoustic impedances in such regions were in a reasonable range of values. By the same analysis, the displacements of partially floated cells were also successfully calculated. The elapsed day transitions of the compensated acoustic impedances and displacements were successfully evaluated. In the process of differentiation, actin fibers comprising the cytoskeleton are supposed to loosen in order to induce cellular fusion. In addition, the progress in cell differentiation accompanied by a change into a three-dimensional structure can partially be assessed by the displacement between a cell and a cultured film. Hence, we believe that cell differentiation can be evaluated using an ultrasonic microscope.

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.

Dynamic quantitative analysis of adherent cell cultures by means of lens-free video microscopy

NASA Astrophysics Data System (ADS)

Allier, C.; Vincent, R.; Navarro, F.; Menneteau, M.; Ghenim, L.; Gidrol, X.; Bordy, T.; Hervé, L.; Cioni, O.; Bardin, S.; Bornens, M.; Usson, Y.; Morales, S.

2018-02-01

We present our implementation of lens-free video microscopy setup for the monitoring of adherent cell cultures. We use a multi-wavelength LED illumination together with a dedicated holographic reconstruction algorithm that allows for an efficient removal of twin images from the reconstructed phase image for densities up to those of confluent cell cultures (>500 cells/mm2). We thereby demonstrate that lens-free video microscopy, with a large field of view ( 30 mm2) can enable us to capture the images of thousands of cells simultaneously and directly inside the incubator. It is then possible to trace and quantify single cells along several cell cycles. We thus prove that lens-free microscopy is a quantitative phase imaging technique enabling estimation of several metrics at the single cell level as a function of time, for example the area, dry mass, maximum thickness, major axis length and aspect ratio of each cell. Combined with cell tracking, it is then possible to extract important parameters such as the initial cell dry mass (just after cell division), the final cell dry mass (just before cell division), the average cell growth rate, and the cell cycle duration. As an example, we discuss the monitoring of a HeLa cell cultures which provided us with a data-set featuring more than 10 000 cell cycle tracks and more than 2x106 cell morphological measurements in a single time-lapse.

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

PubMed Central

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

2008-01-01

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

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

PubMed

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

2008-07-16

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

Potential of ultraviolet wide-field imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes.

PubMed

Wüstner, Daniel; Brewer, Jonathan R; Bagatolli, Luis; Sage, Daniel

2011-01-01

Dehydroergosterol (DHE) is an intrinsically fluorescent sterol with absorption/emission in the ultraviolet (UV) region and biophysical properties similar to those of cholesterol. We compared the potential of UV-sensitive low-light-level wide-field (UV-WF) imaging with that of multiphoton (MP) excitation microscopy to monitor DHE in living cells. Significantly reduced photobleaching in MP microscopy of DHE enabled us to acquire three-dimensional z-stacks of DHE-stained cells and to obtain high-resolution maps of DHE in surface ruffles, nanotubes, and the apical membrane of epithelial cells. We found that the lateral resolution of MP microscopy is ∼1.5-fold higher than that of UV-WF deconvolution microscopy, allowing for improved spatiotemporal analysis of plasma membrane sterol distribution. Surface intensity patterns of DHE with a diameter of 0.2 μm persisting over several minutes could be resolved by MP time-lapse microscopy. Diffusion coefficients of 0.25-μm-diameter endocytic vesicles containing DHE were determined by MP spatiotemporal image correlation spectroscopy. The requirement of extremely high laser power for visualization of DHE by MP microscopy made this method less potent for multicolor applications with organelle markers like green fluorescent protein-tagged proteins. The signal-to-noise ratio obtainable by UV-WF imaging could be significantly improved by pixelwise bleach rate fitting and calculation of an amplitude image from the decay model and by frame averaging after pixelwise bleaching correction of the image stacks. We conclude that UV-WF imaging and MP microscopy of DHE provide complementary information regarding membrane distribution and intracellular targeting of sterols. © 2010 Wiley-Liss, Inc.

Quantitative 4D analyses of epithelial folding during Drosophila gastrulation.

PubMed

Khan, Zia; Wang, Yu-Chiun; Wieschaus, Eric F; Kaschube, Matthias

2014-07-01

Understanding the cellular and mechanical processes that underlie the shape changes of individual cells and their collective behaviors in a tissue during dynamic and complex morphogenetic events is currently one of the major frontiers in developmental biology. The advent of high-speed time-lapse microscopy and its use in monitoring the cellular events in fluorescently labeled developing organisms demonstrate tremendous promise in establishing detailed descriptions of these events and could potentially provide a foundation for subsequent hypothesis-driven research strategies. However, obtaining quantitative measurements of dynamic shapes and behaviors of cells and tissues in a rapidly developing metazoan embryo using time-lapse 3D microscopy remains technically challenging, with the main hurdle being the shortage of robust imaging processing and analysis tools. We have developed EDGE4D, a software tool for segmenting and tracking membrane-labeled cells using multi-photon microscopy data. Our results demonstrate that EDGE4D enables quantification of the dynamics of cell shape changes, cell interfaces and neighbor relations at single-cell resolution during a complex epithelial folding event in the early Drosophila embryo. We expect this tool to be broadly useful for the analysis of epithelial cell geometries and movements in a wide variety of developmental contexts. © 2014. Published by The Company of Biologists Ltd.

Quantification of endocytosis using a folate functionalized silica hollow nanoshell platform

PubMed Central

Sandoval, Sergio; Mendez, Natalie; Alfaro, Jesus G.; Yang, Jian; Aschemeyer, Sharraya; Liberman, Alex; Trogler, William C.; Kummel, Andrew C.

2015-01-01

Abstract. A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N-hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900  μg, respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization. PMID:26315280

Calcium phosphate nanoparticles as versatile carrier for small and large molecules across cell membranes

NASA Astrophysics Data System (ADS)

Sokolova, Viktoriya; Rotan, Olga; Klesing, Jan; Nalbant, Perihan; Buer, Jan; Knuschke, Torben; Westendorf, Astrid M.; Epple, Matthias

2012-06-01

The successful transport of molecules across the cell membrane is a key point in biology and medicine. In most cases, molecules alone cannot penetrate the cell membrane, therefore an efficient carrier is needed. Calcium phosphate nanoparticles (diameter: 100-250 nm, depending on the functionalization) were loaded with fluorescent oligonucleotides, peptide, proteins, antibodies, polymers or porphyrins and characterized by dynamic light scattering, nanoparticle tracking analysis and scanning electron microscopy. Any excess of molecules was removed by ultracentrifugation, and the dissolved molecules at the same concentration were used as control. The uptake of such fluorescence-labeled nanoparticles into HeLa cells was monitored by fluorescence microscopy and confocal laser scanning microscopy. Calcium phosphate nanoparticles were able to transport all molecules across the cell membrane, whereas the dissolved molecules alone were taken up only to a very small extent or even not at all.

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.

Tracking of cell nuclei for assessment of in vitro uptake kinetics in ultrasound-mediated drug delivery using fibered confocal fluorescence microscopy.

PubMed

Derieppe, Marc; de Senneville, Baudouin Denis; Kuijf, Hugo; Moonen, Chrit; Bos, Clemens

2014-10-01

Previously, we demonstrated the feasibility to monitor ultrasound-mediated uptake of a cell-impermeable model drug in real time with fibered confocal fluorescence microscopy. Here, we present a complete post-processing methodology, which corrects for cell displacements, to improve the accuracy of pharmacokinetic parameter estimation. Nucleus detection was performed based on the radial symmetry transform algorithm. Cell tracking used an iterative closest point approach. Pharmacokinetic parameters were calculated by fitting a two-compartment model to the time-intensity curves of individual cells. Cells were tracked successfully, improving time-intensity curve accuracy and pharmacokinetic parameter estimation. With tracking, 93 % of the 370 nuclei showed a fluorescence signal variation that was well-described by a two-compartment model. In addition, parameter distributions were narrower, thus increasing precision. Dedicated image analysis was implemented and enabled studying ultrasound-mediated model drug uptake kinetics in hundreds of cells per experiment, using fiber-based confocal fluorescence microscopy.

Cell invasion in the spheroid sprouting assay: a spatial organisation analysis adaptable to cell behaviour.

PubMed

Blacher, Silvia; Erpicum, Charlotte; Lenoir, Bénédicte; Paupert, Jenny; Moraes, Gustavo; Ormenese, Sandra; Bullinger, Eric; Noel, Agnès

2014-01-01

The endothelial cell spheroid assay provides a suitable in vitro model to study (lymph) angiogenesis and test pro- and anti-(lymph) angiogenic factors or drugs. Usually, the extent of cell invasion, observed through optical microscopy, is measured. The present study proposes the spatial distribution of migrated cells as a new descriptor of the (lymph) angiogenic response. The utility of this novel method rests with its capacity to locally characterise spheroid structure, allowing not only the investigation of single and collective cell invasion but also the evolution of the spheroid core itself. Moreover, the proposed method can be applied to 2D-projected spheroid images obtained by optical microscopy, as well as to 3D images acquired by confocal microscopy. To validate the proposed methodology, endothelial cell invasion was evaluated under different experimental conditions. The results were compared with widely used global parameters. The comparison shows that our method prevents local spheroid modifications from being overlooked and leading to the possible misinterpretation of results.

Development of image analysis software for quantification of viable cells in microchips.

PubMed

Georg, Maximilian; Fernández-Cabada, Tamara; Bourguignon, Natalia; Karp, Paola; Peñaherrera, Ana B; Helguera, Gustavo; Lerner, Betiana; Pérez, Maximiliano S; Mertelsmann, Roland

2018-01-01

Over the past few years, image analysis has emerged as a powerful tool for analyzing various cell biology parameters in an unprecedented and highly specific manner. The amount of data that is generated requires automated methods for the processing and analysis of all the resulting information. The software available so far are suitable for the processing of fluorescence and phase contrast images, but often do not provide good results from transmission light microscopy images, due to the intrinsic variation of the acquisition of images technique itself (adjustment of brightness / contrast, for instance) and the variability between image acquisition introduced by operators / equipment. In this contribution, it has been presented an image processing software, Python based image analysis for cell growth (PIACG), that is able to calculate the total area of the well occupied by cells with fusiform and rounded morphology in response to different concentrations of fetal bovine serum in microfluidic chips, from microscopy images in transmission light, in a highly efficient way.

Raman and coherent anti-Stokes Raman scattering microscopy studies of changes in lipid content and composition in hormone-treated breast and prostate cancer cells

NASA Astrophysics Data System (ADS)

Potcoava, Mariana C.; Futia, Gregory L.; Aughenbaugh, Jessica; Schlaepfer, Isabel R.; Gibson, Emily A.

2014-11-01

Increasing interest in the role of lipids in cancer cell proliferation and resistance to drug therapies has motivated the need to develop better tools for cellular lipid analysis. Quantification of lipids in cells is typically done by destructive chromatography protocols that do not provide spatial information on lipid distribution and prevent dynamic live cell studies. Methods that allow the analysis of lipid content in live cells are therefore of great importance. Using micro-Raman spectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy, we generated a lipid profile for breast (T47D, MDA-MB-231) and prostate (LNCaP, PC3) cancer cells upon exposure to medroxyprogesterone acetate (MPA) and synthetic androgen R1881. Combining Raman spectra with CARS imaging, we can study the process of hormone-mediated lipogenesis. Our results show that hormone-treated cancer cells T47D and LNCaP have an increased number and size of intracellular lipid droplets and higher degree of saturation than untreated cells. MDA-MB-231 and PC3 cancer cells showed no significant changes upon treatment. Principal component analysis with linear discriminant analysis of the Raman spectra was able to differentiate between cancer cells that were treated with MPA, R1881, and untreated.

Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria.

PubMed

Karbowski, M; Kurono, C; Wozniak, M; Ostrowski, M; Teranishi, M; Soji, T; Wakabayashi, T

1999-02-04

Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6, 6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.

Comparison of manual & automated analysis methods for corneal endothelial cell density measurements by specular microscopy.

PubMed

Huang, Jianyan; Maram, Jyotsna; Tepelus, Tudor C; Modak, Cristina; Marion, Ken; Sadda, SriniVas R; Chopra, Vikas; Lee, Olivia L

2017-08-07

To determine the reliability of corneal endothelial cell density (ECD) obtained by automated specular microscopy versus that of validated manual methods and factors that predict such reliability. Sharp central images from 94 control and 106 glaucomatous eyes were captured with Konan specular microscope NSP-9900. All images were analyzed by trained graders using Konan CellChek Software, employing the fully- and semi-automated methods as well as Center Method. Images with low cell count (input cells number <100) and/or guttata were compared with the Center and Flex-Center Methods. ECDs were compared and absolute error was used to assess variation. The effect on ECD of age, cell count, cell size, and cell size variation was evaluated. No significant difference was observed between the Center and Flex-Center Methods in corneas with guttata (p=0.48) or low ECD (p=0.11). No difference (p=0.32) was observed in ECD of normal controls <40 yrs old between the fully-automated method and manual Center Method. However, in older controls and glaucomatous eyes, ECD was overestimated by the fully-automated method (p=0.034) and semi-automated method (p=0.025) as compared to manual method. Our findings show that automated analysis significantly overestimates ECD in the eyes with high polymegathism and/or large cell size, compared to the manual method. Therefore, we discourage reliance upon the fully-automated method alone to perform specular microscopy analysis, particularly if an accurate ECD value is imperative. Copyright © 2017. Published by Elsevier España, S.L.U.

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

In vivo laser confocal microscopic analysis of murine cornea and lens microstructures.

PubMed

Yuasa, Masashi; Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2008-01-01

The purpose of the current study is to investigate in vivo microstructures of anterior segments of normal murine eyes by new-generation in vivo laser confocal microscopy. Twenty-six corneas and lenses from 13 mice were analyzed by in vivo laser confocal microscopy. Murine corneal superficial cells formed a polygonal cell pattern, with a mean cell density of 577 +/- 115 cells/mm2 (mean +/- standard deviation). Corneal basal epithelial cells had dark cytoplasm and were closely organized (9,312 +/- 1,777 cells/mm2). Sub-basal nerve fiber bundles were arranged in a whorl pattern, with both clockwise and counter-clockwise patterns. In the stroma, keratocytes were observed as numerous reflective stellate structures. The endothelial cells were organized in a honeycomb pattern (2,463 +/- 292 cells/mm2). Deeper inside the eye, murine lens epithelial cells were organized in a regular pattern (4,168 +/- 636 cells/mm2) and numerous lens fibers were observed. In vivo laser confocal microscopy can provide high-resolution images of all corneal layers and lens structures of mice without sacrificing animals or tissue preparation.

Extracting archaeal populations from iron oxidizing systems

NASA Astrophysics Data System (ADS)

Whitmore, L. M.; Hutchison, J.; Chrisler, W.; Jay, Z.; Moran, J.; Inskeep, W.; Kreuzer, H.

2013-12-01

Unique environments in Yellowstone National Park offer exceptional conditions for studying microorganisms in extreme and constrained systems. However, samples from some extreme systems often contain inorganic components that pose complications during microbial and molecular analysis. Several archaeal species are found in acidic, geothermal ferric-oxyhydroxide mats; these species have been shown to adhere to mineral surfaces in flocculated colonies. For optimal microbial analysis, (microscopy, flow cytometry, genomic extractions, proteomic analysis, stable isotope analysis, and others), improved techniques are needed to better facilitate cell detachment and separation from mineral surfaces. As a requirement, these techniques must preserve cell structure while simultaneously minimizing organic carryover to downstream analysis. Several methods have been developed for removing sediments from mixed prokaryotic populations, including ultra-centrifugation, nycodenz gradient, sucrose cushions, and cell straining. In this study we conduct a comparative analysis of mechanisms used to detach archaeal cell populations from the mineral interface. Specifically, we evaluated mechanical and chemical approaches for cell separation and homogenization. Methods were compared using confocal microscopy, flow cytometry analyses, and real-time PCR detection. The methodology and approaches identified will be used to optimize biomass collection from environmental specimens or isolates grown with solid phases.

Extending the knowledge in histochemistry and cell biology.

PubMed

Heupel, Wolfgang-Moritz; Drenckhahn, Detlev

2010-01-01

Central to modern Histochemistry and Cell Biology stands the need for visualization of cellular and molecular processes. In the past several years, a variety of techniques has been achieved bridging traditional light microscopy, fluorescence microscopy and electron microscopy with powerful software-based post-processing and computer modeling. Researchers now have various tools available to investigate problems of interest from bird's- up to worm's-eye of view, focusing on tissues, cells, proteins or finally single molecules. Applications of new approaches in combination with well-established traditional techniques of mRNA, DNA or protein analysis have led to enlightening and prudent studies which have paved the way toward a better understanding of not only physiological but also pathological processes in the field of cell biology. This review is intended to summarize articles standing for the progress made in "histo-biochemical" techniques and their manifold applications.

Background fluorescence estimation and vesicle segmentation in live cell imaging with conditional random fields.

PubMed

Pécot, Thierry; Bouthemy, Patrick; Boulanger, Jérôme; Chessel, Anatole; Bardin, Sabine; Salamero, Jean; Kervrann, Charles

2015-02-01

Image analysis applied to fluorescence live cell microscopy has become a key tool in molecular biology since it enables to characterize biological processes in space and time at the subcellular level. In fluorescence microscopy imaging, the moving tagged structures of interest, such as vesicles, appear as bright spots over a static or nonstatic background. In this paper, we consider the problem of vesicle segmentation and time-varying background estimation at the cellular scale. The main idea is to formulate the joint segmentation-estimation problem in the general conditional random field framework. Furthermore, segmentation of vesicles and background estimation are alternatively performed by energy minimization using a min cut-max flow algorithm. The proposed approach relies on a detection measure computed from intensity contrasts between neighboring blocks in fluorescence microscopy images. This approach permits analysis of either 2D + time or 3D + time data. We demonstrate the performance of the so-called C-CRAFT through an experimental comparison with the state-of-the-art methods in fluorescence video-microscopy. We also use this method to characterize the spatial and temporal distribution of Rab6 transport carriers at the cell periphery for two different specific adhesion geometries.

Multicell migration tracking within angiogenic networks by deep learning-based segmentation and augmented Bayesian filtering.

PubMed

Wang, Mengmeng; Ong, Lee-Ling Sharon; Dauwels, Justin; Asada, H Harry

2018-04-01

Cell migration is a key feature for living organisms. Image analysis tools are useful in studying cell migration in three-dimensional (3-D) in vitro environments. We consider angiogenic vessels formed in 3-D microfluidic devices (MFDs) and develop an image analysis system to extract cell behaviors from experimental phase-contrast microscopy image sequences. The proposed system initializes tracks with the end-point confocal nuclei coordinates. We apply convolutional neural networks to detect cell candidates and combine backward Kalman filtering with multiple hypothesis tracking to link the cell candidates at each time step. These hypotheses incorporate prior knowledge on vessel formation and cell proliferation rates. The association accuracy reaches 86.4% for the proposed algorithm, indicating that the proposed system is able to associate cells more accurately than existing approaches. Cell culture experiments in 3-D MFDs have shown considerable promise for improving biology research. The proposed system is expected to be a useful quantitative tool for potential microscopy problems of MFDs.

Confocal laser scanning microscopic photoconversion: a new method to stabilize fluorescently labeled cellular elements for electron microscopic analysis.

PubMed

Colello, Raymond J; Tozer, Jordan; Henderson, Scott C

2012-01-01

Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low-resolution cell targeting. Accordingly, we have developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We have found that this method greatly reduces photoconversion times, as compared to conventional wide field microscopy. Moreover, region-of-interest scanning capabilities of a confocal microscope facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This reduces the area of the cell exposed to light energy, thereby reducing the ultrastructural damage common to this process when widefield microscopes are employed. © 2012 by John Wiley & Sons, Inc.

Experimental and theoretical analysis for improved microscope design of optical projection tomographic microscopy.

PubMed

Coe, Ryan L; Seibel, Eric J

2013-09-01

We present theoretical and experimental results of axial displacement of objects relative to a fixed condenser focal plane (FP) in optical projection tomographic microscopy (OPTM). OPTM produces three-dimensional, reconstructed images of single cells from two-dimensional projections. The cell rotates in a microcapillary to acquire projections from different perspectives where the objective FP is scanned through the cell while the condenser FP remains fixed at the center of the microcapillary. This work uses a combination of experimental and theoretical methods to improve the OPTM instrument design.

A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy

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

Verhertbruggen, Yves; Walker, Jesse L.; Guillon, Fabienne

Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of thesemore » three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy.« less

A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy

DOE PAGES

Verhertbruggen, Yves; Walker, Jesse L.; Guillon, Fabienne; ...

2017-08-29

Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of thesemore » three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy.« less

A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy

PubMed Central

Verhertbruggen, Yves; Walker, Jesse L.; Guillon, Fabienne; Scheller, Henrik V.

2017-01-01

Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of these three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy. PMID:28900439

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

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

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

2012-05-11

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

An automated approach for single-cell tracking in epifluorescence microscopy applied to E. coli growth analysis on microfluidics biochips

NASA Astrophysics Data System (ADS)

Fetita, Catalin; Kirov, Boris; Jaramillo, Alfonso; Lefevre, Christophe

2012-03-01

With the accumulation of knowledge for the intimate molecular mechanisms governing the processes inside the living cells in the later years, the ability to characterize the performance of elementary genetic circuits and parts at the single-cell level is becoming of crucial importance. Biological science is arriving to the point where it can develop hypothesis for the action of each molecule participating in the biochemical reactions and need proper techniques to test those hypothesis. Microfluidics is emerging as the technology that combined with high-magnification microscopy will allow for the long-term single-cell level observation of bacterial physiology. In this study we design, build and characterize the gene dynamics of genetic circuits as one of the basic parts governing programmed cell behavior. We use E. coli as model organism and grow it in microfluidics chips, which we observe with epifluorescence microscopy. One of the most invaluable segments of this technology is the consequent image processing, since it allows for the automated analysis of vast amount of single-cell observation and the fast and easy derivation of conclusions based on that data. Specifically, we are interested in promoter activity as function of time. We expect it to be oscillatory and for that we use GFP (green fluorescent protein) as a reporter in our genetic circuits. In this paper, an automated framework for single-cell tracking in phase-contrast microscopy is developed, combining 2D segmentation of cell time frames and graph-based reconstruction of their spatiotemporal evolution with fast tracking of the associated fluorescence signal. The results obtained on the investigated biological database are presented and discussed.

Sequential processing of quantitative phase images for the study of cell behaviour in real-time digital holographic microscopy.

PubMed

Zikmund, T; Kvasnica, L; Týč, M; Křížová, A; Colláková, J; Chmelík, R

2014-11-01

Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time-lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long-term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least-squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Automated quantitative cytological analysis using portable microfluidic microscopy.

PubMed

Jagannadh, Veerendra Kalyan; Murthy, Rashmi Sreeramachandra; Srinivasan, Rajesh; Gorthi, Sai Siva

2016-06-01

In this article, a portable microfluidic microscopy based approach for automated cytological investigations is presented. Inexpensive optical and electronic components have been used to construct a simple microfluidic microscopy system. In contrast to the conventional slide-based methods, the presented method employs microfluidics to enable automated sample handling and image acquisition. The approach involves the use of simple in-suspension staining and automated image acquisition to enable quantitative cytological analysis of samples. The applicability of the presented approach to research in cellular biology is shown by performing an automated cell viability assessment on a given population of yeast cells. Further, the relevance of the presented approach to clinical diagnosis and prognosis has been demonstrated by performing detection and differential assessment of malaria infection in a given sample. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Automatic microscopy for mitotic cell location.

NASA Technical Reports Server (NTRS)

Herron, J.; Ranshaw, R.; Castle, J.; Wald, N.

1972-01-01

Advances are reported in the development of an automatic microscope with which to locate hematologic or other cells in mitosis for subsequent chromosome analysis. The system under development is designed to perform the functions of: slide scanning to locate metaphase cells; conversion of images of selected cells into binary form; and on-line computer analysis of the digitized image for significant cytogenetic data. Cell detection criteria are evaluated using a test sample of 100 mitotic cells and 100 artifacts.

Studies by immune electron microscopy of hepatitis B surface antigen in PLC/PRF/5 cells.

PubMed

Shibayama, T; Watanabe, T; Kojima, H; Yoshikawa, A; Watanabe, S; Kamimura, T; Suzuki, S; Ichida, F

1984-01-01

Electron microscopic studies of the morphology of hepatitis B surface antigen (HBsAg) produced by PLC/PRF/5 cells in vitro were carried out. Aggregates of 20-nm spherical particles in 3-day culture supernatants were observed by immune electron microscopy (IEM). Aggregates of tubular structures were found with IEM in the extracts of the cells. Tubular structures 18 to 22 nm in diameter were seen by electron microscopy (EM) in the cisternae of the endoplasmic reticulum in 2-3% of the cells. The tubular structures in the cytoplasm and extracts of PLC/PRF/5 cells resembled those observed in the hepatocytes of human carriers of hepatitis B virus (HBV). Intracellular localization of HBsAg in PLC/PRF/5 cells by direct peroxidase-conjugated antibody staining was observed on the tubular structures and the cisternal wall, which contained these structures. Rotation technique analysis indicated that the tubular structures were composed of 11 or 12 subunits.

Use of direct fluorescence labeling and confocal microscopy to determine the biodistribution of two protein therapeutics, Cerezyme and Ceredase.

PubMed

Piepenhagen, Peter A; Vanpatten, Scott; Hughes, Heather; Waire, James; Murray, James; Andrews, Laura; Edmunds, Tim; O'Callaghan, Michael; Thurberg, Beth L

2010-07-01

Efficient targeting of therapeutic reagents to tissues and cell types of interest is critical to achieving therapeutic efficacy and avoiding unwanted side effects due to offtarget uptake. To increase assay efficiency and reduce the number of animals used per experiment during preclinical development, we used a combination of direct fluorescence labeling and confocal microscopy to simultaneously examine the biodistribution of two therapeutic proteins, Cerezyme and Ceredase, in the same animals. We show that the fluorescent tags do not interfere with protein uptake and localization. We are able to detect Cerezyme and Ceredase in intact cells and organs and demonstrate colocalization within target cells using confocal microscopy. In addition, the relative amount of protein internalized by different cell types can be quantified using cell type-specific markers and morphometric analysis. This approach provides an easy and straightforward means of assessing the tissue and cell type-specific biodistribution of multiple protein therapeutics in target organs using a minimal number of animals. (c) 2009 Wiley-Liss, Inc.

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.

Combined use of atomic force microscopy, X-ray photoelectron spectroscopy, and secondary ion mass spectrometry for cell surface analysis.

PubMed

Dague, Etienne; Delcorte, Arnaud; Latgé, Jean-Paul; Dufrêne, Yves F

2008-04-01

Understanding the surface properties of microbial cells is a major challenge of current microbiological research and a key to efficiently exploit them in biotechnology. Here, we used three advanced surface analysis techniques with different sensitivity, probing depth, and lateral resolution, that is, in situ atomic force microscopy, X-ray photoelectron spectroscopy, and secondary ion mass spectrometry, to gain insight into the surface properties of the conidia of the human fungal pathogen Aspergillus fumigatus. We show that the native ultrastructure, surface protein and polysaccharide concentrations, and amino acid composition of three mutants affected in hydrophobin production are markedly different from those of the wild-type, thereby providing novel insight into the cell wall architecture of A. fumigatus. The results demonstrate the power of using multiple complementary techniques for probing microbial cell surfaces.

Refractive index measurements of single, spherical cells using digital holographic microscopy.

PubMed

Schürmann, Mirjam; Scholze, Jana; Müller, Paul; Chan, Chii J; Ekpenyong, Andrew E; Chalut, Kevin J; Guck, Jochen

2015-01-01

In this chapter, we introduce digital holographic microscopy (DHM) as a marker-free method to determine the refractive index of single, spherical cells in suspension. The refractive index is a conclusive measure in a biological context. Cell conditions, such as differentiation or infection, are known to yield significant changes in the refractive index. Furthermore, the refractive index of biological tissue determines the way it interacts with light. Besides the biological relevance of this interaction in the retina, a lot of methods used in biology, including microscopy, rely on light-tissue or light-cell interactions. Hence, determining the refractive index of cells using DHM is valuable in many biological applications. This chapter covers the main topics that are important for the implementation of DHM: setup, sample preparation, and analysis. First, the optical setup is described in detail including notes and suggestions for the implementation. Following that, a protocol for the sample and measurement preparation is explained. In the analysis section, an algorithm for the determination of quantitative phase maps is described. Subsequently, all intermediate steps for the calculation of the refractive index of suspended cells are presented, exploiting their spherical shape. In the last section, a discussion of possible extensions to the setup, further measurement configurations, and additional analysis methods are given. Throughout this chapter, we describe a simple, robust, and thus easily reproducible implementation of DHM. The different possibilities for extensions show the diverse fields of application for this technique. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Timmermans, F. J.; Otto, C.

New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemicallymore » or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.« less

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.

Analysis of Peroxisome Biogenesis in Pollen by Confocal Microscopy and Transmission Electron Microscopy.

PubMed

Jia, Peng-Fei; Li, Hong-Ju; Yang, Wei-Cai

2017-01-01

Peroxisome is an essential single-membrane bound organelle in most eukaryotic cells and functions in diverse cellular processes. De novo formation, division, and turnover of peroxisomes contribute to its biogenesis, morphology, and population regulation. In plants, peroxisome plays multiple roles, including metabolism, development, and stress response. Defective peroxisome biogenesis and development retard plant growth, adaption, and reproduction. Through tracing the subcellular localization of fluorescent reporter tagged matrix protein of peroxisome, fluorescence microscopy is a reliable and fast way to detect peroxisome biogenesis. Further fine-structural observation of peroxisome by TEM enables researchers to observe the detailed ultrastructure of its morphology and spatial contact with other organelles. Pollen grain is a specialized structure where two small sperm cells are enclosed in the cytoplasm of a large vegetative cell. Two features make pollen grain a good system to study peroxisome biogenesis: indispensable requirement of peroxisome for germination on the stigma and homogeneity. Here, we describe the methods of studying peroxisome biogenesis in Arabidopsis pollen grains by fluorescent live-imaging with confocal laser scanning microscopy (CLSM) and by DAB-staining based transmission electron microscopy (TEM).

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

NASA Astrophysics Data System (ADS)

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

2017-06-01

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

Lensfree On-Chip Microscopy and Tomography for Bio-Medical Applications

PubMed Central

Isikman, Serhan O.; Bishara, Waheb; Mudanyali, Onur; Sencan, Ikbal; Su, Ting-Wei; Tseng, Derek; Yaglidere, Oguzhan; Sikora, Uzair; Ozcan, Aydogan

2012-01-01

Lensfree on-chip holographic microscopy is an emerging technique that offers imaging of biological specimens over a large field-of-view without using any lenses or bulky optical components. Lending itself to a compact, cost-effective and mechanically robust architecture, lensfree on-chip holographic microscopy can offer an alternative toolset addressing some of the emerging needs of microscopic analysis and diagnostics in low-resource settings, especially for telemedicine applications. In this review, we summarize the latest achievements in lensfree optical microscopy based on partially coherent on-chip holography, including portable telemedicine microscopy, cell-phone based microscopy and field-portable optical tomographic microscopy. We also discuss some of the future directions for telemedicine microscopy and its prospects to help combat various global health challenges. PMID:24478572

Biological applications of phase-contrast electron microscopy.

PubMed

Nagayama, Kuniaki

2014-01-01

Here, I review the principles and applications of phase-contrast electron microscopy using phase plates. First, I develop the principle of phase contrast based on a minimal model of microscopy, introducing a double Fourier-transform process to mathematically formulate the image formation. Next, I explain four phase-contrast (PC) schemes, defocus PC, Zernike PC, Hilbert differential contrast, and schlieren optics, as image-filtering processes in the context of the minimal model, with particular emphases on the Zernike PC and corresponding Zernike phase plates. Finally, I review applications of Zernike PC cryo-electron microscopy to biological systems such as protein molecules, virus particles, and cells, including single-particle analysis to delineate three-dimensional (3D) structures of protein and virus particles and cryo-electron tomography to reconstruct 3D images of complex protein systems and cells.

Infrared Microspectroscopy: A Multiple-Screening Platform for Investigating Single-Cell Biochemical Perturbations upon Prion Infection

PubMed Central

2011-01-01

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrPSc) possesses the capability to convert the host-encoded cellular isoform of the prion protein, PrPC, into nascent PrPSc. The present work aims at providing novel insight into cellular response upon prion infection evidenced by synchrotron radiation infrared microspectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations undergone by prion infected mouse hypothalamic GT1-1 cells at the cellular and subcellular level. A decrement in total cellular protein content upon prion infection was identified by infrared (IR) whole-cell spectra and validated by bicinchoninic acid assay and single-cell volume analysis by atomic force microscopy (AFM). Hierarchical cluster analysis (HCA) of IR data discriminated between infected and uninfected cells and allowed to deduce an increment of lysosomal bodies within the cytoplasm of infected GT1-1 cells, a hypothesis further confirmed by SR-IRMS at subcellular spatial resolution and fluorescent microscopy. The purpose of this work, therefore, consists of proposing IRMS as a powerful multiscreening platform, drawing on the synergy with conventional biological assays and microscopy techniques in order to increase the accuracy of investigations performed at the single-cell level. PMID:22778865

Infrared microspectroscopy: a multiple-screening platform for investigating single-cell biochemical perturbations upon prion infection.

PubMed

Didonna, Alessandro; Vaccari, Lisa; Bek, Alpan; Legname, Giuseppe

2011-03-16

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrP(Sc)) possesses the capability to convert the host-encoded cellular isoform of the prion protein, PrP(C), into nascent PrP(Sc). The present work aims at providing novel insight into cellular response upon prion infection evidenced by synchrotron radiation infrared microspectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations undergone by prion infected mouse hypothalamic GT1-1 cells at the cellular and subcellular level. A decrement in total cellular protein content upon prion infection was identified by infrared (IR) whole-cell spectra and validated by bicinchoninic acid assay and single-cell volume analysis by atomic force microscopy (AFM). Hierarchical cluster analysis (HCA) of IR data discriminated between infected and uninfected cells and allowed to deduce an increment of lysosomal bodies within the cytoplasm of infected GT1-1 cells, a hypothesis further confirmed by SR-IRMS at subcellular spatial resolution and fluorescent microscopy. The purpose of this work, therefore, consists of proposing IRMS as a powerful multiscreening platform, drawing on the synergy with conventional biological assays and microscopy techniques in order to increase the accuracy of investigations performed at the single-cell level.

Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain.

PubMed

Hezel, Marcus; Ebrahimi, Fahim; Koch, Marco; Dehghani, Faramarz

2012-10-01

Immunohistochemical visualization of antigens in specimen has evolved to an indispensable technique in biomedical research for investigations of cell morphology and pathology both in bright field and fluorescence microscopy. While there are couple of staining methods that reveal entire cytoarchitecture in bright field microscopy such as Nissl or hemalaun-eosin, there are still limitations in visualizations of cytoarchitecture in fluorescence microscopy. The present study reports a simple staining method that provides the required illustration of cell allocations and cellular composition in fluorescence microscopy in adult and in developing rodent central nervous system using the fluorophore propidium iodide (PI, 5μg/mL). PI is a well-accepted marker for degenerating cells when applied prior to fixation (pre-fixation PI staining). Here, PI was added to the sections after the fixation (post-fixation PI staining). This revised labeling procedure led to similar cytoarchitectural staining patterns in fluorescence microscopy as observed with hemalaun in bright field microscopy. This finding was proven in organotypic hippocampal slice cultures (OHSC) and brain sections obtained from different postnatal developmental stages. Excitotoxically lesioned OHSC subjected to pre-fixation PI staining merely showed brightly labeled condensed nuclei of degenerating neurons. In contrast, post-fixation PI staining additionally revealed extensive labeling of neuronal cell bodies and glial cells within the OHSC, thus allowing visualization of stratification of neuronal layers and cell morphology. Furthermore, post-fixation PI staining was combined with NeuN, calbindin, calretinin, glial fibrillary acidic protein or Griffonia simplicifolia isolectin B4 (IB(4)) in post natal (p1 and p9) and adult rats. In early post-natal brain sections almost all mentioned cellular markers led to an incomplete staining of the native cell organization and resulted in an inaccurate estimation of cell morphology when compared to adult brains. In contrast, post-fixation PI staining allowed investigation of the whole cytoarchitecture independent of the developmental stage. Taken together, post-fixation PI staining provides a detailed insight in the morphology of both developing and adult brain tissues in fluorescence microscopy. Copyright © 2012 Elsevier Ltd. All rights reserved.

Extracting microtubule networks from superresolution single-molecule localization microscopy data

PubMed Central

Zhang, Zhen; Nishimura, Yukako; Kanchanawong, Pakorn

2017-01-01

Microtubule filaments form ubiquitous networks that specify spatial organization in cells. However, quantitative analysis of microtubule networks is hampered by their complex architecture, limiting insights into the interplay between their organization and cellular functions. Although superresolution microscopy has greatly facilitated high-resolution imaging of microtubule filaments, extraction of complete filament networks from such data sets is challenging. Here we describe a computational tool for automated retrieval of microtubule filaments from single-molecule-localization–based superresolution microscopy images. We present a user-friendly, graphically interfaced implementation and a quantitative analysis of microtubule network architecture phenotypes in fibroblasts. PMID:27852898

[Effects of infrasound therapy on proliferation, apoptosis and ultrastructure of human B lymphoma Raji cells].

PubMed

Bao, Yong; Fan, Jian-Zhong; Li, Ke; Li, Chuan; Yang, Jun-Feng

2008-06-01

To investigate the effect of infrasound therapy on the proliferation, apoptosis and ultrastructure of human B lymphoma Raji cells. Human B lymphoma Raji cells were exposed to infrasound treatment for 15, 30, 60, 90 and 120 min and cultured subsequently for 24 or 48 h. MTT assay, flow cytometry analysis, and electron microscopy were performed to examine the proliferative status, cell apoptosis and ultrastructural changes of the exposed cells, respectively. MTT assay revealed no significant changes in the proliferation of the cells exposed to infrasound treatment (P>0.05), nor did flow cytometry analysis identified significant variation in the cell apoptosis (P>0.05). Scanning electron microscopy, however, identified shortened or reduced cell processes and microvilli on the surface of the cells with infrasound exposure and a subsequent 24-hour culture, and the cell membrane surface became smooth. Under transmission electron microscope, the cells with infrasound treatment presented with significantly reduced microvilli, and the cell nuclei appeared homogeneous, with cytoplasmic budding and losses after a 48-hour culture. Infrasound less than 90 dB does not obviously affect the proliferation and apoptosis of Raji cells, but may directly cause cell ultrastructural changes such as reduction of the cell processes.

Morphological Analysis of Human Induced Pluripotent Stem Cells During Induced Differentiation and Reverse Programming

PubMed Central

Magniez, Aurélie; Oudrhiri, Noufissa; Féraud, Olivier; Bacci, Josette; Gobbo, Emilie; Proust, Stéphanie; Turhan, Ali G.

2014-01-01

Abstract The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report, human mesenchymal cells (hMSCs) generated from the human ES cell line H9, were reprogrammed back to induced pluripotent state using Oct-4, Sox2, Nanog, and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes, lipid droplets, glycogen, scarce reticulum) and nuclear levels (features of nuclear plasticity, presence of euchromatin, reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal–epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming. PMID:25371857

Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy

DOE PAGES

Jin, Qiaoling; Paunesku, Tatjana; Lai, Barry; ...

2016-08-31

Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with freshmore » media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. Lastly, when chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.« less

Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy

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

Jin, Qiaoling; Paunesku, Tatjana; Lai, Barry

Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with freshmore » media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. Lastly, when chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.« less

Visualizing Viral Infection In Vivo by Multi-Photon Intravital Microscopy.

PubMed

Sewald, Xaver

2018-06-20

Viral pathogens have adapted to the host organism to exploit the cellular machinery for virus replication and to modulate the host cells for efficient systemic dissemination and immune evasion. Much of our knowledge of the effects that virus infections have on cells originates from in vitro imaging studies using experimental culture systems consisting of cell lines and primary cells. Recently, intravital microscopy using multi-photon excitation of fluorophores has been applied to observe virus dissemination and pathogenesis in real-time under physiological conditions in living organisms. Critical steps during viral infection and pathogenesis could be studied by direct visualization of fluorescent virus particles, virus-infected cells, and the immune response to viral infection. In this review, I summarize the latest research on in vivo studies of viral infections using multi-photon intravital microscopy (MP-IVM). Initially, the underlying principle of multi-photon microscopy is introduced and experimental challenges during microsurgical animal preparation and fluorescent labeling strategies for intravital imaging are discussed. I will further highlight recent studies that combine MP-IVM with optogenetic tools and transcriptional analysis as a powerful approach to extend the significance of in vivo imaging studies of viral pathogens.

Nanoscale live cell optical imaging of the dynamics of intracellular microvesicles in neural cells.

PubMed

Lee, Sohee; Heo, Chaejeong; Suh, Minah; Lee, Young Hee

2013-11-01

Recent advances in biotechnology and imaging technology have provided great opportunities to investigate cellular dynamics. Conventional imaging methods such as transmission electron microscopy, scanning electron microscopy, and atomic force microscopy are powerful techniques for cellular imaging, even at the nanoscale level. However, these techniques have limitations applications in live cell imaging because of the experimental preparation required, namely cell fixation, and the innately small field of view. In this study, we developed a nanoscale optical imaging (NOI) system that combines a conventional optical microscope with a high resolution dark-field condenser (Cytoviva, Inc.) and halogen illuminator. The NOI system's maximum resolution for live cell imaging is around 100 nm. We utilized NOI to investigate the dynamics of intracellular microvesicles of neural cells without immunocytological analysis. In particular, we studied direct, active random, and moderate random dynamic motions of intracellular microvesicles and visualized lysosomal vesicle changes after treatment of cells with a lysosomal inhibitor (NH4Cl). Our results indicate that the NOI system is a feasible, high-resolution optical imaging system for live small organelles that does not require complicated optics or immunocytological staining processes.

Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2011-04-13

The intracellular uptake of 30 nm diameter gold nanoparticles (Au-NPs) was studied at the nanoscale in pristine eukaryotic cells. Live COS-7 cells were maintained in a microfluidic chamber and imaged using scanning transmission electron microscopy. A quantitative image analysis showed that Au-NPs bound to the membranes of vesicles, possibly lysosomes, and occupied 67% of the available surface area. The vesicles accumulated to form a micrometer-sized cluster after 24 h of incubation. Two clusters were analyzed and found to consist of 117 ± 9 and 164 ± 4 NP-filled vesicles.

The genome-wide expression profile of Curcuma longa-treated cisplatin-stimulated HEK293 cells

PubMed Central

Sohn, Sung-Hwa; Ko, Eunjung; Chung, Hwan-Suck; Lee, Eun-Young; Kim, Sung-Hoon; Shin, Minkyu; Hong, Moochang; Bae, Hyunsu

2010-01-01

AIM The rhizome of turmeric, Curcuma longa (CL), is a herbal medicine used in many traditional prescriptions. It has previously been shown that CL treatment showed greater than 47% recovery from cisplatin-induced cell damage in human kidney HEK 293 cells. This study was conducted to evaluate the recovery mechanisms of CL that occur during cisplatin induced nephrotoxicity by examining the genome wide mRNA expression profiles of HEK 293 -cells. METHOD Recovery mechanisms of CL that occur during cisplatin-induced nephrotoxicity were determined by microarray, real-time PCR, immunofluorescent confocal microscopy and Western blot analysis. RESULTS The results of microarray analysis and real-time PCR revealed that NFκB pathway-related genes and apoptosis-related genes were down-regulated in CL-treated HEK 293 cells. In addition, immunofluorescent confocal microscopy and Western blot analysis revealed that NFκB p65 nuclear translocation was inhibited in CL-treated HEK 293 cells. Therefore, the mechanism responsible for the effects of CL on HEK 293 cells is closely associated with regulation of the NFκB pathway. CONCLUSION CL possesses novel therapeutic agents that can be used for the prevention or treatment of cisplatin-induced renal disorders. PMID:20840446

A Series of Zn(II) Terpyridine-Based Nitrate Complexes as Two-Photon Fluorescent Probe for Identifying Apoptotic and Living Cells via Subcellular Immigration.

PubMed

Liu, Dandan; Zhang, Mingzhu; Du, Wei; Hu, Lei; Li, Fei; Tian, Xiaohe; Wang, Aidong; Zhang, Qiong; Zhang, Zhongping; Wu, Jieying; Tian, Yupeng

2018-06-19

Two-photon active probe to label apoptotic cells plays a significant role in biological systems. However, discrimination of live/apoptotic cells at subcellular level under microscopy remains unachieved. Here, three novel Zn(II) terpyridine-based nitrate complexes (C1-C3) containing different pull/push units were designed. The structures of the ligands and their corresponding Zn(II) complexes were confirmed by single-crystal X-ray diffraction analysis. On the basis of the comprehensive comparison, C3 had a suitable two-photon absorption cross section in the near-infrared wavelength and good biocompatibility. Under two-photon confocal microscopy and transmission electron microscopy, it is found that C3 could target mitochondria in living cells but immigrate into the nucleolus during the apoptotic process. This dual-functional probe (C3) not only offers a valuable image tool but also acts as an indicator for cell mortality at subcellular level in a real-time manner.

TLM-Quant: an open-source pipeline for visualization and quantification of gene expression heterogeneity in growing microbial cells.

PubMed

Piersma, Sjouke; Denham, Emma L; Drulhe, Samuel; Tonk, Rudi H J; Schwikowski, Benno; van Dijl, Jan Maarten

2013-01-01

Gene expression heterogeneity is a key driver for microbial adaptation to fluctuating environmental conditions, cell differentiation and the evolution of species. This phenomenon has therefore enormous implications, not only for life in general, but also for biotechnological applications where unwanted subpopulations of non-producing cells can emerge in large-scale fermentations. Only time-lapse fluorescence microscopy allows real-time measurements of gene expression heterogeneity. A major limitation in the analysis of time-lapse microscopy data is the lack of fast, cost-effective, open, simple and adaptable protocols. Here we describe TLM-Quant, a semi-automatic pipeline for the analysis of time-lapse fluorescence microscopy data that enables the user to visualize and quantify gene expression heterogeneity. Importantly, our pipeline builds on the open-source packages ImageJ and R. To validate TLM-Quant, we selected three possible scenarios, namely homogeneous expression, highly 'noisy' heterogeneous expression, and bistable heterogeneous expression in the Gram-positive bacterium Bacillus subtilis. This bacterium is both a paradigm for systems-level studies on gene expression and a highly appreciated biotechnological 'cell factory'. We conclude that the temporal resolution of such analyses with TLM-Quant is only limited by the numbers of recorded images.

Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.

PubMed

Reipert, S; Reipert, B M; Allen, T D

1994-09-01

The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scanning electron microscope.

Automated fluorescent miscroscopic image analysis of PTBP1 expression in glioma

PubMed Central

Becker, Aline; Elder, Brad; Puduvalli, Vinay; Winter, Jessica; Gurcan, Metin

2017-01-01

Multiplexed immunofluorescent testing has not entered into diagnostic neuropathology due to the presence of several technical barriers, amongst which includes autofluorescence. This study presents the implementation of a methodology capable of overcoming the visual challenges of fluorescent microscopy for diagnostic neuropathology by using automated digital image analysis, with long term goal of providing unbiased quantitative analyses of multiplexed biomarkers for solid tissue neuropathology. In this study, we validated PTBP1, a putative biomarker for glioma, and tested the extent to which immunofluorescent microscopy combined with automated and unbiased image analysis would permit the utility of PTBP1 as a biomarker to distinguish diagnostically challenging surgical biopsies. As a paradigm, we utilized second resections from patients diagnosed either with reactive brain changes (pseudoprogression) and recurrent glioblastoma (true progression). Our image analysis workflow was capable of removing background autofluorescence and permitted quantification of DAPI-PTBP1 positive cells. PTBP1-positive nuclei, and the mean intensity value of PTBP1 signal in cells. Traditional pathological interpretation was unable to distinguish between groups due to unacceptably high discordance rates amongst expert neuropathologists. Our data demonstrated that recurrent glioblastoma showed more DAPI-PTBP1 positive cells and a higher mean intensity value of PTBP1 signal compared to resections from second surgeries that showed only reactive gliosis. Our work demonstrates the potential of utilizing automated image analysis to overcome the challenges of implementing fluorescent microscopy in diagnostic neuropathology. PMID:28282372

Automated processing of label-free Raman microscope images of macrophage cells with standardized regression for high-throughput analysis.

PubMed

Milewski, Robert J; Kumagai, Yutaro; Fujita, Katsumasa; Standley, Daron M; Smith, Nicholas I

2010-11-19

Macrophages represent the front lines of our immune system; they recognize and engulf pathogens or foreign particles thus initiating the immune response. Imaging macrophages presents unique challenges, as most optical techniques require labeling or staining of the cellular compartments in order to resolve organelles, and such stains or labels have the potential to perturb the cell, particularly in cases where incomplete information exists regarding the precise cellular reaction under observation. Label-free imaging techniques such as Raman microscopy are thus valuable tools for studying the transformations that occur in immune cells upon activation, both on the molecular and organelle levels. Due to extremely low signal levels, however, Raman microscopy requires sophisticated image processing techniques for noise reduction and signal extraction. To date, efficient, automated algorithms for resolving sub-cellular features in noisy, multi-dimensional image sets have not been explored extensively. We show that hybrid z-score normalization and standard regression (Z-LSR) can highlight the spectral differences within the cell and provide image contrast dependent on spectral content. In contrast to typical Raman imaging processing methods using multivariate analysis, such as single value decomposition (SVD), our implementation of the Z-LSR method can operate nearly in real-time. In spite of its computational simplicity, Z-LSR can automatically remove background and bias in the signal, improve the resolution of spatially distributed spectral differences and enable sub-cellular features to be resolved in Raman microscopy images of mouse macrophage cells. Significantly, the Z-LSR processed images automatically exhibited subcellular architectures whereas SVD, in general, requires human assistance in selecting the components of interest. The computational efficiency of Z-LSR enables automated resolution of sub-cellular features in large Raman microscopy data sets without compromise in image quality or information loss in associated spectra. These results motivate further use of label free microscopy techniques in real-time imaging of live immune cells.

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

PubMed Central

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

2018-01-01

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

Repeatability and reproducibility of intracellular molar concentration assessed by synchrotron-based x-ray fluorescence microscopy

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

Merolle, L., E-mail: lucia.merolle@elettra.eu; Gianoncelli, A.; Malucelli, E., E-mail: emil.malucelli@unibo.it

2016-01-28

Elemental analysis of biological sample can give information about content and distribution of elements essential for human life or trace elements whose absence is the cause of abnormal biological function or development. However, biological systems contain an ensemble of cells with heterogeneous chemistry and elemental content; therefore, accurate characterization of samples with high cellular heterogeneity may only be achieved by analyzing single cells. Powerful methods in molecular biology are abundant, among them X-Ray microscopy based on synchrotron light source has gaining increasing attention thanks to its extremely sensitivity. However, reproducibility and repeatability of these measurements is one of the majormore » obstacles in achieving a statistical significance in single cells population analysis. In this study, we compared the elemental content of human colon adenocarcinoma cells obtained by three distinct accesses to synchrotron radiation light.« less

Analysis of effect of nanoporous alumina substrate coated with polypyrrole nanowire on cell morphology based on AFM topography.

PubMed

El-Said, Waleed Ahmed; Yea, Cheol-Heon; Jung, Mi; Kim, Hyuncheol; Choi, Jeong-Woo

2010-05-01

In this study, in situ electrochemical synthesis of polypyrrole nanowires with nanoporous alumina template was described. The formation of highly ordered porous alumina substrate was demonstrated with Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM). In addition, Fourier transform infrared analysis confirmed that polypyrrole (PP) nanowires were synthesized by direct electrochemical oxidation of pyrrole. HeLa cancer cells and HMCF normal cells were immobilized on the polypyrrole nanowires/nanoporous alumina substrates to determine the effects of the substrate on the cell morphology, adhesion and proliferation as well as the biocompatibility of the substrate. Cell adhesion and proliferation were characterized using a standard MTT assay. The effects of the polypyrrole nanowires/nanoporous alumina substrate on the cell morphology were studied by AFM. The nanoporous alumina coated with polypyrrole nanowires was found to exhibit better cell adhesion and proliferation than polystyrene petridish, aluminum foil, 1st anodized and uncoated 2nd anodized alumina substrate. This study showed the potential of the polypyrrole nanowires/nanoporous alumina substrate as biocompatibility electroactive polymer substrate for both healthy and cancer cell cultures applications.

Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D)

PubMed Central

Li, Weizhe; Germain, Ronald N.

2017-01-01

Organ homeostasis, cellular differentiation, signal relay, and in situ function all depend on the spatial organization of cells in complex tissues. For this reason, comprehensive, high-resolution mapping of cell positioning, phenotypic identity, and functional state in the context of macroscale tissue structure is critical to a deeper understanding of diverse biological processes. Here we report an easy to use method, clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency for most organs, preserves cellular morphology and protein fluorescence, and is robustly compatible with antibody-based immunolabeling. This enhanced signal quality and capacity for extensive probe multiplexing permits quantitative analysis of distinct, highly intermixed cell populations in intact Ce3D-treated tissues via 3D histo-cytometry. We use this technology to demonstrate large-volume, high-resolution microscopy of diverse cell types in lymphoid and nonlymphoid organs, as well as to perform quantitative analysis of the composition and tissue distribution of multiple cell populations in lymphoid tissues. Combined with histo-cytometry, Ce3D provides a comprehensive strategy for volumetric quantitative imaging and analysis that bridges the gap between conventional section imaging and disassociation-based techniques. PMID:28808033

Analysis of mitosis and antimitotic drug responses in tumors by in vivo microscopy and single-cell pharmacodynamics.

PubMed

Orth, James D; Kohler, Rainer H; Foijer, Floris; Sorger, Peter K; Weissleder, Ralph; Mitchison, Timothy J

2011-07-01

Cancer relies upon frequent or abnormal cell division, but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the antimitotic drug paclitaxel (Ptx) was the same in tumors as in cell culture, observed the impact of Ptx on the tumor response as a whole, and evaluated the single-cell pharmacodynamics (PD) of Ptx (by in vivo PD microscopy). Mitotic initiation was generally less frequent in tumors than in cell culture, but subsequently it proceeded normally. Ptx treatment caused spindle assembly defects and mitotic arrest, followed by slippage from mitotic arrest, multinucleation, and apoptosis. Compared with cell culture, the peak mitotic index in tumors exposed to Ptx was lower and the tumor cells survived longer after mitotic arrest, becoming multinucleated rather than dying directly from mitotic arrest. Thus, the tumor microenvironment was much less proapoptotic than cell culture. The morphologies associated with mitotic arrest were dose and time dependent, thereby providing a semiquantitative, single-cell measure of PD. Although many tumor cells did not progress through Ptx-induced mitotic arrest, tumor significantly regressed in the model. Our findings show that in vivo microscopy offers a useful tool to visualize mitosis during tumor progression, drug responses, and cell fate at the single-cell level. ©2011 AACR.

Passive microrheology of normal and cancer cells after ML7 treatment by atomic force microscopy

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

Lyapunova, Elena, E-mail: lyapunova@icmm.ru; Ural Federal University, Kuibyishev Str. 48, Ekaterinburg, 620000; Nikituk, Alexander, E-mail: nas@icmm.ru

Mechanical properties of living cancer and normal thyroidal cells were investigated by atomic force microscopy (AFM). Cell mechanics was compared before and after treatment with ML7, which is known to reduce myosin activity and induce softening of cell structures. We recorded force curves with extended dwell time of 6 seconds in contact at maximum forces from 500 pN to 1 nN. Data were analyzed within different frameworks: Hertz fit was applied in order to evaluate differences in Young’s moduli among cell types and conditions, while the fluctuations of the cantilever in contact with cells were analyzed with both conventional algorithmsmore » (probability density function and power spectral density) and multifractal detrended fluctuation analysis (MF-DFA). We found that cancer cells were softer than normal cells and ML7 had a substantial softening effect on normal cells, but only a marginal one on cancer cells. Moreover, we observed that all recorded signals for normal and cancer cells were monofractal with small differences between their scaling parameters. Finally, the applicability of wavelet-based methods of data analysis for the discrimination of different cell types is discussed.« less

Automated microscopy for high-content RNAi screening

PubMed Central

2010-01-01

Fluorescence microscopy is one of the most powerful tools to investigate complex cellular processes such as cell division, cell motility, or intracellular trafficking. The availability of RNA interference (RNAi) technology and automated microscopy has opened the possibility to perform cellular imaging in functional genomics and other large-scale applications. Although imaging often dramatically increases the content of a screening assay, it poses new challenges to achieve accurate quantitative annotation and therefore needs to be carefully adjusted to the specific needs of individual screening applications. In this review, we discuss principles of assay design, large-scale RNAi, microscope automation, and computational data analysis. We highlight strategies for imaging-based RNAi screening adapted to different library and assay designs. PMID:20176920

Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ.

PubMed

Bharat, Tanmay A M; Hoffmann, Patrick C; Kukulski, Wanda

2018-04-10

Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-fluorescence microscopy with electron cryo-tomography of vitreous sections into a coherent correlative microscopy workflow, ideal for detection and structural analysis of elusive protein assemblies in situ. We used this workflow to address an open question on BAR-domain coating of yeast plasma membrane compartments known as eisosomes. BAR domains can sense or induce membrane curvature, and form scaffold-like membrane coats in vitro. Our results demonstrate that in cells, the BAR protein Pil1 localizes to eisosomes of varying membrane curvature. Sub-tomogram analysis revealed a dense protein coat on curved eisosomes, which was not present on shallow eisosomes, indicating that while BAR domains can assemble at shallow membranes in vivo, scaffold formation is tightly coupled to curvature generation. Copyright © 2018 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd.. All rights reserved.

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

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.

Live morphological analysis of taxol-induced cytoplasmic vacuolization [corrected] in human lung adenocarcinoma cells.

PubMed

Wang, Xiao-Ping; Chen, Tong-Sheng; Sun, Lei; Cai, Ji-Ye; Wu, Ming-Qian; Mok, Martin

2008-12-01

Taxol (paclitaxel), one of the most active cancer chemotherapeutic agents, can cause programmed cell death (PCD) and cytoplasmic vacuolization. The objective of this study was to analyze the morphological characteristics induced by taxol. Human lung adenocarcinoma (ASTC-a-1) cells were exposed to various concentration of taxol. CCK-8 was used to assay the cell viability. Atomic force microscopy (AFM), plasmid transfection and confocal fluorescence microscopy were performed to image the cells morphological change induced by taxol. Fluorescence resonance energy transfer (FRET) was used to monitor the caspase-3 activation in living cells during taxol-induced cell death. Cells treated with taxol exhibited significant swelling and cytoplasmic vacuolization which may be due to endoplasmic reticulum (ER) vacuolization. Caspase-3 was not activated during taxol-induced cytoplasmic vacuolization and cell death. These findings suggest that taxol induces caspase-3-independent cytoplasmic vacuolization, cell swelling and cell death through ER vacuolization.

Intravital Microscopic Interrogation of Peripheral Taste Sensation

NASA Astrophysics Data System (ADS)

Choi, Myunghwan; Lee, Woei Ming; Yun, Seok Hyun

2015-03-01

Intravital microscopy is a powerful tool in neuroscience but has not been adapted to the taste sensory organ due to anatomical constraint. Here we developed an imaging window to facilitate microscopic access to the murine tongue in vivo. Real-time two-photon microscopy allowed the visualization of three-dimensional microanatomy of the intact tongue mucosa and functional activity of taste cells in response to topically administered tastants in live mice. Video microscopy also showed the calcium activity of taste cells elicited by small-sized tastants in the blood circulation. Molecular kinetic analysis suggested that intravascular taste sensation takes place at the microvilli on the apical side of taste cells after diffusion of the molecules through the pericellular capillaries and tight junctions in the taste bud. Our results demonstrate the capabilities and utilities of the new tool for taste research in vivo.

Intravital microscopic interrogation of peripheral taste sensation.

PubMed

Choi, Myunghwan; Lee, Woei Ming; Yun, Seok Hyun

2015-03-02

Intravital microscopy is a powerful tool in neuroscience but has not been adapted to the taste sensory organ due to anatomical constraint. Here we developed an imaging window to facilitate microscopic access to the murine tongue in vivo. Real-time two-photon microscopy allowed the visualization of three-dimensional microanatomy of the intact tongue mucosa and functional activity of taste cells in response to topically administered tastants in live mice. Video microscopy also showed the calcium activity of taste cells elicited by small-sized tastants in the blood circulation. Molecular kinetic analysis suggested that intravascular taste sensation takes place at the microvilli on the apical side of taste cells after diffusion of the molecules through the pericellular capillaries and tight junctions in the taste bud. Our results demonstrate the capabilities and utilities of the new tool for taste research in vivo.

Helium ion microscopy and ultra-high-resolution scanning electron microscopy analysis of membrane-extracted cells reveals novel characteristics of the cytoskeleton of Giardia intestinalis.

PubMed

Gadelha, Ana Paula Rocha; Benchimol, Marlene; de Souza, Wanderley

2015-06-01

Giardia intestinalis presents a complex microtubular cytoskeleton formed by specialized structures, such as the adhesive disk, four pairs of flagella, the funis and the median body. The ultrastructural organization of the Giardia cytoskeleton has been analyzed using different microscopic techniques, including high-resolution scanning electron microscopy. Recent advances in scanning microscopy technology have opened a new venue for the characterization of cellular structures and include scanning probe microscopy techniques such as ultra-high-resolution scanning electron microscopy (UHRSEM) and helium ion microscopy (HIM). Here, we studied the organization of the cytoskeleton of G. intestinalis trophozoites using UHRSEM and HIM in membrane-extracted cells. The results revealed a number of new cytoskeletal elements associated with the lateral crest and the dorsal surface of the parasite. The fine structure of the banded collar was also observed. The marginal plates were seen linked to a network of filaments, which were continuous with filaments parallel to the main cell axis. Cytoplasmic filaments that supported the internal structures were seen by the first time. Using anti-actin antibody, we observed a labeling in these filamentous structures. Taken together, these data revealed new surface characteristics of the cytoskeleton of G. intestinalis and may contribute to an improved understanding of the structural organization of trophozoites. Copyright © 2015 Elsevier Inc. All rights reserved.

Contribution of high-resolution correlative imaging techniques in the study of the liver sieve in three-dimensions.

PubMed

Braet, Filip; Wisse, Eddie; Bomans, Paul; Frederik, Peter; Geerts, Willie; Koster, Abraham; Soon, Lilian; Ringer, Simon

2007-03-01

Correlative microscopy has become increasingly important for the analysis of the structure, function, and dynamics of cells. This is largely due to the result of recent advances in light-, probe-, laser- and various electron microscopy techniques that facilitate three-dimensional studies. Furthermore, the improved understanding in the past decade of imaging cell compartments in the third dimension has resulted largely from the availability of powerful computers, fast high-resolution CCD cameras, specifically developed imaging analysis software, and various probes designed for labeling living and or fixed cells. In this paper, we review different correlative high-resolution imaging methodologies and how these microscopy techniques facilitated the accumulation of new insights in the morpho-functional and structural organization of the hepatic sieve. Various aspects of hepatic endothelial fenestrae regarding their structure, origin, dynamics, and formation will be explored throughout this paper by comparing the results of confocal laser scanning-, correlative fluorescence and scanning electron-, atomic force-, and whole-mount electron microscopy. Furthermore, the recent advances of vitrifying cells with the vitrobot in combination with the glove box for the preparation of cells for cryo-electron microscopic investigation will be discussed. Finally, the first transmission electron tomography data of the liver sieve in three-dimensions are presented. The obtained data unambiguously show the involvement of special domains in the de novo formation and disappearance of hepatic fenestrae, and focuses future research into the (supra)molecular structure of the fenestrae-forming center, defenestration center and fenestrae-, and sieve plate cytoskeleton ring by using advanced cryo-electron tomography. (c) 2007 Wiley-Liss, Inc.

Optofluidic Cell Selection from Complex Microbial Communities for Single-Genome Analysis

PubMed Central

Landry, Zachary C.; Giovanonni, Stephen J.; Quake, Stephen R.; Blainey, Paul C.

2013-01-01

Genetic analysis of single cells is emerging as a powerful approach for studies of heterogeneous cell populations. Indeed, the notion of homogeneous cell populations is receding as approaches to resolve genetic and phenotypic variation between single cells are applied throughout the life sciences. A key step in single-cell genomic analysis today is the physical isolation of individual cells from heterogeneous populations, particularly microbial populations, which often exhibit high diversity. Here, we detail the construction and use of instrumentation for optical trapping inside microfluidic devices to select individual cells for analysis by methods including nucleic acid sequencing. This approach has unique advantages for analyses of rare community members, cells with irregular morphologies, small quantity samples, and studies that employ advanced optical microscopy. PMID:24060116

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.

Voltage controlled nano-injection system for single-cell surgery

PubMed Central

Seger, R. Adam; Actis, Paolo; Penfold, Catherine; Maalouf, Michelle; Vilozny, Boaz; Pourmand, Nader

2015-01-01

Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes. PMID:22899383

Voltage controlled nano-injection system for single-cell surgery.

PubMed

Adam Seger, R; Actis, Paolo; Penfold, Catherine; Maalouf, Michelle; Vilozny, Boaz; Pourmand, Nader

2012-09-28

Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes.

Depth-resolved cellular microrheology using HiLo microscopy.

PubMed

Michaelson, Jarett; Choi, Heejin; So, Peter; Huang, Hayden

2012-06-01

It is increasingly important to measure cell mechanical properties in three-dimensional environments. Particle tracking microrheology (PTM) can measure cellular viscoelastic properties; however, out-of-plane data can introduce artifacts into these measurements. We developed a technique that employs HiLo microscopy to reduce out-of-plane contributions. This method eliminated signals from 90% of probes 0.5 μm or further from the focal plane, while retaining all in-plane probes. We used this technique to characterize live-cell bilayers and found that there were significant, frequency-dependent changes to the extracted cell moduli when compared to conventional analysis. Our results indicate that removal of out-of-plane information is vital for accurate assessments of cell mechanical properties.

Comparison of flow cytometry, fluorescence microscopy and spectrofluorometry for analysis of gene electrotransfer efficiency.

PubMed

Marjanovič, Igor; Kandušer, Maša; Miklavčič, Damijan; Keber, Mateja Manček; Pavlin, Mojca

2014-12-01

In this study, we compared three different methods used for quantification of gene electrotransfer efficiency: fluorescence microscopy, flow cytometry and spectrofluorometry. We used CHO and B16 cells in a suspension and plasmid coding for GFP. The aim of this study was to compare and analyse the results obtained by fluorescence microscopy, flow cytometry and spectrofluorometry and in addition to analyse the applicability of spectrofluorometry for quantifying gene electrotransfer on cells in a suspension. Our results show that all the three methods detected similar critical electric field strength, around 0.55 kV/cm for both cell lines. Moreover, results obtained on CHO cells showed that the total fluorescence intensity and percentage of transfection exhibit similar increase in response to increase electric field strength for all the three methods. For B16 cells, there was a good correlation at low electric field strengths, but at high field strengths, flow cytometer results deviated from results obtained by fluorescence microscope and spectrofluorometer. Our study showed that all the three methods detected similar critical electric field strengths and high correlations of results were obtained except for B16 cells at high electric field strengths. The results also demonstrated that flow cytometry measures higher values of percentage transfection compared to microscopy. Furthermore, we have demonstrated that spectrofluorometry can be used as a simple and consistent method to determine gene electrotransfer efficiency on cells in a suspension.

Nonlinear dynamic phase contrast microscopy for microfluidic and microbiological applications

NASA Astrophysics Data System (ADS)

Denz, C.; Holtmann, F.; Woerdemann, M.; Oevermann, M.

2008-08-01

In live sciences, the observation and analysis of moving living cells, molecular motors or motion of micro- and nano-objects is a current field of research. At the same time, microfluidic innovations are needed for biological and medical applications on a micro- and nano-scale. Conventional microscopy techniques are reaching considerable limits with respect to these issues. A promising approach for this challenge is nonlinear dynamic phase contrast microscopy. It is an alternative full field approach that allows to detect motion as well as phase changes of living unstained micro-objects in real-time, thereby being marker free, without contact and non destructive, i.e. fully biocompatible. The generality of this system allows it to be combined with several other microscope techniques such as conventional bright field or fluorescence microscopy. In this article we will present the dynamic phase contrast technique and its applications in analysis of micro organismic dynamics, micro flow velocimetry and micro-mixing analysis.

Automatic detection and analysis of cell motility in phase-contrast time-lapse images using a combination of maximally stable extremal regions and Kalman filter approaches.

PubMed

Kaakinen, M; Huttunen, S; Paavolainen, L; Marjomäki, V; Heikkilä, J; Eklund, L

2014-01-01

Phase-contrast illumination is simple and most commonly used microscopic method to observe nonstained living cells. Automatic cell segmentation and motion analysis provide tools to analyze single cell motility in large cell populations. However, the challenge is to find a sophisticated method that is sufficiently accurate to generate reliable results, robust to function under the wide range of illumination conditions encountered in phase-contrast microscopy, and also computationally light for efficient analysis of large number of cells and image frames. To develop better automatic tools for analysis of low magnification phase-contrast images in time-lapse cell migration movies, we investigated the performance of cell segmentation method that is based on the intrinsic properties of maximally stable extremal regions (MSER). MSER was found to be reliable and effective in a wide range of experimental conditions. When compared to the commonly used segmentation approaches, MSER required negligible preoptimization steps thus dramatically reducing the computation time. To analyze cell migration characteristics in time-lapse movies, the MSER-based automatic cell detection was accompanied by a Kalman filter multiobject tracker that efficiently tracked individual cells even in confluent cell populations. This allowed quantitative cell motion analysis resulting in accurate measurements of the migration magnitude and direction of individual cells, as well as characteristics of collective migration of cell groups. Our results demonstrate that MSER accompanied by temporal data association is a powerful tool for accurate and reliable analysis of the dynamic behaviour of cells in phase-contrast image sequences. These techniques tolerate varying and nonoptimal imaging conditions and due to their relatively light computational requirements they should help to resolve problems in computationally demanding and often time-consuming large-scale dynamical analysis of cultured cells. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

Nanovesicles released by Dictyostelium cells: a potential carrier for drug delivery.

PubMed

Lavialle, Françoise; Deshayes, Sophie; Gonnet, Florence; Larquet, Eric; Kruglik, Sergei G; Boisset, Nicolas; Daniel, Régis; Alfsen, Annette; Tatischeff, Irène

2009-10-01

Nanovesicles released by Dictyostelium discoideum cells grown in the presence of the DNA-specific dye Hoechst 33342 have been previously shown to mediate the transfer of the dye into the nuclei of Hoechst-resistant cells. The present investigation extends this work by conducting experiments in the presence of hypericin, a fluorescent therapeutic photosensitizer assayed for antitumoral photodynamic therapy. Nanovesicles released by Dictyostelium cells exhibit an averaged diameter between 50 and 150 nm, as measured by transmission cryoelectron microscopy. A proteomic analysis reveals a predominance of actin and actin-related proteins. The detection of a lysosomal membrane protein (LIMP II) indicates that these vesicles are likely generated in the late endosomal compartment. The use of the hypericin-containing nanovesicles as nanodevices for in vitro drug delivery was investigated by fluorescence microscopy. The observed signal was almost exclusively located in the perinuclear area of two human cell lines, skin fibroblasts (HS68) and cervix carcinoma (HeLa) cells. Studies by confocal microscopy with specific markers of cell organelles, provided evidence that hypericin was accumulated in the Golgi apparatus. All these data shed a new light on in vitro drug delivery by using cell-released vesicles as carriers.

Systems microscopy: an emerging strategy for the life sciences.

PubMed

Lock, John G; Strömblad, Staffan

2010-05-01

Dynamic cellular processes occurring in time and space are fundamental to all physiology and disease. To understand complex and dynamic cellular processes therefore demands the capacity to record and integrate quantitative multiparametric data from the four spatiotemporal dimensions within which living cells self-organize, and to subsequently use these data for the mathematical modeling of cellular systems. To this end, a raft of complementary developments in automated fluorescence microscopy, cell microarray platforms, quantitative image analysis and data mining, combined with multivariate statistics and computational modeling, now coalesce to produce a new research strategy, "systems microscopy", which facilitates systems biology analyses of living cells. Systems microscopy provides the crucial capacities to simultaneously extract and interrogate multiparametric quantitative data at resolution levels ranging from the molecular to the cellular, thereby elucidating a more comprehensive and richly integrated understanding of complex and dynamic cellular systems. The unique capacities of systems microscopy suggest that it will become a vital cornerstone of systems biology, and here we describe the current status and future prospects of this emerging field, as well as outlining some of the key challenges that remain to be overcome. Copyright 2010 Elsevier Inc. All rights reserved.

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

Analyzing the dynamics of cell cycle processes from fixed samples through ergodic principles.

PubMed

Wheeler, Richard John

2015-11-05

Tools to analyze cyclical cellular processes, particularly the cell cycle, are of broad value for cell biology. Cell cycle synchronization and live-cell time-lapse observation are widely used to analyze these processes but are not available for many systems. Simple mathematical methods built on the ergodic principle are a well-established, widely applicable, and powerful alternative analysis approach, although they are less widely used. These methods extract data about the dynamics of a cyclical process from a single time-point "snapshot" of a population of cells progressing through the cycle asynchronously. Here, I demonstrate application of these simple mathematical methods to analysis of basic cyclical processes--cycles including a division event, cell populations undergoing unicellular aging, and cell cycles with multiple fission (schizogony)--as well as recent advances that allow detailed mapping of the cell cycle from continuously changing properties of the cell such as size and DNA content. This includes examples using existing data from mammalian, yeast, and unicellular eukaryotic parasite cell biology. Through the ongoing advances in high-throughput cell analysis by light microscopy, electron microscopy, and flow cytometry, these mathematical methods are becoming ever more important and are a powerful complementary method to traditional synchronization and time-lapse cell cycle analysis methods. © 2015 Wheeler. 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).

Crystals seen on CSF microscopy in a case of suspected subarachnoid haemorrhage

PubMed Central

Weiand, Daniel; Hanning, Ian; Mouhamadou, Moussa; Wearmouth, Debbie

2015-01-01

Although crystals are rarely identified on cerebrospinal fluid (CSF) microscopy, their presence can be of significant diagnostic value. We report a case of oxalate crystals seen on CSF microscopy of a 43-year-old woman. The patient presented with headaches, nausea and vomiting. CT of the head showed a small focus of hyper-density, suspicious of haemorrhage, in the right side of the pontine cistern. CSF cell count was within the normal range. Although no organisms were seen on microscopy, copious oxalate crystals were seen. The same crystals were seen on microscopy of CSF collected in a fluoride oxalate container used for glucose analysis. A follow-up contrast-enhanced CT angiogram did not demonstrate any abnormalities. It transpired that excess CSF had been collected into a fluoride oxalate container. This had subsequently been decanted into a plain container for microbiological analysis. Correct specimen collection should be emphasised when teaching lumbar puncture technique. PMID:26139652

Towards native-state imaging in biological context in the electron microscope

PubMed Central

Weston, Anne E.; Armer, Hannah E. J.

2009-01-01

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context. PMID:19916039

Micro patterned surfaces: an effective tool for long term digital holographic microscopy cell imaging

NASA Astrophysics Data System (ADS)

Mues, Sarah; Lilge, Inga; Schönherr, Holger; Kemper, Björn; Schnekenburger, Jürgen

2017-02-01

The major problem of Digital Holographic Microscopy (DHM) long term live cell imaging is that over time most of the tracked cells move out of the image area and other ones move in. Therefore, most of the cells are lost for the evaluation of individual cellular processes. Here, we present an effective solution for this crucial problem of long-term microscopic live cell analysis. We have generated functionalized slides containing areas of 250 μm per 200 μm. These micropatterned biointerfaces consist of passivating polyaclrylamide brushes (PAAm). Inner areas are backfilled with octadecanthiol (ODT), which allows cell attachment. The fouling properties of these surfaces are highly controllable and therefore the defined areas designed for the size our microscopic image areas were effective in keeping all cells inside the rectangles over the selected imaging period.

Midbody Accumulation in Breast Cancer Stem Cells

DTIC Science & Technology

2011-08-01

transit amplifying or differentiating cells. These results suggest that MBds are in almost exclusively in stem cells and putative breast cancer stem...confer tumor-like properties to these cells. We were unable to establish GFP-MKLP1 breast cancer cell lines for this analysis for some reason that we...and nonpolarized cells (Fig. 1c, d). Immuno- electron microscopy confirmed this localization and revealed ultrastructural features characteristic of

Fungus-mediated biological synthesis of gold nanoparticles: potential in detection of liver cancer

PubMed Central

Chauhan, Arun; Zubair, Swaleha; Tufail, Saba; Sherwani, Asif; Sajid, Mohammad; Raman, Suri C; Azam, Amir; Owais, Mohammad

2011-01-01

Background Nanomaterials are considered to be the pre-eminent component of the rapidly advancing field of nanotechnology. However, developments in the biologically inspired synthesis of nanoparticles are still in their infancy and consequently attracting the attention of material scientists throughout the world. Keeping in mind the fact that microorganism-assisted synthesis of nanoparticles is a safe and economically viable prospect, in the current study we report Candida albicans-mediated biological synthesis of gold nanoparticles. Methods and results Transmission electron microscopy, atomic force microscopy, and various spectrophotometric analyses were performed to characterize the gold nanoparticles. The morphology of the synthesized gold particles depended on the abundance of C. albicans cytosolic extract. Transmission electron microscopy, nanophox particle analysis, and atomic force microscopy revealed the size of spherical gold nanoparticles to be in the range of 20–40 nm and nonspherical gold particles were found to be 60–80 nm. We also evaluated the potential of biogenic gold nanoparticles to probe liver cancer cells by conjugating them with liver cancer cell surface-specific antibodies. The antibody-conjugated gold particles were found to bind specifically to the surface antigens of the cancer cells. Conclusion The antibody-conjugated gold particles synthesized in this study could successfully differentiate normal cell populations from cancerous cells. PMID:22072868

Analysis of incomplete excisions of basal-cell carcinomas after breadloaf microscopy compared with 3D-microscopy: a prospective randomized and blinded study.

PubMed

Boehringer, Alexandra; Adam, Patrick; Schnabl, Saskia; Häfner, Hans-Martin; Breuninger, Helmut

2015-08-01

Basal-cell carcinomas may show irregular, asymmetric subclinical growth. This study analyzed the efficacy of 'breadloaf' microscopy (serial sectioning) and three-dimensional (3D) microscopy in detecting positive tumor margins. Two hundred eighty-three (283) tumors (51.2%) were put into the breadloaf microscopy group; 270 tumors (48.8%) into the 3D microscopy group. The position of any detected tumor outgrowths was identified in clock face fashion. The time required for cutting and embedding the specimens and the examination of the microscopic slides was measured. Patient/tumor characteristics and surgical margins did not differ significantly. Tumor outgrowths at the excision margin were found in 62 of 283 cases (21.9%) in the breadloaf microscopy group and in 115 of 270 cases (42.6%) in the 3D microscopy group, constituting a highly significant difference (p < 0.001). This difference held true with incomplete excision of fibrosing (infiltrative/sclerosing/morpheaform) tumors [32.9% in the breadloaf microscopy group and 57.5% in the 3D microscopy group (p = 0.003)] and also with solid (nodular) tumors [16.1 and 34.2%, respectively (p < 0.001)]. The mean overall examination time required showed no important difference. In summary, for detection of tumor outgrowths, 3D microscopy has almost twice the sensitivity of breadloaf microscopy, particularly in the situation of aggressive/infiltrative carcinomas. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Bacteria on Urine Microscopy Is Not Associated with Systemic Infection in Patients with Obstructing Urolithiasis.

PubMed

Cheung, Felix; Loeb, Charles A; Croglio, Michael P; Waltzer, Wayne C; Weissbart, Steven J

2017-09-01

Determining whether bacterial presence in urine microscopy represents infection is important as ureteral stent placement is indicated in patients with obstructing urolithiasis and infection. We aim to investigate whether the presence of bacteria on urine microscopy is associated with other markers of infection in patients with obstructing urolithiasis presenting to the emergency room. We performed a cross-sectional study of 199 patients with obstructing urolithiasis and divided patients into two groups according to the presence of bacteria on urine microscopy. The primary outcome was serum white blood cell count and secondary outcomes were objective fever, subjective fever, tachycardia, pyuria, and final urine culture. Univariate and multivariate analysis were used to assess whether the presence of bacteria on microscopy was associated with other markers of infection. The study included 72 patients in the bacteriuria group and 127 without bacteriuria. On univariate analysis, the presence of bacteria was not associated with leukocytosis, objective fever, or subjective fever, but it was associated with gender (p < 0.001), pyuria (p < 0.001), positive nitrites (p = 0.001), positive leukocyte esterase (p < 0.001), and squamous epithelial cells (p = 0.002). In a multilinear regression model including the presence of squamous cells, age, and sex, the presence of bacteriuria was not related to serum white blood cell count (coefficient -0.47; 95% confidence interval [CI] -1.1, 0.2; p = 0.17), heart rate (coefficient 0.85; 95% CI -2.5, 4.2; p = 0.62), presence of subjective or objective fever (odds ratio [OR] 1.5; 95% CI 0.8, 3.1; p = 0.18), or the presence of squamous epithelial cells (coefficient -4.4; 95% CI -10, 1.2; p = 0.12). However, the presence of bacteriuria was related to only the degree of pyuria (coefficient 16.4; 95% CI 9.6, 23.3; p < 0.001). Bacteria on urine microscopy is not associated with other markers of systemic infection and may largely represent a contaminant. Renal colic may be a risk factor for providing a contaminated urine specimen.

Separating grain-boundary and bulk recombination with time-resolved photoluminescence microscopy

DOE PAGES

Kuciauskas, Darius; Lu, Dingyuan; Grover, Sachit; ...

2017-12-04

Two-photon excitation (2PE) microscopy allows contactless and non-destructive cross-sectional analysis of grain-boundary (GB) and grain-interior (GI) properties in polycrystalline solar cells, with measurements of doping uniformity, space-charge field distribution, and carrier dynamics in different regions of the device. Using 2PE time-resolved microscopy, we analyzed charge-carrier lifetimes near the GBs and in the GI of polycrystalline thin-film CdTe solar cells doped with As. When the grain radius is larger than the minority-carrier diffusion length, GI lifetimes are interpreted as the bulk lifetimes ..tau..B, and GB recombination velocity SGB is extracted by comparing recombination rates in the GI and near GBs. Inmore » As-doped CdTe solar cells, we find ..tau..B = 1.0-2.4 ns and S GB = (1-4) x 10 5 cm/s. The results imply the potential to improve solar cell voltage via GB passivation and reduced recombination center concentration in the GI.« less

Separating grain-boundary and bulk recombination with time-resolved photoluminescence microscopy

NASA Astrophysics Data System (ADS)

Kuciauskas, Darius; Lu, Dingyuan; Grover, Sachit; Xiong, Gang; Gloeckler, Markus

2017-12-01

Two-photon excitation (2PE) microscopy allows contactless and non-destructive cross-sectional analysis of grain-boundary (GB) and grain-interior (GI) properties in polycrystalline solar cells, with measurements of doping uniformity, space-charge field distribution, and carrier dynamics in different regions of the device. Using 2PE time-resolved microscopy, we analyzed charge-carrier lifetimes near the GBs and in the GI of polycrystalline thin-film CdTe solar cells doped with As. When the grain radius is larger than the minority-carrier diffusion length, GI lifetimes are interpreted as the bulk lifetimes τB, and GB recombination velocity SGB is extracted by comparing recombination rates in the GI and near GBs. In As-doped CdTe solar cells, we find τB = 1.0-2.4 ns and SGB = (1-4) × 105 cm/s. The results imply the potential to improve solar cell voltage via GB passivation and reduced recombination center concentration in the GI.

Separating grain-boundary and bulk recombination with time-resolved photoluminescence microscopy

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

Kuciauskas, Darius; Lu, Dingyuan; Grover, Sachit

Two-photon excitation (2PE) microscopy allows contactless and non-destructive cross-sectional analysis of grain-boundary (GB) and grain-interior (GI) properties in polycrystalline solar cells, with measurements of doping uniformity, space-charge field distribution, and carrier dynamics in different regions of the device. Using 2PE time-resolved microscopy, we analyzed charge-carrier lifetimes near the GBs and in the GI of polycrystalline thin-film CdTe solar cells doped with As. When the grain radius is larger than the minority-carrier diffusion length, GI lifetimes are interpreted as the bulk lifetimes ..tau..B, and GB recombination velocity SGB is extracted by comparing recombination rates in the GI and near GBs. Inmore » As-doped CdTe solar cells, we find ..tau..B = 1.0-2.4 ns and S GB = (1-4) x 10 5 cm/s. The results imply the potential to improve solar cell voltage via GB passivation and reduced recombination center concentration in the GI.« less

Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

PubMed

Loussert Fonta, Celine; Leis, Andrew; Mathisen, Cliff; Bouvier, David S; Blanchard, Willy; Volterra, Andrea; Lich, Ben; Humbel, Bruno M

2015-01-01

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Protective effects of edaravone against cisplatin-induced hair cell damage in zebrafish.

PubMed

Hong, Seok Jin; Im, Gi Jung; Chang, Jiwon; Chae, Sung Won; Lee, Seung Hoon; Kwon, Soon Young; Jung, Hak Hyun; Chung, Ah Young; Park, Hae Chul; Choi, June

2013-06-01

Edaravone is known to have a potent free radical scavenging effect. The objective of the present study was to evaluate the effects of edaravone on cisplatin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). Five day post-fertilization zebrafish larvae were exposed to 1000 μM cisplatin and 50 μM, 100 μM, 250 μM, 500 μM, 750 μM, and 1000 μM concentrations of edaravone for 4h. Hair cells within neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed by fluorescence microscopy and confocal microscopy (n=10). Hair cell survival was calculated as a percentage of the hair cells in the control group that were not exposed to cisplatin. Ultrastructural changes were evaluated using scanning electron microscopy and transmission electron microscopy. Edaravone protected cisplatin-induced hair cell loss of neuromasts (edaravone 750 μM: 8.7 ± 1.5 cells, cisplatin 1000 μM only: 3.7 ± 0.9 cells; n=10, p<0.0001) and decreased the Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction. Structures of mitochondria and hair cell within neuromasts in ultrastructural analysis were preserved in zebrafish exposed to 1000 μM cisplatin and 750 μM edaravone for 4h. Edaravone attenuated cisplatin-induced hair cell damage in zebrafish. The results of the current study suggest that cisplatin induces apoptosis, and the apoptotic cell death can be prevented by treatment with edaravone in zebrafish. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis.

PubMed

Sugimoto, K; Williamson, R E; Wasteneys, G O

2000-12-01

This article explores root epidermal cell elongation and its dependence on two structural elements of cells, cortical microtubules and cellulose microfibrils. The recent identification of Arabidopsis morphology mutants with putative cell wall or cytoskeletal defects demands a procedure for examining and comparing wall architecture and microtubule organization patterns in this species. We developed methods to examine cellulose microfibrils by field emission scanning electron microscopy and microtubules by immunofluorescence in essentially intact roots. We were able to compare cellulose microfibril and microtubule alignment patterns at equivalent stages of cell expansion. Field emission scanning electron microscopy revealed that Arabidopsis root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Our analysis showed that microtubules and microfibrils have similar orientation only during the initial phase of elongation growth. Microtubule patterns deviate from a predominantly transverse orientation while cells are still expanding, whereas cellulose microfibrils remain transverse until well after expansion finishes. We also observed microtubule-microfibril alignment discord before cells enter their elongation phase. This study and the new technology it presents provide a starting point for further investigations on the physical properties of cell walls and their mechanisms of assembly.

Endocytosis and interaction of poly (amidoamine) dendrimers with Caco-2 cells.

PubMed

Kitchens, Kelly M; Foraker, Amy B; Kolhatkar, Rohit B; Swaan, Peter W; Ghandehari, Hamidreza

2007-11-01

To investigate the internalization and subcellular trafficking of fluorescently labeled poly (amidoamine) (PAMAM) dendrimers in intestinal cell monolayers. PAMAM dendrimers with positive or negative surface charge were conjugated to fluorescein isothiocyanate (FITC) and visualized for colocalization with endocytosis markers using confocal microscopy. Effect of concentration, generation and charge on the morphology of microvilli was observed using transmission electron microscopy. Both cationic and anionic PAMAM dendrimers internalized within 20 min, and differentially colocalized with endocytosis markers clathrin, EEA-1, and LAMP-1. Transmission electron microscopy analysis showed a concentration-, generation- and surface charge-dependent effect on microvilli morphology. These studies provide visual evidence that endocytic mechanism(s) contribute to the internalization and subcellular trafficking of PAMAM dendrimers across the intestinal cells, and that appropriate selection of PAMAM dendrimers based on surface charge, concentration and generation number allows the application of these polymers for oral drug delivery.

Microfluidic microscopy-assisted label-free approach for cancer screening: automated microfluidic cytology for cancer screening.

PubMed

Jagannadh, Veerendra Kalyan; Gopakumar, G; Subrahmanyam, Gorthi R K Sai; Gorthi, Sai Siva

2017-05-01

Each year, about 7-8 million deaths occur due to cancer around the world. More than half of the cancer-related deaths occur in the less-developed parts of the world. Cancer mortality rate can be reduced with early detection and subsequent treatment of the disease. In this paper, we introduce a microfluidic microscopy-based cost-effective and label-free approach for identification of cancerous cells. We outline a diagnostic framework for the same and detail an instrumentation layout. We have employed classical computer vision techniques such as 2D principal component analysis-based cell type representation followed by support vector machine-based classification. Analogous to criminal face recognition systems implemented with help of surveillance cameras, a signature-based approach for cancerous cell identification using microfluidic microscopy surveillance is demonstrated. Such a platform would facilitate affordable mass screening camps in the developing countries and therefore help decrease cancer mortality rate.

Automated classification of cell morphology by coherence-controlled holographic microscopy

NASA Astrophysics Data System (ADS)

Strbkova, Lenka; Zicha, Daniel; Vesely, Pavel; Chmelik, Radim

2017-08-01

In the last few years, classification of cells by machine learning has become frequently used in biology. However, most of the approaches are based on morphometric (MO) features, which are not quantitative in terms of cell mass. This may result in poor classification accuracy. Here, we study the potential contribution of coherence-controlled holographic microscopy enabling quantitative phase imaging for the classification of cell morphologies. We compare our approach with the commonly used method based on MO features. We tested both classification approaches in an experiment with nutritionally deprived cancer tissue cells, while employing several supervised machine learning algorithms. Most of the classifiers provided higher performance when quantitative phase features were employed. Based on the results, it can be concluded that the quantitative phase features played an important role in improving the performance of the classification. The methodology could be valuable help in refining the monitoring of live cells in an automated fashion. We believe that coherence-controlled holographic microscopy, as a tool for quantitative phase imaging, offers all preconditions for the accurate automated analysis of live cell behavior while enabling noninvasive label-free imaging with sufficient contrast and high-spatiotemporal phase sensitivity.

Automated classification of cell morphology by coherence-controlled holographic microscopy.

PubMed

Strbkova, Lenka; Zicha, Daniel; Vesely, Pavel; Chmelik, Radim

2017-08-01

In the last few years, classification of cells by machine learning has become frequently used in biology. However, most of the approaches are based on morphometric (MO) features, which are not quantitative in terms of cell mass. This may result in poor classification accuracy. Here, we study the potential contribution of coherence-controlled holographic microscopy enabling quantitative phase imaging for the classification of cell morphologies. We compare our approach with the commonly used method based on MO features. We tested both classification approaches in an experiment with nutritionally deprived cancer tissue cells, while employing several supervised machine learning algorithms. Most of the classifiers provided higher performance when quantitative phase features were employed. Based on the results, it can be concluded that the quantitative phase features played an important role in improving the performance of the classification. The methodology could be valuable help in refining the monitoring of live cells in an automated fashion. We believe that coherence-controlled holographic microscopy, as a tool for quantitative phase imaging, offers all preconditions for the accurate automated analysis of live cell behavior while enabling noninvasive label-free imaging with sufficient contrast and high-spatiotemporal phase sensitivity. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

A novel method for isolating podocytes using magnetic activated cell sorting.

