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Sample records for cell microscopy segmentation

  1. Adaptive Cell Segmentation and Tracking for Volumetric Confocal Microscopy Images of a Developing Plant Meristem

    Institute of Scientific and Technical Information of China (English)

    Min Liu; Anirban Chakraborty; Damanpreet Singh; Ram Kishor Yadav; Gopi Meenakshisundaram; G. Venugopala Reddy; Amit Roy-Chowdhury

    2011-01-01

    Automated segmentation and tracking of cells in actively developing tissues can provide high-throughput and quantitative spatiotemporal measurements of a range of cell behaviors; cell expansion and cell-division kinetics leading to a better understanding of the underlying dynamics of morphogenesis.Here,we have studied the problem of constructing cell lineages in time-lapse volumetric image stacks obtained using Confocal Laser Scanning Microscopy (CLSM).The novel contribution of the work lies in its ability to segment and track cells in densely packed tissue,the shoot apical meristem (SAM),through the use of a close-loop,adaptive segmentation,and tracking approach.The tracking output acts as an indicator of the quality of segmentation and,in turn,the segmentation can be improved to obtain better tracking results.We construct an optimization function that minimizes the segmentation error,which is,in turn,estimated from the tracking results.This adaptive approach significantly improves both tracking and segmentation when compared to an open loop framework in which segmentation and tracking modules operate separately.

  2. Cell motility dynamics: a novel segmentation algorithm to quantify multi-cellular bright field microscopy images.

    Directory of Open Access Journals (Sweden)

    Assaf Zaritsky

    Full Text Available Confocal microscopy analysis of fluorescence and morphology is becoming the standard tool in cell biology and molecular imaging. Accurate quantification algorithms are required to enhance the understanding of different biological phenomena. We present a novel approach based on image-segmentation of multi-cellular regions in bright field images demonstrating enhanced quantitative analyses and better understanding of cell motility. We present MultiCellSeg, a segmentation algorithm to separate between multi-cellular and background regions for bright field images, which is based on classification of local patches within an image: a cascade of Support Vector Machines (SVMs is applied using basic image features. Post processing includes additional classification and graph-cut segmentation to reclassify erroneous regions and refine the segmentation. This approach leads to a parameter-free and robust algorithm. Comparison to an alternative algorithm on wound healing assay images demonstrates its superiority. The proposed approach was used to evaluate common cell migration models such as wound healing and scatter assay. It was applied to quantify the acceleration effect of Hepatocyte growth factor/scatter factor (HGF/SF on healing rate in a time lapse confocal microscopy wound healing assay and demonstrated that the healing rate is linear in both treated and untreated cells, and that HGF/SF accelerates the healing rate by approximately two-fold. A novel fully automated, accurate, zero-parameters method to classify and score scatter-assay images was developed and demonstrated that multi-cellular texture is an excellent descriptor to measure HGF/SF-induced cell scattering. We show that exploitation of textural information from differential interference contrast (DIC images on the multi-cellular level can prove beneficial for the analyses of wound healing and scatter assays. The proposed approach is generic and can be used alone or alongside traditional

  3. Cell segmentation for division rate estimation in computerized video time-lapse microscopy

    Science.gov (United States)

    He, Weijun; Wang, Xiaoxu; Metaxas, Dimitris; Mathew, Robin; White, Eileen

    2007-02-01

    The automated estimation of cell division rate plays an important role in the evaluation of a gene function in high throughput biomedical research. Using Computerized Video Time-Lapse (CVTL) microcopy , it is possible to follow a large number of cells in their physiological conditions for several generations. However analysis of this large volume data is complicated due to cell to cell contacts in a high density population. We approach this problem by segmenting out cells or cell clusters through a learning method. The feature of a pixel is represented by the intensity and gradient information in a small surrounding sub-window. Curve evolution techniques are used to accurately find the cell or cell cluster boundary. With the assumption that the average cell size is the same in each frame, we can use the cell area to estimate the cell division rate. Our segmentation results are compared to manually-defined ground truth. Both recall and precision measures for segmentation accuracy are above 95%.

  4. Real-Time Three-Dimensional Cell Segmentation in Large-Scale Microscopy Data of Developing Embryos.

    Science.gov (United States)

    Stegmaier, Johannes; Amat, Fernando; Lemon, William C; McDole, Katie; Wan, Yinan; Teodoro, George; Mikut, Ralf; Keller, Philipp J

    2016-01-25

    We present the Real-time Accurate Cell-shape Extractor (RACE), a high-throughput image analysis framework for automated three-dimensional cell segmentation in large-scale images. RACE is 55-330 times faster and 2-5 times more accurate than state-of-the-art methods. We demonstrate the generality of RACE by extracting cell-shape information from entire Drosophila, zebrafish, and mouse embryos imaged with confocal and light-sheet microscopes. Using RACE, we automatically reconstructed cellular-resolution tissue anisotropy maps across developing Drosophila embryos and quantified differences in cell-shape dynamics in wild-type and mutant embryos. We furthermore integrated RACE with our framework for automated cell lineaging and performed joint segmentation and cell tracking in entire Drosophila embryos. RACE processed these terabyte-sized datasets on a single computer within 1.4 days. RACE is easy to use, as it requires adjustment of only three parameters, takes full advantage of state-of-the-art multi-core processors and graphics cards, and is available as open-source software for Windows, Linux, and Mac OS. PMID:26812020

  5. Segmentation and learning in the quantitative analysis of microscopy images

    Science.gov (United States)

    Ruggiero, Christy; Ross, Amy; Porter, Reid

    2015-02-01

    In material science and bio-medical domains the quantity and quality of microscopy images is rapidly increasing and there is a great need to automatically detect, delineate and quantify particles, grains, cells, neurons and other functional "objects" within these images. These are challenging problems for image processing because of the variability in object appearance that inevitably arises in real world image acquisition and analysis. One of the most promising (and practical) ways to address these challenges is interactive image segmentation. These algorithms are designed to incorporate input from a human operator to tailor the segmentation method to the image at hand. Interactive image segmentation is now a key tool in a wide range of applications in microscopy and elsewhere. Historically, interactive image segmentation algorithms have tailored segmentation on an image-by-image basis, and information derived from operator input is not transferred between images. But recently there has been increasing interest to use machine learning in segmentation to provide interactive tools that accumulate and learn from the operator input over longer periods of time. These new learning algorithms reduce the need for operator input over time, and can potentially provide a more dynamic balance between customization and automation for different applications. This paper reviews the state of the art in this area, provides a unified view of these algorithms, and compares the segmentation performance of various design choices.

  6. Understanding the optics to aid microscopy image segmentation.

    Science.gov (United States)

    Yin, Zhaozheng; Li, Kang; Kanade, Takeo; Chen, Mei

    2010-01-01

    Image segmentation is essential for many automated microscopy image analysis systems. Rather than treating microscopy images as general natural images and rushing into the image processing warehouse for solutions, we propose to study a microscope's optical properties to model its image formation process first using phase contrast microscopy as an exemplar. It turns out that the phase contrast imaging system can be relatively well explained by a linear imaging model. Using this model, we formulate a quadratic optimization function with sparseness and smoothness regularizations to restore the "authentic" phase contrast images that directly correspond to specimen's optical path length without phase contrast artifacts such as halo and shade-off. With artifacts removed, high quality segmentation can be achieved by simply thresholding the restored images. The imaging model and restoration method are quantitatively evaluated on two sequences with thousands of cells captured over several days. PMID:20879233

  7. General Purpose Segmentation for Microorganisms in Microscopy Images

    DEFF Research Database (Denmark)

    Jensen, Sebastian H. Nesgaard; Moeslund, Thomas B.; Rankl, Christian

    2014-01-01

    In this paper, we propose an approach for achieving generalized segmentation of microorganisms in mi- croscopy images. It employs a pixel-wise classification strategy based on local features. Multilayer percep- trons are utilized for classification of the local features and is trained for each...... specific segmentation problem using supervised learning. This approach was tested on five different segmentation problems in bright field, differential interference contrast, fluorescence and laser confocal scanning microscopy. In all instance good results were achieved with the segmentation quality...

  8. Understanding the Phase Contrast Optics to Restore Artifact-free Microscopy Images for Segmentation

    OpenAIRE

    Yin, Zhaozheng; Kanade, Takeo; Chen, Mei

    2012-01-01

    Phase contrast, a noninvasive microscopy imaging technique, is widely used to capture time-lapse images to monitor the behavior of transparent cells without staining or altering them. Due to the optical principle, phase contrast microscopy images contain artifacts such as the halo and shade-off that hinder image segmentation, a critical step in automated microscopy image analysis. Rather than treating phase contrast microscopy images as general natural images and applying generic image proces...

  9. Advanced Microscopy of Microbial Cells

    DEFF Research Database (Denmark)

    Haagensen, Janus Anders Juul; Regenberg, Birgitte; Sternberg, Claus

    2011-01-01

    Growing awareness of heterogeneity in cells of microbial populations has emphasized the importance of advanced microscopy for visualization and understanding of the molecular mechanisms underlying cell-to-cell variation. In this review, we highlight some of the recent advances in confocal...... microscopy, super-resolution optical microscopy (STED, SIM, PALM) as well as atomic force microscopy and Raman spectroscopy. Using examples of bistability in microbial populations as well as biofilm development and differentiation in bacterial and yeast consortia, we demonstrate the importance of microscopy...

  10. Advanced microscopy of microbial cells

    DEFF Research Database (Denmark)

    Haagensen, Janus Anders Juul; Regenberg, Birgitte; Sternberg, Claus

    2011-01-01

    Growing awareness of heterogeneity in cells of microbial populations has emphasized the importance of advanced microscopy for visualization and understanding of the molecular mechanisms underlying cell-to-cell variation. In this review, we highlight some of the recent advances in confocal...... microscopy, super-resolution optical microscopy (STED, SIM, PALM) as well as atomic force microscopy and Raman spectroscopy. Using examples of bistability in microbial populations as well as biofilm development and differentiation in bacterial and yeast consortia, we demonstrate the importance of microscopy...

  11. Embryonic Heart Morphogenesis from Confocal Microscopy Imaging and Automatic Segmentation

    Directory of Open Access Journals (Sweden)

    Hongda Mao

    2013-01-01

    Full Text Available Embryonic heart morphogenesis (EHM is a complex and dynamic process where the heart transforms from a single tube into a four-chambered pump. This process is of great biological and clinical interest but is still poorly understood for two main reasons. On the one hand, the existing imaging modalities for investigating EHM suffered from either limited penetration depth or limited spatial resolution. On the other hand, current works typically adopted manual segmentation, which was tedious, subjective, and time consuming considering the complexity of developing heart geometry and the large size of images. In this paper, we propose to utilize confocal microscopy imaging with tissue optical immersion clearing technique to image the heart at different stages of development for EHM study. The imaging method is able to produce high spatial resolution images and achieve large penetration depth at the same time. Furthermore, we propose a novel convex active contour model for automatic image segmentation. The model has the ability to deal with intensity fall-off in depth which is characterized by confocal microscopy images. We acquired the images of embryonic quail hearts from day 6 to day 14 of incubation for EHM study. The experimental results were promising and provided us with an insight view of early heart growth pattern and also paved the road for data-driven heart growth modeling.

  12. Robust supervised segmentation of neuropathology whole-slide microscopy images.

    Science.gov (United States)

    Vandenberghe, Michel E; Balbastre, Yaël; Souedet, Nicolas; Hérard, Anne-Sophie; Dhenain, Marc; Frouin, Frédérique; Delzescaux, Thierry

    2015-08-01

    Alzheimer's disease is characterized by brain pathological aggregates such as Aβ plaques and neurofibrillary tangles which trigger neuroinflammation and participate to neuronal loss. Quantification of these pathological markers on histological sections is widely performed to study the disease and to evaluate new therapies. However, segmentation of neuropathology images presents difficulties inherent to histology (presence of debris, tissue folding, non-specific staining) as well as specific challenges (sparse staining, irregular shape of the lesions). Here, we present a supervised classification approach for the robust pixel-level classification of large neuropathology whole slide images. We propose a weighted form of Random Forest in order to fit nonlinear decision boundaries that take into account class imbalance. Both color and texture descriptors were used as predictors and model selection was performed via a leave-one-image-out cross-validation scheme. Our method showed superior results compared to the current state of the art method when applied to the segmentation of Aβ plaques and neurofibrillary tangles in a human brain sample. Furthermore, using parallel computing, our approach easily scales-up to large gigabyte-sized images. To show this, we segmented a whole brain histology dataset of a mouse model of Alzheimer's disease. This demonstrates our method relevance as a routine tool for whole slide microscopy images analysis in clinical and preclinical research settings. PMID:26737134

  13. Understanding the phase contrast optics to restore artifact-free microscopy images for segmentation.

    Science.gov (United States)

    Yin, Zhaozheng; Kanade, Takeo; Chen, Mei

    2012-07-01

    Phase contrast, a noninvasive microscopy imaging technique, is widely used to capture time-lapse images to monitor the behavior of transparent cells without staining or altering them. Due to the optical principle, phase contrast microscopy images contain artifacts such as the halo and shade-off that hinder image segmentation, a critical step in automated microscopy image analysis. Rather than treating phase contrast microscopy images as general natural images and applying generic image processing techniques on them, we propose to study the optical properties of the phase contrast microscope to model its image formation process. The phase contrast imaging system can be approximated by a linear imaging model. Based on this model and input image properties, we formulate a regularized quadratic cost function to restore artifact-free phase contrast images that directly correspond to the specimen's optical path length. With artifacts removed, high quality segmentation can be achieved by simply thresholding the restored images. The imaging model and restoration method are quantitatively evaluated on microscopy image sequences with thousands of cells captured over several days. We also demonstrate that accurate restoration lays the foundation for high performance in cell detection and tracking. PMID:22386070

  14. Observation of posterior corneal vesicles with in vivo confocal microscopy and anterior segment OCT

    Directory of Open Access Journals (Sweden)

    Ryou Watanabe

    2010-10-01

    Full Text Available Ryou Watanabe, Toru Nakazawa, Nobuo FuseDepartment of Ophthalmology, Tohoku University Graduate School of Medicine, Sendai, JapanAbstract: The histopathology of posterior corneal vesicles (PCV has not yet been revealed. A 15-year-old girl, who was diagnosed by slit-lamp microscopy as PCV, was examined using specular microscopy, in vivo confocal microscopy, and anterior segment OCT (optical coherence tomography. Anterior segment OCT showed that the thickness of both corneas was within normal limits. At the same time, in vivo confocal microscopy revealed endothelial cells in the rounded dark areas, acellular hyporeflective layers on the Descemet’s membrane, and hyperreflective linear lesions. These findings were not reported previously by slit-lamp and specular microscopy. The abnormal findings only existed at the Descemet’s membrane and corneal endothelial layer. Previous reports dealing with posterior polymorphous dystrophy (PPMD examined using in vivo confocal microscopy reported almost the same findings, suggesting that PCV and PPMD may be the same at the microstructural level.Keywords: cornea, Descemet’s membrane, imaging

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

    Science.gov (United States)

    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. PMID:25465844

  16. Interactive cell segmentation based on phase contrast optics.

    Science.gov (United States)

    Su, Hang; Su, Zhou; Zheng, Shibao; Yang, Hua; Wei, Sha

    2014-01-01

    Cell segmentation in phase contrast microscopy images lays a crucial foundation for numerous subsequent computer-aided cell image analysis, but it encounters many unsolved challenges due to image qualities and artifacts caused by phase contrast optics. Addressing the unsolved challenges, the authors propose an interactive cell segmentation scheme over phase retardation features. After partitioning the images into phase homogeneous atoms, human annotations are propagated to unlabeled atoms over an affinity graph that is learned based on discrimination analysis. Then, an active query strategy is proposed for which the most informative unlabeled atom is selected for annotation, which is also propagated to the other unlabeled atoms. Cell segmentation converges to quality results after several rounds of interactions involving both the user's intentions and characteristics of image features. Experimental results demonstrate that cells with different optical properties are well segmented via the proposed approach.

  17. Atomic Force Microscopy Based Cell Shape Index

    Science.gov (United States)

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

    2013-03-01

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

  18. Microscopy Image Browser: A Platform for Segmentation and Analysis of Multidimensional Datasets.

    Directory of Open Access Journals (Sweden)

    Ilya Belevich

    2016-01-01

    Full Text Available Understanding the structure-function relationship of cells and organelles in their natural context requires multidimensional imaging. As techniques for multimodal 3-D imaging have become more accessible, effective processing, visualization, and analysis of large datasets are posing a bottleneck for the workflow. Here, we present a new software package for high-performance segmentation and image processing of multidimensional datasets that improves and facilitates the full utilization and quantitative analysis of acquired data, which is freely available from a dedicated website. The open-source environment enables modification and insertion of new plug-ins to customize the program for specific needs. We provide practical examples of program features used for processing, segmentation and analysis of light and electron microscopy datasets, and detailed tutorials to enable users to rapidly and thoroughly learn how to use the program.

  19. Liquid Cell Transmission Electron Microscopy

    Science.gov (United States)

    Liao, Hong-Gang; Zheng, Haimei

    2016-05-01

    Liquid cell transmission electron microscopy (TEM) has attracted significant interest in recent years. With nanofabricated liquid cells, it has been possible to image through liquids using TEM with subnanometer resolution, and many previously unseen materials dynamics have been revealed. Liquid cell TEM has been applied to many areas of research, ranging from chemistry to physics, materials science, and biology. So far, topics of study include nanoparticle growth and assembly, electrochemical deposition and lithiation for batteries, tracking and manipulation of nanoparticles, catalysis, and imaging of biological materials. In this article, we first review the development of liquid cell TEM and then highlight progress in various areas of research. In the study of nanoparticle growth, the electron beam can serve both as the illumination source for imaging and as the input energy for reactions. However, many other research topics require the control of electron beam effects to minimize electron beam damage. We discuss efforts to understand electron beam-liquid matter interactions. Finally, we provide a perspective on future challenges and opportunities in liquid cell TEM.

  20. Adaptive segmentation of nuclei in H&S stained tendon microscopy

    Science.gov (United States)

    Chuang, Bo-I.; Wu, Po-Ting; Hsu, Jian-Han; Jou, I.-Ming; Su, Fong-Chin; Sun, Yung-Nien

    2015-12-01

    Tendiopathy is a popular clinical issue in recent years. In most cases like trigger finger or tennis elbow, the pathology change can be observed under H and E stained tendon microscopy. However, the qualitative analysis is too subjective and thus the results heavily depend on the observers. We develop an automatic segmentation procedure which segments and counts the nuclei in H and E stained tendon microscopy fast and precisely. This procedure first determines the complexity of images and then segments the nuclei from the image. For the complex images, the proposed method adopts sampling-based thresholding to segment the nuclei. While for the simple images, the Laplacian-based thresholding is employed to re-segment the nuclei more accurately. In the experiments, the proposed method is compared with the experts outlined results. The nuclei number of proposed method is closed to the experts counted, and the processing time of proposed method is much faster than the experts'.

  1. 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

    CERN Document Server

    Wollman, Adam J M; Foster, Simon; Leake, Mark C

    2016-01-01

    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 morphological...

  2. Quantifying local heterogeneity of in vivo transport dynamics using stochastic scanning multiphoton multifocal microscopy and segmented spatiotemporal image correlation spectroscopy

    Science.gov (United States)

    Kim, Hee Y.; Jureller, Justin E.; Kuznetsov, Andrey; Philipson, Louis H.; Scherer, Norbert F.

    2008-02-01

    Elucidating the mechanisms of insulin granule trafficking in pancreatic β-cells is a critical step in understanding Type II Diabetes and abnormal insulin secretion. In this paper, rapid-sampling stochastic scanning multiphoton multifocal microscopy (SS-MMM) was developed to capture fast insulin granule dynamics in vivo. Stochastic scanning of (a diffractive optic generated) 10×10 hexagonal array of foci with a galvanometer yields a uniformly sampled image with fewer spatio-temporal artifacts than obtained by conventional or multibeam raster scanning. In addition, segmented spatio-temporal image correlation spectroscopy (Segmented STICS) was developed to extract dynamics of insulin granules from the image sequences. Measurements we conducted on MIN6 cells, which exhibit an order of magnitude lower granule number density, allow comparison of particle tracking with Segmented-STICS. Segmentation of the images into 8×8 pixel segments (similar to a size of one granule) allows some amount of spatial averaging, which can reduce the computation time required to calculate the correlation function, yet retains information about the local spatial heterogeneity of transport. This allows the correlation analysis to quantify the dynamics within each of the segments producing a "map" of the localized properties of the cell. The results obtained from Segmented STICS are compared with dynamics determined from particle tracking analysis of the same images. The resulting range of diffusion coefficients of insulin granules are comparable to previously published values indicating that SS-MMM and segmented- STICS will be useful to address the imaging challenges presented by β-cells, particularly the extremely large number density of granules.

  3. TRANSMISSION ELECTRON MICROSCOPY OF SEGMENTED POLYURETHANES WITH RUTHENIUM TETROXIDE AS A STAINING AGENT

    Institute of Scientific and Technical Information of China (English)

    XIAO Fengfei; CHEN Shouxi; JIN Yongze; SHI Lianghe; XU Mao

    1991-01-01

    Microphase separation and lamellar structure of segmented polyether- and polyester-polyurethanes have been investigated by means of transmission electron microscopy with the ruthenium tetroxide staining technique. The results show that the RuO4 staining technique is simpler and may give better image contrast than other staining methods for this polymer. Microphase separation and lamellar structure of segmented polyether- and polyester-polyurethanes were directly observed and discussed.

  4. Vessel segmentation analysis of ischemic stroke images acquired with photoacoustic microscopy

    Science.gov (United States)

    Soetikno, Brian; Hu, Song; Gonzales, Ernie; Zhong, Qiaonan; Maslov, Konstantin; Lee, Jin-Moo; Wang, Lihong V.

    2012-02-01

    We have applied optical-resolution photoacoustic microscopy (OR-PAM) for longitudinal monitoring of cerebral metabolism through the intact skull of mice before, during, and up to 72 hours after a 1-hour transient middle cerebral artery occlusion (tMCAO). The high spatial resolution of OR-PAM enabled us to develop vessel segmentation techniques for segment-wise analysis of cerebrovascular responses.

  5. Segmentation and Tracking of Neural Stem Cell

    Institute of Scientific and Technical Information of China (English)

    TANG Chun-ming; ZHAO Chun-hui; Ewert Bengtsson

    2005-01-01

    In order to understand the development of stem cells into specialized mature cells it is necessary to study the growth of cells in culture. For this purpose it is very useful to have an efficient computerized cell tracking system. In this paper a prototype system for tracking neural stem cells in a sequence of images is described. In order to get reliable tracking results it is important to have good and robust segmentation of the cells. To achieve this we have implemented three levels of segmentation. The primary level, applied to all frames, is based on fuzzy threshold and watershed segmentation of a fuzzy gray weighted distance transformed image.The second level, applied to difficult frames where the first algorithm seems to have failed, is based on a fast geometric active contour model based on the level set algorithm. Finally, the automatic segmentation result on the crucial first frame can be interactively inspected and corrected. Visual inspection and correction can also be applied to other frames but this is generally not needed. For the tracking all cells are classified into inactive, active, dividing and clustered cells. Different algorithms are used to deal with the different cell categories. A special backtracking step is used to automatically correct for some common errors that appear in the initial forward tracking process.

  6. Segmented cell testing for cathode parameter investigation

    Science.gov (United States)

    Tanasini, Pietro; Schuler, J. Andreas; Wuillemin, Zacharie; Ameur, Myriam L. Ben; Comninellis, Christos; Van herle, Jan

    The increasing quality and durability of solid oxide fuel cells (SOFCs) state-of-the-art materials renders the long-term testing of fuel cells difficult since considerably long equipment times are needed to obtain valuable results. Moreover, reproducibility issues are common due to the high sensitivity of the performance and degradation on the testing conditions. An original segmented cell configuration has been adopted in order to carry out four tests in parallel, thus decreasing the total experimental time and ensuring the same operating conditions for the four segments. The investigation has been performed on both anode-supported cells and symmetrical Lanthanum-Strontium Manganite-Yttria-stabilized Zirconia (LSM-YSZ) electrolyte-supported cells. In separate tests, the influence of variables like cathode thickness, current density and cathode composition on performance and degradation have been explored on anode-supported cells. Furthermore, the effect of chromium poisoning has been studied on electrolyte-supported symmetric cells by contacting one segment with a chromium-iron interconnect material. Long-term polarization of the segments is controlled with a multi-channel galvanostatic device designed in-house. Electrochemical characterization has been performed through electrochemical impedance spectroscopy (EIS) at different H 2 partial pressures, temperatures and bias current, effectively demonstrating the direct impact of each studied variable on the cell performance and degradation behavior. Segmented cell testing has been proven to be an effective strategy to achieve better reproducibility for SOFC measurements since it avoids the inevitable fluctuations found in a series of successively run tests. Moreover, simultaneous testing increased n-fold the data output per experiment, implying a considerable economy of time.

  7. Atomic force microscopy in cell biology

    Institute of Scientific and Technical Information of China (English)

    LU Zhexue; ZHANG Zhiling; PANG Daiwen

    2005-01-01

    The history, characteristic, operation modes and coupling techniques of atomic force microscopy (AFM) are introduced. Then the application in cell biology is reviewed in four aspects: cell immobilization methods, cell imaging, force spectrum study and cell manipulation. And the prospect of AFM application in cell biology is discussed.

  8. Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy.

    Science.gov (United States)

    Liv, Nalan; van Oosten Slingeland, Daan S B; Baudoin, Jean-Pierre; Kruit, Pieter; Piston, David W; Hoogenboom, Jacob P

    2016-01-26

    We present an approach toward dynamic nanoimaging: live fluorescence of cells encapsulated in a bionanoreactor is complemented with in situ scanning electron microscopy (SEM) on an integrated microscope. This allows us to take SEM snapshots on-demand, that is, at a specific location in time, at a desired region of interest, guided by the dynamic fluorescence imaging. We show that this approach enables direct visualization, with EM resolution, of the distribution of bioconjugated quantum dots on cellular extensions during uptake and internalization.

  9. An Automatic Indirect Immunofluorescence Cell Segmentation System

    Directory of Open Access Journals (Sweden)

    Yung-Kuan Chan

    2014-01-01

    Full Text Available Indirect immunofluorescence (IIF with HEp-2 cells has been used for the detection of antinuclear autoantibodies (ANA in systemic autoimmune diseases. The ANA testing allows us to scan a broad range of autoantibody entities and to describe them by distinct fluorescence patterns. Automatic inspection for fluorescence patterns in an IIF image can assist physicians, without relevant experience, in making correct diagnosis. How to segment the cells from an IIF image is essential in developing an automatic inspection system for ANA testing. This paper focuses on the cell detection and segmentation; an efficient method is proposed for automatically detecting the cells with fluorescence pattern in an IIF image. Cell culture is a process in which cells grow under control. Cell counting technology plays an important role in measuring the cell density in a culture tank. Moreover, assessing medium suitability, determining population doubling times, and monitoring cell growth in cultures all require a means of quantifying cell population. The proposed method also can be used to count the cells from an image taken under a fluorescence microscope.

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

    Science.gov (United States)

    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.

  11. Probe microscopy: Scanning below the cell surface

    Science.gov (United States)

    Sahin, Ozgur

    2008-08-01

    Conventional atomic force microscopy probes only the surface of specimens. A related technique called scanning near-field ultrasonic holography can now image nanoparticles buried below the surfaces of cells, which could prove useful in nanotoxicology.

  12. Cell reactions with biomaterials: the microscopies

    Directory of Open Access Journals (Sweden)

    Curtis A. S.G.

    2001-01-01

    Full Text Available The methods and results of optical microscopy that can be used to observe cell reactions to biomaterials are Interference Reflection Microscopy (IRM, Total Internal Reflection Fluorescence Microscopy (TIRFM, Surface Plasmon Resonance Microscopy (SPRM and Forster Resonance Energy Transfer Microscopy (FRETM and Standing Wave Fluorescence Microscopy. The last three are new developments, which have not yet been fully perfected. TIRFM and SPRM are evanescent wave methods. The physics of these methods depend upon optical phenomena at interfaces. All these methods give information on the dimensions of the gap between cell and the substratum to which it is adhering and thus are especially suited to work with biomaterials. IRM and FRETM can be used on opaque surfaces though image interpretation is especially difficult for IRM on a reflecting opaque surface. These methods are compared with several electron microscopical methods for studying cell adhesion to substrata. These methods all yield fairly consistent results and show that the cell to substratum distance on many materials is in the range 5 to 30 nm. The area of contact relative to the total projected area of the cell may vary from a few per cent to close to 100% depending on the cell type and substratum. These methods show that those discrete contact areas well known as focal contacts are frequently present. The results of FRETM suggest that the separation from the substratum even in a focal contact is about 5 nm.

  13. [Segmental testicular infarction in sickle cell anemia].

    Science.gov (United States)

    Mueller, F E

    2014-05-01

    Vascular occlusions are the clinical indicators of sickle cell disease and in urology they can lead to papillary necrosis, renal infarction or priapism. Segmental testicular infarction in patients with sickle cell disease is a rare event and only a few cases have been reported. We present a 25-year-old man with right testicular pain increasing over 3 days and sickle cell disease. Ultrasound of the right scrotum presented an inhomogeneous, mainly hypoechegenic mass with a hyperechogenic margin and no sign of blood flow. A partial orchiectomy was performed with total enucleation of the lesion, which was histologically diagnosed as benign hemorrhagic necrotic testicular tissue.

  14. Automatic segmentation and classification of mycobacterium tuberculosis with conventional light microscopy

    Science.gov (United States)

    Xu, Chao; Zhou, Dongxiang; Zhai, Yongping; Liu, Yunhui

    2015-12-01

    This paper realizes the automatic segmentation and classification of Mycobacterium tuberculosis with conventional light microscopy. First, the candidate bacillus objects are segmented by the marker-based watershed transform. The markers are obtained by an adaptive threshold segmentation based on the adaptive scale Gaussian filter. The scale of the Gaussian filter is determined according to the color model of the bacillus objects. Then the candidate objects are extracted integrally after region merging and contaminations elimination. Second, the shape features of the bacillus objects are characterized by the Hu moments, compactness, eccentricity, and roughness, which are used to classify the single, touching and non-bacillus objects. We evaluated the logistic regression, random forest, and intersection kernel support vector machines classifiers in classifying the bacillus objects respectively. Experimental results demonstrate that the proposed method yields to high robustness and accuracy. The logistic regression classifier performs best with an accuracy of 91.68%.

  15. Improved Methods for Fluorescence Microscopy Detection of Macromolecules at the Axon Initial Segment

    Science.gov (United States)

    Alshammari, Musaad A.; Alshammari, Tahani K.; Laezza, Fernanda

    2016-01-01

    The axonal initial segment (AIS) is the subcellular compartment required for initiation of the action potential in neurons. Scaffolding and regulatory proteins at the AIS cluster with ion channels ensuring the integrity of electrical signaling. Interference with the configuration of this protein network can lead to profound effects on neuronal polarity, excitability, cell-to-cell connectivity and brain circuit plasticity. As such, the ability to visualize AIS components with precision provides an invaluable opportunity for parsing out key molecular determinants of neuronal function. Fluorescence-based immunolabeling is a sensitive method for morphological and molecular characterization of fine structures in neurons. Yet, even when combined with confocal microscopy, detection of AIS elements with immunofluorescence has been limited by the loss of antigenicity caused by fixative materials. This technical barrier has posed significant limitations in detecting AIS components alone or in combination with other markers. Here, we designed improved protocols targeted to confocal immunofluorescence detection of the AIS marker fibroblast growth factor 14 (FGF14) in combination with the cytoskeletal-associated protein Ankyrin-G, the scaffolding protein βIV-spectrin, voltage-gated Na+ (Nav) channels (especially the Nav1.6 isoform) and critical cell type-specific neuronal markers such as parvalbumin, calbindin, and NeuN in the mouse brain. Notably, we demonstrate that intracardiac perfusion of animals with a commercially available solution containing 1% formaldehyde and 0.5% methanol, followed by brief fixation with cold acetone is an optimal and sensitive protocol for FGF14 and other AIS marker detection that guarantees excellent tissue integrity. With variations in the procedure, we also significantly improved the detection of Nav1.6, a Nav isoform known for its fixative-sensitivity. Overall, this study provides an ensemble of immunohistochemical recipes that permit

  16. Improved methods for fluorescence microscopy detection of macromolecules at the axon initial segment

    Directory of Open Access Journals (Sweden)

    Musaad A Alshammari

    2016-02-01

    Full Text Available The axonal initial segment (AIS is the subcellular compartment required for initiation of the action potential in neurons. Scaffolding and regulatory proteins at the AIS cluster with ion channels ensuring the integrity of electrical signaling. Interference with the configuration of this protein network can lead to profound effects on neuronal polarity, excitability, cell-to-cell connectivity and brain circuit plasticity. As such, the ability to visualize AIS components with precision provides an invaluable opportunity for parsing out key molecular determinants of neuronal function. Fluorescence-based immunolabeling is a sensitive method for morphological and molecular characterization of fine structures in neurons. Yet, even when combined with confocal microscopy, detection of AIS elements with immunofluorescence has been limited by the loss of antigenicity caused by fixative materials. This technical barrier has posed significant limitations in detecting AIS components alone or in combination with other markers. Here, we designed improved protocols targeted to confocal immunofluorescence detection of the AIS marker fibroblast growth factor 14 (FGF14 in combination with the cytoskeletal-associated protein Ankyrin-G, the scaffolding protein βIV-spectrin, voltage-gated Na+ (Nav channels (especially the Nav1.6 isoform and critical cell type-specific neuronal markers such as parvalbumin, calbindin, and NeuN in the mouse brain. Notably, we demonstrate that intracardiac perfusion of animals with a commercially available solution containing 1% formaldehyde and 0.5% methanol, followed by brief fixation with cold acetone is an optimal and sensitive protocol for FGF14 and other AIS marker detection that guarantees excellent tissue integrity. With variations in the procedure, we also significantly improved the detection of Nav1.6, a Nav isoform known for its fixative-sensitivity. Overall, this study provides an ensemble of immunohistochemical recipes that

  17. Improved Methods for Fluorescence Microscopy Detection of Macromolecules at the Axon Initial Segment.

    Science.gov (United States)

    Alshammari, Musaad A; Alshammari, Tahani K; Laezza, Fernanda

    2016-01-01

    The axonal initial segment (AIS) is the subcellular compartment required for initiation of the action potential in neurons. Scaffolding and regulatory proteins at the AIS cluster with ion channels ensuring the integrity of electrical signaling. Interference with the configuration of this protein network can lead to profound effects on neuronal polarity, excitability, cell-to-cell connectivity and brain circuit plasticity. As such, the ability to visualize AIS components with precision provides an invaluable opportunity for parsing out key molecular determinants of neuronal function. Fluorescence-based immunolabeling is a sensitive method for morphological and molecular characterization of fine structures in neurons. Yet, even when combined with confocal microscopy, detection of AIS elements with immunofluorescence has been limited by the loss of antigenicity caused by fixative materials. This technical barrier has posed significant limitations in detecting AIS components alone or in combination with other markers. Here, we designed improved protocols targeted to confocal immunofluorescence detection of the AIS marker fibroblast growth factor 14 (FGF14) in combination with the cytoskeletal-associated protein Ankyrin-G, the scaffolding protein βIV-spectrin, voltage-gated Na(+) (Nav) channels (especially the Nav1.6 isoform) and critical cell type-specific neuronal markers such as parvalbumin, calbindin, and NeuN in the mouse brain. Notably, we demonstrate that intracardiac perfusion of animals with a commercially available solution containing 1% formaldehyde and 0.5% methanol, followed by brief fixation with cold acetone is an optimal and sensitive protocol for FGF14 and other AIS marker detection that guarantees excellent tissue integrity. With variations in the procedure, we also significantly improved the detection of Nav1.6, a Nav isoform known for its fixative-sensitivity. Overall, this study provides an ensemble of immunohistochemical recipes that permit

  18. Bright-field cell image segmentation by principal component pursuit with an Ncut penalization

    Science.gov (United States)

    Chen, Yuehuan; Wan, Justin W. L.

    2015-03-01

    Segmentation of cells in time-lapse bright-field microscopic images is crucial in understanding cell behaviours for oncological research. However, the complex nature of the cells makes it difficult to segment cells accurately. Furthermore, poor contrast, broken cell boundaries and the halo artifact pose additional challenges to this problem. Standard segmentation techniques such as edged-based methods, watershed, or active contours result in poor segmentation. Other existing methods for bright-field images cannot provide good results without localized segmentation steps. In this paper, we present two robust mathematical models to segment bright-field cells automatically for the entire image. These models treat cell image segmentation as a background subtraction problem, which can be formulated as a Principal Component Pursuit (PCP) problem. Our first segmentation model is formulated as a PCP with nonnegative constraints. We exploit the sparse component of the PCP solution for identifying the cell pixels. However, there is no control on the quality of the sparse component and the nonzero entries can scatter all over the image, resulting in a noisy segmentation. The second model is an improvement of the first model by combining PCP with spectral clustering. Seemingly unrelated approaches, we combine the two techniques by incorporating normalized-cut in the PCP as a measure for the quality of the segmentation. These two models have been applied to a set of C2C12 cells obtained from bright-field microscopy. Experimental results demonstrate that the proposed models are effective in segmenting cells from bright-field images.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kwak, In Hae; Son, Minjun [Physics Department, University of Florida, P.O. Box 118440, Gainesville, FL 32611-8440 (United States); Hagen, Stephen J., E-mail: sjhagen@ufl.edu [Physics Department, University of Florida, P.O. Box 118440, Gainesville, FL 32611-8440 (United States)

    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 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.

  20. Quantification of plant cell coupling with live-cell microscopy

    DEFF Research Database (Denmark)

    Liesche, Johannes; Schulz, Alexander

    2015-01-01

    by confocal microscopy, loaded tracer is activated by UV illumination in a target cell and its spread to neighboring cells monitored. When combined with high-speed acquisition by resonant scanning or spinning disc confocal microscopy, the high signal-to-noise ratio of photoactivation allows collection...

  1. Quantification of Plant Cell Coupling with Live-Cell Microscopy

    DEFF Research Database (Denmark)

    Liesche, Johannes; Schulz, Alexander

    2015-01-01

    by confocal microscopy, loaded tracer is activated by UV illumination in a target cell and its spread to neighboring cells monitored. When combined with high-speed acquisition by resonant scanning or spinning disc confocal microscopy, the high signal-to-noise ratio of photoactivation allows collection...

  2. A method for the evaluation of thousands of automated 3D stem cell segmentations.

    Science.gov (United States)

    Bajcsy, P; Simon, M; Florczyk, S J; Simon, C G; Juba, D; Brady, M C

    2015-12-01

    There is no segmentation method that performs perfectly with any dataset in comparison to human segmentation. Evaluation procedures for segmentation algorithms become critical for their selection. The problems associated with segmentation performance evaluations and visual verification of segmentation results are exaggerated when dealing with thousands of three-dimensional (3D) image volumes because of the amount of computation and manual inputs needed. We address the problem of evaluating 3D segmentation performance when segmentation is applied to thousands of confocal microscopy images (z-stacks). Our approach is to incorporate experimental imaging and geometrical criteria, and map them into computationally efficient segmentation algorithms that can be applied to a very large number of z-stacks. This is an alternative approach to considering existing segmentation methods and evaluating most state-of-the-art algorithms. We designed a methodology for 3D segmentation performance characterization that consists of design, evaluation and verification steps. The characterization integrates manual inputs from projected surrogate 'ground truth' of statistically representative samples and from visual inspection into the evaluation. The novelty of the methodology lies in (1) designing candidate segmentation algorithms by mapping imaging and geometrical criteria into algorithmic steps, and constructing plausible segmentation algorithms with respect to the order of algorithmic steps and their parameters, (2) evaluating segmentation accuracy using samples drawn from probability distribution estimates of candidate segmentations and (3) minimizing human labour needed to create surrogate 'truth' by approximating z-stack segmentations with 2D contours from three orthogonal z-stack projections and by developing visual verification tools. We demonstrate the methodology by applying it to a dataset of 1253 mesenchymal stem cells. The cells reside on 10 different types of biomaterial

  3. In Vivo Confocal Microscopy and Anterior Segment Optic Coherence Tomography Findings in Ocular Ochronosis

    Directory of Open Access Journals (Sweden)

    Elif Demirkilinc Biler

    2015-01-01

    Full Text Available Purpose. To report clinical and in vivo confocal microscopy (IVCM findings of two patients with ocular ochronosis secondary due to alkaptonuria. Materials and Methods. Complete ophthalmologic examinations, including IVCM (HRT II/Rostock Cornea Module, Heidelberg, Germany, anterior segment optical coherence tomography (AS-OCT (Topcon 3D spectral-domain OCT 2000, Topcon Medical Systems, Paramus, NJ, USA, corneal topography (Pentacam, OCULUS Optikgeräte GmbH, Wetzlar, Germany, and anterior segment photography, were performed. Results. Biomicroscopic examination showed bilateral darkly pigmented lesions of the nasal and temporal conjunctiva and episclera in both patients. In vivo confocal microscopy of the lesions revealed prominent degenerative changes, including vacuoles and fragmentation of collagen fibers in the affected conjunctival lamina propria and episclera. Hyperreflective pigment granules in different shapes were demonstrated in the substantia propria beneath the basement membrane. AS-OCT of Case 1 demonstrated hyporeflective areas. Fundus examination was within normal limits in both patients, except tilted optic discs with peripapillary atrophy in one of the patients. Corneal topography, thickness, and macular OCT were normal bilaterally in both cases. Conclusion. The degenerative and anatomic changes due to ochronotic pigment deposition in alkaptonuria can be demonstrated in detail with IVCM and AS-OCT. Confocal microscopic analysis in ocular ochronosis may serve as a useful adjunct in diagnosis and monitoring of the disease progression.

  4. Minimum cell connection and separation in line segment arrangements

    CERN Document Server

    Alt, Helmut; Giannopoulos, Panos; Knauer, Christian

    2011-01-01

    We study the complexity of the following cell connection and separation problems in segment arrangements. Given a set of straight-line segments in the plane and two points a and b: (i) compute the minimum number of segments one needs to remove so that there is a path connecting a to b that does not intersect any of the remaining segments; (ii) compute the minimum number of segments one needs to remove so that the arrangement induced by the remaining segments has a single cell; (iii) compute the minimum number of segments one needs to retain so that any path connecting a to b intersects some of the retained segments. We show that problems (i) and (ii) are NP-hard, while problem (iii) is polynomial-time solvable. We also discuss special polynomial-time and fixed-parameter tractable cases.

  5. Nanonet Force Microscopy for Measuring Cell Forces.

    Science.gov (United States)

    Sheets, Kevin; Wang, Ji; Zhao, Wei; Kapania, Rakesh; Nain, Amrinder S

    2016-07-12

    The influence of physical forces exerted by or felt by cells on cell shape, migration, and cytoskeleton arrangement is now widely acknowledged and hypothesized to occur due to modulation of cellular inside-out forces in response to changes in the external fibrous environment (outside-in). Our previous work using the non-electrospinning Spinneret-based Tunable Engineered Parameters' suspended fibers has revealed that cells are able to sense and respond to changes in fiber curvature and structural stiffness as evidenced by alterations to focal adhesion cluster lengths. Here, we present the development and application of a suspended nanonet platform for measuring C2C12 mouse myoblast forces attached to fibers of three diameters (250, 400, and 800 nm) representing a wide range of structural stiffness (3-50 nN/μm). The nanonet force microscopy platform measures cell adhesion forces in response to symmetric and asymmetric external perturbation in single and cyclic modes. We find that contractility-based, inside-out forces are evenly distributed at the edges of the cell, and that forces are dependent on fiber structural stiffness. Additionally, external perturbation in symmetric and asymmetric modes biases cell-fiber failure location without affecting the outside-in forces of cell-fiber adhesion. We then extend the platform to measure forces of (1) cell-cell junctions, (2) single cells undergoing cyclic perturbation in the presence of drugs, and (3) cancerous single-cells transitioning from a blebbing to a pseudopodial morphology. PMID:27410747

  6. A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images.

    Science.gov (United States)

    Afshar, Yaser; Sbalzarini, Ivo F

    2016-01-01

    Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 10(10) pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments. PMID:27046144

  7. Spectro-Microscopy of Living Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Klaus Harter; Alfred J. Meixner; Frank Schleifenbaum

    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 lowexpressed 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 F(o)rster 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

  8. A combined watershed and level set method for segmentation of brightfield cell images

    Science.gov (United States)

    Tse, Shutong; Bradbury, Laura; Wan, Justin W. L.; Djambazian, Haig; Sladek, Robert; Hudson, Thomas

    2009-02-01

    Segmentation of brightfield cell images from microscopy is challenging in several ways. The contrast between cells and the background is low. Cells are usually surrounded by "halo", an optical artifact common in brightfield images. Also, cell divisions occur frequently, which raises the issue of topological change to segmentation. In this paper, we present a robust segmentation method based on the watershed and level set methods. Instead of heuristically locate where the initial markers for watershed should be, we apply a multiphase level set marker extraction to determine regions inside a cell. In contrast with the standard level set segmentation where only one level set function is used, we apply multiple level set functions (usually 3) to capture the different intensity levels in a cell image. This is particularly important to be able to distinguish regions of similar but different intensity levels in low contrast images. All the pixels obtained will be used as an initial marker for watershed. The region growing process of watershed will capture the rest of the cell until it hits the halo which serves as a "wall" to stop the expansion. By using these relatively large number of points as markers together with watershed, we show that the low contrast cell boundary can be captured correctly. Furthermore, we present a technique for watershed and level set to detect cell division automatically with no special human attention. Finally, we present segmentation results of C2C12 cells in brightfield images to illustrate the effectiveness of our method.