PubMed

Murakami, Ayumi; Oshiro, Hisashi; Kanzaki, Seiichi; Yamaguchi, Akira; Yamanaka, Shoji; Furuya, Mitsuko; Miura, Satoshi; Kanno, Hiroshi; Nagashima, Yoji; Aoki, Ichiro; Nagahama, Kiyotaka

2010-12-01

A large body of accumulated data has now revealed that podocytes play a major role in the development of proteinuria. However, the mechanisms of podocyte injury, leading to foot process effacement and proteinuria, are still unclear partly due to the current lack of an appropriate strategy for preparing podocytes. In this study, we have developed a novel method of rapid isolation of podocytes from mice using magnetic activated cell sorting with an anti-nephrin antibody. After endothelial cell depletion using anti-CD31 antibody, nephrin-positive cells were prepared from mouse kidneys using magnetic activated cell sorting with polyclonal rabbit anti-nephrin antibody. Purity of the positively sorted cells was determined by confocal microscopy and fluorescence-activated cell sorting (FACS) analysis. Expression profiles of podocyte-specific molecules in the sorted fractions were characterized by qualitative PCR and immunoblot analysis. Nephrin-positive cells, isolated from mouse kidneys within 6 h, showed dual positivity for synaptopodin and rabbit IgG on confocal microscopy. FACS analysis revealed that the purity of the positively sorted fractions was ∼75%. The nephrin-positive cells sorted by this approach showed a significantly higher expression of podocyte-specific molecules compared with nephrin-negative fractions. These data strongly suggest that our novel method for isolating podocytes has great utility for various downstream applications such as genomic analysis, proteomics and transcriptomics to elucidate molecular profiling of podocyte biology in vivo compared with conventional methods as our approach requires only several hours to complete and no tissue culture.

Label-Free, High Resolution, Multi-Modal Light Microscopy for Discrimination of Live Stem Cell Differentiation Status.

PubMed

Zhang, Jing; Moradi, Emilia; Somekh, Michael G; Mather, Melissa L

2018-01-15

A label-free microscopy method for assessing the differentiation status of stem cells is presented with potential application for characterization of therapeutic stem cell populations. The microscopy system is capable of characterizing live cells based on the use of evanescent wave microscopy and quantitative phase contrast (QPC) microscopy. The capability of the microscopy system is demonstrated by studying the differentiation of live immortalised neonatal mouse neural stem cells over a 15 day time course. Metrics extracted from microscope images are assessed and images compared with results from endpoint immuno-staining studies to illustrate the system's performance. Results demonstrate the potential of the microscopy system as a valuable tool for cell biologists to readily identify the differentiation status of unlabelled live cells.

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.

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.

Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.

PubMed

Subach, Fedor V; Patterson, George H; Renz, Malte; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V

2010-05-12

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

A new protocol for functional analysis of adipogenesis using reverse transfection technology and time-lapse video microscopy.

PubMed

Grönniger, Elke; Wessel, Sonja; Kühn, Sonja Christin; Söhle, Jörn; Wenck, Horst; Stäb, Franz; Winnefeld, Marc

2010-07-01

Since the worldwide increase in obesity represents a growing challenge for healthcare systems, research focusing on fat cell metabolism has become a focal point of interest. Here, we describe a small interfering RNA (siRNA)-technology-based screening method to study fat cell differentiation in human primary preadipocytes that could be further developed towards an automated middle-throughput screening procedure. First, we established optimal conditions for the reverse transfection of human primary preadipocytes demonstrating that an efficient reverse transfection of preadipocytes is technically feasible. Aligning the processes of reverse transfection and fat cell differentiation utilizing peroxisome proliferator-activated receptor gamma (PPAR gamma)-siRNA, we showed that preadipocyte differentiation was suppressed by knock-down of PPAR gamma, the key regulator of fat cell differentiation. The use of fluorescently labelled fatty acids in combination with fluorescence time-lapse microscopy over a longer period of time enabled us to quantify the PPAR gamma phenotype. Additionally, our data demonstrate that reverse transfection of human cultured preadipocytes with TIP60 (HIV-1 Tat-interacting protein 60)-siRNA lead to a TIP60 knock-down and subsequently inhibits fat cell differentiation, suggesting a role of this protein in human adipogenesis. In conclusion, we established a protocol that allows for an efficient functional and time-dependent analysis by quantitative time-lapse microscopy to identify novel adipogenesis-associated genes.

TLM-Quant: An Open-Source Pipeline for Visualization and Quantification of Gene Expression Heterogeneity in Growing Microbial Cells

PubMed Central

Piersma, Sjouke; Denham, Emma L.; Drulhe, Samuel; Tonk, Rudi H. J.; Schwikowski, Benno; van Dijl, Jan Maarten

2013-01-01

Gene expression heterogeneity is a key driver for microbial adaptation to fluctuating environmental conditions, cell differentiation and the evolution of species. This phenomenon has therefore enormous implications, not only for life in general, but also for biotechnological applications where unwanted subpopulations of non-producing cells can emerge in large-scale fermentations. Only time-lapse fluorescence microscopy allows real-time measurements of gene expression heterogeneity. A major limitation in the analysis of time-lapse microscopy data is the lack of fast, cost-effective, open, simple and adaptable protocols. Here we describe TLM-Quant, a semi-automatic pipeline for the analysis of time-lapse fluorescence microscopy data that enables the user to visualize and quantify gene expression heterogeneity. Importantly, our pipeline builds on the open-source packages ImageJ and R. To validate TLM-Quant, we selected three possible scenarios, namely homogeneous expression, highly ‘noisy’ heterogeneous expression, and bistable heterogeneous expression in the Gram-positive bacterium Bacillus subtilis. This bacterium is both a paradigm for systems-level studies on gene expression and a highly appreciated biotechnological ‘cell factory’. We conclude that the temporal resolution of such analyses with TLM-Quant is only limited by the numbers of recorded images. PMID:23874729

Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

PubMed

Mas, Abraham; Amenós, Montse; Lois, L Maria

2016-01-01

Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

Tracking colliding cells in vivo microscopy.

PubMed

Nguyen, Nhat H; Keller, Steven; Norris, Eric; Huynh, Toan T; Clemens, Mark G; Shin, Min C

2011-08-01

Leukocyte motion represents an important component in the innate immune response to infection. Intravital microscopy is a powerful tool as it enables in vivo imaging of leukocyte motion. Under inflammatory conditions, leukocytes may exhibit various motion behaviors, such as flowing, rolling, and adhering. With many leukocytes moving at a wide range of speeds, collisions occur. These collisions result in abrupt changes in the motion and appearance of leukocytes. Manual analysis is tedious, error prone,time consuming, and could introduce technician-related bias. Automatic tracking is also challenging due to the noise inherent in in vivo images and abrupt changes in motion and appearance due to collision. This paper presents a method to automatically track multiple cells undergoing collisions by modeling the appearance and motion for each collision state and testing collision hypotheses of possible transitions between states. The tracking results are demonstrated using in vivo intravital microscopy image sequences.We demonstrate that 1)71% of colliding cells are correctly tracked; (2) the improvement of the proposed method is enhanced when the duration of collision increases; and (3) given good detection results, the proposed method can correctly track 88% of colliding cells. The method minimizes the tracking failures under collisions and, therefore, allows more robust analysis in the study of leukocyte behaviors responding to inflammatory conditions.

Impact of fipronil on the mushroom bodies of the stingless bee Scaptotrigona postica.

PubMed

Jacob, Cynthia R O; Soares, Hellen M; Nocelli, Roberta C F; Malaspina, Osmar

2015-01-01

Studies on stingless bees are scarce, and little is known about these insects, especially regarding the effects of contamination by neurotoxic insecticides, which can cause damage to important structures of the insect brain. This study evaluated the morphological changes in the intrinsic neurons of the protocerebral mushroom bodies (Kenyon cells) of the stingless bee Scaptotrigona postica after exposure to different doses of fipronil, using light microscopy and transmission electron microscopy. This region of the brain was selected for analysis because of its importance as a sensory integration centre. In both oral and topical treatments, Kenyon cells presented pyknotic profiles, suggesting cell death. Statistical analysis showed significant differences among doses and exposure times. Transmission electron microscopy revealed changes in the nucleus and cellular organelles. Depending on the dose, the characteristics observed suggested apoptotosis or necrosis. This study demonstrates the toxic effects of fipronil. An increase in the number of pyknotic profiles of Kenyon cells of mushroom bodies was observed even at the sublethal doses of 0.27 ng AI bee(-1) and 0.24 ng AI µL(-1) in the topical and oral treatments respectively. Also, differences in the number of pyknotic profiles were dose and time dependent. © 2014 Society of Chemical Industry.

MitoGen: A Framework for Generating 3D Synthetic Time-Lapse Sequences of Cell Populations in Fluorescence Microscopy.

PubMed

Svoboda, David; Ulman, Vladimir

2017-01-01

The proper analysis of biological microscopy images is an important and complex task. Therefore, it requires verification of all steps involved in the process, including image segmentation and tracking algorithms. It is generally better to verify algorithms with computer-generated ground truth datasets, which, compared to manually annotated data, nowadays have reached high quality and can be produced in large quantities even for 3D time-lapse image sequences. Here, we propose a novel framework, called MitoGen, which is capable of generating ground truth datasets with fully 3D time-lapse sequences of synthetic fluorescence-stained cell populations. MitoGen shows biologically justified cell motility, shape and texture changes as well as cell divisions. Standard fluorescence microscopy phenomena such as photobleaching, blur with real point spread function (PSF), and several types of noise, are simulated to obtain realistic images. The MitoGen framework is scalable in both space and time. MitoGen generates visually plausible data that shows good agreement with real data in terms of image descriptors and mean square displacement (MSD) trajectory analysis. Additionally, it is also shown in this paper that four publicly available segmentation and tracking algorithms exhibit similar performance on both real and MitoGen-generated data. The implementation of MitoGen is freely available.

Gold-FISH: A correlative approach to microscopic imaging of single microbial cells in environmental samples

NASA Astrophysics Data System (ADS)

Schmidt, Hannes; Seki, David; Woebken, Dagmar; Eickhorst, Thilo

2017-04-01

Fluorescence in situ hybridization (FISH) is routinely used for the phylogenetic identification, detection, and quantification of single microbial cells environmental microbiology. Oligonucleotide probes that match the 16S rRNA sequence of target organisms are generally applied and the resulting signals are visualized via fluorescence microscopy. Consequently, the detection of the microbial cells of interest is limited by the resolution and the sensitivity of light microscopy where objects smaller than 0.2 µm can hardly be represented. Visualizing microbial cells at magnifications beyond light microscopy, however, can provide information on the composition and potential complexity of microbial habitats - the actual sites of nutrient cycling in soil and sediments. We present a recently developed technique that combines (1) the phylogenetic identification and detection of individual microorganisms by epifluorescence microscopy, with (2) the in situ localization of gold-labelled target cells on an ultrastructural level by SEM. Based on 16S rRNA targeted in situ hybridization combined with catalyzed reporter deposition, a streptavidin conjugate labeled with a fluorescent dye and nanogold particles is introduced into whole microbial cells. A two-step visualization process including an autometallographic enhancement of nanogold particles then allows for either fluorescence or electron microscopy, or a correlative application thereof. We will present applications of the Gold-FISH protocol to samples of marine sediments, agricultural soils, and plant roots. The detection and enumeration of bacterial cells in soil and sediment samples was comparable to CARD-FISH applications via fluorescence microscopy. Examples of microbe-surface interaction analysis will be presented on the basis of bacteria colonizing the rhizoplane of rice roots. In principle, Gold-FISH can be performed on any material to give a snapshot of microbe-surface interactions and provides a promising tool for the acquisition of correlative information on microorganisms within their respective habitats.

Graphene liquid cells for multi-technique analysis of biological cells in water environment

NASA Astrophysics Data System (ADS)

Matruglio, A.; Zucchiatti, P.; Birarda, G.; Marmiroli, B.; D'Amico, F.; Kocabas, C.; Kiskinova, M.; Vaccari, L.

2018-05-01

In-cell exploration of biomolecular constituents is the new frontier of cellular biology that will allow full access to structure-activity correlation of biomolecules, overcoming the limitations imposed by dissecting the cellular milieu. However, the presence of water, which is a very strong IR absorber and incompatible with the vacuum working conditions of all analytical methods using soft x-rays and electrons, poses severe constraint to perform important imaging and spectroscopic analyses under physiological conditions. Recent advances to separate the sample compartment in liquid cell are based on electron and photon transparent but molecular-impermeable graphene membranes. This strategy has opened a unique opportunity to explore technological materials under realistic operation conditions using various types of electron microscopy. However, the widespread of the graphene liquid cell applications is still impeded by the lack of well-established approaches for their massive production. We report on the first preliminary results for the fabrication of reproducible graphene liquid cells appropriate for the analysis of biological specimens in their natural hydrated environment with several crucial analytical techniques, namely FTIR microscopy, Raman spectroscopy, AFM, SEM and TEM.

Toward single cell traction microscopy within 3D collagen matrices

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

Hall, Matthew S.; Long, Rong; Feng, Xinzeng

Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives onmore » the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels.« less

Nanoscale nuclear architecture for cancer diagnosis beyond pathology via spatial-domain low-coherence quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Wang, Pin; Bista, Rajan K.; Khalbuss, Walid E.; Qiu, Wei; Uttam, Shikhar; Staton, Kevin; Zhang, Lin; Brentnall, Teresa A.; Brand, Randall E.; Liu, Yang

2010-11-01

Definitive diagnosis of malignancy is often challenging due to limited availability of human cell or tissue samples and morphological similarity with certain benign conditions. Our recently developed novel technology-spatial-domain low-coherence quantitative phase microscopy (SL-QPM)-overcomes the technical difficulties and enables us to obtain quantitative information about cell nuclear architectural characteristics with nanoscale sensitivity. We explore its ability to improve the identification of malignancy, especially in cytopathologically non-cancerous-appearing cells. We perform proof-of-concept experiments with an animal model of colorectal carcinogenesis-APCMin mouse model and human cytology specimens of colorectal cancer. We show the ability of in situ nanoscale nuclear architectural characteristics in identifying cancerous cells, especially in those labeled as ``indeterminate or normal'' by expert cytopathologists. Our approach is based on the quantitative analysis of the cell nucleus on the original cytology slides without additional processing, which can be readily applied in a conventional clinical setting. Our simple and practical optical microscopy technique may lead to the development of novel methods for early detection of cancer.

Depth-resolved cellular microrheology using HiLo microscopy

PubMed Central

Michaelson, Jarett; Choi, Heejin; So, Peter; Huang, Hayden

2012-01-01

It is increasingly important to measure cell mechanical properties in three-dimensional environments. Particle tracking microrheology (PTM) can measure cellular viscoelastic properties; however, out-of-plane data can introduce artifacts into these measurements. We developed a technique that employs HiLo microscopy to reduce out-of-plane contributions. This method eliminated signals from 90% of probes 0.5 μm or further from the focal plane, while retaining all in-plane probes. We used this technique to characterize live-cell bilayers and found that there were significant, frequency-dependent changes to the extracted cell moduli when compared to conventional analysis. Our results indicate that removal of out-of-plane information is vital for accurate assessments of cell mechanical properties. PMID:22741071

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

Segmentation of fluorescence microscopy images for quantitative analysis of cell nuclear architecture.

PubMed

Russell, Richard A; Adams, Niall M; Stephens, David A; Batty, Elizabeth; Jensen, Kirsten; Freemont, Paul S

2009-04-22

Considerable advances in microscopy, biophysics, and cell biology have provided a wealth of imaging data describing the functional organization of the cell nucleus. Until recently, cell nuclear architecture has largely been assessed by subjective visual inspection of fluorescently labeled components imaged by the optical microscope. This approach is inadequate to fully quantify spatial associations, especially when the patterns are indistinct, irregular, or highly punctate. Accurate image processing techniques as well as statistical and computational tools are thus necessary to interpret this data if meaningful spatial-function relationships are to be established. Here, we have developed a thresholding algorithm, stable count thresholding (SCT), to segment nuclear compartments in confocal laser scanning microscopy image stacks to facilitate objective and quantitative analysis of the three-dimensional organization of these objects using formal statistical methods. We validate the efficacy and performance of the SCT algorithm using real images of immunofluorescently stained nuclear compartments and fluorescent beads as well as simulated images. In all three cases, the SCT algorithm delivers a segmentation that is far better than standard thresholding methods, and more importantly, is comparable to manual thresholding results. By applying the SCT algorithm and statistical analysis, we quantify the spatial configuration of promyelocytic leukemia nuclear bodies with respect to irregular-shaped SC35 domains. We show that the compartments are closer than expected under a null model for their spatial point distribution, and furthermore that their spatial association varies according to cell state. The methods reported are general and can readily be applied to quantify the spatial interactions of other nuclear compartments.

Segmentation of Fluorescence Microscopy Images for Quantitative Analysis of Cell Nuclear Architecture

PubMed Central

Russell, Richard A.; Adams, Niall M.; Stephens, David A.; Batty, Elizabeth; Jensen, Kirsten; Freemont, Paul S.

2009-01-01

Abstract Considerable advances in microscopy, biophysics, and cell biology have provided a wealth of imaging data describing the functional organization of the cell nucleus. Until recently, cell nuclear architecture has largely been assessed by subjective visual inspection of fluorescently labeled components imaged by the optical microscope. This approach is inadequate to fully quantify spatial associations, especially when the patterns are indistinct, irregular, or highly punctate. Accurate image processing techniques as well as statistical and computational tools are thus necessary to interpret this data if meaningful spatial-function relationships are to be established. Here, we have developed a thresholding algorithm, stable count thresholding (SCT), to segment nuclear compartments in confocal laser scanning microscopy image stacks to facilitate objective and quantitative analysis of the three-dimensional organization of these objects using formal statistical methods. We validate the efficacy and performance of the SCT algorithm using real images of immunofluorescently stained nuclear compartments and fluorescent beads as well as simulated images. In all three cases, the SCT algorithm delivers a segmentation that is far better than standard thresholding methods, and more importantly, is comparable to manual thresholding results. By applying the SCT algorithm and statistical analysis, we quantify the spatial configuration of promyelocytic leukemia nuclear bodies with respect to irregular-shaped SC35 domains. We show that the compartments are closer than expected under a null model for their spatial point distribution, and furthermore that their spatial association varies according to cell state. The methods reported are general and can readily be applied to quantify the spatial interactions of other nuclear compartments. PMID:19383481

Variable-angle epifluorescence microscopy characterizes protein dynamics in the vicinity of plasma membrane in plant cells.

PubMed

Chen, Tong; Ji, Dongchao; Tian, Shiping

2018-03-14

The assembly of protein complexes and compositional lipid patterning act together to endow cells with the plasticity required to maintain compositional heterogeneity with respect to individual proteins. Hence, the applications for imaging protein localization and dynamics require high accuracy, particularly at high spatio-temporal level. We provided experimental data for the applications of Variable-Angle Epifluorescence Microscopy (VAEM) in dissecting protein dynamics in plant cells. The VAEM-based co-localization analysis took penetration depth and incident angle into consideration. Besides direct overlap of dual-color fluorescence signals, the co-localization analysis was carried out quantitatively in combination with the methodology for calculating puncta distance and protein proximity index. Besides, simultaneous VAEM tracking of cytoskeletal dynamics provided more insights into coordinated responses of actin filaments and microtubules. Moreover, lateral motility of membrane proteins was analyzed by calculating diffusion coefficients and kymograph analysis, which represented an alternative method for examining protein motility. The present study presented experimental evidence on illustrating the use of VAEM in tracking and dissecting protein dynamics, dissecting endosomal dynamics, cell structure assembly along with membrane microdomain and protein motility in intact plant cells.

Atomic Force Microscopy in Characterizing Cell Mechanics for Biomedical Applications: A Review.

PubMed

Li, Mi; Dang, Dan; Liu, Lianqing; Xi, Ning; Wang, Yuechao

2017-09-01

Cell mechanics is a novel label-free biomarker for indicating cell states and pathological changes. The advent of atomic force microscopy (AFM) provides a powerful tool for quantifying the mechanical properties of single living cells in aqueous conditions. The wide use of AFM in characterizing cell mechanics in the past two decades has yielded remarkable novel insights in understanding the development and progression of certain diseases, such as cancer, showing the huge potential of cell mechanics for practical applications in the field of biomedicine. In this paper, we reviewed the utilization of AFM to characterize cell mechanics. First, the principle and method of AFM single-cell mechanical analysis was presented, along with the mechanical responses of cells to representative external stimuli measured by AFM. Next, the unique changes of cell mechanics in two types of physiological processes (stem cell differentiation, cancer metastasis) revealed by AFM were summarized. After that, the molecular mechanisms guiding cell mechanics were analyzed. Finally the challenges and future directions were discussed.

Curcumin inhibits growth potential by G1 cell cycle arrest and induces apoptosis in p53-mutated COLO 320DM human colon adenocarcinoma cells.

PubMed

Dasiram, Jade Dhananjay; Ganesan, Ramamoorthi; Kannan, Janani; Kotteeswaran, Venkatesan; Sivalingam, Nageswaran

2017-02-01

Curcumin, a natural polyphenolic compound and it is isolated from the rhizome of Curcuma longa, have been reported to possess anticancer effect against stage I and II colon cancer. However, the effect of curcumin on colon cancer at Dukes' type C metastatic stage III remains still unclear. In the present study, we have investigated the anticancer effects of curcumin on p53 mutated COLO 320DM human colon adenocarcinoma cells derived from Dukes' type C metastatic stage. The cellular viability and proliferation were assessed by trypan blue exclusion assay and MTT assay, respectively. The cytotoxicity effect was examined by lactate dehydrogenase (LDH) cytotoxicity assay. Apoptosis was analyzed by DNA fragmentation analysis, Hoechst and propidium iodide double fluorescent staining and confocal microscopy analysis. Cell cycle distribution was performed by flow cytometry analysis. Here we have observed that curcumin treatment significantly inhibited the cellular viability and proliferation potential of p53 mutated COLO 320DM cells in a dose- and time-dependent manner. In addition, curcumin treatment showed no cytotoxic effects to the COLO 320DM cells. DNA fragmentation analysis, Hoechst and propidium iodide double fluorescent staining and confocal microscopy analysis revealed that curcumin treatment induced apoptosis in COLO 320DM cells. Furthermore, curcumin caused cell cycle arrest at the G1 phase, decreased the cell population in the S phase and induced apoptosis in COLO 320DM colon adenocarcinoma cells. Together, these data suggest that curcumin exerts anticancer effects and induces apoptosis in p53 mutated COLO 320DM human colon adenocarcinoma cells derived from Dukes' type C metastatic stage. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Phenol homeostasis is ensured in vanilla fruit by storage under solid form in a new chloroplast-derived organelle, the phenyloplast

PubMed Central

Conéjéro, Geneviève

2014-01-01

A multiple cell imaging approach combining immunofluorescence by confocal microscopy, fluorescence spectral analysis by multiphotonic microscopy, and transmission electron microscopy identified the site of accumulation of 4-O-(3-methoxybenzaldehyde) β-d-glucoside, a phenol glucoside massively stockpiled by vanilla fruit. The glucoside is sufficiently abundant to be detected by spectral analysis of its autofluorescence. The convergent results obtained by these different techniques demonstrated that the phenol glucoside accumulates in the inner volume of redifferentiating chloroplasts as solid amorphous deposits, thus ensuring phenylglucoside cell homeostasis. Redifferentiation starts with the generation of loculi between thylakoid membranes which are progressively filled with the glucoside until a fully matured organelle is obtained. This peculiar mode of storage of a phenolic secondary metabolite is suspected to occur in other plants and its generalization in the Plantae could be considered. This new chloroplast-derived organelle is referred to as a ‘phenyloplast’. PMID:24683183

Phenol homeostasis is ensured in vanilla fruit by storage under solid form in a new chloroplast-derived organelle, the phenyloplast.

PubMed

Brillouet, Jean-Marc; Verdeil, Jean-Luc; Odoux, Eric; Lartaud, Marc; Grisoni, Michel; Conéjéro, Geneviève

2014-06-01

A multiple cell imaging approach combining immunofluorescence by confocal microscopy, fluorescence spectral analysis by multiphotonic microscopy, and transmission electron microscopy identified the site of accumulation of 4-O-(3-methoxybenzaldehyde) β-d-glucoside, a phenol glucoside massively stockpiled by vanilla fruit. The glucoside is sufficiently abundant to be detected by spectral analysis of its autofluorescence. The convergent results obtained by these different techniques demonstrated that the phenol glucoside accumulates in the inner volume of redifferentiating chloroplasts as solid amorphous deposits, thus ensuring phenylglucoside cell homeostasis. Redifferentiation starts with the generation of loculi between thylakoid membranes which are progressively filled with the glucoside until a fully matured organelle is obtained. This peculiar mode of storage of a phenolic secondary metabolite is suspected to occur in other plants and its generalization in the Plantae could be considered. This new chloroplast-derived organelle is referred to as a 'phenyloplast'. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Label-free distinguishing between neurons and glial cells based on two-photon excited fluorescence signal of neuron perinuclear granules

NASA Astrophysics Data System (ADS)

Du, Huiping; Jiang, Liwei; Wang, Xingfu; Liu, Gaoqiang; Wang, Shu; Zheng, Liqin; Li, Lianhuang; Zhuo, Shuangmu; Zhu, Xiaoqin; Chen, Jianxin

2016-08-01

Neurons and glial cells are two critical cell types of brain tissue. Their accurate identification is important for the diagnosis of psychiatric disorders such as depression and schizophrenia. In this paper, distinguishing between neurons and glial cells by using the two-photon excited fluorescence (TPEF) signals of intracellular intrinsic sources was performed. TPEF microscopy combined with TUJ-1 and GFAP immunostaining and quantitative image analysis demonstrated that the perinuclear granules of neurons in the TPEF images of brain tissue and the primary cultured cortical cells were a unique characteristic of neurons compared to glial cells which can become a quantitative feature to distinguish neurons from glial cells. With the development of miniaturized TPEF microscope (‘two-photon fiberscopes’) imaging devices, TPEF microscopy can be developed into an effective diagnostic and monitoring tool for psychiatric disorders such as depression and schizophrenia.

Comparative analysis of the effect of low-dimensional alumina structures on cell lines L929 and Neuro-2a

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

Fomenko, A. N., E-mail: alserova@ispms.tsc.ru; Korovin, M. S., E-mail: msk@ispms.tsc.ru

The paper presents the toxicity evaluation of nanostructures on the basis of alumina of different shape (nanofibers, nanoplates, nanosheets, nanosheet agglomerates) and with similar physical and chemical properties (particle size, specific surface area, phase composition, and zeta potential). The nanostructures were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), low-temperature nitrogen adsorption, and dynamic light scattering (DLS). The cytotoxicity of nanostructures was estimated using L929 fibroblast cells and Neuro-2a tumor cells. It has been found that the L929 cells are less subject to the influence of alumina nanoparticles than the Neuro-2a tumor cells. Probably, themore » differences in the proliferation activity of normal and tumor cells in contact with the synthesized nanostructures are due to a change in the pH of the cell microenvironment.« less

Comparative analysis of the effect of low-dimensional alumina structures on cell lines L929 and Neuro-2a

NASA Astrophysics Data System (ADS)

Fomenko, A. N.; Korovin, M. S.

2016-08-01

The paper presents the toxicity evaluation of nanostructures on the basis of alumina of different shape (nanofibers, nanoplates, nanosheets, nanosheet agglomerates) and with similar physical and chemical properties (particle size, specific surface area, phase composition, and zeta potential). The nanostructures were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), low-temperature nitrogen adsorption, and dynamic light scattering (DLS). The cytotoxicity of nanostructures was estimated using L929 fibroblast cells and Neuro-2a tumor cells. It has been found that the L929 cells are less subject to the influence of alumina nanoparticles than the Neuro-2a tumor cells. Probably, the differences in the proliferation activity of normal and tumor cells in contact with the synthesized nanostructures are due to a change in the pH of the cell microenvironment.

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

PubMed Central

Lewis, Leanne E.; Bain, Judith M.; Okai, Blessing; Gow, Neil A.R.; Erwig, Lars Peter

2013-01-01

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10. In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms. PMID:23329139

5D imaging via light sheet microscopy reveals cell dynamics during the eye-antenna disc primordium formation in Drosophila

NASA Astrophysics Data System (ADS)

Huang, Yu Shan; Ku, Hui Yu; Tsai, Yun Chi; Chang, Chin Hao; Pao, Sih Hua; Sun, Y. Henry; Chiou, Arthur

2017-03-01

5D images of engrailed (en) and eye gone (eyg) gene expressions during the course of the eye-antenna disc primordium (EADP) formation of Drosophila embryos from embryonic stages 13 through 16 were recorded via light sheet microscopy and analyzed to reveal the cell dynamics involved in the development of the EADP. Detailed analysis of the time-lapsed images revealed the process of EADP formation and its invagination trajectory, which involved an inversion of the EADP anterior-posterior axis relative to the body. Furthermore, analysis of the en-expression pattern in the EADP provided strong evidence that the EADP is derived from one of the en-expressing head segments.

Retracing in correlative light electron microscopy: where is my object of interest?

PubMed

Hodgson, Lorna; Nam, David; Mantell, Judith; Achim, Alin; Verkade, Paul

2014-01-01

Correlative light electron microscopy (CLEM) combines the strengths of light and electron microscopy in a single experiment. There are many ways to perform a CLEM experiment and a variety of microscopy modalities can be combined either on separate instruments or as completely integrated solutions. In general, however, a CLEM experiment can be divided into three parts: probes, processing, and analysis. Most of the existing technologies are focussed around the development and use of probes or describe processing methodologies that explain or circumvent some of the compromises that need to be made when performing both light and electron microscopy on the same sample. So far, relatively little attention has been paid to the analysis part of CLEM experiments. Although it is an essential part of each CLEM experiment, it is usually a cumbersome manual process. Here, we briefly discuss each of the three above-mentioned steps, with a focus on the analysis part. We will also introduce an automated registration algorithm that can be applied to the analysis stage to enable the accurate registration of LM and EM images. This facilitates tracing back the right cell/object seen in the light microscope in the EM. © 2014 Elsevier Inc. All rights reserved.

Yining Zeng | NREL

Science.gov Websites

pretreatment conditions and biological digestion methods, which might not be detected by large-scale ) "Coherent Raman Microscopy Analysis of Plant Cell Walls," Biomass Conversion: Methods and Protocols, Methods in Molecular Biology (2012) "Chemical, Ultrastructural and Supramolecular Analysis

Classification of M1/M2-polarized human macrophages by label-free hyperspectral reflectance confocal microscopy and multivariate analysis.

PubMed

Bertani, Francesca R; Mozetic, Pamela; Fioramonti, Marco; Iuliani, Michele; Ribelli, Giulia; Pantano, Francesco; Santini, Daniele; Tonini, Giuseppe; Trombetta, Marcella; Businaro, Luca; Selci, Stefano; Rainer, Alberto

2017-08-21

The possibility of detecting and classifying living cells in a label-free and non-invasive manner holds significant theranostic potential. In this work, Hyperspectral Imaging (HSI) has been successfully applied to the analysis of macrophagic polarization, given its central role in several pathological settings, including the regulation of tumour microenvironment. Human monocyte derived macrophages have been investigated using hyperspectral reflectance confocal microscopy, and hyperspectral datasets have been analysed in terms of M1 vs. M2 polarization by Principal Components Analysis (PCA). Following PCA, Linear Discriminant Analysis has been implemented for semi-automatic classification of macrophagic polarization from HSI data. Our results confirm the possibility to perform single-cell-level in vitro classification of M1 vs. M2 macrophages in a non-invasive and label-free manner with a high accuracy (above 98% for cells deriving from the same donor), supporting the idea of applying the technique to the study of complex interacting cellular systems, such in the case of tumour-immunity in vitro models.

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.

PubMed

Yang, Yang; Guan, Xiangming

2017-05-01

Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY's reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY's reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY's reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY's reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy. Graphical Abstract Live cell imaging and quantification of non-protein thiols by TBOP.

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 chemical functionality in plant cell walls

DOE PAGES

Zeng, Yining; Himmel, Michael E.; Ding, Shi-You

2017-11-30

Understanding plant cell wall cross-linking chemistry and polymeric architecture is key to the efficient utilization of biomass in all prospects from rational genetic modification to downstream chemical and biological conversion to produce fuels and value chemicals. In fact, the bulk properties of cell wall recalcitrance are collectively determined by its chemical features over a wide range of length scales from tissue, cellular to polymeric architectures. Microscopic visualization of cell walls from the nanometer to the micrometer scale offers an in situ approach to study their chemical functionality considering its spatial and chemical complexity, particularly the capabilities of characterizing biomass non-destructivelymore » and in real-time during conversion processes. Microscopic characterization has revealed heterogeneity in the distribution of chemical features, which would otherwise be hidden in bulk analysis. Key microscopic features include cell wall type, wall layering, and wall composition - especially cellulose and lignin distributions. Microscopic tools, such as atomic force microscopy, stimulated Raman scattering microscopy, and fluorescence microscopy, have been applied to investigations of cell wall structure and chemistry from the native wall to wall treated by thermal chemical pretreatment and enzymatic hydrolysis. While advancing our current understanding of plant cell wall recalcitrance and deconstruction, microscopic tools with improved spatial resolution will steadily enhance our fundamental understanding of cell wall function.« less

Visualizing chemical functionality in plant cell walls

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

Zeng, Yining; Himmel, Michael E.; Ding, Shi-You

Understanding plant cell wall cross-linking chemistry and polymeric architecture is key to the efficient utilization of biomass in all prospects from rational genetic modification to downstream chemical and biological conversion to produce fuels and value chemicals. In fact, the bulk properties of cell wall recalcitrance are collectively determined by its chemical features over a wide range of length scales from tissue, cellular to polymeric architectures. Microscopic visualization of cell walls from the nanometer to the micrometer scale offers an in situ approach to study their chemical functionality considering its spatial and chemical complexity, particularly the capabilities of characterizing biomass non-destructivelymore » and in real-time during conversion processes. Microscopic characterization has revealed heterogeneity in the distribution of chemical features, which would otherwise be hidden in bulk analysis. Key microscopic features include cell wall type, wall layering, and wall composition - especially cellulose and lignin distributions. Microscopic tools, such as atomic force microscopy, stimulated Raman scattering microscopy, and fluorescence microscopy, have been applied to investigations of cell wall structure and chemistry from the native wall to wall treated by thermal chemical pretreatment and enzymatic hydrolysis. While advancing our current understanding of plant cell wall recalcitrance and deconstruction, microscopic tools with improved spatial resolution will steadily enhance our fundamental understanding of cell wall function.« less

Visualizing chemical functionality in plant cell walls.

PubMed

Zeng, Yining; Himmel, Michael E; Ding, Shi-You

2017-01-01

Understanding plant cell wall cross-linking chemistry and polymeric architecture is key to the efficient utilization of biomass in all prospects from rational genetic modification to downstream chemical and biological conversion to produce fuels and value chemicals. In fact, the bulk properties of cell wall recalcitrance are collectively determined by its chemical features over a wide range of length scales from tissue, cellular to polymeric architectures. Microscopic visualization of cell walls from the nanometer to the micrometer scale offers an in situ approach to study their chemical functionality considering its spatial and chemical complexity, particularly the capabilities of characterizing biomass non-destructively and in real-time during conversion processes. Microscopic characterization has revealed heterogeneity in the distribution of chemical features, which would otherwise be hidden in bulk analysis. Key microscopic features include cell wall type, wall layering, and wall composition-especially cellulose and lignin distributions. Microscopic tools, such as atomic force microscopy, stimulated Raman scattering microscopy, and fluorescence microscopy, have been applied to investigations of cell wall structure and chemistry from the native wall to wall treated by thermal chemical pretreatment and enzymatic hydrolysis. While advancing our current understanding of plant cell wall recalcitrance and deconstruction, microscopic tools with improved spatial resolution will steadily enhance our fundamental understanding of cell wall function.

Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells

PubMed Central

Stagge, Franziska; Mitronova, Gyuzel Y.; Belov, Vladimir N.; Wurm, Christian A.; Jakobs, Stefan

2013-01-01

Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6′-carboxy isomers and not the 5′-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of 1H NMR spectra to discriminate between 6′- and 5′-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae. PMID:24205303

Theoretical Analysis of Novel Quasi-3D Microscopy of Cell Deformation

PubMed Central

Qiu, Jun; Baik, Andrew D.; Lu, X. Lucas; Hillman, Elizabeth M. C.; Zhuang, Zhuo; Guo, X. Edward

2012-01-01

A novel quasi-three-dimensional (quasi-3D) microscopy technique has been developed to enable visualization of a cell under dynamic loading in two orthogonal planes simultaneously. The three-dimensional (3D) dynamics of the mechanical behavior of a cell under fluid flow can be examined at a high temporal resolution. In this study, a numerical model of a fluorescently dyed cell was created in 3D space, and the cell was subjected to uniaxial deformation or unidirectional fluid shear flow via finite element analysis (FEA). Therefore, the intracellular deformation in the simulated cells was exactly prescribed. Two-dimensional fluorescent images simulating the quasi-3D technique were created from the cell and its deformed states in 3D space using a point-spread function (PSF) and a convolution operation. These simulated original and deformed images were processed by a digital image correlation technique to calculate quasi-3D-based intracellular strains. The calculated strains were compared to the prescribed strains, thus providing a theoretical basis for the measurement of the accuracy of quasi-3D and wide-field microscopy-based intracellular strain measurements against the true 3D strains. The signal-to-noise ratio (SNR) of the simulated quasi-3D images was also modulated using additive Gaussian noise, and a minimum SNR of 12 was needed to recover the prescribed strains using digital image correlation. Our computational study demonstrated that quasi-3D strain measurements closely recovered the true 3D strains in uniform and fluid flow cellular strain states to within 5% strain error. PMID:22707985

Antitumor Activity of Ethanolic Extract of Dendrobium formosum in T-Cell Lymphoma: An In Vitro and In Vivo Study

PubMed Central

Prasad, Ritika; Koch, Biplob

2014-01-01

Dendrobium, a genus of orchid, was found to possess useful therapeutic activities like anticancer, hypoglycaemic, antimicrobial, immunomodulatory, hepatoprotective, antioxidant, and neuroprotective activities. The study was aimed to evaluate the anticancer property of the ethanolic extract of Dendrobium formosum on Dalton's lymphoma. In vitro cytotoxicity was determined by MTT assay, apoptosis was determined by fluorescence microscopy, and cell cycle progression was analysed using flow cytometry; in vivo antitumor activity was performed in Dalton's lymphoma bearing mice. The IC50 value of ethanolic extract was obtained at 350 μg/mL in Dalton's lymphoma cells. Fluorescence microscopy analysis showed significant increase in apoptotic cell death in dose- and time-dependent manner which was further confirmed through the resulting DNA fragmentation. Further, flow cytometry analysis showed that the ethanolic extract arrests the cells in G2/M phase of the cell cycle. The in vivo anticancer activity study illustrates significant increase in the survival time of Dalton's lymphoma bearing mice on treatment with ethanolic extract when compared to control. These results substantiate the antitumor properties of ethanolic extract of Dendrobium formosum and suggest an alternative in treatment of cancer. Further studies are required regarding the isolation and characterization of bioactive components along with the analysis of molecular mechanism involved. PMID:24959588

Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques.

PubMed

Paddock, Stephen W; Eliceiri, Kevin W

2014-01-01

Confocal microscopy is an established light microscopical technique for imaging fluorescently labeled specimens with significant three-dimensional structure. Applications of confocal microscopy in the biomedical sciences include the imaging of the spatial distribution of macromolecules in either fixed or living cells, the automated collection of 3D data, the imaging of multiple labeled specimens and the measurement of physiological events in living cells. The laser scanning confocal microscope continues to be chosen for most routine work although a number of instruments have been developed for more specific applications. Significant improvements have been made to all areas of the confocal approach, not only to the instruments themselves, but also to the protocols of specimen preparation, to the analysis, the display, the reproduction, sharing and management of confocal images using bioinformatics techniques.

Seeing through walls at the nanoscale: Microwave microscopy of enclosed objects and processes in liquids

DOE PAGES

Velmurugan, Jeyavel; Kalinin, Sergei V.; Kolmakov, Andrei; ...

2016-02-11

Here, noninvasive in situ nanoscale imaging in liquid environments is a current imperative in the analysis of delicate biomedical objects and electrochemical processes at reactive liquid–solid interfaces. Microwaves of a few gigahertz frequencies offer photons with energies of ≈10 μeV, which can affect neither electronic states nor chemical bonds in condensed matter. Here, we describe an implementation of scanning near-field microwave microscopy for imaging in liquids using ultrathin molecular impermeable membranes separating scanning probes from samples enclosed in environmental cells. We imaged a model electroplating reaction as well as individual live cells. Through a side-by-side comparison of the microwave imagingmore » with scanning electron microscopy, we demonstrate the advantage of microwaves for artifact-free imaging.« less

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.

Ultrastructural analysis of testicular tissue and sperm by transmission and scanning electron microscopy.

PubMed

Chemes, Hector E

2013-01-01

Transmission electron microscopy (TEM) studies have provided the basis for an in-depth understanding of the cell biology and normal functioning of the testis and male gametes and have opened the way to characterize the functional role played by specific organelles in spermatogenesis and sperm function. The development of the scanning electron microscope (SEM) extended these boundaries to the recognition of cell and organ surface features and the architectural array of cells and tissues. The merging of immunocytochemical and histochemical approaches with electron microscopy has completed a series of technical improvements that integrate structural and functional features to provide a broad understanding of cell biology in health and disease. With these advances the detailed study of the intricate structural and molecular organization as well as the chemical composition of cellular organelles is now possible. Immunocytochemistry is used to identify proteins or other components and localize them in specific cells or organelles with high specificity and sensitivity, and histochemistry can be used to understand their function (i.e., enzyme activity). When these techniques are used in conjunction with electron microscopy their resolving power is further increased to subcellular levels. In the present chapter we will describe in detail various ultrastructural techniques that are now available for basic or translational research in reproductive biology and reproductive medicine. These include TEM, ultrastructural immunocytochemistry, ultrastructural histochemistry, and SEM.

Composition analysis of a polymer electrolyte membrane fuel cell microporous layer using scanning transmission X-ray microscopy and near edge X-ray absorption fine structure analysis

NASA Astrophysics Data System (ADS)

George, Michael G.; Wang, Jian; Banerjee, Rupak; Bazylak, Aimy

2016-03-01

The novel application of scanning transmission X-ray microscopy (STXM) to the microporous layer (MPL) of a polymer electrolyte membrane fuel cell is investigated. A spatially resolved chemical component distribution map is obtained for the MPL of a commercially available SGL 25 BC sample. This is achieved with near edge X-ray absorption fine structure spectroscopic analysis. Prior to analysis the sample is embedded in non-reactive epoxy and ultra-microtomed to a thickness of 100 nm. Polytetrafluoroethylene (PTFE), carbon particle agglomerates, and supporting epoxy resin distributions are identified and reconstructed for a scanning area of 6 μm × 6 μm. It is observed that the spatial distribution of PTFE is strongly correlated to the carbon particle agglomerations. Additionally, agglomerate structures of PTFE are identified, possibly indicating the presence of a unique mesostructure in the MPL. STXM analysis is presented as a useful technique for the investigation of chemical species distributions in the MPL.

Quantifying phosphoric acid in high-temperature polymer electrolyte fuel cell components by X-ray tomographic microscopy.

PubMed

Eberhardt, S H; Marone, F; Stampanoni, M; Büchi, F N; Schmidt, T J

2014-11-01

Synchrotron-based X-ray tomographic microscopy is investigated for imaging the local distribution and concentration of phosphoric acid in high-temperature polymer electrolyte fuel cells. Phosphoric acid fills the pores of the macro- and microporous fuel cell components. Its concentration in the fuel cell varies over a wide range (40-100 wt% H3PO4). This renders the quantification and concentration determination challenging. The problem is solved by using propagation-based phase contrast imaging and a referencing method. Fuel cell components with known acid concentrations were used to correlate greyscale values and acid concentrations. Thus calibration curves were established for the gas diffusion layer, catalyst layer and membrane in a non-operating fuel cell. The non-destructive imaging methodology was verified by comparing image-based values for acid content and concentration in the gas diffusion layer with those from chemical analysis.

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.

Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

PubMed Central

Chakraborty, Paramita; Bjork, Per; Källberg, Eva; Olsson, Anders; Riva, Matteo; Mörgelin, Matthias; Liberg, David; Ivars, Fredrik; Leanderson, Tomas

2015-01-01

We show here, by using surface biotinylation, followed by Western blotting or surface plasmon resonance analysis, that very low levels of S100A8 and/or S100A9 can be detected on the surface of THP-1 cells or freshly isolated human monocytes. This was supported by immune-electron microscopy where we observed membrane-associated expression of the proteins restricted to small patches. By using confocal microscopy we could determine that S100A8 and S100A9 protein in THP-1 cells or freshly isolated human monocytes was mostly present in vesicular structures. This finding was confirmed using immune-electron microscopy. Subcellular fractionation and confocal microscopy showed that these vesicular structures are mainly early endosomes and endolysosomes. Our subsequent studies showed that accumulation of S100A8 and S100A9 in the endolysosomal compartment is associated with induction of their release from the cells. Furthermore, an inhibitor of lysosomal activity could modulate the release of S100A8 and S100A9 in the extracellular milieu. Our current results suggest that the S100A8 and S100A9 proteins are primarily associated with certain kinds of cytosolic vesicles and may be secreted via an endolysosomal pathway. PMID:26661255

Synthesis and characterization of polyamidoamine dendrimer-coated multi-walled carbon nanotubes and their application in gene delivery systems

NASA Astrophysics Data System (ADS)

Pan, Bifeng; Cui, Daxiang; Xu, Ping; Ozkan, Cengiz; Feng, Gao; Ozkan, Mihri; Huang, Tuo; Chu, Bingfeng; Li, Qing; He, Rong; Hu, Guohan

2009-03-01

With the aim of improving the amount and delivery efficiency of genes taken by carbon nanotubes into human cancer cells, different generations of polyamidoamine dendrimer modified multi-walled carbon nanotubes (dMNTs) were fabricated, and characterized by high-resolution transmission electron microscopy, atomic force microscopy, x-ray photoelectron spectroscopy, Raman spectroscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis, revealing the presence of dendrimer capped on the surface of carbon nanotubes. The dMNTs fully conjugated with FITC-labeled antisense c-myc oligonucleotides (asODN), those resultant asODN-dMNTs composites were incubated with human breast cancer cell line MCF-7 cells and MDA-MB-435 cells, and liver cancer cell line HepG2 cells, and confirmed to enter into tumor cells within 15 min by laser confocal microscopy. These composites inhibited the cell growth in time- and dose-dependent means, and down-regulated the expression of the c-myc gene and C-Myc protein. Compared with the composites of CNT-NH2-asODN and dendrimer-asODN, no. 5 generation of dendrimer-modified MNT-asODN composites exhibit maximal transfection efficiencies and inhibition effects on tumor cells. The intracellular gene transport and uptake via dMNTs should be generic for the mammalian cell lines. The dMNTs have potentials in applications such as gene or drug delivery for cancer therapy and molecular imaging.

A cellular uptake and cytotoxicity properties study of gallic acid-loaded mesoporous silica nanoparticles on Caco-2 cells

NASA Astrophysics Data System (ADS)

Rashidi, Ladan; Vasheghani-Farahani, Ebrahim; Soleimani, Masoud; Atashi, Amir; Rostami, Khosrow; Gangi, Fariba; Fallahpour, Masoud; Tahouri, Mohammad Taher

2014-03-01

In this study, the effects of intracellular delivery of various concentrations of gallic acid (GA) as a semistable antioxidant, gallic acid-loaded mesoporous silica nanoparticles (MSNs-GA), and cellular uptake of nanoparticles into Caco-2 cells were investigated. MSNs were synthesized and loaded with GA, then characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, N2 adsorption isotherms, X-ray diffraction, and thermal gravimetric analysis. The cytotoxicity of MSNs and MSNs-GA at low and high concentrations were studied by means of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry. MSNs did not show significant toxicity in various concentrations (0-500 μg/ml) on Caco-2 cells. For MSNs-GA, cell viability was reduced as a function of incubation time and different concentrations of nanoparticles. The in vitro GA release from MSNs-GA exhibited the same antitumor properties as free GA on Caco-2 cells. Flow cytometry results confirmed those obtained using MTT assay. TEM and fluorescent microscopy confirmed the internalization of MSNs by Caco-2 cells through nonspecific cellular uptake. MSNs can easily internalize into Caco-2 cells without deleterious effects on cell viability. The cell viability of Caco-2 cells was affected during MSNs-GA uptake. MSNs could be designed as suitable nanocarriers for antioxidants delivery.

Simulated single molecule microscopy with SMeagol.

PubMed

Lindén, Martin; Ćurić, Vladimir; Boucharin, Alexis; Fange, David; Elf, Johan

2016-08-01

SMeagol is a software tool to simulate highly realistic microscopy data based on spatial systems biology models, in order to facilitate development, validation and optimization of advanced analysis methods for live cell single molecule microscopy data. SMeagol runs on Matlab R2014 and later, and uses compiled binaries in C for reaction-diffusion simulations. Documentation, source code and binaries for Mac OS, Windows and Ubuntu Linux can be downloaded from http://smeagol.sourceforge.net johan.elf@icm.uu.se Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Live cell refractometry based on non-SPR microparticle sensor.

PubMed

Liu, Chang; Chen, David D Y; Yu, Lirong; Luo, Yong

2013-06-01

Unlike the nanoparticles with surface plasmon resonance, the optical response of polystyrene microparticles (PSMPs) is insensitive to the chemical components of the surrounding medium under the wavelength-dependent differential interference contrast microscopy. This fact is exploited for the measurement of the refractive index of cytoplasm in this study. PSMPs of 400 nm in diameter were loaded into the cell to contact cytoplasm seamlessly, and the refractive index information of cytoplasm could be extracted by differential interference contrast microscopy operated at 420 nm illumination wavelength through the contrast analysis of PSMPs images. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Atomic Force Microscopy of Photosystem II and Its Unit Cell Clustering Quantitatively Delineate the Mesoscale Variability in Arabidopsis Thylakoids

PubMed Central

Onoa, Bibiana; Schneider, Anna R.; Brooks, Matthew D.; Grob, Patricia; Nogales, Eva; Geissler, Phillip L.; Niyogi, Krishna K.; Bustamante, Carlos

2014-01-01

Photoautotrophic organisms efficiently regulate absorption of light energy to sustain photochemistry while promoting photoprotection. Photoprotection is achieved in part by triggering a series of dissipative processes termed non-photochemical quenching (NPQ), which depend on the re-organization of photosystem (PS) II supercomplexes in thylakoid membranes. Using atomic force microscopy, we characterized the structural attributes of grana thylakoids from Arabidopsis thaliana to correlate differences in PSII organization with the role of SOQ1, a recently discovered thylakoid protein that prevents formation of a slowly reversible NPQ state. We developed a statistical image analysis suite to discriminate disordered from crystalline particles and classify crystalline arrays according to their unit cell properties. Through detailed analysis of the local organization of PSII supercomplexes in ordered and disordered phases, we found evidence that interactions among light-harvesting antenna complexes are weakened in the absence of SOQ1, inducing protein rearrangements that favor larger separations between PSII complexes in the majority (disordered) phase and reshaping the PSII crystallization landscape. The features we observe are distinct from known protein rearrangements associated with NPQ, providing further support for a role of SOQ1 in a novel NPQ pathway. The particle clustering and unit cell methodology developed here is generalizable to multiple types of microscopy and will enable unbiased analysis and comparison of large data sets. PMID:25007326

Atomic Force Microscopy of Photosystem II and Its Unit Cell Clustering Quantitatively Delineate the Mesoscale Variability in Arabidopsis Thylakoids

DOE PAGES

Onoa, Bibiana; Schneider, Anna R.; Brooks, Matthew D.; ...

2014-07-09

Photoautotrophic organisms efficiently regulate absorption of light energy to sustain photochemistry while promoting photoprotection. Photoprotection is achieved in part by triggering a series of dissipative processes termed non-photochemical quenching (NPQ), which depend on the re-organization of photosystem (PS) II supercomplexes in thylakoid membranes. Using atomic force microscopy, we characterized the structural attributes of grana thylakoids from Arabidopsis thaliana to correlate differences in PSII organization with the role of SOQ1, a recently discovered thylakoid protein that prevents formation of a slowly reversible NPQ state. We developed a statistical image analysis suite to discriminate disordered from crystalline particles and classify crystalline arraysmore » according to their unit cell properties. Through detailed analysis of the local organization of PSII supercomplexes in ordered and disordered phases, we found evidence that interactions among light-harvesting antenna complexes are weakened in the absence of SOQ1, inducing protein rearrangements that favor larger separations between PSII complexes in the majority (disordered) phase and reshaping the PSII crystallization landscape. The features we observe are distinct from known protein rearrangements associated with NPQ, providing further support for a role of SOQ1 in a novel NPQ pathway. The particle clustering and unit cell methodology developed here is generalizable to multiple types of microscopy and will enable unbiased analysis and comparison of large data sets.« less

Light Microscopy at Maximal Precision

NASA Astrophysics Data System (ADS)

Bierbaum, Matthew; Leahy, Brian D.; Alemi, Alexander A.; Cohen, Itai; Sethna, James P.

2017-10-01

Microscopy is the workhorse of the physical and life sciences, producing crisp images of everything from atoms to cells well beyond the capabilities of the human eye. However, the analysis of these images is frequently little more accurate than manual marking. Here, we revolutionize the analysis of microscopy images, extracting all the useful information theoretically contained in a complex microscope image. Using a generic, methodological approach, we extract the information by fitting experimental images with a detailed optical model of the microscope, a method we call parameter extraction from reconstructing images (PERI). As a proof of principle, we demonstrate this approach with a confocal image of colloidal spheres, improving measurements of particle positions and radii by 10-100 times over current methods and attaining the maximum possible accuracy. With this unprecedented accuracy, we measure nanometer-scale colloidal interactions in dense suspensions solely with light microscopy, a previously impossible feat. Our approach is generic and applicable to imaging methods from brightfield to electron microscopy, where we expect accuracies of 1 nm and 0.1 pm, respectively.

Sample to answer visualization pipeline for low-cost point-of-care blood cell counting

NASA Astrophysics Data System (ADS)

Smith, Suzanne; Naidoo, Thegaran; Davies, Emlyn; Fourie, Louis; Nxumalo, Zandile; Swart, Hein; Marais, Philip; Land, Kevin; Roux, Pieter

2015-03-01

We present a visualization pipeline from sample to answer for point-of-care blood cell counting applications. Effective and low-cost point-of-care medical diagnostic tests provide developing countries and rural communities with accessible healthcare solutions [1], and can be particularly beneficial for blood cell count tests, which are often the starting point in the process of diagnosing a patient [2]. The initial focus of this work is on total white and red blood cell counts, using a microfluidic cartridge [3] for sample processing. Analysis of the processed samples has been implemented by means of two main optical visualization systems developed in-house: 1) a fluidic operation analysis system using high speed video data to determine volumes, mixing efficiency and flow rates, and 2) a microscopy analysis system to investigate homogeneity and concentration of blood cells. Fluidic parameters were derived from the optical flow [4] as well as color-based segmentation of the different fluids using a hue-saturation-value (HSV) color space. Cell count estimates were obtained using automated microscopy analysis and were compared to a widely accepted manual method for cell counting using a hemocytometer [5]. The results using the first iteration microfluidic device [3] showed that the most simple - and thus low-cost - approach for microfluidic component implementation was not adequate as compared to techniques based on manual cell counting principles. An improved microfluidic design has been developed to incorporate enhanced mixing and metering components, which together with this work provides the foundation on which to successfully implement automated, rapid and low-cost blood cell counting tests.

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

PubMed

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

2018-02-01

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

Cellular distribution of uranium after acute exposure of renal epithelial cells: SEM, TEM and nuclear microscopy analysis

NASA Astrophysics Data System (ADS)

Carrière, Marie; Gouget, Barbara; Gallien, Jean-Paul; Avoscan, Laure; Gobin, Renée; Verbavatz, Jean-Marc; Khodja, Hicham

2005-04-01

The major health effect of uranium exposure has been reported to be chemical kidney toxicity, functional and histological damages being mainly observed in proximal tubule cells. Uranium enters the proximal tubule as uranyl-bicarbonate or uranyl-citrate complexes. The aim of our research is to investigate the mechanisms of uranium toxicity, intracellular accumulation and repartition after acute intoxication of rat renal proximal tubule epithelial cells, as a function of its chemical form. Microscopic observations of renal epithelial cells after acute exposure to uranyl-bicarbonate showing the presence of intracellular precipitates as thin needles of uranyl-phosphate localized in cell lysosomes have been published. However the initial site of precipitates formation has not been identified yet: they could either be formed outside the cells before internalization, or directly inside the cells. Uranium solubility as a function and initial concentration was specified by ICP-MS analysis of culture media. In parallel, uranium uptake and distribution in cell monolayers exposed to U-bicarbonate was investigated by nuclear microprobe analyses. Finally, the presence of uranium precipitates was tested out by scanning electron microscopic observations (SEM), while extracellular and/or intracellular precipitates were observed on thin sections of cells by transmission electron microscopy (TEM).

An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology

PubMed Central

Littlejohn, George R.; Mansfield, Jessica C.; Christmas, Jacqueline T.; Witterick, Eleanor; Fricker, Mark D.; Grant, Murray R.; Smirnoff, Nicholas; Everson, Richard M.; Moger, Julian; Love, John

2014-01-01

Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximize the information gained from advances in fluorescent protein labeling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the PFCs, PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using four advanced modes of microscopy: laser scanning confocal microscopy (LSCM), two-photon fluorescence microscopy, second harmonic generation microscopy, and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image PFCs directly in the mesophyll and thereby easily delimit the “negative space” within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells. PMID:24795734

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.

Fully Hydrated Yeast Cells Imaged with Electron Microscopy

PubMed Central

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

2011-01-01

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

Fully hydrated yeast cells imaged with electron microscopy.

PubMed

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

2011-05-18

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

Morphometric, quantitative, and three-dimensional analysis of the heart muscle fibers of old rats: transmission electron microscopy and high-resolution scanning electron microscopy methods.

PubMed

Cury, Diego Pulzatto; Dias, Fernando José; Sosthenes, Marcia Consentino Kronka; Dos Santos Haemmerle, Carlos Alexandre; Ogawa, Koichi; Da Silva, Marcelo Cavenaghi Pereira; Mardegan Issa, João Paulo; Iyomasa, Mamie Mizusaki; Watanabe, Ii-Sei

2013-02-01

This research investigated the morphological, morphometric, and ultrastructural cardiomyocyte characteristics of male Wistar rats at 18 months of age. The animals were euthanized using an overdose of anesthesia (ketamine and xylazine, 150/10 mg/kg) and perfused transcardially, after which samples were collected for light microscopy, transmission electron microscopy, and high-resolution scanning electron microscopy. The results showed that cardiomyocyte arrangement was disposed parallel between the mitochondria and the A-, I-, and H-bands and their M- and Z-lines from the sarcomere. The sarcomere junction areas had intercalated disks, a specific structure of heart muscle. The ultrastructural analysis revealed several mitochondria of various sizes and shapes intermingled between the blood capillaries and their endothelial cells; some red cells inside vessels are noted. The muscle cell sarcolemma could be observed associated with the described structures. The cardiomyocytes of old rats presented an average sarcomere length of 2.071 ± 0.09 μm, a mitochondrial volume density (Vv) of 0.3383, a mitochondrial average area of 0.537 ± 0.278 μm(2), a mitochondrial average length of 1.024 ± 0.352 μm, an average mitochondrial cristae thickness of 0.038 ± 0.09 μm and a ratio of mitochondrial greater length/lesser length of 1.929 ± 0.965. Of the observed mitochondrial shapes, 23.4% were rounded, 45.3% were elongated, and 31.1% had irregular profiles. In this study, we analyzed the morphology and morphometry of cardiomyocytes in old rats, focusing on mitochondria. These data are important for researchers who focus the changes in cardiac tissue, especially changes owing to pathologies and drug administration that may or may not be correlated with aging. Copyright © 2012 Wiley Periodicals, Inc.

Quantitative PCR Coupled with Melt Curve Analysis for Detection of Selected Pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean Sea▿

PubMed Central

Andree, Karl B.; Fernández-Tejedor, Margarita; Elandaloussi, Laurence M.; Quijano-Scheggia, Sonia; Sampedro, Nagore; Garcés, Esther; Camp, Jordi; Diogène, Jorge

2011-01-01

The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy. PMID:21193668

Advanced techniques for in situ analysis of the biofilm matrix (structure, composition, dynamics) by means of laser scanning microscopy.

PubMed

Neu, Thomas R; Lawrence, John R

2014-01-01

The extracellular constituents in bioaggregates and biofilms can be imaged four dimensionally by using laser scanning microscopy. In this protocol we provide guidance on how to examine the various extracellular compartments in between microbial cells and communities associated with interfaces. The current options for fluorescence staining of matrix compounds and extracellular microhabitats are presented. Furthermore, practical aspects are discussed and useful notes are added. The chapter ends with a brief introduction to other approaches for EPS analysis and an outlook for future needs.

Green synthesis of multifunctional carbon dots from coriander leaves and their potential application as antioxidants, sensors and bioimaging agents.

PubMed

Sachdev, Abhay; Gopinath, P

2015-06-21

In the present study, a facile one-step hydrothermal treatment of coriander leaves for preparing carbon dots (CDs) has been reported. Optical and structural properties of the CDs have been extensively studied by UV-visible and fluorescence spectroscopic, microscopic (transmission electron microscopy, scanning electron microscopy) and X-ray diffraction techniques. Surface functionality and composition of the CDs have been illustrated by elemental analysis and Fourier transform infrared spectroscopy (FTIR). Quenching of the fluorescence of the CDs in the presence of metal ions is of prime significance, hence CDs have been used as a fluorescence probe for sensitive and selective detection of Fe(3+) ions. Eventually, biocompatibility and bioimaging aspects of CDs have been evaluated on lung normal (L-132) and cancer (A549) cell lines. Qualitative analysis of cellular uptake of CDs has been pursued through fluorescence microscopy, while quantitative analysis using a flow cytometer provided an insight into the concentration and cell-type dependent uptake of CDs. The article further investigates the antioxidant activity of CDs. Therefore, we have validated the practicality of CDs obtained from a herbal carbon source for versatile applications.

microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling

NASA Astrophysics Data System (ADS)

Comi, Troy J.; Neumann, Elizabeth K.; Do, Thanh D.; Sweedler, Jonathan V.

2017-09-01

Image-guided mass spectrometry (MS) profiling provides a facile framework for analyzing samples ranging from single cells to tissue sections. The fundamental workflow utilizes a whole-slide microscopy image to select targets of interest, determine their spatial locations, and subsequently perform MS analysis at those locations. Improving upon prior reported methodology, a software package was developed for working with microscopy images. microMS, for microscopy-guided mass spectrometry, allows the user to select and profile diverse samples using a variety of target patterns and mass analyzers. Written in Python, the program provides an intuitive graphical user interface to simplify image-guided MS for novice users. The class hierarchy of instrument interactions permits integration of new MS systems while retaining the feature-rich image analysis framework. microMS is a versatile platform for performing targeted profiling experiments using a series of mass spectrometers. The flexibility in mass analyzers greatly simplifies serial analyses of the same targets by different instruments. The current capabilities of microMS are presented, and its application for off-line analysis of single cells on three distinct instruments is demonstrated. The software has been made freely available for research purposes. [Figure not available: see fulltext.

microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling.

PubMed

Comi, Troy J; Neumann, Elizabeth K; Do, Thanh D; Sweedler, Jonathan V

2017-09-01

Image-guided mass spectrometry (MS) profiling provides a facile framework for analyzing samples ranging from single cells to tissue sections. The fundamental workflow utilizes a whole-slide microscopy image to select targets of interest, determine their spatial locations, and subsequently perform MS analysis at those locations. Improving upon prior reported methodology, a software package was developed for working with microscopy images. microMS, for microscopy-guided mass spectrometry, allows the user to select and profile diverse samples using a variety of target patterns and mass analyzers. Written in Python, the program provides an intuitive graphical user interface to simplify image-guided MS for novice users. The class hierarchy of instrument interactions permits integration of new MS systems while retaining the feature-rich image analysis framework. microMS is a versatile platform for performing targeted profiling experiments using a series of mass spectrometers. The flexibility in mass analyzers greatly simplifies serial analyses of the same targets by different instruments. The current capabilities of microMS are presented, and its application for off-line analysis of single cells on three distinct instruments is demonstrated. The software has been made freely available for research purposes. Graphical Abstract ᅟ.

Inhomogeneity of Cellulose Microfibril Assembly in Plant Cell Walls Revealed with Sum Frequency Generation Microscopy.

PubMed

Huang, Shixin; Makarem, Mohamadamin; Kiemle, Sarah N; Hamedi, Hossein; Sau, Moujhuri; Cosgrove, Daniel J; Kim, Seong H

2018-05-17

Sum frequency generation (SFG) vibrational spectroscopy can selectively detect and analyze noncentrosymmetric components interspersed in amorphous matrices; this principle has been used for studies of nanoscale structure and mesoscale assembly of cellulose in plant cell walls. However, the spectral information averaged over a large area or volume cannot provide regiospecific or tissue-specific information of different cells in plants. This study demonstrates spatially resolved SFG analysis and imaging by combining a broad-band SFG spectroscopy system with an optical microscope. The system was designed to irradiate both narrow-band 800 nm and broad-band tunable IR beams through a single reflective objective lens, but from opposite sides of the surface normal direction of the sample. The developed technique was used to reveal inhomogeneous distributions of cellulose microfibrils within single cell walls, such as cotton fibers and onion epidermis as well as among different tissues in Arabidopsis inflorescence stems and bamboo culms. SFG microscopy can be used for vibrational spectroscopic imaging of other biological systems in complement to conventional Fourier transform infrared spectroscopy and confocal Raman microscopy.

Synthesis of strongly fluorescent molybdenum disulfide nanosheets for cell-targeted labeling.

PubMed

Wang, Nan; Wei, Fang; Qi, Yuhang; Li, Hongxiang; Lu, Xin; Zhao, Guoqiang; Xu, Qun

2014-11-26

MoS2 nanosheets with polydispersity of the lateral dimensions from natural mineral molybdenite have been prepared in the emulsions microenvironment built by the water/surfactant/CO2 system. The size, thickness, and atomic structure are characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), and laser-scattering particle size analysis. Meanwhile, by the analysis of photoluminescence spectroscopy and microscope, the MoS2 nanosheets with smaller lateral dimensions exhibit extraordinary photoluminescence properties different from those with relatively larger lateral dimensions. The discovery of the excitation dependent photoluminescence for MoS2 nanosheets makes them potentially of interests for the applications in optoelectronics and biology. Moreover, we demonstrate that the fabricated MoS2 nanosheets can be a nontoxic fluorescent label for cell-targeted labeling application.

Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy.

PubMed

Hirano, Yu; Kodama, Mikie; Shibuya, Masahiro; Maki, Yoshiyuki; Komatsu, Yasuo

2014-02-15

The contractile behavior of cardiomyocytes can be monitored by measuring their action potentials, and the analysis is essential for screening the safety of potential drugs. However, immobilizing cardiac cells on a specific electrode is considerably complicated. In this study, we demonstrate that scanning electrochemical microscopy (SECM) can be used to analyze rapid topographic changes in beating cardiomyocytes in a standard culture dish. Various cardiomyocyte contraction parameters and oxygen consumption based on cell respiration could be determined from SECM data. We also confirmed that cellular changes induced by adding the cardiotonic agent digoxin were conveniently monitored by this SECM system. These results show that SECM can be a potentially powerful tool for use in drug development for cardiovascular diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

Automated method for the rapid and precise estimation of adherent cell culture characteristics from phase contrast microscopy images.

PubMed

Jaccard, Nicolas; Griffin, Lewis D; Keser, Ana; Macown, Rhys J; Super, Alexandre; Veraitch, Farlan S; Szita, Nicolas

2014-03-01

The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (<1 s per 1,208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Automated Method for the Rapid and Precise Estimation of Adherent Cell Culture Characteristics from Phase Contrast Microscopy Images

PubMed Central

Jaccard, Nicolas; Griffin, Lewis D; Keser, Ana; Macown, Rhys J; Super, Alexandre; Veraitch, Farlan S; Szita, Nicolas

2014-01-01

The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (<1 s per 1,208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license. Biotechnol. Bioeng. 2014;111: 504–517. © 2013 Wiley Periodicals, Inc. PMID:24037521

A new 3D tracking method for cell mechanics investigation exploiting the capabilities of digital holography in microscopy

NASA Astrophysics Data System (ADS)

Miccio, L.; Memmolo, P.; Merola, F.; Fusco, S.; Netti, P. A.; Ferraro, P.

2014-03-01

A method for 3D tracking has been developed exploiting Digital Holography features in Microscopy (DHM). In the framework of self-consistent platform for manipulation and measurement of biological specimen we use DHM for quantitative and completely label free analysis of samples with low amplitude contrast. Tracking capability extend the potentiality of DHM allowing to monitor the motion of appropriate probes and correlate it with sample properties. Complete 3D tracking has been obtained for the probes avoiding the amplitude refocusing in traditional tracking processes. Moreover, in biology and biomedical research fields one of the main topic is the understanding of morphology and mechanics of cells and microorganisms. Biological samples present low amplitude contrast that limits the information that can be retrieved through optical bright-field microscope measurements. The main effect on light propagating in such objects is in phase. This is known as phase-retardation or phase-shift. DHM is an innovative and alternative approach in microscopy, it's a good candidate for no-invasive and complete specimen analysis because its main characteristic is the possibility to discern between intensity and phase information performing quantitative mapping of the Optical Path Length. In this paper, the flexibility of DH is employed to analyze cell mechanics of unstained cells subjected to appropriate stimuli. DHM is used to measure all the parameters useful to understand the deformations induced by external and controlled stresses on in-vitro cells. Our configuration allows 3D tracking of micro-particles and, simultaneously, furnish quantitative phase-contrast maps. Experimental results are presented and discussed for in vitro cells.

Adhesion modification of neural stem cells induced by nanoscale ripple patterns

NASA Astrophysics Data System (ADS)

Pedraz, P.; Casado, S.; Rodriguez, V.; Giordano, M. C.; Buatier de Mongeot, F.; Ayuso-Sacido, A.; Gnecco, E.

2016-03-01

We have studied the influence of anisotropic nanopatterns (ripples) on the adhesion and morphology of mouse neural stem cells (C17.2) on glass substrates using cell viability assay, optical microscopy and atomic force microscopy. The ripples were produced by defocused ion beam sputtering with inert Ar ions, which physically remove atoms from the surface at the energy of 800 eV. The ripple periodicity (∼200 nm) is comparable to the thickness of the cytoplasmatic microspikes (filopodia) which link the stem cells to the substrate. All methods show that the cell adhesion is significantly lowered compared to the same type of cells on flat glass surfaces. Furthermore, the AFM analysis reveals that the filopodia tend to be trapped parallel or perpendicular to the ripples, which limits the spreading of the stem cell on the rippled substrate. This opens the perspective of controlling the micro-adhesion of stem cells and the orientation of their filopodia by tuning the anisotropic substrate morphology without chemical reactions occurring at the surface.

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

In vivo laser confocal microscopy findings in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2012-01-01

The purpose of this study was to investigate pathological changes of the corneal cell layer in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy by in vivo laser corneal confocal microscopy. Two patients were evaluated using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT 2-RCM). The affected corneal areas of both patients were examined. Image analysis was performed to identify corneal epithelial and stromal deposits correlated with this dystrophy. Variously shaped (linear, multilaminar, curvilinear, ring-shape, geographic) highly reflective materials were observed in the "map" area, mainly in the basal epithelial cell layer. In "fingerprint" lesions, multiple linear and curvilinear hyporeflective lines were observed. Additionally, in the affected corneas, infiltration of possible Langerhans cells and other inflammatory cells was observed as highly reflective Langerhans cell-like or dot images. Finally, needle-shaped materials were observed in one patient. HRT 2-RCM laser confocal microscopy is capable of identifying corneal microstructural changes related to map-dot-fingerprint corneal dystrophy in vivo. The technique may be useful in elucidating the pathogenesis and natural course of map-dot-fingerprint corneal dystrophy and other similar basement membrane abnormalities.

Folate-grafted boron nitride nanotubes: possible exploitation in cancer therapy.

PubMed

Ferreira, Tiago H; Marino, Attilio; Rocca, Antonella; Liakos, Ioannis; Nitti, Simone; Athanassiou, Athanassia; Mattoli, Virgilio; Mazzolai, Barbara; de Sousa, Edesia M B; Ciofani, Gianni

2015-03-15

Boron nitride nanotubes (BNNTs) have generated considerable interest among the scientific community because of their unique physical and chemical properties. They present good chemical inertness, high thermal stability, and optimal resistance to oxidation, that make them ideal candidates for biomedical applications, in particular as nanovectors for drug, gene and protein delivery into the cells. In this study, BNNTs were prepared through a synthesis based on a chemical vapor deposition (CVD) method, and thereafter chemically functionalized with folic acid. The obtained nanostructures have been characterized by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The characterization showed efficiently functionalized BNNTs of length of about 1 μm. Furthermore, confocal laser microscopy demonstrated that our nanotubes can be fluorescently-traced under appropriate excitation. Thanks to this property, it has been possible to investigate their internalization by HeLa cells through confocal microscopy, demonstrating that the BNNT up-take clearly increases after the functionalization with folate, a result confirmed by inductively coupled plasma (ICP) assessment of boron content inside the treated cell cultures. Copyright © 2015 Elsevier B.V. All rights reserved.

Green synthesis, characterization, and anticancer activity of hyaluronan/zinc oxide nanocomposite

PubMed Central

Namvar, Farideh; Azizi, Susan; Rahman, Heshu Sulaiman; Mohamad, Rosfarizan; Rasedee, Abdullah; Soltani, Mozhgan; Rahim, Raha Abdul

2016-01-01

The study describes an in situ green biosynthesis of zinc oxide nanocomposite using the seaweed Sargassum muticum water extract and hyaluronan biopolymer. The morphology and optical properties of the hyaluronan/zinc oxide (HA/ZnO) nanocomposite were determined by Fourier transform infrared spectroscopy, X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy, and ultraviolet–vis analysis. Electron microscopy and X-ray diffraction analysis showed that the zinc oxide nanoparticles were polydispersed with a mean size of 10.2±1.5 nm. The nanoparticles were mostly hexagonal in crystalline form. The HA/ZnO nanocomposite showed the absorption properties in the ultraviolet zone that is ascribed to the band gap of zinc oxide nanocomposite. In the cytotoxicity study, cancer cells, pancreatic adenocarcinoma (PANC-1), ovarian adenocarcinoma (CaOV-3), colonic adenocarcinoma (COLO205), and acute promyelocytic leukemia (HL-60) cells were treated with HA/ZnO nanocomposite. At 72 hours of treatment, the half maximal inhibitory concentration (IC50) value via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was 10.8±0.3 μg/mL, 15.4±1.2 μg/mL, 12.1±0.9 μg/mL, and 6.25±0.5 μg/mL for the PANC-1, CaOV-3, COLO-205, and HL-60 cells, respectively, showing that the composite is most toxic to the HL-60 cells. On the other hand, HA/ZnO nanocomposite treatment for 72 hours did not cause toxicity to the normal human lung fibroblast (MRC-5) cell line. Using fluorescent dyes and flow cytometry analysis, HA/ZnO nanocomposite caused G2/M cell cycle arrest and stimulated apoptosis-related increase in caspase-3 and -7 activities of the HL-60 cells. Thus, the study shows that the HA/ZnO nanocomposite produced through green synthesis has great potential to be developed into an efficacious therapeutic agent for cancers. PMID:27555781

Ultrananocrystalline diamond-coated nanoporous membranes support SK-N-SH neuroblastoma endothelial cell attachment.

PubMed

Yang, Kai-Hung; Nguyen, Alexander K; Goering, Peter L; Sumant, Anirudha V; Narayan, Roger J

2018-06-06

Ultrananocrystalline diamond (UNCD) has been demonstrated to have attractive features for biomedical applications and can be combined with nanoporous membranes for applications in drug delivery systems, biosensing, immunoisolation and single molecule analysis. In this study, free-standing nanoporous UNCD membranes with pore sizes of 100 or 400 nm were fabricated by directly depositing ultrathin UNCD films on nanoporous silicon nitride membranes and then etching away silicon nitride using reactive ion etching. Successful deposition of UNCD on the substrate with a novel process was confirmed with Raman spectroscopy, X-ray photoelectron spectroscopy, cross-section scanning electron microscopy (SEM) and transmission electron microscopy. Both sample types exhibited uniform geometry and maintained a clear hexagonal pore arrangement. Cellular attachment of SK-N-SH neuroblastoma endothelial cells was examined using confocal microscopy and SEM. Attachment of SK-N-SH cells onto UNCD membranes on both porous regions and solid surfaces was shown, indicating the potential use of UNCD membranes in biomedical applications such as biosensors and tissue engineering scaffolds.

Analysis of live cell images: Methods, tools and opportunities.

PubMed

Nketia, Thomas A; Sailem, Heba; Rohde, Gustavo; Machiraju, Raghu; Rittscher, Jens

2017-02-15

Advances in optical microscopy, biosensors and cell culturing technologies have transformed live cell imaging. Thanks to these advances live cell imaging plays an increasingly important role in basic biology research as well as at all stages of drug development. Image analysis methods are needed to extract quantitative information from these vast and complex data sets. The aim of this review is to provide an overview of available image analysis methods for live cell imaging, in particular required preprocessing image segmentation, cell tracking and data visualisation methods. The potential opportunities recent advances in machine learning, especially deep learning, and computer vision provide are being discussed. This review includes overview of the different available software packages and toolkits. Copyright © 2017. Published by Elsevier Inc.

Characterization of dominant giant rod-shaped magnetotactic bacteria from a low tide zone of the China Sea

NASA Astrophysics Data System (ADS)

Teng, Zhaojie; Zhang, Wenyan; Chen, Yiran; Pan, Hongmiao; Xiao, Tian; Wu, Long-Fei

2017-08-01

Magnetotactic bacteria are a group of Gram-negative bacteria that synthesize magnetic crystals, enabling them to navigate in relation to magnetic field lines. Morphologies of magnetotactic bacteria include spirillum, coccoid, rod, vibrio, and multicellular morphotypes. The coccid shape is generally the most abundant morphotype among magnetotactic bacteria. Here we describe a species of giant rod-shaped magnetotactic bacteria (designated QR-1) collected from sediment in the low tide zone of Huiquan Bay (Yellow Sea, China). This morphotype accounted for 90% of the magnetotactic bacteria collected, and the only taxonomic group which was detected in the sampling site. Microscopy analysis revealed that QR-1 cells averaged (6.71±1.03)×(1.54±0.20) μm in size, and contained in each cell 42-146 magnetosomes that are arranged in a bundle formed one to four chains along the long axis of the cell. The QR-1 cells displayed axial magnetotaxis with an average velocity of 70±28 μm/s. Transmission electron microscopy based analysis showed that QR-1 cells had two tufts of flagella at each end. Phylogenetic analysis of the 16S rRNA genes revealed that QR-1 together with three other rod-shaped uncultivated magnetotactic bacteria are clustered into a deep branch of Alphaproteobacteria.

Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes.

PubMed

Lemcke, Heiko; Peukert, Janine; Voronina, Natalia; Skorska, Anna; Steinhoff, Gustav; David, Robert

2016-09-01

Small antisense RNAs like miRNA and siRNA are of crucial importance in cardiac physiology, pathology and, moreover, can be applied as therapeutic agents for the treatment of cardiovascular diseases. Identification of novel strategies for miRNA/siRNA therapy requires a comprehensive understanding of the underlying mechanisms. Emerging data suggest that small RNAs are transferred between cells via gap junctions and provoke gene regulatory effects in the recipient cell. To elucidate the role of miRNA/siRNA as signalling molecules, suitable tools are required that will allow the analysis of these small RNAs at the cellular level. In the present study, we applied 3 dimensional fluorescence recovery after photo bleaching microscopy (3D-FRAP) to visualise and quantify the gap junctional exchange of small RNAs between neonatal cardiomyocytes in real time. Cardiomyocytes were transfected with labelled miRNA and subjected to FRAP microscopy. Interestingly, we observed recovery rates of 21% already after 13min, indicating strong intercellular shuttling of miRNA, which was significantly reduced when connexin43 was knocked down. Flow cytometry analysis confirmed our FRAP results. Furthermore, using an EGFP/siRNA reporter construct we demonstrated that the intercellular transfer does not affect proper functioning of small RNAs, leading to marker gene silencing in the recipient cell. Our results show that 3D-FRAP microscopy is a straightforward, non-invasive live cell imaging technique to evaluate the GJ-dependent shuttling of small RNAs with high spatio-temporal resolution. Moreover, the data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation where small RNAs act as signalling molecules within the intercellular network. Copyright © 2016 Elsevier Ltd. All rights reserved.

Segmentation and Quantitative Analysis of Apoptosis of Chinese Hamster Ovary Cells from Fluorescence Microscopy Images.

PubMed

Du, Yuncheng; Budman, Hector M; Duever, Thomas A

2017-06-01

Accurate and fast quantitative analysis of living cells from fluorescence microscopy images is useful for evaluating experimental outcomes and cell culture protocols. An algorithm is developed in this work to automatically segment and distinguish apoptotic cells from normal cells. The algorithm involves three steps consisting of two segmentation steps and a classification step. The segmentation steps are: (i) a coarse segmentation, combining a range filter with a marching square method, is used as a prefiltering step to provide the approximate positions of cells within a two-dimensional matrix used to store cells' images and the count of the number of cells for a given image; and (ii) a fine segmentation step using the Active Contours Without Edges method is applied to the boundaries of cells identified in the coarse segmentation step. Although this basic two-step approach provides accurate edges when the cells in a given image are sparsely distributed, the occurrence of clusters of cells in high cell density samples requires further processing. Hence, a novel algorithm for clusters is developed to identify the edges of cells within clusters and to approximate their morphological features. Based on the segmentation results, a support vector machine classifier that uses three morphological features: the mean value of pixel intensities in the cellular regions, the variance of pixel intensities in the vicinity of cell boundaries, and the lengths of the boundaries, is developed for distinguishing apoptotic cells from normal cells. The algorithm is shown to be efficient in terms of computational time, quantitative analysis, and differentiation accuracy, as compared with the use of the active contours method without the proposed preliminary coarse segmentation step.

Intracellular inclusion bodies in 14 patients with B cell lymphoproliferative disorders.

PubMed Central

Peters, O; Thielemans, C; Steenssens, L; De Waele, M; Hijmans, W; Van Camp, B

1984-01-01

Two types of intracytoplasmic inclusion were detected by immunofluorescence microscopy in 12 patients with chronic lymphocytic leukaemia and two patients with a leukaemic phase of well differentiated lymphocytic lymphoma. Further analysis with light- and electron microscopy, showed that most inclusion bodies were rod-like crystalline structures. However, in three patients they consisted of amorphous vesicular precipitates. Immunological studies revealed the presence of immunoglobulins of the same class and type at the cell surface as well as in the inclusion bodies. The monoclonal immunoglobulins were all of lambda type except in two cases. The origin of immunoglobulin inclusion bodies in B cell malignancies is discussed in relation to published data and our own observation in one patient followed during treatment. Images PMID:6323543

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.

Imaging and reconstruction of cell cortex structures near the cell surface

NASA Astrophysics Data System (ADS)

Jin, Luhong; Zhou, Xiaoxu; Xiu, Peng; Luo, Wei; Huang, Yujia; Yu, Feng; Kuang, Cuifang; Sun, Yonghong; Liu, Xu; Xu, Yingke

2017-11-01

Total internal reflection fluorescence microscopy (TIRFM) provides high optical sectioning capability and superb signal-to-noise ratio for imaging of cell cortex structures. The development of multi-angle (MA)-TIRFM permits high axial resolution imaging and reconstruction of cellular structures near the cell surface. Cytoskeleton is composed of a network of filaments, which are important for maintenance of cell function. The high-resolution imaging and quantitative analysis of filament organization would contribute to our understanding of cytoskeleton regulation in cell. Here, we used a custom-developed MA-TIRFM setup, together with stochastic photobleaching and single molecule localization method, to enhance the lateral resolution of TIRFM imaging to about 100 nm. In addition, we proposed novel methods to perform filament segmentation and 3D reconstruction from MA-TIRFM images. Furthermore, we applied these methods to study the 3D localization of cortical actin and microtubule structures in U373 cancer cells. Our results showed that cortical actins localize ∼ 27 nm closer to the plasma membrane when compared with microtubules. We found that treatment of cells with chemotherapy drugs nocodazole and cytochalasin B disassembles cytoskeletal network and induces the reorganization of filaments towards the cell periphery. In summary, this study provides feasible approaches for 3D imaging and analyzing cell surface distribution of cytoskeletal network. Our established microscopy platform and image analysis toolkits would facilitate the study of cytoskeletal network in cells.

Differentiating Pseudomonas sp. strain ADP cells in suspensions and biofilms using Raman spectroscopy and scanning electron microscopy.

PubMed

Henry, Victoria A; Jessop, Julie L P; Peeples, Tonya L

2017-02-01

High quality spectra of Pseudomonas sp. strain ADP in the planktonic and biofilm state were obtained using Raman microspectroscopy. These spectra enabled the identification of key differences between free and biofilm cells in the fingerprint region of Raman spectra in the nucleic acid, carbohydrate, and protein regions. Scanning electron microscopy (SEM) enabled detailed visualization of ADP biofilm with confirmation of associated extracellular matrix structure. Following extraction and Raman analysis of extracellular polymeric substances, Raman spectral differences between free and biofilm cells were largely attributed to the contribution of extracellular matrix components produced in mature biofilms. Raman spectroscopy complemented with SEM proves to be useful in distinguishing physiological properties among cells of the same species. Graphical Abstract Raman spectroscopy complemented with SEM proves to be useful in distinguishing physiological properties among cells of the same species.

Two-photon confocal microscopy in wound healing

NASA Astrophysics Data System (ADS)

Navarro, Fernando A.; So, Peter T. C.; Driessen, Antoine; Kropf, Nina; Park, Christine S.; Huertas, Juan C.; Lee, Hoon B.; Orgill, Dennis P.

2001-04-01

Advances in histopathology and immunohistochemistry have allowed for precise microanatomic detail of tissues. Two Photon Confocal Microscopy (TPCM) is a new technology useful in non-destructive analysis of tissue. Laser light excites the natural florophores, NAD(P)H and NADP+ and the scattering patterns of the emitted light are analyzed to reconstruct microanatomic features. Guinea pig skin was studied using TPCM and skin preparation methods including chemical depilation and tape striping. Results of TPCM were compared with conventional hematoxylin and eosin microscopy. Two-dimensional images were rendered from the three dimensional reconstructions. Images of deeper layers including basal cells and the dermo-epidermal junction improved after removing the stratum corneum with chemical depilation or tape stripping. TCPM allows good resolution of corneocytes, basal cells and collagen fibers and shows promise as a non-destructive method to study wound healing.

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.

Image analysis driven single-cell analytics for systems microbiology.

PubMed

Balomenos, Athanasios D; Tsakanikas, Panagiotis; Aspridou, Zafiro; Tampakaki, Anastasia P; Koutsoumanis, Konstantinos P; Manolakos, Elias S

2017-04-04

Time-lapse microscopy is an essential tool for capturing and correlating bacterial morphology and gene expression dynamics at single-cell resolution. However state-of-the-art computational methods are limited in terms of the complexity of cell movies that they can analyze and lack of automation. The proposed Bacterial image analysis driven Single Cell Analytics (BaSCA) computational pipeline addresses these limitations thus enabling high throughput systems microbiology. BaSCA can segment and track multiple bacterial colonies and single-cells, as they grow and divide over time (cell segmentation and lineage tree construction) to give rise to dense communities with thousands of interacting cells in the field of view. It combines advanced image processing and machine learning methods to deliver very accurate bacterial cell segmentation and tracking (F-measure over 95%) even when processing images of imperfect quality with several overcrowded colonies in the field of view. In addition, BaSCA extracts on the fly a plethora of single-cell properties, which get organized into a database summarizing the analysis of the cell movie. We present alternative ways to analyze and visually explore the spatiotemporal evolution of single-cell properties in order to understand trends and epigenetic effects across cell generations. The robustness of BaSCA is demonstrated across different imaging modalities and microscopy types. BaSCA can be used to analyze accurately and efficiently cell movies both at a high resolution (single-cell level) and at a large scale (communities with many dense colonies) as needed to shed light on e.g. how bacterial community effects and epigenetic information transfer play a role on important phenomena for human health, such as biofilm formation, persisters' emergence etc. Moreover, it enables studying the role of single-cell stochasticity without losing sight of community effects that may drive it.

Investigation of podosome ring protein arrangement using localization microscopy images.

PubMed

Staszowska, Adela D; Fox-Roberts, Patrick; Foxall, Elizabeth; Jones, Gareth E; Cox, Susan

2017-02-15

Podosomes are adhesive structures formed on the plasma membrane abutting the extracellular matrix of macrophages, osteoclasts, and dendritic cells. They consist of an f-actin core and a ring structure composed of integrins and integrin-associated proteins. The podosome ring plays a major role in adhesion to the underlying extracellular matrix, but its detailed structure is poorly understood. Recently, it has become possible to study the nano-scale structure of podosome rings using localization microscopy. Unlike traditional microscopy images, localization microscopy images are reconstructed using discrete points, meaning that standard image analysis methods cannot be applied. Here, we present a pipeline for podosome identification, protein position calculation, and creating a podosome ring model for use with localization microscopy data. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy.

PubMed

Dukes, Madeline J; Ramachandra, Ranjan; Baudoin, Jean-Pierre; Gray Jerome, W; de Jonge, Niels

2011-06-01

Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells. Copyright © 2011 Elsevier Inc. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2015-03-01

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

Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3 nm precision using aberration-corrected scanning transmission electron microscopy

PubMed Central

Dukes, Madeline J.; Ramachandra, Ranjan; Baudoin, Jean-Pierre; Jerome, W. Gray; de Jonge, Niels

2011-01-01

Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3 nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under the electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells. PMID:21440635

Microscopic analysis of cell death by metabolic stress-induced autophagy in prostate cancer

NASA Astrophysics Data System (ADS)

Changou, Chun; Cheng, R. Holland; Bold, Richard; Kung, Hsing-Jien; Chuang, Frank Y. S.

2013-02-01

Autophagy is an intracellular recycling mechanism that helps cells to survive against environmental stress and nutritional starvation. We have recently shown that prostate cancers undergo metabolic stress and caspase-independent cell death following exposure to arginine deiminase (ADI, an enzyme that degrades arginine in tissue). The aims of our current investigation into the application of ADI as a novel cancer therapy are to identify the components mediating tumor cell death, and to determine the role of autophagy (stimulated by ADI and/or rapamycin) on cell death. Using advanced fluorescence microscopy techniques including 3D deconvolution and superresolution structured-illumination microscopy (SIM), we show that prostate tumor cells that are killed after exposure to ADI for extended periods, exhibit a morphology that is distinct from caspase-dependent apoptosis; and that autophagosomes forming as a result of ADI stimulation contain DAPI-stained nuclear material. Fluorescence imaging (as well as cryo-electron microscopy) show a breakdown of both the inner and outer nuclear membranes at the interface between the cell nucleus and aggregated autophagolysosomes. Finally, the addition of N-acetyl cysteine (or NAC, a scavenger for reactive oxygen species) effectively abolishes the appearance of autophagolysosomes containing nuclear material. We hope to continue this research to understand the processes that govern the survival or death of these tumor cells, in order to develop methods to improve the efficacy of cancer pharmacotherapy.

High-speed stimulated Raman scattering microscopy for studying the metabolic diversity of motile Euglena gracilis

NASA Astrophysics Data System (ADS)

Suzuki, Y.; Wakisaka, Y.; Iwata, O.; Nakashima, A.; Ito, T.; Hirose, M.; Domon, R.; Sugawara, M.; Tsumura, N.; Watarai, H.; Shimobaba, T.; Suzuki, K.; Goda, K.; Ozeki, Y.