  9. Cell shape identification using digital holographic microscopy

    CERN Document Server

    Zakrisson, Johan; Andersson, Magnus

    2015-01-01

    We present a cost-effective, simple and fast digital holographic microscopy method based upon Rayleigh-Sommerfeld back propagation for identification of the geometrical shape of a cell. The method was tested using synthetic hologram images generated by ray-tracing software and from experimental images of semi-transparent spherical beads and living red blood cells. Our results show that by only using the real part of the back-reconstructed amplitude the proposed method can provide information of the geometrical shape of the object and at the same time accurately determine the axial position of the object under study. The proposed method can be used in flow chamber assays for pathophysiological studies where fast morphological changes of cells are studied in high numbers and at different heights.

  10. A new level set model for cell image segmentation

    Science.gov (United States)

    Ma, Jing-Feng; Hou, Kai; Bao, Shang-Lian; Chen, Chun

    2011-02-01

    In this paper we first determine three phases of cell images: background, cytoplasm and nucleolus according to the general physical characteristics of cell images, and then develop a variational model, based on these characteristics, to segment nucleolus and cytoplasm from their relatively complicated backgrounds. In the meantime, the preprocessing obtained information of cell images using the OTSU algorithm is used to initialize the level set function in the model, which can speed up the segmentation and present satisfactory results in cell image processing.

  11. A new level set model for cell image segmentation

    Institute of Scientific and Technical Information of China (English)

    Ma Jing-Feng; Hou Kai; Bao Shang-Lian; Chen Chun

    2011-01-01

    In this paper we first determine three phases of cell images: background, cytoplasm and nucleolus according to the general physical characteristics of cell images, and then develop a variational model, based on these characteristics, to segment nucleolus and cytoplasm from their relatively complicated backgrounds. In the meantime, the preprocessing obtained information of cell images using the OTSU algorithm is used to initialize the level set function in the model, which can speed up the segmentation and present satisfactory results in cell image processing.

  12. Rapid and Semi-Automated Extraction of Neuronal Cell Bodies and Nuclei from Electron Microscopy Image Stacks

    Science.gov (United States)

    Holcomb, Paul S.; Morehead, Michael; Doretto, Gianfranco; Chen, Peter; Berg, Stuart; Plaza, Stephen; Spirou, George

    2016-01-01

    Connectomics—the study of how neurons wire together in the brain—is at the forefront of modern neuroscience research. However, many connectomics studies are limited by the time and precision needed to correctly segment large volumes of electron microscopy (EM) image data. We present here a semi-automated segmentation pipeline using freely available software that can significantly decrease segmentation time for extracting both nuclei and cell bodies from EM image volumes. PMID:27259933

  13. Investigating cell mechanics with atomic force microscopy.

    Science.gov (United States)

    Haase, Kristina; Pelling, Andrew E

    2015-03-01

    Transmission of mechanical force is crucial for normal cell development and functioning. However, the process of mechanotransduction cannot be studied in isolation from cell mechanics. Thus, in order to understand how cells 'feel', we must first understand how they deform and recover from physical perturbations. Owing to its versatility, atomic force microscopy (AFM) has become a popular tool to study intrinsic cellular mechanical properties. Used to directly manipulate and examine whole and subcellular reactions, AFM allows for top-down and reconstitutive approaches to mechanical characterization. These studies show that the responses of cells and their components are complex, and largely depend on the magnitude and time scale of loading. In this review, we generally describe the mechanotransductive process through discussion of well-known mechanosensors. We then focus on discussion of recent examples where AFM is used to specifically probe the elastic and inelastic responses of single cells undergoing deformation. We present a brief overview of classical and current models often used to characterize observed cellular phenomena in response to force. Both simple mechanistic models and complex nonlinear models have been used to describe the observed cellular behaviours, however a unifying description of cell mechanics has not yet been resolved. PMID:25589563

  14. Nuclear microscopy of rat colon epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Ren, M., E-mail: phyrenmq@nus.edu.sg [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Rajendran, Reshmi [Lab of Molecular Imaging, Singapore Bioimaging Consotium, 11 Biopolis Way, 02-02 Helios, Singapore 138667 (Singapore); Ng, Mary [Department of Pharmacology, National University of Singapore (Singapore); Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Jenner, Andrew Michael [Illawara Health and Medical Research Institute (IHMRI), University of Wollongong, NSW 2522 (Australia)

    2011-10-15

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  15. Impact of image segmentation on high-content screening data quality for SK-BR-3 cells

    Directory of Open Access Journals (Sweden)

    Li Yizheng

    2007-09-01

    Full Text Available Abstract Background High content screening (HCS is a powerful method for the exploration of cellular signalling and morphology that is rapidly being adopted in cancer research. HCS uses automated microscopy to collect images of cultured cells. The images are subjected to segmentation algorithms to identify cellular structures and quantitate their morphology, for hundreds to millions of individual cells. However, image analysis may be imperfect, especially for "HCS-unfriendly" cell lines whose morphology is not well handled by current image segmentation algorithms. We asked if segmentation errors were common for a clinically relevant cell line, if such errors had measurable effects on the data, and if HCS data could be improved by automated identification of well-segmented cells. Results Cases of poor cell body segmentation occurred frequently for the SK-BR-3 cell line. We trained classifiers to identify SK-BR-3 cells that were well segmented. On an independent test set created by human review of cell images, our optimal support-vector machine classifier identified well-segmented cells with 81% accuracy. The dose responses of morphological features were measurably different in well- and poorly-segmented populations. Elimination of the poorly-segmented cell population increased the purity of DNA content distributions, while appropriately retaining biological heterogeneity, and simultaneously increasing our ability to resolve specific morphological changes in perturbed cells. Conclusion Image segmentation has a measurable impact on HCS data. The application of a multivariate shape-based filter to identify well-segmented cells improved HCS data quality for an HCS-unfriendly cell line, and could be a valuable post-processing step for some HCS datasets.

  16. Characterisation of cell-wall polysaccharides from mandarin segment membranes

    NARCIS (Netherlands)

    Coll-Almela, L.; Saura-Lopez, D.; Laencina-Sanchez, J.; Schols, H.A.; Voragen, A.G.J.; Ros-García, J.M.

    2015-01-01

    In an attempt to develop a process of enzymatic peeling of mandarin segments suitable for use on an industrial scale, the cell wall fraction of the segment membrane of Satsuma mandarin fruits was extracted to obtain a chelating agent-soluble pectin fraction (ChSS), a dilute sodium hydroxide-soluble

  17. Automated detection and segmentation of synaptic contacts in nearly isotropic serial electron microscopy images.

    Directory of Open Access Journals (Sweden)

    Anna Kreshuk

    Full Text Available We describe a protocol for fully automated detection and segmentation of asymmetric, presumed excitatory, synapses in serial electron microscopy images of the adult mammalian cerebral cortex, taken with the focused ion beam, scanning electron microscope (FIB/SEM. The procedure is based on interactive machine learning and only requires a few labeled synapses for training. The statistical learning is performed on geometrical features of 3D neighborhoods of each voxel and can fully exploit the high z-resolution of the data. On a quantitative validation dataset of 111 synapses in 409 images of 1948×1342 pixels with manual annotations by three independent experts the error rate of the algorithm was found to be comparable to that of the experts (0.92 recall at 0.89 precision. Our software offers a convenient interface for labeling the training data and the possibility to visualize and proofread the results in 3D. The source code, the test dataset and the ground truth annotation are freely available on the website http://www.ilastik.org/synapse-detection.

  18. Automated microscopy system for peripheral blood cells

    Science.gov (United States)

    Boev, Sergei F.; Sazonov, Vladimir V.; Kozinets, Gennady I.; Pogorelov, Valery M.; Gusev, Alexander A.; Korobova, Farida V.; Vinogradov, Alexander G.; Verdenskaya, Natalya V.; Ivanova, Irina A.

    2000-11-01

    The report describes the instrument ASPBS (Automated Screening of Peripheral Blood Cells) designed for an automated analysis of dry blood smears. The instrument is based on computer microscopy and uses dry blood smears prepared according to the standard Romanovskii-Giemza procedure. In comparison with the well-known flow cytometry systems, our instrument provides more detailed information and offers an opporunity of visualizing final results. The basic performances of the instrument are given. Software of this instrument is based on digital image processing and image recognition procedures. It is pointed out that the instrument can be used as a fairly universal tool in scientific research, public demonstrations, in medical treatment, and in medical education. The principle used as the basis of the instrument appeared adequate for creating an instrument version serviceable even during space flights where standard manual procedures and flow cytometry systems fail. The benefit of the use of the instrument in clinical laboratories is described.

  19. Automatic segmentation of HeLa cell images

    CERN Document Server

    Urban, Jan

    2011-01-01

    In this work, the possibilities for segmentation of cells from their background and each other in digital image were tested, combined and improoved. Lot of images with young, adult and mixture cells were able to prove the quality of described algorithms. Proper segmentation is one of the main task of image analysis and steps order differ from work to work, depending on input images. Reply for biologicaly given question was looking for in this work, including filtration, details emphasizing, segmentation and sphericity computing. Order of algorithms and way to searching for them was also described. Some questions and ideas for further work were mentioned in the conclusion part.

  20. Quantitative tracking of tumor cells in phase-contrast microscopy exploiting halo artifact pattern

    Science.gov (United States)

    Kang, Mi-Sun; Song, Soo-Min; Lee, Hana; Kim, Myoung-Hee

    2012-03-01

    Tumor cell morphology is closely related to its invasiveness characteristics and migratory behaviors. An invasive tumor cell has a highly irregular shape, whereas a spherical cell is non-metastatic. Thus, quantitative analysis of cell features is crucial to determine tumor malignancy or to test the efficacy of anticancer treatment. We use phase-contrast microscopy to analyze single cell morphology and to monitor its change because it enables observation of long-term activity of living cells without photobleaching and phototoxicity, which is common in other fluorescence-labeled microscopy. Despite this advantage, there are image-level drawbacks to phase-contrast microscopy, such as local light effect and contrast interference ring, among others. Thus, we first applied a local filter to compensate for non-uniform illumination. Then, we used intensity distribution information to detect the cell boundary. In phase-contrast microscopy images, the cell normally appears as a dark region surrounded by a bright halo. As the halo artifact around the cell body is minimal and has an asymmetric diffusion pattern, we calculated the cross-sectional plane that intersected the center of each cell and was orthogonal to the first principal axis. Then, we extracted the dark cell region by level set. However, a dense population of cultured cells still rendered single-cell analysis difficult. Finally, we measured roundness and size to classify tumor cells into malignant and benign groups. We validated segmentation accuracy by comparing our findings with manually obtained results.

  1. A quick guide to light microscopy in cell biology

    Science.gov (United States)

    Thorn, Kurt

    2016-01-01

    Light microscopy is a key tool in modern cell biology. Light microscopy has several features that make it ideally suited for imaging biology in living cells: the resolution is well-matched to the sizes of subcellular structures, a diverse range of available fluorescent probes makes it possible to mark proteins, organelles, and other structures for imaging, and the relatively nonperturbing nature of light means that living cells can be imaged for long periods of time to follow their dynamics. Here I provide a brief introduction to using light microscopy in cell biology, with particular emphasis on factors to be considered when starting microscopy experiments. PMID:26768859

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

    Science.gov (United States)

    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.

  3. Accurate Morphology Preserving Segmentation of Overlapping Cells based on Active Contours.

    Science.gov (United States)

    Molnar, Csaba; Jermyn, Ian H; Kato, Zoltan; Rahkama, Vesa; Östling, Päivi; Mikkonen, Piia; Pietiäinen, Vilja; Horvath, Peter

    2016-01-01

    The identification of fluorescently stained cell nuclei is the basis of cell detection, segmentation, and feature extraction in high content microscopy experiments. The nuclear morphology of single cells is also one of the essential indicators of phenotypic variation. However, the cells used in experiments can lose their contact inhibition, and can therefore pile up on top of each other, making the detection of single cells extremely challenging using current segmentation methods. The model we present here can detect cell nuclei and their morphology even in high-confluency cell cultures with many overlapping cell nuclei. We combine the "gas of near circles" active contour model, which favors circular shapes but allows slight variations around them, with a new data model. This captures a common property of many microscopic imaging techniques: the intensities from superposed nuclei are additive, so that two overlapping nuclei, for example, have a total intensity that is approximately double the intensity of a single nucleus. We demonstrate the power of our method on microscopic images of cells, comparing the results with those obtained from a widely used approach, and with manual image segmentations by experts. PMID:27561654

  4. UV laser mediated cell selective destruction by confocal microscopy

    Directory of Open Access Journals (Sweden)

    Giangrande Angela

    2008-04-01

    Full Text Available Abstract Analysis of cell-cell interactions, cell function and cell lineages greatly benefits selective destruction techniques, which, at present, rely on dedicated, high energy, pulsed lasers and are limited to cells that are detectable by conventional microscopy. We present here a high resolution/sensitivity technique based on confocal microscopy and relying on commonly used UV lasers. Coupling this technique with time-lapse enables the destruction and following of any cell(s in any pattern(s in living animals as well as in cell culture systems.

  5. Espina: A Tool for the Automated Segmentation and Counting of Synapses in Large Stacks of Electron Microscopy Images

    Science.gov (United States)

    Morales, Juan; Alonso-Nanclares, Lidia; Rodríguez, José-Rodrigo; DeFelipe, Javier; Rodríguez, Ángel; Merchán-Pérez, Ángel

    2011-01-01

    The synapses in the cerebral cortex can be classified into two main types, Gray's type I and type II, which correspond to asymmetric (mostly glutamatergic excitatory) and symmetric (inhibitory GABAergic) synapses, respectively. Hence, the quantification and identification of their different types and the proportions in which they are found, is extraordinarily important in terms of brain function. The ideal approach to calculate the number of synapses per unit volume is to analyze 3D samples reconstructed from serial sections. However, obtaining serial sections by transmission electron microscopy is an extremely time consuming and technically demanding task. Using focused ion beam/scanning electron microscope microscopy, we recently showed that virtually all synapses can be accurately identified as asymmetric or symmetric synapses when they are visualized, reconstructed, and quantified from large 3D tissue samples obtained in an automated manner. Nevertheless, the analysis, segmentation, and quantification of synapses is still a labor intensive procedure. Thus, novel solutions are currently necessary to deal with the large volume of data that is being generated by automated 3D electron microscopy. Accordingly, we have developed ESPINA, a software tool that performs the automated segmentation and counting of synapses in a reconstructed 3D volume of the cerebral cortex, and that greatly facilitates and accelerates these processes. PMID:21633491

  6. ESPINA: a tool for the automated segmentation and counting of synapses in large stacks of electron microscopy images

    Directory of Open Access Journals (Sweden)

    Juan eMorales

    2011-03-01

    Full Text Available The synapses in the cerebral cortex can be classified into two main types, Gray’s type I and type II, which correspond to asymmetric (mostly glutamatergic excitatory and symmetric (inhibitory GABAergic synapses, respectively. Hence, the quantification and identification of their different types and the proportions in which they are found, is extraordinarily important in terms of brain function. The ideal approach to calculate the number of synapses per unit volume is to analyze three-dimensional samples reconstructed from serial sections. However, obtaining serial sections by transmission electron microscopy is an extremely time consuming and technically demanding task. Using FIB/SEM microscopy, we recently showed that virtually all synapses can be accurately identified as asymmetric or symmetric synapses when they are visualized, reconstructed and quantified from large three-dimensional tissue samples obtained in an automated manner. Nevertheless, the analysis, segmentation and quantification of synapses is still a labor intensive procedure. Thus, novel solutions are currently necessary to deal with the large volume of data that is being generated by automated 3D electron microscopy. Accordingly, we have developed ESPINA, a software tool that performs the automated segmentation and counting of synapses in a reconstructed 3D volume of the cerebral cortex, and that greatly facilitates and accelerates these processes.

  7. Super-resolution Microscopy in Plant Cell Imaging.

    Science.gov (United States)

    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.

  8. Nuclear microscopy of sperm cell elemental structure

    International Nuclear Information System (INIS)

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility. (orig.)

  9. Active Appearance Segmentation for Intensity Inhomogeneity in Light Sheet Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Jensen, Casper Bo; Lyksborg, Mark; Hecksher-Sørensen, J.;

    2016-01-01

    -one-out approach on images with minimal imperfections where the left out images are corrupted by a simulated bias field and segmented using the AAM. Secondly we test the model on LSFM images with common acquisition problems. In both experiments the proposed approach outperforms the often used AAM implementation...

  10. Basal Cell Carcinoma in Type 2 Segmental Darier's Disease

    Directory of Open Access Journals (Sweden)

    Lynne Robertson

    2012-01-01

    Full Text Available Background. Darier's disease (DD, also known as Keratosis Follicularis or Darier-White disease, is a rare disorder of keratinization. DD can present as a generalized autosomal dominant condition as well as a localized or segmental postzygotic condition (Vázquez et al., 2002. Clinical features of DD include greasy, warty papules and plaques on seborrheic areas, dystrophic nails, palmo-plantar pits, and papules on the dorsum of the hands and feet. Objective. We report a case of basal cell carcinoma developing in a patient with type 2 segmental DD. Conclusion. According to the current literature, Type 2 segmental disease is a rare presentation of Darier's disease with only 8 previous cases reported to date. In addition, nonmelanoma skin cancer (NMSC arising from DD is rarely reported; however, there may be an association between DD and risk of carcinogenesis.

  11. Exploring Neural Cell Dynamics with Digital Holographic Microscopy

    KAUST Repository

    Marquet, Pierre

    2013-04-21

    In this talk, I will present how digital holographic microscopy, as a powerful quantitative phase technique, can non-invasively measure cell dynamics and especially resolve local neuronal network activity through simultaneous multiple site optical recording.

  12. Transmission electron microscopy and atomic force microscopy characterization of nickel deposition on bacterial cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Recently bacterial cells have become attractive biological templates for the fabrication of metal nano- structures or nanomaterials due to their inherent small size, various standard geometrical shapes and abundant source. In this paper, nickel-coated bacterial cells (gram-negative bacteria of Escherichia coli) were fabricated via electroless chemical plating. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) characterization results reveal evident morphological difference between bacterial cells before and after deposition with nickel. The bare cells with smooth surface presented transverse outspreading effect at mica surface. Great changes took place in surface roughness for those bacterial cells after metallization. A large number of nickel nanoparticles were observed to be equably distributed at bacterial surface after activation and subsequent metallization. Furthermore, ultra thin section analytic results validated the presence and uniformity of thin nickel coating at bacterial surface after metallization.

  13. Probing stem cell differentiation using atomic force microscopy

    Science.gov (United States)

    Liang, Xiaobin; Shi, Xuetao; Ostrovidov, Serge; Wu, Hongkai; Nakajima, Ken

    2016-03-01

    A real-time method using atomic force microscopy (AFM) was developed to probe stem cell differentiation by measuring the mechanical properties of cells and the extracellular matrix (ECM). The mechanical properties of stem cells and their ECMs can be used to clearly distinguish specific stem cell-differentiated lineages. It is clear that AFM is a facile and useful tool for monitoring the differentiation of stem cells in a non-invasive manner.

  14. Analysis of mixed cell cultures with quantitative digital holographic phase microscopy

    Science.gov (United States)

    Kemper, Björn; Wibbeling, Jana; Ketelhut, Steffi

    2014-05-01

    In order to study, for example, the influence of pharmaceuticals or pathogens on different cell types under identical measurement conditions and to analyze interactions between different cellular specimens a minimally-invasive quantitative observation of mixed cell cultures is of particular interest. Quantitative phase microscopy (QPM) provides high resolution detection of optical path length changes that is suitable for stain-free minimally-invasive live cell analysis. Due to low light intensities for object illumination, QPM minimizes the interaction with the sample and is in particular suitable for long term time-lapse investigations, e.g., for the detection of cell morphology alterations due to drugs and toxins. Furthermore, QPM has been demonstrated to be a versatile tool for the quantification of cellular growth, the extraction morphological parameters and cell motility. We studied the feasibility of QPM for the analysis of mixed cell cultures. It was explored if quantitative phase images provide sufficient information to distinguish between different cell types and to extract cell specific parameters. For the experiments quantitative phase imaging with digital holographic microscopy (DHM) was utilized. Mixed cell cultures with different types of human pancreatic tumor cells were observed with quantitative DHM phase contrast up to 35 h. The obtained series of quantitative phase images were evaluated by adapted algorithms for image segmentation. From the segmented images the cellular dry mass and the mean cell thickness were calculated and used in the further analysis as parameters to quantify the reliability the measurement principle. The obtained results demonstrate that it is possible to characterize the growth of cell types with different morphologies in a mixed cell culture separately by consideration of specimen size and cell thickness in the evaluation of quantitative DHM phase images.

  15. [Polar coordinates representation based leukocyte segmentation of microscopic cell images].

    Science.gov (United States)

    Gu, Guanghua; Cui, Dong; Hao, Lianwang

    2010-12-01

    We propose an algorithm for segmentation of the overlapped leukocyte in the microscopic cell image. The histogram of the saturation channel in the cell image is smoothed to obtain the meaningful global valley point by the fingerprint smoothing method, and then the nucleus can be segmented. A circular region, containing the entire regions of the leukocyte, is marked off according to the equivalent sectional radius of the nucleus. Then, the edge of the overlapped leukocyte is represented by polar coordinates. The overlapped region by the change of the polar angle of the edge pixels is determined, and the closed edge of the leukocyte integrating the gradient information of the overlapped region is reconstructed. Finally, the leukocyte is exactly extracted. The experimental results show that our method has good performance in terms of recall ratio, precision ratio and pixel error ratio. PMID:21374971

  16. Waveguide evanescent field fluorescence microscopy & its application in cell biology

    Science.gov (United States)

    Hassanzadeh, Abdollah

    There are many powerful microscopy technologies available for the investigation of bulk materials as well as for thin film samples. Nevertheless, for imaging an interface, especially live cells on a substrate and ultra thin-films, only Total Internal Reflection Fluorescence (TIRF) microscopy is available. This TIRF microscopy allows imaging without interference of the bulk. Various approaches are employed in fluorescence microscopy applications to restrict the excitation and detection of fluorophores to a thin region of the specimen. Elimination of background fluorescence from outside the focal plane can dramatically improve the signal-to-noise ratio, and consequently, the spatial resolution of the features or events of interest. TIRF microscopy is an evanescent field based microscopy. In this method, fluorescent dyes are only excited within an evanescent field: roughly within 100 nm above a glass coverslip. This will allow imaging surface and interfacial issues of the glass coverslip and an adjacent material. Waveguide evanescent field fluorescence (WEFF) microscopy is a new development for imaging cell-substrate interactions in real time and in vitro. It is an alternative to TIRF microscopy. In this method the light is coupled into a waveguide via an optical grating. The coupled light propagates as a waveguide mode and exhibits an evanescent field on top of the waveguide. This can be used as a surface-bound illumination source to excite fluorophores. This evanescent field serves as an extremely powerful tool for quality control of thin films, to study cell-substrate contacts, and investigating the effect of external agents and drugs on the cell-substrate interaction in real time and in vitro. This new method has been established and optimized to minimize non-uniformity, scattering and photo bleaching issues. Visualizing and quantifying of the cell-substrates and solid thin films have been carried out by WEFF microscopy. The images of the cell-substrate interface

  17. Segmental identity and cerebellar granule cell induction in rhombomere 1

    Directory of Open Access Journals (Sweden)

    Bell Esther

    2004-06-01

    Full Text Available Abstract Background Cerebellar granule cell precursors are specifically generated within the hindbrain segment, rhombomere 1, which is bounded rostrally by the midbrain/hindbrain isthmus and caudally by the boundary of the Hoxa2 expression domain. While graded signals from the isthmus have a demonstrable patterning role within this region, the significance of segmental identity for neuronal specification within rhombomere 1 is unexplored. We examined the response of granule cell precursors to the overexpression of Hoxa2, which normally determines patterns of development specific to the hindbrain. How much does the development of the cerebellum, a midbrain/hindbrain structure, reflect its neuromeric origin as a hindbrain segment? Results We show that a Gbx2-positive, Otx2-/Hoxa2-negative territory corresponding to rhombomere 1 forms prior to an identifiable isthmic organiser. Early global overexpression of Hoxa2 at embryonic day 0 has no effect on the expression of isthmic signalling molecules or the allocation of rhombomere 1 territory, but selectively results in the loss of granule cell markers at embryonic day 6 and the depletion of cell bodies from the external granule cell layer. By comparison the trochlear nucleus and locus coeruleus form normally in ventral rhombomere 1 under these conditions. Microsurgery, coupled with electroporation, to target Hoxa2 overexpression to rhombic lip precursors, reveals a profound, autonomous respecification of migration. Rhombic lip derivatives, normally destined to occupy the external granule cell layer, violate the cerebellar boundary to form a ventrolateral nucleus in a position comparable to that occupied by rhombic lip derived neurons in rhombomere 2. Conclusions Different overexpression strategies reveal that the recognition of migration cues by granule cell precursors is dependent on their identity as rhombomere 1 derivatives. Segmental patterning cues operate autonomously within the rhombic lip

  18. Segmentation, Reconstruction, and Analysis of Blood Thrombus Formation in 3D 2-Photon Microscopy Images

    Directory of Open Access Journals (Sweden)

    Xu Zhiliang

    2010-01-01

    Full Text Available We study the problem of segmenting, reconstructing, and analyzing the structure growth of thrombi (clots in blood vessels in vivo based on 2-photon microscopic image data. First, we develop an algorithm for segmenting clots in 3D microscopic images based on density-based clustering and methods for dealing with imaging artifacts. Next, we apply the union-of-balls (or alpha-shape algorithm to reconstruct the boundary of clots in 3D. Finally, we perform experimental studies and analysis on the reconstructed clots and obtain quantitative data of thrombus growth and structures. We conduct experiments on laser-induced injuries in vessels of two types of mice (the wild type and the type with low levels of coagulation factor VII and analyze and compare the developing clot structures based on their reconstructed clots from image data. The results we obtain are of biomedical significance. Our quantitative analysis of the clot composition leads to better understanding of the thrombus development, and is valuable to the modeling and verification of computational simulation of thrombogenesis.

  19. Helium Ion Microscopy Visualizes Lipid Nanodomains in Mammalian Cells.

    Science.gov (United States)

    Schürmann, Matthias; Frese, Natalie; Beyer, André; Heimann, Peter; Widera, Darius; Mönkemöller, Viola; Huser, Thomas; Kaltschmidt, Barbara; Kaltschmidt, Christian; Gölzhäuser, Armin

    2015-11-18

    Cell membranes are composed of 2D bilayers of amphipathic lipids, which allow a lateral movement of the respective membrane components. These components are arranged in an inhomogeneous manner as transient micro- and nanodomains, which are believed to be crucially involved in the regulation of signal transduction pathways in mammalian cells. Because of their small size (diameter 10-200 nm), membrane nanodomains cannot be directly imaged using conventional light microscopy. Here, direct visualization of cell membrane nanodomains by helium ion microscopy (HIM) is presented. It is shown that HIM is capable to image biological specimens without any conductive coating and that HIM images clearly allow the identification of nanodomains in the ultrastructure of membranes with 1.5 nm resolution. The shape of these nanodomains is preserved by fixation of the surrounding unsaturated fatty acids while saturated fatty acids inside the nanodomains are selectively removed. Atomic force microscopy, fluorescence microscopy, 3D structured illumination microscopy, and direct stochastic optical reconstruction microscopy provide additional evidence that the structures in the HIM images of cell membranes originate from membrane nanodomains. The nanodomains observed by HIM have an average diameter of 20 nm and are densely arranged with a minimal nearest neighbor distance of ≈ 15 nm.

  20. Helium Ion Microscopy Visualizes Lipid Nanodomains in Mammalian Cells.

    Science.gov (United States)

    Schürmann, Matthias; Frese, Natalie; Beyer, André; Heimann, Peter; Widera, Darius; Mönkemöller, Viola; Huser, Thomas; Kaltschmidt, Barbara; Kaltschmidt, Christian; Gölzhäuser, Armin

    2015-11-18

    Cell membranes are composed of 2D bilayers of amphipathic lipids, which allow a lateral movement of the respective membrane components. These components are arranged in an inhomogeneous manner as transient micro- and nanodomains, which are believed to be crucially involved in the regulation of signal transduction pathways in mammalian cells. Because of their small size (diameter 10-200 nm), membrane nanodomains cannot be directly imaged using conventional light microscopy. Here, direct visualization of cell membrane nanodomains by helium ion microscopy (HIM) is presented. It is shown that HIM is capable to image biological specimens without any conductive coating and that HIM images clearly allow the identification of nanodomains in the ultrastructure of membranes with 1.5 nm resolution. The shape of these nanodomains is preserved by fixation of the surrounding unsaturated fatty acids while saturated fatty acids inside the nanodomains are selectively removed. Atomic force microscopy, fluorescence microscopy, 3D structured illumination microscopy, and direct stochastic optical reconstruction microscopy provide additional evidence that the structures in the HIM images of cell membranes originate from membrane nanodomains. The nanodomains observed by HIM have an average diameter of 20 nm and are densely arranged with a minimal nearest neighbor distance of ≈ 15 nm. PMID:26436577

  1. Osteoblast Adhesion of Breast Cancer Cells with Scanning Acoustic Microscopy

    Science.gov (United States)

    Miyasaka, C.; Mercer, R. R.; Mastro, A. M.

    Conditioned medium was collected from a bone-metastatic breast cancer cell line, MDA-MB-231, and cultured with an immature osteoblast cell line, MC3T3-E1. Under these conditions the osteoblasts acquired a changed morphology and appeared to adhere in a different way to the substrate and to each other. To characterize cellular adhesion, MC3T3-E1 osteoblasts were cultured with or without MDA-MB-231 conditioned medium for two days. With mechanical scanning acoustic reflection microscopy, we were able to detect a change in the adhesive condition of the interface between the cell and the substrate, but not with optical microscopy

  2. Exploring neural cell dynamics with digital holographic microscopy

    KAUST Repository

    Marquet, Pierre

    2013-07-11

    In this review, we summarize how the new concept of digital optics applied to the field of holographic microscopy has allowed the development of a reliable and flexible digital holographic quantitative phase microscopy (DH-QPM) technique at the nanoscale particularly suitable for cell imaging. Particular emphasis is placed on the original biological ormation provided by the quantitative phase signal. We present the most relevant DH-QPM applications in the field of cell biology, including automated cell counts, recognition, classification, three-dimensional tracking, discrimination between physiological and pathophysiological states, and the study of cell membrane fluctuations at the nanoscale. In the last part, original results show how DH-QPM can address two important issues in the field of neurobiology, namely, multiple-site optical recording of neuronal activity and noninvasive visualization of dendritic spine dynamics resulting from a full digital holographic microscopy tomographic approach. Copyright © 2013 by Annual Reviews.

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

    Science.gov (United States)

    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.

  4. RBCs and Parasites Segmentation from Thin Smear Blood Cell Images

    Directory of Open Access Journals (Sweden)

    Vishal V. Panchbhai

    2012-09-01

    Full Text Available Manually examine the blood smear for the detection of malaria parasite consumes lot of time for trend pathologists. As the computational power increases, the role of automatic visual inspection becomes more important. An automated system is therefore needed to complete as much work as possible for the identification of malaria parasites. The given scheme based on used of RGB color space, G layer processing, and segmentation of Red Blood Cells (RBC as well as cell parasites by auto-thresholding with offset value and use of morphological processing. The work compare with the manual results obtained from the pathology lab, based on total RBC count and cells parasite count. The designed system successfully detects malaria parasites and RBC cells in thin smear image.

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

    Science.gov (United States)

    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. PMID:24037521

  6. Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM

    Energy Technology Data Exchange (ETDEWEB)

    Hirano, Kazumi; Kinoshita, Takaaki [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577 (Japan); Uemura, Takeshi [Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Molecular and Cellular Physiology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Motohashi, Hozumi [Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-cho, Aoba-ku, Sendai 980-8575 (Japan); Watanabe, Yohei; Ebihara, Tatsuhiko [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Nishiyama, Hidetoshi [JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558 (Japan); Sato, Mari [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Suga, Mitsuo [JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558 (Japan); Maruyama, Yuusuke; Tsuji, Noriko M. [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Yamamoto, Masayuki [Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575 (Japan); Nishihara, Shoko, E-mail: shoko@soka.ac.jp [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577 (Japan); Sato, Chikara, E-mail: ti-sato@aist.go.jp [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan)

    2014-08-01

    Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future. - Highlights: • In situ correlative light electron microscopy of samples in open solution by ASEM. • Primary cultures for in-solution CLEM by developing SiN-film coating methods • First visualization of fluorescent magnetic beads in aqueous solution by CLEM. • Presynaptic induction of neurons by GluRδ2-N-terminus-coated beads studied by CLEM. • Axonal partitioning, bacterial phagocytosis, platelet formation imaged by CLEM.

  7. Analysis of Immunolabeled Cells by Atomic Force Microscopy, Optical Microscopy, and Flow Cytometry

    OpenAIRE

    Neagu, C.; Werf, van der, W.; Putman, C.A.J.; Kraan, Y.M.; Grooth, de, B.G.; Hulst, van der, R.W.M.; Greve, J de

    1994-01-01

    In this study we investigated the applicability of the (silver- enhanced) immunogold labeling method for atomic force microscopy. Human lymphocytes were labeled with anti-CD3 conjugated to fluorescein isothiocyanate and a secondary antibody (goat anti-mouse) linked with 1- or 30-nm colloidal gold particles. Silver enhancement was applied o­n these labeled cells to increase the size of the labels. In a setup combining an inverted optical microscope and a stand-alone atomic force microscope, a ...

  8. Automated Segmentation of Skin Strata in Reflectance Confocal Microscopy Depth Stacks.

    Science.gov (United States)

    Hames, Samuel C; Ardigò, Marco; Soyer, H Peter; Bradley, Andrew P; Prow, Tarl W

    2016-01-01

    Reflectance confocal microscopy (RCM) is a powerful tool for in-vivo examination of a variety of skin diseases. However, current use of RCM depends on qualitative examination by a human expert to look for specific features in the different strata of the skin. Developing approaches to quantify features in RCM imagery requires an automated understanding of what anatomical strata is present in a given en-face section. This work presents an automated approach using a bag of features approach to represent en-face sections and a logistic regression classifier to classify sections into one of four classes (stratum corneum, viable epidermis, dermal-epidermal junction and papillary dermis). This approach was developed and tested using a dataset of 308 depth stacks from 54 volunteers in two age groups (20-30 and 50-70 years of age). The classification accuracy on the test set was 85.6%. The mean absolute error in determining the interface depth for each of the stratum corneum/viable epidermis, viable epidermis/dermal-epidermal junction and dermal-epidermal junction/papillary dermis interfaces were 3.1 μm, 6.0 μm and 5.5 μm respectively. The probabilities predicted by the classifier in the test set showed that the classifier learned an effective model of the anatomy of human skin. PMID:27088865

  9. Automated Segmentation of Skin Strata in Reflectance Confocal Microscopy Depth Stacks

    Science.gov (United States)

    Hames, Samuel C.; Ardigò, Marco; Soyer, H. Peter; Bradley, Andrew P.; Prow, Tarl W.

    2016-01-01

    Reflectance confocal microscopy (RCM) is a powerful tool for in-vivo examination of a variety of skin diseases. However, current use of RCM depends on qualitative examination by a human expert to look for specific features in the different strata of the skin. Developing approaches to quantify features in RCM imagery requires an automated understanding of what anatomical strata is present in a given en-face section. This work presents an automated approach using a bag of features approach to represent en-face sections and a logistic regression classifier to classify sections into one of four classes (stratum corneum, viable epidermis, dermal-epidermal junction and papillary dermis). This approach was developed and tested using a dataset of 308 depth stacks from 54 volunteers in two age groups (20–30 and 50–70 years of age). The classification accuracy on the test set was 85.6%. The mean absolute error in determining the interface depth for each of the stratum corneum/viable epidermis, viable epidermis/dermal-epidermal junction and dermal-epidermal junction/papillary dermis interfaces were 3.1 μm, 6.0 μm and 5.5 μm respectively. The probabilities predicted by the classifier in the test set showed that the classifier learned an effective model of the anatomy of human skin. PMID:27088865

  10. Automated Segmentation of Skin Strata in Reflectance Confocal Microscopy Depth Stacks.

    Directory of Open Access Journals (Sweden)

    Samuel C Hames

    Full Text Available Reflectance confocal microscopy (RCM is a powerful tool for in-vivo examination of a variety of skin diseases. However, current use of RCM depends on qualitative examination by a human expert to look for specific features in the different strata of the skin. Developing approaches to quantify features in RCM imagery requires an automated understanding of what anatomical strata is present in a given en-face section. This work presents an automated approach using a bag of features approach to represent en-face sections and a logistic regression classifier to classify sections into one of four classes (stratum corneum, viable epidermis, dermal-epidermal junction and papillary dermis. This approach was developed and tested using a dataset of 308 depth stacks from 54 volunteers in two age groups (20-30 and 50-70 years of age. The classification accuracy on the test set was 85.6%. The mean absolute error in determining the interface depth for each of the stratum corneum/viable epidermis, viable epidermis/dermal-epidermal junction and dermal-epidermal junction/papillary dermis interfaces were 3.1 μm, 6.0 μm and 5.5 μm respectively. The probabilities predicted by the classifier in the test set showed that the classifier learned an effective model of the anatomy of human skin.

  11. ANALYSIS OF ENDOPLASMIC RETICULUM OF TOBACCO CELLS USING CONFOCAL MICROSCOPY

    Directory of Open Access Journals (Sweden)

    Barbora Radochová

    2011-05-01

    Full Text Available Image analysis techniques for preprocessing, segmentation and estimation of geometrical characteristics of fiber-like structures from 2-D or 3-D images captured by a confocal microscope are presented. Methods are demonstrated on fiber-like biological structure: endoplasmic reticulum (ER of tobacco cells. In the presented analysis of 2-D images of ER before and after the treatment of latrunculin B, ER and ER tubules were segmented and the area density of ER as well as the length density of ER tubules in the cell cortical layer were estimated by automatic image analysis algorithms. Images of 3-D arrangement of ER were reconstructed and rendered by various visualization techniques.

  12. Immunogold labels: cell-surface markers in atomic force microscopy

    NARCIS (Netherlands)

    Putman, Constant A.J.; Grooth, de Bart G.; Hansma, Paul K.; Hulst, van Niek F.; Greve, Jan

    1993-01-01

    The feasibility of using immunogold labels as cell-surface markers in atomic force microscopy is shown in this paper. The atomic force microscope (AFM) was used to image the surface of immunogold-labeled human lymphocytes. The lymphocytes were isolated from whole blood and labeled by an indirect imm

  13. Automatic choroid cells segmentation and counting based on approximate convexity and concavity of chain code in fluorescence microscopic image

    Science.gov (United States)

    Lu, Weihua; Chen, Xinjian; Zhu, Weifang; Yang, Lei; Cao, Zhaoyuan; Chen, Haoyu

    2015-03-01

    In this paper, we proposed a method based on the Freeman chain code to segment and count rhesus choroid-retinal vascular endothelial cells (RF/6A) automatically for fluorescence microscopy images. The proposed method consists of four main steps. First, a threshold filter and morphological transform were applied to reduce the noise. Second, the boundary information was used to generate the Freeman chain codes. Third, the concave points were found based on the relationship between the difference of the chain code and the curvature. Finally, cells segmentation and counting were completed based on the characteristics of the number of the concave points, the area and shape of the cells. The proposed method was tested on 100 fluorescence microscopic cell images, and the average true positive rate (TPR) is 98.13% and the average false positive rate (FPR) is 4.47%, respectively. The preliminary results showed the feasibility and efficiency of the proposed method.

  14. Migratory behaviour of tumour cells: a scanning electron microscopy study

    Directory of Open Access Journals (Sweden)

    Giuseppina Bozzuto

    2015-06-01

    Full Text Available BACKGROUND: Tumour cells utilize different migration strategies to invade surrounding tissues and elude anticancer treatments. It is therefore important to understand the mechanisms underlying migration process, in order to aid the development of therapies aimed at blocking the dissemination of cancer cells. AIMS: In this study tumour cell lines of different histological origin were analysed by combining 2D and 3D in vitro assays, biochemical tests and high resolution imaging by scanning electron microscopy (SEM in order to look insight strategies adopted by tumour cells to invade extracellular matrix. RESULTS: Quantitative (computer-assisted colour camera equipped-light microscopy and qualitative analysis (SEM indicated that the most aggressive tumour cells adopt an "individual" behaviour. The analysis of intracellular signalling demonstrated that the highest invasive potential was associated with the activation of AKT, ERK, FAK and ERM proteins. The "individual" behaviour was positively related to the expression of VLA-2 and inversely related with the E-cadherin expression. CONCLUSIONS: The combination of 2D and 3D in vitro assays, biochemical tests and ultrastructural investigations proved to be a suitable test for the investigation of tumour cell migration and invasion. The high resolution imaging by SEM highlighted the interrelationships between cells in different migratory behaviours of tumour cells.

  15. Raman microscopy of individual living human embryonic stem cells

    DEFF Research Database (Denmark)

    Novikov, Sergey M.; Beermann, Jonas; Bozhevolnyi, Sergey I.;

    2010-01-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing...... cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal...

  16. High resolution scanning electron microscopy of cells using dielectrophoresis.

    Directory of Open Access Journals (Sweden)

    Shi-Yang Tang

    Full Text Available Ultrastructural analysis of cells can reveal valuable information about their morphological, physiological, and biochemical characteristics. Scanning electron microscopy (SEM has been widely used to provide high-resolution images from the surface of biological samples. However, samples need to be dehydrated and coated with conductive materials for SEM imaging. Besides, immobilizing non-adherent cells during processing and analysis is challenging and requires complex fixation protocols. In this work, we developed a novel dielectrophoresis based microfluidic platform for interfacing non-adherent cells with high-resolution SEM at low vacuum mode. The system enables rapid immobilization and dehydration of samples without deposition of chemical residues over the cell surface. Moreover, it enables the on-chip chemical stimulation and fixation of immobilized cells with minimum dislodgement. These advantages were demonstrated for comparing the morphological changes of non-budding and budding yeast cells following Lyticase treatment.

  17. Quantitative aspects of digital microscopy applied to cellular localization of heparin in smooth muscle cells

    Science.gov (United States)

    Johnston, Richard F.; Hanzel, David K.; Stack, Bob; Brandley, Brian; Castellot, John

    1995-04-01

    High Resolution digital acquisition allows a great deal of flexibility in the types of questions that can be directed to microscopic samples. To eliminate subjective bias and provide quantitative results we have approached microscopy with an automated digital format. This mode can return quantitative data at high resolution over large fields. The digital format makes accessible data including [data segmentation]: multispectral colocalization, seeding and connectivity, particle size and shape distribution and population analysis. We have begun a program to investigate this approach using the confocal microscope. Scanning larger fields-of-view at lower spatial resolutions (e.g., low magnification objective) defines large maps that allow alignment of high spatial resolution (diffraction limited) sampling. The [objective] selection of the field-of-view with low spatial resolution reduces the subjective nature of the selection of a 'typical staining pattern'. High resolution digital scanning in three dimensions contribute both to the 'objective' nature of the analysis and allow for quantitation of characteristics not historically available/accessible. The complex carbohydrate heparin is implicated in tumor growth and wound healing by affecting angiogenesis, cell proliferation and motility. The internal localization of heparin within vascular cells appears to be a good predictor of the sensitivity of those cells to the action of heparin. Cells resistant to the antiproliferative action of heparin are able to sequester the heparin in large vacuoles whereas those cells sensitive to the carbohydrate do not exhibit these structures. We have applied our approach to QUANTITATIVE DIGITAL MICROSCOPY to the analysis of intracellular heparin distribution.

  18. Immunogold labels: cell-surface markers in atomic force microscopy

    OpenAIRE

    Putman, Constant A.J.; Grooth, de, B.G.; Hansma, Paul K.; Hulst, van der, R.W.M.; Greve, Jan

    1993-01-01

    The feasibility of using immunogold labels as cell-surface markers in atomic force microscopy is shown in this paper. The atomic force microscope (AFM) was used to image the surface of immunogold-labeled human lymphocytes. The lymphocytes were isolated from whole blood and labeled by an indirect immunolabeling method using the monoclonal antibody anti-CD3 and a secondary antibody (Goat-anti-Mouse) linked to 30 nm colloidal gold particles. Some of the samples were enhanced by silver deposition...