2017-02-01

Microalgae have been receiving great attention for their ability to produce biomaterials that are applicable for food supplements, drugs, biodegradable plastics, and biofuels. Among such microalgae, Euglena gracilis has become a popular species by virtue of its capability of accumulating useful metabolites including paramylon and lipids. In order to maximize the production of desired metabolites, it is essential to find ideal culturing conditions and to develop efficient methods for genetic transformation. To achieve this, understanding and controlling cell-to-cell variations in response to external stress is essential, with chemically specific analysis of microalgal cells including E. gracilis. However, conventional analytical tools such as fluorescence microscopy and spontaneous Raman scattering are not suitable for evaluation of diverse populations of motile microalgae, being restricted either by the requirement for fluorescent labels or a limited imaging speed, respectively. Here we demonstrate video-rate label-free metabolite imaging of live E. gracilis using stimulated Raman scattering (SRS) - an optical spectroscopic method for probing the vibrational signatures of molecules with orders of magnitude higher sensitivity than spontaneous Raman scattering. Our SRS's highspeed image acquisition (27 metabolite images per second) allows for population analysis of live E. gracilis cells cultured under nitrogen-deficiency - a technique for promoting the accumulation of paramylon and lipids within the cell body. Thus, our SRS system's fast imaging capability enables quantification and analysis of previously unresolvable cell-to-cell variations in the metabolite accumulation of large motile E. gracilis cell populations.

Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells.

PubMed

Wojcik, Michal; Hauser, Margaret; Li, Wan; Moon, Seonah; Xu, Ke

2015-06-11

The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results.

Three-dimensional bright-field scanning transmission electron microscopy elucidate novel nanostructure in microbial biofilms.

PubMed

Hickey, William J; Shetty, Ameesha R; Massey, Randall J; Toso, Daniel B; Austin, Jotham

2017-01-01

Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright-field scanning transmission electron microscopy (BF-STEM) and in the present report BF-STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon-degrading soil bacterium, Delftia acidovorans Cs1-4. Dual-axis BF-STEM enabled high-resolution 3-D tomographic recontructions (6-10 nm) visualization of thick (1250 and 1500 nm) sections. The 3-D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF-STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual-axis BF-STEM tomography to obtain high-resolution 3-D images of novel nanostructures in bacterial biofilms. Future work with dual-axis BF-STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

General Staining and Segmentation Procedures for High Content Imaging and Analysis.

PubMed

Chambers, Kevin M; Mandavilli, Bhaskar S; Dolman, Nick J; Janes, Michael S

2018-01-01

Automated quantitative fluorescence microscopy, also known as high content imaging (HCI), is a rapidly growing analytical approach in cell biology. Because automated image analysis relies heavily on robust demarcation of cells and subcellular regions, reliable methods for labeling cells is a critical component of the HCI workflow. Labeling of cells for image segmentation is typically performed with fluorescent probes that bind DNA for nuclear-based cell demarcation or with those which react with proteins for image analysis based on whole cell staining. These reagents, along with instrument and software settings, play an important role in the successful segmentation of cells in a population for automated and quantitative image analysis. In this chapter, we describe standard procedures for labeling and image segmentation in both live and fixed cell samples. The chapter will also provide troubleshooting guidelines for some of the common problems associated with these aspects of HCI.

Magnetite nanoparticles functionalized with α-tocopheryl succinate (α-TOS) promote selective cervical cancer cell death

NASA Astrophysics Data System (ADS)

Angulo-Molina, Aracely; Méndez-Rojas, Miguel Ángel; Palacios-Hernández, Teresa; Contreras-López, Oscar Edel; Hirata-Flores, Gustavo Alonso; Flores-Alonso, Juan Carlos; Merino-Contreras, Saul; Valenzuela, Olivia; Hernández, Jesús; Reyes-Leyva, Julio

2014-08-01

The vitamin E analog α-tocopheryl succinate (α-TOS) selectively induces apoptosis in several cancer cells, but it is sensitive to esterases present in cervical cancer cells. Magnetite nanoparticles (Nps) were prepared by a reduction-coprecipitation method; their surface was silanized and conjugated to α-TOS to enhance its resistance. Morphology, size, and crystal structure were analyzed by scanning electron microscopy, transmission electron microscopy, and selected area electron diffraction. Chemical composition was analyzed by energy-dispersive X-ray spectroscopy; functional groups were determined by Fourier transform infrared spectroscopy; and α-TOS content was estimated by thermogravimetric analysis. The cytotoxic activity of α-TOS-Nps was evaluated in non-malignant fibroblasts and cervical cancer cells by means of the colorimetric MTT viability test. Intracellular localization was identified by confocal laser scanning microscopy. Characterization of α-TOS-Nps revealed sphere-like Nps with 15 nm average size, formed by mineral and organic constituents with high stability. α-TOS-Nps were internalized in the nucleus and selectively affected the viability of cervical cancer cells in a dose- and time-dependent manner but were biocompatible with non-malignant fibroblasts. In conclusion, functionalization of magnetite Nps protected the cytotoxic activity of α-TOS in non-sensitive cervical cancer cells.

Proof-of-principle for SERS imaging of Aspergillus nidulans hyphae using in vivo synthesis of gold nanoparticles.

PubMed

Prusinkiewicz, Martin A; Farazkhorasani, Fatemeh; Dynes, James J; Wang, Jian; Gough, Kathleen M; Kaminskyj, Susan G W

2012-11-07

High spatial resolution methods to assess the physiology of growing cells should permit analysis of fungal biochemical composition. Whole colony methods cannot capture the details of physiology and organism-environment interaction, in part because the structure, function and composition of fungal hyphae vary within individual cells depending on their distance from the growing apex. Surface Enhanced Raman Scattering (SERS) can provide chemical information on materials that are in close contact with appropriate metal substrates, such as nanopatterned gold surfaces and gold nanoparticles (AuNPs). Since nanoparticles can be generated by living cells, we have created conditions for AuNP formation within and on the surface of Aspergillus nidulans hyphae in order to explore their potential for SERS analysis. AuNP distribution and composition have been assessed by UV-Vis spectroscopy, fluorescence light microscopy, transmission electron microscopy, and scanning transmission X-ray microscopy. AuNPs were often associated with hyphal walls, both in the peripheral cytoplasm and on the outer wall surface. Interpretation of SERS spectra is challenging, and will require validation for the diversity of organic molecules present. Here, we show proof-of-principle that it is possible to generate SERS spectra from nanoparticles grown in situ by living hyphae.

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

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.

Measurement of marine picoplankton cell size by using a cooled, charge-coupled device camera with image-analyzed fluorescence microscopy.

PubMed Central

Viles, C L; Sieracki, M E

1992-01-01

Accurate measurement of the biomass and size distribution of picoplankton cells (0.2 to 2.0 microns) is paramount in characterizing their contribution to the oceanic food web and global biogeochemical cycling. Image-analyzed fluorescence microscopy, usually based on video camera technology, allows detailed measurements of individual cells to be taken. The application of an imaging system employing a cooled, slow-scan charge-coupled device (CCD) camera to automated counting and sizing of individual picoplankton cells from natural marine samples is described. A slow-scan CCD-based camera was compared to a video camera and was superior for detecting and sizing very small, dim particles such as fluorochrome-stained bacteria. Several edge detection methods for accurately measuring picoplankton cells were evaluated. Standard fluorescent microspheres and a Sargasso Sea surface water picoplankton population were used in the evaluation. Global thresholding was inappropriate for these samples. Methods used previously in image analysis of nanoplankton cells (2 to 20 microns) also did not work well with the smaller picoplankton cells. A method combining an edge detector and an adaptive edge strength operator worked best for rapidly generating accurate cell sizes. A complete sample analysis of more than 1,000 cells averages about 50 min and yields size, shape, and fluorescence data for each cell. With this system, the entire size range of picoplankton can be counted and measured. Images PMID:1610183

Assessing resolution in live cell structured illumination microscopy

NASA Astrophysics Data System (ADS)

Pospíšil, Jakub; Fliegel, Karel; Klíma, Miloš

2017-12-01

Structured Illumination Microscopy (SIM) is a powerful super-resolution technique, which is able to enhance the resolution of optical microscope beyond the Abbe diffraction limit. In the last decade, numerous SIM methods that achieve the resolution of 100 nm in the lateral dimension have been developed. The SIM setups with new high-speed cameras and illumination pattern generators allow rapid acquisition of the live specimen. Therefore, SIM is widely used for investigation of the live structures in molecular and live cell biology. Quantitative evaluation of resolution enhancement in a real sample is essential to describe the efficiency of super-resolution microscopy technique. However, measuring the resolution of a live cell sample is a challenging task. Based on our experimental findings, the widely used Fourier ring correlation (FRC) method does not seem to be well suited for measuring the resolution of SIM live cell video sequences. Therefore, the resolution assessing methods based on Fourier spectrum analysis are often used. We introduce a measure based on circular average power spectral density (PSDca) estimated from a single SIM image (one video frame). PSDca describes the distribution of the power of a signal with respect to its spatial frequency. Spatial resolution corresponds to the cut-off frequency in Fourier space. In order to estimate the cut-off frequency from a noisy signal, we use a spectral subtraction method for noise suppression. In the future, this resolution assessment approach might prove useful also for single-molecule localization microscopy (SMLM) live cell imaging.

Skeletal stem cell and bone implant interactions are enhanced by LASER titanium modification

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

Sisti, Karin E., E-mail: karinellensisti@gmail.com; Biomaterials Group, Institute of Chemistry, São Paulo State University; Federal University of Mato Grosso do Sul

Purpose: To evaluate the osteo-regenerative potential of Titanium (Ti) modified by Light Amplification by Stimulated Emission of Radiation (LASER) beam (Yb-YAG) upon culture with human Skeletal Stem Cells (hSSCs{sup 1}). Methods: Human skeletal cell populations were isolated from the bone marrow of haematologically normal patients undergoing primary total hip replacement following appropriate consent. STRO-1{sup +} hSSC{sup 1} function was examined for 10 days across four groups using Ti discs: i) machined Ti surface group in basal media (Mb{sup 2}), ii) machined Ti surface group in osteogenic media (Mo{sup 3}), iii) LASER-modified Ti group in basal media (Lb{sup 4}) and, iv)more » LASER-modified Ti group in osteogenic media (Lo{sup 5}). Molecular analysis and qRT-PCR as well as functional analysis including biochemistry (DNA, Alkaline Phosphatase (ALP{sup 6}) specific activity), live/dead immunostaining (Cell Tracker Green (CTG{sup 7})/Ethidium Homodimer-1 (EH-1{sup 8})), and fluorescence staining (for vinculin and phalloidin) were undertaken. Inverted, confocal and Scanning Electron Microscopy (SEM) approaches were used to characterise cell adherence, proliferation, and phenotype. Results: Enhanced cell spreading and morphological rearrangement, including focal adhesions were observed following culture of hSSCs{sup 1} on LASER surfaces in both basal and osteogenic conditions. Biochemical analysis demonstrated enhanced ALP{sup 6} specific activity on the hSSCs{sup 1}-seeded on LASER-modified surface in basal culture media. Molecular analysis demonstrated enhanced ALP{sup 6} and osteopontin expression on titanium LASER treated surfaces in basal conditions. SEM, inverted microscopy and confocal laser scanning microscopy confirmed extensive proliferation and migration of human bone marrow stromal cells on all surfaces evaluated. Conclusions: LASER-modified Ti surfaces modify the behaviour of hSSCs.{sup 1} In particular, SSC{sup 1} adhesion, osteogenic gene expression, cell morphology and cytoskeleton structure were affected. The current studies show Ti LASER modification can enhance the osseointegration between Ti and skeletal cells, with important implications for orthopaedic application. - Highlights: • Bone stem cells on LASER Ti surface display enhanced cell growth and viability. • Bone stem cells on LASER Ti surface exhibit marked biocompatibility. • Human bone stem cells on LASER Ti surface exhibit altered morphology. • LASER Ti enhance osteogenic differentiation of human bone skeletal stem cells. • LASER Ti provides a unique approach to enhance osseointegration with the material.« less

Analytical electron microscopy as a powerful tool in plant cell biology: examples using electron energy loss spectroscopy and X-ray microanalysis.

PubMed

Lichtenberger, O; Neumann, D

1997-08-01

Energy filtering transmission electron microscopy in combination with energy dispersive X-ray analysis (EDX) and quantumchemical calculations opens new possibilities for elemental and bone analysis at the ultrastructural level. The possibilities and limitations of these methods, applied to botanical samples, are discussed and some examples are given. Ca-oxalate crystals in plant cell vacuoles show a specific C K-edge in the electron energy loss spectrum (EELS), which allows a more reliable identification than light microscopical or cytochemical methods. In some dicots crystalline inclusions can be observed in different cell compartments, which are identified as silicon dioxide or calcium silicate by the fine structure of the Si L2,3-edge. Their formation is discussed on the basis of EEL-spectra and quantumchemical calculations. Examples concerning heavy metal detoxification are given for some tolerant plants. In Minuartia Zn is bound as Zn-silicate in cell walls; Armeria accumulates Cu in leaf idioblasts by chelation with phenolic compounds and Cd is precipitated as CdS/phytochelatin-complexes in tomato.

Iron- and 4-hydroxy-2-alkylquinoline-containing periplasmic inclusion bodies of Pseudomonas aeruginosa: A chemical analysis

USGS Publications Warehouse

Royt, P.W.; Honeychuck, R.V.; Pant, R.R.; Rogers, M.L.; Asher, L.V.; Lloyd, J.R.; Carlos, W.E.; Belkin, H.E.; Patwardhan, S.

2007-01-01

Dark aggregated particles were seen on pellets of iron-rich, mid-logarithmic phase Pseudomonas aeruginosa. Transmission electron microscopy of these cells showed inclusion bodies in periplasmic vacuoles. Aggregated particles isolated from the spent medium of these cells contained iron as indicated by atomic absorption spectroscopy and by electron paramagnetic resonance spectroscopy that revealed Fe3+. Scanning electron microscopy/energy dispersive X-ray analysis of whole cells revealed the presence of iron-containing particles beneath the surface of the cell, indicating that the isolated aggregates were the intracellular inclusion bodies. Collectively, mass spectroscopy and nuclear magnetic resonance spectroscopy of the isolated inclusion bodies revealed the presence of 3,4-dihydroxy-2-heptylquinoline which is the Pseudomonas quinolone signaling compound (PQS) and an iron chelator; 4-hydroxy-2-heptylquinoline (pseudan VII), which is an iron chelator, antibacterial compound and precursor of PQS; 4-hydroxy-2-nonylquinoline (pseudan IX) which is an iron chelator and antibacterial compound; 4-hydroxy-2-methylquinoline (pseudan I), and 4-hydroxy-2-nonylquinoline N-oxide. ?? 2006 Elsevier Inc. All rights reserved.

Physalis angulata induces in vitro differentiation of murine bone marrow cells into macrophages.

PubMed

da Silva, Bruno José Martins; Rodrigues, Ana Paula D; Farias, Luis Henrique S; Hage, Amanda Anastácia P; Do Nascimento, Jose Luiz M; Silva, Edilene O

2014-10-03

The bone marrow is a hematopoietic tissue that, in the presence of cytokines and growth factors, generates all of the circulating blood cells. These cells are important for protecting the organism against pathogens and for establishing an effective immune response. Previous studies have shown immunomodulatory effects of different products isolated from plant extracts. This study aimed to evaluate the immunomodulatory properties of aqueous Physalis angulata (AEPa) extract on the differentiation of bone marrow cells. Increased cellular area, higher spreading ability and several cytoplasmatic projections were observed in the treated cells, using optical microscopy, suggesting cell differentiation. Furthermore, AEPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. The ultrastructural analysis by Transmission Electron Microscopy of treated cells showed spreading ability, high number of cytoplasmatic projections and increase of autophagic vacuoles. Moreover, a high level of LC3b expression by treated cells was detected by flow cytometry, suggesting an autophagic process. Cell surface expression of F4/80 and CD11b also indicated that AEPa may stimulate differentiation of bone marrow cells mainly into macrophages. In addition, AEPa did not differentiate cells into dendritic cells, as assessed by CD11c analysis. Furthermore, no cytotoxic effects were observed in the cells treated with AEPa. Results demonstrate that AEPa promotes the differentiation of bone marrow cells, particularly into macrophages and may hold promise as an immunomodulating agent.

Physalis angulata induces in vitro differentiation of murine bone marrow cells into macrophages

PubMed Central

2014-01-01

Background The bone marrow is a hematopoietic tissue that, in the presence of cytokines and growth factors, generates all of the circulating blood cells. These cells are important for protecting the organism against pathogens and for establishing an effective immune response. Previous studies have shown immunomodulatory effects of different products isolated from plant extracts. This study aimed to evaluate the immunomodulatory properties of aqueous Physalis angulata (AEPa) extract on the differentiation of bone marrow cells. Results Increased cellular area, higher spreading ability and several cytoplasmatic projections were observed in the treated cells, using optical microscopy, suggesting cell differentiation. Furthermore, AEPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. The ultrastructural analysis by Transmission Electron Microscopy of treated cells showed spreading ability, high number of cytoplasmatic projections and increase of autophagic vacuoles. Moreover, a high level of LC3b expression by treated cells was detected by flow cytometry, suggesting an autophagic process. Cell surface expression of F4/80 and CD11b also indicated that AEPa may stimulate differentiation of bone marrow cells mainly into macrophages. In addition, AEPa did not differentiate cells into dendritic cells, as assessed by CD11c analysis. Furthermore, no cytotoxic effects were observed in the cells treated with AEPa. Conclusion Results demonstrate that AEPa promotes the differentiation of bone marrow cells, particularly into macrophages and may hold promise as an immunomodulating agent. PMID:25281406

[A STUDY OF THE ISOLATED BACTERIOPHAGE ΦAB-SP7 ADSORPTION ON THE CELL SURFACE OF THE AZOSPIRILLUM BRASILENSE SP7].

PubMed

Guliy, O I; Karavaeva, O A; Velikov, V A; Sokolov, O I; Pavily, S A; Larionova, O S; Burov, A M; Ignatov, O V

2016-01-01

The bacteriophage ΦAb-Sp7 was isolated from the cells of the Azospirillum brasilense Sp7. The morphology, size of the gram-negative colonies, and range of lytic activity against other strains and species of the genus Azospirillum was tested. The isolated phage DNA was examined using electrophoretic and restriction analysis, and the size of the genome were established. The electron microscopy. resuIts show that the phage (capsid) has a strand-like form. The electron microscopy study of the bacteriophage ΦAb-Sp7 adsorption on the A. brasilense Sp7 bacterial surface was performed.

Building cell models and simulations from microscope images.

PubMed

Murphy, Robert F

2016-03-01

The use of fluorescence microscopy has undergone a major revolution over the past twenty years, both with the development of dramatic new technologies and with the widespread adoption of image analysis and machine learning methods. Many open source software tools provide the ability to use these methods in a wide range of studies, and many molecular and cellular phenotypes can now be automatically distinguished. This article presents the next major challenge in microscopy automation, the creation of accurate models of cell organization directly from images, and reviews the progress that has been made towards this challenge. Copyright © 2015 Elsevier Inc. All rights reserved.

Scientist, Single Cell Analysis Facility | Center for Cancer Research

Cancer.gov

The Cancer Research Technology Program (CRTP) develops and implements emerging technology, cancer biology expertise and research capabilities to accomplish NCI research objectives.  The CRTP is an outward-facing, multi-disciplinary hub purposed to enable the external cancer research community and provides dedicated support to NCI’s intramural Center for Cancer Research (CCR).  The dedicated units provide electron microscopy, protein characterization, protein expression, optical microscopy and nextGen sequencing. These research efforts are an integral part of CCR at the Frederick National Laboratory for Cancer Research (FNLCR).  CRTP scientists also work collaboratively with intramural NCI investigators to provide research technologies and expertise. KEY ROLES AND RESPONSIBILITIES We are seeking a highly motivated Scientist II to join the newly established Single Cell Analysis Facility (SCAF) of the Center for Cancer Research (CCR) at NCI. The SCAF will house state of the art single cell sequencing technologies including 10xGenomics Chromium, BD Genomics Rhapsody, DEPPArray, and other emerging single cell technologies. The Scientist: Will interact with close to 200 laboratories within the CCR to design and carry out single cell experiments for cancer research Will work on single cell isolation/preparation from various tissues and cells and related NexGen sequencing library preparation Is expected to author publications in peer reviewed scientific journals

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 Quantification and Integrative Analysis of 2D and 3D Mitochondrial Shape and Network Properties

PubMed Central

Nikolaisen, Julie; Nilsson, Linn I. H.; Pettersen, Ina K. N.; Willems, Peter H. G. M.; Lorens, James B.; Koopman, Werner J. H.; Tronstad, Karl J.

2014-01-01

Mitochondrial morphology and function are coupled in healthy cells, during pathological conditions and (adaptation to) endogenous and exogenous stress. In this sense mitochondrial shape can range from small globular compartments to complex filamentous networks, even within the same cell. Understanding how mitochondrial morphological changes (i.e. “mitochondrial dynamics”) are linked to cellular (patho) physiology is currently the subject of intense study and requires detailed quantitative information. During the last decade, various computational approaches have been developed for automated 2-dimensional (2D) analysis of mitochondrial morphology and number in microscopy images. Although these strategies are well suited for analysis of adhering cells with a flat morphology they are not applicable for thicker cells, which require a three-dimensional (3D) image acquisition and analysis procedure. Here we developed and validated an automated image analysis algorithm allowing simultaneous 3D quantification of mitochondrial morphology and network properties in human endothelial cells (HUVECs). Cells expressing a mitochondria-targeted green fluorescence protein (mitoGFP) were visualized by 3D confocal microscopy and mitochondrial morphology was quantified using both the established 2D method and the new 3D strategy. We demonstrate that both analyses can be used to characterize and discriminate between various mitochondrial morphologies and network properties. However, the results from 2D and 3D analysis were not equivalent when filamentous mitochondria in normal HUVECs were compared with circular/spherical mitochondria in metabolically stressed HUVECs treated with rotenone (ROT). 2D quantification suggested that metabolic stress induced mitochondrial fragmentation and loss of biomass. In contrast, 3D analysis revealed that the mitochondrial network structure was dissolved without affecting the amount and size of the organelles. Thus, our results demonstrate that 3D imaging and quantification are crucial for proper understanding of mitochondrial shape and topology in non-flat cells. In summary, we here present an integrative method for unbiased 3D quantification of mitochondrial shape and network properties in mammalian cells. PMID:24988307

Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

PubMed

Dukes, Madeline J; Peckys, Diana B; de Jonge, Niels

2010-07-27

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

PubMed Central

Dukes, Madeline J.; Peckys, Diana B.; de Jonge, Niels

2010-01-01

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs. PMID:20550177

Phytosynthesis of silver nanoparticles using Artemisia marschalliana Sprengel aerial part extract and assessment of their antioxidant, anticancer, and antibacterial properties

PubMed Central

Salehi, Soheil; Shandiz, Seyed Ataollah Sadat; Ghanbar, Farinaz; Darvish, Mohammad Raouf; Ardestani, Mehdi Shafiee; Mirzaie, Amir; Jafari, Mohsen

2016-01-01

A rapid phytosynthesis of silver nanoparticles (AgNPs) using an extract from the aerial parts of Artemisia marschalliana Sprengel was investigated in this study. The synthesized AgNPs using A. marschalliana extract was analyzed by UV–visible spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy and further characterized by transmission electron microscopy, scanning electron microscopy, zeta potential, and energy-dispersive spectroscopy. Characteristic absorption bands of AgNPs were found near 430 nm in the UV–vis spectrum. Energy-dispersive spectroscopy analysis of AgNPs in the energy range 2–4 keV confirmed the silver signal due to surface plasmon resonance. Scanning electron microscopy and transmission electron microscopy results revealed that the AgNPs were mostly spherical with an average size ranging from 5 nm to 50 nm. The zeta potential value of −31 mV confirmed the stability of the AgNPs. AgNPs produced using the aqueous A. marschalliana extract might serve as a potent in vitro antioxidant, as revealed by 2,2-diphenyl-1-picryl hydrazyl assay. The present study demonstrates the anticancer properties of phytosynthesized AgNPs against human gastric carcinoma AGS cells. AgNPs exerted a dose-dependent inhibitory effect on the viability of cells. Real-time polymerase chain reaction was used for the investigation of Bax and Bcl-2 gene expression in cancer and normal cell lines. Our findings show that the mRNA levels of pro-apoptotic Bax gene expression were significantly upregulated, while the expression of anti-apoptotic Bcl-2 was declined in cells treated with AgNPs compared to normal cells. In addition, flow cytometric analysis showed that the number of early and late apoptotic AGS cells was significantly enhanced following treatment with AgNPs as compared to untreated cells. In addition, the AgNPs showed strong antibacterial properties against tested pathogenic bacteria such as Staphylococcus aureus, Bacillus cereus, Acinetobacter baumannii, and Pseudomonas aeruginosa. Based on the obtained data, we suggest that phytosynthesized AgNPs are good alternatives in the treatment of diseases because of the presence of bioactive agents. PMID:27199558

Non-thermal plasma mills bacteria: Scanning electron microscopy observations

NASA Astrophysics Data System (ADS)

Lunov, O.; Churpita, O.; Zablotskii, V.; Deyneka, I. G.; Meshkovskii, I. K.; Jäger, A.; Syková, E.; Kubinová, Š.; Dejneka, A.

2015-02-01

Non-thermal plasmas hold great promise for a variety of biomedical applications. To ensure safe clinical application of plasma, a rigorous analysis of plasma-induced effects on cell functions is required. Yet mechanisms of bacteria deactivation by non-thermal plasma remain largely unknown. We therefore analyzed the influence of low-temperature atmospheric plasma on Gram-positive and Gram-negative bacteria. Using scanning electron microscopy, we demonstrate that both Gram-positive and Gram-negative bacteria strains in a minute were completely destroyed by helium plasma. In contrast, mesenchymal stem cells (MSCs) were not affected by the same treatment. Furthermore, histopathological analysis of hematoxylin and eosin-stained rat skin sections from plasma-treated animals did not reveal any abnormalities in comparison to control ones. We discuss possible physical mechanisms leading to the shred of bacteria under non-thermal plasma irradiation. Our findings disclose how helium plasma destroys bacteria and demonstrates the safe use of plasma treatment for MSCs and skin cells, highlighting the favorability of plasma applications for chronic wound therapy.

Malignant mixed müllerian tumors. An ultrastructural and immunohistochemical analysis with histogenetic considerations.

PubMed

Geisinger, K R; Dabbs, D J; Marshall, R B

1987-05-15

In the female genital tract, malignant mixed müllerian tumors (MMTs) are uncommon neoplasms of uncertain histogenesis. We have examined 11 MMTs by both electron microscopy (EM) and immunoperoxidase techniques (IPX). Eight were of endometrial, two were of ovarian, and one of tubal origins. The IPX analysis included monoclonal antibodies to keratin (k) and vimentin (v) and a polyclonal antibody to myoglobin. Carcinomatous elements were always keratin positive (K+) and were focally positive for vimentin in six tumors. Homologous stromal sarcoma cells were vimentin positive (V+) and in three tumors were focally K+. Ultrastructurally, the epithelial cells were not highly differentiated and the sarcomatous elements generally resembled normal proliferative-phase stromal cells. The epithelial and stromal elements were separated by a thin basal lamina that only rarely and focally had discontinuities. No transitional cellular forms were identified. A definite positive myoglobin reaction was seen in two of the four neoplasms in which rhabdomyoblasts were identified by light microscopy. Myofilaments were identified by electron microscope in three neoplasms.

Insight into plant cell wall chemistry and structure by combination of multiphoton microscopy with Raman imaging.

PubMed

Heiner, Zsuzsanna; Zeise, Ingrid; Elbaum, Rivka; Kneipp, Janina

2018-04-01

Spontaneous Raman scattering microspectroscopy, second harmonic generation (SHG) and 2-photon excited fluorescence (2PF) were used in combination to characterize the morphology together with the chemical composition of the cell wall in native plant tissues. As the data obtained with unstained sections of Sorghum bicolor root and leaf tissues illustrate, nonresonant as well as pre-resonant Raman microscopy in combination with hyperspectral analysis reveals details about the distribution and composition of the major cell wall constituents. Multivariate analysis of the Raman data allows separation of different tissue regions, specifically the endodermis, xylem and lumen. The orientation of cellulose microfibrils is obtained from polarization-resolved SHG signals. Furthermore, 2-photon autofluorescence images can be used to image lignification. The combined compositional, morphological and orientational information in the proposed coupling of SHG, Raman imaging and 2PF presents an extension of existing vibrational microspectroscopic imaging and multiphoton microscopic approaches not only for plant tissues. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Non-thermal plasma mills bacteria: Scanning electron microscopy observations

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

Lunov, O., E-mail: lunov@fzu.cz; Churpita, O.; Zablotskii, V.

2015-02-02

Non-thermal plasmas hold great promise for a variety of biomedical applications. To ensure safe clinical application of plasma, a rigorous analysis of plasma-induced effects on cell functions is required. Yet mechanisms of bacteria deactivation by non-thermal plasma remain largely unknown. We therefore analyzed the influence of low-temperature atmospheric plasma on Gram-positive and Gram-negative bacteria. Using scanning electron microscopy, we demonstrate that both Gram-positive and Gram-negative bacteria strains in a minute were completely destroyed by helium plasma. In contrast, mesenchymal stem cells (MSCs) were not affected by the same treatment. Furthermore, histopathological analysis of hematoxylin and eosin–stained rat skin sections frommore » plasma–treated animals did not reveal any abnormalities in comparison to control ones. We discuss possible physical mechanisms leading to the shred of bacteria under non-thermal plasma irradiation. Our findings disclose how helium plasma destroys bacteria and demonstrates the safe use of plasma treatment for MSCs and skin cells, highlighting the favorability of plasma applications for chronic wound therapy.« less

Quantitative Analysis of Rat Dorsal Root Ganglion Neurons Cultured on Microelectrode Arrays Based on Fluorescence Microscopy Image Processing.

PubMed

Mari, João Fernando; Saito, José Hiroki; Neves, Amanda Ferreira; Lotufo, Celina Monteiro da Cruz; Destro-Filho, João-Batista; Nicoletti, Maria do Carmo

2015-12-01

Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.

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

PubMed

Peckys, Diana B; de Jonge, Niels

2014-04-01

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

New Frontiers and Challenges for Single-Cell Electrochemical Analysis.

PubMed

Zhang, Jingjing; Zhou, Junyu; Pan, Rongrong; Jiang, Dechen; Burgess, James D; Chen, Hong-Yuan

2018-02-23

Previous measurements of cell populations might obscure many important cellular differences, and new strategies for single-cell analyses are urgently needed to re-examine these fundamental biological principles for better diagnosis and treatment of diseases. Electrochemistry is a robust technique for the analysis of single living cells that has the advantages of minor interruption of cellular activity and provides the capability of high spatiotemporal resolution. The achievements of the past 30 years have revealed significant information about the exocytotic events of single cells to elucidate the mechanisms of cellular activity. Currently, the rapid developments of micro/nanofabrication and optoelectronic technologies drive the development of multifunctional electrodes and novel electrochemical approaches with higher resolution for single cells. In this Perspective, three new frontiers in this field, namely, electrochemical microscopy, intracellular analysis, and single-cell analysis in a biological system (i.e., neocortex and retina), are reviewed. The unique features and remaining challenges of these techniques are discussed.

A national facility for biological cryo-electron microscopy

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

Saibil, Helen R., E-mail: h.saibil@mail.cryst.bbk.ac.uk; Grünewald, Kay; Stuart, David I.

2015-01-01

This review provides a brief update on the use of cryo-electron microscopy for integrated structural biology, along with an overview of the plans for the UK national facility for electron microscopy being built at the Diamond synchrotron. Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided ofmore » the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback.« less

Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

PubMed

Svitkina, Tatyana M

2017-05-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platinum Replica Electron Microscopy: Imaging the Cytoskeleton Globally and Locally

PubMed Central

SVITKINA, Tatyana M.

2017-01-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the “comfort zones” of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. PMID:28323208

Photocatalytic activity against azo dye and cytotoxicity on MCF-7 cell lines of zirconium oxide nanoparticle mediated using leaves of Lagerstroemia speciosa.

PubMed

Sai Saraswathi, V; Santhakumar, K

2017-04-01

Metal oxide nanoparticles are gaining interest in recent years. The present paper explains about the green synthesis of zirconium oxide nanoparticles (ZrO NPs) mediated from the leaves of Lagerstroemia speciosa. The prepared ZrO NPs were characterized by UV-vis spectroscopy, FT-IR, X-ray diffraction analysis (XRD), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray spectroscopy (EDX) and Thermogravimetric Analysis (TGA). The photocatalytic activity of ZrO NPs was studied for azo dye by exposing to sunlight. The azo dye was degraded up to 94.58%. Also the ZrO NPs were studied for in vitro cytotoxicity activity against breast cancer cell lines-MCF-7 and evaluated by MTT assay. The cell morphological changes were recorded by light microscope. The cells viability was seen at 500μg/mL when compared against control. Hence the research highlights, that the method was simple, eco-friendly towards environment by phytoremediation activity of the azo dye and cytotoxicity activity against MCF-7 cell lines. Hence the present paper may help to further explore the metal nanoparticle for its potential applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Combining Raman spectroscopy and digital holographic microscopy for label-free classification of human immune cells (Conference Presentation)

NASA Astrophysics Data System (ADS)

McReynolds, Naomi; Cooke, Fiona G. M.; Chen, Mingzhou; Powis, Simon J.; Dholakia, Kishan

2017-02-01

Moving towards label-free techniques for cell identification is essential for many clinical and research applications. Raman spectroscopy and digital holographic microscopy (DHM) are both label-free, non-destructive optical techniques capable of providing complimentary information. We demonstrate a multi-modal system which may simultaneously take Raman spectra and DHM images to provide both a molecular and a morphological description of our sample. In this study we use Raman spectroscopy and DHM to discriminate between three immune cell populations CD4+ T cells, B cells, and monocytes, which together comprise key functional immune cell subsets in immune responses to invading pathogens. Various parameters that may be used to describe the phase images are also examined such as pixel value histograms or texture analysis. Using our system it is possible to consider each technique individually or in combination. Principal component analysis is used on the data set to discriminate between cell types and leave-one-out cross-validation is used to estimate the efficiency of our method. Raman spectroscopy provides specific chemical information but requires relatively long acquisition times, combining this with a faster modality such as DHM could help achieve faster throughput rates. The combination of these two complimentary optical techniques provides a wealth of information for cell characterisation which is a step towards achieving label free technology for the identification of human immune cells.

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-speed vibrational imaging and spectral analysis of lipid bodies by compound Raman microscopy.

PubMed

Slipchenko, Mikhail N; Le, Thuc T; Chen, Hongtao; Cheng, Ji-Xin

2009-05-28

Cells store excess energy in the form of cytoplasmic lipid droplets. At present, it is unclear how different types of fatty acids contribute to the formation of lipid droplets. We describe a compound Raman microscope capable of both high-speed chemical imaging and quantitative spectral analysis on the same platform. We used a picosecond laser source to perform coherent Raman scattering imaging of a biological sample and confocal Raman spectral analysis at points of interest. The potential of the compound Raman microscope was evaluated on lipid bodies of cultured cells and live animals. Our data indicate that the in vivo fat contains much more unsaturated fatty acids (FAs) than the fat formed via de novo synthesis in 3T3-L1 cells. Furthermore, in vivo analysis of subcutaneous adipocytes and glands revealed a dramatic difference not only in the unsaturation level but also in the thermodynamic state of FAs inside their lipid bodies. Additionally, the compound Raman microscope allows tracking of the cellular uptake of a specific fatty acid and its abundance in nascent cytoplasmic lipid droplets. The high-speed vibrational imaging and spectral analysis capability renders compound Raman microscopy an indispensible analytical tool for the study of lipid-droplet biology.

Scanning and transmission electron microscopic analysis of ampullary segment of oviduct during estrous cycle in caprines.

PubMed

Sharma, R K; Singh, R; Bhardwaj, J K

2015-01-01

The ampullary segment of the mammalian oviduct provides suitable milieu for fertilization and development of zygote before implantation into uterus. It is, therefore, in the present study, the cyclic changes in the morphology of ampullary segment of goat oviduct were studied during follicular and luteal phases using scanning and transmission electron microscopy techniques. Topographical analysis revealed the presence of uniformly ciliated ampullary epithelia, concealing apical processes of non-ciliated cells along with bulbous secretory cells during follicular phase. The luteal phase was marked with decline in number of ciliated cells with increased occurrence of secretory cells. The ultrastructure analysis has demonstrated the presence of indented nuclear membrane, supranuclear cytoplasm, secretory granules, rough endoplasmic reticulum, large lipid droplets, apically located glycogen masses, oval shaped mitochondria in the secretory cells. The ciliated cells were characterized by the presence of elongated nuclei, abundant smooth endoplasmic reticulum, oval or spherical shaped mitochondria with crecentric cristae during follicular phase. However, in the luteal phase, secretory cells were possessing highly indented nucleus with diffused electron dense chromatin, hyaline nucleosol, increased number of lipid droplets. The ciliated cells had numerous fibrous granules and basal bodies. The parallel use of scanning and transmission electron microscopy techniques has enabled us to examine the cyclic and hormone dependent changes occurring in the topography and fine structure of epithelium of ampullary segment and its cells during different reproductive phases that will be great help in understanding major bottle neck that limits success rate in vitro fertilization and embryo transfer technology. © Wiley Periodicals, Inc.

Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment. © 2014 Elsevier Inc. All rights reserved.

Comparison of segmentation algorithms for fluorescence microscopy images of cells.

PubMed

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

2011-07-01

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

Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells.

PubMed

Christakou, Athanasia E; Ohlin, Mathias; Önfelt, Björn; Wiklund, Martin

2015-08-07

We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in a suspension into a single aggregate in each well of the microplate and, by this, nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to monitor cell culture from production to analysis of 3D tissue or tumor models using microscopy in one single microplate device.

Correlated Light and Electron Microscopy/Electron Tomography of Mitochondria In Situ

PubMed Central

Perkins, Guy A.; Sun, Mei G.; Frey, Terrence G.

2009-01-01

Three-dimensional light microscopy and three-dimensional electron microscopy (electron tomography) separately provide very powerful tools to study cellular structure and physiology, including the structure and physiology of mitochondria. Fluorescence microscopy allows one to study processes in live cells with specific labels and stains that follow the movement of labeled proteins and changes within cellular compartments but does not have sufficient resolution to define the ultrastructure of intracellular organelles such as mitochondria. Electron microscopy and electron tomography provide the highest resolution currently available to study mitochondrial ultrastructure but cannot follow processes in living cells. We describe the combination of these two techniques in which fluorescence confocal microscopy is used to study structural and physiologic changes in mitochondria within apoptotic HeLa cells to define the apoptotic timeframe. Cells can then be selected at various stages of the apoptotic timeframe for examination at higher resolution by electron microscopy and electron tomography. This is a form of “virtual” 4-dimensional electron microscopy that has revealed interesting structural changes in the mitochondria of HeLa cells during apoptosis. The same techniques can be applied, with modification, to study other dynamic processes within cells in other experimental contexts. PMID:19348881

N-Acetylcysteine Amide Protects Against Oxidative Stress–Induced Microparticle Release From Human Retinal Pigment Epithelial Cells

PubMed Central

Carver, Kyle A.; Yang, Dongli

2016-01-01

Purpose Oxidative stress is a major factor involved in retinal pigment epithelium (RPE) apoptosis that underlies AMD. Drusen, extracellular lipid- and protein-containing deposits, are strongly associated with the development of AMD. Cell-derived microparticles (MPs) are small membrane-bound vesicles shed from cells. The purpose of this study was to determine if oxidative stress drives MP release from RPE cells, to assess whether these MPs carry membrane complement regulatory proteins (mCRPs: CD46, CD55, and CD59), and to evaluate the effects of a thiol antioxidant on oxidative stress–induced MP release. Methods Retinal pigment epithelium cells isolated from human donor eyes were cultured and treated with hydrogen peroxide (H2O2) to induce oxidative stress. Isolated MPs were fixed for transmission electron microscopy or processed for component analysis by flow cytometry, Western blot analysis, and confocal microscopy. Results Transmission electron microscopy showed that MPs ranged in diameter from 100 to 1000 nm. H2O2 treatment led to time- and dose-dependent elevations in MPs with externalized phosphatidylserine and phosphatidylethanolamine, known markers of MPs. These increases were strongly correlated to RPE apoptosis. Oxidative stress significantly increased the release of mCRP-positive MPs, which were prevented by a thiol antioxidant, N-acetylcysteine amide (NACA). Conclusions This is the first evidence that oxidative stress induces cultured human RPE cells to release MPs that carry mCRPs on their surface. The levels of released MPs are strongly correlated with RPE apoptosis. N-acetylcysteine amide prevents oxidative stress–induced effects. Our findings indicate that oxidative stress reduces mCRPs on the RPE surface through releasing MPs. PMID:26842754

Large-scale time-lapse microscopy of Oct4 expression in human embryonic stem cell colonies.

PubMed

Bhadriraju, Kiran; Halter, Michael; Amelot, Julien; Bajcsy, Peter; Chalfoun, Joe; Vandecreme, Antoine; Mallon, Barbara S; Park, Kye-Yoon; Sista, Subhash; Elliott, John T; Plant, Anne L

2016-07-01

Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling, we examined >680 colonies from 3 different preparations of cells over 5days each, generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies, correlation of colony characteristics with Oct4 expression, and identification of rare events. Copyright © 2016. Published by Elsevier B.V.

Whole-Cell Analysis of Low-Density Lipoprotein Uptake by Macrophages Using STEM Tomography

PubMed Central

Baudoin, Jean-Pierre; Jerome, W. Gray; Kübel, Christian; de Jonge, Niels

2013-01-01

Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters. PMID:23383042

Detection of intracellular glutathione using ThiolTracker violet stain and fluorescence microscopy.

PubMed

Mandavilli, Bhaskar S; Janes, Michael S

2010-07-01

Glutathione plays an important role in protecting mammalian cells from oxidative stress and cell death. Because reduced glutathione (GSH) represents the large majority of intracellular free thiols, cell-permeant, thiol-reactive fluorescent probes represent potentially useful indicators of intracellular GSH. The ThiolTracker Violet stain (a registered trademark of Invitrogen) is a bright fluorescent probe that is highly reactive to thiols and can be used as a convenient and effective indicator of intracellular GSH and general redox status by a variety of detection modalities. While this probe has been validated in flow cytometry and microplate fluorimetry assays, the following method will describe details on the use of the ThiolTracker Violet dye in traditional fluorescence microscopy, as well as high-content imaging and analysis.

Intracellular localization analysis of npAu-PpIX in HeLa cells using specific dyes and confocal microscopy

NASA Astrophysics Data System (ADS)

Roblero-Bartolón, Victoria Gabriela; Maldonado-Alvarado, Elizabeth; Galván-Mendoza, José Iván; Ramón-Gallegos, Eva

2012-10-01

Cervical carcinoma (CC) represents the second leading cause of cancer death in Mexican women. No conventional treatments are being developed such as photodynamic therapy (PDT), involving the simultaneous presence of a photosensitizer (Ps), light of a specific wavelength and tissue oxygen. On the other hand, it has seen that the use of gold nanoparticles coupled to protoporphyrin IX increases the effectiveness of PDT. The aim of this study was to determine the site of accumulation of the conjugate npAu-PpIX in cells of cervical cancer by the use of specific dyes and confocal microscopy. The results indicate that the gold nanoparticles coupled to protoporphyrin IX are accumulated in both the cytoplasm and nucleus of HeLa cells.

Surface plasmon enhanced cell microscopy with blocked random spatial activation

NASA Astrophysics Data System (ADS)

Son, Taehwang; Oh, Youngjin; Lee, Wonju; Yang, Heejin; Kim, Donghyun

2016-03-01

We present surface plasmon enhanced fluorescence microscopy with random spatial sampling using patterned block of silver nanoislands. Rigorous coupled wave analysis was performed to confirm near-field localization on nanoislands. Random nanoislands were fabricated in silver by temperature annealing. By analyzing random near-field distribution, average size of localized fields was found to be on the order of 135 nm. Randomly localized near-fields were used to spatially sample F-actin of J774 cells (mouse macrophage cell-line). Image deconvolution algorithm based on linear imaging theory was established for stochastic estimation of fluorescent molecular distribution. The alignment between near-field distribution and raw image was performed by the patterned block. The achieved resolution is dependent upon factors including the size of localized fields and estimated to be 100-150 nm.

Investigation of autofocus algorithms for brightfield microscopy of unstained cells

NASA Astrophysics Data System (ADS)

Wu, Shu Yu; Dugan, Nazim; Hennelly, Bryan M.

2014-05-01

In the past decade there has been significant interest in image processing for brightfield cell microscopy. Much of the previous research on image processing for microscopy has focused on fluorescence microscopy, including cell counting, cell tracking, cell segmentation and autofocusing. Fluorescence microscopy provides functional image information that involves the use of labels in the form of chemical stains or dyes. For some applications, where the biochemical integrity of the cell is required to remain unchanged so that sensitive chemical testing can later be applied, it is necessary to avoid staining. For this reason the challenge of processing images of unstained cells has become a topic of increasing attention. These cells are often effectively transparent and appear to have a homogenous intensity profile when they are in focus. Bright field microscopy is the most universally available and most widely used form of optical microscopy and for this reason we are interested in investigating image processing of unstained cells recorded using a standard bright field microscope. In this paper we investigate the application of a range of different autofocus metrics applied to unstained bladder cancer cell lines using a standard inverted bright field microscope with microscope objectives that have high magnification and numerical aperture. We present a number of conclusions on the optimum metrics and the manner in which they should be applied for this application.

Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system

PubMed Central

Lerner, Natalie; Avissar, Sofia

2017-01-01

Purpose Canonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. Our goal was to gain insight into the influence of non-pigmented ciliary epithelium (NPCE)-derived exosomes on Wnt signaling by trabecular meshwork (TM) cells. The potential impact of exosomes on Wnt signaling in the ocular drainage system remains poorly understood. Methods Exosomes isolated from media collected from cultured NPCE cells by differential ultracentrifugation were characterized by dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA), sucrose density gradient migration and transmission electron microscopy (TEM). The cellular target specificity of the NPCE-derived exosomes was investigated by confocal microscopy-based monitoring of the uptake of DiD-labeled exosomes over time, as compared to uptake by various cell lines. Changes in Wnt protein levels in TM cells induced by NPCE exosomes were evaluated by Western blot. Results Exosomes derived from NPCE cells were purified and detected as small rounded 50–140 nm membrane vesicles, as defined by DLS, NTA, TRPS and TEM. Western blot analysis indicated that the nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were found in sucrose density fractions typical of exosomes (1.118–1.188 g/mL sucrose). Using confocal microscopy, we demonstrated time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3β and reduced β-catenin levels. Finally, we found that treatment of NTM cells with exosomes resulted in a greater than 2-fold decrease in the level of β-catenin in the cytosolic fraction. In contrast, no remarkable difference in the amount of β-catenin in the nuclear fraction was noted, relative to the control. Conclusions The data suggest that NPCE cells release exosome-like vesicles and that these nanoparticles affect canonical Wnt signaling in TM cells. These findings may have therapeutic relevance since canonical Wnt pathway is involved in intra-ocular pressure regulation. Further understanding of NPCE-derived exosome-responsive signaling pathways may reveal new targets for pharmacological intervention within the drainage system as a target for glaucoma therapy. PMID:28241021

Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system.

PubMed

Lerner, Natalie; Avissar, Sofia; Beit-Yannai, Elie

2017-01-01

Canonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. Our goal was to gain insight into the influence of non-pigmented ciliary epithelium (NPCE)-derived exosomes on Wnt signaling by trabecular meshwork (TM) cells. The potential impact of exosomes on Wnt signaling in the ocular drainage system remains poorly understood. Exosomes isolated from media collected from cultured NPCE cells by differential ultracentrifugation were characterized by dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA), sucrose density gradient migration and transmission electron microscopy (TEM). The cellular target specificity of the NPCE-derived exosomes was investigated by confocal microscopy-based monitoring of the uptake of DiD-labeled exosomes over time, as compared to uptake by various cell lines. Changes in Wnt protein levels in TM cells induced by NPCE exosomes were evaluated by Western blot. Exosomes derived from NPCE cells were purified and detected as small rounded 50-140 nm membrane vesicles, as defined by DLS, NTA, TRPS and TEM. Western blot analysis indicated that the nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were found in sucrose density fractions typical of exosomes (1.118-1.188 g/mL sucrose). Using confocal microscopy, we demonstrated time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3β and reduced β-catenin levels. Finally, we found that treatment of NTM cells with exosomes resulted in a greater than 2-fold decrease in the level of β-catenin in the cytosolic fraction. In contrast, no remarkable difference in the amount of β-catenin in the nuclear fraction was noted, relative to the control. The data suggest that NPCE cells release exosome-like vesicles and that these nanoparticles affect canonical Wnt signaling in TM cells. These findings may have therapeutic relevance since canonical Wnt pathway is involved in intra-ocular pressure regulation. Further understanding of NPCE-derived exosome-responsive signaling pathways may reveal new targets for pharmacological intervention within the drainage system as a target for glaucoma therapy.

Microvesicle- and exosome-mediated drug delivery enhances the cytotoxicity of Paclitaxel in autologous prostate cancer cells.

PubMed

Saari, Heikki; Lázaro-Ibáñez, Elisa; Viitala, Tapani; Vuorimaa-Laukkanen, Elina; Siljander, Pia; Yliperttula, Marjo

2015-12-28

Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate intercellular communication by delivering molecular information between cells. In this study, we investigated the effectiveness of two different populations of EVs (microvesicle- and exosome-enriched) as carriers of Paclitaxel to autologous prostate cancer cells. EVs were isolated from LNCaP- and PC-3 prostate cancer cell cultures using differential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and Western blot. The uptake of microvesicles and exosomes by the autologous prostate cancer cells was assessed by flow cytometry and confocal microscopy. The EVs were loaded with Paclitaxel and the effectiveness of EV-mediated drug delivery was assessed with viability assays. The distribution of EVs and EV-delivered Paclitaxel in cells was inspected by confocal microscopy. Our main finding was that the loading of Paclitaxel to autologous prostate cancer cell-derived EVs increased its cytotoxic effect. This capacity was independent of the EV population and the cell line tested. Although the EVs without the drug increased cancer cell viability, the net effect of enhanced cytotoxicity remained. Both EV populations delivered Paclitaxel to the recipient cells through endocytosis, leading to the release of the drug from within the cells. The removal of EV surface proteins did not affect exosomes, while the drug delivery mediated by microvesicles was partially inhibited. Cancer cell-derived EVs can be used as effective carriers of Paclitaxel to their parental cells, bringing the drug into the cells through an endocytic pathway and increasing its cytotoxicity. However, due to the increased cell viability, the use of cancer cell-derived EVs must be further investigated before any clinical applications can be designed. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Ultrastructural Analysis of Different Human Mesenchymal Stem Cells After in Vitro Expansion: A Technical Review

PubMed Central

Danišovič, Ľ.; Majidi, A.; Varga, I.

2015-01-01

Transmission electron microscopy reveals ultrastructural details of cells, and it is a valuable method for studying cell organelles. That is why we used this method for detailed morphological description of different adult tissue-derived stem cells, focusing on the morphological signs of their functions (proteosynthetic activity, exchange with external environment, etc.) and their comparison. Preparing a specimen from the cell culture suitable for transmission electron microscopy is, however, much more challenging than routine tissue processing for normal histological examination. There are several issues that need to be solved while working with cell pellets instead of solid tissue. Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells from different adult tissues, with applications to stem cell biology and regenerative medicine. Since we are working with population of cells that was obtained after many days of passaging, very efficient and gentle procedures are highly necessary. We demonstrated that our semi-conservative approach regarding to histological techniques and processing of cells for transmission electron microscopy is a well reproducible procedure which results in quality pictures and images of cell populations with minimum distortions and artifacts. We also commented about riskiest steps and histochemical issues (e.g., precise pH, temperature) while preparing the specimen. We bring full and detailed procedures of fixation, post-fixation, infiltration, embedding, polymerization and contrasting of cell obtained from in vitro cell and tissue cultures, with modifications according to our experience. All this steps are essential for us to know more about adult stem cells derived from different sources or about other random cell populations. The knowledge about detailed ultra-structure of adult stem cells cultured in vitro are also essential for their using in regenerative medicine and tissue engineering. PMID:26708176

The distinguishing effects of low-intensity electromagnetic radiation of different extremely high frequencies on Enterococcus hirae: growth rate inhibition and scanning electron microscopy analysis.

PubMed

Hovnanyan, K; Kalantaryan, V; Trchounian, A

2017-09-01

A low-intensity electromagnetic field of extremely high frequency has inhibitory and stimulatory effects on bacteria, including Enterococcus hirae. It was shown that the low-intensity (the incident power density of 0·06 mW cm -2 ) electromagnetic field at the frequencies of 51·8 GHz and 53 GHz inhibited E. hirae ATCC 9790 bacterial growth rate; a stronger effect was observed with 53 GHz, regardless of exposure duration (0·5 h, 1 h or 2 h). Scanning electron microscopy analysis of these effects has been done; the cells were of spherical shape. Electromagnetic field at 53 GHz, but not 51·8 GHz, changed the cell size-the diameter was enlarged 1·3 fold at 53 GHz. These results suggest the difference in mechanisms of action on bacteria for electromagnetic fields at 51·8 GHz and 53 GHz. A stronger inhibitory effect of low-intensity electromagnetic field on Enterococcus hirae ATCC 9790 bacterial growth rate was observed with 53 GHz vs 51·8 GHz, regardless of exposure duration. Scanning electron microscopy analysis showed that almost all irradiated cells in the population have spherical shapes similar to nonirradiated ones, but they have increased diameters in case of irradiated cells at 53 GHz, but not 51·8 GHz. The results are novel, showing distinguishing effects of low-intensity electromagnetic field of different frequencies. They could be applied in treatment of food and different products in medicine and veterinary, where E. hirae plays an important role. © 2017 The Society for Applied Microbiology.

Necrosis and apoptosis pathways of cell death at photodynamic treatment in vitro as revealed by digital holographic microscopy

NASA Astrophysics Data System (ADS)

Semenova, I. V.; Belashov, A. V.; Belyaeva, T. N.; Kornilova, E. S.; Salova, A. V.; Zhikhoreva, A. A.; Vasyutinskii, O. S.

2018-02-01

Monitoring of variations in morphological characteristics of cultured HeLa cells after photodynamic treatment with Radachlorin photosensitizer is performed by means of digital holographic microscopy. The observed dose-dependent post-treatment variations of phase shift evidence threshold effect of photodynamic treatment and allow for distinguishing between necrotic or apoptotic pathways of cell death. Results obtained by holographic microscopy were confirmed by means of far-field optical microscopy and confocal fluorescence microscopy with commonly used test assays.

Super-resolution Microscopy in Plant Cell Imaging.

PubMed

Komis, George; Šamajová, Olga; Ovečka, Miroslav; Šamaj, Jozef

2015-12-01

Although the development of super-resolution microscopy methods dates back to 1994, relevant applications in plant cell imaging only started to emerge in 2010. Since then, the principal super-resolution methods, including structured-illumination microscopy (SIM), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED), have been implemented in plant cell research. However, progress has been limited due to the challenging properties of plant material. Here we summarize the basic principles of existing super-resolution methods and provide examples of applications in plant science. The limitations imposed by the nature of plant material are reviewed and the potential for future applications in plant cell imaging is highlighted. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluation of TgH(CX3CR1-EGFP) mice implanted with mCherry-GL261 cells as an in vivo model for morphometrical analysis of glioma-microglia interaction.

PubMed

Resende, Fernando F B; Bai, Xianshu; Del Bel, Elaine Aparecida; Kirchhoff, Frank; Scheller, Anja; Titze-de-Almeida, Ricardo

2016-02-08

Glioblastoma multiforme is the most aggressive brain tumor. Microglia are prominent cells within glioma tissue and play important roles in tumor biology. This work presents an animal model designed for the study of microglial cell morphology in situ during gliomagenesis. It also allows a quantitative morphometrical analysis of microglial cells during their activation by glioma cells. The animal model associates the following cell types: 1- mCherry red fluorescent GL261 glioma cells and; 2- EGFP fluorescent microglia, present in the TgH(CX3CR1-EGFP) mouse line. First, mCherry-GL261 glioma cells were implanted in the brain cortex of TgH(CX3CR1-EGFP) mice. Epifluorescence - and confocal laser-scanning microscopy were employed for analysis of fixed tissue sections, whereas two-photon laser-scanning microscopy (2P-LSM) was used to track tumor cells and microglia in the brain of living animals. Implanted mCherry-GL261 cells successfully developed brain tumors. They mimic the aggressive behavior found in human disease, with a rapid increase in size and the presence of secondary tumors apart from the injection site. As tumor grows, mCherry-GL261 cells progressively lost their original shape, adopting a heterogeneous and diffuse morphology at 14-18 d. Soma size increased from 10-52 μm. At this point, we focused on the kinetics of microglial access to glioma tissues. 2P-LSM revealed an intense microgliosis in brain areas already shortly after tumor implantation, i.e. at 30 min. By confocal microscopy, we found clusters of microglial cells around the tumor mass in the first 3 days. Then cells infiltrated the tumor area, where they remained during all the time points studied, from 6-18 days. Microglia in contact with glioma cells also present changes in cell morphology, from a ramified to an amoeboid shape. Cell bodies enlarged from 366 ± 0.0 μm(2), in quiescent microglia, to 1310 ± 146.0 μm(2), and the cell processes became shortened. The GL261/CX3CR1 mouse model reported here is a valuable tool for imaging of microglial cells during glioma growth, either in fixed tissue sections or living animals. Remarkable advantages are the use of immunocompetent animals and the simplified imaging method without the need of immunohistochemical procedures.

Analysis of interphase node proteins in fission yeast by quantitative and superresolution fluorescence microscopy

PubMed Central

Akamatsu, Matthew; Lin, Yu; Bewersdorf, Joerg; Pollard, Thomas D.

2017-01-01

We used quantitative confocal microscopy and FPALM superresolution microscopy of live fission yeast to investigate the structures and assembly of two types of interphase nodes—multiprotein complexes associated with the plasma membrane that merge together and mature into the precursors of the cytokinetic contractile ring. During the long G2 phase of the cell cycle, seven different interphase node proteins maintain constant concentrations as they accumulate in proportion to cell volume. During mitosis, the total numbers of type 1 node proteins (cell cycle kinases Cdr1p, Cdr2p, Wee1p, and anillin Mid1p) are constant even when the nodes disassemble. Quantitative measurements provide strong evidence that both types of nodes have defined sizes and numbers of constituent proteins, as observed for cytokinesis nodes. Type 1 nodes assemble in two phases—a burst at the end of mitosis, followed by steady increase during interphase to double the initial number. Type 2 nodes containing Blt1p, Rho-GEF Gef2p, and kinesin Klp8p remain intact throughout the cell cycle and are constituents of the contractile ring. They are released from the contractile ring as it disassembles and then associate with type 1 nodes around the equator of the cell during interphase. PMID:28539404

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

In vivo laser confocal microscopy findings in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy

PubMed Central

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2012-01-01

Background: The purpose of this study was to investigate pathological changes of the corneal cell layer in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy by in vivo laser corneal confocal microscopy. Methods: Two patients were evaluated using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT 2-RCM). The affected corneal areas of both patients were examined. Image analysis was performed to identify corneal epithelial and stromal deposits correlated with this dystrophy. Results: Variously shaped (linear, multilaminar, curvilinear, ring-shape, geographic) highly reflective materials were observed in the “map” area, mainly in the basal epithelial cell layer. In “fingerprint” lesions, multiple linear and curvilinear hyporeflective lines were observed. Additionally, in the affected corneas, infiltration of possible Langerhans cells and other inflammatory cells was observed as highly reflective Langerhans cell-like or dot images. Finally, needle-shaped materials were observed in one patient. Conclusion: HRT 2-RCM laser confocal microscopy is capable of identifying corneal microstructural changes related to map-dot-fingerprint corneal dystrophy in vivo. The technique may be useful in elucidating the pathogenesis and natural course of map-dot-fingerprint corneal dystrophy and other similar basement membrane abnormalities. PMID:22888214

Use of the second harmonic generation microscopy to evaluate chondrogenic differentiation of mesenchymal stem cells for cartilage repair

NASA Astrophysics Data System (ADS)

Bordeaux-Rego, P.; Baratti, M. O.; Duarte, A. S. S.; Ribeiro, T. B.; Andreoli-Risso, M. F.; Vidal, B.; Miranda, J. B.; Adur, J.; de Thomaz, A. A.; Pelegati, V. B.; Costa, F. F.; Carvalho, H. F.; Cesar, C. L.; Luzo, A.; Olalla Saad, S. T.

2012-03-01

Articular cartilage injury remains one of the major concerns in orthopedic surgery. Mesenchymal stem cell (MSC) transplantation has been introduced to avoid some of the side effects and complications of current techniques.. With the aim to evaluate chondrogenic differentiation of mesenchymal stem cells, we used Second Harmonic Generation (SHG) microscopy to analyze the aggregation and orientation of collagen fibrils in the hyaline cartilage of rabbit knees. The experiment was performed using implants with type II collagen hydrogel (a biomaterial that mimics the microenvironment of the cartilage), one implant containing MSC and one other without MSC (control). After 10 weeks, the rabbit knees were dissected and fibril collagen distribution and spatial organization in the extracellular matrix of the lesions were verified by SHG. The result showed significant differences, whereas in histological sections of the cartilaginous lesions with MSC the collagen fibers are organized and regular; in the control sections the collagen fibers are more irregular, with absence of cells. A macroscopic analysis of the lesions confirmed this difference, showing a greater percentage of lesions filling in knees treated with MSC than in the knees used as controls. This study demonstrates that SHG microscopy will be an excellent tool to help in the evaluation of the effectiveness of MSC-based cell therapy for cartilage repair.

Quantitative Microscopic Analysis of Plasma Membrane Receptor Dynamics in Living Plant Cells.

PubMed

Luo, Yu; Russinova, Eugenia

2017-01-01

Plasma membrane-localized receptors are essential for cellular communication and signal transduction. In Arabidopsis thaliana, BRASSINOSTEROID INSENSITIVE1 (BRI1) is one of the receptors that is activated by binding to its ligand, the brassinosteroid (BR) hormone, at the cell surface to regulate diverse plant developmental processes. The availability of BRI1 in the plasma membrane is related to its signaling output and is known to be controlled by the dynamic endomembrane trafficking. Advances in fluorescence labeling and confocal microscopy techniques enabled us to gain a better understanding of plasma membrane receptor dynamics in living cells. Here we describe different quantitative microscopy methods to monitor the relative steady-state levels of the BRI1 protein in the plasma membrane of root epidermal cells and its relative exocytosis and recycling rates. The methods can be applied also to analyze similar dynamics of other plasma membrane-localized receptors.

Quantitative evaluation of bone resorption activity of osteoclast-like cells by measuring calcium phosphate resorbing area using incubator-facilitated and video-enhanced microscopy.

PubMed

Morimoto, Yoshitaka; Hoshino, Hironobu; Sakurai, Takashi; Terakawa, Susumu; Nagano, Akira

2009-04-01

Quantitative evaluation of the ability of bone resorption activity in live osteoclast-like cells (OCLs) has not yet been reported on. In this study, we observed the sequential morphological change of OCLs and measured the resorbing calcium phosphate (CP) area made by OCLs alone and with the addition of elcatonin utilizing incubator facilitated video-enhanced microscopy. OCLs, which were obtained from a coculture of ddy-mouse osteoblastic cells and bone marrow cells, were cultured on CP-coated quartz cover slips. The CP-free area increased constantly in the OCLs alone, whereas it did not increase after the addition of elcatonin. This study showed that analysis of the resorbed areas under the OCL body using this method enables the sequential quantitative evaluation of the bone resorption activity and the effect of several therapeutic agents on bone resorption in vitro.

Confocal laser scanning microscopy coupled to a spectrofluorometric detector as a rapid tool for determining the in vivo effect of metals on phototrophic bacteria.

PubMed

Burnat, Mireia; Diestra, Elia; Esteve, Isabel; Solé, Antonio

2010-01-01

In this paper, we determine for the first time the in vivo effect of heavy metals in a phototrophic bacterium. We used Confocal Laser Scanning Microscopy coupled to a spectrofluorometric detector as a rapid technique to measure pigment response to heavy-metal exposure. To this end, we selected lead and copper (toxic and essential metals) and Microcoleus sp. as the phototrophic bacterium because it would be feasible to see this cyanobacterium as a good biomarker, since it covers large extensions of coastal sediments. The results obtained demonstrate that, while cells are still viable, pigment peak decreases whereas metal concentration increases (from 0.1 to 1 mM Pb). Pigments are totally degraded when cultures were polluted with lead and copper at the maximum doses used (25 mM Pb(NO(3))(2) and 10 mM CuSO(4)). The aim of this study was also to identify the place of metal accumulation in Microcoleus cells. Element analysis of this cyanobacterium in the above mentioned conditions determined by Energy Dispersive X-ray microanalysis (EDX) coupled to Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), shows that Pb (but not Cu) accumulates externally and internally in cells.

Lulo cell line derived from Lutzomyia longipalpis (Diptera: Psychodidae): a novel model to assay Leishmania spp. and vector interaction.

PubMed

Côrtes, Luzia Mc; Silva, Roger Mm; Pereira, Bernardo As; Guerra, Camila; Zapata, Angela C; Bello, Felio J; Finkelstein, Léa C; Madeira, Maria F; Brazil, Reginaldo P; Côrte-Real, Suzana; Alves, Carlos R

2011-11-14

Leishmania (Vianna) braziliensis, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) chagasi are important parasites in the scenario of leishmaniasis in Brazil. During the life cycle of these parasites, the promastigote forms adhere to the midgut epithelial microvillii of phlebotomine insects to avoid being secreted along with digestive products. Lulo cells are a potential model that will help to understand the features of this adhesion phenomenon. Here, we analyze the interaction between Leishmania spp. promastigotes and Lulo cells in vitro, specifically focusing on adhesion events occurring between three Leishmania species and this cell line. Confluent monolayers of Lulo cells were incubated with promastigotes and adhesion was assessed using both light microscopy and scanning electron microscopy. The results indicate that species from the subgenera Leishmania and Viannia have great potential to adhere to Lulo cells. The highest adherence rate was observed for L. (L.) chagasi after 24 h of incubation with Lulo cells (27.3 ± 1.8% of cells with adhered promastigotes), followed by L. (L.) amazonensis (16.0 ± 0.7%) and L. (V.) braziliensis (3.0 ± 0.7%), both after 48 h. In the ultrastructural analysis, promastigote adherence was also assessed by scanning electron microscopy, showing that, for parasites from both subgenera, adhesion occurs by both the body and the flagellum. The interaction of Lulo cells with Leishmania (L.) chagasi showed the participation of cytoplasmic projections from the former closely associating the parasites with the cells. We present evidence that Lulo cells can be useful in studies of insect-parasite interactions for Leishmania species.

Electron Microscopy of Ebola Virus-Infected Cells.

PubMed

Noda, Takeshi

2017-01-01

Ebola virus (EBOV) replicates in host cells, where both viral and cellular components show morphological changes during the process of viral replication from entry to budding. These steps in the replication cycle can be studied using electron microscopy (EM), including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), which is one of the most useful methods for visualizing EBOV particles and EBOV-infected cells at the ultrastructural level. This chapter describes conventional methods for EM sample preparation of cultured cells infected with EBOV.

Ultrastructural analysis of oral exfoliated epithelial cells of tobacco smokers and betel nut chewers: A scanning electron microscopy study.

PubMed

Khan, Sameera Shamim; Shreedhar, Balasundari; Kamboj, Mala

2016-01-01

The study was undertaken to correlate epithelial surface pattern changes of oral exfoliated cells of tobacco smokers and betel nut chewers and also to compare them with patients of oral squamous cell carcinoma (OSCC) and healthy individuals. In this cross-sectional study, a total of fifty persons were included in the study, out of which thirty formed the study group (15 each tobacco smokers and betel nut chewers) and twenty formed the control group (ten each of OSCC patients - positive control and ten normal buccal mucosa - negative control). Their oral exfoliated cells were scraped, fixed, and studied under scanning electron microscope (SEM). The statistical analysis was determined using ANOVA, Tukey honestly significant difference, Chi-square test, and statistical SPASS software, P < 0.05. OSCC, Individual cell modifications, intercellular relationships and surface characteristics observed by scanning electron microscopy between OSCC, tobacco smokers, betel nut chewers compared to normal oral mucosa have been tabulated. In normal oral mucosa, cell surface morphology depends on the state of keratinization of the tissue. Thus, it could prove helpful in detecting any carcinomatous change at its incipient stage and also give an insight into the ultra-structural details of cellular differentiations in epithelial tissues.

Synthesis and characterization of nanostructured CaSiO3 biomaterial

NASA Astrophysics Data System (ADS)

Jagadale, Pramod N.; Kulal, Shivaji R.; Joshi, Meghanath G.; Jagtap, Pramod P.; Khetre, Sanjay M.; Bamane, Sambhaji R.

2013-04-01

Here we report a successful preparation of nanostructured calcium silicate by wet chemical approach. The synthesized sample was characterized by various physico-chemical methods. Thermal stability was investigated using thermo-gravimetric and differential thermal analysis (TG-DTA). Structural characterization of the sample was carried out by the X-ray diffraction technique (XRD) which confirmed its single phase hexagonal structure. Transmission electron microscopy (TEM) was used to study the nanostructure of the ceramics while homogeneous grain distribution was revealed by scanning electron microscopy studies (SEM). The elemental analysis data obtained from energy dispersive X-ray spectroscopy (EDAX) were in close agreement with the starting composition used for the synthesis. Superhydrophilic nature of CaSiO3 was investigated at room temperature by sessile drop technique. Effect of porous nanosized CaSiO3 on early adhesion and proliferation of human bone marrow mesenchymal stem cells (BMMSCs) and cord blood mesenchymal stem (CBMSCs) cells was measured in vitro. MTT cytotoxicity test and cell adhesion test showed that the material had good biocompatibility and promoted cell viability and cell proliferation. It has been stated that the cell viability and proliferation are significantly affected by time and concentration of CaSiO3. These findings indicate that the CaSiO3 ceramics has good biocompatibility and that it is promising as a biomaterial.

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

PubMed

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

2018-03-01

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

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

PubMed Central

Puchner, Elias M.; Walter, Jessica M.; Kasper, Robert; Huang, Bo; Lim, Wendell A.

2013-01-01

Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes. PMID:24043832

Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice.

PubMed

Joshi, Molishree; Keith Pittman, H; Haisch, Carl; Verbanac, Kathryn

2008-09-01

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.

Classification of Normal and Apoptotic Cells from Fluorescence Microscopy Images Using Generalized Polynomial Chaos and Level Set Function.

PubMed

Du, Yuncheng; Budman, Hector M; Duever, Thomas A

2016-06-01

Accurate automated quantitative analysis of living cells based on fluorescence microscopy images can be very useful for fast evaluation of experimental outcomes and cell culture protocols. In this work, an algorithm is developed for fast differentiation of normal and apoptotic viable Chinese hamster ovary (CHO) cells. For effective segmentation of cell images, a stochastic segmentation algorithm is developed by combining a generalized polynomial chaos expansion with a level set function-based segmentation algorithm. This approach provides a probabilistic description of the segmented cellular regions along the boundary, from which it is possible to calculate morphological changes related to apoptosis, i.e., the curvature and length of a cell's boundary. These features are then used as inputs to a support vector machine (SVM) classifier that is trained to distinguish between normal and apoptotic viable states of CHO cell images. The use of morphological features obtained from the stochastic level set segmentation of cell images in combination with the trained SVM classifier is more efficient in terms of differentiation accuracy as compared with the original deterministic level set method.

Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions.

PubMed

Wolters, Manuel; Zobiak, Bernd; Nauth, Theresa; Aepfelbacher, Martin

2015-10-13

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

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

PubMed

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

2009-03-01

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

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

PubMed Central

Avti, Pramod K; Sitharaman, Balaji

2012-01-01

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

Accurate phase measurements for thick spherical objects using optical quadrature microscopy

NASA Astrophysics Data System (ADS)

Warger, William C., II; DiMarzio, Charles A.

2009-02-01

In vitro fertilization (IVF) procedures have resulted in the birth of over three million babies since 1978. Yet the live birth rate in the United States was only 34% in 2005, with 32% of the successful pregnancies resulting in multiple births. These multiple pregnancies were directly attributed to the transfer of multiple embryos to increase the probability that a single, healthy embryo was included. Current viability markers used for IVF, such as the cell number, symmetry, size, and fragmentation, are analyzed qualitatively with differential interference contrast (DIC) microscopy. However, this method is not ideal for quantitative measures beyond the 8-cell stage of development because the cells overlap and obstruct the view within and below the cluster of cells. We have developed the phase-subtraction cell-counting method that uses the combination of DIC and optical quadrature microscopy (OQM) to count the number of cells accurately in live mouse embryos beyond the 8-cell stage. We have also created a preliminary analysis to measure the cell symmetry, size, and fragmentation quantitatively by analyzing the relative dry mass from the OQM image in conjunction with the phase-subtraction count. In this paper, we will discuss the characterization of OQM with respect to measuring the phase accurately for spherical samples that are much larger than the depth of field. Once fully characterized and verified with human embryos, this methodology could provide the means for a more accurate method to score embryo viability.

Myofibroblasts in interstitial lung diseases show diverse electron microscopic and invasive features.

PubMed

Karvonen, Henna M; Lehtonen, Siri T; Sormunen, Raija T; Harju, Terttu H; Lappi-Blanco, Elisa; Bloigu, Risto S; Kaarteenaho, Riitta L

2012-09-01

The characteristic features of myofibroblasts in various lung disorders are poorly understood. We have evaluated the ultrastructure and invasive capacities of myofibroblasts cultured from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples from patients with different types of lung diseases. Cells were cultured from samples of BAL fluid collected from 51 patients that had undergone bronchoscopy and BAL for diagnostic purposes. The cells were visualized by transmission electron microscopy and immunoelectron microscopy to achieve ultrastructural localization of alpha-smooth muscle actin (α-SMA) and fibronectin. The levels of α-SMA protein and mRNA and fibronectin mRNA were measured by western blot and quantitative real-time reverse transcriptase polymerase chain reaction. The invasive capacities of the cells were evaluated. The cultured cells were either fibroblasts or myofibroblasts. The structure of the fibronexus, and the amounts of intracellular actin, extracellular fibronectin and cell junctions of myofibroblasts varied in different diseases. In electron and immunoelectron microscopy, cells cultured from interstitial lung diseases (ILDs) expressed more actin filaments and α-SMA than normal lung. The invasive capacity of the cells obtained from patients with idiopathic pulmonary fibrosis was higher than that from patients with other type of ILDs. Cells expressing more actin filaments had a higher invasion capacity. It is concluded that electron and immunoelectron microscopic studies of myofibroblasts can reveal differential features in various diseases. An analysis of myofibroblasts cultured from diagnostic BAL fluid samples may represent a new kind of tool for diagnostics and research into lung diseases.

[The estimation of possibilities for the application of the laser capture microdissection technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA].

PubMed

Ivanov, P L; Leonov, S N; Zemskova, E Iu

2012-01-01

The present study was designed to estimate the possibilities of application of the laser capture microdissection (LCM) technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA. The experimental method employed for the purpose was the multiplex multilocus analysis of autosomal DNA polymorphism in the preparations of buccal epitheliocytes obtained by LCM. The key principles of the study were the application of physical methods for contrast enhancement of the micropreparations (such as phase-contrast microscopy and dark-field microscopy) and PCR-compatible cell lysis. Genotyping was carried out with the use of AmpFISTR Minifiler TM PCR Amplification Kits ("Applied Biosynthesis", USA). It was shown that the technique employed in the present study ensures reliable genotyping of human chromosomal DNA in the pooled preparations containing 10-20 dissected diploid cells each. This result fairly well agrees with the calculated sensitivity of the method. A few practical recommendations are offered.

Multi-color electron microscopy by element-guided identification of cells, organelles and molecules.

PubMed

Scotuzzi, Marijke; Kuipers, Jeroen; Wensveen, Dasha I; de Boer, Pascal; Hagen, Kees C W; Hoogenboom, Jacob P; Giepmans, Ben N G

2017-04-07

Cellular complexity is unraveled at nanometer resolution using electron microscopy (EM), but interpretation of macromolecular functionality is hampered by the difficulty in interpreting grey-scale images and the unidentified molecular content. We perform large-scale EM on mammalian tissue complemented with energy-dispersive X-ray analysis (EDX) to allow EM-data analysis based on elemental composition. Endogenous elements, labels (gold and cadmium-based nanoparticles) as well as stains are analyzed at ultrastructural resolution. This provides a wide palette of colors to paint the traditional grey-scale EM images for composition-based interpretation. Our proof-of-principle application of EM-EDX reveals that endocrine and exocrine vesicles exist in single cells in Islets of Langerhans. This highlights how elemental mapping reveals unbiased biomedical relevant information. Broad application of EM-EDX will further allow experimental analysis on large-scale tissue using endogenous elements, multiple stains, and multiple markers and thus brings nanometer-scale 'color-EM' as a promising tool to unravel molecular (de)regulation in biomedicine.

Multi-color electron microscopy by element-guided identification of cells, organelles and molecules

PubMed Central

Scotuzzi, Marijke; Kuipers, Jeroen; Wensveen, Dasha I.; de Boer, Pascal; Hagen, Kees (C.) W.; Hoogenboom, Jacob P.; Giepmans, Ben N. G.

2017-01-01

Cellular complexity is unraveled at nanometer resolution using electron microscopy (EM), but interpretation of macromolecular functionality is hampered by the difficulty in interpreting grey-scale images and the unidentified molecular content. We perform large-scale EM on mammalian tissue complemented with energy-dispersive X-ray analysis (EDX) to allow EM-data analysis based on elemental composition. Endogenous elements, labels (gold and cadmium-based nanoparticles) as well as stains are analyzed at ultrastructural resolution. This provides a wide palette of colors to paint the traditional grey-scale EM images for composition-based interpretation. Our proof-of-principle application of EM-EDX reveals that endocrine and exocrine vesicles exist in single cells in Islets of Langerhans. This highlights how elemental mapping reveals unbiased biomedical relevant information. Broad application of EM-EDX will further allow experimental analysis on large-scale tissue using endogenous elements, multiple stains, and multiple markers and thus brings nanometer-scale ‘color-EM’ as a promising tool to unravel molecular (de)regulation in biomedicine. PMID:28387351

Automated cell analysis tool for a genome-wide RNAi screen with support vector machine based supervised learning

NASA Astrophysics Data System (ADS)

Remmele, Steffen; Ritzerfeld, Julia; Nickel, Walter; Hesser, Jürgen

2011-03-01

RNAi-based high-throughput microscopy screens have become an important tool in biological sciences in order to decrypt mostly unknown biological functions of human genes. However, manual analysis is impossible for such screens since the amount of image data sets can often be in the hundred thousands. Reliable automated tools are thus required to analyse the fluorescence microscopy image data sets usually containing two or more reaction channels. The herein presented image analysis tool is designed to analyse an RNAi screen investigating the intracellular trafficking and targeting of acylated Src kinases. In this specific screen, a data set consists of three reaction channels and the investigated cells can appear in different phenotypes. The main issue of the image processing task is an automatic cell segmentation which has to be robust and accurate for all different phenotypes and a successive phenotype classification. The cell segmentation is done in two steps by segmenting the cell nuclei first and then using a classifier-enhanced region growing on basis of the cell nuclei to segment the cells. The classification of the cells is realized by a support vector machine which has to be trained manually using supervised learning. Furthermore, the tool is brightness invariant allowing different staining quality and it provides a quality control that copes with typical defects during preparation and acquisition. A first version of the tool has already been successfully applied for an RNAi-screen containing three hundred thousand image data sets and the SVM extended version is designed for additional screens.

Live-cell confocal microscopy and quantitative 4D image analysis of anchor cell invasion through the basement membrane in C. elegans

PubMed Central

Kelley, Laura C.; Wang, Zheng; Hagedorn, Elliott J.; Wang, Lin; Shen, Wanqing; Lei, Shijun; Johnson, Sam A.; Sherwood, David R.

2018-01-01

Cell invasion through basement membrane (BM) barriers is crucial during development, leukocyte trafficking, and for the spread of cancer. Despite its importance in normal and diseased states, the mechanisms that direct invasion are poorly understood, in large part because of the inability to visualize dynamic cell-basement membrane interactions in vivo. This protocol describes multi-channel time-lapse confocal imaging of anchor cell invasion in live C. elegans. Methods presented include outline slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min), and quantitative analysis (variable timing). Images acquired enable direct measurement of invasive dynamics including invadopodia formation, cell membrane protrusions, and BM removal. This protocol can be combined with genetic analysis, molecular activity probes, and optogenetic approaches to uncover molecular mechanisms underlying cell invasion. These methods can also be readily adapted for real-time analysis of cell migration, basement membrane turnover, and cell membrane dynamics by any worm laboratory. PMID:28880279

Plant cell wall characterization using scanning probe microscopy techniques

PubMed Central

Yarbrough, John M; Himmel, Michael E; Ding, Shi-You

2009-01-01

Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy. PMID:19703302

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

PubMed

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

2010-08-01

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

Unraveling micro- and nanoscale degradation processes during operation of high-temperature polymer-electrolyte-membrane fuel cells

NASA Astrophysics Data System (ADS)

Hengge, K.; Heinzl, C.; Perchthaler, M.; Varley, D.; Lochner, T.; Scheu, C.

2017-10-01

The work in hand presents an electron microscopy based in-depth study of micro- and nanoscale degradation processes that take place during the operation of high-temperature polymer-electrolyte-membrane fuel cells (HT-PEMFCs). Carbon supported Pt particles were used as cathodic catalyst material and the bimetallic, carbon supported Pt/Ru system was applied as anode. As membrane, cross-linked polybenzimidazole was used. Scanning electron microscopy analysis of cross-sections of as-prepared and long-term operated membrane-electrode-assemblies revealed insight into micrometer scale degradation processes: operation-caused catalyst redistribution and thinning of the membrane and electrodes. Transmission electron microscopy investigations were performed to unravel the nanometer scale phenomena: a band of Pt and Pt/Ru nanoparticles was detected in the membrane adjacent to the cathode catalyst layer. Quantification of the elemental composition of several individual nanoparticles and the overall band area revealed that they stem from both anode and cathode catalyst layers. The results presented do not demonstrate any catastrophic failure but rather intermediate states during fuel cell operation and indications to proceed with targeted HT-PEMFC optimization.

Cryogenic coherent X-ray diffraction imaging of biological samples at SACLA: a correlative approach with cryo-electron and light microscopy.

PubMed

Takayama, Yuki; Yonekura, Koji

2016-03-01

Coherent X-ray diffraction imaging at cryogenic temperature (cryo-CXDI) allows the analysis of internal structures of unstained, non-crystalline, whole biological samples in micrometre to sub-micrometre dimensions. Targets include cells and cell organelles. This approach involves preparing frozen-hydrated samples under controlled humidity, transferring the samples to a cryo-stage inside a vacuum chamber of a diffractometer, and then exposing the samples to coherent X-rays. Since 2012, cryo-coherent diffraction imaging (CDI) experiments have been carried out with the X-ray free-electron laser (XFEL) at the SPring-8 Ångstrom Compact free-electron LAser (SACLA) facility in Japan. Complementary use of cryo-electron microscopy and/or light microscopy is highly beneficial for both pre-checking samples and studying the integrity or nature of the sample. This article reports the authors' experience in cryo-XFEL-CDI of biological cells and organelles at SACLA, and describes an attempt towards reliable and higher-resolution reconstructions, including signal enhancement with strong scatterers and Patterson-search phasing.

CIAN - Cell Imaging and Analysis Network at the Biology Department of McGill University

PubMed Central

Lacoste, J.; Lesage, G.; Bunnell, S.; Han, H.; Küster-Schöck, E.

2010-01-01

CF-31 The Cell Imaging and Analysis Network (CIAN) provides services and tools to researchers in the field of cell biology from within or outside Montreal's McGill University community. CIAN is composed of six scientific platforms: Cell Imaging (confocal and fluorescence microscopy), Proteomics (2-D protein gel electrophoresis and DiGE, fluorescent protein analysis), Automation and High throughput screening (Pinning robot and liquid handler), Protein Expression for Antibody Production, Genomics (real-time PCR), and Data storage and analysis (cluster, server, and workstations). Users submit project proposals, and can obtain training and consultation in any aspect of the facility, or initiate projects with the full-service platforms. CIAN is designed to facilitate training, enhance interactions, as well as share and maintain resources and expertise.

Super-resolution atomic force photoactivated microscopy of biological samples (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lee, Seunghyun; Kim, Hyemin; Shin, Seungjun; Doh, Junsang; Kim, Chulhong

2017-03-01

Optical microscopy (OM) and photoacoustic microscopy (PAM) have previously been used to image the optical absorption of intercellular features of biological cells. However, the optical diffraction limit ( 200 nm) makes it difficult for these modalities to image nanoscale inner cell structures and the distribution of internal cell components. Although super-resolution fluorescence microscopy, such as stimulated emission depletion microscopy (STED) and stochastic optical reconstruction microscopy (STORM), has successfully performed nanoscale biological imaging, these modalities require the use of exogenous fluorescence agents, which are unfavorable for biological samples. Our newly developed atomic force photoactivated microscopy (AFPM) can provide optical absorption images with nanoscale lateral resolution without any exogenous contrast agents. AFPM combines conventional atomic force microscopy (AFM) and an optical excitation system, and simultaneously provides multiple contrasts, such as the topography and magnitude of optical absorption. AFPM can detect the intrinsic optical absorption of samples with 8 nm lateral resolution, easily overcoming the diffraction limit. Using the label-free AFPM system, we have successfully imaged the optical absorption properties of a single melanoma cell (B16F10) and a rosette leaf epidermal cell of Arabidopsis (ecotype Columbia (Col-0)) with nanoscale lateral resolution. The remarkable images show the melanosome distribution of a melanoma cell and the biological structures of a plant cell. AFPM provides superior imaging of optical absorption with a nanoscale lateral resolution, and it promises to become widely used in biological and chemical research.

In vivo flow cytometry for blood cell analysis using differential epi-detection of forward scattered light

NASA Astrophysics Data System (ADS)

Paudel, Hari P.; Jung, Yookyung; Raphael, Anthony; Alt, Clemens; Wu, Juwell; Runnels, Judith; Lin, Charles P.

2018-02-01

The present standard of blood cell analysis is an invasive procedure requiring the extraction of patient's blood, followed by ex-vivo analysis using a flow cytometer or a hemocytometer. We are developing a noninvasive optical technique that alleviates the need for blood extraction. For in-vivo blood analysis we need a high speed, high resolution and high contrast label-free imaging technique. In this proceeding report, we reported a label-free method based on differential epi-detection of forward scattered light, a method inspired by Jerome Mertz's oblique back-illumination microscopy (OBM) (Ford et al, Nat. Meth. 9(12) 2012). The differential epi-detection of forward light gives phase contrast image at diffraction-limited resolution. Unlike reflection confocal microscopy (RCM), which detects only sharp refractive index variation and suffers from speckle noise, this technique is suitable for detection of subtle variation of refractive index in biological tissue and it provides the shape and the size of cells. A custom built high speed electronic detection circuit board produces a real-time differential signal which yields image contrast based on phase gradient in the sample. We recorded blood flow in-vivo at 17.2k lines per second in line scan mode, or 30 frames per second (full frame), or 120 frame per second (quarter frame) in frame scan mode. The image contrast and speed of line scan data recording show the potential of the system for noninvasive blood cell analysis.

Design of a bioresorbable polymeric scaffold for osteoblast culture

NASA Astrophysics Data System (ADS)

Ditaranto, Vincent M., Jr.

Bioresorbable polymeric scaffolds were designed for the purpose of growing rat osteosarcoma cells (ROS 17/2.8) using the compression molding method. The material used in the construction of the scaffolds was a mixture of polycaprolactone (PCL), Hydroxyapatite (HA), Glycerin (GL) and salt (NaCl) for porosity. The concentration of the several materials utilized, was determined by volume. Past research at the University of Massachusetts Lowell (UML) has successfully utilized the compression molding method for the construction of scaffolds, but was unable to accomplish the goal of long term cell survival and complete cellular proliferation throughout a three dimensional scaffold. This research investigated various concentrations of the materials and molding temperatures used for the manufacture of scaffolds in order to improve the scaffold design and address those issues. The design of the scaffold using the compression molding process is detailed in the Method and Materials section of this thesis. The porogen (salt) used for porosity was suspected as a possible source of contamination causing cell apoptosis in past studies. This research addressed the issues for cell survival and proliferation throughout a three dimensional scaffold. The leaching of the salt was one major design modification. This research successfully used ultrasonic leaching in addition to the passive method. Prior to cell culture, the scaffolds were irradiated to 2.75 Mrad, with cobalt-60 gamma radionuclide. The tissue culture consisted of two trials: (1) cell culture in scaffolds cleaned with passive leaching; (2) cell culture with scaffolds cleaned with ultrasonic leaching. Cell survival and proliferation was accomplished only with the addition of ultrasonic leaching of the scaffolds. Analysis of the scaffolds included Scanning Electron Microscopy (SEM), Nikon light microscopy and x-ray mapping of the calcium, sodium and chloride ion distribution. The cells were analyzed by Environmental Scanning Electron Microscopy (ESEM) and Nikon light microscopy. The high magnification of ESEM up to 60,000 x revealed an unexpected discovery. The osteoblasts appeared to be remodeling the PCL scaffold shown in the last two figures of this research.

Two-Photon Microscopy Analysis of Gold Nanoparticle Uptake in 3D Cell Spheroids.

PubMed

Rane, Tushar D; Armani, Andrea M

2016-01-01

Nanomaterials can be synthesized from a wide range of material systems in numerous morphologies, creating an extremely diverse portfolio. As result of this tunability, these materials are emerging as a new class of nanotherapeutics and imaging agents. One particularly interesting nanomaterial is the gold nanoparticle. Due to its inherent biocompatibility and tunable photothermal behavior, it has made a rapid transition from the lab setting to in vivo testing. In most nanotherapeutic applications, the efficacy of the agent is directly related to the target of interest. However, the optimization of the AuNP size and shape for efficacy in vitro, prior to testing in in vivo models of a disease, has been largely limited to two dimensional monolayers of cells. Two dimensional cell cultures are unable to reproduce conditions experienced by AuNP in the body. In this article, we systematically investigate the effect of different properties of AuNP on the penetration depth into 3D cell spheroids using two-photon microscopy. The 3D spheroids are formed from the HCT116 cell line, a colorectal carcinoma cell line. In addition to studying different sizes and shapes of AuNPs, we also study the effect of an oligo surface chemistry. There is a significant difference between AuNP uptake profiles in the 2D monolayers of cells as compared to the 3D cell spheroids. Additionally, the range of sizes and shapes studied here also exhibit marked differences in uptake penetration depth and efficacy. Finally, our results demonstrate that two-photon microscopy enables quantitative AuNP localization and concentration data to be obtained at the single spheroid level without fluorescent labeling of the AuNP, thus, providing a viable technique for large scale screening of AuNP properties in 3D cell spheroids as compared to tedious and time consuming techniques like electron microscopy.

Thermal stress cycling of GaAs solar cells

NASA Technical Reports Server (NTRS)

Francis, Robert W.

1987-01-01

Thermal stress cycling was performed on gallium arsenide solar cells to investigate their electrical, mechanical, and structural integrity. Cells were cycled under low Earth orbit (LEO) simulated temperature conditions in vacuum. Cell evaluations consisted of power output values, spectral response, optical microscopy and ion microprobe mass analysis, and depth profiles on both front surface inter-grid areas and metallization contact grid lines. Cells were examined for degradation after 500, 5,000, 10,000 and 15,245 thermal cycles. No indication of performance degradation was found for any vendor's cell lot.

Following damage, the majority of bone marrow-derived airway cells express an epithelial marker.

PubMed

MacPherson, Heather; Keir, Pamela A; Edwards, Carol J; Webb, Sheila; Dorin, Julia R

2006-12-19

Adult-derived bone marrow stem cells are capable of reconstituting the haematopoietic system. However there is ongoing debate in the literature as to whether bone marrow derived cells have the ability to populate other tissues and express tissue specific markers. The airway has been an organ of major interest and was one of the first where this was demonstrated. We have previously demonstrated that the mouse airway can be repopulated by side population bone marrow transplanted cells. Here we investigate the frequency and phenotypic nature of these bone marrow derived cells. Female mice were engrafted with male whole bone marrow or side population (SP) cells and subjected to detergent-induced damage after 3 months. Donor cells were identified by Y chromosome fluorescence in situ hybridisation and their phenotype was assessed by immunohistochemistry on the same sections. Slides were visualised by a combination of widefield and deconvolved microscopy and whole cells were analysed on cytospin preparations. The frequencies of engraftment of male cells in the airway of mice that show this (9/10), range from 1.0-1.6% with whole marrow and 0.6-1.5% with SP cells. Undamaged controls have only between 0.1 and 0.2% male cells in the trachea. By widefield microscopy analysis we find 60.2% (53/88) of male donor derived cells express cytokeratins as a marker of epithelial cells. These results were reinforced using deconvolved microscopy and scored by two independent investigators. In addition cytospin analysis of cells dissociated from the damaged trachea of engrafted mice also reveals donor derived Y chromosome positive cells that are immunopositive for cytokeratin. Using cytokeratin and the universal haematopoietic marker CD45 immunohistochemistry, we find the donor derived cells fall into four phenotypic classes. We do not detect cytokeratin positive cells in whole bone marrow using cytokeratin immunostaining and we do not detect any cytokeratin mRNA in SP or bone marrow samples by RT-PCR. The appearance of bone marrow derived cells in the tracheal epithelium is enriched by detergent-induced tissue damage and the majority of these cells express an epithelial marker. The cytokeratin positive donor derived cells in the tracheal epithelium are not present in the injected donor cells and must have acquired this novel phenotype in vivo.