  19. Nanomechanics of Cells and Biomaterials Studied by Atomic Force Microscopy.

    Science.gov (United States)

    Kilpatrick, Jason I; Revenko, Irène; Rodriguez, Brian J

    2015-11-18

    The behavior and mechanical properties of cells are strongly dependent on the biochemical and biomechanical properties of their microenvironment. Thus, understanding the mechanical properties of cells, extracellular matrices, and biomaterials is key to understanding cell function and to develop new materials with tailored mechanical properties for tissue engineering and regenerative medicine applications. Atomic force microscopy (AFM) has emerged as an indispensable technique for measuring the mechanical properties of biomaterials and cells with high spatial resolution and force sensitivity within physiologically relevant environments and timescales in the kPa to GPa elastic modulus range. The growing interest in this field of bionanomechanics has been accompanied by an expanding array of models to describe the complexity of indentation of hierarchical biological samples. Furthermore, the integration of AFM with optical microscopy techniques has further opened the door to a wide range of mechanotransduction studies. In recent years, new multidimensional and multiharmonic AFM approaches for mapping mechanical properties have been developed, which allow the rapid determination of, for example, cell elasticity. This Progress Report provides an introduction and practical guide to making AFM-based nanomechanical measurements of cells and surfaces for tissue engineering applications.

  20. Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

    Directory of Open Access Journals (Sweden)

    Kazuo Yamagata

    Full Text Available Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.

  1. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    Science.gov (United States)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  2. Mechanical property quantification of endothelial cells using scanning acoustic microscopy

    Science.gov (United States)

    Shelke, A.; Brand, S.; Kundu, T.; Bereiter-Hahn, J.; Blase, C.

    2012-04-01

    The mechanical properties of cells reflect dynamic changes of cellular organization which occur during physiologic activities like cell movement, cell volume regulation or cell division. Thus the study of cell mechanical properties can yield important information for understanding these physiologic activities. Endothelial cells form the thin inner lining of blood vessels in the cardiovascular system and are thus exposed to shear stress as well as tensile stress caused by the pulsatile blood flow. Endothelial dysfunction might occur due to reduced resistance to mechanical stress and is an initial step in the development of cardiovascular disease like, e.g., atherosclerosis. Therefore we investigated the mechanical properties of primary human endothelial cells (HUVEC) of different age using scanning acoustic microscopy at 1.2 GHz. The HUVECs are classified as young (tD 90 h) cells depending upon the generation time for the population doubling of the culture (tD). Longitudinal sound velocity and geometrical properties of cells (thickness) were determined using the material signature curve V(z) method for variable culture condition along spatial coordinates. The plane wave technique with normal incidence is assumed to solve two-dimensional wave equation. The size of the cells is modeled using multilayered (solid-fluid) system. The propagation of transversal wave and surface acoustic wave are neglected in soft matter analysis. The biomechanical properties of HUVEC cells are quantified in an age dependent manner.

  3. Zero Crossing Edge Detection and Contour Tracing for Segmentation of Cervical Cell Nucleus .

    Directory of Open Access Journals (Sweden)

    B.V. Ramesh

    1993-07-01

    Full Text Available To automate the process of screening of normal and abnormal cervical cells, there is a need for automatic segmentation of the nucleus of these cells. This paper presents an algorithm using the Laplacian of Gaussian operator and contour tracer to segment the nucleus from the background. The algorithm has been tested on different kinds of images of cervical cells.

  4. Automatic detection of cell divisions (mitosis) in live-imaging microscopy images using Convolutional Neural Networks.

    Science.gov (United States)

    Shkolyar, Anat; Gefen, Amit; Benayahu, Dafna; Greenspan, Hayit

    2015-08-01

    We propose a semi-automated pipeline for the detection of possible cell divisions in live-imaging microscopy and the classification of these mitosis candidates using a Convolutional Neural Network (CNN). We use time-lapse images of NIH3T3 scratch assay cultures, extract patches around bright candidate regions that then undergo segmentation and binarization, followed by a classification of the binary patches into either containing or not containing cell division. The classification is performed by training a Convolutional Neural Network on a specially constructed database. We show strong results of AUC = 0.91 and F-score = 0.89, competitive with state-of-the-art methods in this field.

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

    Directory of Open Access Journals (Sweden)

    Salmon Patrick

    2010-07-01

    Full Text Available Abstract Background 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. Results 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. Conclusions 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

  6. Electron microscopy study of antioxidant interaction with bacterial cells

    Science.gov (United States)

    Plotnikov, Oleg P.; Novikova, Olga V.; Konnov, Nikolai P.; Korsukov, Vladimir N.; Gunkin, Ivan F.; Volkov, Uryi P.

    2000-10-01

    To maintain native microorganisms genotype and phenotype features a lyophylization technique is widely used. However in this case cells are affected by influences of vacuum and low temperature that cause a part of the cells population to be destruction. Another factor reduced microorganisms vitality is formation of reactive oxygen forms that damage certain biological targets (such as DNA, membranes etc.) Recently to raise microorganism's resistance against adverse condition natural and synthetic antioxidants are used. Antioxidant- are antagonists of free radicals. Introduction of antioxidants in protective medium for lyophylization increase bacteria storage life about 2,0-4,8 fold in comparison with reference samples. In the article the main results of our investigation of antioxidants interaction with microorganism cells is described. As bacteria cells we use vaccine strain yersinia pestis EV, that were grown for 48 h at 28 degree(s)C on the Hottinger agar (pH 7,2). Antioxidants are inserted on the agar surface in specimen under test. To investigate a localization of antioxidants for electron microscopy investigation, thallium organic antioxidants were used. The thallium organic compounds have an antioxidant features if thallium is in low concentration (about 1(mu) g/ml). The localization of the thallium organic antioxidants on bacteria Y. pestis EV is visible in electron microscopy images, thallium being heavy metal with high electron density. The negatively stained bacteria and bacteria thin sections with thallium organic compounds were investigated by means of transmission electron microscopy. The localization of the thallium organic compounds is clearly visible in electron micrographs as small dark spots with size about 10-80nm. Probably mechanisms of interaction of antioxidants with bacteria cells are discussed.

  7. Early cell death detection with digital holographic microscopy.

    Directory of Open Access Journals (Sweden)

    Nicolas Pavillon

    Full Text Available BACKGROUND: Digital holography provides a non-invasive measurement of the quantitative phase shifts induced by cells in culture, which can be related to cell volume changes. It has been shown previously that regulation of cell volume, in particular as it relates to ionic homeostasis, is crucially involved in the activation/inactivation of the cell death processes. We thus present here an application of digital holographic microscopy (DHM dedicated to early and label-free detection of cell death. METHODS AND FINDINGS: We provide quantitative measurements of phase signal obtained on mouse cortical neurons, and caused by early neuronal cell volume regulation triggered by excitotoxic concentrations of L-glutamate. We show that the efficiency of this early regulation of cell volume detected by DHM, is correlated with the occurrence of subsequent neuronal death assessed with the widely accepted trypan blue method for detection of cell viability. CONCLUSIONS: The determination of the phase signal by DHM provides a simple and rapid optical method for the early detection of cell death.

  8. On measuring cell confluence in phase contrast microscopy

    Science.gov (United States)

    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.

  9. Practical fabrication of microfluidic platforms for live-cell microscopy.

    Science.gov (United States)

    Lorusso, Daniel; Nikolov, Hristo N; Milner, Jaques S; Ochotny, Noelle M; Sims, Stephen M; Dixon, S Jeffrey; Holdsworth, David W

    2016-10-01

    We describe a simple fabrication technique - targeted towards non-specialists - that allows for the production of leak-proof polydimethylsiloxane (PDMS) microfluidic devices that are compatible with live-cell microscopy. Thin PDMS base membranes were spin-coated onto a glass-bottom cell culture dish and then partially cured via microwave irradiation. PDMS chips were generated using a replica molding technique, and then sealed to the PDMS base membrane by microwave irradiation. Once a mold was generated, devices could be rapidly fabricated within hours. Fibronectin pre-treatment of the PDMS improved cell attachment. Coupling the device to programmable pumps allowed application of precise fluid flow rates through the channels. The transparency and minimal thickness of the device enabled compatibility with inverted light microscopy techniques (e.g. phase-contrast, fluorescence imaging, etc.). The key benefits of this technique are the use of standard laboratory equipment during fabrication and ease of implementation, helping to extend applications in live-cell microfluidics for scientists outside the engineering and core microdevice communities. PMID:27523472

  10. A spectral k-means approach to bright-field cell image segmentation.

    Science.gov (United States)

    Bradbury, Laura; Wan, Justin W L

    2010-01-01

    Automatic segmentation of bright-field cell images is important to cell biologists, but difficult to complete due to the complex nature of the cells in bright-field images (poor contrast, broken halo, missing boundaries). Standard approaches such as level set segmentation and active contours work well for fluorescent images where cells appear as round shape, but become less effective when optical artifacts such as halo exist in bright-field images. In this paper, we present a robust segmentation method which combines the spectral and k-means clustering techniques to locate cells in bright-field images. This approach models an image as a matrix graph and segment different regions of the image by computing the appropriate eigenvectors of the matrix graph and using the k-means algorithm. We illustrate the effectiveness of the method by segmentation results of C2C12 (muscle) cells in bright-field images. PMID:21096019

  11. [High resolution scanning electron microscopy of isolated outer hair cells].

    Science.gov (United States)

    Koitschev, A; Müller, H

    1996-11-01

    Isolated hair cell preparations have gained wide acceptance as a model for studying physiological and molecular properties of the sensory cells involved in the hearing process. Ultrastructural details, such as stereocilia links, lateral membrane substructure or synaptic links are of crucial importance for normal sensory transduction. For this reason, we developed a high-resolution scanning electron microscopy (SEM) procedure to study the surface of isolated hair cells. Cells were mechanically and/or enzymatically separated, isolated and immobilized on cover slips by alcian blue and fixed by 2% glutardialdehyde or 1% OsO4. After dehydration, preparations were critical point-dried and sputter-coated with gold-palladium (2-4 nm). Up to 5 nm resolution was achieved. Optimal fixation kept the cells in their typical cylindrical forms. Preservation of the stereocilia and the apical plates of the outer hair cells depended strongly on the fixation process. Tip- and side-links were observed only sporadically because of the aggressive preparation procedure. The lateral plasma membranes of the cell bodies showed regular granular structures of 5-7 nm diameter at maximal magnification. The granular structure of the cell membrane seemed to correspond to putative transmembrane proteins believed to generate membrane-based motility. The remnants of the nerve endings and/or supporting cells usually covered the cell base. The preservation of the cells was better when enzymatic isolation was omitted. The technique used allowed for high resolution ultrastructural examination of isolated hair cells and, when combined with immunological labeling, may permit the identification of proteins at a molecular level. PMID:9064297

  12. Raman microscopy of individual living human embryonic stem cells

    Science.gov (United States)

    Novikov, S. M.; Beermann, J.; Bozhevolnyi, S. I.; Harkness, L. M.; Kassem, M.

    2010-04-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal scanning Raman microscope (Alpha300R) from Witec and sub-μm spatially resolved Raman images were obtained using a 532 nm excitation wavelength.

  13. Time-resolved local strain tracking microscopy for cell mechanics

    Science.gov (United States)

    Aydin, O.; Aksoy, B.; Akalin, O. B.; Bayraktar, H.; Alaca, B. E.

    2016-02-01

    A uniaxial cell stretching technique to measure time-resolved local substrate strain while simultaneously imaging adherent cells is presented. The experimental setup comprises a uniaxial stretcher platform compatible with inverted microscopy and transparent elastomer samples with embedded fluorescent beads. This integration enables the acquisition of real-time spatiotemporal data, which is then processed using a single-particle tracking algorithm to track the positions of fluorescent beads for the subsequent computation of local strain. The present local strain tracking method is demonstrated using polydimethylsiloxane (PDMS) samples of rectangular and dogbone geometries. The comparison of experimental results and finite element simulations for the two sample geometries illustrates the capability of the present system to accurately quantify local deformation even when the strain distribution is non-uniform over the sample. For a regular dogbone sample, the experimentally obtained value of local strain at the center of the sample is 77%, while the average strain calculated using the applied cross-head displacement is 48%. This observation indicates that considerable errors may arise when cross-head measurement is utilized to estimate strain in the case of non-uniform sample geometry. Finally, the compatibility of the proposed platform with biological samples is tested using a unibody PDMS sample with a well to contain cells and culture media. HeLa S3 cells are plated on collagen-coated samples and cell adhesion and proliferation are observed. Samples with adherent cells are then stretched to demonstrate simultaneous cell imaging and tracking of embedded fluorescent beads.

  14. Autonomous Image Segmentation using Density-Adaptive Dendritic Cell Algorithm

    Directory of Open Access Journals (Sweden)

    Vishwambhar Pathak

    2013-08-01

    Full Text Available Contemporary image processing based applications like medical diagnosis automation and analysis of satellite imagery include autonomous image segmentation as inevitable facility. The research done shows the efficiency of an adaptive evolutionary algorithm based on immune system dynamics for the task of autonomous image segmentation. The recognition dynamics of immune-kernels modeled with infinite Gaussian mixture models exhibit the capability to automatically determine appropriate number of segments in presence of noise. In addition, the model using representative density-kernel-parameters processes the information with much reduced space requirements. Experiments conducted with synthetic images as well as real images recorded assured convergence and optimal autonomous model estimation. The segmentation results tested in terms of PBM-index values have been found comparable to those of the Fuzzy C-Means (FCM for the same number of segments as generated by our algorithm.

  15. Surface plasmon enhanced cell microscopy with blocked random spatial activation

    Science.gov (United States)

    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.

  16. Cell imaging by transient fluorescence detected infrared microscopy

    Science.gov (United States)

    Ohmori, Tsutomu; Sakai, Makoto; Ishihara, Miya; Kikuchi, Makoto; Fujii, Masaaki

    2008-02-01

    Transient fluorescence detected infrared (TFD-IR) microscopy was developed to overcome the diffraction limit of infrared (IR) light without a near-field system. This microscopic technique is based on TFD-IR spectroscopy, which converts information on IR absorption to fluorescence intensity by further electronic excitation of vibrationally excited molecules by a probing UV/visible light. Roots of Arabidopsis thaliana and living A549 cells with fluorescent dyes were chosen as samples. In the measurements using the TFD-IR microscope, tunable IR picosecond laser pulses were used in the wavelength range from 2700 to 3700 nm, corresponding to CH, NH, and OH stretching modes. Fluorescence images of the root cells of A. thaliana by the TFD-IR scheme were obtained with super-resolution compared with the resolution of conventional IR microscopy. The resolution is estimated to be less than 2.6 μm by fitting of a gaussian function. However, the TFD-IR images were dominated mainly by the fluorescent dyes because they were almost the same as a conventional fluorescence image. To investigate other contributions hidden by that of fluorescent dyes, we plotted the fluorescence intensity in several 5 μm squares at various IR wavelengths, called a TFD-IR spectrum. For root cells of A. thaliana, the TFD-IR spectra show shapes similar to those of a conventional IR absorption spectrum of the fluorescent dye. Therefore, the TFD-IR images are not due to the cellular components. For an A549 cell, the TFD-IR spectra were different from a conventional IR absorption spectrum of fluorescent dyes in the wavelength region shorter than 3100 nm. We speculate that the spectral difference is due to the cellular components, possibly assigned to the combination band related to amino groups of cellular components bonded covalently to the fluorescent dyes.

  17. Label-Free Digital Quantification of Lipid Droplets in Single Cells by Stimulated Raman Microscopy on a Microfluidic Platform.

    Science.gov (United States)

    Cao, Chen; Zhou, Dong; Chen, Tao; Streets, Aaron M; Huang, Yanyi

    2016-05-01

    Quantitative characterization of a single-cell phenotype remains challenging. We combined a scalable microfluidic array of parallel cell culture chambers and stimulated Raman scattering (SRS) microscopy to quantitatively characterize the response of lipid droplet (LD) formation to free-fatty-acid stimuli with single-LD resolution at the single-cell level. By enabling the systematic live-cell imaging with SRS microscopy in a microfluidic device, we were able to quantify the morphology of over a thousand live cells in 10 different chemical environments and with 8 replicates for each culture condition, in a single experiment, and without relying on fluorescent labeling. We developed an image processing pipeline for cell segmentation and LD morphology quantification using dual-channel SRS images. This allows us to construct distributions of the morphological parameters of LDs in the cellular population and expose the vast phenotypic heterogeneity among genetically similar cells. Specifically, this approach provides an analytical tool for quantitatively investigating LD morphology in live cells in situ. With this high-throughput, high-resolution, and label-free method, we found that LD growth dynamics showed considerable cell to cell variation. Lipid accumulation in nonadipocyte cells is mainly reflected in the increase of LD number, as opposed to an increase in their size or lipid concentration. Our method allows statistical single-cell quantification of the LD distribution for further investigation of lipid metabolism and dynamic behavior, and also extends the possibility to couple with other "omics" technologies in the future. PMID:27041129

  18. Segmental Neurofibromatosis

    Directory of Open Access Journals (Sweden)

    Yesudian Devakar

    1997-01-01

    Full Text Available Segmental neurofibromatosis is a rare variant of neurofibromatosis in which the lesions are confined to one segment or dermatome of the body. They resemble classical neurofibromas in their morphology, histopathology and electron microscopy. However, systemic associations are usually absent. We report one such case with these classical features.

  19. Comparative methods for PET image segmentation in pharyngolaryngeal squamous cell carcinoma

    NARCIS (Netherlands)

    Zaidi, Habib; Abdoli, Mehrsima; Fuentes, Carolina Llina; El Naqa, Issam M.

    2012-01-01

    Several methods have been proposed for the segmentation of F-18-FDG uptake in PET. In this study, we assessed the performance of four categories of F-18-FDG PET image segmentation techniques in pharyngolaryngeal squamous cell carcinoma using clinical studies where the surgical specimen served as the

  20. Liquid flow cells having graphene on nitride for microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Adiga, Vivekananda P.; Dunn, Gabriel; Zettl, Alexander K.; Alivisatos, A. Paul

    2016-09-20

    This disclosure provides systems, methods, and apparatus related to liquid flow cells for microscopy. In one aspect, a device includes a substrate having a first and a second oxide layer disposed on surfaces of the substrate. A first and a second nitride layer are disposed on the first and second oxide layers, respectively. A cavity is defined in the first oxide layer, the first nitride layer, and the substrate, with the cavity including a third nitride layer disposed on walls of the substrate and the second oxide layer that define the cavity. A channel is defined in the second oxide layer. An inlet port and an outlet port are defined in the second nitride layer and in fluid communication with the channel. A plurality of viewports is defined in the second nitride layer. A first graphene sheet is disposed on the second nitride layer covering the plurality of viewports.

  1. Microscopy studies on pronton exchange membrane fuel cell electrodes with different ionomer contents

    DEFF Research Database (Denmark)

    Ma, Shuang; Solterbeck, Claus Henning; Odgaard, Madeleine;

    2009-01-01

    Proton Exchange Membrane (PEM) fuel cell electrodes with different ionomer contents were studied with various microscopic techniques. The morphology and surface potential were examined by Atomic Force Microscopy (AFM) and Kelvin Probe Microscopy (KPM), respectively. The particulate nature...

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

    Science.gov (United States)

    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 (Source-code for MATLAB and ImageJ is freely available under a permissive open-source license.

  3. Subventricular zone cell migration: lessons from quantitative 2-photon microscopy

    Directory of Open Access Journals (Sweden)

    Rachel eJames

    2011-03-01

    Full Text Available Neuroblasts born in the adult subventricular zone (SVZ migrate long distances in the rostral migratory stream (RMS to the olfactory bulbs where they integrate into circuitry as functional interneurons. As very little was known about the dynamic parameters of SVZ neuroblast migration, we used two-photon time-lapse microscopy to analyze migration in acute slices. This involved analyzing 3-dimensional stacks of images over time and uncovered several novel aspects of SVZ migration: chains remain stable, cells can be immotile for extensive periods, morphology does not necessarily correlate with motility, neuroblasts exhibit local exploratory motility, dorsoventral migration occurs throughout the striatal SVZ and neuroblasts turn at distinctive angles. We investigated these novel findings in the SVZ and RMS from the population to the single cell level. In this review we also discuss some technical considerations when setting up a two-photon microscopic imaging system. Throughout the review we identify several unsolved questions about SVZ neuroblast migration that might be addressed with current or emerging techniques.

  4. Image segmentation of em bryonic plant cell using pulse-coupled neural networks

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Traditional image segmentation algorithms exhibit weak performance for plant cells which have complex structure. On the other hand, pulse-coupled neural network (PCNN) based on Eckhorn's model of the cat visual cortex should be suitable to the segmentation of plant cell image.But the present theories cannot explain the relationship between the parameters of PCNN mathematical model and the effect of segmentation. Satisfactory results usually require time-consuming selection of experimental parameters. Meanwhile, in a proper, selected parametric model, the number of iteration determines the segmented effect evaluated by visual judgment, which decreases the efficiency of image segmentation. To avoid these flaws, this note proposes a new PCNN algorithm for automatically segmenting plant embryonic cell image based on the maximum entropy principle. The algorithm produces a desirable result. In addition, a model with proper parameters can automatically determine the number of iteration, avoid visual judgment, enhance the speed of segmentation and will be utilized subsequently by accurate quantitative analysis of micro-molecules of plant cell. So this algorithm is valuable for theoretical investigation and application of PCNN.``

  5. A fully automatic framework for cell segmentation on non-confocal adaptive optics images

    Science.gov (United States)

    Liu, Jianfei; Dubra, Alfredo; Tam, Johnny

    2016-03-01

    By the time most retinal diseases are diagnosed, macroscopic irreversible cellular loss has already occurred. Earlier detection of subtle structural changes at the single photoreceptor level is now possible, using the adaptive optics scanning light ophthalmoscope (AOSLO). This work aims to develop a fully automatic segmentation framework to extract cell boundaries from non-confocal split-detection AOSLO images of the cone photoreceptor mosaic in the living human eye. Significant challenges include anisotropy, heterogeneous cell regions arising from shading effects, and low contrast between cells and background. To overcome these challenges, we propose the use of: 1) multi-scale Hessian response to detect heterogeneous cell regions, 2) convex hulls to create boundary templates, and 3) circularlyconstrained geodesic active contours to refine cell boundaries. We acquired images from three healthy subjects at eccentric retinal regions and manually contoured cells to generate ground-truth for evaluating segmentation accuracy. Dice coefficient, relative absolute area difference, and average contour distance were 82±2%, 11±6%, and 2.0±0.2 pixels (Mean±SD), respectively. We find that strong shading effects from vessels are a main factor that causes cell oversegmentation and false segmentation of non-cell regions. Our segmentation algorithm can automatically and accurately segment photoreceptor cells on non-confocal AOSLO images, which is the first step in longitudinal tracking of cellular changes in the individual eye over the time course of disease progression.

  6. Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy

    OpenAIRE

    Peckys, Diana B.; Jean-Pierre Baudoin; Magdalena Eder; Ulf Werner; Niels de Jonge

    2013-01-01

    Imaging single epidermal growth factor receptors (EGFR) in intact cells is presently limited by the available microscopy methods. Environmental scanning electron microscopy (ESEM) of whole cells in hydrated state in combination with specific labeling with gold nanoparticles was used to localize activated EGFRs in the plasma membranes of COS7 and A549 cells. The use of a scanning transmission electron microscopy (STEM) detector yielded a spatial resolution of 3 nm, sufficient to identify the l...

  7. Hybrid Confocal Raman Fluorescence Microscopy on Single Cells Using Semiconductor Quantum Dots

    NARCIS (Netherlands)

    Manen, van Henk-Jan; Otto, Cees

    2007-01-01

    We have overcome the traditional incompatibility of Raman microscopy with fluorescence microscopy by exploiting the optical properties of semiconductor fluorescent quantum dots (QDs). Here we present a hybrid Raman fluorescence spectral imaging approach for single-cell microscopy applications. We sh

  8. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells.

    Science.gov (United States)

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-09-22

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

  9. Segmenting and counting of wall-pasted cells based on gabor filter.

    Science.gov (United States)

    Sun, Nongliang; Xu, Saicong; Cao, Maoyong; Li, Jing

    2005-01-01

    Correctly counting the live cells plays a great role in the ectogenetic anti-virus experiment. According to the irregular shape and arbitrary size of the wall pasted Hela cells overlapping each other, we propose a scheme to segment and count the cells using Gabor filter with different parameters and Morphological operation. Experiments reveal that filters with different parameters will lead to different results and a better segmentation will be achieved based on the characteristics of cells and optimal parameters. Large amount of experiment results show that this algorithm can successfully segment the cells and the accuracy arrives at 99.3%. This scheme based on image analysis and pattern recognition can overcome some disadvantages of traditional approaches, shortening anti-virus experimental period and reducing experimental cost. PMID:17282957

  10. Cytosolic pH gradients in cultured neuronal cell lines studied by laser scanning confocal microscopy, real-time confocal microscopy, and spectral imaging microscopy

    Science.gov (United States)

    Sanchez-Armass, Sergio; Sennoune, Souad; Martinez, Gloria M.; Ortega, Filiberta; Martinez-Zaguilan, Raul

    2002-06-01

    Changes in intracellular pH are important for the regulation of many physiological processes including: cell growth and differentiation, exocytosis, synaptic transmission, cell motility and invasion, to name a few. In pathological states such as cancer and diabetes, pH regulation is known to be altered. Nevertheless the physiological and pathological significance of this ion, there are still many gaps in our knowledge. The advent of fluorescent pH probes to monitor this ion, has substantially accelerated its study. New advances in the methods of detection of this ion by fluorescence-based approaches have also helped us to understand more about the regulation of cytosolic pH. This study evaluates the usefulness of real time confocal imaging microscopy, laser scanning confocal microscopy, and spectral imaging microscopy to the study of pH. These approaches exhibit unsurpassed temporal, spatial, and spectral resolution and are complementary. We employed cell lines derived from the brain exhibiting soma and dendrites. The existence of cell polarity suggests that the different protein composition/micro environment in discrete subcellular domains may affect the properties of fluorescent ion indicators. We performed in situ calibration of pH probes in discrete cellular regions of the neuronal cell lines to eliminate any bias in data interpretation because of differences in cell thickness/micro environment. We show that there are distinct in situ calibration parameters in different cellular domains. These indicate that in situ titrations in discrete cellular domains are needed to assign pH values. We concluded that there are distinct pH micro domains in discrete cellular regions of neuronal cell lines.

  11. A Framework for White Blood Cell Segmentation in Microscopic Blood Images Using Digital Image Processing

    OpenAIRE

    Seman Zainina; Abdul Kahar Badrul; Sadeghian Farnoosh; Ramli Abdul; Saripan M-Iqbal

    2009-01-01

    Abstract Evaluation of blood smear is a commonly clinical test these days. Most of the time, the hematologists are interested on white blood cells (WBCs) only. Digital image processing techniques can help them in their analysis and diagnosis. For example, disease like acute leukemia is detected based on the amount and condition of the WBC. The main objective of this paper is to segment the WBC to its two dominant elements: nucleus and cytoplasm. The segmentation is conducted using a proposed ...

  12. Laser ablation of basal cell carcinomas guided by confocal microscopy

    Science.gov (United States)

    Sierra, Heidy; Cordova, Miguel; Nehal, Kishwer; Rossi, Anthony; Chen, Chih-Shan Jason; Rajadhyaksha, Milind

    2016-02-01

    Laser ablation offers precise and fast removal of superficial and early nodular types of basal cell carcinomas (BCCs). Nevertheless, the lack of histological confirmation has been a limitation. Reflectance confocal microscopy (RCM) imaging combined with a contrast agent can offer cellular-level histology-like feedback to detect the presence (or absence) of residual BCC directly on the patient. We conducted an ex vivo bench-top study to provide a set of effective ablation parameters (fluence, number of passes) to remove superficial BCCs while also controlling thermal coagulation post-ablation to allow uptake of contrast agent. The results for an Er:YAG laser (2.9 um and pulse duration 250us) show that with 6 passes of 25 J/cm2, thermal coagulation can be effectively controlled, to allow both the uptake of acetic acid (contrast agent) and detection of residual (or absence) BCCs. Confirmation was provided with histological examination. An initial in vivo study on 35 patients shows that the uptake of contrast agent aluminum chloride) and imaging quality is similar to that observed in the ex vivo study. The detection of the presence of residual tumor or complete clearance was confirmed in 10 wounds with (additional) histology and in 25 lesions with follow-up imaging. Our results indicate that resolution is sufficient but further development and use of appropriate contrast agent are necessary to improve sensitivity and specificity. Advances in RCM technology for imaging of lateral and deep margins directly on the patient may provide less invasive, faster and less expensive image-guided approaches for treatment of BCCs.

  13. A multi-stage random forest classifier for phase contrast cell segmentation.

    Science.gov (United States)

    Essa, Ehab; Xie, Xianghua; Errington, Rachel J; White, Nick

    2015-01-01

    We present a machine learning based approach to automatically detect and segment cells in phase contrast images. The proposed method consists of a multi-stage classification scheme based on random forest (RF) classifier. Both low level and mid level image features are used to determine meaningful cell regions. Pixel-wise RF classification is first carried out to categorize pixels into 4 classes (dark cell, bright cell, halo artifact, and background) and generate a probability map for cell regions. K-means clustering is then applied on the probability map to group similar pixels into candidate cell regions. Finally, cell validation is performed by another RF to verify the candidate cell regions. The proposed method has been tested on U2-OS human osteosarcoma phase contrast images. The experimental results show better performance of the proposed method with precision 92.96% and recall 96.63% compared to a state-of-the-art segmentation technique. PMID:26737137

  14. Partitioning histopathological images: an integrated framework for supervised color-texture segmentation and cell splitting.

    Science.gov (United States)

    Kong, Hui; Gurcan, Metin; Belkacem-Boussaid, Kamel

    2011-09-01

    For quantitative analysis of histopathological images, such as the lymphoma grading systems, quantification of features is usually carried out on single cells before categorizing them by classification algorithms. To this end, we propose an integrated framework consisting of a novel supervised cell-image segmentation algorithm and a new touching-cell splitting method. For the segmentation part, we segment the cell regions from the other areas by classifying the image pixels into either cell or extra-cellular category. Instead of using pixel color intensities, the color-texture extracted at the local neighborhood of each pixel is utilized as the input to our classification algorithm. The color-texture at each pixel is extracted by local Fourier transform (LFT) from a new color space, the most discriminant color space (MDC). The MDC color space is optimized to be a linear combination of the original RGB color space so that the extracted LFT texture features in the MDC color space can achieve most discrimination in terms of classification (segmentation) performance. To speed up the texture feature extraction process, we develop an efficient LFT extraction algorithm based on image shifting and image integral. For the splitting part, given a connected component of the segmentation map, we initially differentiate whether it is a touching-cell clump or a single nontouching cell. The differentiation is mainly based on the distance between the most likely radial-symmetry center and the geometrical center of the connected component. The boundaries of touching-cell clumps are smoothed out by Fourier shape descriptor before carrying out an iterative, concave-point and radial-symmetry based splitting algorithm. To test the validity, effectiveness and efficiency of the framework, it is applied to follicular lymphoma pathological images, which exhibit complex background and extracellular texture with nonuniform illumination condition. For comparison purposes, the results of the

  15. Probing the compressibility of tumor cell nuclei by combined atomic force-confocal microscopy

    NARCIS (Netherlands)

    Krause, M.; Riet, J. te; Wolf, K. van der

    2013-01-01

    The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness toge

  16. Quantitative detection of gold nanoparticles on individual, unstained cancer cells by scanning electron microscopy

    NARCIS (Netherlands)

    L. Hartsuiker; P. van Es; W. Petersen; T.G. van Leeuwen; L.W.M.M. Terstappen; C. Otto

    2011-01-01

    Gold nanoparticles are rapidly emerging for use in biomedical applications. Characterization of the interaction and delivery of nanoparticles to cells through microscopy is important. Scanning electron microscopes have the intrinsic resolution to visualize gold nanoparticles on cells. A novel sample

  17. Locally resolved measurements in a segmented HTPEM fuel cell with straight flow-fields

    Energy Technology Data Exchange (ETDEWEB)

    Siegel, C. [Zentrum fuer BrennstoffzellenTechnik (ZBT), GmbH (Centre for Fuel Cell Technology), Carl-Benz-Str. 201, D-47057 Duisburg (Germany); University of Duisburg-Essen, Institut fuer Energie- und Umweltverfahrenstechnik, Lotharstr. 1, D-47048 Duisburg (Germany); Siegel Schleimer Ingenieurs-Conseils s.a r.l. - Engineering and research, 2A, rue d' Ehlerange, L-3918 Mondercange (Luxembourg); Bandlamudi, G.; Heinzel, A. [Zentrum fuer BrennstoffzellenTechnik (ZBT), GmbH (Centre for Fuel Cell Technology), Carl-Benz-Str. 201, D-47057 Duisburg (Germany); University of Duisburg-Essen, Institut fuer Energie- und Umweltverfahrenstechnik, Lotharstr. 1, D-47048 Duisburg (Germany); Filusch, F. [Zentrum fuer BrennstoffzellenTechnik (ZBT), GmbH (Centre for Fuel Cell Technology), Carl-Benz-Str. 201, D-47057 Duisburg (Germany)

    2011-08-15

    Significant advances have been reported in building and testing of high-temperature polymer electrolyte membrane (HTPEM) fuel cells and stacks during recent years. Quantity distribution measurement techniques (e.g. current density, temperature and electrochemical impedance spectroscopy (EIS)) using segmented cells are commonly used to characterise low-temperature PEM (LTPEM) fuel cells. Performing these measurements at higher temperatures is more difficult and a relatively new process. For this study, a fully operational segmented HTPEM fuel cell using a straight flow-field configuration was designed, constructed and tested. The cathode side bipolar half-plate consisted of 36 exchangeable segments, whereas, the anode side bipolar half-plate was not segmented. The cell was operated at various operating temperatures with various anode gas compositions and air (no backpressure). In addition to the experimental results, a simple computational fluid dynamics model based on COMSOL Multiphysics {sup registered} 3.5a was used to support the observed behaviour during segmented measurements. The computational domain consisted of the cathode side gas channels and the porous media. All of the boundary conditions and gas properties were defined in a manner similar to the experimental investigations. Some of the theoretical results were compared to the experimental results and conclusions were drawn. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  18. The Influence of Physical and Physiological Cues on Atomic Force Microscopy-Based Cell Stiffness Assessment

    OpenAIRE

    Yu-Wei Chiou; Hsiu-Kuan Lin; Ming-Jer Tang; Hsi-Hui Lin; Ming-Long Yeh

    2013-01-01

    Atomic force microscopy provides a novel technique for differentiating the mechanical properties of various cell types. Cell elasticity is abundantly used to represent the structural strength of cells in different conditions. In this study, we are interested in whether physical or physiological cues affect cell elasticity in Atomic force microscopy (AFM)-based assessments. The physical cues include the geometry of the AFM tips, the indenting force and the operating temperature of the AFM. All...

  19. 3D Imaging of mammalian cells with ion-abrasion scanning electron microscopy

    OpenAIRE

    Heymann, Jurgen A. W.; Shi, Dan; Kim, Sang; Bliss, Donald; Milne, Jacqueline L. S.; Subramaniam, Sriram

    2008-01-01

    Understanding the hierarchical organization of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. We are using ion-abrasion scanning electron microscopy (IA-SEM) to visualize this hierarchical organization in an approach that combines focused ion-beam milling with scanning electron microscopy. Here, we extend our previous studies on imaging yeast cells to image subcellular architecture in human melanoma cells and mela...

  20. Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins.

    Directory of Open Access Journals (Sweden)

    Jenna L Mueller

    Full Text Available To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features.TISSUE EXCISED FROM A GENETICALLY ENGINEERED MOUSE MODEL OF SARCOMA WAS IMAGED USING A SUBCELLULAR RESOLUTION MICROENDOSCOPE AFTER TOPICAL APPLICATION OF A FLUORESCENT ANATOMICAL CONTRAST AGENT: acriflavine. An algorithm based on sparse component analysis (SCA and the circle transform (CT was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma.Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity. For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach.The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.

  1. Effect of auxin on Golgi-mediated cell wall synthesis in pea stem segments

    Energy Technology Data Exchange (ETDEWEB)

    Brummell, D.A.; Maclachlan, G.A.

    1986-04-01

    Stem segments of 7 day-old etiolated Pisum sativum seedlings were abraded using carborundum powder. Batches of segments were pulsed in (/sup 3/H) glucose followed by a chase in cold glucose in the presence or absence of 1AA, then homogenized by chopping with a razor blade. A rate-zonal centrifugation on a linear sucrose gradient was used to separate dictyosomes and secretory vesicles, and membrane-bound radioactivity determined as a measure of Golgi material in the cytoplasm. The amount of membrane-bound radioactivity was increased in tissues treated with 1AA for 30 min, indicative of an enhanced Golgi content in such segments. This increase thus precedes the sustained increase in auxin-stimulated growth of stem segments which occurs around 35-45 min after exposure to auxin and which is thought to be due to increased cell wall synthesis.

  2. Use of scanning electron microscopy to monitor nanofibre/cell interaction in digestive epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Millaku, Agron, E-mail: agron.mi@hotmail.com [Limnos-Company for Applied Ecology Ltd, Podlimbarskega 31, 1000 Ljubljana (Slovenia); Drobne, Damjana [University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana (Slovenia); Centre of Excellence, Advanced Materials and Technologies for the Future (CO NAMASTE), Jamova cesta 39, 1000 Ljubljana (Slovenia); Centre of Excellence, Nanoscience and Nanotechnology (Nanocentre), Jamova cesta 39, 1000 Ljubljana (Slovenia); Torkar, Matjaz [Institute of Metals and Technology IMT, Lepi pot 11, 1000 Ljubljana (Slovenia); Jožef Stefan Institute, Condensed Matter Physics Department, Jamova cesta 39, 1000 Ljubljana (Slovenia); Novak, Sara [University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana (Slovenia); Remškar, Maja [Jožef Stefan Institute, Condensed Matter Physics Department, Jamova cesta 39, 1000 Ljubljana (Slovenia); Pipan-Tkalec, Živa [University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana (Slovenia)

    2013-09-15

    Graphical abstract: Scanning electron microscopy is particularly well suited to the observation of nanofibre/cell interaction in the endothelial cells lining the hepatopancreas. (a) Tungsten oxide nanofibres, (b) test organism Porcellio scaber and schematic appearance of digestive tubes, (c) digestive tube (hepatopancreas) prepared for SEM investigation, (d) digestive gland cells (C) with nanofibres (NF) embedded in the cell membrane and (e) nanofibres inserted deeply in the cells and damaged nanofibres due to peristalsis. -- Highlights: • Tungsten oxide nanofibres react physically with digestive gland epithelial cells in Porcellio scaber. • Physical peristaltic forces of lead to insertion of nanofibres into the cells. • No toxic responses as measured by conventional toxicity biomarkers were detected. • Physical interactions were observed in a majority of the investigated animals. -- Abstract: We provide data obtained by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) on the interaction of ingested tungsten nanofibers with epithelial cells of the digestive tubes of a test organism Porcellio scaber. Conventional toxicity endpoints including feeding behaviour, weight loss and mortality were also measured in each investigated animal. No toxicity was detected in any of exposed animals after 14 days of feeding on tungsten nanofiber dosed food, but when nanofibers enter the digestive system they can react with epithelial cells of the digestive tubes, becoming physically inserted into the cells. In this way, nanofibers can injure the epithelial cells of digestive gland tubes when they are ingested with food. Our SEM data suggest that peristaltic forces may have an important role, not predicted by in vitro experiments, in the interactions of nanomaterials with digestive intestinal cells.

  3. Use of scanning electron microscopy to monitor nanofibre/cell interaction in digestive epithelial cells

    International Nuclear Information System (INIS)

    Graphical abstract: Scanning electron microscopy is particularly well suited to the observation of nanofibre/cell interaction in the endothelial cells lining the hepatopancreas. (a) Tungsten oxide nanofibres, (b) test organism Porcellio scaber and schematic appearance of digestive tubes, (c) digestive tube (hepatopancreas) prepared for SEM investigation, (d) digestive gland cells (C) with nanofibres (NF) embedded in the cell membrane and (e) nanofibres inserted deeply in the cells and damaged nanofibres due to peristalsis. -- Highlights: • Tungsten oxide nanofibres react physically with digestive gland epithelial cells in Porcellio scaber. • Physical peristaltic forces of lead to insertion of nanofibres into the cells. • No toxic responses as measured by conventional toxicity biomarkers were detected. • Physical interactions were observed in a majority of the investigated animals. -- Abstract: We provide data obtained by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) on the interaction of ingested tungsten nanofibers with epithelial cells of the digestive tubes of a test organism Porcellio scaber. Conventional toxicity endpoints including feeding behaviour, weight loss and mortality were also measured in each investigated animal. No toxicity was detected in any of exposed animals after 14 days of feeding on tungsten nanofiber dosed food, but when nanofibers enter the digestive system they can react with epithelial cells of the digestive tubes, becoming physically inserted into the cells. In this way, nanofibers can injure the epithelial cells of digestive gland tubes when they are ingested with food. Our SEM data suggest that peristaltic forces may have an important role, not predicted by in vitro experiments, in the interactions of nanomaterials with digestive intestinal cells

  4. A multi-cell, multi-scale model of vertebrate segmentation and somite formation.

    Directory of Open Access Journals (Sweden)

    Susan D Hester

    2011-10-01

    Full Text Available Somitogenesis, the formation of the body's primary segmental structure common to all vertebrate development, requires coordination between biological mechanisms at several scales. Explaining how these mechanisms interact across scales and how events are coordinated in space and time is necessary for a complete understanding of somitogenesis and its evolutionary flexibility. So far, mechanisms of somitogenesis have been studied independently. To test the consistency, integrability and combined explanatory power of current prevailing hypotheses, we built an integrated clock-and-wavefront model including submodels of the intracellular segmentation clock, intercellular segmentation-clock coupling via Delta/Notch signaling, an FGF8 determination front, delayed differentiation, clock-wavefront readout, and differential-cell-cell-adhesion-driven cell sorting. We identify inconsistencies between existing submodels and gaps in the current understanding of somitogenesis mechanisms, and propose novel submodels and extensions of existing submodels where necessary. For reasonable initial conditions, 2D simulations of our model robustly generate spatially and temporally regular somites, realistic dynamic morphologies and spontaneous emergence of anterior-traveling stripes of Lfng. We show that these traveling stripes are pseudo-waves rather than true propagating waves. Our model is flexible enough to generate interspecies-like variation in somite size in response to changes in the PSM growth rate and segmentation-clock period, and in the number and width of Lfng stripes in response to changes in the PSM growth rate, segmentation-clock period and PSM length.

  5. Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana

    OpenAIRE

    Kim Myung K; Hao Huaiqin; Fan Lusheng; Ash William M; Wan Yinglang; Lin Jinxing

    2011-01-01

    Abstract Background Total internal reflection fluorescence microscopy (TIRFM) is a powerful tool for observing fluorescently labeled molecules on the plasma membrane surface of animal cells. However, the utility of TIRFM in plant cell studies has been limited by the fact that plants have cell walls, thick peripheral layers surrounding the plasma membrane. Recently, a new technique known as variable-angle epifluorescence microscopy (VAEM) was developed to circumvent this problem. However, the ...

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

    OpenAIRE

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

    2016-01-01

    Background 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. Methods Spiked cell l...

  7. Segmentation and Analysis of Cancer Cells in Blood Samples

    Directory of Open Access Journals (Sweden)

    Arjun Nelikanti

    2015-10-01

    Full Text Available Blood cancer is an umbrella term for cancers that affect the blood, bone marrow and lymphatic system. Acute Lymphoblastic Leukemia (ALL is one of the kinds of blood cancer which can be affected at any age in the humans. The analysis of peripheral blood samples is an important test in the procedures for the diagnosis of leukemia. In this paper the blood sample images are used and implementing a clustering algorithm for detection of the cancer cells. This paper also implements morphological operations and feature extraction techniques using MATLAB for the analysis of cancer cells in the images.