Following damage, the majority of bone marrow-derived airway cells express an epithelial marker

PubMed Central

MacPherson, Heather; Keir, Pamela A; Edwards, Carol J; Webb, Sheila; Dorin, Julia R

2006-01-01

Background Adult-derived bone marrow stem cells are capable of reconstituting the haematopoietic system. However there is ongoing debate in the literature as to whether bone marrow derived cells have the ability to populate other tissues and express tissue specific markers. The airway has been an organ of major interest and was one of the first where this was demonstrated. We have previously demonstrated that the mouse airway can be repopulated by side population bone marrow transplanted cells. Here we investigate the frequency and phenotypic nature of these bone marrow derived cells. Methods Female mice were engrafted with male whole bone marrow or side population (SP) cells and subjected to detergent-induced damage after 3 months. Donor cells were identified by Y chromosome fluorescence in situ hybridisation and their phenotype was assessed by immunohistochemistry on the same sections. Slides were visualised by a combination of widefield and deconvolved microscopy and whole cells were analysed on cytospin preparations. Results The frequencies of engraftment of male cells in the airway of mice that show this (9/10), range from 1.0 – 1.6% with whole marrow and 0.6 – 1.5% with SP cells. Undamaged controls have only between 0.1 and 0.2% male cells in the trachea. By widefield microscopy analysis we find 60.2% (53/88) of male donor derived cells express cytokeratins as a marker of epithelial cells. These results were reinforced using deconvolved microscopy and scored by two independent investigators. In addition cytospin analysis of cells dissociated from the damaged trachea of engrafted mice also reveals donor derived Y chromosome positive cells that are immunopositive for cytokeratin. Using cytokeratin and the universal haematopoietic marker CD45 immunohistochemistry, we find the donor derived cells fall into four phenotypic classes. We do not detect cytokeratin positive cells in whole bone marrow using cytokeratin immunostaining and we do not detect any cytokeratin mRNA in SP or bone marrow samples by RT-PCR. Conclusion The appearance of bone marrow derived cells in the tracheal epithelium is enriched by detergent-induced tissue damage and the majority of these cells express an epithelial marker. The cytokeratin positive donor derived cells in the tracheal epithelium are not present in the injected donor cells and must have acquired this novel phenotype in vivo. PMID:17177981

Destructive physical analysis results of Ni/H2 cells cycled in LEO regime

NASA Technical Reports Server (NTRS)

Lim, Hong S.; Zelter, Gabriela R.; Smithrick, John J.; Hall, Stephen W.

1991-01-01

Six 48-Ah individual pressure vessel (IPV) Ni/H2 cells containing 26 and 31 percent KOH electrolyte were life cycle tested in low Earth orbit. All three cells containing 31 percent KOH failed (3729, 4165, and 11,355 cycles), while those with 26 percent KOH were cycled over 14,000 times in the continuing test. Destructive physical analysis (DPA) of the failed cells included visual inspections, measurements of electrode thickness, scanning electron microscopy, chemical analysis, and measurements of nickel electrode capacity in an electrolyte flooded cell. The cycling failure was due to a decrease of nickel electrode capacity. As possible causes of the capacity decrease, researchers observed electrode expansion, rupture, and corrosion of the nickel electrode substrate, active material redistribution, and accumulation of electrochemically undischargeable active material with cycling.

Confocal microscopy to guide laser ablation of basal cell carinoma: a preliminary feasibility study

NASA Astrophysics Data System (ADS)

Larson, Bjorg A.; Sierra, Heidy; Chen, Jason; Rajadhyaksha, Milind

2013-03-01

Laser ablation may be a promising method for removal of skin lesions, with the potential for better cosmetic outcomes and reduced scarring and infection. An obstacle to implementing laser ablation is that the treatment leaves no tissue for histopathological analysis. Pre-operative and intra-operative mapping of BCCs using confocal microscopy may guide the ablation of the tumor until all tumor is removed. We demonstrate preliminary feasibility of confocal microscopy to guide laser ablation of BCCs in freshly excised tissue from Mohs surgery. A 2940 nm Er:YAG laser provides efficient ablation of tumor with reduced thermal damage to the surrounding tissue.

Improved Osteoblast and Chondrocyte Adhesion and Viability by Surface-Modified Ti6Al4V Alloy with Anodized TiO2 Nanotubes Using a Super-Oxidative Solution

PubMed Central

Beltrán-Partida, Ernesto; Moreno-Ulloa, Aldo; Valdez-Salas, Benjamín; Velasquillo, Cristina; Carrillo, Monica; Escamilla, Alan; Valdez, Ernesto; Villarreal, Francisco

2015-01-01

Titanium (Ti) and its alloys are amongst the most commonly-used biomaterials in orthopedic and dental applications. The Ti-aluminum-vanadium alloy (Ti6Al4V) is widely used as a biomaterial for these applications by virtue of its favorable properties, such as high tensile strength, good biocompatibility and excellent corrosion resistance. TiO2 nanotube (NTs) layers formed by anodization on Ti6Al4V alloy have been shown to improve osteoblast adhesion and function when compared to non-anodized material. In his study, NTs were grown on a Ti6Al4V alloy by anodic oxidation for 5 min using a super-oxidative aqueous solution, and their in vitro biocompatibility was investigated in pig periosteal osteoblasts and cartilage chondrocytes. Scanning electron microscopy (SEM), energy dispersion X-ray analysis (EDX) and atomic force microscopy (AFM) were used to characterize the materials. Cell morphology was analyzed by SEM and AFM. Cell viability was examined by fluorescence microscopy. Cell adhesion was evaluated by nuclei staining and cell number quantification by fluorescence microscopy. The average diameter of the NTs was 80 nm. The results demonstrate improved cell adhesion and viability at Day 1 and Day 3 of cell growth on the nanostructured material as compared to the non-anodized alloy. In conclusion, this study evidences the suitability of NTs grown on Ti6Al4V alloy using a super-oxidative water and a short anodization process to enhance the adhesion and viability of osteoblasts and chondrocytes. The results warrant further investigation for its use as medical implant materials. PMID:28787976

Improved Osteoblast and Chondrocyte Adhesion and Viability by Surface-Modified Ti6Al4V Alloy with Anodized TiO₂ Nanotubes Using a Super-Oxidative Solution.

PubMed

Beltrán-Partida, Ernesto; Moreno-Ulloa, Aldo; Valdez-Salas, Benjamín; Velasquillo, Cristina; Carrillo, Monica; Escamilla, Alan; Valdez, Ernesto; Villarreal, Francisco

2015-03-02

Titanium (Ti) and its alloys are amongst the most commonly-used biomaterials in orthopedic and dental applications. The Ti-aluminum-vanadium alloy (Ti6Al4V) is widely used as a biomaterial for these applications by virtue of its favorable properties, such as high tensile strength, good biocompatibility and excellent corrosion resistance. TiO₂ nanotube (NTs) layers formed by anodization on Ti6Al4V alloy have been shown to improve osteoblast adhesion and function when compared to non-anodized material. In his study, NTs were grown on a Ti6Al4V alloy by anodic oxidation for 5 min using a super-oxidative aqueous solution, and their in vitro biocompatibility was investigated in pig periosteal osteoblasts and cartilage chondrocytes. Scanning electron microscopy (SEM), energy dispersion X-ray analysis (EDX) and atomic force microscopy (AFM) were used to characterize the materials. Cell morphology was analyzed by SEM and AFM. Cell viability was examined by fluorescence microscopy. Cell adhesion was evaluated by nuclei staining and cell number quantification by fluorescence microscopy. The average diameter of the NTs was 80 nm. The results demonstrate improved cell adhesion and viability at Day 1 and Day 3 of cell growth on the nanostructured material as compared to the non-anodized alloy. In conclusion, this study evidences the suitability of NTs grown on Ti6Al4V alloy using a super-oxidative water and a short anodization process to enhance the adhesion and viability of osteoblasts and chondrocytes. The results warrant further investigation for its use as medical implant materials.

[Response of HeLa cells to mitomycine C. III. The analysis of nucleoli of mother and daughter cells].

PubMed

Petrov, Iu P; Neguliaev, Iu A; Tsupkina, N V

2014-01-01

The comparative analysis of the number of nucleoli in cells of the established HeLa-M line was carried out before and after exposure to mitomycin C in a concentration of 10 μg/ml for 2 h. Using time-lapse microscopy, nucleoli in mother and their respective daughter cells were computed. It has been shown that the average number of nucleoli per cell is generally higher in daughter cells than in mother cells, and a standard deviation, on the contrary, decreases. An average number of nucleoli in daughter cells, whose mother cells had been treated with mitomycin C, was higher than in corresponding cells of control group. The separate analysis has been performed for the cells having from 1 to 4 nucleoli. Nonrandom complete coincidence of the number of nucleoli in mather and daughter cells has been typicaly shown for about 1/7 of the total cell population. Mitomycin C reduces this value of about 1.5 times.

Analysis of defect structure in silicon. Characterization of SEMIX material. Silicon sheet growth development for the large area silicon sheet task of the low-cost solar array project

NASA Technical Reports Server (NTRS)

Natesh, R.; Stringfellow, G. B.; Virkar, A. V.; Dunn, J.; Guyer, T.

1983-01-01

Statistically significant quantitative structural imperfection measurements were made on samples from ubiquitous crystalline process (UCP) Ingot 5848 - 13C. Important correlation was obtained between defect densities, cell efficiency, and diffusion length. Grain boundary substructure displayed a strong influence on the conversion efficiency of solar cells from Semix material. Quantitative microscopy measurements gave statistically significant information compared to other microanalytical techniques. A surface preparation technique to obtain proper contrast of structural defects suitable for quantimet quantitative image analyzer (QTM) analysis was perfected and is used routinely. The relationships between hole mobility and grain boundary density was determined. Mobility was measured using the van der Pauw technique, and grain boundary density was measured using quantitative microscopy technique. Mobility was found to decrease with increasing grain boundary density.

Diatomite silica nanoparticles for drug delivery

NASA Astrophysics Data System (ADS)

Ruggiero, Immacolata; Terracciano, Monica; Martucci, Nicola M.; De Stefano, Luca; Migliaccio, Nunzia; Tatè, Rosarita; Rendina, Ivo; Arcari, Paolo; Lamberti, Annalisa; Rea, Ilaria

2014-07-01

Diatomite is a natural fossil material of sedimentary origin, constituted by fragments of diatom siliceous skeletons. In this preliminary work, the properties of diatomite nanoparticles as potential system for the delivery of drugs in cancer cells were exploited. A purification procedure, based on thermal treatments in strong acid solutions, was used to remove inorganic and organic impurities from diatomite and to make them a safe material for medical applications. The micrometric diatomite powder was reduced in nanoparticles by mechanical crushing, sonication, and filtering. Morphological analysis performed by dynamic light scattering and transmission electron microscopy reveals a particles size included between 100 and 300 nm. Diatomite nanoparticles were functionalized by 3-aminopropyltriethoxysilane and labeled by tetramethylrhodamine isothiocyanate. Different concentrations of chemically modified nanoparticles were incubated with cancer cells and confocal microscopy was performed. Imaging analysis showed an efficient cellular uptake and homogeneous distribution of nanoparticles in cytoplasm and nucleus, thus suggesting their potentiality as nanocarriers for drug delivery.

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

PubMed

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

2015-11-01

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

Identification of Foreign Particles in Human Tissues using Raman Microscopy.

PubMed

Campion, Alan; Smith, Kenneth J; Fedulov, Alexey V; Gregory, David; Fan, Yuwei; Godleski, John J

2018-06-12

The precise identification of foreign particles in tissue for patient care and research has been studied using polarized light microscopy, electron microscopy with X-ray analysis, and electron diffraction. The goal of this study was to unambiguously identify particles in tissues using a combina-tion of polarized light microscopy and Raman microscopy, which provides chemical composition and microstructural characterization of complex materials with submicron spatial resolution. We designed a model system of stained and unstained cells that contained birefringent talc particles, and systematically investigated the influence of slide and coverslip materials, laser wavelengths, and mounting media on the Raman spectra ob-tained. Hematoxylin and eosin stained slides did not produce useful results because of fluorescence interference from the stains. Unstained cell samples prepared with standard slides and coverslips produce high quality Raman spectra when excited at 532 nm; the spectra are uniquely as-signed to talc. We also obtain high quality Raman spectra specific for talc in unstained tissue samples (pleural tissue following talc pleurodesis and ovarian tissue following long-term perineal talc exposure). Raman microscopy is sufficiently sensitive and compositionally selective to identify particles as small as one micron in diameter. Among commonly used coverslip mounting media, Cytoseal 60 is recommended; Permount was unacceptable due to intense background interference. Raman spectra have been catalogued for thousands of substances, which suggests that this approach is likely to be successful in identifying other particles of interest in tissues, potentially making Raman microscopy a powerful new tool in pathology.

Lens-free microscopy of cerebrospinal fluid for the laboratory diagnosis of meningitis

NASA Astrophysics Data System (ADS)

Delacroix, Robin; Morel, Sophie Nhu An; Hervé, Lionel; Bordy, Thomas; Blandin, Pierre; Dinten, Jean-Marc; Drancourt, Michel; Allier, Cédric

2018-02-01

The cytology of the cerebrospinal fluid is traditionally performed by an operator (physician, biologist) by means of a conventional light microscope. The operator visually counts the leukocytes (white blood cells) present in a sample of cerebrospinal fluid (10 μl). It is a tedious job and the result is operator-dependent. Here in order to circumvent the limitations of manual counting, we approach the question of numeration of erythrocytes and leukocytes for the cytological diagnosis of meningitis by means of lens-free microscopy. In a first step, a prospective counts of leukocytes was performed by five different operators using conventional optical microscopy. The visual counting yielded an overall 16.7% misclassification of 72 cerebrospinal fluid specimens in meningitis/non-meningitis categories using a 10 leukocyte/μL cut-off. In a second step, the lens-free microscopy algorithm was adapted step-by-step for counting cerebrospinal fluid cells and discriminating leukocytes from erythrocytes. The optimization of the automatic lens-free counting was based on the prospective analysis of 215 cerebrospinal fluid specimens. The optimized algorithm yielded a 100% sensitivity and a 86% specificity compared to confirmed diagnostics. In a third step, a blind lens-free microscopic analysis of 116 cerebrospinal fluid specimens, including six cases of microbiology confirmed infectious meningitis, yielded a 100% sensitivity and a 79% specificity. Adapted lens-free microscopy is thus emerging as an operator-independent technique for the rapid numeration of leukocytes and erythrocytes in cerebrospinal fluid. In particular, this technique is well suited to the rapid diagnosis of meningitis at point-of-care laboratories.

Endocytic pathways involved in PLGA nanoparticle uptake by grapevine cells and role of cell wall and membrane in size selection.

PubMed

Palocci, Cleofe; Valletta, Alessio; Chronopoulou, Laura; Donati, Livia; Bramosanti, Marco; Brasili, Elisa; Baldan, Barbara; Pasqua, Gabriella

2017-12-01

PLGA NPs' cell uptake involves different endocytic pathways. Clathrin-independent endocytosis is the main internalization route. The cell wall plays a more prominent role than the plasma membrane in NPs' size selection. In the last years, many studies on absorption and cell uptake of nanoparticles by plants have been conducted, but the understanding of the internalization mechanisms is still largely unknown. In this study, polydispersed and monodispersed poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs) were synthesized, and a strategy combining the use of transmission electron microscopy (TEM), confocal analysis, fluorescently labeled PLGA NPs, a probe for endocytic vesicles (FM4-64), and endocytosis inhibitors (i.e., wortmannin, ikarugamycin, and salicylic acid) was employed to shed light on PLGA NP cell uptake in grapevine cultured cells and to assess the role of the cell wall and plasma membrane in size selection of PLGA NPs. The ability of PLGA NPs to cross the cell wall and membrane was confirmed by TEM and fluorescence microscopy. A strong adhesion of PLGA NPs to the outer side of the cell wall was observed, presumably due to electrostatic interactions. Confocal microscopy and treatment with endocytosis inhibitors suggested the involvement of both clathrin-dependent and clathrin-independent endocytosis in cell uptake of PLGA NPs and the latter appeared to be the main internalization pathway. Experiments on grapevine protoplasts revealed that the cell wall plays a more prominent role than the plasma membrane in size selection of PLGA NPs. While the cell wall prevents the uptake of PLGA NPs with diameters over 50 nm, the plasma membrane can be crossed by PLGA NPs with a diameter of 500-600 nm.

Serial block face scanning electron microscopy--the future of cell ultrastructure imaging.

PubMed

Hughes, Louise; Hawes, Chris; Monteith, Sandy; Vaughan, Sue

2014-03-01

One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells.

PubMed

Nadadhur, Aishwarya G; Emperador Melero, Javier; Meijer, Marieke; Schut, Desiree; Jacobs, Gerbren; Li, Ka Wan; Hjorth, J J Johannes; Meredith, Rhiannon M; Toonen, Ruud F; Van Kesteren, Ronald E; Smit, August B; Verhage, Matthijs; Heine, Vivi M

2017-01-01

Generation of neuronal cultures from induced pluripotent stem cells (hiPSCs) serve the studies of human brain disorders. However we lack neuronal networks with balanced excitatory-inhibitory activities, which are suitable for single cell analysis. We generated low-density networks of hPSC-derived GABAergic and glutamatergic cortical neurons. We used two different co-culture models with astrocytes. We show that these cultures have balanced excitatory-inhibitory synaptic identities using confocal microscopy, electrophysiological recordings, calcium imaging and mRNA analysis. These simple and robust protocols offer the opportunity for single-cell to multi-level analysis of patient hiPSC-derived cortical excitatory-inhibitory networks; thereby creating advanced tools to study disease mechanisms underlying neurodevelopmental disorders.

Bulk Heterojunction Solar Cell with Nitrogen-Doped Carbon Nanotubes in the Active Layer: Effect of Nanocomposite Synthesis Technique on Photovoltaic Properties

PubMed Central

Keru, Godfrey; Ndungu, Patrick G.; Mola, Genene T.; Nyamori, Vincent O.

2015-01-01

Nanocomposites of poly(3-hexylthiophene) (P3HT) and nitrogen-doped carbon nanotubes (N-CNTs) have been synthesized by two methods; specifically, direct solution mixing and in situ polymerization. The nanocomposites were characterized by means of transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray dispersive spectroscopy, UV-Vis spectrophotometry, photoluminescence spectrophotometry (PL), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, thermogravimetric analysis, and dispersive surface energy analysis. The nanocomposites were used in the active layer of a bulk heterojunction organic solar cell with the composition ITO/PEDOT:PSS/P3HT:N-CNTS:PCBM/LiF/Al. TEM and SEM analysis showed that the polymer successfully wrapped the N-CNTs. FTIR results indicated good π-π interaction within the nanocomposite synthesized by in situ polymerization as opposed to samples made by direct solution mixing. Dispersive surface energies of the N-CNTs and nanocomposites supported the fact that polymer covered the N-CNTs well. J-V analysis show that good devices were formed from the two nanocomposites, however, the in situ polymerization nanocomposite showed better photovoltaic characteristics.

Analysis of the 3D structure og agglutinated erythrocyte using CellScan and confocal microscopy: characterization by FLIM-FRET

NASA Astrophysics Data System (ADS)

Riquelme, Bibiana D.; Dumas, Dominique; Valverde de Rasia, Juana; Rasia, Rodolfo J.; Stoltz, Jean Francois

2003-10-01

We report the adhesion of human erythrocyte membranes mediated by monoclonal antibodies anti-glycophorin. The distribution of the linked antibodies on membrane was identified with selective fluorescence labels. To analyze the antibody distribution on interfacial region between two cells agglutinated and on its surface, three types of fluorescence marked strategy were evaluated. The 3D images were obtained in a CellScan and Confocal Laser Scanning Microscopy CLSM. We considered the FRET signal to characterize the agglutination of Red Blood Cells (RBC) by specific monoclonal antibodies (anti-glycophorin A or B). The fluorescence labeling demonstrated that distribution of antibody on erythrocyte membranes is not homogeneous. The fluorescence intensity on contact region in the agglutinated is bigger than the intensity on exterior surface. Tentatively, we interpreted these intensity differences in terms of the mobility of antibody linked to the glycocalix on cell surface. Such mobility has a large consequence in the morphology of cellular agglutinated.

New insights into FtsZ rearrangements during the cell division of Escherichia coli from single-molecule localization microscopy of fixed cells.

PubMed

Vedyaykin, Alexey D; Vishnyakov, Innokentii E; Polinovskaya, Vasilisa S; Khodorkovskii, Mikhail A; Sabantsev, Anton V

2016-06-01

FtsZ - a prokaryotic tubulin homolog - is one of the central components of bacterial division machinery. At the early stage of cytokinesis FtsZ forms the so-called Z-ring at mid-cell that guides septum formation. Many approaches were used to resolve the structure of the Z-ring, however, researchers are still far from consensus on this question. We utilized single-molecule localization microscopy (SMLM) in combination with immunofluorescence staining to visualize FtsZ in Esherichia coli fixed cells that were grown under slow and fast growth conditions. This approach allowed us to obtain images of FtsZ structures at different stages of cell division and accurately measure Z-ring dimensions. Analysis of these images demonstrated that Z-ring thickness increases during constriction, starting at about 70 nm at the beginning of division and increasing by approximately 25% half-way through constriction. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Exploring the Dynamics of Cell Processes through Simulations of Fluorescence Microscopy Experiments

PubMed Central

Angiolini, Juan; Plachta, Nicolas; Mocskos, Esteban; Levi, Valeria

2015-01-01

Fluorescence correlation spectroscopy (FCS) methods are powerful tools for unveiling the dynamical organization of cells. For simple cases, such as molecules passively moving in a homogeneous media, FCS analysis yields analytical functions that can be fitted to the experimental data to recover the phenomenological rate parameters. Unfortunately, many dynamical processes in cells do not follow these simple models, and in many instances it is not possible to obtain an analytical function through a theoretical analysis of a more complex model. In such cases, experimental analysis can be combined with Monte Carlo simulations to aid in interpretation of the data. In response to this need, we developed a method called FERNET (Fluorescence Emission Recipes and Numerical routines Toolkit) based on Monte Carlo simulations and the MCell-Blender platform, which was designed to treat the reaction-diffusion problem under realistic scenarios. This method enables us to set complex geometries of the simulation space, distribute molecules among different compartments, and define interspecies reactions with selected kinetic constants, diffusion coefficients, and species brightness. We apply this method to simulate single- and multiple-point FCS, photon-counting histogram analysis, raster image correlation spectroscopy, and two-color fluorescence cross-correlation spectroscopy. We believe that this new program could be very useful for predicting and understanding the output of fluorescence microscopy experiments. PMID:26039162

Biosorption and biotransformation of chromium by Serratia sp. isolated from tannery effluent.

PubMed

Srivastava, Shaili; Thakur, Indu Shekhar

2012-01-01

A bacterium isolated from soil and sediment ofa leather tanning mill's effluent was identified as Serratia sp. by the analysis of 16S rDNA. Scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDX) and transmission electron microscopy (TEM) were used to assess morphological changes and confirm chromium biosorption in Serratia sp. both in a shake-flask culture containing chromium and in a tannery wastewater. The SEMEDX and the elemental analysis of the chromate-containing samples confirmed the binding of chromium with the bacterial biomass. The TEM exhibited chromium accumulation throughout the bacterial cell, with some granular deposits in the cell periphery and in the cytoplasm. X-ray diffraction analysis (XRD) was used to quantify the chromium and to determine the chemical nature of the metal-microbe interaction. The XRD data showed the crystalline character of the precipitates, which consisted of mainly calcium chromium oxide, chromium fluoride phosphate and related organo-Cr(III) complex crystals. The XRD data also revealed a strong involvement of cellular carboxyl and phosphate groups in chromium binding by the bacterial biomass. The results of the study indicated that a combined mechanism of ion-exchange, complexation, croprecipitation and immobilization was involved in the biosorption of chromium by bacterial cells in contaminated environments.

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

Concepts in Light Microscopy of Viruses

PubMed Central

Witte, Robert; Georgi, Fanny

2018-01-01

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research. PMID:29670029

Concepts in Light Microscopy of Viruses.

PubMed

Witte, Robert; Andriasyan, Vardan; Georgi, Fanny; Yakimovich, Artur; Greber, Urs F

2018-04-18

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research.

Multispectral plasmon coupling microscopy and its application in bio-imaging

NASA Astrophysics Data System (ADS)

Wang, Hongyun

A broad range of cellular activities, including receptor mediated endocytosis, signaling and receptor clustering, involve multi-body interactions between different cellular functionalities. Many of these interactions are dynamic in nature, making optical tools the method of choice for their investigation. Conventional optical microscopy has a resolution about 300nm, limited by the diffraction of light, which is insufficient to explore processes that occur on nanometer or tens of nanometer length scales. The aim of this thesis is to develop and validate a plasmon coupling microscopy (PCM), which utilizes the distance dependent spectral properties of coupled noble metal nanoparticles (NPs) to resolve distance changes between NP labels on deeply sub-diffraction length scales. This colorimetric approach is augmented with a polarization sensitive analysis of the scattered light of individual dimers to monitor simultaneously distance and orientation changes. The distance dependent polarization anisotropy in discrete dimers is investigated experimentally and theoretically. The performed analysis reveals that the polarization anisotropy is robust even against relatively large refractive index changes. The polarization sensitive PCM is then applied to characterize the lateral spatial organization of mammalian plasma membranes by analyzing the translational and rotational motion as well as the extension of discrete NP dimers during their diffusion on lysed HeLa cell membranes. The membrane is found to be compartmentalized with typical domain sizes on the order of 70nm. The functionality of plasmon coupling based imaging method is expanded further by developing a multispectral imaging modality for a quantitative analysis of the plasmon coupling between many noble metal immunolabels in a large field of view simultaneously. This approach provides information about the spatial organization of the silver nanoparticle labels and thus of targeted EGF receptor densities on the surface of epidermoid carcinoma cells (A431). Finally, multispectral plasmon coupling microscopy is applied to investigate the uptake and subsequent intracellular spatial distribution of silver nanoparticles in murine macrophage cells (J774A.1). The studies reveal that NP uptake is mediated by scavenger receptors and that the intracellular NP association and distribution are heterogeneous among cells in a cellular ensemble. The heterogeneity is demonstrated to be correlated with the maturation status of the macrophages.

Characterization of malaria infected blood cells by scanning confocal laser and acoustic vector contrast microscopy

NASA Astrophysics Data System (ADS)

Ahmed Mohamed, E. T.; Schubert, S.; Gilberger, T. W.; Kamanyi, A., Jr.; Wannemacher, R.; Grill, W.

2006-03-01

Acoustic and optical multiple contrast microscopy has been employed in order to explore characterizable parameters of red blood cells, including cells infected by the parasite Plasmodium falciparum, in order to investigate cellular modifications caused by the infection and to identify possible detection schemes for disease monitoring. Imaging schemes were based on fluorescence, optical transmission, optical reflection, and amplitude and phase of ultrasound reflected from the cells. Contrast variations observed in acoustic microscopy, but not in optical microscopy, were tentatively ascribed to changes caused by the infection.

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

NASA Astrophysics Data System (ADS)

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

2009-09-01

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

Rapid fibroblast activation in mammalian cells induced by silicon nanowire arrays.

PubMed

Ha, Qing; Yang, Gao; Ao, Zhuo; Han, Dong; Niu, Fenglan; Wang, Shutao

2014-07-21

Activated tumor-associated fibroblasts (TAFs) with abundant fibroblast activation protein (FAP) expression attract tremendous attention in tumor progression studies. In this work, we report a rapid 24 h FAP activation method for fibroblasts using silicon nanowires (SiNWs) as culture substrates instead of growth factors or chemokines. In contrast with cells cultured on flat silicon which rarely express FAP, SiNW cultivated cells exhibit FAP levels similar to those found in cancerous tissue. We demonstrated that activated cells grown on SiNWs maintain their viability and proliferation in a time-dependent manner. Moreover, environmental scanning electron microscopy (ESEM) and focused ion beam and scanning electron microscopy (FIB-SEM) analysis clearly revealed that activated cells on SiNWs adapt to the structure of their substrates by filling inter-wire cavities via filopodia in contrast to cells cultured on flat silicon which spread freely. We further illustrated that the expression of FAP was rarely detected in activated cells after being re-cultured in Petri dishes, suggesting that the unique structure of SiNWs may have a certain influence on FAP activation.

STED microscopy: A simplified method for liver sinusoidal endothelial fenestrae analysis.

PubMed

Martino, Julie Di; Mascalchi, Patrice; Legros, Philippe; Lacomme, Sabrina; Gontier, Etienne; Bioulac-Sage, Paulette; Balabaud, Charles; Moreau, Violaine; Saltel, Frédéric

2018-05-29

Liver sinusoidal endothelial cells (LSECs) possess fenestrae, open transcellular pores with an average diameter of 100nm. These fenestrae allow for the exchange between blood and hepatocytes. Alterations in their number or diameter in liver diseases have important implications for hepatic microcirculation and function. Although decades of studies, fenestrae are still observed into fixed cells and we have poor knowledge of their dynamics. Using stimulated emission depletion (STED) super-resolution microscopy, we have established a faster and simplest method to observe and quantify fenestrae. Indeed, using cytochalasin D, an actin depolymerizing agent known to promote fenestrae formation we measure the increase of fenestrae number. We adapted this methodology to develop an automated method to study fenestrae dynamics. Moreover, with two colors STED analysis we shown that this approach could be useful to study LSECs fenestrae molecular composition. Our approach demonstrate that STED microscopy is suitable for LSEC fenestrae study. This new way of analyzing LSEC fenestrae will allow for expedited investigation of their dynamics, molecular composition and functions to better understand their function in liver pathophysiology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Nonlinear optical microscopy: use of second harmonic generation and two-photon microscopy for automated quantitative liver fibrosis studies.

PubMed

Sun, Wanxin; Chang, Shi; Tai, Dean C S; Tan, Nancy; Xiao, Guangfa; Tang, Huihuan; Yu, Hanry

2008-01-01

Liver fibrosis is associated with an abnormal increase in an extracellular matrix in chronic liver diseases. Quantitative characterization of fibrillar collagen in intact tissue is essential for both fibrosis studies and clinical applications. Commonly used methods, histological staining followed by either semiquantitative or computerized image analysis, have limited sensitivity, accuracy, and operator-dependent variations. The fibrillar collagen in sinusoids of normal livers could be observed through second-harmonic generation (SHG) microscopy. The two-photon excited fluorescence (TPEF) images, recorded simultaneously with SHG, clearly revealed the hepatocyte morphology. We have systematically optimized the parameters for the quantitative SHG/TPEF imaging of liver tissue and developed fully automated image analysis algorithms to extract the information of collagen changes and cell necrosis. Subtle changes in the distribution and amount of collagen and cell morphology are quantitatively characterized in SHG/TPEF images. By comparing to traditional staining, such as Masson's trichrome and Sirius red, SHG/TPEF is a sensitive quantitative tool for automated collagen characterization in liver tissue. Our system allows for enhanced detection and quantification of sinusoidal collagen fibers in fibrosis research and clinical diagnostics.

Ultrastructural and mechanical changes in tubular epithelial cells by angiotensin II and aldosterone as observed with atomic force microscopy.

PubMed

Quan, Fu-Shi; Jeong, Kyung Hwan; Lee, Gi-Ja

2018-07-01

Tubular epithelial cells (TECs) play an important pathophysiological role in the promotion of renal fibrosis. Quantitative analysis of the mechanical changes in TECs may be helpful in evaluating novel pharmacological strategies. Atomic force microscopy (AFM) is a common nanotechnology tool used for imaging and measuring interaction forces in biological systems. In this study, we used AFM to study ultrastructural and mechanical changes in TECs mediated by the renin-angiotensin-aldosterone system. We quantitatively analyzed changes in the mechanical properties of TECs using three extrinsic factors, namely, chemical fixation, angiotensin II (AT II), and aldosterone (AD). Fixed TECs were 11 times stiffer at the cell body and 3 times stiffer at the cell-cell junction compared to live TECs. After stimulation with AT II, live TECs were four times stiffer at the junctional area than at the cell body, while fixed TECs after AT II stimulation were approximately two times stiffer at the both cell body and cell-cell junction compared to fixed unstimulated TECs. Fixed TECs also reflected changes in the mechanical properties of TECs at the cell body region after AD stimulation. Together, our results suggest that cell stiffness at the cell body region may serve as an effective index for evaluating drugs and stimulation, regardless of whether the cells are live or fixed at the time of analysis. In addition, studying the changes to the intrinsic mechanical property of TECs after application of external stimuli may be useful for investigating pathophysiologic mechanisms and effective therapeutic strategies for renal injury. Copyright © 2018 Elsevier Ltd. All rights reserved.

Analysis of defect structure in silicon. Characterization of samples from UCP ingot 5848-13C

NASA Technical Reports Server (NTRS)

Natesh, R.; Guyer, T.; Stringfellow, G. B.

1982-01-01

Statistically significant quantitative structural imperfection measurements were made on samples from ubiquitous crystalline process (UCP) Ingot 5848 - 13 C. Important trends were noticed between the measured data, cell efficiency, and diffusion length. Grain boundary substructure appears to have an important effect on the conversion efficiency of solar cells from Semix material. Quantitative microscopy measurements give statistically significant information compared to other microanalytical techniques. A surface preparation technique to obtain proper contrast of structural defects suitable for QTM analysis was perfected.

A novel tracing method for the segmentation of cell wall networks.

PubMed

De Vylder, Jonas; Rooms, Filip; Dhondt, Stijn; Inze, Dirk; Philips, Wilfried

2013-01-01

Cell wall networks are a common subject of research in biology, which are important for plant growth analysis, organ studies, etc. In order to automate the detection of individual cells in such cell wall networks, we propose a new segmentation algorithm. The proposed method is a network tracing algorithm, exploiting the prior knowledge of the network structure. The method is applicable on multiple microscopy modalities such as fluorescence, but also for images captured using non invasive microscopes such as differential interference contrast (DIC) microscopes.

Hybrid microscopy of human carotid atheroma by means of optical-resolution optoacoustic and non-linear optical microscopy

NASA Astrophysics Data System (ADS)

Seeger, Markus; Karlas, Angelos; Soliman, Dominik; Pelisek, Jaroslav; Ntziachristos, Vasilis

2017-03-01

Carotid atheromatosis is causally related to stroke, a leading cause of disability and death. We present the analysis of a human carotid atheroma using a novel hybrid microscopy system that combines optical-resolution optoacoustic (photoacoustic) microscopy and several non-linear optical microscopy modalities (second and third harmonic generation, as well as, two-photon excitation fluorescence) to achieve a multimodal examination of the extracted tissue within the same imaging framework. Our system enables the label-free investigation of atheromatous human carotid tissue with a resolution of about 1 μm and allows for the congruent interrogation of plaque morphology and clinically relevant constituents such as red blood cells, collagen, and elastin. Our data reveal mutual interactions between blood embeddings and connective tissue within the atheroma, offering comprehensive insights into its stage of evolution and severity, and potentially facilitating the further development of diagnostic tools, as well as treatment strategies.

JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells.

PubMed

McMurtry, Vanity; Saavedra, Joseph E; Nieves-Alicea, René; Simeone, Ann-Marie; Keefer, Larry K; Tari, Ana M

2011-04-01

Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

Implementation of stimulated Raman scattering microscopy for single cell analysis

NASA Astrophysics Data System (ADS)

D'Arco, Annalisa; Ferrara, Maria Antonietta; Indolfi, Maurizio; Tufano, Vitaliano; Sirleto, Luigi

2017-05-01

In this work, we present successfully realization of a nonlinear microscope, not purchasable in commerce, based on stimulated Raman scattering. It is obtained by the integration of a femtosecond SRS spectroscopic setup with an inverted research microscope equipped with a scanning unit. Taking account of strength of vibrational contrast of SRS, it provides label-free imaging of single cell analysis. Validation tests on images of polystyrene beads are reported to demonstrate the feasibility of the approach. In order to test the microscope on biological structures, we report and discuss the label-free images of lipid droplets inside fixed adipocyte cells.

Analysis of plastid number, size, and distribution in Arabidopsis plants by light and fluorescence microscopy.

PubMed

Pyke, Kevin

2011-01-01

Methods are described which allow one to observe chloroplasts in mesophyll cells from leaves of Arabidopsis, determine their number per cell, measure their area, and determine a value for chloroplast coverage inside mesophyll cells. Non-green plastids can also be imaged either by using staining, or by exploiting fluorescent proteins targeted to the plastid in non-green parts of the plant, such as the roots, in transgenic Arabidopsis.

Fluorescence Live Cell Imaging

PubMed Central

Ettinger, Andreas

2014-01-01

Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio, and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein constructs by spinning disk confocal microscopy. PMID:24974023

On measuring cell confluence in phase contrast microscopy

NASA Astrophysics Data System (ADS)

Dempsey, K. P.; Richardson, J. B.; Lam, K. P.

2014-03-01

A principal focus highlighting recent advances in cell based therapies concerns the development of effective treatments for osteoarthritis. Earlier clinicaltrials have shown that 80% of patients receiving mesenchymal stem cell(MSC) based treatment have improved their quality of life by alleviating pain whilst extending the life of their natural joints. The current challenge facing researchers is to identify the biological differences between the treatments that have worked and those which have shown little improvement. One possible candidate for the difference in treatment prognosis is an examination of the proliferation of the ( type) cells as they grow. To further understanding of the proliferation and differentiation of MSC, non-invasive live cell imaging techniques have been developed which capture important cell events and dynamics in cell divisions over an extended period of time. An automated image analysis procedure capable of tracking cell confluence over time has also been implemented, providing an objective and realistic estimation of cell growth within continuous live cell cultures. The proposed algorithm accounts for the halo artefacts that occur in phase microscopy. In addition to a favourable run-time performance, the method was also validated using continuous live MSC cultures, with consistent and meaningful results.

Graph Theory-Based Analysis of the Lymph Node Fibroblastic Reticular Cell Network.

PubMed

Novkovic, Mario; Onder, Lucas; Bocharov, Gennady; Ludewig, Burkhard

2017-01-01

Secondary lymphoid organs have developed segregated niches that are able to initiate and maintain effective immune responses. Such global organization requires tight control of diverse cellular components, specifically those that regulate lymphocyte trafficking. Fibroblastic reticular cells (FRCs) form a densely interconnected network in lymph nodes and provide key factors necessary for T cell migration and retention, and foster subsequent interactions between T cells and dendritic cells. Development of integrative systems biology approaches has made it possible to elucidate this multilevel complexity of the immune system. Here, we present a graph theory-based analysis of the FRC network in murine lymph nodes, where generation of the network topology is performed using high-resolution confocal microscopy and 3D reconstruction. This approach facilitates the analysis of physical cell-to-cell connectivity, and estimation of topological robustness and global behavior of the network when it is subjected to perturbation in silico.

A multi-phenotypic imaging screen to identify bacterial effectors by exogenous expression in a HeLa cell line.

PubMed

Collins, Adam; Huett, Alan

2018-05-15

We present a high-content screen (HCS) for the simultaneous analysis of multiple phenotypes in HeLa cells expressing an autophagy reporter (mcherry-LC3) and one of 224 GFP-fused proteins from the Crohn's Disease (CD)-associated bacterium, Adherent Invasive E. coli (AIEC) strain LF82. Using automated confocal microscopy and image analysis (CellProfiler), we localised GFP fusions within cells, and monitored their effects upon autophagy (an important innate cellular defence mechanism), cellular and nuclear morphology, and the actin cytoskeleton. This data will provide an atlas for the localisation of 224 AIEC proteins within human cells, as well as a dataset to analyse their effects upon many aspects of host cell morphology. We also describe an open-source, automated, image-analysis workflow to identify bacterial effectors and their roles via the perturbations induced in reporter cell lines when candidate effectors are exogenously expressed.

Methods for the Detection of Autophagy in Mammalian Cells

PubMed Central

Zhang, Ziyan; Singh, Rajat; Aschner, Michael

2016-01-01

Macroautophagy (hereafter referred to as autophagy) is a degradation pathway that delivers cytoplasmic materials to lysosomes via double-membraned vesicles designated autophagosomes. Cytoplasmic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes, where the cargo is degraded. Autophagy is a crucial mechanism involved in many aspects of cell function, including cellular metabolism and energy balance; and alterations in autophagy have been linked to various human pathological processes. Thus, methods that accurately measure autophagic activity are necessary. In this unit, we introduce several approaches to analyze autophagy in mammalian cells, including immunoblotting analysis of LC3 and p62, detection of autophagosome formation by fluorescence microscopy, and monitoring autophagosome maturation by tandem mRFP-GFP fluorescence microscopy. Overall, we recommend a combined use of multiple methods to accurately assess the autophagic activity in any given biological setting. PMID:27479363

Quantitative investigation of red blood cell three-dimensional geometric and chemical changes in the storage lesion using digital holographic microscopy.

PubMed

Jaferzadeh, Keyvan; Moon, Inkyu

2015-11-01

Quantitative phase information obtained by digital holographic microscopy (DHM) can provide new insight into the functions and morphology of single red blood cells (RBCs). Since the functionality of a RBC is related to its three-dimensional (3-D) shape, quantitative 3-D geometric changes induced by storage time can help hematologists realize its optimal functionality period. We quantitatively investigate RBC 3-D geometric changes in the storage lesion using DHM. Our experimental results show that the substantial geometric transformation of the biconcave-shaped RBCs to the spherocyte occurs due to RBC storage lesion. This transformation leads to progressive loss of cell surface area, surface-to-volume ratio, and functionality of RBCs. Furthermore, our quantitative analysis shows that there are significant correlations between chemical and morphological properties of RBCs.

Study of electromechanical and mechanical properties of bacteria using force microscopy

NASA Astrophysics Data System (ADS)

Reukov, Vladimir; Thompson, Gary; Nikiforov, Maxim; Guo, Senli; Ovchinnikov, Oleg; Jesse, Stephen; Kalinin, Sergei; Vertegel, Alexey

2010-03-01

The application of scanning probe microscopy (SPM) to biological systems has evolved over the past decade into a multimodal and spectroscopic instrument that provides multiple information channels at each spatial pixel acquired. Recently, functional recognition imaging based on differing electromechanical properties between Gram negative and Gram positive bacteria was achieved using artificial neural network analysis of band excitation piezoresponse force microscopy (BEPFM) data. The immediate goal of this project was to study mechanical and electromechanical properties of bacterial systems physiologically-relevant solutions using Band-width Excitation Piezoresponce Force Microscopy (BE PFM) in combination with Force Mapping. Electromechanical imaging in physiological environments will improve the versatility of functional recognition imaging and open the way for application of the rapid BEPFM line mode method to other living cell systems.

Widely Accepted Modern Views of Cell Structure are Fundamentally Correct: A Reply to Hillman and Sartory.

ERIC Educational Resources Information Center

Michell, R. H.; And Others

1982-01-01

Argues against Hillman and Sartory's views of cell structure (EJ242880), pointing out that they are erroneous in their analysis of the motives and working methods of the scientific community, in their total rejection of information coming from electron microscopy, and in their claim that lysosomes and endoplasmic reticulum are imaginary. (DC)

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.

Rapid, portable and cost-effective yeast cell viability and concentration analysis using lensfree on-chip microscopy and machine learning.

PubMed

Feizi, Alborz; Zhang, Yibo; Greenbaum, Alon; Guziak, Alex; Luong, Michelle; Chan, Raymond Yan Lok; Berg, Brandon; Ozkan, Haydar; Luo, Wei; Wu, Michael; Wu, Yichen; Ozcan, Aydogan

2016-11-01

Monitoring yeast cell viability and concentration is important in brewing, baking and biofuel production. However, existing methods of measuring viability and concentration are relatively bulky, tedious and expensive. Here we demonstrate a compact and cost-effective automatic yeast analysis platform (AYAP), which can rapidly measure cell concentration and viability. AYAP is based on digital in-line holography and on-chip microscopy and rapidly images a large field-of-view of 22.5 mm 2 . This lens-free microscope weighs 70 g and utilizes a partially-coherent illumination source and an opto-electronic image sensor chip. A touch-screen user interface based on a tablet-PC is developed to reconstruct the holographic shadows captured by the image sensor chip and use a support vector machine (SVM) model to automatically classify live and dead cells in a yeast sample stained with methylene blue. In order to quantify its accuracy, we varied the viability and concentration of the cells and compared AYAP's performance with a fluorescence exclusion staining based gold-standard using regression analysis. The results agree very well with this gold-standard method and no significant difference was observed between the two methods within a concentration range of 1.4 × 10 5 to 1.4 × 10 6 cells per mL, providing a dynamic range suitable for various applications. This lensfree computational imaging technology that is coupled with machine learning algorithms would be useful for cost-effective and rapid quantification of cell viability and density even in field and resource-poor settings.

Structural and biological evaluation of a multifunctional SWCNT-AgNPs-DNA/PVA bio-nanofilm.