  8. Imaging and quantitative data acquisition of biological cell walls with Atomic Force Microscopy and Scanning Acoustic Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tittmann, B. R. [Penn State; Xi, X. [Penn State

    2014-09-01

    This chapter demonstrates the feasibility of Atomic Force Microscopy (AFM) and High Frequency Scanning Acoustic Microscopy (HF-SAM) as tools to characterize biological tissues. Both the AFM and the SAM have shown to provide imaging (with different resolution) and quantitative elasticity measuring abilities. Plant cell walls with minimal disturbance and under conditions of their native state have been examined with these two kinds of microscopy. After descriptions of both the SAM and AFM, their special features and the typical sample preparation is discussed. The sample preparation is focused here on epidermal peels of onion scales and celery epidermis cells which were sectioned for the AFM to visualize the inner surface (closest to the plasma membrane) of the outer epidermal wall. The nm-wide cellulose microfibrils orientation and multilayer structure were clearly observed. The microfibril orientation and alignment tend to be more organized in older scales compared with younger scales. The onion epidermis cell wall was also used as a test analog to study cell wall elasticity by the AFM nanoindentation and the SAM V(z) feature. The novelty in this work was to demonstrate the capability of these two techniques to analyze isolated, single layered plant cell walls in their natural state. AFM nanoindentation was also used to probe the effects of Ethylenediaminetetraacetic acid (EDTA), and calcium ion treatment to modify pectin networks in cell walls. The results suggest a significant modulus increase in the calcium ion treatment and a slight decrease in EDTA treatment. To complement the AFM measurements, the HF-SAM was used to obtain the V(z) signatures of the onion epidermis. These measurements were focused on documenting the effect of pectinase enzyme treatment. The results indicate a significant change in the V(z) signature curves with time into the enzyme treatment. Thus AFM and HF-SAM open the door to a systematic nondestructive structure and mechanical property

  9. Spatial Modulation Microscopy for Real-Time Imaging of Plasmonic Nanoparticles and Cells

    CERN Document Server

    Fairbairn, N; Carter, R; Fernandes, R; Kanaras, A G; Elliott, T J; Somekh, M G; Pitter, M C; Muskens, O L

    2012-01-01

    Spatial modulation microscopy is a technique originally developed for quantitative spectroscopy of individual nano-objects. Here, a parallel implementation of the spatial modulation microscopy technique is demonstrated based on a line detector capable of demodulation at kHz frequencies. The capabilities of the imaging system are shown using an array of plasmonic nanoantennas and dendritic cells incubated with gold nanoparticles.

  10. Segmentation of breast cancer cells positive 1+ and 3+ immunohistochemistry

    Science.gov (United States)

    Labellapansa, Ause; Muhimmah, Izzati; Indrayanti

    2016-03-01

    Breast cancer is a disease occurs as a result of uncontrolled cells growth. One examination method of breast cancer cells is using Immunohistochemistry (IHC) to determine status of Human Epidermal Growth Factor Receptor2 (HER2) protein. This study helps anatomic pathologist to determine HER2 scores using image processing techniques to obtain HER2 overexpression positive area percentages of 1+ and 3+ scores. This is done because the score of 0 is HER2 negative cells and 2+ scores have equivocal results, which means it could not be determined whether it is necessary to give targeted therapy or not. HER2 overexpression positive area percentage is done by dividing the area with a HER2 positive tumor area. To obtain better tumor area, repair is done by eliminating lymphocytes area which is not tumor area using morphological opening. Results of 10 images IHC scores of 1+ and 3+ and 10 IHC images testing without losing lymphocytes area in tumor area, has proven that the system has been able to provide an overall correct classification in accordance with the experts analysis. However by doing operation to remove non-tumor areas, classification can be done correctly 100% for scores of 3+ and 65% for scores of 1+.

  11. Common histological patterns in glomerular epithelial cells in secondary focal segmental glomerulosclerosis

    NARCIS (Netherlands)

    Kuppe, C.; Grone, H.J.; Ostendorf, T.; Kuppevelt, T.H. van; Boor, P.; Floege, J.; Smeets, B.; Moeller, M.J.

    2015-01-01

    Parietal epithelial cells (PECs) are involved in the development of sclerotic lesions in primary focal and segmental glomerulosclerosis (FSGS). Here, the role of PECs was explored in the more common secondary FSGS lesions in 68 patient biopsies, diagnosed with 11 different frequently or rarely encou

  12. Voltammetric scanning electrochemical cell microscopy: dynamic imaging of hydrazine electro-oxidation on platinum electrodes

    NARCIS (Netherlands)

    Chen, C.-H.; Jacobse, L.; McKelvey, K.; Lai, S.C.S.; Koper, M.T.M.; Unwin, P.R.

    2015-01-01

    Voltammetric scanning electrochemical cell microscopy (SECCM) incorporates cyclic voltammetry measurements in the SECCM imaging protocol, by recording electrochemical currents in a wide potential window at each pixel in a map. This provides much more information compared to traditional fixed potenti

  13. Coherent anti-Stokes Raman scattering microscopy of human smooth muscle cells in bioengineered tissue scaffolds

    Science.gov (United States)

    Brackmann, Christian; Esguerra, Maricris; Olausson, Daniel; Delbro, Dick; Krettek, Alexandra; Gatenholm, Paul; Enejder, Annika

    2011-02-01

    The integration of living, human smooth muscle cells in biosynthesized cellulose scaffolds was monitored by nonlinear microscopy toward contractile artificial blood vessels. Combined coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy was applied for studies of the cell interaction with the biopolymer network. CARS microscopy probing CH2-groups at 2845 cm-1 permitted three-dimensional imaging of the cells with high contrast for lipid-rich intracellular structures. SHG microscopy visualized the fibers of the cellulose scaffold, together with a small signal obtained from the cytoplasmic myosin of the muscle cells. From the overlay images we conclude a close interaction between cells and cellulose fibers. We followed the cell migration into the three-dimensional structure, illustrating that while the cells submerge into the scaffold they extrude filopodia on top of the surface. A comparison between compact and porous scaffolds reveals a migration depth of <10 μm for the former, whereas the porous type shows cells further submerged into the cellulose. Thus, the scaffold architecture determines the degree of cell integration. We conclude that the unique ability of nonlinear microscopy to visualize the three-dimensional composition of living, soft matter makes it an ideal instrument within tissue engineering.

  14. OSTEOBLAST ADHESION OF BREAST CANCER CELLS WITH SCANNING ACOUSTIC MICROSCOPY

    Energy Technology Data Exchange (ETDEWEB)

    Chiaki Miyasaka; Robyn R. Mercer; Andrea M. Mastro; Ken L. Telschow

    2005-03-01

    Breast cancer frequently metastasizes to the bone. Upon colonizing bone tissue, the cancer cells stimulate osteoclasts (cells that break bone down), resulting in large lesions in the bone. The breast cancer cells also affect osteoblasts (cells that build new bone). Conditioned medium was collected from a bone-metastatic breast cancer cell line, MDA-MB-231, and cultured with an immature osteoblast cell line, MC3T3-E1. Under these conditions the osteoblasts acquired a changed morphology and appeared to adherer in a different way to the substrate and to each other. To characterize cell adhesion, MC3T3-E1 osteoblasts were cultured with or without MDA-MB-231 conditioned medium for two days, and then assayed with a mechanical scanning acoustic reflection microscope (SAM). The SAM indicated that in normal medium the MC3T3-E1 osteoblasts were firmly attached to their plastic substrate. However, MC3T3-E1 cells cultured with MDA-MB-231 conditioned medium displayed both an abnormal shape and poor adhesion at the substrate interface. The cells were fixed and stained to visualize cytoskeletal components using optical microscopic techniques. We were not able to observe these differences until the cells were quite confluent after 7 days of culture. However, using the SAM, we were able to detect these changes within 2 days of culture with MDA-MB-231 conditioned medium

  15. Comparative methods for PET image segmentation in pharyngolaryngeal squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zaidi, Habib [Geneva University Hospital, Division of Nuclear Medicine and Molecular Imaging, Geneva (Switzerland); Geneva University, Geneva Neuroscience Center, Geneva (Switzerland); University of Groningen, Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, Groningen (Netherlands); Abdoli, Mehrsima [University of Groningen, Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, Groningen (Netherlands); Fuentes, Carolina Llina [Geneva University Hospital, Division of Nuclear Medicine and Molecular Imaging, Geneva (Switzerland); Naqa, Issam M.El [McGill University, Department of Medical Physics, Montreal (Canada)

    2012-05-15

    Several methods have been proposed for the segmentation of {sup 18}F-FDG uptake in PET. In this study, we assessed the performance of four categories of {sup 18}F-FDG PET image segmentation techniques in pharyngolaryngeal squamous cell carcinoma using clinical studies where the surgical specimen served as the benchmark. Nine PET image segmentation techniques were compared including: five thresholding methods; the level set technique (active contour); the stochastic expectation-maximization approach; fuzzy clustering-based segmentation (FCM); and a variant of FCM, the spatial wavelet-based algorithm (FCM-SW) which incorporates spatial information during the segmentation process, thus allowing the handling of uptake in heterogeneous lesions. These algorithms were evaluated using clinical studies in which the segmentation results were compared to the 3-D biological tumour volume (BTV) defined by histology in PET images of seven patients with T3-T4 laryngeal squamous cell carcinoma who underwent a total laryngectomy. The macroscopic tumour specimens were collected ''en bloc'', frozen and cut into 1.7- to 2-mm thick slices, then digitized for use as reference. The clinical results suggested that four of the thresholding methods and expectation-maximization overestimated the average tumour volume, while a contrast-oriented thresholding method, the level set technique and the FCM-SW algorithm underestimated it, with the FCM-SW algorithm providing relatively the highest accuracy in terms of volume determination (-5.9 {+-} 11.9%) and overlap index. The mean overlap index varied between 0.27 and 0.54 for the different image segmentation techniques. The FCM-SW segmentation technique showed the best compromise in terms of 3-D overlap index and statistical analysis results with values of 0.54 (0.26-0.72) for the overlap index. The BTVs delineated using the FCM-SW segmentation technique were seemingly the most accurate and approximated closely the 3-D BTVs

  16. Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy and Live Cell Imaging

    Science.gov (United States)

    Celler, Katherine; Fujita, Miki; Kawamura, Eiko; Ambrose, Chris; Herburger, Klaus; Wasteneys, Geoffrey O.

    2016-01-01

    Microtubules are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labelling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography. PMID:26498784

  17. High resolution surface plasmon microscopy for cell imaging

    Science.gov (United States)

    Argoul, F.; Monier, K.; Roland, T.; Elezgaray, J.; Berguiga, L.

    2010-04-01

    We introduce a new non-labeling high resolution microscopy method for cellular imaging. This method called SSPM (Scanning Surface Plasmon Microscopy) pushes down the resolution limit of surface plasmon resonance imaging (SPRi) to sub-micronic scales. High resolution SPRi is obtained by the surface plasmon lauching with a high numerical aperture objective lens. The advantages of SPPM compared to other high resolution SPRi's rely on three aspects; (i) the interferometric detection of the back reflected light after plasmon excitation, (ii) the twodimensional scanning of the sample for image reconstruction, (iii) the radial polarization of light, enhancing both resolution and sensitivity. This microscope can afford a lateral resolution of - 150 nm in liquid environment and - 200 nm in air. We present in this paper images of IMR90 fibroblasts obtained with SSPM in dried environment. Internal compartments such as nucleus, nucleolus, mitochondria, cellular and nuclear membrane can be recognized without labelling. We propose an interpretation of the ability of SSPM to reveal high index contrast zones by a local decomposition of the V (Z) function describing the response of the SSPM.

  18. Stress relaxation microscopy (STREM): Imaging mechanical force decay in cells

    CERN Document Server

    Moreno-Flores, Susana; Vivanco, Maria dM; Toca-Herrera, Jose Luis

    2009-01-01

    We have developed a novel scanning probe-based methodology to study cell biomechanics. The time dependence of the force exerted by the cell surface on a scanning probe at constant local deformation has been used to extract local relaxational responses. The generalized Maxwell viscoelastic model that accounts for multi relaxations fully describes the mechanical behaviour of the cell surface that exhibits a bimodal relaxation. Within the range of tested forces (0.1-4 nN) a slow and a fast relaxation with characteristic times of 0.1 and 1s have been detected and assigned to rearrangements in the cell membrane and cytoskeleton cortex, respectively. Relaxation time mapping allows to simultaneously detect non-uniformities in membrane and cytoskeletal mechanical behaviour and can be used as both identifying and diagnosing tools for cell type and cell disease.

  19. Planar patch-clamp force microscopy on living cells

    Energy Technology Data Exchange (ETDEWEB)

    Pamir, Evren [Center for Nano Science, Ludwig-Maximilians University, Amalienstr 54, 80799 Munich (Germany); George, Michael; Fertig, Niels [Nanion Technologies GmbH, Erzgiessereistr. 4, 80335 Munich (Germany); Benoit, Martin [Center for Nano Science, Ludwig-Maximilians University, Amalienstr 54, 80799 Munich (Germany)], E-mail: martin.benoit@physik.uni-muenchen.de

    2008-05-15

    Here we report a new combination of the patch-clamp technique with the atomic force microscope (AFM). A planar patch-clamp chip microstructured from borosilicate glass was used as a support for mechanical probing of living cells. The setup not only allows for immobilizing even a non-adherent cell for measurements of its mechanical properties, but also for simultaneously measuring the electrophysiological properties of a single cell. As a proof of principle experiment we measured the voltage-induced membrane movement of HEK293 and Jurkat cells in the whole-cell voltage clamp configuration. The results of these measurements are in good agreement with previous studies. By using the planar patch-clamp chip for immobilization, the AFM not only can image non-adhering cells, but also gets easily access to an electrophysiologically controlled cellular probe at low vibrational noise.

  20. In Situ Ecophysiology of Microbial Biofilm Communities Analyzed by CMEIAS Computer-Assisted Microscopy at Single-Cell Resolution

    Directory of Open Access Journals (Sweden)

    Youssef G. Yanni

    2013-06-01

    Full Text Available This paper describes the utility of CMEIAS (Center for Microbial Ecology Image Analysis System computer-assisted microscopy to extract data from accurately segmented images that provide 63 different insights into the ecophysiology of microbial populations and communities within biofilms and other habitats. Topics include quantitative assessments of: (i morphological diversity as an indicator of impacts that substratum physicochemistries have on biofilm community structure and dominance-rarity relationships among populations; (ii morphotype-specific distributions of biovolume body size that relate microbial allometric scaling, metabolic activity and growth physiology; (iii fractal geometry of optimal cellular positioning for efficient utilization of allocated nutrient resources; (iv morphotype-specific stress responses to starvation, environmental disturbance and bacteriovory predation; (v patterns of spatial distribution indicating positive and negative cell–cell interactions affecting their colonization behavior; and (vi significant methodological improvements to increase the accuracy of color-discriminated ecophysiology, e.g., differentiation of cell viability based on cell membrane integrity, cellular respiratory activity, phylogenetically differentiated substrate utilization, and N-acyl homoserine lactone-mediated cell–cell communication by bacteria while colonizing plant roots. The intensity of these ecophysiological attributes commonly varies at the individual cell level, emphasizing the importance of analyzing them at single-cell resolution and the proper spatial scale at which they occur in situ.

  1. Segmentation of White Blood Cell from Acute Lymphoblastic Leukemia Images Using Dual-Threshold Method

    Directory of Open Access Journals (Sweden)

    Yan Li

    2016-01-01

    Full Text Available We propose a dual-threshold method based on a strategic combination of RGB and HSV color space for white blood cell (WBC segmentation. The proposed method consists of three main parts: preprocessing, threshold segmentation, and postprocessing. In the preprocessing part, we get two images for further processing: one contrast-stretched gray image and one H component image from transformed HSV color space. In the threshold segmentation part, a dual-threshold method is proposed for improving the conventional single-threshold approaches and a golden section search method is used for determining the optimal thresholds. For the postprocessing part, mathematical morphology and median filtering are utilized to denoise and remove incomplete WBCs. The proposed method was tested in segmenting the lymphoblasts on a public Acute Lymphoblastic Leukemia (ALL image dataset. The results show that the performance of the proposed method is better than single-threshold approach independently performed in RGB and HSV color space and the overall single WBC segmentation accuracy reaches 97.85%, showing a good prospect in subsequent lymphoblast classification and ALL diagnosis.

  2. Cell volume and geometric parameters determination in living cells using confocal microscopy and 3D reconstruction

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: David Hevia, Aida Rodriguez-Garcia, Marta Alonso-Gervós, Isabel Quirós-González, Henar M Cimadevilla, Carmen Gómez-Cordovés, Rosa M Sainz & Juan C Mayo ### Abstract The protocol reported here describes a simple, easy, fast and reproducible method aimed to know the geometric parameters of living cells based on confocal laser scanning microscopy combined with 3D reconstruction software. Briefly, the method is based on intrinsic fluorescence properties of acridine orange (AO...

  3. Nuclear area measurement on viable cells, using confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Townsend, K.M.S.; Marsden, S.J. (Medical Research Council, Harwell (United Kingdom). Radiobiological Research Unit)

    1992-04-01

    The authors describe a rapid procedure for the accurate measurement of nuclear areas on unperturbed living cells as used in radiobiological experiments, using the confocal laser scanning microscope. The microdosimetric interpretation of radiobiological data requires precise information on the nuclear area of cells as irradiated with high-LET radiation. (author).

  4. A novel role for MuSK and non-canonical Wnt signaling during segmental neural crest cell migration.

    Science.gov (United States)

    Banerjee, Santanu; Gordon, Laura; Donn, Thomas M; Berti, Caterina; Moens, Cecilia B; Burden, Steven J; Granato, Michael

    2011-08-01

    Trunk neural crest cells delaminate from the dorsal neural tube as an uninterrupted sheet; however, they convert into segmentally organized streams before migrating through the somitic territory. These neural crest cell streams join the segmental trajectories of pathfinding spinal motor axons, suggesting that interactions between these two cell types might be important for neural crest cell migration. Here, we show that in the zebrafish embryo migration of both neural crest cells and motor axons is temporally synchronized and spatially restricted to the center of the somite, but that motor axons are dispensable for segmental neural crest cell migration. Instead, we find that muscle-specific receptor kinase (MuSK) and its putative ligand Wnt11r are crucial for restricting neural crest cell migration to the center of each somite. Moreover, we find that blocking planar cell polarity (PCP) signaling in somitic muscle cells also results in non-segmental neural crest cell migration. Using an F-actin biosensor we show that in the absence of MuSK neural crest cells fail to retract non-productive leading edges, resulting in non-segmental migration. Finally, we show that MuSK knockout mice display similar neural crest cell migration defects, suggesting a novel, evolutionarily conserved role for MuSK in neural crest migration. We propose that a Wnt11r-MuSK dependent, PCP-like pathway restricts neural crest cells to their segmental path.

  5. Live Cell Microscopy-Based RNAi Screening in the Moss Physcomitrella patens.

    Science.gov (United States)

    Miki, Tomohiro; Nakaoka, Yuki; Goshima, Gohta

    2016-01-01

    RNA interference (RNAi) is a powerful technique enabling the identification of the genes involved in a certain cellular process. Here, we discuss protocols for microscopy-based RNAi screening in protonemal cells of the moss Physcomitrella patens, an emerging model system for plant cell biology. Our method is characterized by the use of conditional (inducible) RNAi vectors, transgenic moss lines in which the RNAi vector is integrated, and time-lapse fluorescent microscopy. This method allows for effective and efficient screening of >100 genes involved in various cellular processes such as mitotic cell division, organelle distribution, or cell growth. PMID:27581297

  6. Scanning electrochemical microscopy of living cells. 3. Rhodobacter sphaeroides.

    Science.gov (United States)

    Cai, Chenxin; Liu, Biao; Mirkin, Michael V; Frank, Harry A; Rusling, James F

    2002-01-01

    The scanning electrochemical microscope (SECM) was used to probe the redox activity of individual purple bacteria (Rhodobacter sphaeroides). The approaches developed in our previous studies of mammalian cells were expanded to measure the rates and investigate the pathway of transmembrane charge transfer in bacteria. The two groups of redox mediators (i.e., hydrophilic and hydrophobic redox species) were used to shuttle the electrons between the SECM tip electrode in solution and the redox centers inside the cell. The analysis of the dependencies of the measured rate constant on formal potential and concentration of mediator species in solution yielded information about the permeability of the outer cell membrane to different ionic species and intracellular redox properties. The maps of redox reactivity of the cell surface were obtained with a micrometer or submicrometer spatial resolution. PMID:11795778

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

    Science.gov (United States)

    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.

  8. Impression cytology and in vivo confocal microscopy in corneas with total limbal stem cell deficiency

    Directory of Open Access Journals (Sweden)

    Aline Lütz de Araújo

    2013-10-01

    Full Text Available PURPOSES: To describe corneal changes seen on in vivo confocal microscopy in patients with total limbal stem cell deficiency and to correlate them with cytological findings. METHODS: A prospective case series including 13 eyes (8 patients with total limbal deficiency was carried out. Stem cell deficiency was diagnosed clinically and by corneal impression cytology. Confocal images of the central cornea were taken with the Heidelberg Retina Tomograph II, Rostock Corneal Module (Heidelberg Engineering, Heidelberg, Germany. RESULTS: Impression cytology of the cornea revealed conjunctival epithelial cells and goblet cells in all cases. In vivo confocal microscopy showed disruption of normal layers of the corneal epithelium in all eyes. Confocal images showed cells with characteristics of conjunctival epithelium at the cornea in 76.9% of the total. These findings on confocal microscopy were compatible to limbal stem cell deficiency. Additionally, goblet cells, squamous metaplasia, inflammatory cells and dendritic cells were observed. The sub-basal nerve plexus was not identified in any of the corneas. Corneal neovessels were observed at the epithelium and stroma. All cases showed diffuse hyper-reflective images of the stroma corresponding to opacity of the tissue. CONCLUSIONS: Limbal stem cell deficiency had been confirmed by impression cytology in all cases, and 76.9% of the cases could also be diagnosed by in vivo confocal microscopy through the conjunctival epithelial cell visualization on the corneal surface. Frequent confocal microscopy findings were abnormal cells at the cornea (conjunctival epithelial, goblet and inflammatory cells, corneal neovessels and diffuse hyper-reflection of the stroma.

  9. Rice dwarf phytoreovirus segment S6-encoded nonstructural protein has a cell-to-cell movement function.

    Science.gov (United States)

    Li, Yi; Bao, Yi M; Wei, Chun H; Kang, Zhen S; Zhong, Yong W; Mao, Peng; Wu, Gang; Chen, Zhang L; Schiemann, Joachim; Nelson, Richard S

    2004-05-01

    Rice dwarf virus (RDV) is a member of the genus Phytoreovirus, which is composed of viruses with segmented double-stranded RNA genomes. Proteins that support the intercellular movement of these viruses in the host have not been identified. Microprojectile bombardment was used to determine which open reading frames (ORFs) support intercellular movement of a heterologous virus. A plasmid containing an infectious clone of Potato virus X (PVX) defective in cell-to-cell movement and expressing either beta-glucuronidase or green fluorescent protein (GFP) was used for cobombardment with plasmids containing ORFs from RDV gene segments S1 through S12 onto leaves of Nicotiana benthamiana. Cell-to-cell movement of the movement-defective PVX was restored by cobombardment with a plasmid containing S6. In the absence of S6, no other gene segment supported movement. Identical results were obtained with Nicotiana tabacum, a host that allows fewer viruses to infect and spread within its tissue. S6 supported the cell-to-cell movement of the movement-defective PVX in sink and source leaves of N. benthamiana. A mutant S6 lacking the translation start codon did not complement the cell-to-cell movement of the movement-defective PVX. An S6 protein product (Pns6)-enhanced GFP fusion was observed near or within cell walls of epidermal cells from N. tabacum. By immunocytochemistry, unfused Pns6 was localized to plasmodesmata in rice leaves infected with RDV. S6 thus encodes a protein with characteristics identical to those of other viral proteins required for the cell-to-cell movement of their genome and therefore is likely required for the cell-to-cell movement of RDV.

  10. Regulatory T Cells Vary over Bleeding Segments in Asthmatic and non-Asthmatic Women

    OpenAIRE

    Wegienka, Ganesa; Bobbitt, Kevin R.; Woodcroft, Kimberley J.; Havstad, Suzanne

    2011-01-01

    Sex hormones may play an important role in observed gender differences in asthma incidence and severity. Regulatory T cells (Treg cells) are presumed to be involved in asthma and may vary with hormone levels. To investigate the effects of sex hormones on levels of Treg cells (percentage of CD4+CD25+Foxp3+ lymphocytes that are CD127), a cohort of 13 women (6 with and 7 without an asthma diagnosis) had blood drawn multiple times over the course of a bleeding segment (bleeding interval plus the ...

  11. Enlightening intracellular complexity of living cells with quantitative phase microscopy

    Science.gov (United States)

    Martinez Torres, C.; Laperrousaz, B.; Berguiga, L.; Boyer Provera, E.; Elezgaray, J.; Nicolini, F. E.; Maguer-Satta, V.; Arneodo, A.; Argoul, F.

    2016-03-01

    The internal distribution of refractive indices (RIs) of a living cell is much more complex than usually admitted in multi-shell models. The reconstruction of RI maps from single phase images has rarely been achieved for several reasons: (i) we still have very little knowledge of the impact of internal macromolecular complexes on the local RI and (ii) phase changes produced by light propagation through the sample are mixed with diffraction effects by internal cell bodies. We propose the implementation a 2D wavelet-based contour chain detection method to distinguish internal boundaries thanks to their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are morphological indicators for distinguishing cells of different origins and to follow their transformation in pathologic situations. We use this method to compare non adherent blood cells from primary and laboratory culture origins, in healthy and pathological situations (chronic myelogenous leukaemia). In a second part of this presentation, we concentrate on the temporal dynamics of the phase contour chains and we discuss the spectral decomposition of their dynamics in both health and disease.

  12. Glycoproteins of coated pits, cell junctions, and the entire cell surface revealed by monoclonal antibodies and immunoelectron microscopy

    OpenAIRE

    1983-01-01

    Topographical descriptions of three major plasma membrane glycoproteins of murine 3T3 cells were obtained by immunoelectron microscopy with monoclonal antibodies. A glycoprotein of Mr 80,000 was distributed throughout the total cell surface. A second of Mr 90,000 was concentrated in coated pits, and a third of Mr 100,000 was localized at cell junctions.

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

    Science.gov (United States)

    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.

  14. Waveguide evanescent field fluorescence microscopy: Thin film fluorescence intensities and its application in cell biology

    Science.gov (United States)

    Hassanzadeh, Abdollah; Nitsche, Michael; Mittler, Silvia; Armstrong, Souzan; Dixon, Jeff; Langbein, Uwe

    2008-06-01

    We demonstrate an inexpensive alternative to total internal reflection fluorescence microscopy. A method for imaging ultrathin films and living cells located on waveguides—illuminated with their evanescent fields—is introduced. An extensive analysis of ion-exchanged waveguides focusing on their application as microscopy substrates for studying interfacial phenomena is presented. Experimental results are in excellent agreement with the simulations. As an application osteoblasts (bone matrix forming cells) and ultrathin Langmuir-Blodgett films were imaged. The fluorescence intensity has been used to determine the cell attachment.

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

    International Nuclear Information System (INIS)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei, E-mail: biehzw@nus.edu.sg [Optical Bioimaging Laboratory, Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117576 (Singapore)

    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.

  17. Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-Soo; Torelli, Marco; Hamers, Robert J.; Murphy, Catherine; Orr, Galya; Haynes, Christy L.

    2014-01-01

    A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

  18. Atomic force microscopy as analytical tool to study physico-mechanical properties of intestinal cells

    Directory of Open Access Journals (Sweden)

    Christa Schimpel

    2015-07-01

    Full Text Available The small intestine is a complex system that carries out various functions. The main function of enterocytes is absorption of nutrients, whereas membranous cells (M cells are responsible for delivering antigens/foreign substances to the mucosal lymphoid tissues. However, to get a fundamental understanding of how cellular structures contribute to physiological processes, precise knowledge about surface morphologies, cytoskeleton organizations and biomechanical properties is necessary. Atomic force microscopy (AFM was used here as a powerful tool to study surface topographies of Caco-2 cells and M cells. Furthermore, cell elasticity (i.e., the mechanical response of a cell on a tip indentation, was elucidated by force curve measurements. Besides elasticity, adhesion was evaluated by recording the attraction and repulsion forces between the tip and the cell surface. Organization of F-actin networks were investigated via phalloidin labeling and visualization was performed with confocal laser scanning fluorescence microscopy (CLSM and scanning electron microscopy (SEM. The results of these various experimental techniques revealed significant differences in the cytoskeleton/microvilli arrangements and F-actin organization. Caco-2 cells displayed densely packed F-actin bundles covering the entire cell surface, indicating the formation of a well-differentiated brush border. In contrast, in M cells actins were arranged as short and/or truncated thin villi, only available at the cell edge. The elasticity of M cells was 1.7-fold higher compared to Caco-2 cells and increased significantly from the cell periphery to the nuclear region. Since elasticity can be directly linked to cell adhesion, M cells showed higher adhesion forces than Caco-2 cells. The combination of distinct experimental techniques shows that morphological differences between Caco-2 cells and M cells correlate with mechanical cell properties and provide useful information to understand

  19. Imaging immune response of skin mast cells in vivo with two-photon microscopy

    Science.gov (United States)

    Li, Chunqiang; Pastila, Riikka K.; Lin, Charles P.

    2012-02-01

    Intravital multiphoton microscopy has provided insightful information of the dynamic process of immune cells in vivo. However, the use of exogenous labeling agents limits its applications. There is no method to perform functional imaging of mast cells, a population of innate tissue-resident immune cells. Mast cells are widely recognized as the effector cells in allergy. Recently their roles as immunoregulatory cells in certain innate and adaptive immune responses are being actively investigated. Here we report in vivo mouse skin mast cells imaging with two-photon microscopy using endogenous tryptophan as the fluorophore. We studied the following processes. 1) Mast cells degranulation, the first step in the mast cell activation process in which the granules are released into peripheral tissue to trigger downstream reactions. 2) Mast cell reconstitution, a procedure commonly used to study mast cells functioning by comparing the data from wild type mice, mast cell-deficient mice, and mast-cell deficient mice reconstituted with bone marrow-derived mast cells (BMMCs). Imaging the BMMCs engraftment in tissue reveals the mast cells development and the efficiency of BMMCs reconstitution. We observed the reconstitution process for 6 weeks in the ear skin of mast cell-deficient Kit wsh/ w-sh mice by two-photon imaging. Our finding is the first instance of imaging mast cells in vivo with endogenous contrast.

  20. Cystine uptake by cultured cells originating from dog proximal tubule segments

    International Nuclear Information System (INIS)

    Large numbers of kidney epithelial cells were cultured successfully from isolated dog proximal tubule segments. Cells in primary culture and in first passage retained the cystine-dibasic amino acid co-transporter system which is found in vivo and in freshly isolated proximal tubule segments. In contrast to other cultured cells, the cystine-glutamate anti-porter was absent in primary cultures. However, this anti-porter system seemed to be developing in cells in first passage. The intracellular ratio of cysteine:reduced glutathione (CSH:GSH) was maintained at 1:36 in both primary cultures and in low passage cells. Incubation of cells in primary culture for 5 min at 37 degrees C with 0.025 mM [35S]L-cystine resulted in incorporation of approximately 36 and 8.5% of the label into intracellular CSH and GSH, respectively. These cultured cells, therefore, seem to be an excellent model system for the eventual elucidation of (a) the inticacies of cystine metabolism and (b) regulation of (1) the cystine-dibasic amino acid co-transporter system and (2) the development of the cysteine-glutamate anti-porter system

  1. In vivo reflectance confocal microscopy features of a large cell acanthoma: report of a case.

    Science.gov (United States)

    Shahriari, Neda; Grant-Kels, Jane M; Rabinovitz, Harold S; Oliviero, Margaret; Scope, Alon

    2016-07-01

    Reflectance confocal microscopy (RCM) is an FDA approved noninvasive optical imaging technique that acquires cellular level-resolution skin images in vivo. Herein, we report a case of histopathologically proven large cell acanthoma (LCA) whose RCM features simulate those of squamous cell carcinoma in situ. PMID:27648388

  2. Meniscus confined fabrication of multidimensional conducting polymer nanostructures with scanning electrochemical cell microscopy (SECCM).

    Science.gov (United States)

    McKelvey, Kim; O'Connell, Michael A; Unwin, Patrick R

    2013-04-14

    Scanning electrochemical cell microscopy (SECCM) is demonstrated as a new approach for the construction of extended multi-dimensional conducting polymer (polyaniline) nanostructures, making use of a mobile dual-channel theta pipette cell to control and monitor the location, rate and extent of electropolymerisation.

  3. In vivo reflectance confocal microscopy features of a large cell acanthoma: report of a case.

    Science.gov (United States)

    Shahriari, Neda; Grant-Kels, Jane M; Rabinovitz, Harold S; Oliviero, Margaret; Scope, Alon

    2016-07-01

    Reflectance confocal microscopy (RCM) is an FDA approved noninvasive optical imaging technique that acquires cellular level-resolution skin images in vivo. Herein, we report a case of histopathologically proven large cell acanthoma (LCA) whose RCM features simulate those of squamous cell carcinoma in situ.

  4. 2. Brazilian Congress on Cell Biology and 7. Brazilian Colloquium on Electron Microscopy - Abstracts

    International Nuclear Information System (INIS)

    Immunology, virology, bacteriology, genetics and protozoology are some of the subjects treated in the 2. Brazilian Congress on Cell Biology. Studies using radioisotopic techniques and ultrastructural cytological studies are presented. Use of optical - and electron microscopy in some of these studies is discussed. In the 7. Brazilian Colloquium on Electron Microscopy, the application of this technique to materials science is discussed (failure analysis in metallurgy, energy dispersion X-ray analysis, etc). (I.C.R.)

  5. Microrheology of cells with magnetic force modulation atomic force microscopy.

    Science.gov (United States)

    Rebêlo, L M; de Sousa, J S; Mendes Filho, J; Schäpe, J; Doschke, H; Radmacher, M

    2014-04-01

    We propose a magnetic force modulation method to measure the stiffness and viscosity of living cells using a modified AFM apparatus. An oscillating magnetic field makes a magnetic cantilever oscillate in contact with the sample, producing a small AC indentation. By comparing the amplitude of the free cantilever motion (A0) with the motion of the cantilever in contact with the sample (A1), we determine the sample stiffness and viscosity. To test the method, the frequency-dependent stiffness of 3T3 fibroblasts was determined as a power law k(s)(f) = α + β(f/f¯)(γ) (α = 7.6 × 10(-4) N m(-1), β = 1.0 × 10(-4) N m(-1), f¯ = 1 Hz, γ = 0.6), where the coefficient γ = 0.6 is in good agreement with rheological data of actin solutions with concentrations similar to those in cells. The method also allows estimation of the internal friction of the cells. In particular we found an average damping coefficient of 75.1 μN s m(-1) for indentation depths ranging between 1.0 μm and 2.0 μm. PMID:24651941

  6. Visual-servoing optical microscopy

    Science.gov (United States)

    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.

  7. Multimodal label-free growth and morphology characterization of different cell types in a single culture with quantitative digital holographic phase microscopy

    Science.gov (United States)

    Kemper, Björn; Wibbeling, Jana; Kastl, Lena; Schnekenburger, Jürgen; Ketelhut, Steffi

    2015-03-01

    For the analysis of the impact of pharmaceuticals or pathogens on different cellular phenotypes under identical measurement conditions and to analyze interactions between different cellular specimens a minimally-invasive quantitative observation of different cell types in a single culture is of particular interest. Digital holographic microscopy (DHM), a var-iant of quantitative phase microscopy (QPM), provides high resolution detection of optical path length changes that is suitable for stain-free minimally-invasive live cell analysis. Due to low light intensities for object illumination, QPM minimizes the interaction with the sample and has been demonstrated in particular to be suitable for long-term time-lapse investigations, e.g., for the detection of cell morphology alterations due to drugs and toxins. Furthermore, QPM has been demonstrated to be a versatile tool for the quantification of cellular growth and motility. Thus, we studied the feasibility of QPM for the analysis of mixed cell cultures and explored if quantitative phase images provide sufficient information to distinguish between different cell types and to extract cell specific parameters. For the experiments quantitative phase imaging with DHM was utilized. Mixed cell cultures with different cell types were observed with quantitative DHM phase contrast up to 35 h. The obtained series of quantitative phase images were evaluated by adapted algorithms for image segmentation. From the segmented images the area covered by the cells, the cellular dry mass and the mean cell thickness were calculated and used in the further analysis as parameters to quantify the reliability of the measurement principle. The obtained results demonstrate that it is possible to characterize the growth of cell types with different mor-phology features separately in a single culture.

  8. Interfacial energetics approach for analysis of endothelial cell and segmental polyurethane interactions.

    Science.gov (United States)

    Hill, Michael J; Cheah, Calvin; Sarkar, Debanjan

    2016-08-01

    Understanding the physicochemical interactions between endothelial cells and biomaterials is vital for regenerative medicine applications. Particularly, physical interactions between the substratum interface and spontaneously deposited biomacromolecules as well as between the induced biomolecular interface and the cell in terms of surface energetics are important factors to regulate cellular functions. In this study, we examined the physical interactions between endothelial cells and segmental polyurethanes (PUs) using l-tyrosine based PUs to examine the structure-property relations in terms of PU surface energies and endothelial cell organization. Since, contact angle analysis used to probe surface energetics provides incomplete interpretation and understanding of the physical interactions, we sought a combinatorial surface energetics approach utilizing water contact angle, Zisman's critical surface tension (CST), Kaelble's numerical method, and van Oss-Good-Chaudhury theory (vOGCT), and applied to both substrata and serum adsorbed matrix to correlate human umbilical vein endothelial cell (HUVEC) behavior with surface energetics of l-tyrosine based PU surfaces. We determined that, while water contact angle of substratum or adsorbed matrix did not correlate well with HUVEC behavior, overall higher polarity according to the numerical method as well as Lewis base character of the substratum explained increased HUVEC interaction and monolayer formation as opposed to organization into networks. Cell interaction was also interpreted in terms of the combined effects of substratum and adsorbed matrix polarity and Lewis acid-base character to determine the effect of PU segments. PMID:27065449

  9. The ePetri dish, an on-chip cell imaging platform based on subpixel perspective sweeping microscopy (SPSM)

    OpenAIRE

    Zheng, Guoan; Lee, Seung Ah; Antebi, Yaron; Elowitz, Michael B.; Yang, Changhuei

    2011-01-01

    We report a chip-scale lensless wide-field-of-view microscopy imaging technique, subpixel perspective sweeping microscopy, which can render microscopy images of growing or confluent cell cultures autonomously. We demonstrate that this technology can be used to build smart Petri dish platforms, termed ePetri, for cell culture experiments. This technique leverages the recent broad and cheap availability of high performance image sensor chips to provide a low-cost and automated microscopy soluti...

  10. Bioluminescence Microscopy as a Method to Measure Single Cell Androgen Receptor Activity Heterogeneous Responses to Antiandrogens

    Science.gov (United States)

    Jain, Pallavi; Neveu, Bertrand; Velot, Lauriane; Wu, Lily; Fradet, Yves; Pouliot, Frédéric

    2016-01-01

    Cancer cell heterogeneity is well-documented. Therefore, techniques to monitor single cell heterogeneous responses to treatment are needed. We developed a highly translational and quantitative bioluminescence microscopy method to measure single cell androgen receptor (AR) activity modulation by antiandrogens from fluid biopsies. We showed that this assay can detect heterogeneous cellular response to drug treatment and that the sum of single cell AR activity can mirror the response in the whole cell population. This method may thus be used to monitor heterogeneous dynamic treatment responses in cancer cells. PMID:27678181

  11. Cell morphology classification in phase contrast microscopy image reducing halo artifact

    Science.gov (United States)

    Kang, Mi-Sun; Song, Soo-Min; Lee, Hana; Kim, Myoung-Hee

    2012-03-01

    Since the morphology of tumor cells is a good indicator of their invasiveness, we used time-lapse phase-contrast microscopy to examine the morphology of tumor cells. This technique enables long-term observation of the activity of live cells without photobleaching and phototoxicity which is common in other fluorescence-labeled microscopy. However, it does have certain drawbacks in terms of imaging. Therefore, we first corrected for non-uniform illumination artifacts and then we use intensity distribution information to detect cell boundary. In phase contrast microscopy image, cell is normally appeared as dark region surrounded by bright halo ring. Due to halo artifact is minimal around the cell body and has non-symmetric diffusion pattern, we calculate cross sectional plane which intersects center of each cell and orthogonal to first principal axis. Then, we extract dark cell region by analyzing intensity profile curve considering local bright peak as halo area. Finally, we examined cell morphology to classify tumor cells as malignant and benign.

  12. A simple way to identify non-viable cells within living plant tissue using confocal microscopy

    Directory of Open Access Journals (Sweden)

    Truernit Elisabeth

    2008-06-01

    Full Text Available Abstract Background Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. Results Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy. The dyes were selective for non-viable cells and showed very little background staining in living cells. Simultaneous detection of SYTOX dye and fluorescent protein (e.g. GFP fluorescence was possible. Conclusion The fluorescent SYTOX dyes are useful for an easy and quick first assay of plant cell viability in living plant samples using fluorescence and confocal laser-scanning microscopy.

  13. Scanning electron microscopy of cells and tissues under fully hydrated conditions.

    Science.gov (United States)

    Thiberge, Stephan; Nechushtan, Amotz; Sprinzak, David; Gileadi, Opher; Behar, Vered; Zik, Ory; Chowers, Yehuda; Michaeli, Shulamit; Schlessinger, Joseph; Moses, Elisha

    2004-03-01

    A capability for scanning electron microscopy of wet biological specimens is presented. A membrane that is transparent to electrons protects the fully hydrated sample from the vacuum. The result is a hybrid technique combining the ease of use and ability to see into cells of optical microscopy with the higher resolution of electron microscopy. The resolution of low-contrast materials is approximately 100 nm, whereas in high-contrast materials the resolution can reach 10 nm. Standard immunogold techniques and heavy-metal stains can be applied and viewed in the fluid to improve the contrast. Images present a striking combination of whole-cell morphology with a wealth of internal details. A possibility for direct inspection of tissue slices transpires, imaging only the external layer of cells. Simultaneous imaging with photons excited by the electrons incorporates data on material distribution, indicating a potential for multilabeling and specific scintillating markers.

  14. A simple way to identify non-viable cells within living plant tissue using confocal microscopy

    OpenAIRE

    Truernit Elisabeth; Haseloff Jim

    2008-01-01

    Abstract Background Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. Results Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy....

  15. Cellular mechanisms of alpha herpesvirus egress: live cell fluorescence microscopy of pseudorabies virus exocytosis.

    OpenAIRE

    Hogue, Ian B.; Jens B Bosse; Jiun-Ruey Hu; Thiberge, Stephan Y.; Enquist, Lynn W.

    2014-01-01

    Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluo...

  16. Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

    Science.gov (United States)

    Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Skalli, Omar; Estraño, Carlos E

    2016-01-01

    Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes. PMID:27515076

  17. Computational modeling of STED microscopy through multiple biological cells under one- and two-photon excitation

    Science.gov (United States)

    Mark, Andrew E.; Davis, Mitchell A.; Starosta, Matthew S.; Dunn, Andrew K.

    2015-03-01

    While superresolution optical microscopy techniques afford enhanced resolution for biological applications, they have largely been used to study structures in isolated cells. We use the FDTD method to simulate the propagation of focused beams for STED microscopy through multiple biological cells. We model depletion beams that provide 2D and 3D confinement of the fluorescence spot and assess the effective PSF of the system as a function of focal depth. We compare the relative size of the STED effective PSF under one- and two-photon excitation. PSF calculations suggest that imaging is possible up to the maximum simulation depth if the fluorescence emission remains detectable.

  18. Dental pulp stem cells (DPSCs) differentiation study by confocal Raman microscopy

    Science.gov (United States)

    Salehi, H.; Collart-Dutilleul, P.-Y.; Gergely, C.; Cuisinier, F. J. G.

    2014-03-01

    Regenerative medicine brings a huge application for Mesenchymal stem cells such as Dental Pulp Stem Cells (DPSCs). Confocal Raman microscopy, a non-invasive, label free , real time and high spatial resolution imaging technique is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800-3000 cm-1 region (C-H stretching) and 960 cm-1 peak (phosphate PO4 3-) were collected. In Dental Pulp Stem Cells 21st day differentiated in buffer solution, phosphate peaks ν1 PO4 3- (first vibrational mode) at 960cm-1 and ν2 PO4 3- at 430cm-1 and ν4 PO4 3- at 585cm-1 are obviously present. Confocal Raman microscopy enables the detection of cell differentiation and it can be used to investigate clinical stem cell research.

  19. Live-Cell Bioorthogonal Chemical Imaging: Stimulated Raman Scattering Microscopy of Vibrational Probes.

    Science.gov (United States)

    Wei, Lu; Hu, Fanghao; Chen, Zhixing; Shen, Yihui; Zhang, Luyuan; Min, Wei

    2016-08-16

    Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. In particular, fluorescence microscopy with the expanding choices of fluorescent probes has provided a comprehensive toolkit to tag and visualize various molecules of interest with exquisite specificity and high sensitivity. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because common fluorescent labels, which are relatively bulky, could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, a bioorthogonal chemical imaging platform has recently been introduced. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes and stable isotopes), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, and biocompatibility for imaging small biomolecules in live systems. In this Account, we review recent technical achievements for visualizing a broad spectrum of small biomolecules, including ribonucleosides and deoxyribonucleosides, amino acids, fatty acids, choline, glucose, cholesterol, and small-molecule drugs in live biological systems ranging from individual cells to animal tissues and model organisms. Importantly, this platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, we discuss further chemical and spectroscopic strategies for multicolor bioorthogonal chemical imaging, a valuable technique in the era of "omics". As a unique tool for biological discovery, this platform has been applied to

  20. Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

    Science.gov (United States)

    Uphoff, Stephan

    2016-01-01

    Summary The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types. PMID:27283312

  1. Morphological Measurement of Living Cells in Methanol with Digital Holographic Microscopy

    Directory of Open Access Journals (Sweden)

    Yunxin Wang

    2013-01-01

    Full Text Available Cell morphology is the research foundation in many applications related to the estimation of cell status, drug response, and toxicity screening. In biomedical field, the quantitative phase detection is an inevitable trend for living cells. In this paper, the morphological change of HeLa cells treated with methanol of different concentrations is detected using digital holographic microscopy. The compact image-plane digital holographic system is designed based on fiber elements. The quantitative phase image of living cells is obtained in combination with numerical analysis. The statistical analysis shows that the area and average optical thickness of HeLa cells treated with 12.5% or 25% methanol reduce significantly, which indicates that the methanol with lower concentration could cause cellular shrinkage. The area of HeLa cells treated with 50% methanol is similar to that of normal cells (P>0.05, which reveals the fixative effect of methanol with higher concentration. The maximum optical thickness of the cells treated with 12.5%, 25%, and 50% methanol is greater than that of untreated cells, which implies the pyknosis of HeLa cells under the effect of methanol. All of the results demonstrate that digital holographic microscopy has supplied a noninvasive imaging alternative to measure the morphological change of label-free living cells.

  2. 3D measurements of live cells via digital holographic microscopy and terahertz spectroscopy

    Science.gov (United States)

    Park, Jun Yong; Oser, Dorian; Iapozzuto, Peter; Norbury, Sean; Mahajan, Supriya; Khmaladze, Alexander; Sharikova, Anna

    2016-03-01

    This is a study of the central nervous system (CNS) cells, including brain micro vascular endothelial cells (BMV) that constitute the blood brain barrier, and C6 glial cells that are the predominant cell in the brain. The cells are exposed to various chemicals by non-invasive, label-free methods. Digital holographic microscopy (DHM) is a technique that records an interference pattern between an object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information, and 3D images are obtained. The measurement of cell cultures by digital holographic microscopy yields information about cell death mechanisms, since these processes are correlated with individual cell volume. Our in-house DHM combines a visible (red) laser source with a conventional microscope base, and LabVIEW-run data processing. Terahertz spectral signatures are associated with structural changes in molecules and provide complementary information about cells. Both CNS cells BMV and C6 cells are treated with the drug "Methamphetamine" (METH), which induces apoptosis in neuronal cells and exhibits decrease in cell volume, a characteristic of cells undergoing apoptosis (induced cell death). METH can cause CNS cell death by cross-talk between mitochondria-, endoplasmic reticulum-, and receptor-mediated apoptotic events, all of which results in drug induced changes in neuroplasticity and significant neuropathology. Doxorubicin (DOX), a popular anticancer drug, is used as a control. We observe that METH treatment resulted in more pronounced cell volume shrinkage in both the BMV and C6 cells, as compared to DOX-induced cell apoptosis.

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

    NARCIS (Netherlands)

    Pluk, H.; Stokes, D.J.; Lich, B.; Wieringa, B.; Fransen, J.A.M.

    2009-01-01

    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 elect

  4. Single Molecule Localization Microscopy of Mammalian Cell Nuclei on the Nanoscale.

    Science.gov (United States)

    Szczurek, Aleksander; Xing, Jun; Birk, Udo J; Cremer, Christoph

    2016-01-01

    Nuclear texture analysis is a well-established method of cellular pathology. It is hampered, however, by the limits of conventional light microscopy (ca. 200 nm). These limits have been overcome by a variety of super-resolution approaches. An especially promising approach to chromatin texture analysis is single molecule localization microscopy (SMLM) as it provides the highest resolution using fluorescent based methods. At the present state of the art, using fixed whole cell samples and standard DNA dyes, a structural resolution of chromatin in the 50-100 nm range is obtained using SMLM. We highlight how the combination of localization microscopy with standard fluorophores opens the avenue to a plethora of studies including the spatial distribution of DNA and associated proteins in eukaryotic cell nuclei with the potential to elucidate the functional organization of chromatin. These views are based on our experience as well as on recently published research in this field. PMID:27446198

  5. Single Molecule Localization Microscopy of Mammalian Cell Nuclei on the Nanoscale

    Science.gov (United States)

    Szczurek, Aleksander; Xing, Jun; Birk, Udo J.; Cremer, Christoph

    2016-01-01

    Nuclear texture analysis is a well-established method of cellular pathology. It is hampered, however, by the limits of conventional light microscopy (ca. 200 nm). These limits have been overcome by a variety of super-resolution approaches. An especially promising approach to chromatin texture analysis is single molecule localization microscopy (SMLM) as it provides the highest resolution using fluorescent based methods. At the present state of the art, using fixed whole cell samples and standard DNA dyes, a structural resolution of chromatin in the 50–100 nm range is obtained using SMLM. We highlight how the combination of localization microscopy with standard fluorophores opens the avenue to a plethora of studies including the spatial distribution of DNA and associated proteins in eukaryotic cell nuclei with the potential to elucidate the functional organization of chromatin. These views are based on our experience as well as on recently published research in this field. PMID:27446198

  6. Darkfield-Confocal Microscopy detection of nanoscale particle internalization by human lung cells

    Directory of Open Access Journals (Sweden)

    Samet James M

    2011-01-01

    Full Text Available Abstract Background Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP. Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm. Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane. Results Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal and scattered transmitted light (Darkfield along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO2 particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependant internalization process. Conclusions DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron

  7. Analysis of Vero cell growth behavior on microcarrier by means of environmental scanning electron microscopy

    Institute of Scientific and Technical Information of China (English)

    邵曼君; 姜蕾; 丛威; 欧阳藩

    2002-01-01

    By using environmental scanning electron microscopy, the morphological changes of Vero cells attached to and grown on the microcarrier Cytodex-3 were observed, and their behavior of adhesion, spreading and proliferation was analyzed. The effect of exogenous fibronectin/ laminin on adhesion and spreading of MCC/Vero cell was studied. The images of ESEM showed that expansion of cell growth was directed toward vacancy space. The growth curve and cell concentration change during the whole culture process were obtained from the statistical counting method based on ESEM images and the crystal violet method. The growth rate of Vero cells increases with increasing the concentration of cell inoculation, that is, the specific growth rate increases quickly with increasing the concentration of cell inoculation. When serum concentration in medium #199 ranged from 5% to 10%, experimental results indicated that serum concentration is one of the important factors influencing cell growth, particularly in the cell adhesion and spreading stage.

  8. Observations of xenon gas-treated barley cells in solution by atomic force microscopy.

    Science.gov (United States)

    Yoshino, T; Sotome, I; Ohtani, T; Isobe, S; Oshita, S; Maekawa, T

    2000-01-01

    Barley cells cut from a sprout were exposed to either air or high-pressure xenon gas for 3 days and the surface of those cells was observed by atomic force microscopy (AFM) to examine the effect of the gas treatment. This method enabled the direct observation of the fresh surface of the barley cells in solution at high resolution. The cuticle layer was preserved on the primary cell wall of 0.48 MPa xenon gas-treated barley cells, while air-treated barley cells lost the cuticle layer from the primary cell wall. These findings indicate that the high-pressure xenon gas treatment is effective to preserve the cuticle layer attached to the primary cell wall. AFM is a powerful tool for the observation of the surface structure of living plant cells in solution. PMID:11108038

  9. Analysis of Vero cell growth behavior on microcarrier by means of environmental scanning electron microscopy.

    Science.gov (United States)

    Shao, Manjun; Jiang, Lei; Cong, Wei; Ouyang, Fan

    2002-04-01

    By using environmental scanning electron microscopy, the morphological changes of Vero cells attached to and grown on the microcarrier Cytodex-3 were observed, and their behavior of adhesion, spreading and proliferation was analyzed. The effect of exogenous fibronectin/ laminin on adhesion and spreading of MCC/Vero cell was studied. The images of ESEM showed that expansion of cell growth was directed toward vacancy space. The growth curve and cell concentration change during the whole culture process were obtained from the statistical counting method based on ESEM images and the crystal violet method. The growth rate of Vero cells increases with increasing the concentration of cell inoculation, that is, the specific growth rate increases quickly with increasing the concentration of cell inoculation. When serum concentration in medium #199 ranged from 5% to 10%, experimental results indicated that serum concentration is one of the important factors influencing cell growth, particularly in the cell adhesion and spreading stage. PMID:18763074

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  12. LOCALIZATION OF BRANCHING ENZYME IN POTATO-TUBER CELLS WITH THE USE OF IMMUNOELECTRON MICROSCOPY

    NARCIS (Netherlands)

    KRAM, AM; OOSTERGETEL, GT; VANBRUGGEN, EFJ

    1993-01-01

    Potato branching enzyme, a key enzyme in the biosynthesis of starch, was localized in amyloplasts in starch-storage cells of potato (Solanum tuberosum L) with the use of immunogold electron microscopy. Branching enzyme was found in the amyloplast stroma, concentrated at the interface of the stroma a

  13. Chip-based optical microscopy for imaging membrane sieve plates of liver scavenger cells

    Science.gov (United States)

    Helle, Øystein I.; Øie, Cristina I.; McCourt, Peter; Ahluwalia, Balpreet S.

    2015-08-01

    The evanescent field on top of optical waveguides is used to image membrane network and sieve-plates of liver endothelial cells. In waveguide excitation, the evanescent field is dominant only near the surface (~100-150 nm) providing a default optical sectioning by illuminating fluorophores in close proximity to the surface and thus benefiting higher signal-to-noise ratio. The sieve plates of liver sinusoidal endothelial cells are present on the cell membrane, thus near-field waveguide chip-based microscopy configuration is preferred over epi-fluorescence. The waveguide chip is compatible with optical fiber components allowing easy multiplexing to different wavelengths. In this paper, we will discuss the challenges and opportunities provided by integrated optical microscopy for imaging cell membranes.

  14. Combined ion conductance and fluorescence confocal microscopy for biological cell membrane transport studies

    Science.gov (United States)

    Shevchuk, A. I.; Novak, P.; Velazquez, M. A.; Fleming, T. P.; Korchev, Y. E.

    2013-09-01

    Optical visualization of nanoscale morphological changes taking place in living biological cells during such important processes as endo- and exocytosis is challenging due to the low refractive index of lipid membranes. In this paper we summarize and discuss advances in the powerful combination of two complementary live imaging techniques, ion conductance and fluorescence confocal microscopy, that allows cell membrane topography to be related with molecular-specific fluorescence at high spatial and temporal resolution. We demonstrate the feasibility of the use of ion conductance microscopy to image apical plasma membrane of mouse embryo trophoblast outgrowth cells at a resolution sufficient to depict single endocytic pits. This opens the possibility to study individual endocytic events in embryo trophoblast outgrowth cells where endocytosis plays a crucial role during early stages of embryo development.

  15. Hierarchical Mergence Approach to Cell Detection in Phase Contrast Microscopy Images

    Directory of Open Access Journals (Sweden)

    Lei Chen

    2014-01-01

    Full Text Available Phase contrast microscope is one of the most universally used instruments to observe long-term cell movements in different solutions. Most of classic segmentation methods consider a homogeneous patch as an object, while the recorded cell images have rich details and a lot of small inhomogeneous patches, as well as some artifacts, which can impede the applications. To tackle these challenges, this paper presents a hierarchical mergence approach (HMA to extract homogeneous patches out and heuristically add them up. Initially, the maximum region of interest (ROI, in which only cell events exist, is drawn by using gradient information as a mask. Then, different levels of blurring based on kernel or grayscale morphological operations are applied to the whole image to produce reference images. Next, each of unconnected regions in the mask is applied with Otsu method independently according to different reference images. Consequently, the segmentation result is generated by the combination of usable patches in all informative layers. The proposed approach is more than simply a fusion of the basic segmentation methods, but a well-organized strategy that integrates these basic methods. Experiments demonstrate that the proposed method outperforms previous methods within our datasets.

  16. Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells

    OpenAIRE

    Mauzeroll, Janine; Bard, Allen J.

    2004-01-01

    The uptake of menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy. The progression of the in vitro reaction between menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV–visible) behavior. By observing the scanning electrochemical microscope tip current of yeast cells suspended in a menadione-containing solution,...

  17. Label-free detection of anticancer drug paclitaxel in living cells by confocal Raman microscopy

    Science.gov (United States)

    Salehi, H.; Derely, L.; Vegh, A.-G.; Durand, J.-C.; Gergely, C.; Larroque, C.; Fauroux, M.-A.; Cuisinier, F. J. G.

    2013-03-01

    Confocal Raman microscopy, a non-invasive, label-free, and high spatial resolution imaging technique is employed to trace the anticancer drug paclitaxel in living Michigan Cancer Foundation-7 (MCF-7) cells. The Raman images were treated by K-mean cluster analysis to detect the drug in cells. Distribution of paclitaxel in cells is verified by calculating the correlation coefficient between the reference spectrum of the drug and the whole Raman image spectra. A time dependent gradual diffusion of paclitaxel all over the cell is observed suggesting a complementary picture of the pharmaceutical action of this drug based on rapid binding of free tubulin to crystallized paclitaxel.

  18. Intracellular accumulation and dissolution of silver nanoparticles in L-929 fibroblast cells using live cell time-lapse microscopy.

    Science.gov (United States)

    Wildt, Bridget E; Celedon, Alfredo; Maurer, Elizabeth I; Casey, Brendan J; Nagy, Amber M; Hussain, Saber M; Goering, Peter L

    2016-08-01

    Cytotoxicity assessments of nanomaterials, such as silver nanoparticles, are challenging due to interferences with test reagents and indicators as well uncertainties in dosing as a result of the complex nature of nanoparticle intracellular accumulation. Furthermore, current theories suggest that silver nanoparticle cytotoxicity is a result of silver nanoparticle dissolution and subsequent ion release. This study introduces a novel technique, nanoparticle associated cytotoxicity microscopy analysis (NACMA), which combines fluorescence microscopy detection using ethidium homodimer-1, a cell permeability marker that binds to DNA after a cell membrane is compromised (a classical dead-cell indicator dye), with live cell time-lapse microscopy and image analysis to simultaneously investigate silver nanoparticle accumulation and cytotoxicity in L-929 fibroblast cells. Results of this method are consistent with traditional methods of assessing cytotoxicity and nanoparticle accumulation. Studies conducted on 10, 50, 100 and 200 nm silver nanoparticles reveal size dependent cytotoxicity with particularly high cytotoxicity from 10 nm particles. In addition, NACMA results, when combined with transmission electron microscopy imaging, reveal direct evidence of intracellular silver ion dissolution and possible nanoparticle reformation within cells for all silver nanoparticle sizes. PMID:26643278

  19. Evaluation of Filtering Bleb Function after Trabeculectomy with Mitomycin C Using Biomicroscopy, Anterior Segment Optical Coherence Tomography and In Vivo Confocal Microscopy

    Directory of Open Access Journals (Sweden)

    Suzan Güven Yılmaz

    2015-08-01

    Full Text Available Objectives: To analyze and assess compatibility of trabeculectomy filtering bleb characteristics and appearances using biomicroscopy, anterior segment optical coherence tomography (AS-OCT and in vivo confocal microscopy (IVCM. Materials and Methods: Twenty-eight eyes of 28 patients who underwent glaucoma filtering surgery with mitomycin C in our clinic between 2009 and 2013 were evaluated. Morphological appearances of the blebs on slit-lamp biomicroscopy were defined according to the Moorfields bleb classification system. For the internal tissue assessment of blebs, AS-OCT and IVCM were performed. Bleb biometric parameters such as length, height and bleb wall thickness were assessed by AS-OCT; conjunctival epithelial-stromal cyst, structural network of conjunctival stroma and vascularisation were examined with IVCM. The relation between biomicroscopic morphological staging and bleb characteristics detected on AS-OCT and IVCM were assessed. Results: The mean age of the 28 patients (16 male, 12 female was 57.2±15.9 (19 to 79 years. The mean time elapsed between surgery and examination was 29.2±19.2 (6 to 68 months. According to biomicroscopic appearance, 17 (60.7% blebs were functional (13 diffuse, 4 microcystic, whereas 11 (39.3% blebs were non-functional (9 flat, 2 encapsulated. In the comparison of non-functional and functional blebs, functional blebs were found to be superior in terms of biometric parameters on AS-OCT assessment (p<0.05. Higher number of epithelial and stromal cysts and less vascularisation were detected by IVCM in functional blebs when compared with non-functional blebs (p<0.05. Conclusion: Biomicroscopic appearances and characteristics on AS-OCT and IVCM of filtration blebs are consistent with each other. Besides biomicroscopic examination, which is an easy and practical method for determining bleb morphology, cross-sectional images obtained by AS-OCT and IVCM provide objective data regarding internal structure and

  20. Tumor cell survival dependence on helical tomotherapy, continuous arc and segmented dose delivery

    Science.gov (United States)

    Yang, Wensha; Wang, Li; Larner, James; Read, Paul; Benedict, Stan; Sheng, Ke

    2009-11-01

    The temporal pattern of radiation delivery has been shown to influence the tumor cell survival fractions for the same radiation dose. To study the effect more specifically for state of the art rotational radiation delivery modalities, 2 Gy of radiation dose was delivered to H460 lung carcinoma, PC3 prostate cancer cells and MCF-7 breast tumor cells by helical tomotherapy (HT), seven-field LINAC (7F), and continuous dose delivery (CDD) over 2 min that simulates volumetric rotational arc therapy. Cell survival was measured by the clonogenic assay. The number of viable H460 cell colonies was 23.2 ± 14.4% and 27.7 ± 15.6% lower when irradiated by CDD compared with HT and 7F, respectively, and the corresponding values were 36.8 ± 18.9% and 35.3 ± 18.9% lower for MCF7 cells (p < 0.01). The survival of PC3 was also lower when irradiated by CDD than by HT or 7F but the difference was not as significant (p = 0.06 and 0.04, respectively). The higher survival fraction from HT delivery was unexpected because 90% of the 2 Gy was delivered in less than 1 min at a significantly higher dose rate than the other two delivery techniques. The results suggest that continuous dose delivery at a constant dose rate results in superior in vitro tumor cell killing compared with prolonged, segmented or variable dose rate delivery.

  1. Tumor cell survival dependence on helical tomotherapy, continuous arc and segmented dose delivery

    Energy Technology Data Exchange (ETDEWEB)

    Yang Wensha; Wang Li; Larner, James; Read, Paul; Benedict, Stan; Sheng Ke [Department of Radiation Oncology, University of Virginia, VA (United States)], E-mail: ks2mc@virginia.edu

    2009-11-07

    The temporal pattern of radiation delivery has been shown to influence the tumor cell survival fractions for the same radiation dose. To study the effect more specifically for state of the art rotational radiation delivery modalities, 2 Gy of radiation dose was delivered to H460 lung carcinoma, PC3 prostate cancer cells and MCF-7 breast tumor cells by helical tomotherapy (HT), seven-field LINAC (7F), and continuous dose delivery (CDD) over 2 min that simulates volumetric rotational arc therapy. Cell survival was measured by the clonogenic assay. The number of viable H460 cell colonies was 23.2 {+-} 14.4% and 27.7 {+-} 15.6% lower when irradiated by CDD compared with HT and 7F, respectively, and the corresponding values were 36.8 {+-} 18.9% and 35.3 {+-} 18.9% lower for MCF7 cells (p < 0.01). The survival of PC3 was also lower when irradiated by CDD than by HT or 7F but the difference was not as significant (p = 0.06 and 0.04, respectively). The higher survival fraction from HT delivery was unexpected because 90% of the 2 Gy was delivered in less than 1 min at a significantly higher dose rate than the other two delivery techniques. The results suggest that continuous dose delivery at a constant dose rate results in superior in vitro tumor cell killing compared with prolonged, segmented or variable dose rate delivery.

  2. An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

    Science.gov (United States)

    Bohórquez, Diego V; Samsa, Leigh A; Roholt, Andrew; Medicetty, Satish; Chandra, Rashmi; Liddle, Rodger A

    2014-01-01

    The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells. PMID:24587096

  3. An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

    Directory of Open Access Journals (Sweden)

    Diego V Bohórquez

    Full Text Available The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM. Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

  4. Effect of cold plasma on glial cell morphology studied by atomic force microscopy.

    Directory of Open Access Journals (Sweden)

    Nina Recek

    Full Text Available The atomic force microscope (AFM is broadly used to study the morphology of cells. The morphological characteristics and differences of the cell membrane between normal human astrocytes and glial tumor cells are not well explored. Following treatment with cold atmospheric plasma, evaluation of the selective effect of plasma on cell viability of tumor cells is poorly understood and requires further evaluation. Using AFM we imaged morphology of glial cells before and after cold atmospheric plasma treatment. To look more closely at the effect of plasma on cell membrane, high resolution imaging was used. We report the differences between normal human astrocytes and human glioblastoma cells by considering the membrane surface details. Our data, obtained for the first time on these cells using atomic force microscopy, argue for an architectural feature on the cell membrane, i.e. brush layers, different in normal human astrocytes as compared to glioblastoma cells. The brush layer disappears from the cell membrane surface of normal E6/E7 cells and is maintained in the glioblastoma U87 cells after plasma treatment.

  5. Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy

    Science.gov (United States)

    Miller, Mark J.; Wei, Sindy H.; Cahalan, Michael D.; Parker, Ian

    2003-03-01

    The recirculation of T cells between the blood and secondary lymphoid organs requires that T cells are motile and sensitive to tissue-specific signals. T cell motility has been studied in vitro, but the migratory behavior of individual T cells in vivo has remained enigmatic. Here, using intravital two-photon laser microscopy, we imaged the locomotion and trafficking of naïve CD4+ T cells in the inguinal lymph nodes of anesthetized mice. Intravital recordings deep within the lymph node showed T cells flowing rapidly in the microvasculature and captured individual homing events. Within the diffuse cortex, T cells displayed robust motility with an average velocity of 11 μm·min1. T cells cycled between states of low and high motility roughly every 2 min, achieving peak velocities >25 μm·min1. An analysis of T cell migration in 3D space revealed a default trafficking program analogous to a random walk. Our results show that naïve T cells do not migrate collectively, as they might under the direction of pervasive chemokine gradients. Instead, they appear to migrate as autonomous agents, each cell taking an independent trafficking path. Our results call into question the role of chemokine gradients for basal T cell trafficking within T cell areas and suggest that antigen detection may result from a stochastic process through which a random walk facilitates contact with antigen-presenting dendritic cells.

  6. Solid oxide fuel cell anode image segmentation based on a novel quantum-inspired fuzzy clustering

    Science.gov (United States)

    Fu, Xiaowei; Xiang, Yuhan; Chen, Li; Xu, Xin; Li, Xi

    2015-12-01

    High quality microstructure modeling can optimize the design of fuel cells. For three-phase accurate identification of Solid Oxide Fuel Cell (SOFC) microstructure, this paper proposes a novel image segmentation method on YSZ/Ni anode Optical Microscopic (OM) images. According to Quantum Signal Processing (QSP), the proposed approach exploits a quantum-inspired adaptive fuzziness factor to adaptively estimate the energy function in the fuzzy system based on Markov Random Filed (MRF). Before defuzzification, a quantum-inspired probability distribution based on distance and gray correction is proposed, which can adaptively adjust the inaccurate probability estimation of uncertain points caused by noises and edge points. In this study, the proposed method improves accuracy and effectiveness of three-phase identification on the micro-investigation. It provides firm foundation to investigate the microstructural evolution and its related properties.

  7. Live cell imaging based on surface plasmon-enhanced fluorescence microscopy using random nanostructures

    Science.gov (United States)

    Oh, Youngjin; Lee, Wonju; Son, Taehwang; Kim, Sook Young; Shin, Jeon-Soo; Kim, Donghyun

    2014-02-01

    Localized surface plasmon enhanced microscopy based on nanoislands of random spatial distribution was demonstrated for imaging live cells and molecular interactions. Nanoislands were produced without lithography by high temperature annealing under various processing conditions. The localization of near-field distribution that is associated with localized surface plasmon on metallic random nanoislands was analyzed theoretically and experimentally in comparison with periodic nanostructures. For experimental validation in live cell imaging, mouse macrophage-like cell line stained with Alexa Fluor 488 was prepared on nanoislands. The results suggest the possibility of attaining the imaging resolution on the order of 80 nm.

  8. Combination of Small Molecule Microarray and Confocal Microscopy Techniques for Live Cell Staining Fluorescent Dye Discovery

    Directory of Open Access Journals (Sweden)

    Attila Bokros

    2013-08-01

    Full Text Available Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS methods. In the present study a combination of small molecule microarray (SMM prescreening and confocal laser scanning microscopy (CLSM was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment-specific, cell-permeable (or plasma membrane-targeted fluorochromes were identified. Their cytotoxicity was tested and found that between 1–10 micromolar range, they were non-toxic even during long-term incubations.

  9. The influence of physical and physiological cues on atomic force microscopy-based cell stiffness assessment.

    Directory of Open Access Journals (Sweden)

    Yu-Wei Chiou

    Full Text Available Atomic force microscopy provides a novel technique for differentiating the mechanical properties of various cell types. Cell elasticity is abundantly used to represent the structural strength of cells in different conditions. In this study, we are interested in whether physical or physiological cues affect cell elasticity in Atomic force microscopy (AFM-based assessments. The physical cues include the geometry of the AFM tips, the indenting force and the operating temperature of the AFM. All of these cues show a significant influence on the cell elasticity assessment. Sharp AFM tips create a two-fold increase in the value of the effective Young's modulus (E(eff relative to that of the blunt tips. Higher indenting force at the same loading rate generates higher estimated cell elasticity. Increasing the operation temperature of the AFM leads to decreases in the cell stiffness because the structure of actin filaments becomes disorganized. The physiological cues include the presence of fetal bovine serum or extracellular matrix-coated surfaces, the culture passage number, and the culture density. Both fetal bovine serum and the extracellular matrix are critical for cells to maintain the integrity of actin filaments and consequently exhibit higher elasticity. Unlike primary cells, mouse kidney progenitor cells can be passaged and maintain their morphology and elasticity for a very long period without a senescence phenotype. Finally, cell elasticity increases with increasing culture density only in MDCK epithelial cells. In summary, for researchers who use AFM to assess cell elasticity, our results provide basic and significant information about the suitable selection of physical and physiological cues.

  10. Investigation of multi-junction solar cells using electrostatic force microscopy methods

    International Nuclear Information System (INIS)

    Multi-junction III–V solar cells are designed to have a much broader absorption of the solar spectrum than Si-based or single junctions, thus yield the highest conversion. The conversion efficiency can be further scaled with sun concentration. The ability of high conversion efficiencies makes multi-junction prime candidates for fine-tuning explorations aimed at getting closer to the theoretical efficiencies. In this paper, we report on electrostatic force microscopy (EFM) measurements of the built-in potential of multi-junction III–V semiconductor-based solar cells. Kelvin probe force microscopy (KPFM) was employed to qualitatively study the width and electrical properties of individual junctions, i.e., built-in potential, activity, and thickness of the p–n junctions. In addition, the voltage drops across individual solar cell p–n junctions were measured using Kelvin probe microscopy under various operation conditions: dark; illuminated; short-circuit; and biased. We present a method which enables the measurement of a working structure, while focusing on the electrical characteristics of an individual junction by virtue of selecting the spectral range of the illumination used. We show that these pragmatic studies can provide a feedback to improve photovoltaic device design, particularly of operation under a current mismatched situation. This new analysis technique offers additional insights into behavior of the multi-junction solar cell and shows promise for further progress in this field. - Highlights: • We explore the electronic structure of III–V based high efficiency solar cells. • Qualitative study of the solar cell operation characteristics is presented. • Quantitative study of the electrostatic landscape of operational high efficiency devices is presented. • Precise identification of the epitaxially grown p–n and tunnel junctions in the multi-junction solar cell. • Influence of illumination conditions and cell biasing on each p

  11. Intravital two-photon microscopy of immune cell dynamics in corneal lymphatic vessels.

    Directory of Open Access Journals (Sweden)

    Philipp Steven

    Full Text Available BACKGROUND: The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years. METHODOLOGY/PRINCIPAL FINDINGS: We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM. Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1-5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces. CONCLUSIONS: To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible

  12. Investigation of multi-junction solar cells using electrostatic force microscopy methods

    Energy Technology Data Exchange (ETDEWEB)

    Moczała, M., E-mail: magdalena.moczala@pwr.wroc.pl [Wrocław University of Technology, Faculty of Microsystem Electronics and Photonics, Division of Metrology of Micro- and Nanostructures, ul. Z. Janiszewskiego 11/17, 50-372 Wrocław (Poland); Sosa, N.; Topol, A. [IBM Thomas J. Watson Research Center, P.O. Box 218, Yorktown Heights, NY 10598 (United States); Gotszalk, T. [Wrocław University of Technology, Faculty of Microsystem Electronics and Photonics, Division of Metrology of Micro- and Nanostructures, ul. Z. Janiszewskiego 11/17, 50-372 Wrocław (Poland)

    2014-06-01

    Multi-junction III–V solar cells are designed to have a much broader absorption of the solar spectrum than Si-based or single junctions, thus yield the highest conversion. The conversion efficiency can be further scaled with sun concentration. The ability of high conversion efficiencies makes multi-junction prime candidates for fine-tuning explorations aimed at getting closer to the theoretical efficiencies. In this paper, we report on electrostatic force microscopy (EFM) measurements of the built-in potential of multi-junction III–V semiconductor-based solar cells. Kelvin probe force microscopy (KPFM) was employed to qualitatively study the width and electrical properties of individual junctions, i.e., built-in potential, activity, and thickness of the p–n junctions. In addition, the voltage drops across individual solar cell p–n junctions were measured using Kelvin probe microscopy under various operation conditions: dark; illuminated; short-circuit; and biased. We present a method which enables the measurement of a working structure, while focusing on the electrical characteristics of an individual junction by virtue of selecting the spectral range of the illumination used. We show that these pragmatic studies can provide a feedback to improve photovoltaic device design, particularly of operation under a current mismatched situation. This new analysis technique offers additional insights into behavior of the multi-junction solar cell and shows promise for further progress in this field. - Highlights: • We explore the electronic structure of III–V based high efficiency solar cells. • Qualitative study of the solar cell operation characteristics is presented. • Quantitative study of the electrostatic landscape of operational high efficiency devices is presented. • Precise identification of the epitaxially grown p–n and tunnel junctions in the multi-junction solar cell. • Influence of illumination conditions and cell biasing on each p

  13. Light sheet microscopy for tracking single molecules on the apical surface of living cells.

    Science.gov (United States)

    Li, Yu; Hu, Ying; Cang, Hu

    2013-12-12

    Single particle tracking is a powerful tool to study single molecule dynamics in living biological samples. However, current tracking techniques, which are based mainly on epifluorescence, confocal, or TIRF microscopy, have difficulties in tracking single molecules on the apical surface of a cell. We present here a three-dimensional (3D) single particle tracking technique that is based on prism coupled light-sheet microscopy (PCLSM). This novel design provides a signal-to-noise ratio comparable to confocal microscopy while it has the capability of illuminating at arbitrary depth. We demonstrate tracking of single EGF molcules on the apical surface of live cell membranes from their binding to EGF receptors until they are internalized or photobleached. We found that EGF exhibits multiple diffusion behaviors on live A549 cell membranes. At room temperature, the average diffusion coefficient of EGF on A549 cells was measured to be 0.13 μm(2)/s. Depletion of cellular cholesterol with methyl-β-cyclodextrin leads to a broader distribution of diffusion coefficients and an increase of the average diffusion coefficient at room temperature. This light-sheet based 3D single particle tracking technique solves the technique difficulty of tracking single particles on apical membranes and is able to document the whole "lifetime" of a particle from binding till photobleaching or internalization. PMID:23895420

  14. Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Kim Myung K

    2011-09-01

    Full Text Available Abstract Background Total internal reflection fluorescence microscopy (TIRFM is a powerful tool for observing fluorescently labeled molecules on the plasma membrane surface of animal cells. However, the utility of TIRFM in plant cell studies has been limited by the fact that plants have cell walls, thick peripheral layers surrounding the plasma membrane. Recently, a new technique known as variable-angle epifluorescence microscopy (VAEM was developed to circumvent this problem. However, the lack of a detailed analysis of the optical principles underlying VAEM has limited its applications in plant-cell biology. Results Here, we present theoretical and experimental evidence supporting the use of variable-angle TIRFM in observations of intact plant cells. We show that when total internal reflection occurs at the cell wall/cytosol interface with an appropriate angle of incidence, an evanescent wave field of constant depth is produced inside the cytosol. Results of experimental TIRFM observations of the dynamic behaviors of phototropin 1 (a membrane receptor protein and clathrin light chain (a vesicle coat protein support our theoretical analysis. Conclusions These findings demonstrate that variable-angle TIRFM is appropriate for quantitative live imaging of cells in intact tissues of Arabidopsis thaliana.

  15. Atomic force microscopy analysis of progenitor corneal epithelial cells fractionated by a rapid centrifugation isolation technique.

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    Full Text Available PURPOSE: To investigate the use of atomic force microscopy (AFM to image the three groups of corneal epithelial cells fractionated by a novel rapid centrifugation isolation technique. METHODS: Epithelial cells harvested from primary cultures of rabbit limbal rings were centrifuged onto uncoated dishes, first at 1400 rpm and then at 1800 rpm. The adherent cells after centrifugation at 1400 rpm (ATC1, the adherent cells at 1800 rpm (ATC2 and the non-adherent cells at 1800 rpm (NAC were investigated for BrdU retention and were subjected to contact mode AFM and Transmission Electron Microscopy (TEM. RESULTS: Compared with unfractionated cells, the ATC1 group, accounting for about 10% of the whole population, was enriched in BrdU label-retaining cells. There were dramatic overall shape, surface membrane and intra-cellular ultrastructure differences noted among ATC1, ATC2 and NAC populations. The whole cell roughness measurements were 21.1±1.5 nm, 79.5±3.4 nm and 103±4.6 nm for the ATC1, ATC2 and NAC groups, respectively. The mero-nucleus roughness measurements were 34.2±1.7 nm, 13.0±0.8 nm and 8.5±0.5 nm in the ATC1, ATC2 and NAC populations, respectively. CONCLUSIONS: AFM was found to be a good tool for distinguishing among the three groups of cells. BrdU label retention, the AFM parameters and TEM together suggest that the ATC1, ATC2 and NAC populations may be progenitor corneal epithelial cells, transit amplifying cells and terminal differentiation cells, respectively.

  16. Total 3D imaging of phase objects using defocusing microscopy: application to red blood cells

    CERN Document Server

    Roma, P M S; Amaral, F T; Agero, U; Mesquita, O N

    2014-01-01

    We present Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total 3D imaging of transparent objects. By total 3D imaging we mean the determination of the actual shapes of the upper and lower surfaces of a phase object. We propose a new methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic and hypertonic solutions. For each situation the shape of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of RBCs surfaces due to adhesion on the glass-substrate. The axial resolution of our technique allowed us to image surface-membranes separated by distances as small as 300 nm. Finally, we determine volume, superficial area, sphericity index and RBCs refractive index for each osmotic condition.

  17. Environmental cell assembly for use in for use in spectroscopy and microscopy applications

    Science.gov (United States)

    Stowe, Ashley Clinton; Smyrl, Norman; Hallman, Jr., Russell L.

    2014-09-02

    An environmental cell assembly for use in microscopy and spectroscopy applications, including: an environmentally sealed body assembly configured to selectively hold and contain a sample; a plurality of ports manufactured into one or more surfaces of the body assembly for one or more of evacuating the body assembly and injecting a gas into or removing a gas from the body assembly; a port manufactured into a surface of the body assembly for receiving a translating stage configured to move the sample within the body assembly; and a port manufactured into a surface of the body assembly for receiving one or more lenses utilized in a microscopy or spectroscopy application; wherein the one or more lenses are disposed adjacent the sample without intervening structures disposed there between. The cell assembly also includes a port manufactured into a surface of the body assembly for retaining a window and providing visualization of the sample.

  18. Observation of Insulin Exocytosis by a Pancreatic (3 Cell Line with Total Internal Reflection Fluorescence Microscopy

    Institute of Scientific and Technical Information of China (English)

    Zhao-ying Fu; Ya-ping Wang; Yu Chen

    2011-01-01

    @@ INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radio-immunoassay.However,these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis.In recent years,an imaging technique known as total internal reflection fluorescence (TIRF) microscopy has been employed to study insulin secretion.1-4 This imaging technique can explore events taking place near or on live cell membrane,such as secretory granule movement,exocytosis,vesicle content release,and membrane fusion.5-10 In the present paper,we applied TIRF microscopy to the observation of insulin exocytosis by the pancreatic β cell line Ins-1.

  19. Imaging Gold Nanoparticles in Living Cells Environments using Heterodyne Digital Holographic Microscopy

    CERN Document Server

    Warnasooriya, Nilanthi; Bun, Philippe; Tessier, Gilles; Coppey-Moisan, Maite; Desbiolles, Pierre; Atlan, Michael; Abboud, Marie; Gross, Michel

    2009-01-01

    This paper describes an imaging microscopic technique based on heterodyne digital holography where subwavelength-sized gold colloids can be imaged in cell environment. Surface cellular receptors of 3T3 mouse fibroblasts are labeled with 40 nm gold nanoparticles, and the biological specimen is imaged in a total internal reflection configuration with holographic microscopy. Due to a higher scattering efficiency of the gold nanoparticles versus that of cellular structures, accurate localization of a gold marker is obtained within a 3D mapping of the entire sample's scattered field, with a lateral precision of 5 nm and 100 nm in the x,y and in the z directions respectively, demonstrating the ability of holographic microscopy to locate nanoparticles in living cells environments.

  20. Localization of bleomycin in a single living cell using three-photon excitation microscopy

    Science.gov (United States)

    Abraham, Anil T.; Brautigan, David L.; Hecht, Sidney M.; Periasamy, Ammasi

    2001-04-01

    Bleomycin has been used in the clinic as a chemotherapeutic agent for the treatment of several neoplasms, including non-Hodgkins lymphomas, squamous cell carcinomas, and testicular tumors. The effectiveness of bleomycin is believed to be derived from its ability to bind and oxidatively cleave DNA in the presence of a iron cofactor in vivo. A substantial amount of data on BLM has been collected, there is little information concerning the effects of bleomycin in living cells. In order to obtain data pertinent to the effects of BLM in intact cells, we have exploited the intrinsic fluorescence property of bleomycin to monitor the uptake of the drug in mammalian cells. We employed two light microscopy techniques, a wide-field and three-photon excitation (760 nm) fluorescence microscopy. Treatment of HeLa cells with bleomycin resulted in rapid to localization within the cells. In addition data collected from the wide field experiments, three-photon excitation of BLM which considerably reduced the phototoxic effect compared with UV light excitation in the wide-field microscopy indicated co-localization of the drug to regions of the cytoplasm occupied by the endoplasmic reticulum probe, DiOC5. The data clearly indicates that the cellular uptake of bleomycin after one minute includes the nucleus as well as in cytoplasm. Contrary to previous studies, which indicate chromosomal DNA as the target of bleomycin, the current findings suggest that the drug is distributed to many areas within the cell, including the endoplasmic reticulum, an organelle that is known to contain ribonucleic acids.

  1. Investigation of integrin expression on the surface of osteoblast-like cells by atomic force microscopy

    International Nuclear Information System (INIS)

    The transforming growth factor β1 (TGF-β1) is a human cytokine which has been demonstrated to modulate cell surface integrin repertoire. In this work integrin expression in response to TGF-β1 stimulation has been investigated on the surface of human osteoblast-like cells. We used atomic force microscopy (AFM) and confocal laser scanning microscopy to assess integrin expression and to evaluate their distribution over the dorsal side of the plasma membrane. AFM probes have been covalently functionalised with monoclonal antibodies specific to the β1 integrin subunit. Force curves have been collected in order to obtain maps of the interaction between the immobilized antibody and the respective cell membrane receptors. Adhesion peaks have been automatically detected by means of an ad hoc developed data analysis software. The specificity of the detected interactions has been assessed by adding free antibody in the solution and monitoring the dramatic decrease in the recorded interactions. In addition, the effect of TGF-β1 treatment on both the fluorescence signal and the adhesion events has been tested. The level of expression of the β1 integrin subunit was enhanced by TGF-β1. As a further analysis, the adhesion force of the single living cells to the substrate was measured by laterally pushing the cell with the AFM tip and measuring the force necessary to displace it. The treatment with TGF-β1 resulted in a decrease of the cell/substrate adhesion force. Results obtained by AFM have been validated by confocal laser scanning microscopy thus demonstrating the high potential of the AFM technique for the investigation of cell surface receptors distribution and trafficking at the nanoscale.

  2. Investigation into local cell mechanics by atomic force microscopy mapping and optical tweezer vertical indentation.

    Science.gov (United States)

    Coceano, G; Yousafzai, M S; Ma, W; Ndoye, F; Venturelli, L; Hussain, I; Bonin, S; Niemela, J; Scoles, G; Cojoc, D; Ferrari, E

    2016-02-12

    Investigating the mechanical properties of cells could reveal a potential source of label-free markers of cancer progression, based on measurable viscoelastic parameters. The Young's modulus has proved to be the most thoroughly studied so far, however, even for the same cell type, the elastic modulus reported in different studies spans a wide range of values, mainly due to the application of different experimental conditions. This complicates the reliable use of elasticity for the mechanical phenotyping of cells. Here we combine two complementary techniques, atomic force microscopy (AFM) and optical tweezer microscopy (OTM), providing a comprehensive mechanical comparison of three human breast cell lines: normal myoepithelial (HBL-100), luminal breast cancer (MCF-7) and basal breast cancer (MDA-MB-231) cells. The elastic modulus was measured locally by AFM and OTM on single cells, using similar indentation approaches but different measurement parameters. Peak force tapping AFM was employed at nanonewton forces and high loading rates to draw a viscoelastic map of each cell and the results indicated that the region on top of the nucleus provided the most meaningful results. OTM was employed at those locations at piconewton forces and low loading rates, to measure the elastic modulus in a real elastic regime and rule out the contribution of viscous forces typical of AFM. When measured by either AFM or OTM, the cell lines' elasticity trend was similar for the aggressive MDA-MB-231 cells, which were found to be significantly softer than the other two cell types in both measurements. However, when comparing HBL-100 and MCF-7 cells, we found significant differences only when using OTM. PMID:26683826

  3. Investigation into local cell mechanics by atomic force microscopy mapping and optical tweezer vertical indentation

    Science.gov (United States)

    Coceano, G.; Yousafzai, M. S.; Ma, W.; Ndoye, F.; Venturelli, L.; Hussain, I.; Bonin, S.; Niemela, J.; Scoles, G.; Cojoc, D.; Ferrari, E.

    2016-02-01

    Investigating the mechanical properties of cells could reveal a potential source of label-free markers of cancer progression, based on measurable viscoelastic parameters. The Young’s modulus has proved to be the most thoroughly studied so far, however, even for the same cell type, the elastic modulus reported in different studies spans a wide range of values, mainly due to the application of different experimental conditions. This complicates the reliable use of elasticity for the mechanical phenotyping of cells. Here we combine two complementary techniques, atomic force microscopy (AFM) and optical tweezer microscopy (OTM), providing a comprehensive mechanical comparison of three human breast cell lines: normal myoepithelial (HBL-100), luminal breast cancer (MCF-7) and basal breast cancer (MDA-MB-231) cells. The elastic modulus was measured locally by AFM and OTM on single cells, using similar indentation approaches but different measurement parameters. Peak force tapping AFM was employed at nanonewton forces and high loading rates to draw a viscoelastic map of each cell and the results indicated that the region on top of the nucleus provided the most meaningful results. OTM was employed at those locations at piconewton forces and low loading rates, to measure the elastic modulus in a real elastic regime and rule out the contribution of viscous forces typical of AFM. When measured by either AFM or OTM, the cell lines’ elasticity trend was similar for the aggressive MDA-MB-231 cells, which were found to be significantly softer than the other two cell types in both measurements. However, when comparing HBL-100 and MCF-7 cells, we found significant differences only when using OTM.

  4. Long-term live cell microscopy studies of lipid droplet fusion dynamics in adipocytes[S

    OpenAIRE

    Jüngst, Christian; Klein, Matthias; Zumbusch, Andreas

    2013-01-01

    During the adipogenic differentiation process of mesenchymal stem cells, lipid droplets (LDs) grow slowly by transferring lipids between each other. Recent findings hint at the possibility that a fusion pore is involved. In this study, we analyze lipid transfer data obtained in long-term label-free microscopy studies in the framework of a Hagen-Poiseuille model. The data obtained show a LD fusion process in which the lipid transfer directionality depends on the size difference between LDs, wh...

  5. Fluorescein Punctate Staining Traced to Superficial Corneal Epithelial Cells by Impression Cytology and Confocal Microscopy

    OpenAIRE

    Mokhtarzadeh, Maryam; Casey, Richard; Glasgow, Ben J.