PubMed

Subbiah, Ramesh P; Lee, Haisung; Veerapandian, Murugan; Sadhasivam, Sathya; Seo, Soo-Won; Yun, Kyusik

2011-04-01

A bio-nanofilm consisting of a tetrad nanomaterial (nanotubes, nanoparticles, DNA, polymer) was fabricated utilizing in situ reduction and noncovalent interactions and it displayed effective antibacterial activity and biocompatibility. This bio-nanofilm was composed of homogenous silver nanoparticles (AgNPs) coated on single-walled carbon nanotubes (SWCNTs), which were later hybridized with DNA and stabilized in poly(vinyl alcohol) (PVA) in the presence of a surfactant with the aid of ultrasonication. Electron microscopy and bio-AFM (atomic force microscopy) images were used to assess the morphology of the nanocomposite (NC) structure. Functionalization and fabrication were examined using FT-Raman spectroscopy by analyzing the functional changes in the bio-nanofilm before and after fabrication. UV-visible spectroscopy and X-ray powder diffraction (XRD) confirmed that AgNPs were present in the final NC on the basis of its surface plasmon resonance (370 nm) and crystal planes. Thermal gravimetric analysis was used to measure the percentage weight loss of SWCNT (17.5%) and final SWCNT-AgNPs-DNA/PVA (47.7%). The antimicrobial efficiency of the bio-nanofilm was evaluated against major pathogenic organisms. Bactericidal ratios, zone of inhibition, and minimum inhibitory concentration were examined against gram positive and gram negative bacteria. A preliminary cytotoxicity analysis was conducted using A549 lung cancer cells and IMR-90 fibroblast cells. Confocal laser microscopy, bio-AFM, and field emission scanning electron microscopy (FE-SEM) images demonstrated that the NCs were successfully taken up by the cells. These combined results indicate that this bio-nanofilm was biocompatible and displayed antimicrobial activity. Thus, this novel bio-nanofilm holds great promise for use as a multifunctional tool in burn therapy, tissue engineering, and other biomedical applications.

Normalized Polarization Ratios for the Analysis of Cell Polarity

PubMed Central

Shimoni, Raz; Pham, Kim; Yassin, Mohammed; Ludford-Menting, Mandy J.; Gu, Min; Russell, Sarah M.

2014-01-01

The quantification and analysis of molecular localization in living cells is increasingly important for elucidating biological pathways, and new methods are rapidly emerging. The quantification of cell polarity has generated much interest recently, and ratiometric analysis of fluorescence microscopy images provides one means to quantify cell polarity. However, detection of fluorescence, and the ratiometric measurement, is likely to be sensitive to acquisition settings and image processing parameters. Using imaging of EGFP-expressing cells and computer simulations of variations in fluorescence ratios, we characterized the dependence of ratiometric measurements on processing parameters. This analysis showed that image settings alter polarization measurements; and that clustered localization is more susceptible to artifacts than homogeneous localization. To correct for such inconsistencies, we developed and validated a method for choosing the most appropriate analysis settings, and for incorporating internal controls to ensure fidelity of polarity measurements. This approach is applicable to testing polarity in all cells where the axis of polarity is known. PMID:24963926

Boundary segmentation for fluorescence microscopy using steerable filters

NASA Astrophysics Data System (ADS)

Ho, David Joon; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

2017-02-01

Fluorescence microscopy is used to image multiple subcellular structures in living cells which are not readily observed using conventional optical microscopy. Moreover, two-photon microscopy is widely used to image structures deeper in tissue. Recent advancement in fluorescence microscopy has enabled the generation of large data sets of images at different depths, times, and spectral channels. Thus, automatic object segmentation is necessary since manual segmentation would be inefficient and biased. However, automatic segmentation is still a challenging problem as regions of interest may not have well defined boundaries as well as non-uniform pixel intensities. This paper describes a method for segmenting tubular structures in fluorescence microscopy images of rat kidney and liver samples using adaptive histogram equalization, foreground/background segmentation, steerable filters to capture directional tendencies, and connected-component analysis. The results from several data sets demonstrate that our method can segment tubular boundaries successfully. Moreover, our method has better performance when compared to other popular image segmentation methods when using ground truth data obtained via manual segmentation.

4Pi-confocal microscopy of live cells

NASA Astrophysics Data System (ADS)

Bahlmann, Karsten; Jakobs, Stefan; Hell, Stefan W.

2002-06-01

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

Superresolution imaging of Drosophila tissues using expansion microscopy.

PubMed

Jiang, Nan; Kim, Hyeon-Jin; Chozinski, Tyler J; Azpurua, Jorge E; Eaton, Benjamin A; Vaughan, Joshua C; Parrish, Jay Z

2018-06-15

The limited resolving power of conventional diffraction-limited microscopy hinders analysis of small, densely packed structural elements in cells. Expansion microscopy (ExM) provides an elegant solution to this problem, allowing for increased resolution with standard microscopes via physical expansion of the specimen in a swellable polymer hydrogel. Here, we apply, validate, and optimize ExM protocols that enable the study of Drosophila embryos, larval brains, and larval and adult body walls. We achieve a lateral resolution of ∼70 nm in Drosophila tissues using a standard confocal microscope, and we use ExM to analyze fine intracellular structures and intercellular interactions. First, we find that ExM reveals features of presynaptic active zone (AZ) structure that are observable with other superresolution imaging techniques but not with standard confocal microscopy. We further show that synapses known to exhibit age-dependent changes in activity also exhibit age-dependent changes in AZ structure. Finally, we use the significantly improved axial resolution of ExM to show that dendrites of somatosensory neurons are inserted into epithelial cells at a higher frequency than previously reported in confocal microscopy studies. Altogether, our study provides a foundation for the application of ExM to Drosophila tissues and underscores the importance of tissue-specific optimization of ExM procedures.

Photoacoustic microscopy of single cells employing an intensity-modulated diode laser

NASA Astrophysics Data System (ADS)

Langer, Gregor; Buchegger, Bianca; Jacak, Jaroslaw; Dasa, Manoj Kumar; Klar, Thomas A.; Berer, Thomas

2018-02-01

In this work, we employ frequency-domain photoacoustic microscopy to obtain photoacoustic images of labeled and unlabeled cells. The photoacoustic microscope is based on an intensity-modulated diode laser in combination with a focused piezo-composite transducer and allows imaging of labeled cells without severe photo-bleaching. We demonstrate that frequency-domain photoacoustic microscopy realized with a diode laser is capable of recording photoacoustic images of single cells with sub-µm resolution. As examples, we present images of undyed human red blood cells, stained human epithelial cells, and stained yeast cells.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy.

PubMed

Fuchs, Felix M; Raguse, Marina; Fiebrandt, Marcel; Madela, Kazimierz; Awakowicz, Peter; Laue, Michael; Stapelmann, Katharina; Moeller, Ralf

2017-11-30

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Single-molecule fluorescence microscopy review: shedding new light on old problems

PubMed Central

Shashkova, Sviatlana

2017-01-01

Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called ‘green revolution’, has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called ‘super-resolution’ fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques. PMID:28694303

Lab-on-a-chip technologies for proteomic analysis from isolated cells.

PubMed

Sedgwick, H; Caron, F; Monaghan, P B; Kolch, W; Cooper, J M

2008-10-06

Lab-on-a-chip systems offer a versatile environment in which low numbers of cells and molecules can be manipulated, captured, detected and analysed. We describe here a microfluidic device that allows the isolation, electroporation and lysis of single cells. A431 human epithelial carcinoma cells, expressing a green fluorescent protein-labelled actin, were trapped by dielectrophoresis within an integrated lab-on-a-chip device containing saw-tooth microelectrodes. Using these same trapping electrodes, on-chip electroporation was performed, resulting in cell lysis. Protein release was monitored by confocal fluorescence microscopy.

Robust analysis method for acoustic properties of biological specimens measured by acoustic microscopy

NASA Astrophysics Data System (ADS)

Arakawa, Mototaka; Mori, Shohei; Kanai, Hiroshi; Nagaoka, Ryo; Horie, Miki; Kobayashi, Kazuto; Saijo, Yoshifumi

2018-07-01

We proposed a robust analysis method for the acoustic properties of biological specimens measured by acoustic microscopy. Reflected pulse signals from the substrate and specimen were converted into frequency domains to obtain sound speed and thickness. To obtain the average acoustic properties of the specimen, parabolic approximation was performed to determine the frequency at which the amplitude of the normalized spectrum became maximum or minimum, considering the sound speed and thickness of the specimens and the operating frequency of the ultrasonic device used. The proposed method was demonstrated for a specimen of malignant melanoma of the skin by using acoustic microscopy attaching a concave transducer with a center frequency of 80 MHz. The variations in sound speed and thickness analyzed by the proposed method were markedly smaller than those analyzed by the method based on an autoregressive model. The proposed method is useful for the analysis of the acoustic properties of bilogical tissues or cells.

SEM Analysis of Surface Impact on Biofilm Antibiotic Treatment.

PubMed

Gomes, Luciana Calheiros; Mergulhão, Filipe José

2017-01-01

The aim of this work was to use scanning electron microscopy (SEM) to investigate the effect of ampicillin treatment on Escherichia coli biofilms formed on two surface materials with different properties, silicone (SIL) and glass (GLA). Epifluorescence microscopy (EM) was initially used to assess biofilm formation and killing efficiency on both surfaces. This technique showed that higher bacterial colonization was obtained in the hydrophobic SIL than in the hydrophilic GLA. It has also shown that higher biofilm inactivation was attained for GLA after the antibiotic treatment (7-log reduction versus 1-log reduction for SIL). Due to its high resolution and magnification, SEM enabled a more detailed analysis of the antibiotic effect on biofilm cells, complementing the killing efficiency information obtained by EM. SEM micrographs revealed that ampicillin-treated cells have an elongated form when compared to untreated cells. Additionally, it has shown that different materials induced different levels of elongation on cells exposed to antibiotic. Biofilms formed on GLA showed a 37% higher elongation than those formed on SIL. Importantly, cell elongation was related to viability since ampicillin had a higher bactericidal effect on GLA-formed biofilms. These findings raise the possibility of using SEM for understanding the efficacy of antimicrobial treatments by observation of biofilm morphology.

3D membrane segmentation and quantification of intact thick cells using cryo soft X-ray transmission microscopy: A pilot study

PubMed Central

Klementieva, Oxana; Werner, Stephan; Guttmann, Peter; Pratsch, Christoph; Cladera, Josep

2017-01-01

Structural analysis of biological membranes is important for understanding cell and sub-cellular organelle function as well as their interaction with the surrounding environment. Imaging of whole cells in three dimension at high spatial resolution remains a significant challenge, particularly for thick cells. Cryo-transmission soft X-ray microscopy (cryo-TXM) has recently gained popularity to image, in 3D, intact thick cells (∼10μm) with details of sub-cellular architecture and organization in near-native state. This paper reports a new tool to segment and quantify structural changes of biological membranes in 3D from cryo-TXM images by tracking an initial 2D contour along the third axis of the microscope, through a multi-scale ridge detection followed by an active contours-based model, with a subsequent refinement along the other two axes. A quantitative metric that assesses the grayscale profiles perpendicular to the membrane surfaces is introduced and shown to be linearly related to the membrane thickness. Our methodology has been validated on synthetic phantoms using realistic microscope properties and structure dimensions, as well as on real cryo-TXM data. Results demonstrate the validity of our algorithms for cryo-TXM data analysis. PMID:28376110

The use of fluorescence microscopy and image analysis for rapid detection of non-producing revertant cells of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002.

PubMed

Schulze, Katja; Lang, Imke; Enke, Heike; Grohme, Diana; Frohme, Marcus

2015-04-17

Ethanol production via genetically engineered cyanobacteria is a promising solution for the production of biofuels. Through the introduction of a pyruvate decarboxylase and alcohol dehydrogenase direct ethanol production becomes possible within the cells. However, during cultivation genetic instability can lead to mutations and thus loss of ethanol production. Cells then revert back to the wild type phenotype. A method for a rapid and simple detection of these non-producing revertant cells in an ethanol producing cell population is an important quality control measure in order to predict genetic stability and the longevity of a producing culture. Several comparable cultivation experiments revealed a difference in the pigmentation for non-producing and producing cells: the accessory pigment phycocyanin (PC) is reduced in case of the ethanol producer, resulting in a yellowish appearance of the culture. Microarray and western blot studies of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002 confirmed this PC reduction on the level of RNA and protein. Based on these findings we developed a method for fluorescence microscopy in order to distinguish producing and non-producing cells with respect to their pigmentation phenotype. By applying a specific filter set the emitted fluorescence of a producer cell with a reduced PC content appeared orange. The emitted fluorescence of a non-producing cell with a wt pigmentation phenotype was detected in red, and dead cells in green. In an automated process multiple images of each sample were taken and analyzed with a plugin for the image analysis software ImageJ to identify dead (green), non-producing (red) and producing (orange) cells. The results of the presented validation experiments revealed a good identification with 98 % red cells in the wt sample and 90 % orange cells in the producer sample. The detected wt pigmentation phenotype (red cells) in the producer sample were either not fully induced yet (in 48 h induced cultures) or already reverted to a non-producing cells (in long-term photobioreactor cultivations), emphasizing the sensitivity and resolution of the method. The fluorescence microscopy method displays a useful technique for a rapid detection of non-producing single cells in an ethanol producing cell population.

(Machine-)Learning to analyze in vivo microscopy: Support vector machines.

PubMed

Wang, Michael F Z; Fernandez-Gonzalez, Rodrigo

2017-11-01

The development of new microscopy techniques for super-resolved, long-term monitoring of cellular and subcellular dynamics in living organisms is revealing new fundamental aspects of tissue development and repair. However, new microscopy approaches present several challenges. In addition to unprecedented requirements for data storage, the analysis of high resolution, time-lapse images is too complex to be done manually. Machine learning techniques are ideally suited for the (semi-)automated analysis of multidimensional image data. In particular, support vector machines (SVMs), have emerged as an efficient method to analyze microscopy images obtained from animals. Here, we discuss the use of SVMs to analyze in vivo microscopy data. We introduce the mathematical framework behind SVMs, and we describe the metrics used by SVMs and other machine learning approaches to classify image data. We discuss the influence of different SVM parameters in the context of an algorithm for cell segmentation and tracking. Finally, we describe how the application of SVMs has been critical to study protein localization in yeast screens, for lineage tracing in C. elegans, or to determine the developmental stage of Drosophila embryos to investigate gene expression dynamics. We propose that SVMs will become central tools in the analysis of the complex image data that novel microscopy modalities have made possible. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid fibroblast activation in mammalian cells induced by silicon nanowire arrays

NASA Astrophysics Data System (ADS)

Ha, Qing; Yang, Gao; Ao, Zhuo; Han, Dong; Niu, Fenglan; Wang, Shutao

2014-06-01

Activated tumor-associated fibroblasts (TAFs) with abundant fibroblast activation protein (FAP) expression attract tremendous attention in tumor progression studies. In this work, we report a rapid 24 h FAP activation method for fibroblasts using silicon nanowires (SiNWs) as culture substrates instead of growth factors or chemokines. In contrast with cells cultured on flat silicon which rarely express FAP, SiNW cultivated cells exhibit FAP levels similar to those found in cancerous tissue. We demonstrated that activated cells grown on SiNWs maintain their viability and proliferation in a time-dependent manner. Moreover, environmental scanning electron microscopy (ESEM) and focused ion beam and scanning electron microscopy (FIB-SEM) analysis clearly revealed that activated cells on SiNWs adapt to the structure of their substrates by filling inter-wire cavities via filopodia in contrast to cells cultured on flat silicon which spread freely. We further illustrated that the expression of FAP was rarely detected in activated cells after being re-cultured in Petri dishes, suggesting that the unique structure of SiNWs may have a certain influence on FAP activation.Activated tumor-associated fibroblasts (TAFs) with abundant fibroblast activation protein (FAP) expression attract tremendous attention in tumor progression studies. In this work, we report a rapid 24 h FAP activation method for fibroblasts using silicon nanowires (SiNWs) as culture substrates instead of growth factors or chemokines. In contrast with cells cultured on flat silicon which rarely express FAP, SiNW cultivated cells exhibit FAP levels similar to those found in cancerous tissue. We demonstrated that activated cells grown on SiNWs maintain their viability and proliferation in a time-dependent manner. Moreover, environmental scanning electron microscopy (ESEM) and focused ion beam and scanning electron microscopy (FIB-SEM) analysis clearly revealed that activated cells on SiNWs adapt to the structure of their substrates by filling inter-wire cavities via filopodia in contrast to cells cultured on flat silicon which spread freely. We further illustrated that the expression of FAP was rarely detected in activated cells after being re-cultured in Petri dishes, suggesting that the unique structure of SiNWs may have a certain influence on FAP activation. Electronic supplementary information (ESI) available: (1) ESEM cross-sectional view images of the flat silicon and SiNW substrates. (2) Bright field morphology images of fibroblasts cultured in Petri dishes. (3) FIB/SEM 52° tilt images of fibroblasts cultured on SiNW 2 and SiNW 3. (4) Immunofluorescence images of FAP expression in fibroblasts re-cultured in Petri dishes after detachment from flat silicon and a series of SiNW substrates. (5) ESEM images of cells re-cultured in Petri dishes after detachment from each group. See DOI: 10.1039/c4nr01415d

Thermal and pH responsive polymer-tethered multifunctional magnetic nanoparticles for targeted delivery of anticancer drug.

PubMed

Sahoo, Banalata; Devi, K Sanjana P; Banerjee, Rakesh; Maiti, Tapas K; Pramanik, Panchanan; Dhara, Dibakar

2013-05-01

Targeted and efficient delivery of therapeutics to tumor cells is one of the key issues in cancer therapy. In the present work, we report a temperature and pH dual responsive core-shell nanoparticles comprising smart polymer shell coated on magnetic nanoparticles as an anticancer drug carrier and cancer cell-specific targeting agent. Magnetite nanoparticles (MNPs), prepared by a simple coprecipitation method, was surface modified by introducing amine groups using 3-aminopropyltriethoxysilane. Dual-responsive poly(N-isopropylacrylamide)-block-poly(acrylic acid) copolymer, synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, was then attached to the amine-functionalized MNPs via EDC/NHS method. Further, to accomplish cancer-specific targeting properties, folic acid was tethered to the surface of the nanoparticles. Thereafter, rhodamine B isothiocyanate was conjugated to endow fluorescent property to the MNPs required for cellular imaging applications. The nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), selected area electron diffraction (SAED), field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDX), thermogravimetric analysis (TGA), zeta potential, vibrating sample magnetometer (VSM), X-ray photoelectron spectroscopy (XPS) measurements, and FTIR, UV-vis spectral analysis. Doxorubicin (DOX), an anticancer drug used for the present study, was loaded into the nanoparticles and its release behavior was subsequently studied. Result showed a sustained release of DOX preferentially at the desired lysosomal pH and temperature condition. The biological activity of the DOX-loaded MNPs was studied by MTT assay, fluorescence microscopy, and apoptosis. Intracellular-uptake studies revealed preferential uptake of these nanoparticles into cancer cells (HeLa cells) compared to normal fibroblast cells (L929 cells). The in vitro apoptosis study revealed that the DOX-loaded nanoparticles caused significant death to the HeLa cells. These nanoparticles were capable of target specific release of the loaded drug in response to pH and temperature and hence may serve as a potential drug carrier for in vivo applications.

Imaging of single retinal ganglion cell with differential interference contrast microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Oh, Juyeong; Kim, Yu Jeong; Kim, Chul-Ki; Lee, Taik Jin; Seo, Mina; Lee, Seok; Woo, Deok Ha; Jun, Seong Chan; Park, Ki-Ho; Kim, Seok Hwan; Kim, Jae Hun

2017-02-01

Glaucoma is a progressive optic neuropathy, characterized by the selective loss of retinal ganglion cells (RGCs). Therefore, monitoring the change of number or morphology of RGC is essential for the early detection as well as investigation of pathophysiology of glaucoma. Since RGC layer is transparent and hyporeflective, the direct optical visualization of RGCs has not been successful so far. Therefore, glaucoma evaluation mostly depends on indirect diagnostic methods such as the evaluation of optic disc morphology or retinal nerve fiber layer thickness measurement by optical coherence tomography. We have previously demonstrated single photoreceptor cell imaging with differential interference contrast (DIC) microscopy. Herein, we successfully visualized single RGC using DIC microscopy. Since RGC layer is much less reflective than photoreceptor layer, various techniques including the control of light wavelength and bandwidth using a tunable band pass filter were adopted to reduce the chromatic aberration in z-axis for higher and clearer resolution. To verify that the imaged cells were the RGCs, the flat-mounted retina of Sprague-Dawley rat, in which the RGCs were retrogradely labeled with fluorescence, was observed by both fluorescence and DIC microscopies for direct comparison. We have confirmed that the cell images obtained by fluorescence microscopy were perfectly matched with cell images by DIC microscopy. As conclusion, we have visualized single RGC with DIC microscopy, and confirmed with fluorescence microscopy.

Lulo cell line derived from Lutzomyia longipalpis (Diptera: Psychodidae): a novel model to assay Leishmania spp. and vector interaction

PubMed Central

2011-01-01

Background Leishmania (Vianna) braziliensis, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) chagasi are important parasites in the scenario of leishmaniasis in Brazil. During the life cycle of these parasites, the promastigote forms adhere to the midgut epithelial microvillii of phlebotomine insects to avoid being secreted along with digestive products. Lulo cells are a potential model that will help to understand the features of this adhesion phenomenon. Here, we analyze the interaction between Leishmania spp. promastigotes and Lulo cells in vitro, specifically focusing on adhesion events occurring between three Leishmania species and this cell line. Methods Confluent monolayers of Lulo cells were incubated with promastigotes and adhesion was assessed using both light microscopy and scanning electron microscopy. Findings The results indicate that species from the subgenera Leishmania and Viannia have great potential to adhere to Lulo cells. The highest adherence rate was observed for L. (L.) chagasi after 24 h of incubation with Lulo cells (27.3 ± 1.8% of cells with adhered promastigotes), followed by L. (L.) amazonensis (16.0 ± 0.7%) and L. (V.) braziliensis (3.0 ± 0.7%), both after 48 h. In the ultrastructural analysis, promastigote adherence was also assessed by scanning electron microscopy, showing that, for parasites from both subgenera, adhesion occurs by both the body and the flagellum. The interaction of Lulo cells with Leishmania (L.) chagasi showed the participation of cytoplasmic projections from the former closely associating the parasites with the cells. Conclusions We present evidence that Lulo cells can be useful in studies of insect-parasite interactions for Leishmania species. PMID:22082050

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

Quantitative Confocal Microscopy Analysis as a Basis for Search and Study of Potassium Kv1.x Channel Blockers

NASA Astrophysics Data System (ADS)

Feofanov, Alexey V.; Kudryashova, Kseniya S.; Nekrasova, Oksana V.; Vassilevski, Alexander A.; Kuzmenkov, Alexey I.; Korolkova, Yuliya V.; Grishin, Eugene V.; Kirpichnikov, Mikhail P.

Artificial KcsA-Kv1.x (x = 1, 3) receptors were recently designed by transferring the ligand-binding site from human Kv1.x voltage-gated potassium channels into corresponding domain of the bacterial KscA channel. We found that KcsA-Kv1.x receptors expressed in E. coli cells are embedded into cell membrane and bind ligands when the cells are transformed to spheroplasts. We supposed that E. coli spheroplasts with membrane-embedded KcsA-Kv1.x and fluorescently labeled ligand agitoxin-2 (R-AgTx2) can be used as elements of an advanced analytical system for search and study of Kv1-channel blockers. To realize this idea, special procedures were developed for measurement and quantitative treatment of fluorescence signals obtained from spheroplast membrane using confocal laser scanning microscopy (CLSM). The worked out analytical "mix and read" systems supported by quantitative CLSM analysis were demonstrated to be reliable alternative to radioligand and electrophysiology techniques in the search and study of selective Kv1.x channel blockers of high scientific and medical importance.

Aging results in copper accumulations in glial fibrillary acidic protein-positive cells in the subventricular zone.

PubMed

Pushkar, Yulia; Robison, Gregory; Sullivan, Brendan; Fu, Sherleen X; Kohne, Meghan; Jiang, Wendy; Rohr, Sven; Lai, Barry; Marcus, Matthew A; Zakharova, Taisiya; Zheng, Wei

2013-10-01

Analysis of rodent brains with X-ray fluorescence (XRF) microscopy combined with immunohistochemistry allowed us to demonstrate that local Cu concentrations are thousands of times higher in the glia of the subventricular zone (SVZ) than in other cells. Using XRF microscopy with subcellular resolution and intracellular X-ray absorption spectroscopy we determined the copper (I) oxidation state and the sulfur ligand environment. Cu K-edge X-ray absorption near edge spectroscopy is consistent with Cu being bound as a multimetallic Cu-S cluster similar to one present in Cu-metallothionein. Analysis of age-related changes show that Cu content in astrocytes of the SVZ increases fourfold from 3 weeks to 9 months, while Cu concentration in other brain areas remain essentially constant. This increase in Cu correlates with a decrease in adult neurogenesis assessed using the Ki67 marker (both, however, can be age-related effects). We demonstrate that the Cu distribution and age-related concentration changes in the brain are highly cell specific. © 2013 The Anatomical Society and John Wiley & Sons Ltd.

CdTe-TiO2 nanocomposite: an impeder of bacterial growth and biofilm

NASA Astrophysics Data System (ADS)

Gholap, Haribhau; Patil, Rajendra; Yadav, Prasad; Banpurkar, Arun; Ogale, Satishchandra; Gade, Wasudeo

2013-05-01

The resurgence of infectious diseases and associated issues related to antibiotic resistance has raised enormous challenges which may possibly be confronted primarily by nanotechnology routes. One key need of critical significance in this context is the development of an agent capable of inhibiting quorum sensing mediated biofilm formation in pathogenic organisms. In this work we examine the possible use of a nanocomposite, CdTe-TiO2, as an impeder of growth and biofilm. In the presence of CdTe-TiO2, scanning electron microscopy (SEM) analysis shows exposed cells without the surrounding matrix. Confocal laser scanning microscopy shows spatially distributed fluorescence, a typical indication of an impeded biofilm, as opposed to the control which shows matrix-covered cells and continuous fluorescence, typical of biofilm formation. Quantitatively, the inhibition of biofilm was ˜57%. CdTe-TiO2 also exhibits good antibacterial properties against Gram positive and Gram negative organisms by virtue of the generation of reactive oxygen species inside the cells, reflected by a ruptured appearance in the SEM analysis.

Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations.

PubMed

Engelmann, Péter; Hayashi, Yuya; Bodó, Kornélia; Ernszt, Dávid; Somogyi, Ildikó; Steib, Anita; Orbán, József; Pollák, Edit; Nyitrai, Miklós; Németh, Péter; Molnár, László

2016-12-01

Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

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

2017-01-01

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

New approaches in renal microscopy: volumetric imaging and superresolution microscopy.

PubMed

Kim, Alfred H J; Suleiman, Hani; Shaw, Andrey S

2016-05-01

Histologic and electron microscopic analysis of the kidney has provided tremendous insight into structures such as the glomerulus and nephron. Recent advances in imaging, such as deep volumetric approaches and superresolution microscopy, have the capacity to dramatically enhance our current understanding of the structure and function of the kidney. Volumetric imaging can generate images millimeters below the surface of the intact kidney. Superresolution microscopy breaks the diffraction barrier inherent in traditional light microscopy, enabling the visualization of fine structures. Here, we describe new approaches to deep volumetric and superresolution microscopy of the kidney. Rapid advances in lasers, microscopic objectives, and tissue preparation have transformed our ability to deep volumetric image the kidney. Innovations in sample preparation have allowed for superresolution imaging with electron microscopy correlation, providing unprecedented insight into the structures within the glomerulus. Technological advances in imaging have revolutionized our capacity to image both large volumes of tissue and the finest structural details of a cell. These new advances have the potential to provide additional profound observations into the normal and pathologic functions of the kidney.

Single-cell analysis of radiotracers' uptake by fluorescence microscopy: direct and droplet approach

NASA Astrophysics Data System (ADS)

Gallina, M. E.; Kim, T. J.; Vasquez, J.; Tuerkcan, S.; Abbyad, P.; Pratx, G.

2017-02-01

Radionuclides are used for sensitive and specific detection of small molecules in vivo and in vitro. Recently, radioluminescence microscopy extended their use to single-cell studies. Here we propose a new single-cell radioisotopic assay that improves throughput while adding sorting capabilities. The new method uses fluorescence-based sensor for revealing single-cell interactions with radioactive molecular markers. This study focuses on comparing two different experimental approaches. Several probes were tested and Dihydrorhodamine 123 was selected as the best compromise between sensitivity, brightness and stability. The sensor was incorporated either directly within the cell cytoplasm (direct approach), or it was coencapsulated with radiolabeled single-cells in oil-dispersed water droplets (droplet approach). Both approaches successfully activated the fluorescence signal following cellular uptake of 18F-fluorodeoxyglucose (FDG) and external Xrays exposure. The direct approach offered single-cell resolution and longtime stability ( > 20 hours), moreover it could discriminate FDG uptake at labelling concentration as low as 300 μCi/ml. In cells incubated with Dihydrorhodamine 123 after exposure to high radiation doses (8-16 Gy), the fluorescence signal was found to increase with the depletion of ROS quenchers. On the other side, the droplet approach required higher labelling concentrations (1.00 mCi/ml), and, at the current state of art, three cells per droplet are necessary to produce a fluorescent signal. This approach, however, is independent on cellular oxidative stress and, with further improvements, will be more suitable for studying heterogeneous populations. We anticipate this technology to pave the way for the analysis of single-cell interactions with radiomarkers by radiofluorogenic-activated single-cell sorting.

Ultrastructural characterization of the new NG97ht human-derived glioma cell line using two different electron microscopy technical procedures.

PubMed

Machado, Camila Maria Longo; Zorzeto, Tatiane Queiroz; Bianco, Juares E Romero; Rosa, Renata Giardini; Genari, Selma Candelaria; Joazeiro, Paulo Pinto; Verinaud, Liana

2009-04-01

On the basis of transmission electron microscopy observations in tumor cell lines, oncologists have made innumerous diagnostic and therapeutical progresses. Following this path, the UNICAMP immunopathologies laboratory established the NG97 cell line derived from a human astrocytoma grade III, which when injected to the athymic nude mouse flank developed a grade IV astrocytoma. In this study, we focused on ultrastructural characterization of the NG97 cells after being recovered from xenotransplant (NG97ht). These cells in culture were assayed by two different electron microscopy procedures to characterize ultrastructures related to grade IV astrocytomas and to observe their structures through cell subcultivation. Additionally, comparative morphological descriptions of different cell passages in these technical procedures could be a useful tool for improving electron microscopy cell lineage protocols. Results from many cell passage observations showed ultrastructural similarities, which suggest malignant and glioblastoma phenotypes. In the first procedure, NG97ht cells were harvested and then incorporated into agarose before subjecting them to electron microscopy protocols, whereas in the second one, monolayer cells grew first on cover slides. Comparison among protocols revealed that organelles, cytoplasmatic extensions, spatial conformation of filopodia, and cell attachment to substrate were more preserved in the second procedure. Furthermore, in this latter procedure, a unique ellipsoidal structure was observed, which was already described when dealing with gliosarcoma cell line elsewhere. Therefore, these analyses demonstrated a morphological characterization of a new NG97ht cell line using electron transmission microscopy. Moreover, it has been shown that the second procedure provides more detailed information compared with the first.

Quantitative super-resolution single molecule microscopy dataset of YFP-tagged growth factor receptors.

PubMed

Lukeš, Tomáš; Pospíšil, Jakub; Fliegel, Karel; Lasser, Theo; Hagen, Guy M

2018-03-01

Super-resolution single molecule localization microscopy (SMLM) is a method for achieving resolution beyond the classical limit in optical microscopes (approx. 200 nm laterally). Yellow fluorescent protein (YFP) has been used for super-resolution single molecule localization microscopy, but less frequently than other fluorescent probes. Working with YFP in SMLM is a challenge because a lower number of photons are emitted per molecule compared with organic dyes, which are more commonly used. Publically available experimental data can facilitate development of new data analysis algorithms. Four complete, freely available single molecule super-resolution microscopy datasets on YFP-tagged growth factor receptors expressed in a human cell line are presented, including both raw and analyzed data. We report methods for sample preparation, for data acquisition, and for data analysis, as well as examples of the acquired images. We also analyzed the SMLM datasets using a different method: super-resolution optical fluctuation imaging (SOFI). The 2 modes of analysis offer complementary information about the sample. A fifth single molecule super-resolution microscopy dataset acquired with the dye Alexa 532 is included for comparison purposes. This dataset has potential for extensive reuse. Complete raw data from SMLM experiments have typically not been published. The YFP data exhibit low signal-to-noise ratios, making data analysis a challenge. These datasets will be useful to investigators developing their own algorithms for SMLM, SOFI, and related methods. The data will also be useful for researchers investigating growth factor receptors such as ErbB3.

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

NASA Astrophysics Data System (ADS)

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

2016-10-01

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

Frontiers in Fluctuation Spectroscopy: Measuring protein dynamics and protein spatio-temporal connectivity

NASA Astrophysics Data System (ADS)

Digman, Michelle

Fluorescence fluctuation spectroscopy has evolved from single point detection of molecular diffusion to a family of microscopy imaging correlation tools (i.e. ICS, RICS, STICS, and kICS) useful in deriving spatial-temporal dynamics of proteins in living cells The advantage of the imaging techniques is the simultaneous measurement of all points in an image with a frame rate that is increasingly becoming faster with better sensitivity cameras and new microscopy modalities such as the sheet illumination technique. A new frontier in this area is now emerging towards a high level of mapping diffusion rates and protein dynamics in the 2 and 3 dimensions. In this talk, I will discuss the evolution of fluctuation analysis from the single point source to mapping diffusion in whole cells and the technology behind this technique. In particular, new methods of analysis exploit correlation of molecular fluctuations originating from measurement of fluctuation correlations at distant points (pair correlation analysis) and methods that exploit spatial averaging of fluctuations in small regions (iMSD). For example the pair correlation fluctuation (pCF) analyses done between adjacent pixels in all possible radial directions provide a window into anisotropic molecular diffusion. Similar to the connectivity atlas of neuronal connections from the MRI diffusion tensor imaging these new tools will be used to map the connectome of protein diffusion in living cells. For biological reaction-diffusion systems, live single cell spatial-temporal analysis of protein dynamics provides a mean to observe stochastic biochemical signaling in the context of the intracellular environment which may lead to better understanding of cancer cell invasion, stem cell differentiation and other fundamental biological processes. National Institutes of Health Grant P41-RRO3155.

3D-printed PCL scaffolds for the cultivation of mesenchymal stem cells.

PubMed

Steffens, Daniela; Rezende, Rodrigo Alvarenga; Santi, Bruna; Pereira, Frederico David Alencar de Sena; Inforçatti Neto, Paulo; da Silva, Jorge Vicente Lopes; Pranke, Patricia

2016-04-06

Tissue engineering is a field which is currently under a great deal of investigation for the development and/or restoration of tissue and organs, through the combination of cell therapy with biomaterials. Rapid prototyping or additive manufacturing is a versatile technology which makes possible the fabrication of three dimensional (3D) structures from a wide range of materials with complex geometry and accuracy, such as scaffolds. The aim of this study has been to investigate the interaction between mesenchymal stem cells with poly (ε-caprolactone) (PCL) biomaterials used for obtaining scaffolds through additive manufacturing. Scanning electron microscopy, confocal microscopy and biological assays were performed to analyse the successful interaction between the cells and the biomaterials. As a result, the number of viable cells attached to the scaffolds was lower when compared to the control group; however, it was possible to observe cells in the scaffolds since day 1 of analysis, with regions of confluence after 21 days of seeding. To conclude, these biomaterials are interesting if used as medical artifacts, principally in tissue with prolonged regeneration time and which requires 3D supports with good mechanical properties.

Phagocytosis of PLGA Microparticles in Rat Peritoneal Exudate Cells: A Time-Dependent Study

NASA Astrophysics Data System (ADS)

Gomes, Anderson De Jesus; Nain Lunardi, Claure; Henrique Caetano, Flávio; Orive Lunardi, Laurelúcia; da Hora Machado, Antonio Eduardo

2006-07-01

With the purpose of enhancing the efficacy of microparticle-encapsulated therapeutic agents, in this study we evaluated the phagocytic ability of rat peritoneal exudate cells and the preferential location of poly(D,L-lactide-co-glycolic acid) (PLGA) microparticles inside these cells. The microparticles used were produced by a solvent evaporation method and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Size distribution analysis using DLS and SEM showed that the particles were spherical, with diameters falling between 0.5 and 1.5 [mu]m. Results from cell adhesion by SEM assay, indicated that the PLGA microparticles are not toxic to cells and do not cause any distinct damage to them as confirmed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Among the large variety of cell populations found in the peritoneal exudates (neutrophils, eosinophils, monocytes, and macrophages), TEM showed that only the latter phagocytosed PLGA microparticles, in a time-dependent manner. The results obtained indicate that the microparticles studied show merits as possible carriers of drugs for intracellular delivery.

Radioluminescence Microscopy: Measuring the Heterogeneous Uptake of Radiotracers in Single Living Cells

PubMed Central

Pratx, Guillem; Chen, Kai; Sun, Conroy; Martin, Lynn; Carpenter, Colin M.; Olcott, Peter D.; Xing, Lei

2012-01-01

Radiotracers play an important role in interrogating molecular processes both in vitro and in vivo. However, current methods are limited to measuring average radiotracer uptake in large cell populations and, as a result, lack the ability to quantify cell-to-cell variations. Here we apply a new technique, termed radioluminescence microscopy, to visualize radiotracer uptake in single living cells, in a standard fluorescence microscopy environment. In this technique, live cells are cultured sparsely on a thin scintillator plate and incubated with a radiotracer. Light produced following beta decay is measured using a highly sensitive microscope. Radioluminescence microscopy revealed strong heterogeneity in the uptake of [18F]fluoro-deoxyglucose (FDG) in single cells, which was found consistent with fluorescence imaging of a glucose analog. We also verified that dynamic uptake of FDG in single cells followed the standard two-tissue compartmental model. Last, we transfected cells with a fusion PET/fluorescence reporter gene and found that uptake of FHBG (a PET radiotracer for transgene expression) coincided with expression of the fluorescent protein. Together, these results indicate that radioluminescence microscopy can visualize radiotracer uptake with single-cell resolution, which may find a use in the precise characterization of radiotracers. PMID:23056276

Fast evaluation of 69 basal cell carcinomas with ex vivo fluorescence confocal microscopy: criteria description, histopathological correlation, and interobserver agreement.

PubMed

Bennàssar, Antoni; Carrera, Cristina; Puig, Susana; Vilalta, Antoni; Malvehy, Josep

2013-07-01

Fluorescence confocal microscopy (FCM) represents a first step toward a rapid "bedside pathology" in the Mohs surgery setting and in other fields of general pathology. To describe and validate FCM criteria for the main basal cell carcinoma (BCC) subtypes and to demonstrate the overall agreement with classic pathologic analysis of hematoxylin-eosin-stained samples. DESIGN A total of 69 BCCs from 66 patients were prospectively imaged using ex vivo FCM. Confocal mosaics were evaluated in real time and compared with classic pathologic analysis. Department of Dermatology, Hospital Clínic of Barcelona, Barcelona, Spain, between November 2010 and July 2011. Patients with BCC attending the Mohs Surgery Unit. Presence or absence of BCC and histological subtype (superficial, nodular, and infiltrating) in the confocal mosaics. Eight criteria for BCC were described, evaluated, and validated. Although there were minor differences among BCC subtypes, the most BCC-defining criteria were peripheral palisading, clefting, nuclear pleomorphism, and presence of stroma. These criteria were validated with independent observers (κ values >0.7 [corrected] for most criteria). We herein propose, describe, and validate FCM criteria for BCC diagnosis. Fluorescence confocal microscopy is an attractive alternative to histopathologic analysis of frozen sections during Mohs surgery because large areas of freshly excised tissue can be assessed in real time without the need for tissue processing while minimizing labor and costs.

Metal Free Graphene Oxide (GO) Nanosheets and Pristine-Single Wall Carbon Nanotubes (p-SWCNTs) Biocompatibility Investigation: A Comparative Study in Different Human Cell Lines.

PubMed

Valentini, Federica; Mari, Emanuela; Zicari, Alessandra; Calcaterra, Andrea; Talamo, Maurizio; Scioli, Maria Giovanna; Orlandi, Augusto; Mardente, Stefania

2018-04-28

The in vitro biocompatibility of Graphene Oxide (GO) nanosheets, which were obtained by the electrochemical exfoliation of graphite electrodes in an electrolytic bath containing salts, was compared with the pristine Single Wall Carbon Nanotubes (p-SWCNTs) under the same experimental conditions in different human cell lines. The cells were treated with different concentrations of GO and SWCNTs for up to 48 h. GO did not induce any significant morphological or functional modifications (demonstrating a high biocompatibility), while SWNCTs were toxic at any concentration used after a few hours of treatment. The cell viability or cytotoxicity were detected by the trypan blue assay and the lactate dehydrogenase LDH quantitative enzymatic test. The Confocal Laser Scanning Microscopy (CLSM) and transmission electron microscopy (TEM) analysis demonstrated the uptake and internalization of GO sheets into cells, which was localized mainly in the cytoplasm. Different results were observed in the same cell lines treated with p-SWCNTs. TEM and CLSM (Confocal Laser Scanning Microscopy) showed that the p-SWCNTs induced vacuolization in the cytoplasm, disruption of cellular architecture and damage to the nuclei. The most important result of this study is our finding of a higher GO biocompatibility compared to the p-SWCNTs in the same cell lines. This means that GO nanosheets, which are obtained by the electrochemical exfoliation of a graphite-based electrode (carried out in saline solutions or other physiological working media) could represent an eligible nanocarrier for drug delivery, gene transfection and molecular cell imaging tests.

Light and electron microscopic analyses of Vasa expression in adult germ cells of the fish medaka.

PubMed

Yuan, Yongming; Li, Mingyou; Hong, Yunhan

2014-07-15

Germ cells of diverse animal species have a unique membrane-less organelle called germ plasm (GP). GP is usually associated with mitochondria and contains RNA binding proteins and mRNAs of germ genes such as vasa. GP has been described as the mitochondrial cloud (MC), intermitochondrial cement (IC) and chromatoid body (CB). The mechanism underlying varying GP structures has remained incompletely understood. Here we report the analysis of GP through light and electron microscopy by using Vasa as a marker in adult male germ cells of the fish medaka (Oryzias latipes). Immunofluorescence light microscopy revealed germ cell-specific Vasa expression. Vasa is the most abundant in mitotic germ cells (oogonia and spermatogonia) and reduced in meiotic germ cells. Vasa in round spermatids exist as a spherical structure reminiscent of CB. Nanogold immunoelectron microscopy revealed subcellular Vasa redistribution in male germ cells. Vasa in spermatogonia concentrates in small areas of the cytoplasm and is surrounded by mitochondria, which is reminiscent of MC. Vasa is intermixed with mitochondria to form IC in primary spermatocytes, appears as the free cement (FC) via separation from mitochondria in secondary spermatocyte and becomes condensed in CB at the caudal pole of round spermatids. During spermatid morphogenesis, Vasa redistributes and forms a second CB that is a ring-like structure surrounding the dense fiber of the flagellum in the midpiece. These structures resemble those described for GP in various species. Thus, Vasa identifies GP and adopts varying structures via dynamic reorganization at different stages of germ cell development. Copyright © 2014 Elsevier B.V. All rights reserved.

Two Genera of Magnetococci with Bean-like Morphology from Intertidal Sediments of the Yellow Sea, China

PubMed Central

Zhang, Wen-Yan; Zhou, Ke; Pan, Hong-Miao; Yue, Hai-Dong; Jiang, Ming

2012-01-01

Magnetotactic bacteria have the unique capacity of being able to swim along geomagnetic field lines. They are Gram-negative bacteria with diverse morphologies and variable phylogenetic relatedness. Here, we describe a group of uncultivated marine magnetococci collected from intertidal sediments of Huiquan Bay in the Yellow Sea. They were coccoid-ovoid in morphology, with an average size of 2.8 ± 0.3 μm by 2.0 ± 0.2 μm. Differential interference contrast microscopy, fluorescence microscopy, and transmission electron microscopy revealed that each cell was apparently composed of two hemispheres. The cells synthesized iron oxide-type magnetosomes that clustered on one side of the cell at the interface between the two hemispheres. In some cells two chains of magnetosomes were observed across the interface. Each cell had two bundles of flagella enveloped in a sheath and displayed north-seeking helical motion. Two 16S rRNA gene sequences having 91.8% identity were obtained, and their authenticity was confirmed by fluorescence in situ hybridization. Phylogenetic analysis revealed that the magnetococci are affiliated with the Alphaproteobacteria and are most closely related to two uncultured magnetococci with sequence identities of 92.7% and 92.4%, respectively. Because they display a >7% sequence divergence to all bacteria reported, the bean-like magnetococci may represent two novel genera. PMID:22660708

Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy

PubMed Central

Yan, Yuling; Petchprayoon, Chutima; Mao, Shu; Marriott, Gerard

2013-01-01

Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens. PMID:23267183

Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments

PubMed Central

Goode, Angela E.; Skepper, Jeremy N.; Thorley, Andrew J.; Seiffert, Joanna M.; Chung, K. Fan; Tetley, Teresa D.; Shaffer, Milo S. P.; Ryan, Mary P.

2015-01-01

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinised. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data. PMID:25606708

Hepatitis C Virus Induces the Mitochondrial Translocation of Parkin and Subsequent Mitophagy

PubMed Central

Kim, Seong-Jun; Syed, Gulam H.; Siddiqui, Aleem

2013-01-01

Hepatitis C Virus (HCV) induces intracellular events that trigger mitochondrial dysfunction and promote host metabolic alterations. Here, we investigated selective autophagic degradation of mitochondria (mitophagy) in HCV-infected cells. HCV infection stimulated Parkin and PINK1 gene expression, induced perinuclear clustering of mitochondria, and promoted mitochondrial translocation of Parkin, an initial event in mitophagy. Liver tissues from chronic HCV patients also exhibited notable levels of Parkin induction. Using multiple strategies involving confocal and electron microscopy, we demonstrated that HCV-infected cells display greater number of mitophagosomes and mitophagolysosomes compared to uninfected cells. HCV-induced mitophagy was evidenced by the colocalization of LC3 puncta with Parkin-associated mitochondria and lysosomes. Ultrastructural analysis by electron microscopy and immunoelectron microscopy also displayed engulfment of damaged mitochondria in double membrane vesicles in HCV-infected cells. The HCV-induced mitophagy occurred irrespective of genotypic differences. Silencing Parkin and PINK1 hindered HCV replication suggesting the functional relevance of mitophagy in HCV propagation. HCV-mediated decline of mitochondrial complex I enzyme activity was rescued by chemical inhibition of mitophagy or by Parkin silencing. Overall our results suggest that HCV induces Parkin-dependent mitophagy, which may have significant contribution in mitochondrial liver injury associated with chronic hepatitis C. PMID:23555273