    2011-01-01

    Punctate fluorescein staining is an important sign in ocular surface disease but its basis is controversial. The common view is that the spots reflect small epithelial defects. In this study, clinicocytologic and histopathologic correlation of punctate stains in dry eye disease was performed. The hyperfluorescent spots were traced from slit lamp examination to confocal microscopy of tissue to reveal that fluorescent superficial epithelial cells are basis of punctate staining.

  6. Automatic cell detection in bright-field microscopy for microbeam irradiation studies

    International Nuclear Information System (INIS)

    Automatic cell detection in bright-field illumination microscopy is challenging due to cells’ inherent optical properties. Applications including individual cell microbeam irradiation demand minimisation of additional cell stressing factors, so contrast-enhancing fluorescence microscopy should be avoided. Additionally, the use of optically non-homogeneous substrates amplifies the problem. This research focuses on the design of a method for automatic cell detection on polypropylene substrate, suitable for microbeam irradiation. In order to fulfil the relative requirements, the Harris corner detector was employed to detect apparent cellular features. These features-corners were clustered based on a dual-clustering technique according to the density of their distribution across the image. Weighted centroids were extracted from the clusters of corners and constituted the targets for irradiation. The proposed method identified more than 88% of the 1,738 V79 Chinese hamster cells examined. Moreover, a processing time of 2.6 s per image fulfilled the requirements for a near real-time cell detection-irradiation system. (paper)

  7. Automatic cell detection in bright-field microscopy for microbeam irradiation studies

    Science.gov (United States)

    Georgantzoglou, A.; Merchant, M. J.; Jeynes, J. C. G.; Wéra, A.-C.; Kirkby, K. J.; Kirkby, N. F.; Jena, R.

    2015-08-01

    Automatic cell detection in bright-field illumination microscopy is challenging due to cells’ inherent optical properties. Applications including individual cell microbeam irradiation demand minimisation of additional cell stressing factors, so contrast-enhancing fluorescence microscopy should be avoided. Additionally, the use of optically non-homogeneous substrates amplifies the problem. This research focuses on the design of a method for automatic cell detection on polypropylene substrate, suitable for microbeam irradiation. In order to fulfil the relative requirements, the Harris corner detector was employed to detect apparent cellular features. These features-corners were clustered based on a dual-clustering technique according to the density of their distribution across the image. Weighted centroids were extracted from the clusters of corners and constituted the targets for irradiation. The proposed method identified more than 88% of the 1,738 V79 Chinese hamster cells examined. Moreover, a processing time of 2.6 s per image fulfilled the requirements for a near real-time cell detection-irradiation system.

  8. Nanoscale Electric Permittivity of Single Bacterial Cells at Gigahertz Frequencies by Scanning Microwave Microscopy.

    Science.gov (United States)

    Biagi, Maria Chiara; Fabregas, Rene; Gramse, Georg; Van Der Hofstadt, Marc; Juárez, Antonio; Kienberger, Ferry; Fumagalli, Laura; Gomila, Gabriel

    2016-01-26

    We quantified the electric permittivity of single bacterial cells at microwave frequencies and nanoscale spatial resolution by means of near-field scanning microwave microscopy. To this end, calibrated complex admittance images have been obtained at ∼19 GHz and analyzed with a methodology that removes the nonlocal topographic cross-talk contributions and thus provides quantifiable intrinsic dielectric images of the bacterial cells. Results for single Escherichia coli cells provide a relative electric permittivity of ∼4 in dry conditions and ∼20 in humid conditions, with no significant loss contributions. Present findings, together with the ability of microwaves to penetrate the cell membrane, open an important avenue in the microwave label-free imaging of single cells with nanoscale spatial resolution.

  9. Analysis of the coagulation of human blood cells on diamond surfaces by atomic force microscopy

    Science.gov (United States)

    Baranauskas, V.; Fontana, M.; Guo, Zhao Jing; Ceragioli, H. J.; Peterlevitz, A. C.

    2004-11-01

    Atomic force microscopy (AFM) was used to study the morphology and coagulation of human blood cells in contact with solid surfaces. Blood was extracted from the veins of healthy adult donors and the samples were used immediately after extraction, deposited either on borosilicate glass or diamond substrates. Some blood samples were anti-coagulated by adding heparin for single cell AFM imaging. No chemicals were used for attaching or immobilizing the cells. The diamond substrates were produced by chemical vapour deposition (CVD diamond) using a hot-filament CVD system fed with ethanol highly diluted in hydrogen. AFM imaging of isolated cells (anti-coagulated by heparin) was only possible on the glass substrates due to the lack of adherence of the cells to the diamond surface. The coagulation results suggest that blood clotting on diamond produces a less rough surface than blood clotting on glass.

  10. Optomechanical properties of cancer cells revealed by light-induced deformation and quantitative phase microscopy

    Science.gov (United States)

    Kastl, Lena; Budde, Björn; Isbach, Michael; Rommel, Christina; Kemper, Björn; Schnekenburger, Jürgen

    2015-05-01

    There is a growing interest in cell biology and clinical diagnostics in label-free, optical techniques as the interaction with the sample is minimized and substances like dyes or fixatives do not affect the investigated cells. Such techniques include digital holographic microscopy (DHM) and the optical stretching by fiber optical two beam traps. DHM enables quantitative phase contrast imaging and thereby the determination of the cellular refractive index, dry mass and the volume, whereas optical cell stretching reveals the deformability of cells. Since optical stretching strongly depends on the optical properties and the shape of the investigated material we combined the usage of fiber optical stretching and DHM for the characterization of pancreatic tumor cells. The risk of tumors is their potential to metastasize, spread through the bloodstream and build distal tumors/metastases. The grade of dedifferentiation in which the cells lose their cell type specific properties is a measure for this metastatic potential. The less differentiated the cells are, the higher is their risk to metastasize. Our results demonstrate that pancreatic tumor cells, which are from the same tumor but vary in their grade of differentiation, show significant differences in their deformability. The retrieved data show that differentiated cells have a higher stiffness than less differentiated cells of the same tumor. Even cells that differ only in the expression of a single tumor suppressor gene which is responsible for cell-cell adhesions can be distinguished by their mechanical properties. Additionally, results from DHM measurements yield that the refractive index shows only few variations, indicating that it does not significantly influence optical cell stretching. The obtained results show a promising new approach for the phenotyping of different cell types, especially in tumor cell characterization and cancer diagnostics.

  11. A novel cell traction force microscopy to study multi-cellular system.

    Directory of Open Access Journals (Sweden)

    Xin Tang

    2014-06-01

    Full Text Available Traction forces exerted by adherent cells on their microenvironment can mediate many critical cellular functions. Accurate quantification of these forces is essential for mechanistic understanding of mechanotransduction. However, most existing methods of quantifying cellular forces are limited to single cells in isolation, whereas most physiological processes are inherently multi-cellular in nature where cell-cell and cell-microenvironment interactions determine the emergent properties of cell clusters. In the present study, a robust finite-element-method-based cell traction force microscopy technique is developed to estimate the traction forces produced by multiple isolated cells as well as cell clusters on soft substrates. The method accounts for the finite thickness of the substrate. Hence, cell cluster size can be larger than substrate thickness. The method allows computing the traction field from the substrate displacements within the cells' and clusters' boundaries. The displacement data outside these boundaries are not necessary. The utility of the method is demonstrated by computing the traction generated by multiple monkey kidney fibroblasts (MKF and human colon cancerous (HCT-8 cells in close proximity, as well as by large clusters. It is found that cells act as individual contractile groups within clusters for generating traction. There may be multiple of such groups in the cluster, or the entire cluster may behave a single group. Individual cells do not form dipoles, but serve as a conduit of force (transmission lines over long distances in the cluster. The cell-cell force can be either tensile or compressive depending on the cell-microenvironment interactions.

  12. N-way FRET microscopy of multiple protein-protein interactions in live cells.

    Directory of Open Access Journals (Sweden)

    Adam D Hoppe

    Full Text Available Fluorescence Resonance Energy Transfer (FRET microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

  13. Field emission scanning electron microscopy (FE-SEM) as an approach for nanoparticle detection inside cells.

    Science.gov (United States)

    Havrdova, M; Polakova, K; Skopalik, J; Vujtek, M; Mokdad, A; Homolkova, M; Tucek, J; Nebesarova, J; Zboril, R

    2014-12-01

    When developing new nanoparticles for bio-applications, it is important to fully characterize the nanoparticle's behavior in biological systems. The most common techniques employed for mapping nanoparticles inside cells include transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). These techniques entail passing an electron beam through a thin specimen. STEM or TEM imaging is often used for the detection of nanoparticles inside cellular organelles. However, lengthy sample preparation is required (i.e., fixation, dehydration, drying, resin embedding, and cutting). In the present work, a new matrix (FTO glass) for biological samples was used and characterized by field emission scanning electron microscopy (FE-SEM) to generate images comparable to those obtained by TEM. Using FE-SEM, nanoparticle images were acquired inside endo/lysosomes without disruption of the cellular shape. Furthermore, the initial steps of nanoparticle incorporation into the cells were captured. In addition, the conductive FTO glass endowed the sample with high stability under the required accelerating voltage. Owing to these features of the sample, further analyses could be performed (material contrast and energy-dispersive X-ray spectroscopy (EDS)), which confirmed the presence of nanoparticles inside the cells. The results showed that FE-SEM can enable detailed characterization of nanoparticles in endosomes without the need for contrast staining or metal coating of the sample. Images showing the intracellular distribution of nanoparticles together with cellular morphology can give important information on the biocompatibility and demonstrate the potential of nanoparticle utilization in medicine.

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Science.gov (United States)

    Lyapunova, Elena; Nikituk, Alexander; Bayandin, Yuriy; Naimark, Oleg; Rianna, Carmela; Radmacher, Manfred

    2016-08-01

    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 algorithms (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.

  16. Enhanced CellClassifier: a multi-class classification tool for microscopy images

    Directory of Open Access Journals (Sweden)

    Horvath Peter

    2010-01-01

    Full Text Available Abstract Background Light microscopy is of central importance in cell biology. The recent introduction of automated high content screening has expanded this technology towards automation of experiments and performing large scale perturbation assays. Nevertheless, evaluation of microscopy data continues to be a bottleneck in many projects. Currently, among open source software, CellProfiler and its extension Analyst are widely used in automated image processing. Even though revolutionizing image analysis in current biology, some routine and many advanced tasks are either not supported or require programming skills of the researcher. This represents a significant obstacle in many biology laboratories. Results We have developed a tool, Enhanced CellClassifier, which circumvents this obstacle. Enhanced CellClassifier starts from images analyzed by CellProfiler, and allows multi-class classification using a Support Vector Machine algorithm. Training of objects can be done by clicking directly "on the microscopy image" in several intuitive training modes. Many routine tasks like out-of focus exclusion and well summary are also supported. Classification results can be integrated with other object measurements including inter-object relationships. This makes a detailed interpretation of the image possible, allowing the differentiation of many complex phenotypes. For the generation of the output, image, well and plate data are dynamically extracted and summarized. The output can be generated as graphs, Excel-files, images with projections of the final analysis and exported as variables. Conclusion Here we describe Enhanced CellClassifier which allows multiple class classification, elucidating complex phenotypes. Our tool is designed for the biologist who wants both, simple and flexible analysis of images without requiring programming skills. This should facilitate the implementation of automated high-content screening.

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

    Science.gov (United States)

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

    2015-10-01

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

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

    Science.gov (United States)

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

    2015-01-01

    Video microscopy offers outstanding capabilities to investigate the dynamics of biological and pathological mechanisms in optimal culture conditions. Contact imaging is one of the simplest imaging architectures to digitally record images of cells due to the absence of any objective between the sample and the image sensor. However, in the framework of in-line holography, other optical components, e.g., an optical filter or a pinhole, are placed underneath the light source in order to illuminate the cells with a coherent or quasi-coherent incident light. In this study, we demonstrate that contact imaging with an incident light of both limited temporal and spatial coherences can be achieved with sufficiently high quality for most applications in cell biology, including monitoring of cell sedimentation, rolling, adhesion, spreading, proliferation, motility, death and detachment. Patterns of cells were recorded at various distances between 0 and 1000 μm from the pixel array of the image sensors. Cells in suspension, just deposited or at mitosis focalise light into photonic nanojets which can be visualised by contact imaging. Light refraction by cells significantly varies during the adhesion process, the cell cycle and among the cell population in connection with every modification in the tridimensional morphology of a cell. PMID:26459014

  19. Fluorescence Dynamics in the Endoplasmic Reticulum of a Live Cell: Time-Resolved Confocal Microscopy.

    Science.gov (United States)

    Ghosh, Shirsendu; Nandi, Somen; Ghosh, Catherine; Bhattacharyya, Kankan

    2016-09-19

    Fluorescence dynamics in the endoplasmic reticulum (ER) of a live non-cancer lung cell (WI38) and a lung cancer cell (A549) are studied by using time-resolved confocal microscopy. To selectively study the organelle, ER, we have used an ER-Tracker dye. From the emission maximum (λmaxem) of the ER-Tracker dye, polarity (i.e. dielectric constant, ϵ) in the ER region of the cells (≈500 nm in WI38 and ≈510 nm in A549) is estimated to be similar to that of chloroform (λmaxem =506 nm, ϵ≈5). The red shift by 10 nm in λmaxem in the cancer cell (A549) suggests a slightly higher polarity compared to the non-cancer cell (WI38). The fluorescence intensity of the ER-Tracker dye exhibits prolonged intermittent oscillations on a timescale of 2-6 seconds for the cancer cell (A549). For the non-cancer cell (WI38), such fluorescence oscillations are much less prominent. The marked fluorescence intensity oscillations in the cancer cell are attributed to enhanced calcium oscillations. The average solvent relaxation time () of the ER region in the lung cancer cell (A549, 250±50 ps) is about four times faster than that in the non-cancer cell (WI38, 1000±50 ps).

  20. [Comparison of cell elasticity analysis methods based on atomic force microscopy indentation].

    Science.gov (United States)

    Wang, Zhe; Hao, Fengtao; Chen, Xiaohu; Yang, Zhouqi; Ding, Chong; Shang, Peng

    2014-10-01

    In order to investigate in greater detail the two methods based on Hertz model for analyzing force-distance curve obtained by atomic force microscopy, we acquired the force-distance curves of Hela and MCF-7 cells by atomic force microscopy (AFM) indentation in this study. After the determination of contact point, Young's modulus in different indentation depth were calculated with two analysis methods of "two point" and "slope fitting". The results showed that the Young's modulus of Hela cell was higher than that of MCF-7 cell,which is in accordance with the F-actin distribution of the two types of cell. We found that the Young's modulus of the cells was decreased with increasing indentation depth and the curve trends by "slope fitting". This indicated that the "slope fitting" method could reduce the error caused by the miscalculation of contact point. The purpose of this study was to provide a guidance for researcher to choose an appropriate method for analyzing AFM indentation force-distance curve. PMID:25764725

  1. Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium-tin-oxide.

    Science.gov (United States)

    Rodighiero, Simona; Torre, Bruno; Sogne, Elisa; Ruffilli, Roberta; Cagnoli, Cinzia; Francolini, Maura; Di Fabrizio, Enzo; Falqui, Andrea

    2015-06-01

    Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. PMID:25810353

  2. Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium-tin-oxide

    KAUST Repository

    Rodighiero, Simona

    2015-03-22

    Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. © 2015 Wiley Periodicals, Inc.

  3. Segmental bronchoprovocation in allergic rhinitis patients affects mast cell and basophil numbers in nasal and bronchial mucosa

    OpenAIRE

    Braunstahl, Gert-Jan; Overbeek, Shelley; Fokkens, Wytske; KleinJan, Alex; McEuen, A.R.; Walls, A F; Hoogsteden, Henk; Prins, Jan-Bas

    2001-01-01

    textabstractMast cells and basophils are cells that play an important role in the initiation and control of allergic inflammation in asthma and rhinitis. This study was undertaken to determine the presence and dynamics of mast cells and basophils in the nasal and bronchial mucosa of allergic rhinitis patients after segmental bronchial provocation (SBP). Eight nonasthmatic, grass pollen-allergic rhinitis patients and eight healthy controls were included. Bronchial and nasal biopsies, as well a...

  4. Topography of Cells Revealed by Variable-Angle Total Internal Reflection Fluorescence Microscopy.

    Science.gov (United States)

    Cardoso Dos Santos, Marcelina; Déturche, Régis; Vézy, Cyrille; Jaffiol, Rodolphe

    2016-09-20

    We propose an improved version of variable-angle total internal reflection fluorescence microscopy (vaTIRFM) adapted to modern TIRF setup. This technique involves the recording of a stack of TIRF images, by gradually increasing the incident angle of the light beam on the sample. A comprehensive theory was developed to extract the membrane/substrate separation distance from fluorescently labeled cell membranes. A straightforward image processing was then established to compute the topography of cells with a nanometric axial resolution, typically 10-20 nm. To highlight the new opportunities offered by vaTIRFM to quantify adhesion process of motile cells, adhesion of MDA-MB-231 cancer cells on glass substrate coated with fibronectin was examined. PMID:27653490

  5. Imaging lipid domains in cell membranes: the advent of super-resolution fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Dylan Myers Owen

    2013-12-01

    Full Text Available The lipid bilayer of model membranes, liposomes reconstituted from cell lipids, and plasma membrane vesicles and spheres can separate into two distinct liquid phases to yield lipid domains with liquid-ordered and liquid-disordered properties. These observations are the basis of the lipid raft hypothesis that postulates the existence of cholesterol-enriched ordered-phase lipid domains in cell membranes that could regulate protein mobility, localization and interaction. Here we review the evidence that nano-scaled lipid complexes and meso-scaled lipid domains exist in cell membranes and how new fluorescence microscopy techniques that overcome the diffraction limit provide new insights into lipid organization in cell membranes.

  6. Picoliter Drop-On-Demand Dispensing for Multiplex Liquid Cell Transmission Electron Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Patterson, Joseph P.; Parent, Lucas R.; Cantlon, Joshua; Eickhoff, Holger; Bared, Guido; Evans, James E.; Gianneschi, Nathan C.

    2016-06-03

    Liquid Cell Transmission Electron Microscopy (LCTEM) provides a unique insight into the dynamics of nanoparticles in solution. Controlling the mixing of multiple solutions within the liquid cells remains a key hurdle in our ability to study nanoparticle formation/modifications through conventional synthesis i.e. by mixing solutions. Here, we report that piezo dispensing techniques, allow for the mixing of multiple solutions directly within the viewing area. This technique deposits pL droplets of various aqueous and organic solutions onto the liquid cell window, prior to assembly of the cell in a fully controlled manner. This proof of concept shows the great potential for using picoliter dispensing in combination with LCTEM to observe nanoparticle synthesis or modification by solution phase mixing and the creation of chemical gradients.

  7. Shape reconstruction and height fluctuations of red blood cells using defocusing microscopy

    CERN Document Server

    Siman, L; Amaral, F T; Agero, U; Mesquita, O N

    2014-01-01

    In this paper the bright-field defocusing microscopy (DM) technique is presented. DM is able to obtain quantitative information of each plane/surface of pure phase objects, as live unlabeled cells, and its application to red blood cells (RBCs) is demonstrated. Based on contrast, simple methods to obtain thickness profile and three dimensional (3D) total reconstruction of RBCs are proposed and the actual height profiles of upper and lower surface-membranes (lipid bilayer$/$cytoskeleton) of discocyte and stomatocyte red cells are presented as examples. In addition, using the mean square contrast fluctuation and modeling the RBC membranes fluctuations spectra as dependent of a bending modulus $(\\kappa_c)$, a surface tension $(\\sigma)$ and a confining potential $(\\gamma)$ term, slowly varying quantities along the cell radius, a genetic algorithm (GA) is used and the radial height fluctuations of each surface-membrane are accessed, separately. The radial behaviors of $\\kappa_c$, $\\sigma$ and $\\gamma$ are also obta...

  8. Atomic Force Microscopy-based Cell Nanostructure for Ligand-conjugated Quantum Dot Endocytosis

    Institute of Scientific and Technical Information of China (English)

    Yun-Long PAN; Ji-Ye CAI; Li QIN; Hao WANG

    2006-01-01

    While it has been well demonstrated that quantum dots (QDs) play an important role in biological labeling both in vitro and in vivo,there is no report describing the cellular nanostructure basis of receptor-mediated endocytosis. Here, nanostructure evolution responses to the endocytosis of transferrin force microscopy (AFM). AFM-based nanostructure analysis demonstrated that the Tf-conjugated QDs were specifically and tightly bound to the cell receptors rrelated with the cell membrane receptor-mediated transduction.Consistently, confocal microscopic and flow cytometry results have demonstrated the specificity and the internalization of Tf-QD is linearly related to time. Moreover, while the nanoparticles on the cell membrane increased, the endocytosis was still nanoparticles did not interfere sterically with the binding and function of receptors. Therefore, ligand-conjugated QDs are potentially useful in biological labeling of cells at a nanometer scale.

  9. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.

    Directory of Open Access Journals (Sweden)

    Xiaokun Shu

    2011-04-01

    Full Text Available Electron microscopy (EM achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator, a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.

  10. Long segmental hyperplasia of interstitial cells of Cajal with giant diverticulum formation.

    Science.gov (United States)

    Xue, Liyan; Qiu, Tian; Song, Ying; Shan, Ling; Liu, Xiuyun; Guo, Lei; Ying, Jianming; Zou, Shuangmei; Shi, Susheng; Polydorides, Alexandros D; Zhao, Xinming; Lu, Ning; Lin, Dongmei

    2013-01-01

    Sporadic gastrointestinal stromal tumors (GISTs) usually form a well-circumscribed mass. In contrast, diffuse interstitial cell of Cajal (ICC) hyperplasia along the Auerbach plexus without a discrete mass may occur in patients with germline mutations in the NF1, c-KIT or PDGFRA genes. However, sporadic, diffuse ICC hyperplasia without c-KIT or PDGFRA mutations has not been reported. We describe herein one such case, forming a giant diverticulum. A 63-year-old woman with no features of Neurofibromatosis 1 (NF1) presented with increasing abdominal pain for more than 30 years. A large, diverticulum-like mass in the ileum was resected. Microscopically, a diffuse proliferation of bland spindle cells was seen extending for 12 cm, replacing the muscularis propria and lined by intact mucosa. The spindle cells were CD117+/CD34+/DOG1+/SMA+/Desmin-/S100-. Mutation analyses did not reveal any mutations in c-KIT or PDGFRA. The lesion had two silent mutations in the NF1 gene. It is rare of the diffuse form of sporadic ICC hyperplasia showing diffuse longitudinal microscopic growth completely replacing the muscularis propria, mimicking diffuse ICC hyperplasia in hereditary GIST syndromes, but without solid components and no c-KIT or PDGFRA gene mutations. This peculiar form of sporadic ICC hyperplasia may be related to intestinal dysmotility in this ileal segment and giant diverticulum formation. PMID:24294389

  11. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis

    Directory of Open Access Journals (Sweden)

    Ludovico eSilvestri

    2015-05-01

    Full Text Available Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all Purkinje cells are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent Purkinje cells. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of Purkinje cells, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of Purkinje cells with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments.

  12. An Entropy-Based Automated Cell Nuclei Segmentation and Quantification: Application in Analysis of Wound Healing Process

    Directory of Open Access Journals (Sweden)

    Varun Oswal

    2013-01-01

    Full Text Available The segmentation and quantification of cell nuclei are two very significant tasks in the analysis of histological images. Accurate results of cell nuclei segmentation are often adapted to a variety of applications such as the detection of cancerous cell nuclei and the observation of overlapping cellular events occurring during wound healing process in the human body. In this paper, an automated entropy-based thresholding system for segmentation and quantification of cell nuclei from histologically stained images has been presented. The proposed translational computation system aims to integrate clinical insight and computational analysis by identifying and segmenting objects of interest within histological images. Objects of interest and background regions are automatically distinguished by dynamically determining 3 optimal threshold values for the 3 color components of an input image. The threshold values are determined by means of entropy computations that are based on probability distributions of the color intensities of pixels and the spatial similarity of pixel intensities within neighborhoods. The effectiveness of the proposed system was tested over 21 histologically stained images containing approximately 1800 cell nuclei, and the overall performance of the algorithm was found to be promising, with high accuracy and precision values.

  13. Experimental validation of atomic force microscopy-based cell elasticity measurements

    Science.gov (United States)

    Harris, Andrew R.; Charras, G. T.

    2011-08-01

    Atomic force microscopy (AFM) is widely used for measuring the elasticity of living cells yielding values ranging from 100 Pa to 100 kPa, much larger than those obtained using bead-tracking microrheology or micropipette aspiration (100-500 Pa). AFM elasticity measurements appear dependent on tip geometry with pyramidal tips yielding elasticities 2-3 fold larger than spherical tips, an effect generally attributed to the larger contact area of spherical tips. In AFM elasticity measurements, experimental force-indentation curves are analyzed using contact mechanics models that infer the tip-cell contact area from the tip geometry and indentation depth. The validity of these assumptions has never been verified. Here we utilize combined AFM-confocal microscopy of epithelial cells expressing a GFP-tagged membrane marker to directly characterize the indentation geometry and measure the indentation depth. Comparison with data derived from AFM force-indentation curves showed that the experimentally measured contact area for spherical tips agrees well with predicted values, whereas for pyramidal tips, the contact area can be grossly underestimated at forces larger than ~ 0.2 nN leading to a greater than two-fold overestimation of elasticity. These data suggest that a re-examination of absolute cellular elasticities reported in the literature may be necessary and we suggest guidelines for avoiding elasticity measurement artefacts introduced by extraneous cantilever-cell contact.

  14. Structural Insight into Cell Wall Architecture of Micanthus sinensis cv. using Correlative Microscopy Approaches.

    Science.gov (United States)

    Ma, Jianfeng; Lv, Xunli; Yang, Shumin; Tian, Genlin; Liu, Xing'e

    2015-10-01

    Structural organization of the plant cell wall is a key parameter for understanding anisotropic plant growth and mechanical behavior. Four imaging platforms were used to investigate the cell wall architecture of Miscanthus sinensis cv. internode tissue. Using transmission electron microscopy with potassium permanganate, we found a great degree of inhomogeneity in the layering structure (4-9 layers) of the sclerenchymatic fiber (Sf). However, the xylem vessel showed a single layer. Atomic force microscopy images revealed that the cellulose microfibrils (Mfs) deposited in the primary wall of the protoxylem vessel (Pxv) were disordered, while the secondary wall was composed of Mfs oriented in parallel in the cross and longitudinal section. Furthermore, Raman spectroscopy images indicated no variation in the Mf orientation of Pxv and the Mfs in Pxv were oriented more perpendicular to the cell axis than that of Sfs. Based on the integrated results, we have proposed an architectural model of Pxv composed of two layers: an outermost primary wall composed of a meshwork of Mfs and inner secondary wall containing parallel Mfs. This proposed model will support future ultrastructural analysis of plant cell walls in heterogeneous tissues, an area of increasing scientific interest particularly for liquid biofuel processing. PMID:26358178

  15. Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology.

    Science.gov (United States)

    Narayan, Kedar; Danielson, Cindy M; Lagarec, Ken; Lowekamp, Bradley C; Coffman, Phil; Laquerre, Alexandre; Phaneuf, Michael W; Hope, Thomas J; Subramaniam, Sriram

    2014-03-01

    Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB-SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce "keyframe imaging" as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a "target map" of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10(9) in a single automated experimental run.

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

    Directory of Open Access Journals (Sweden)

    Elizabeth Kolos

    2015-06-01

    Full Text Available 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.

  17. Experimental validation of atomic force microscopy-based cell elasticity measurements

    Energy Technology Data Exchange (ETDEWEB)

    Harris, Andrew R; Charras, G T, E-mail: g.charras@ucl.ac.uk [London Centre for Nanotechnology, University College London, London WC1H 0AH (United Kingdom)

    2011-08-26

    Atomic force microscopy (AFM) is widely used for measuring the elasticity of living cells yielding values ranging from 100 Pa to 100 kPa, much larger than those obtained using bead-tracking microrheology or micropipette aspiration (100-500 Pa). AFM elasticity measurements appear dependent on tip geometry with pyramidal tips yielding elasticities 2-3 fold larger than spherical tips, an effect generally attributed to the larger contact area of spherical tips. In AFM elasticity measurements, experimental force-indentation curves are analyzed using contact mechanics models that infer the tip-cell contact area from the tip geometry and indentation depth. The validity of these assumptions has never been verified. Here we utilize combined AFM-confocal microscopy of epithelial cells expressing a GFP-tagged membrane marker to directly characterize the indentation geometry and measure the indentation depth. Comparison with data derived from AFM force-indentation curves showed that the experimentally measured contact area for spherical tips agrees well with predicted values, whereas for pyramidal tips, the contact area can be grossly underestimated at forces larger than {approx} 0.2 nN leading to a greater than two-fold overestimation of elasticity. These data suggest that a re-examination of absolute cellular elasticities reported in the literature may be necessary and we suggest guidelines for avoiding elasticity measurement artefacts introduced by extraneous cantilever-cell contact.

  18. Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy.

    Science.gov (United States)

    Zhang, Xi; Zhang, Mingshu; Li, Dong; He, Wenting; Peng, Jianxin; Betzig, Eric; Xu, Pingyong

    2016-09-13

    Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm(2)) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view. PMID:27562163

  19. Confocal microscopy and electrophysiological study of single patient corneal endothelium cell cultures

    Science.gov (United States)

    Tatini, Francesca; Rossi, Francesca; Coppi, Elisabetta; Magni, Giada; Fusco, Irene; Menabuoni, Luca; Pedata, Felicita; Pugliese, Anna Maria; Pini, Roberto

    2016-04-01

    The characterization of the ion channels in corneal endothelial cells and the elucidation of their involvement in corneal pathologies would lead to the identification of new molecular target for pharmacological treatments and to the clarification of corneal physiology. The corneal endothelium is an amitotic cell monolayer with a major role in preserving corneal transparency and in regulating the water and solute flux across the posterior surface of the cornea. Although endothelial cells are non-excitable, they express a range of ion channels, such as voltage-dependent Na+ channels and K+ channels, L-type Ca2 channels and many others. Interestingly, purinergic receptors have been linked to a variety of conditions within the eye but their presence in the endothelium and their role in its pathophysiology is still uncertain. In this study, we were able to extract endothelial cells from single human corneas, thus obtaining primary cultures that represent the peculiarity of each donor. Corneas were from tissues not suitable for transplant in patients. We characterized the endothelial cells by confocal microscopy, both within the intact cornea and in the primary endothelial cells cultures. We also studied the functional role of the purinergic system (adenosine, ATP and their receptors) by means of electrophysiological recordings. The experiments were performed by patch clamp recordings and confocal time-lapse microscopy and our results indicate that the application of purinergic compounds modulates the amplitude of outward currents in the isolated endothelial cells. These findings may lead to the proposal of new therapies for endothelium-related corneal diseases.

  20. Digital holographic microscopy for imaging biophysical changes in cells during migration (Conference Presentation)

    Science.gov (United States)

    Nham, Kien V.; Hur, Dong; Kim, Young-tae; Mohanty, Samarendra K.

    2016-03-01

    It is well known that biochemical changes in cancer cell occur in response to environmental cues and during migration. However, information about changes in the physical properties (e.g., volume, elasticity) of cancer cells during migration and/or in response to physical modulations (confinement and perturbations). We report the use of a near-infrared (NIR) laser microbeam system integrated with a NIR digital holographic microscopy (DHM) to study physical response of cancer cells. The cancer cells were cultured in microfluidic devices and subjected to different physical confinement (controlled by channel geometry), osmolarity changes of extracellular medium and/or laser-induced perturbations. The changes in optical thickness (or phase map) of the cells were monitored with high spatial and temporal resolution during and after the physico-chemical perturbations. A weakly-focused continuous-wave laser microbeam was used to impart radiation pressure on cell membrane and the changes in thickness were monitored using DHM to estimate elasticity. Further, an ultrafast tightly-focused laser microbeam was used to allow extracellular fluid flow into the cell or from the cytoplasm under different osmolarity conditions. Dynamic changes in physical properties of various cells and observed differences in responding to different physical/chemical environment/perturbations will be presented.

  1. TimeLapseAnalyzer: Multi-target analysis for live-cell imaging and time-lapse microscopy

    DEFF Research Database (Denmark)

    Huth, Johannes; Buchholz, Malte; Kraus, Johann M.;

    2011-01-01

    , we developed TimeLapseAnalyzer. Apart from general purpose image enhancements and segmentation procedures, this extensible, self-contained, modular cross-platform package provides dedicated modalities for fast and reliable analysis of multi-target cell tracking, scratch wound healing analysis, cell...

  2. Understanding Alterations in Cell Nano-architecture during Early Carcinogenesis using Optical Microscopy

    Science.gov (United States)

    Damania, Dhwanil

    Carcinogenesis is a complex multi-step process which eventually results in a malignant phenotype that often progresses into a fatal metastatic stage. There are several molecular changes (e.g. DNA methylation, activation of proto-oncogenes, loss of tumor-suppressor genes, histone acetylation) that occur in cells prior to the microscopically detectable morphological alterations. Hence, it is intuitive that these molecular changes should impact various biochemical, biophysical and transport processes within the cell and therefore its nanoscale morphology. Furthermore, recent studies have established that apparently `normal' cells (i.e., away from the actual tumor location) undergo similar genetic/epigenetic changes as the actual cancer cells, giving rise to the phenomenon of field carcinogenesis. Unfortunately, traditional microscopy or histopathology cannot resolve structures below 300 nm due to diffraction-limited resolution. Hence, we developed a novel optical imaging technique, partial wave spectroscopic (PWS) microscopy or optical nanocytology which quantifies the nanoscale refractive-index fluctuations (i.e. mass-density variations such as chromatin compaction) in an optically measured biomarker, disorder strength (Ld). This dissertation proves the nanoscale sensitivity of PWS nanocytology and shows that increase in Ld parallels neoplastic potential of a cell by using standardized cell-lines and animal-models. Based on concept of field carcinogenesis, we employ PWS nanocytology in a multi-center clinical study on approximately 450 patients in four different cancer-types (colon, ovarian, thyroid and lung) and we illustrate that nanoscale disorder increase is a ubiquitous phenomenon across different organs. We further demonstrate the potential of PWS nanocytology in predicting risk for developing future neoplasia. Biologically, we prove that cytoskeletal organization in both nucleus and cytoplasm plays a crucial role in governing L d-differences. Moreover, we

  3. Raman confocal microscopy and AFM combined studies of cancerous cells treated with Paclitaxel

    Science.gov (United States)

    Derely, L.; Collart Dutilleul, P.-Y.; Michotte de Welle, Sylvain; Szabo, V.; Gergely, C.; Cuisinier, F. J. G.

    2011-03-01

    Paclitaxel interferes with the normal function of microtubule breakdown, induces apoptosis in cancer cells and sequesters free tubulin. As this drug acts also on other cell mechanisms it is important to monitor its accumulation in the cell compartments. The intracellular spreading of the drug was followed using a WITEC 300R confocal Raman microscope equipped with a CCD camera. Hence Atomic force microscopy (an MFP3D- Asylum Research AFM) in imaging and force mode was used to determine the morphological and mechanical modifications induced on living cells. These studies were performed on living epithelial MCF-7 breast cancer cells. Paclitaxel was added to cell culture media for 3, 6 and 9 hours. Among the specific paclitaxel Raman bands we selected the one at 1670 cm-1 because it is not superposed by the spectrum of the cells. Confocal Raman images are formed by monitoring this band, the NH2 and the PO4 band. Paclitaxel slightly accumulates in the nucleus forming patches. The drug is also concentrated in the vicinity of the cell membrane and in an area close to the nucleus where proteins accumulate. Our AFM images reveal that the treated cancerous MCF-7 cells keep the same size as the non treated ones, but their shape becomes more oval. Cell's elasticity is also modified: a difference of 2 kPa in the Young Modulus characterizes the treated MCF-7 mammary cancerous cell. Our observations demonstrate that paclitaxel acts not only on microtubules but accumulates also in other cell compartments (nucleus) where microtubules are absent.

  4. Automated identification and location analysis of marked stem cells colonies in optical microscopy images.

    Directory of Open Access Journals (Sweden)

    Vincenzo Paduano

    Full Text Available Embryonic stem cells (ESCs are characterized by two remarkable peculiarities: the capacity to propagate as undifferentiated cells (self-renewal and the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives (pluripotency. Although the majority of ESCs divide without losing the pluripotency, it has become evident that ESC cultures consists of multiple cell populations highlighted by the expression of early germ lineage markers during spontaneous differentiation. Hence, the identification and characterization of ESCs subpopulations represents an efficient approach to improve the comprehension of correlation between gene expression and cell specification status. To study markers of ESCs heterogeneity, we developed an analysis pipeline which can automatically process images of stem cell colonies in optical microscopy. The question we try to address is to find out the statistically significant preferred locations of the marked cells. We tested our algorithm on a set of images of stem cell colonies to analyze the expression pattern of the Zscan4 gene, which was an elite candidate gene to be studied because it is specifically expressed in subpopulation of ESCs. To validate the proposed method we analyzed the behavior of control genes whose pattern had been associated to biological status such as differentiation (EndoA, pluripotency (Pou5f1, and pluripotency fluctuation (Nanog. We found that Zscan4 is not uniformly expressed inside a stem cell colony, and that it tends to be expressed towards the center of the colony, moreover cells expressing Zscan4 cluster each other. This is of significant importance because it allows us to hypothesize a biological status where the cells expressing Zscan4 are preferably associated to the inner of colonies suggesting pluripotent cell status features, and the clustering between themselves suggests either a colony paracrine effect or an early phase of cell specification through proliferation. Also, the

  5. Cell depth imaging by point laser scanning fluorescence microscopy with an optical disk pickup head

    Science.gov (United States)

    Tsai, Rung-Ywan; Chen, Jung-Po; Lee, Yuan-Chin; Chiang, Hung-Chih; Cheng, Chih-Ming; Huang, Chun-Chieh; Huang, Tai-Ting; Cheng, Chung-Ta; Tiao, Golden

    2015-09-01

    A compact, cost-effective, and position-addressable digital laser scanning microscopy (DLSM) instrument is made using a commercially available Blu-ray disc read-only memory (BD-ROM) pickup head. Fluorescent cell images captured by DLSM have resolutions of 0.38 µm. Because of the position-addressable function, multispectral fluorescence cell images are captured using the same sample slide with different excitation laser sources. Specially designed objective lenses with the same working distance as the image-capturing beam are used for the different excitation laser sources. By accurately controlling the tilting angles of the sample slide or by moving the collimator lens of the image-capturing beam, the fluorescence cell images along different depth positions of the sample are obtained. Thus, z-section images with micrometer-depth resolutions are achievable.

  6. Detecting CD20-Rituximab specific interactions on lymphoma cells using atomic force microscopy

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Elucidating the underlying mechanisms of cell physiology is currently an important research topic in life sciences. Atomic force microscopy methods can be used to investigate these molecular mechanisms. In this study, single-molecule force spectroscopy was used to explore the specific recognition between the CD20 antigen and anti-CD20 antibody Rituximab on B lymphoma cells under near-physiological conditions. The CD20-Rituximab specific binding force was measured through tip functionalization. Distribution of CD20 on the B lymphoma cells was visualized three-dimensionally. In addition, the relationship between the intramolecular force and the molecular extension of the CD20-Rituximab complex was analyzed under an external force. These results facilitate further investigation of the mechanism of Rituximab’s anti-cancer effect.

  7. Segmental basal cell naevus syndrome caused by an activating mutation in smoothened.

    Science.gov (United States)

    Khamaysi, Z; Bochner, R; Indelman, M; Magal, L; Avitan-Hersh, E; Sarig, O; Sprecher, E; Bergman, R

    2016-07-01

    Aberrant sonic hedgehog signalling, mostly due to PTCH1 mutations, has been shown to play a central role in the pathogenesis of basal cell carcinoma (BCC), as well as in basal cell naevus syndrome (BCNS). Mutations in smoothened (SMO) encoding a receptor for sonic hedgehog have been reported in sporadic BCCs but not in BCNS. We report a case with multiple BCCs, pits and comedones in a segmental distribution over the upper part of the body, along with other findings compatible with BCNS. Histopathologically, there were different types of BCC. A heterozygous mutation (c.1234C>T, p.L412F) in SMO was detected in three BCCs but not in peripheral blood lymphocytes or the uninvolved skin. These were compatible with the type 1 mosaic form of BCNS. The p.L412F mutation was found experimentally to result in increased SMO transactivating activity, and the patient responded to vismodegib therapy. Activating mutations in SMO may cause BCNS. The identification of a gain-of-function mutation in SMO causing a type 1 mosaic form of BCNS further expands our understanding of the pathogenesis of BCC, with implications for the treatment of these tumours, whether sporadic or inherited. PMID:26822128

  8. Intracellular concentration map of magnesium in whole cells by combined use of X-ray fluorescence microscopy and atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lagomarsino, Stefano, E-mail: stefano.lagomarsino@cnr.it [IPCF-CNR -UOS Roma c/o Dip Fisica Universita' ' Sapienza' , P.le A. Moro, 2 Rome (Italy); Physics Department, Universita' Sapienza, P.le A. Moro, 2 Rome (Italy); Iotti, Stefano [Dipartimento di Medicina Interna, dell' Invecchiamento e Malattie Nefrologiche Universita di Bologna, Via Massarenti, 9 40138 Bologna (Italy); Istituto Nazionale Biostrutture e Biosistemi - Rome (Italy); Farruggia, Giovanna [Dipartimento di Biochimica ' G. Moruzzi' Universita di Bologna, Via Irnerio, 48 40126 Bologna (Italy); Cedola, Alessia [IFN-CNR - V. Cineto Romano, 42 00156 Rome (Italy); Trapani, Valentina [Istituto di Patologia Generale - Universita Cattolica del Sacro Cuore - Facolta di Medicina ' A. Gemelli' L.go F. Vito, 1 00168 Rome (Italy); Fratini, Michela [IFN-CNR - V. Cineto Romano, 42 00156 Rome (Italy); Bukreeva, Inna [IFN-CNR - V. Cineto Romano, 42 00156 Rome (Italy); Shubnikov Institute of Crystallography, Leninskii prospekt 59, Moscow, 119333 (Russian Federation); Notargiacomo, Andrea [IFN-CNR - V. Cineto Romano, 42 00156 Rome (Italy); Mastrototaro, Lucia [Istituto di Patologia Generale - Universita Cattolica del Sacro Cuore - Facolta di Medicina ' A. Gemelli' L.go F. Vito, 1 00168 Rome (Italy); Marraccini, Chiara [Dipartimento di Medicina Interna, dell' Invecchiamento e Malattie Nefrologiche Universita di Bologna, Via Massarenti, 9 40138 Bologna (Italy); and others

    2011-11-15

    We report a novel experimental approach to derive quantitative concentration map of light elements in whole cells by combining two complementary nano-probe methods: X-ray fluorescence microscopy (XRFM) and atomic force microscopy (AFM). The concentration is derived by normalizing point-by-point the elemental (here Mg) spatial distribution obtained by XRFM, by the thickness measured using AFM. The considerable difference between the elemental distribution and the concentration maps indicates that this procedure is essential to obtain reliable information on the role and function of elements in whole cells. - Highlights: Black-Right-Pointing-Pointer X-ray fluorescence and AFM have been measured on the same de-hydrated whole cells. Black-Right-Pointing-Pointer The element distribution has been normalized point-by-point by the cell thickness. Black-Right-Pointing-Pointer The element (Mg) concentration map has been obtained on a whole cell. Black-Right-Pointing-Pointer The element concentration map is quite different from the distribution map. Black-Right-Pointing-Pointer Higher Mg concentration is found in the cell periphery.

  9. Discrimination Between Cervical Cancer Cells and Normal Cervical Cells Based on Longitudinal Elasticity Using Atomic Force Microscopy.

    Science.gov (United States)

    Zhao, Xueqin; Zhong, Yunxin; Ye, Ting; Wang, Dajing; Mao, Bingwei

    2015-12-01

    The mechanical properties of cells are considered promising biomarkers for the early diagnosis of cancer. Recently, atomic force microscopy (AFM)-based nanoindentation technology has been utilized for the examination of cell cortex mechanics in order to distinguish malignant cells from normal cells. However, few attempts to evaluate the biomechanical properties of cells have focused on the quantification of the non-homogeneous longitudinal elasticity of cellular structures. In the present study, we applied a variation of the method of Carl and Schillers to investigate the differences between longitudinal elasticity of human cervical squamous carcinoma cells (CaSki) and normal cervical epithelial cells (CRL2614) using AFM. The results reveal a three-layer heterogeneous structure in the probing volume of both cell types studied. CaSki cells exhibited a lower whole-cell stiffness and a softer nuclei zone compared to the normal counterpart cells. Moreover, a better differentiated cytoskeleton was found in the inner cytoplasm/nuclei zone of the normal CRL2614 cells, whereas a deeper cytoskeletal distribution was observed in the probing volume of the cancerous counterparts. The sensitive cortical panel of CaSki cells, with a modulus of 0.35~0.47 kPa, was located at 237~225 nm; in normal cells, the elasticity was 1.20~1.32 kPa at 113~128 nm. The present improved method may be validated using the conventional Hertz-Sneddon method, which is widely reported in the literature. In conclusion, our results enable the quantification of the heterogeneous longitudinal elasticity of cancer cells, in particular the correlation with the corresponding depth. Preliminary results indicate that our method may potentially be applied to improve the detection of cancerous cells and provide insights into the pathophysiology of the disease. PMID:26666911

  10. Discrimination Between Cervical Cancer Cells and Normal Cervical Cells Based on Longitudinal Elasticity Using Atomic Force Microscopy

    Science.gov (United States)

    Zhao, Xueqin; Zhong, Yunxin; Ye, Ting; Wang, Dajing; Mao, Bingwei

    2015-12-01

    The mechanical properties of cells are considered promising biomarkers for the early diagnosis of cancer. Recently, atomic force microscopy (AFM)-based nanoindentation technology has been utilized for the examination of cell cortex mechanics in order to distinguish malignant cells from normal cells. However, few attempts to evaluate the biomechanical properties of cells have focused on the quantification of the non-homogeneous longitudinal elasticity of cellular structures. In the present study, we applied a variation of the method of Carl and Schillers to investigate the differences between longitudinal elasticity of human cervical squamous carcinoma cells (CaSki) and normal cervical epithelial cells (CRL2614) using AFM. The results reveal a three-layer heterogeneous structure in the probing volume of both cell types studied. CaSki cells exhibited a lower whole-cell stiffness and a softer nuclei zone compared to the normal counterpart cells. Moreover, a better differentiated cytoskeleton was found in the inner cytoplasm/nuclei zone of the normal CRL2614 cells, whereas a deeper cytoskeletal distribution was observed in the probing volume of the cancerous counterparts. The sensitive cortical panel of CaSki cells, with a modulus of 0.35~0.47 kPa, was located at 237~225 nm; in normal cells, the elasticity was 1.20~1.32 kPa at 113~128 nm. The present improved method may be validated using the conventional Hertz-Sneddon method, which is widely reported in the literature. In conclusion, our results enable the quantification of the heterogeneous longitudinal elasticity of cancer cells, in particular the correlation with the corresponding depth. Preliminary results indicate that our method may potentially be applied to improve the detection of cancerous cells and provide insights into the pathophysiology of the disease.

  11. Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells.

    Science.gov (United States)

    Mauzeroll, Janine; Bard, Allen J

    2004-05-25

    The uptake of menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy. The progression of the in vitro reaction between menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV-visible) behavior. By observing the scanning electrochemical microscope tip current of yeast cells suspended in a menadione-containing solution, the export of the conjugate from the cells with time could be measured. Similar experiments were performed on immobilized yeast cell aggregates stressed by a menadione solution. From the export of the menadione-glutathione conjugate detected at a 1-microm-diameter electrode situated 10 microm from the cells, a flux of about 30,000 thiodione molecules per second per cell was extracted. Numerical simulations based on an explicit finite difference method further revealed that the observation of a constant efflux of thiodione from the cells suggested the rate was limited by the uptake of menadione and that the efflux through the glutathione-conjugate pump was at least an order of magnitude faster. PMID:15148374

  12. Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy

    International Nuclear Information System (INIS)

    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 106-3 x 108 labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 106 cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells. (orig.)

  13. Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Daldrup-Link, Heike E. [Department of Radiology, UCSF Medical Center, University of California in San Francisco, 513 Parnassus Ave, CA 94143, San Francisco (United States); Rudelius, Martina; Piontek, Guido; Schlegel, Juergen [Institute of Pathology, Technical University, Munich (Germany); Metz, Stephan; Settles, Marcus; Rummeny, Ernst J. [Department of Radiology, Technical University, Munich (Germany); Pichler, Bernd [Department of Biomedical Engineering, University of California Davis, Davis (United States); Heinzmann, Ulrich [National Research Center for Environment and Health, Technical University, Munich (Germany); Oostendorp, Robert A.J. [3. Clinic of Internal Medicine, Laboratory of Stem Cell Physiology, Technical University, Munich (Germany)

    2004-09-01

    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 10{sup 6}-3 x 10{sup 8} labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 10{sup 6} cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells

  14. Cross-sectional electrostatic force microscopy of thin-film solar cells

    Science.gov (United States)

    Ballif, C.; Moutinho, H. R.; Al-Jassim, M. M.

    2001-01-01

    In a recent work, we showed that atomic force microscopy (AFM) is a powerful technique to image cross sections of polycrystalline thin films. In this work, we apply a modification of AFM, namely, electrostatic force microscopy (EFM), to investigate the electronic properties of cleaved II-VI and multijunction thin-film solar cells. We cleave the devices in such a way that they are still working with their nominal photovoltaic efficiencies and can be polarized for the measurements. This allows us to differentiate between surface effects (work function and surface band bending) and bulk device properties. In the case of polycrystalline CdTe/CdS/SnO2/glass solar cells, we find a drop of the EFM signal in the area of the CdTe/CdS interface (±50 nm). This drop varies in amplitude and sign according to the applied external bias and is compatible with an n-CdS/p-CdTe heterojunction model, thereby invalidating the possibility of a deeply buried n-p CdTe homojunction. In the case of a triple-junction GaInP/GaAs/Ge device, we observe a variation of the EFM signal linked to both the material work-function differences and to the voltage bias applied to the cell. We attempt a qualitative explanation of the results and discuss the implications and difficulties of the EFM technique for the study of such thin-film devices.

  15. Imaging arterial cells, atherosclerosis, and restenosis by multimodal nonlinear optical microscopy

    Science.gov (United States)

    Wang, Han-Wei; Simianu, Vlad; Locker, Matthew J.; Sturek, Michael; Cheng, Ji-Xin

    2008-02-01

    By integrating sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on a coherent anti-Stokes Raman scattering (CARS) microscope platform, multimodal nonlinear optical (NLO) imaging of arteries and atherosclerotic lesions was demonstrated. CARS signals arising from CH II-rich membranes allowed visualization of endothelial cells and smooth muscle cells in a carotid artery. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are rich in CH II bonds in their cross-linking residues. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. The system is capable of identifying different atherosclerotic lesion stages with sub-cellular resolution. The stages of atherosclerosis, such as macrophage infiltration, lipid-laden foam cell accumulation, extracellular lipid distribution, fibrous tissue deposition, plaque establishment, and formation of other complicated lesions could be viewed by our multimodal CARS microscope. Collagen percentages in the region adjacent to coronary artery stents were resolved. High correlation between NLO and histology imaging evidenced the validity of the NLO imaging. The capability of imaging significant components of an arterial wall and distinctive stages of atherosclerosis in a label-free manner suggests the potential application of multimodal nonlinear optical microscopy to monitor the onset and progression of arterial diseases.

  16. Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.

    Science.gov (United States)

    Yang, R; Elankumaran, Y; Hijjawi, N; Ryan, U

    2015-06-01

    A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages.

  17. Rapid recognition and functional analysis of membrane proteins on human cancer cells using atomic force microscopy.

    Science.gov (United States)

    Li, Mi; Xiao, Xiubin; Liu, Lianqing; Xi, Ning; Wang, Yuechao

    2016-09-01

    Understanding the physicochemical properties of cell surface signalling molecules is important for us to uncover the underlying mechanisms that guide the cellular behaviors. Atomic force microscopy (AFM) has become a powerful tool for detecting the molecular interactions on individual cells with nanometer resolution. In this paper, AFM peak force tapping (PFT) imaging mode was applied to rapidly locate and visually map the CD20 molecules on human lymphoma cells using biochemically sensitive tips. First, avidin-biotin system was used to test the effectiveness of using PFT imaging mode to probe the specific molecular interactions. The adhesion images obtained on avidin-coated mica using biotin-tethered tips obviously showed the recognition spots which corresponded to the avidins in the simultaneously obtained topography images. The experiments confirmed the specificity and reproducibility of the recognition results. Then, the established procedure was applied to visualize the nanoscale organization of CD20s on the surface of human lymphoma Raji cells using rituximab (a monoclonal anti-CD20 antibody)-tethered tips. The experiments showed that the recognition spots in the adhesion images corresponded to the specific CD20-rituximab interactions. The cluster sizes of CD20s on lymphoma Raji cells were quantitatively analyzed from the recognition images. Finally, under the guidance of fluorescence recognition, the established procedure was applied to cancer cells from a clinical lymphoma patient. The results showed that there were significant differences between the adhesion images obtained on cancer cells and on normal cells (red blood cell). The CD20 distributions on ten cancer cells from the patient were quantified according to the adhesion images. The experimental results demonstrate the capability of applying PFT imaging to rapidly investigate the nanoscale biophysical properties of native membrane proteins on the cell surface, which is of potential significance in

  18. Voltammetric Scanning Electrochemical Cell Microscopy: Dynamic Imaging of Hydrazine Electro-oxidation on Platinum Electrodes.

    Science.gov (United States)

    Chen, Chang-Hui; Jacobse, Leon; McKelvey, Kim; Lai, Stanley C S; Koper, Marc T M; Unwin, Patrick R

    2015-06-01

    Voltammetric scanning electrochemical cell microscopy (SECCM) incorporates cyclic voltammetry measurements in the SECCM imaging protocol, by recording electrochemical currents in a wide potential window at each pixel in a map. This provides much more information compared to traditional fixed potential imaging. Data can be represented as movies (hundreds of frames) of current (over a surface region) at a series of potentials and are highly revealing of subtle variations in electrode activity. Furthermore, by combining SECCM data with other forms of microscopy, e.g. scanning electron microscopy and electron backscatter diffraction data, it is possible to directly relate the current-voltage characteristics to spatial position and surface structure. In this work we use a "hopping mode", where the SECCM pipet probe is translated toward the surface at a series of positions until meniscus contact. Small amounts of residue left on the surface, upon probe retraction, demark the precise area of each measurement. We use these techniques to study hydrazine oxidation on a polycrystalline platinum substrate both in air and in a deaerated environment. In both cases, the detected faradaic current shows a structural dependence on the surface crystallographic orientation. Significantly, in the presence of oxygen (aerated solution) the electrochemical current decreases strongly for almost all grains (crystallographic orientations). The results highlight the flexibility of voltammetric SECCM for electrochemical imaging and present important implications for hydrazine electroanalysis. PMID:25942527

  19. Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super-Resolution STED Microscopy in Living Cells.

    Science.gov (United States)

    Butkevich, Alexey N; Mitronova, Gyuzel Yu; Sidenstein, Sven C; Klocke, Jessica L; Kamin, Dirk; Meineke, Dirk N H; D'Este, Elisa; Kraemer, Philip-Tobias; Danzl, Johann G; Belov, Vladimir N; Hell, Stefan W

    2016-03-01

    A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye-ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure-property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one- and two-color images of living cells with an optical resolution of 40-60 nm.

  20. Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells

    OpenAIRE

    Hagen, Christoph; Guttmann, Peter; Klupp, Barbara; Werner, Stephan; Rehbein, Stefan; Mettenleiter, Thomas C.; Schneider, Gerd; Grünewald, Kay

    2012-01-01

    Soft X-ray cryo-microscopy/tomography of vitreous samples is becoming a valuable tool in structural cell biology. Within the ‘water-window’ wavelength region (2.34–4.37 nm), it provides absorption contrast images with high signal to noise ratio and resolution of a few tens of nanometer. Soft X-rays with wavelengths close to the K-absorption edge of oxygen penetrate biological samples with thicknesses in the micrometer range. Here, we report on the application of a recently established extensi...

  1. The nematode stoma: Homology of cell architecture with improved understanding by confocal microscopy of labeled cell boundaries.

    Science.gov (United States)

    Jay Burr, A H; Baldwin, James G

    2016-09-01

    Nematode stomas vary widely in the cuticular structures evolved for different feeding strategies, yet the arrangement of the epithelial cell classes that form these structures may be conserved. This article addresses several issues that have impeded the full acceptance of this hypothesis including controversies arising from the structure of the Caenorhabditis elegans stoma. We investigated fluorescent antibody labeling of cell boundaries in conjunction with confocal microscopy as an alternative to transmission electron microscopy (TEM), using MH27 to label apical junctions in C. elegans and two other species. Accurately spaced optical sections collected by the confocal microscope provide a three-dimensional array of pixels (voxels) that, using image-processing software, can be rotated and sectioned at accurately chosen thicknesses and locations. Ribbons of fluorescence clearly identify cell boundaries along the luminal cuticle in C. elegans and Zeldia punctata and less clearly in Bunonema sp. The patterns render cell classes and their relationships readily identifiable. In the C. elegans stoma they correct a misreading of serial TEMs that was not congruent with architecture in other nematodes-the row of marginal cells is now seen to be continuous as in other nematodes, rather than being interrupted by encircling pm1 cells. Also impeding understanding, the reference to certain cell classes as 'epithelial' and others as "muscle" in the C. elegans literature is at variance with muscle expression in most other taxa. For consistent comparison among species, we propose that these cell class descriptors based on function be replaced by topological terms. With these and other confusing concepts and terminology removed, the homology of the cellular architecture among taxa becomes obvious. We provide a corrected description of the cell architecture of the C. elegans stoma and examples of how it is modified in other taxa with different feeding strategies. J. Morphol. 277

  2. The nematode stoma: Homology of cell architecture with improved understanding by confocal microscopy of labeled cell boundaries.

    Science.gov (United States)

    Jay Burr, A H; Baldwin, James G

    2016-09-01

    Nematode stomas vary widely in the cuticular structures evolved for different feeding strategies, yet the arrangement of the epithelial cell classes that form these structures may be conserved. This article addresses several issues that have impeded the full acceptance of this hypothesis including controversies arising from the structure of the Caenorhabditis elegans stoma. We investigated fluorescent antibody labeling of cell boundaries in conjunction with confocal microscopy as an alternative to transmission electron microscopy (TEM), using MH27 to label apical junctions in C. elegans and two other species. Accurately spaced optical sections collected by the confocal microscope provide a three-dimensional array of pixels (voxels) that, using image-processing software, can be rotated and sectioned at accurately chosen thicknesses and locations. Ribbons of fluorescence clearly identify cell boundaries along the luminal cuticle in C. elegans and Zeldia punctata and less clearly in Bunonema sp. The patterns render cell classes and their relationships readily identifiable. In the C. elegans stoma they correct a misreading of serial TEMs that was not congruent with architecture in other nematodes-the row of marginal cells is now seen to be continuous as in other nematodes, rather than being interrupted by encircling pm1 cells. Also impeding understanding, the reference to certain cell classes as 'epithelial' and others as "muscle" in the C. elegans literature is at variance with muscle expression in most other taxa. For consistent comparison among species, we propose that these cell class descriptors based on function be replaced by topological terms. With these and other confusing concepts and terminology removed, the homology of the cellular architecture among taxa becomes obvious. We provide a corrected description of the cell architecture of the C. elegans stoma and examples of how it is modified in other taxa with different feeding strategies. J. Morphol. 277

  3. Study on lipid droplet dynamics in live cells and fluidity changes in model bacterial membranes using optical microscopy techniques

    OpenAIRE

    Wong, Christine Shiang Yee

    2014-01-01

    In this thesis optical microscopy techniques are used to consider aspects of viral and bacterial infections. In part 1, the physical effects of cytomegalovirus on lipid droplet dynamics in live cells are studied; in part 2, the effects of an antimicrobial peptide on the fluidity of model bacterial membranes are studied. The optical microscopy techniques used to study the effects of murine-cytomegalovirus (mCMV) on lipid droplets in live NIH/3T3 fibroblast cells in real-time are...

  4. Evaluation of Corneal Stromal Demarcation Line after Two Different Protocols of Accelerated Corneal Collagen Cross-Linking Procedures Using Anterior Segment Optical Coherence Tomography and Confocal Microscopy

    Directory of Open Access Journals (Sweden)

    Engin Bilge Ozgurhan

    2014-01-01

    Full Text Available Purpose. To evaluate the depth of corneal stromal demarcation line using AS-OCT and confocal microscopy after two different protocols of accelerated corneal collagen cross-linking procedures (CXL. Methods. Patients with keratoconus were divided into two groups. Peschke CXL device (Peschke CCL-VARIO Meditrade GmbH applied UVA light with an intended irradiance of 18.0 mW/cm2 for 5 minutes after applying riboflavin for 20 minutes (group 1 and 30 minutes (group 2. One month postoperatively, corneal stromal demarcation line was measured using AS-OCT and confocal microscopy. Results. This study enrolled 34 eyes of 34 patients (17 eyes in group 1 and 17 eyes in group 2. The mean depth of the corneal stromal demarcation line was 208.64±18.41 μm in group 1 and 240.37±18.89 μm in group 2 measured with AS OCT, while it was 210.29±18.66 μm in group 1 and 239.37±20.07 μm in group 2 measured with confocal microscopy. Corneal stromal demarcation line depth measured with AS OCT or confocal microscopy was significantly deeper in group 2 than group 1 (P<0.01. Conclusion. The group in which riboflavin was applied for 30 minutes showed significantly deeper corneal stromal demarcation line than the group in which riboflavin was applied for 20 minutes.

  5. Spiral conformation of Vibrio cholerae as determined by scanning electron microscopy of elongated cells induced by cephalexin treatment.

    OpenAIRE

    Konishi, H.; Katayama, A.; Ito, T.; Tanaka, S.; Yoshii, Z

    1986-01-01

    The elongated cells of Vibrio spp. induced by cephalexin treatment were examined by scanning electron microscopy. The results showed that Vibrio cholerae has a twisted cell body and a right-handed spiral conformation and that the cell bodies of V. parahaemolyticus and V. alginolyticus are straight rather than curved.

  6. Context based mixture model for cell phase identification in automated fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Zhou Xiaobo

    2007-01-01

    Full Text Available Abstract Background Automated identification of cell cycle phases of individual live cells in a large population captured via automated fluorescence microscopy technique is important for cancer drug discovery and cell cycle studies. Time-lapse fluorescence microscopy images provide an important method to study the cell cycle process under different conditions of perturbation. Existing methods are limited in dealing with such time-lapse data sets while manual analysis is not feasible. This paper presents statistical data analysis and statistical pattern recognition to perform this task. Results The data is generated from Hela H2B GFP cells imaged during a 2-day period with images acquired 15 minutes apart using an automated time-lapse fluorescence microscopy. The patterns are described with four kinds of features, including twelve general features, Haralick texture features, Zernike moment features, and wavelet features. To generate a new set of features with more discriminate power, the commonly used feature reduction techniques are used, which include Principle Component Analysis (PCA, Linear Discriminant Analysis (LDA, Maximum Margin Criterion (MMC, Stepwise Discriminate Analysis based Feature Selection (SDAFS, and Genetic Algorithm based Feature Selection (GAFS. Then, we propose a Context Based Mixture Model (CBMM for dealing with the time-series cell sequence information and compare it to other traditional classifiers: Support Vector Machine (SVM, Neural Network (NN, and K-Nearest Neighbor (KNN. Being a standard practice in machine learning, we systematically compare the performance of a number of common feature reduction techniques and classifiers to select an optimal combination of a feature reduction technique and a classifier. A cellular database containing 100 manually labelled subsequence is built for evaluating the performance of the classifiers. The generalization error is estimated using the cross validation technique. The

  7. Bone Marrow Mononuclear Cell Transplantation Restores Inflammatory Balance of Cytokines after ST Segment Elevation Myocardial Infarction.

    Directory of Open Access Journals (Sweden)

    Kirsi Alestalo

    Full Text Available Acute myocardial infarction (AMI launches an inflammatory response and a repair process to compensate cardiac function. During this process, the balance between proinflammatory and anti-inflammatory cytokines is important for optimal cardiac repair. Stem cell transplantation after AMI improves tissue repair and increases the ventricular ejection fraction. Here, we studied in detail the acute effect of bone marrow mononuclear cell (BMMNC transplantation on proinflammatory and anti-inflammatory cytokines in patients with ST segment elevation myocardial infarction (STEMI.Patients with STEMI treated with thrombolysis followed by percutaneous coronary intervention (PCI were randomly assigned to receive either BMMNC or saline as an intracoronary injection. Cardiac function was evaluated by left ventricle angiogram during the PCI and again after 6 months. The concentrations of 27 cytokines were measured from plasma samples up to 4 days after the PCI and the intracoronary injection.Twenty-six patients (control group, n = 12; BMMNC group, n = 14 from the previously reported FINCELL study (n = 80 were included to this study. At day 2, the change in the proinflammatory cytokines correlated with the change in the anti-inflammatory cytokines in both groups (Kendall's tau, control 0.6; BMMNC 0.7. At day 4, the correlation had completely disappeared in the control group but was preserved in the BMMNC group (Kendall's tau, control 0.3; BMMNC 0.7.BMMNC transplantation is associated with preserved balance between pro- and anti-inflammatory cytokines after STEMI in PCI-treated patients. This may partly explain the favorable effect of stem cell transplantation after AMI.

  8. In situ microscopy for online monitoring of cell concentration in Pichia pastoris cultivations.

    Science.gov (United States)

    Marquard, D; Enders, A; Roth, G; Rinas, U; Scheper, T; Lindner, P

    2016-09-20

    In situ Microscopy (ISM) is an optical non-invasive technique to monitor cells in bioprocesses in real-time. Pichia pastoris is one of the most promising protein expression systems. This yeast combines fast growth on simple media and important eukaryotic features such as glycosylation. In this work, the ISM technology was applied to Pichia pastoris cultivations for online monitoring of the cell concentration during cultivation. Different ISM settings were tested. The acquired images were analyzed with two image processing algorithms. In seven cultivations the cell concentration was monitored by the applied algorithms and offline samples were taken to determine optical density (OD) and dry cell mass (DCM). Cell concentrations up to 74g/L dry cell mass could be analyzed via the ISM. Depending on the algorithm and the ISM settings, an accuracy between 0.3 % and 12 % was achieved. The overall results show that for a robust measurement a combination of the two described algorithms is required. PMID:27485811

  9. Spring constants and adhesive properties of native bacterial biofilm cells measured by atomic force microscopy.

    Science.gov (United States)

    Volle, C B; Ferguson, M A; Aidala, K E; Spain, E M; Núñez, M E

    2008-11-15

    Bacterial biofilms were imaged by atomic force microscopy (AFM), and their elasticity and adhesion to the AFM tip were determined from a series of tip extension and retraction cycles. Though the five bacterial strains studied included both Gram-negative and -positive bacteria and both environmental and laboratory strains, all formed simple biofilms on glass surfaces. Cellular spring constants, determined from the extension portion of the force cycle, varied between 0.16+/-0.01 and 0.41+/-0.01 N/m, where larger spring constants were measured for Gram-positive cells than for Gram-negative cells. The nonlinear regime in the extension curve depended upon the biomolecules on the cell surface: the extension curves for the smooth Gram-negative bacterial strains with the longest lipopolysaccharides on their surface had a larger nonlinear region than the rough bacterial strain with shorter lipopolysaccharides on the surface. Adhesive forces between the retracting silicon nitride tip and the cells varied between cell types in terms of the force components, the distance components, and the number of adhesion events. The Gram-negative cells' adhesion to the tip showed the longest distance components, sometimes more than 1 microm, whereas the shortest distance adhesion events were measured between the two Gram-positive cell types and the tip. Fixation of free-swimming planktonic cells by NHS and EDC perturbed both the elasticity and the adhesive properties of the cells. Here we consider the biochemical meaning of the measured physical properties of simple biofilms and implications to the colonization of surfaces in the first stages of biofilm formation. PMID:18815013

  10. A Rapid and Efficient 2D/3D Nuclear Segmentation Method for Analysis of Early Mouse Embryo and Stem Cell Image Data

    Directory of Open Access Journals (Sweden)

    Xinghua Lou

    2014-03-01

    Full Text Available Segmentation is a fundamental problem that dominates the success of microscopic image analysis. In almost 25 years of cell detection software development, there is still no single piece of commercial software that works well in practice when applied to early mouse embryo or stem cell image data. To address this need, we developed MINS (modular interactive nuclear segmentation as a MATLAB/C++-based segmentation tool tailored for counting cells and fluorescent intensity measurements of 2D and 3D image data. Our aim was to develop a tool that is accurate and efficient yet straightforward and user friendly. The MINS pipeline comprises three major cascaded modules: detection, segmentation, and cell position classification. An extensive evaluation of MINS on both 2D and 3D images, and comparison to related tools, reveals improvements in segmentation accuracy and usability. Thus, its accuracy and ease of use will allow MINS to be implemented for routine single-cell-level image analyses.

  11. What is a segment?

    Science.gov (United States)

    Hannibal, Roberta L; Patel, Nipam H

    2013-12-17

    Animals have been described as segmented for more than 2,000 years, yet a precise definition of segmentation remains elusive. Here we give the history of the definition of segmentation, followed by a discussion on current controversies in defining a segment. While there is a general consensus that segmentation involves the repetition of units along the anterior-posterior (a-p) axis, long-running debates exist over whether a segment can be composed of only one tissue layer, whether the most anterior region of the arthropod head is considered segmented, and whether and how the vertebrate head is segmented. Additionally, we discuss whether a segment can be composed of a single cell in a column of cells, or a single row of cells within a grid of cells. We suggest that 'segmentation' be used in its more general sense, the repetition of units with a-p polarity along the a-p axis, to prevent artificial classification of animals. We further suggest that this general definition be combined with an exact description of what is being studied, as well as a clearly stated hypothesis concerning the specific nature of the potential homology of structures. These suggestions should facilitate dialogue among scientists who study vastly differing segmental structures.

  12. Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues.

    Directory of Open Access Journals (Sweden)

    Roberta Galli

    Full Text Available Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.

  13. Local electromechanical properties of different phenotype models of vascular smooth muscle cells using force microscopy

    Science.gov (United States)

    Thompson, Gary; Reukov, Vladimir; Nikiforov, Maxim; Guo, Senli; Ovchinnikov, Oleg; Jesse, Stephen; Kalinin, Sergei; Vertegel, Alexey

    2010-03-01

    Vascular smooth muscle cells (VSMCs) exist as a spectrum of diverse phenotypes raning between contractile and synthetic, the latter being associated with disease states. Different VSMC phenotypes, modeled using serum-starvation, exhibit characteristic electromechanical responses that can be distinguished using band excitation piezoresponse force microscopy (BEPFM), which maps information at the same rate as the atomic force microscope (AFM) scan performed simultaneously. BEPFM image formation mechanism in the culture medium is determined using excitation steps from 1 mV to 100 V. High voltage improves contrast between cells and collagen-coated substrates. Viscoelasticity from AFM stress relaxation experiments and local elasticity from force maps correlate to BEPFM data providing a map of local mechanical properties on different VSMCs.

  14. Motion artefact detection in structured illumination microscopy for live cell imaging.

    Science.gov (United States)

    Förster, Ronny; Wicker, Kai; Müller, Walter; Jost, Aurélie; Heintzmann, Rainer

    2016-09-19

    The reconstruction process of structured illumination microscopy (SIM) creates substantial artefacts if the specimen has moved during the acquisition. This reduces the applicability of SIM for live cell imaging, because these artefacts cannot always be recognized as such in the final image. A movement is not necessarily visible in the raw data, due to the varying excitation patterns and the photon noise. We present a method to detect motion by extracting and comparing two independent 3D wide-field images out of the standard SIM raw data without needing additional images. Their difference reveals moving objects overlaid with noise, which are distinguished by a probability theory-based analysis. Our algorithm tags motion-artefacts in the final high-resolution image for the first time, preventing the end-user from misinterpreting the data. We show and explain different types of artefacts and demonstrate our algorithm on a living cell.

  15. Calcium dynamics in root cells of Arabidopsis thaliana visualized with selective plane illumination microscopy.

    Directory of Open Access Journals (Sweden)

    Alex Costa

    Full Text Available Selective Plane Illumination Microscopy (SPIM is an imaging technique particularly suited for long term in-vivo analysis of transparent specimens, able to visualize small organs or entire organisms, at cellular and eventually even subcellular resolution. Here we report the application of SPIM in Calcium imaging based on Förster Resonance Energy Transfer (FRET. Transgenic Arabidopsis plants expressing the genetically encoded-FRET-based Ca(2+ probe Cameleon, in the cytosol or nucleus, were used to demonstrate that SPIM enables ratiometric fluorescence imaging at high spatial and temporal resolution, both at tissue and single cell level. The SPIM-FRET technique enabled us to follow nuclear and cytosolic Ca(2+ dynamics in Arabidopsis root tip cells, deep inside the organ, in response to different stimuli. A relevant physiological phenomenon, namely Ca(2+ signal percolation, predicted in previous studies, has been directly visualized.

  16. Identification and automatic segmentation of multiphasic cell growth using a linear hybrid model.

    Science.gov (United States)

    Hartmann, András; Neves, Ana Rute; Lemos, João M; Vinga, Susana

    2016-09-01

    This article considers a new mathematical model for the description of multiphasic cell growth. A linear hybrid model is proposed and it is shown that the two-parameter logistic model with switching parameters can be represented by a Switched affine AutoRegressive model with eXogenous inputs (SARX). The growth phases are modeled as continuous processes, while the switches between the phases are considered to be discrete events triggering a change in growth parameters. This framework provides an easily interpretable model, because the intrinsic behavior is the same along all the phases but with a different parameterization. Another advantage of the hybrid model is that it offers a simpler alternative to recent more complex nonlinear models. The growth phases and parameters from datasets of different microorganisms exhibiting multiphasic growth behavior such as Lactococcus lactis, Streptococcus pneumoniae, and Saccharomyces cerevisiae, were inferred. The segments and parameters obtained from the growth data are close to the ones determined by the experts. The fact that the model could explain the data from three different microorganisms and experiments demonstrates the strength of this modeling approach for multiphasic growth, and presumably other processes consisting of multiple phases. PMID:27424949

  17. Multicolor excitation two-photon microscopy: in vivo imaging of cells and tissues

    Science.gov (United States)

    Li, Dong; Zheng, Wei; Qu, Jianan Y.

    2010-02-01

    Two-photon microscopy based on endogenous fluorescence provides non-invasive imaging of living biological system. Reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), keratin, collagen and elastin are the endogenous fluorophores widely used as the contrast agents for imaging metabolism and morphology of living cells and tissue. The fluorescence of tryptophan, a kind of essential amino acid, conveys the information on cellular protein content, structure and microenvironment. However, it can't be effectively excited by the commonly used Ti:sapphire femtosecond laser. Because each endogenous fluorophore provides limited information, it is desirable to simultaneously excite fluorescence from as many fluorophores as possible to obtain accurate biochemical and morphological information on biomedical samples. In this study, we demonstrate that the supercontinuum generation from a photonic crystal fiber (PCF) excited by an ultrafast source can be used to excite multiple endogenous nonlinear optical signals simultaneously. By employing the spectral lifetime detection capability, this technology provides a unique approach to sense the fine structure, protein distribution and cellular metabolism of cells and tissues in vivo. In particular, with application of acetic acid, a safe contrast agent used for detection cervical cancer for many years, the tryptophan signals reveal cellular morphology and even cell-cell junctions clearly. Moreover, it was found that the pH value dependent lifetime of tryptophan fluorescence could provide the qualitative information on the gradient of pH value in epithelial tissue. Finally, we will demonstrate the potential of our multi-color TPEF microscopy to investigate the early development of cancer in epithelial tissue.

  18. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis.

    Science.gov (United States)

    Silvestri, Ludovico; Paciscopi, Marco; Soda, Paolo; Biamonte, Filippo; Iannello, Giulio; Frasconi, Paolo; Pavone, Francesco S

    2015-01-01

    Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells (PCs) across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all PCs are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent PCs. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of PCs, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of PCs with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments. PMID:26074783

  19. The Use of Atomic Force Microscopy as a Technique for the Identification of Cancerous Cells

    International Nuclear Information System (INIS)

    The monograph presents the use of atomic force microscopy (AFM) as a tool for the identification of cancerous cells by studies of the expression of different types of molecules directly on the surface of living cells. The full quantitative description (that is not accessible by other techniques) performed for a given type of molecular interactions has been obtained by using the following quantities: an unbinding force, probability, rupture length and the effective spring constant taking into account the stiffness of a single complex. All, these parameters were extracted from AFM measurements The analysis of the interaction forces performed by AFM allows the quantitative determination of: i) the static properties of a single molecular complex where its strength of interaction and stiffness of the studied complex can be obtained, ii) dynamic properties, on the basis of which the kinetic properties of the unbinding process can be delivered, and iii) properties of adhesion clusters, where the interrelation between single complexes can be characterized, in particular the mechanism of the unbinding can be obtained. The presented characterization of the interaction force between single molecules demonstrates that atomic force microscopy can be used as exceptional technique to study the expression of molecules on a cell surface. Such measurements are not limited to a typical interactions occurring between single molecules but also it is possible to study the interactions between parts of molecules. The results presented in this monograph point to a novel approach to identify cancer-related changes in a quantitative way what can be used for describing and confirming the pathological state of a single cell. (author)

  20. Correlative microscopy.

    Science.gov (United States)

    Loussert Fonta, Céline; Humbel, Bruno M

    2015-09-01

    In recent years correlative microscopy, combining the power and advantages of different imaging system, e.g., light, electrons, X-ray, NMR, etc., has become an important tool for biomedical research. Among all the possible combinations of techniques, light and electron microscopy, have made an especially big step forward and are being implemented in more and more research labs. Electron microscopy profits from the high spatial resolution, the direct recognition of the cellular ultrastructure and identification of the organelles. It, however, has two severe limitations: the restricted field of view and the fact that no live imaging can be done. On the other hand light microscopy has the advantage of live imaging, following a fluorescently tagged molecule in real time and at lower magnifications the large field of view facilitates the identification and location of sparse individual cells in a large context, e.g., tissue. The combination of these two imaging techniques appears to be a valuable approach to dissect biological events at a submicrometer level. Light microscopy can be used to follow a labelled protein of interest, or a visible organelle such as mitochondria, in time, then the sample is fixed and the exactly same region is investigated by electron microscopy. The time resolution is dependent on the speed of penetration and fixation when chemical fixatives are used and on the reaction time of the operator for cryo-fixation. Light microscopy can also be used to identify cells of interest, e.g., a special cell type in tissue or cells that have been modified by either transfections or RNAi, in a large population of non-modified cells. A further application is to find fluorescence labels in cells on a large section to reduce searching time in the electron microscope. Multiple fluorescence labelling of a series of sections can be correlated with the ultrastructure of the individual sections to get 3D information of the distribution of the marked proteins: array

  1. Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yun

    2008-12-18

    The purpose of this research was to expand the chemiluminescence microscopy applications in live bacterial/mammalian cell imaging and to improve the detection sensitivity for ATP leaking or release events. We first demonstrated that chemiluminescence (CL) imaging can be used to interrogate single bacterial cells. While using a luminometer allows detecting ATP from cell lysate extracted from at least 10 bacterial cells, all previous cell CL detection never reached this sensitivity of single bacteria level. We approached this goal with a different strategy from before: instead of breaking bacterial cell membrane and trying to capture the transiently diluted ATP with the firefly luciferase CL assay, we introduced the firefly luciferase enzyme into bacteria using the modern genetic techniques and placed the CL reaction substrate D-luciferin outside the cells. By damaging the cell membrane with various antibacterial drugs including antibiotics such as Penicillins and bacteriophages, the D-luciferin molecules diffused inside the cell and initiated the reaction that produces CL light. As firefly luciferases are large protein molecules which are retained within the cells before the total rupture and intracellular ATP concentration is high at the millmolar level, the CL reaction of firefly luciferase, ATP and D-luciferin can be kept for a relatively long time within the cells acting as a reaction container to generate enough photons for detection by the extremely sensitive intensified charge coupled device (ICCD) camera. The result was inspiring as various single bacterium lysis and leakage events were monitored with 10-s temporal resolution movies. We also found a new way of enhancing diffusion D-luciferin into cells by dehydrating the bacteria. Then we started with this novel single bacterial CL imaging technique, and applied it for quantifying gene expression levels from individual bacterial cells. Previous published result in single cell gene expression quantification

  2. Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yun [Iowa State Univ., Ames, IA (United States)

    2008-12-18

    The purpose of this research was to expand the chemiluminescence microscopy applications in live bacterial/mammalian cell imaging and to improve the detection sensitivity for ATP leaking or release events. We first demonstrated that chemiluminescence (CL) imaging can be used to interrogate single bacterial cells. While using a luminometer allows detecting ATP from cell lysate extracted from at least 10 bacterial cells, all previous cell CL detection never reached this sensitivity of single bacteria level. We approached this goal with a different strategy from before: instead of breaking bacterial cell membrane and trying to capture the transiently diluted ATP with the firefly luciferase CL assay, we introduced the firefly luciferase enzyme into bacteria using the modern genetic techniques and placed the CL reaction substrate D-luciferin outside the cells. By damaging the cell membrane with various antibacterial drugs including antibiotics such as Penicillins and bacteriophages, the D-luciferin molecules diffused inside the cell and initiated the reaction that produces CL light. As firefly luciferases are large protein molecules which are retained within the cells before the total rupture and intracellular ATP concentration is high at the millmolar level, the CL reaction of firefly luciferase, ATP and D-luciferin can be kept for a relatively long time within the cells acting as a reaction container to generate enough photons for detection by the extremely sensitive intensified charge coupled device (ICCD) camera. The result was inspiring as various single bacterium lysis and leakage events were monitored with 10-s temporal resolution movies. We also found a new way of enhancing diffusion D-luciferin into cells by dehydrating the bacteria. Then we started with this novel single bacterial CL imaging technique, and applied it for quantifying gene expression levels from individual bacterial cells. Previous published result in single cell gene expression quantification

  3. Potential of Cathodoluminescence Microscopy and Spectroscopy for the Detection of Prokaryotic Cells on Minerals

    Science.gov (United States)

    Rommevaux-Jestin, Céline; Ménez, Bénédicte

    2010-11-01

    Detecting mineral-hosted ecosystems to assess the extent and functioning of the biosphere from the surface to deep Earth requires appropriate techniques that provide, beyond the morphological criteria, indubitable clues of the presence of prokaryotic cells. Here, we evaluate the capability of cathodoluminescence microscopy and spectroscopy, implemented on a scanning electron microscope, to identify prokaryotes on mineral surfaces. For this purpose, we used, as a first step, a simple model of either unstained or stained cultivable cells (Escherichia coli, Deinococcus radiodurans) deposited on minerals that are common in the oceanic crust (basaltic glass, amphibole, pyroxene, and magnetite). Our results demonstrate that the detection of cells is possible at the micrometric level on the investigated minerals through the intrinsic fluorescence of their constituting macromolecules (aromatic amino and nucleic acids, coenzymes). This allows us to distinguish biomorph inorganic phases from cells. This easily implemented technique permits an exploration of colonized rock samples. In addition, the range of spectrometric techniques available on a scanning electron microscope can provide additional information on the nature and chemistry of the associated mineral phases, which would lead to a simultaneous characterization of cells, their microhabitats, and a better understanding of their potential relationships.

  4. Fast Stiffness Mapping of Cells Using High-Bandwidth Atomic Force Microscopy.

    Science.gov (United States)

    Wang, Andrew; Vijayraghavan, Karthik; Solgaard, Olav; Butte, Manish J

    2016-01-26

    The cytoskeleton controls cellular morphology and mediates the mechanical interactions between a cell and its environment. Atomic force microscopy (AFM) has the unique capability to map cytoskeletal mechanics and structures with nanometer resolution. However, whole-cell cytomechanical imaging with conventional AFM techniques is limited by low imaging speed. Here, we present fast nanomechanical mapping of cells using high-bandwidth AFM (HB-AFM), where >10(6) nanoindentation measurements were acquired in ∼10 min-a task that would take weeks to finish using conventional AFM. High-bandwidth measurements enabled capture of the entire tip-sample interaction for each tap on cells, engendering a new measurement ("force phase") that exceeds the contrast of conventional tapping mode and enabling spectral visualization of >10 harmonics. The abundance of measurements allowed discovery of subtle cytomechanical features, including the stiffness of fibers of the cellular spectrin network in situ. This approach bridges HB-AFM and high-harmonic imaging and opens future opportunities for measuring the dynamic mechanical properties of living cells. PMID:26554581

  5. Generating and characterizing the mechanical properties of cell-derived matrices using atomic force microscopy.

    Science.gov (United States)

    Tello, Marta; Spenlé, Caroline; Hemmerlé, Joseph; Mercier, Luc; Fabre, Roxane; Allio, Guillaume; Simon-Assmann, Patricia; Goetz, Jacky G

    2016-02-01

    Mechanical interaction between cells and their surrounding extracellular matrix (ECM) controls key processes such as proliferation, differentiation and motility. For many years, two-dimensional (2D) models were used to better understand the interactions between cells and their surrounding ECM. More recently, variation of the mechanical properties of tissues has been reported to play a major role in physiological and pathological scenarios such as cancer progression. The 3D architecture of the ECM finely tunes cellular behavior to perform physiologically relevant tasks. Technical limitations prevented scientists from obtaining accurate assessment of the mechanical properties of physiologically realistic matrices. There is therefore a need for combining the production of high-quality cell-derived 3D matrices (CDMs) and the characterization of their topographical and mechanical properties. Here, we describe methods that allow to accurately measure the young modulus of matrices produced by various cellular types. In the first part, we will describe and review several protocols for generating CDMs matrices from endothelial, epithelial, fibroblastic, muscle and mesenchymal stem cells. We will discuss tools allowing the characterization of the topographical details as well as of the protein content of such CDMs. In a second part, we will report the methodologies that can be used, based on atomic force microscopy, to accurately evaluate the stiffness properties of the CDMs through the quantification of their young modulus. Altogether, such methodologies allow characterizing the stiffness and topography of matrices deposited by the cells, which is key for the understanding of cellular behavior in physiological conditions.

  6. Sub-cellular force microscopy in single normal and cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Babahosseini, H. [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States); Carmichael, B. [Nonlinear Intelligent Structures Laboratory, Department of Mechanical Engineering, University of Alabama, Tuscaloosa, AL 35487-0276 (United States); Strobl, J.S. [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States); Mahmoodi, S.N., E-mail: nmahmoodi@eng.ua.edu [Nonlinear Intelligent Structures Laboratory, Department of Mechanical Engineering, University of Alabama, Tuscaloosa, AL 35487-0276 (United States); Agah, M., E-mail: agah@vt.edu [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States)

    2015-08-07

    This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. - Highlights: • The cells are modeled as a triple-layered structure using Generalized Maxwell model. • The sub-domains include membrane/cortex, cytoplasm/nucleus, and nuclear/integrin. • Biomechanics of corresponding sub-domains are compared among normal and cancer cells. • Viscoelasticity of sub-domains show a decreasing trend from normal to cancer cells. • The decreasing trend becomes most significant in the deeper sub-domain.

  7. Importance and Challenges of Electrochemical in Situ Liquid Cell Electron Microscopy for Energy Conversion Research.

    Science.gov (United States)

    Hodnik, Nejc; Dehm, Gerhard; Mayrhofer, Karl J J

    2016-09-20

    The foreseeable worldwide energy and environmental challenges demand renewable alternative sources, energy conversion, and storage technologies. Therefore, electrochemical energy conversion devices like fuel cells, electrolyzes, and supercapacitors along with photoelectrochemical devices and batteries have high potential to become increasingly important in the near future. Catalytic performance in electrochemical energy conversion results from the tailored properties of complex nanometer-sized metal and metal oxide particles, as well as support nanostructures. Exposed facets, surface defects, and other structural and compositional features of the catalyst nanoparticles affect the electrocatalytic performance to varying degrees. The characterization of the nanometer-size and atomic regime of electrocatalysts and its evolution over time are therefore paramount for an improved understanding and significant optimization of such important technologies like electrolyzers or fuel cells. Transmission electron microscopy (TEM) and scanning transmission electron microscope (STEM) are to a great extent nondestructive characterization tools that provide structural, morphological, and compositional information with nanoscale or even atomic resolution. Due to recent marked advancement in electron microscopy equipment such as aberration corrections and monochromators, such insightful information is now accessible in many institutions around the world and provides huge benefit to everyone using electron microscopy characterization in general. Classical ex situ TEM characterization of random catalyst locations however suffers from two limitations regarding catalysis. First, the necessary low operation pressures in the range of 10(-6) to 10(-9) mbar for TEM are not in line with typical reaction conditions, especially considering electrocatalytic solid-liquid interfaces, so that the active state cannot be assessed. Second, and somewhat related, is the lack of time resolution for the

  8. Probing the mechanical properties of TNF-α stimulated endothelial cell with atomic force microscopy

    Directory of Open Access Journals (Sweden)

    Sei-Young Lee

    2011-01-01

    Full Text Available Sei-Young Lee1,2, Ana-Maria Zaske3, Tommaso Novellino1,4*, Delia Danila3, Mauro Ferrari1,5*, Jodie Conyers3, Paolo Decuzzi1,6*1Department of Nanomedicine and Biomedical Engineering, The University of Texas Medical School at Houston, Houston, TX, USA; 2Department of Mechanical Engineering, The University of Texas at Austin, Austin, TX, USA; 3CeTIR – Center for Translational Injury Research, The University of Texas Health Science Center at Houston, Houston, TX, USA; 4Department of Biomedical Engineering, Biomedical Campus University of Rome, Italy; 5MD Anderson Cancer Center, Houston, TX, USA; 6BioNEM – Center of Bio-Nanotechnology and Engineering for Medicine, University of Magna Graecia, Catanzaro, Italy; *Currently at Department of Nanomedicine and Biomedical Engineering, The Methodist Hospital Research Institute, Houston, TX, USAAbstract: TNF-α (tumor necrosis factor-α is a potent pro-inflammatory cytokine that regulates the permeability of blood and lymphatic vessels. The plasma concentration of TNF-α is elevated (> 1 pg/mL in several pathologies, including rheumatoid arthritis, atherosclerosis, cancer, pre-eclampsia; in obese individuals; and in trauma patients. To test whether circulating TNF-α could induce similar alterations in different districts along the vascular system, three endothelial cell lines, namely HUVEC, HPMEC, and HCAEC, were characterized in terms of 1 mechanical properties, employing atomic force microscopy; 2 cytoskeletal organization, through fluorescence microscopy; and 3 membrane overexpression of adhesion molecules, employing ELISA and immunostaining. Upon stimulation with TNF-α (10 ng/mL for 20 h, for all three endothelial cells, the mechanical stiffness increased by about 50% with a mean apparent elastic modulus of E ~5 ± 0.5 kPa (~3.3 ± 0.35 kPa for the control cells; the density of F-actin filaments increased in the apical and median planes; and the ICAM-1 receptors were overexpressed compared with

  9. Scanning electrochemical microscopy of living cells: different redox activities of nonmetastatic and metastatic human breast cells.

    Science.gov (United States)

    Liu, B; Rotenberg, S A; Mirkin, M V

    2000-08-29

    Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Calpha), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators. PMID:10963658

  10. An automated three-dimensional detection and segmentation method for touching cells by integrating concave points clustering and random walker algorithm.

    Directory of Open Access Journals (Sweden)

    Yong He

    Full Text Available Characterizing cytoarchitecture is crucial for understanding brain functions and neural diseases. In neuroanatomy, it is an important task to accurately extract cell populations' centroids and contours. Recent advances have permitted imaging at single cell resolution for an entire mouse brain using the Nissl staining method. However, it is difficult to precisely segment numerous cells, especially those cells touching each other. As presented herein, we have developed an automated three-dimensional detection and segmentation method applied to the Nissl staining data, with the following two key steps: 1 concave points clustering to determine the seed points of touching cells; and 2 random walker segmentation to obtain cell contours. Also, we have evaluated the performance of our proposed method with several mouse brain datasets, which were captured with the micro-optical sectioning tomography imaging system, and the datasets include closely touching cells. Comparing with traditional detection and segmentation methods, our approach shows promising detection accuracy and high robustness.

  11. Microscopy of hierarchically organized TiO2 photoelectrode for dye solar cells

    Science.gov (United States)

    Eskandar, A.; Mohamed, N. M.

    2015-07-01

    Research on improving the performance of dye solar cells has various aspects of the device being investigated. This paper analyzes the deliberately hierarchized photoelectrode configuration for DSC applications to improve the performance of DSCs. Multiple layers of differently composed TiO2 particle types namely aggregates and nanoparticles were deposited to form a photoelectrode with thickness of about 12 µm. The photoelectrodes were assembled into working DSCs with an active area of 1 cm2. Measurement for solar power conversion performance was measured under 1 sun at AM1.5 spectrum simulated sunlight. Electron microscopy for photoelectrode analysis was conducted using Field Emission Scattering Electron Microscopy with enhanced resolution. External Quantum Efficiency was measured using a purpose built instrument. Kinetics were investigated using the Electrochemical Impedance Spectroscopy (EIS) measurement with a potentiostat. The best performing DSC is of the hierarchically organized photoelectrode with a photoconversion efficiency of 4.58%, an increase of 14% in comparison to the reference samples with fully aggregates configuration. Short circuit current density, Jsc increases by about 2.223 mA cm-2 relative to the blanks. The electron microscopy confirmed expected thickness at around 10 µm and layers forming the photoelectrode being hierarchically deposited with ˜20 nm TiO2 nanoparticles and 450 nm TiO2 aggregates mixture composition. EQE improved especially for visible region of 500-550 nm light wavelengths with 12 % increase in the response of in that region. Improvement to the diffusion coefficient as measured by the EIS contributed to the performance increase of the photoelectrode configuration under investigation.

  12. Microscopy of hierarchically organized TiO{sub 2} photoelectrode for dye solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Eskandar, A., E-mail: aeska07@gmail.com [Department of Electrical and Electronics, Universiti Teknologi PETRONAS, Tronoh, Perak (Malaysia); Mohamed, N. M., E-mail: noranimuti-mohamed@petronas.com.my [Centre of Innovative Nanostructures and Nanodevices, Universiti Teknologi PETRONAS, Tronoh, Perak (Malaysia)

    2015-07-22

    Research on improving the performance of dye solar cells has various aspects of the device being investigated. This paper analyzes the deliberately hierarchized photoelectrode configuration for DSC applications to improve the performance of DSCs. Multiple layers of differently composed TiO{sub 2} particle types namely aggregates and nanoparticles were deposited to form a photoelectrode with thickness of about 12 µm. The photoelectrodes were assembled into working DSCs with an active area of 1 cm{sup 2}. Measurement for solar power conversion performance was measured under 1 sun at AM1.5 spectrum simulated sunlight. Electron microscopy for photoelectrode analysis was conducted using Field Emission Scattering Electron Microscopy with enhanced resolution. External Quantum Efficiency was measured using a purpose built instrument. Kinetics were investigated using the Electrochemical Impedance Spectroscopy (EIS) measurement with a potentiostat. The best performing DSC is of the hierarchically organized photoelectrode with a photoconversion efficiency of 4.58%, an increase of 14% in comparison to the reference samples with fully aggregates configuration. Short circuit current density, Jsc increases by about 2.223 mA cm{sup −2} relative to the blanks. The electron microscopy confirmed expected thickness at around 10 µm and layers forming the photoelectrode being hierarchically deposited with ∼20 nm TiO{sub 2} nanoparticles and 450 nm TiO{sub 2} aggregates mixture composition. EQE improved especially for visible region of 500-550 nm light wavelengths with 12 % increase in the response of in that region. Improvement to the diffusion coefficient as measured by the EIS contributed to the performance increase of the photoelectrode configuration under investigation.

  13. Adhesion of living cells revealed by variable-angle total internal reflection fluorescence microscopy (Conference Presentation)

    Science.gov (United States)

    Cardoso Dos Santos, Marcelina; Vézy, Cyrille; Jaffiol, Rodolphe

    2016-02-01

    Total Internal Reflection Fluorescence Microscopy (TIRFM) is a widespread technique to study cellular process occurring near the contact region with the glass substrate. In this field, determination of the accurate distance from the surface to the plasma membrane constitutes a crucial issue to investigate the physical basis of cellular adhesion process. However, quantitative interpretation of TIRF pictures regarding the distance z between a labeled membrane and the substrate is not trivial. Indeed, the contrast of TIRF images depends on several parameters more and less well known (local concentration of dyes, absorption cross section, angular emission pattern…). The strategy to get around this problem is to exploit a series of TIRF pictures recorded at different incident angles in evanescent regime. This technique called variable-angle TIRF microscopy (vaTIRFM), allowing to map the membrane-substrate separation distance with a nanometric resolution (10-20 nm). vaTIRFM was developed by Burmeister, Truskey and Reichert in the early 1990s with a prism-based TIRF setup [Journal of Microscopy 173, 39-51 (1994)]. We propose a more convenient prismless setup, which uses only a rotatable mirror to adjust precisely the laser beam on the back focal plane of the oil immersion objective (no azimuthal scanning is needed). The series of TIRF images permit us to calculate accurately membrane-surface distances in each pixel. We demonstrate that vaTIRFM are useful to quantify the adhesion of living cells for specific and unspecific membrane-surface interactions, achieved on various functionalized substrates with polymers (BSA, poly-L-lysin) or extracellular matrix proteins (collagen and fibronectin).

  14. Video-rate processing in tomographic phase microscopy of biological cells using CUDA.

    Science.gov (United States)

    Dardikman, Gili; Habaza, Mor; Waller, Laura; Shaked, Natan T

    2016-05-30

    We suggest a new implementation for rapid reconstruction of three-dimensional (3-D) refractive index (RI) maps of biological cells acquired by tomographic phase microscopy (TPM). The TPM computational reconstruction process is extremely time consuming, making the analysis of large data sets unreasonably slow and the real-time 3-D visualization of the results impossible. Our implementation uses new phase extraction, phase unwrapping and Fourier slice algorithms, suitable for efficient CPU or GPU implementations. The experimental setup includes an external off-axis interferometric module connected to an inverted microscope illuminated coherently. We used single cell rotation by micro-manipulation to obtain interferometric projections from 73 viewing angles over a 180° angular range. Our parallel algorithms were implemented using Nvidia's CUDA C platform, running on Nvidia's Tesla K20c GPU. This implementation yields, for the first time to our knowledge, a 3-D reconstruction rate higher than video rate of 25 frames per second for 256 × 256-pixel interferograms with 73 different projection angles (64 × 64 × 64 output). This allows us to calculate additional cellular parameters, while still processing faster than video rate. This technique is expected to find uses for real-time 3-D cell visualization and processing, while yielding fast feedback for medical diagnosis and cell sorting. PMID:27410107

  15. Nanomechanical and topographical imaging of living cells by atomic force microscopy with colloidal probes

    Energy Technology Data Exchange (ETDEWEB)

    Puricelli, Luca; Galluzzi, Massimiliano; Schulte, Carsten; Podestà, Alessandro, E-mail: alessandro.podesta@mi.infn.it; Milani, Paolo [CIMaINa and Department of Physics, Università degli Studi di Milano, Via Celoria 16, 20133 Milano (Italy)

    2015-03-15

    Atomic Force Microscopy (AFM) has a great potential as a tool to characterize mechanical and morphological properties of living cells; these properties have been shown to correlate with cells’ fate and patho-physiological state in view of the development of novel early-diagnostic strategies. Although several reports have described experimental and technical approaches for the characterization of cellular elasticity by means of AFM, a robust and commonly accepted methodology is still lacking. Here, we show that micrometric spherical probes (also known as colloidal probes) are well suited for performing a combined topographic and mechanical analysis of living cells, with spatial resolution suitable for a complete and accurate mapping of cell morphological and elastic properties, and superior reliability and accuracy in the mechanical measurements with respect to conventional and widely used sharp AFM tips. We address a number of issues concerning the nanomechanical analysis, including the applicability of contact mechanical models and the impact of a constrained contact geometry on the measured Young’s modulus (the finite-thickness effect). We have tested our protocol by imaging living PC12 and MDA-MB-231 cells, in order to demonstrate the importance of the correction of the finite-thickness effect and the change in Young’s modulus induced by the action of a cytoskeleton-targeting drug.

  16. Photothermal confocal multicolor microscopy of nanoparticles and nanodrugs in live cells.

    Science.gov (United States)

    Nedosekin, Dmitry A; Foster, Stephen; Nima, Zeid A; Biris, Alexandru S; Galanzha, Ekaterina I; Zharov, Vladimir P

    2015-08-01

    Growing biomedical applications of non-fluorescent nanoparticles (NPs) for molecular imaging, disease diagnosis, drug delivery, and theranostics require new tools for real-time detection of nanomaterials, drug nano-carriers, and NP-drug conjugates (nanodrugs) in complex biological environments without additional labeling. Photothermal (PT) microscopy (PTM) has enormous potential for absorption-based identification and quantification of non-fluorescent molecules and NPs at a single molecule and 1.4 nm gold NP level. Recently, we have developed confocal PTM providing three-dimensional (3D) mapping and spectral identification of multiple chromophores and fluorophores in live cells. Here, we summarize recent advances in the application of confocal multicolor PTM for 3D visualization of single and clustered NPs, alone and in individual cells. In particular, we demonstrate identification of functionalized magnetic and gold-silver NPs, as well as graphene and carbon nanotubes in cancer cells and among blood cells. The potential to use PTM for super-resolution imaging (down to 50 nm), real-time NP tracking, guidance of PT nanotherapy, and multiplex cancer markers targeting, as well as analysis of non-linear PT phenomena and amplification of nanodrug efficacy through NP clustering and nano-bubble formation are also discussed. PMID:26133539

  17. Raman Spectroscopy and Microscopy of Individual Cells andCellular Components

    Energy Technology Data Exchange (ETDEWEB)

    Chan, J; Fore, S; Wachsmann-Hogiu, S; Huser, T

    2008-05-15

    Raman spectroscopy provides the unique opportunity to non-destructively analyze chemical concentrations on the submicron length scale in individual cells without the need for optical labels. This enables the rapid assessment of cellular biochemistry inside living cells, and it allows for their continuous analysis to determine cellular response to external events. Here, we review recent developments in the analysis of single cells, subcellular compartments, and chemical imaging based on Raman spectroscopic techniques. Spontaneous Raman spectroscopy provides for the full spectral assessment of cellular biochemistry, while coherent Raman techniques, such as coherent anti-Stokes Raman scattering is primarily used as an imaging tool comparable to confocal fluorescence microscopy. These techniques are complemented by surface-enhanced Raman spectroscopy, which provides higher sensitivity and local specificity, and also extends the techniques to chemical indicators, i.e. pH sensing. We review the strengths and weaknesses of each technique, demonstrate some of their applications and discuss their potential for future research in cell biology and biomedicine.

  18. Perspectives on low voltage transmission electron microscopy as applied to cell biology.

    Science.gov (United States)

    Bendayan, Moise; Paransky, Eugene

    2014-12-01

    Low voltage transmission electron microscopy (LVTEM) with accelerating voltages as low as 5 kV was applied to cell biology. To take advantage of the increased contrast given by LVTEM, tissue preparation was modified omitting all heavy metals such as osmium, uranium, and lead from the fixation, on block staining and counterstaining. Nonstained ultra-thin tissue sections (40 nm thick) generated highly contrasted images. While the aspect of the cells remains similar to that obtained by conventional TEM, some new substructures were revealed. The pancreatic acinar cells granules present a heterogeneous matrix with partitions corresponding to segregation of their different secretory proteins. Microvilli display their core of microfilaments anchored to the dense top membrane. Mitochondria revealed the presence of distinct particles along their cristea membranes that may correspond to the ATP synthase complexes or oxysomes. The dense nuclear chromatin displays a honey-comb appearance while distinct beads aligned along thin threads were seen in the dispersed chromatin. These new features revealed by LVTEM correlate with structures described or predicted through other approaches. Masking effects due to thickness of the tissue sections and to the presence of heavy metals must have prevented their observation by conventional TEM. Furthermore, the immunogold was adapted to LVTEM revealing nuclear lamin-A at the edge of the dense chromatin ribbons. Combining cytochemistry with LVTEM brings additional advantages to this new approach in cell biology.

  19. Poly(urethane-dimethylsiloxane) copolymers displaying a range of soft segment contents, noncytotoxic chemistry, and nonadherent properties toward endothelial cells.

    Science.gov (United States)

    Stefanović, Ivan S; Djonlagić, Jasna; Tovilović, Gordana; Nestorov, Jelena; Antić, Vesna V; Ostojić, Sanja; Pergal, Marija V

    2015-04-01

    Polyurethane copolymers based on α,ω-dihydroxypropyl poly(dimethylsiloxane) (PDMS) with a range of soft segment contents were prepared by two-stage polymerization, and their microstructures, thermal, thermomechanical, and surface properties, as well as in vitro hemo- and cytocompatibility were evaluated. All utilized characterization methods confirmed the existence of moderately microphase separated structures with the appearance of some microphase mixing between segments as the PDMS (i.e., soft segment) content increased. Copolymers showed higher crystallinity, storage moduli, surface roughness, and surface free energy, but less hydrophobicity with decreasing PDMS content. Biocompatibility of copolymers was evaluated using an endothelial EA.hy926 cell line by direct contact, an extraction method and after pretreatment of copolymers with multicomponent protein mixture, as well as by a competitive protein adsorption assay. Copolymers showed no toxic effect to endothelial cells and all copolymers, except that with the lowest PDMS content, exhibited resistance to endothelial cell adhesion, suggesting their unsuitability for long-term biomedical devices which particularly require re-endothelialization. All copolymers exhibited excellent resistance to fibrinogen adsorption and adsorbed more albumin than fibrinogen in the competitive adsorption assay, suggesting their good hemocompatibility. The noncytotoxic chemistry of these synthesized materials, combined with their nonadherent properties which are inhospitable to cell attachment and growth, underlie the need for further investigations to clarify their potential for use in short-term biomedical devices. PMID:25046378

  20. Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells

    Science.gov (United States)

    Shibata, Mikihiro; Uchihashi, Takayuki; Ando, Toshio; Yasuda, Ryohei

    2015-03-01

    Visualization of morphological dynamics of live cells with nanometer resolution under physiological conditions is highly desired, but challenging. It has been demonstrated that high-speed atomic force microscopy is a powerful technique for visualizing dynamics of biomolecules under physiological conditions. However, application of high-speed atomic force microscopy for imaging larger objects such as live mammalian cells has been complicated because of the collision between the cantilever and samples. Here, we demonstrate that attaching an extremely long (~3 μm) and thin (~5 nm) tip by amorphous carbon to the cantilever allows us to image the surface structure of live cells with the spatiotemporal resolution of nanometers and seconds. We demonstrate that long-tip high-speed atomic force microscopy is capable of imaging morphogenesis of filopodia, membrane ruffles, pit formation, and endocytosis in COS-7, HeLa cells and hippocampal neurons.

  1. Organ-to-Cell-Scale Health Assessment Using Geographical Information System Approaches with Multibeam Scanning Electron Microscopy.

    Science.gov (United States)

    Knothe Tate, Melissa L; Zeidler, Dirk; Pereira, André F; Hageman, Daniel; Garbowski, Tomasz; Mishra, Sanjay; Gardner, Lauren; Knothe, Ulf R

    2016-07-01

    This study combines novel multibeam electron microscopy with a geographical information system approach to create a first, seamless, navigable anatomic map of the human hip and its cellular inhabitants. Using spatial information acquired by localizing relevant map landmarks (e.g. cells, blood vessels), network modeling will enable disease epidemiology studies in populations of cells inhabiting tissues and organs.

  2. Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy.

    Directory of Open Access Journals (Sweden)

    Sangeeta Nath

    Full Text Available INTRODUCTION: Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved. METHOD: Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties. RESULTS: The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii retention and spatial localization of chemical compounds vary within and between each cell line; and (iii the structural similarities of compounds can infer their non-specific binding properties. CONCLUSION: We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented.

  3. Electron microscopy of single-stranded structures in the DNA of competent Haemophilus influenzae cells

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Kupfer, D.M.

    1987-02-01

    Chromosomal DNAs from exponential-phase and competent cells of Haemophilus influenzae were examined by electron microscopy to determine whether the chromosome undergoes structural changes during competence development. Single-stranded gaps and single-stranded tails formed in chromosomal DNA during competence development. The generation of gaps was dependent on the rec-2 function. Since the rec-2 mutant is defective in the translocation of donor DNA, it was inferred that the gaps were involved in the translocation step of transformation. The generation of single-stranded tails was independent of the rec-1 and rec-2 genes. Therefore, these structures were assumed to play no direct role in the interaction of donor and recipient DNAs during transformation. Gaps were preferentially associated with a readily denaturable, possible A + T-rich fraction of the genome. This finding raised the possibility that hot spots for transformation might be associated with A + T-rich DNA.

  4. Live-cell fluorescent microscopy platforms for real-time monitoring of polyplex-cell interaction

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Wu, LinPing; Andersen, Helene;

    2014-01-01

    A myriad of cationic polymeric delivery vehicles are currently being developed with the aim of transporting various forms of nucleic acids to mammalian cells. The complexes between polycations and nucleic acids are referred to as polyplexes. The screening for successful polyplex candidates requir...

  5. Microscopy Images as Interactive Tools in Cell Modeling and Cell Biology Education

    Science.gov (United States)

    Araujo-Jorge, Tania C.; Cardona, Tania S.; Mendes, Claudia L. S.; Henriques-Pons, Andrea; Meirelles, Rosane M. S.; Coutinho, Claudia M. L. M.; Aguiar, Luiz Edmundo V.; Meirelles, Maria de Nazareth L.; de Castro, Solange L.; Barbosa, Helene S.; Luz, Mauricio R. M. P.

    2004-01-01

    The advent of genomics, proteomics, and microarray technology has brought much excitement to science, both in teaching and in learning. The public is eager to know about the processes of life. In the present context of the explosive growth of scientific information, a major challenge of modern cell biology is to popularize basic concepts of…

  6. Toward Fourier interferometry fluorescence excitation/emission imaging of malignant cells combined with photoacoustic microscopy

    Science.gov (United States)

    Kohen, Elli; Hirschberg, Joseph G.; Berry, John P.; Ozkutuk, Nuri; Ornek, Ceren; Monti, Marco; Leblanc, Roger M.; Schachtschabel, Dietrich O.; Haroon, Sumaira

    2003-10-01

    Dual excitation fluorescence imaging has been used as a first step towards multi-wavelength excitation/emission fluorescence spectral imaging. Target cells are transformed keratinocytes, and other osteosarcoma, human breast and color cancer cells. Mitochondrial membrane potential probes, e.g. TMRM (tetramethylrhodamine methyl ester), Mitotracker Green (Molecular Probes, Inc., Eugene OR,USA; a recently synthesized mitochondrial oxygen probe, [PRE,P1"- pyrene butyl)-2-rhodamine ester] allow dual excitation in the UV plus in teh blue-green spectral regions. Also, using the natural endogenous probe NAD(P)H, preliminary results indicate mitochondrial responses to metabolic challenges (e.g. glucose addition), plus changes in mitochonrial distribution and morphology. In terms of application to biomedicine (for diagnostiscs, prognostsics and drug trials) three parameters have been selected in addition to the natural probe NAD(P)H, i.e. vital fluorescence probing of mitochondria, lysosomes and Golgi apparatus. It is hoped that such a multiparameter approach will allow malignant cell characterization and grading. A new area being introduced is the use of similar methodology for biotechnical applications such as the study of the hydrogen-producing alga Chlamydomonas Reinhardtii, and possible agricultural applications, such as Saccharomyces yeast for oenology. Complementation by Photoacoustic Microscopy is also contemplated, to study the internal conversion component which follows the excitation by photons.

  7. Image segmentation and classification of white blood cells with the extreme learning machine and the fast relevance vector machine.

    Science.gov (United States)

    Ravikumar, S

    2016-05-01

    White blood cells (WBCs) or leukocytes are an important part of the body's defense against infectious organisms and foreign substances. WBC segmentation is a challenging issue because of the morphological diversity of WBCs and the complex and uncertain background of blood smear images. The standard ELM classification techniques are used for WBC segmentation. The generalization performance of the ELM classifier has not achieved the maximum nearest accuracy of image segmentation. This paper gives a novel technique for WBC detection based on the fast relevance vector machine (Fast-RVM). Firstly, astonishingly sparse relevance vectors (RVs) are obtained while fitting the histogram by RVM. Next, the relevant required threshold value is directly sifted from these limited RVs. Finally, the entire connective WBC regions are segmented from the original image. The proposed method successfully works for WBC detection, and effectively reduces the effects brought about by illumination and staining. To achieve the maximum accuracy of the RVM classifier, we design a search for the best value of the parameters that tune its discriminant function, and upstream by looking for the best subset of features that feed the classifier. Therefore, this proposed RVM method effectively works for WBC detection, and effectively reduces the computational time and preserves the images.

  8. Localization of Cell Division Protein FtsQ by Immunofluorescence Microscopy in Dividing and Nondividing Cells of Escherichia coli

    Science.gov (United States)

    Buddelmeijer, Nienke; Aarsman, Mirjam E. G.; Kolk, Arend H. J.; Vicente, Miguel; Nanninga, Nanne

    1998-01-01

    The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the γ form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane. PMID:9829918

  9. Axonal elongation through long acellular nerve segments depends on recruitment of phagocytic cells from the near-nerve environment. Electrophysiological and morphological studies in the cat

    DEFF Research Database (Denmark)

    Sørensen, J; Fugleholm, K; Moldovan, M;

    2001-01-01

    or tissue ingrowth, respectively. In a second set of experiments, tibial nerves were crushed and either frozen for 20+20 mm, leaving a 10 mm segment with viable cells in the center (stepping-stone segment) or frozen for 50 mm. These nerves were enclosed in cuffs with 2.0 mm holes corresponding to the viable...

  10. Scanning electrochemical microscopy of living cells: Different redox activities of nonmetastatic and metastatic human breast cells

    OpenAIRE

    Liu, Biao; Rotenberg, Susan A.; Mirkin, Michael V.

    2000-01-01

    Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransform...

  11. Visualizing Cell Architecture and Molecular Location Using Soft X-Ray Tomography and Correlated Cryo-Light Microscopy

    OpenAIRE

    McDermott, Gerry; Le Gros, Mark A.; Larabell, Carolyn A.

    2012-01-01

    Living cells are structured to create a range of microenvironments that support specific chemical reactions and processes. Understanding how cells function therefore requires detailed knowledge of both the subcellular architecture and the location of specific molecules within this framework. Here we review the development of two correlated cellular imaging techniques that fulfill this need. Cells are first imaged using cryogenic fluorescence microscopy to determine the location of molecules o...

  12. Two-photon microscopy for non-invasive, quantitative monitoring of stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    William L Rice

    Full Text Available BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF and second harmonic generation (SHG as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(PH, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(PH and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool

  13. Counting White Blood Cells from a Blood Smear Using Fourier Ptychographic Microscopy.

    Directory of Open Access Journals (Sweden)

    Jaebum Chung

    Full Text Available White blood cell (WBC count is a valuable metric for assisting with diagnosis or prognosis of various diseases such as coronary heart disease, type 2 diabetes, or infection. Counting WBCs can be done either manually or automatically. Automatic methods are capable of counting a large number of cells to give a statistically more accurate reading of the WBC count of a sample, but the specialized equipment tends to be expensive. Manual methods are inexpensive since they only involve a conventional light microscope setup. However, it is more laborious and error-prone because the small field-of-view (FOV of the microscope necessitates mechanical scanning of a specimen for counting an adequate number of WBCs. Here, we investigate the use of Fourier ptychographic microscopy (FPM to bypass these issues of the manual methods. With a 2x objective, FPM can provide a FOV of 120 mm2 with enhanced resolution comparable to that of a 20x objective, which is adequate for non-differentially counting WBCs in just one FOV. A specialist was able to count the WBCs in FPM images with 100% accuracy compared to the count as determined from conventional microscope images. An automatic counting algorithm was also developed to identify WBCs from FPM's captured images with 95% accuracy, paving the way for a cost-effective WBC counting setup with the advantages of both the automatic and manual counting methods.

  14. Mechanical stimulation of individual stereocilia of living cochlear hair cells by atomic force microscopy.

    Science.gov (United States)

    Langer, M G; Koitschev, A; Haase, H; Rexhausen, U; Hörber, J K; Ruppersberg, J P

    2000-02-01

    This paper describes the investigation of elastical properties and imaging of living cochlear hair bundles of inner (IHC) and outer hair cells (OHC) on the level of individual stereocilia. A custom-made AFM-setup was used, allowing to scan the mechano-sensitive structures of the inner ear under direct control of an upright differential interference contrast (DIC) microscope with a water-immersion objective. Scanning electron microscopy (SEM) images of the identical hair bundles obtained after AFM investigation demonstrated that forces up to 1.5 nanonewton (nN) did not cause obvious damage of the surface morphology of the stereocilia. These are the first images of hair bundles of living sensory cells of the organ of Corti by AFM. They display the tips of individual stereocilia and the typical V-shape of ciliary bundles. Since line scans clearly show that slope and force interaction depend on the elastical properties of stereocilia, quantitative stiffness measurements and stimulation of single transduction channels are suggested. PMID:10741679

  15. Electrochemical characterization of sub-micro-gram amounts of organic semiconductors using scanning droplet cell microscopy.

    Science.gov (United States)

    Gasiorowski, Jacek; Mardare, Andrei I; Sariciftci, Niyazi S; Hassel, Achim Walter

    2013-02-15

    Scanning droplet cell microscopy (SDCM) uses a very small electrolyte droplet at the tip of a capillary which comes in contact with the working electrode. This method is particularly interesting for studies on organic semiconductors since it provides localized electrochemical investigations with high reproducibility. One clear advantage of applying SDCM is represented by the very small amounts of material necessary (less than 1 mg). Organic materials can be investigated quickly and inexpensively in electrochemical studies with a high throughput. In the present study, thin layers of poly(3-hexylthiophene) (P3HT), which is one of the most often used material for organic solar cells, were deposited on ITO/glass as working electrodes in SDCM studies. The redox reactions in 0.1 M tetra(n-butyl)ammonium hexafluorophosphate (TBAPF6) dissolved in propylene carbonate were studied by cyclic voltammetry and by electrochemical impedance spectroscopy. Two reversible, distinct oxidation steps of the P3HT were detected and their kinetics were studied in detail. The doping of P3HT increased due to the electrochemical oxidation and had resulted in a decrease of the film resistance by a few orders of magnitude. Due to localization on the sample various parameter combinations can be studied quantitatively and reproducibly. PMID:24926226

  16. Characterizing automotive fuel cell materials by soft x-ray scanning transmission x-ray microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Hitchcock, A. P., E-mail: aph@mcmaster.ca; Lee, V.; Wu, J.; Cooper, G. [Chemistry & Chemical Biology, McMaster University, Hamilton, ON, L8S 4M1 (Canada); West, M. M.; Berejnov, V. [Faculty of Health Sciences Electron Microscopy, McMaster University, Hamilton, ON L8N 3Z5 (Canada); Soboleva, T.; Susac, D.; Stumper, J. [Automotive Fuel Cell Cooperation Corp., Burnaby BC V5J 5J8 (Canada)

    2016-01-28

    Proton-Exchange Membrane Fuel Cell (PEM-FC) based engines are being developed rapidly for near-term implementation in hydrogen fueled, mass production, personal automobiles. Research is focused on understanding and controlling various degradation processes (carbon corrosion, Pt migration, cold start), and reducing cost by reducing or eliminating Pt catalyst. We are using soft X-ray scanning transmission X-ray microscopy (STXM) at the S 2p, C 1s, O 1s and F 1s edges to study a variety of issues related to optimization of PEM-FC materials for automotive applications. A method to efficiently and accurately measure perfluorosulfonic acid distributions was developed and is being used to better understand how different loadings and preparation methods affect the ionomer distribution in the cathode. Progress towards an environmental cell capable of controlling the temperature and humidity of a PEM-FC sample in the STXM is described. Methods for studying the 3D chemical structure of PEM-FC are outlined.

  17. Study of Mast Cells and Granules from Primo Nodes Using Scanning Ionic Conductance Microscopy.

    Science.gov (United States)

    Yoo, Yeong-Yung; Jung, Goo-Eun; Kwon, Hee-Min; Bae, Kyoung-Hee; Cho, Sang-Joon; Soh, Kwang-Sup

    2015-12-01

    Acupuncture points have a notable characteristic in that they have a higher density of mast cells (MCs) compared with nonacupoints in the skin, which is consistent with the augmentation of the immune function by acupuncture treatment. The primo vascular system, which was proposed as the anatomical structure of the acupuncture points and meridians, also has a high density of MCs. We isolated the primo nodes from the surfaces of internal abdominal organs, and the harvested primo nodes were stained with toluidine blue. The MCs were easily recognized by their stained color and their characteristic granules. The MCs were classified into four stages according to the degranulation of histamine granules in the MCs. Using conventional optical microscopes details of the degranulation state of MCs in each stage were not observable. However, we were able to investigate the distribution of the granules on the surfaces of the MCs in each stage, and to demonstrate the height profiles and three-dimensional structures of the MCs without disturbance of the cell membrane by using the scanning ion conductance microscopy. PMID:26742911

  18. In vivo photoacoustic microscopy of human cuticle microvasculature with single-cell resolution

    Science.gov (United States)

    Hsu, Hsun-Chia; Wang, Lidai; Wang, Lihong V.

    2016-05-01

    As a window on the microcirculation, human cuticle capillaries provide rich information about the microvasculature, such as its morphology, density, dimensions, or even blood flow speed. Many imaging technologies have been employed to image human cuticle microvasculature. However, almost none of these techniques can noninvasively observe the process of oxygen release from single red blood cells (RBCs), an observation which can be used to study healthy tissue functionalities or to diagnose, stage, or monitor diseases. For the first time, we adapted single-cell resolution photoacoustic (PA) microscopy (PA flowoxigraphy) to image cuticle capillaries and quantified multiple functional parameters. Our results show more oxygen release in the curved cuticle tip region than in other regions of a cuticle capillary loop, associated with a low of RBC flow speed in the tip region. Further analysis suggests that in addition to the RBC flow speed, other factors, such as the drop of the partial oxygen pressure in the tip region, drive RBCs to release more oxygen in the tip region.

  19. Visualizing Nanoscale Distribution of Corrosion Cells by Open-Loop Electric Potential Microscopy.

    Science.gov (United States)

    Honbo, Kyoko; Ogata, Shoichiro; Kitagawa, Takuya; Okamoto, Takahiro; Kobayashi, Naritaka; Sugimoto, Itto; Shima, Shohei; Fukunaga, Akira; Takatoh, Chikako; Fukuma, Takeshi

    2016-02-23

    Corrosion is a traditional problem but still one of the most serious problems in industry. To reduce the huge economic loss caused by corrosion, tremendous effort has been made to understand, predict and prevent it. Corrosion phenomena are generally explained by the formation of corrosion cells at a metal-electrolyte interface. However, experimental verification of their nanoscale distribution has been a major challenge owing to the lack of a method able to visualize the local potential distribution in an electrolytic solution. In this study, we have investigated the nanoscale corrosion behavior of Cu fine wires and a duplex stainless steel by in situ imaging of local corrosion cells by open-loop electric potential microscopy (OL-EPM). For both materials, potential images obtained by OL-EPM show nanoscale contrasts, where areas of higher and lower potential correspond to anodic areas (i.e., corrosion sites) and cathodic areas, respectively. This imaging capability allows us to investigate the real-time transition of local corrosion sites even when surface structures show little change. This is particularly useful for investigating reactions under surface oxide layers or highly corrosion-resistant materials as demonstrated here. The proposed technique should be applicable to the study of other redox reactions on a battery electrode or a catalytic material. The results presented here open up such future applications of OL-EPM in nanoscale electrochemistry. PMID:26811989

  20. Characterizing automotive fuel cell materials by soft x-ray scanning transmission x-ray microscopy

    International Nuclear Information System (INIS)

    Proton-Exchange Membrane Fuel Cell (PEM-FC) based engines are being developed rapidly for near-term implementation in hydrogen fueled, mass production, personal automobiles. Research is focused on understanding and controlling various degradation processes (carbon corrosion, Pt migration, cold start), and reducing cost by reducing or eliminating Pt catalyst. We are using soft X-ray scanning transmission X-ray microscopy (STXM) at the S 2p, C 1s, O 1s and F 1s edges to study a variety of issues related to optimization of PEM-FC materials for automotive applications. A method to efficiently and accurately measure perfluorosulfonic acid distributions was developed and is being used to better understand how different loadings and preparation methods affect the ionomer distribution in the cathode. Progress towards an environmental cell capable of controlling the temperature and humidity of a PEM-FC sample in the STXM is described. Methods for studying the 3D chemical structure of PEM-FC are outlined

  1. Characterizing automotive fuel cell materials by soft x-ray scanning transmission x-ray microscopy

    Science.gov (United States)

    Hitchcock, A. P.; Lee, V.; Wu, J.; West, M. M.; Cooper, G.; Berejnov, V.; Soboleva, T.; Susac, D.; Stumper, J.

    2016-01-01

    Proton-Exchange Membrane Fuel Cell (PEM-FC) based engines are being developed rapidly for near-term implementation in hydrogen fueled, mass production, personal automobiles. Research is focused on understanding and controlling various degradation processes (carbon corrosion, Pt migration, cold start), and reducing cost by reducing or eliminating Pt catalyst. We are using soft X-ray scanning transmission X-ray microscopy (STXM) at the S 2p, C 1s, O 1s and F 1s edges to study a variety of issues related to optimization of PEM-FC materials for automotive applications. A method to efficiently and accurately measure perfluorosulfonic acid distributions was developed and is being used to better understand how different loadings and preparation methods affect the ionomer distribution in the cathode. Progress towards an environmental cell capable of controlling the temperature and humidity of a PEM-FC sample in the STXM is described. Methods for studying the 3D chemical structure of PEM-FC are outlined.

  2. Reconstructive procedures for segmental resection of bone in giant cell tumors around the knee

    Directory of Open Access Journals (Sweden)

    Aggarwal Aditya

    2007-01-01

    Full Text Available Background: Segmental resection of bone in Giant Cell Tumor (GCT around the knee, in indicated cases, leaves a gap which requires a complex reconstructive procedure. The present study analyzes various reconstructive procedures in terms of morbidity and various complications encountered. Materials and Methods: Thirteen cases (M-six and F-seven; lower end femur-six and upper end tibia -seven of GCT around the knee, radiologically either Campanacci Grade II, Grade II with pathological fracture or Grade III were included. Mean age was 25.6 years (range 19-30 years. Resection arthrodesis with telescoping (shortening over intramedullary nail ( n=5, resection arthrodesis with an intercalary allograft threaded over a long intramedullary nail ( n=3 and resection arthrodesis with intercalary fibular autograft and simultaneous limb lengthening ( n=5 were the procedure performed. Results: Shortening was the major problem following resection arthrodesis with telescoping (shortening over intramedullary nail. Only two patients agreed for subsequent limb lengthening. The rest continued to walk with shortening. Infection was the major problem in all cases of resection arthrodesis with an intercalary allograft threaded over a long intramedullary nail and required multiple drainage procedures. Fusion was achieved after two years in two patients. In the third patient the allograft sequestrated. The patient underwent sequestrectomy, telescoping of fragments and ilizarov fixator application with subsequent limb lengthening. The patient was finally given an ischial weight relieving orthosis, 54 months after the index procedure. After resection arthrodesis with intercalary autograft and simultaneous lengthening the resultant gap (~15cm was partially bridged by intercalary nonvascularized dual fibular strut graft (6-7cm and additional corticocancellous bone graft from ipsilateral patella. Simultaneous limb lengthening with a distal tibial corticotomy was performed on an

  3. Enhanced multi-spectral imaging of live breast cancer cells using immunotargeted gold nanoshells and two-photon excitation microscopy

    International Nuclear Information System (INIS)

    We demonstrate the capability of using immunotargeted gold nanoshells as contrast agents for in vitro two-photon microscopy. The two-photon luminescence properties of different-sized gold nanoshells are first validated using near-infrared excitation at 780 nm. The utility of two-photon microscopy as a tool for imaging live HER2-overexpressing breast cancer cells labeled with anti-HER2-conjugated nanoshells is then explored and imaging results are compared to normal breast cells. Five different imaging channels are simultaneously examined within the emission wavelength range of 451-644 nm. Our results indicate that under near-infrared excitation, superior contrast of SK-BR-3 cancer cells labeled with immunotargeted nanoshells occurs at an emission wavelength ranging from 590 to 644 nm. Luminescence from labeled normal breast cells and autofluorescence from unlabeled cancer and normal cells remain imperceptible under the same conditions

  4. Single molecule narrowfield microscopy of protein-DNA binding dynamics in glucose signal transduction of live yeast cells

    CERN Document Server

    Wollman, Adam J M

    2016-01-01

    Single-molecule narrowfield microscopy is a versatile tool to investigate a diverse range of protein dynamics in live cells and has been extensively used in bacteria. Here, we describe how these methods can be extended to larger eukaryotic, yeast cells, which contain sub-cellular compartments. We describe how to obtain single-molecule microscopy data but also how to analyse these data to track and obtain the stoichiometry of molecular complexes diffusing in the cell. We chose glucose mediated signal transduction of live yeast cells as the system to demonstrate these single-molecule techniques as transcriptional regulation is fundamentally a single molecule problem - a single repressor protein binding a single binding site in the genome can dramatically alter behaviour at the whole cell and population level.

  5. Exploring the limits of optical microscopy: live cell and superresolution fluorescence microscopy of HIV-1 Transfer Between T lymphocytes Across the Virological Synapse

    Science.gov (United States)

    McNerney, Gregory Paul

    Human immunodeficiency virus 1 (HIV-1) is a human retrovirus that efficiently, albeit gradually, overruns the immune system. An already infected T lymphocyte can latch onto another T lymphocyte whereby creating a virological synapse (VS); this junction drives viral assembly and transfer to the target cell in batches in an efficient, protective manor. My Ph.D. doctoral thesis focused on studying this transmission mechanism using advanced optical imaging modalities and the fully infectious fluorescent clone HIV Gag-iGFP. T lymphocytes are non-adherent cells (˜10 um thick) and the viral transmission process is fairly dynamic, hence we employed a custom spinning disk confocal microscope that revealed many interesting characteristics of this cooperative event. This methodology has low throughput as cell contact and transfer is at random. Optical tweezers was then added to the microscope to directly initiate cell contact at will. To assess when viral maturation occurs post-transfer, an optical assay based off of Forster resonance energy transfer was developed to monitor maturation. Structured illumination microscopy was further used to image the process at higher resolution and it showed that viral particles are not entering existing degradative compartments. Non-HIV-1 applications of the optical technologies are also reviewed.

  6. Effects of spaceflight in the adductor longus muscle of rats flown in the Soviet Biosatellite COSMOS 2044. A study employing neural cell adhesion molecule (N-CAM) immunocytochemistry and conventional morphological techniques (light and electron microscopy)

    Science.gov (United States)

    D'Amelio, F.; Daunton, N. G.

    1992-01-01

    The effects of spaceflight upon the "slow" muscle adductor longus were examined in rats flown in the Soviet Biosatellite COSMOS 2044. The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leukocytes and mononuclear cells. Neural cell adhesion molecule immunoreactivity (N-CAM-IR) was seen on the myofiber surface and in regenerating myofibers. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with apparent preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments. The principal electron microscopic changes of the neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles replaced by microtubules and neurofilaments, degeneration of axon terminals, vacant axonal spaces and changes suggestive of axonal sprouting. The present observations suggest that alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

  7. Unconventional Specimen Preparation Techniques Using High Resolution Low Voltage Field Emission Scanning Electron Microscopy to Study Cell Motility, Host Cell Invasion, and Internal Cell Structures in Toxoplasma gondii

    Science.gov (United States)

    Schatten, Heide; Ris, Hans

    2002-04-01

    Apicomplexan parasites employ complex and unconventional mechanisms for cell locomotion, host cell invasion, and cell division that are only poorly understood. While immunofluorescence and conventional transmission electron microscopy have been used to answer questions about the localization of some cytoskeletal proteins and cell organelles, many questions remain unanswered, partly because new methods are needed to study the complex interactions of cytoskeletal proteins and organelles that play a role in cell locomotion, host cell invasion, and cell division. The choice of fixation and preparation methods has proven critical for the analysis of cytoskeletal proteins because of the rapid turnover of actin filaments and the dense spatial organization of the cytoskeleton and its association with the complex membrane system. Here we introduce new methods to study structural aspects of cytoskeletal motility, host cell invasion, and cell division of Toxoplasma gondii, a most suitable laboratory model that is representative of apicomplexan parasites. The novel approach in our experiments is the use of high resolution low voltage field emission scanning electron microscopy (LVFESEM) combined with two new specimen preparation techniques. The first method uses LVFESEM after membrane extraction and stabilization of the cytoskeleton. This method allows viewing of actin filaments which had not been possible with any other method available so far. The second approach of imaging the parasite's ultrastructure and interactions with host cells uses semithick sections (200 nm) that are resin de-embedded (Ris and Malecki, 1993) and imaged with LVFESEM. This method allows analysis of structural detail in the parasite before and after host cell invasion and interactions with the membrane of the parasitophorous vacuole as well as parasite cell division.

  8. Using digital inline holographic microscopy and quantitative phase contrast imaging to assess viability of cultured mammalian cells

    Science.gov (United States)

    Missan, Sergey; Hrytsenko, Olga

    2015-03-01

    Digital inline holographic microscopy was used to record holograms of mammalian cells (HEK293, B16, and E0771) in culture. The holograms have been reconstructed using Octopus software (4Deep inwater imaging) and phase shift maps were unwrapped using the FFT-based phase unwrapping algorithm. The unwrapped phase shifts were used to determine the maximum phase shifts in individual cells. Addition of 0.5 mM H2O2 to cell media produced rapid rounding of cultured cells, followed by cell membrane rupture. The cell morphology changes and cell membrane ruptures were detected in real time and were apparent in the unwrapped phase shift images. The results indicate that quantitative phase contrast imaging produced by the digital inline holographic microscope can be used for the label-free real time automated determination of cell viability and confluence in mammalian cell cultures.

  9. Transmission electron microscopy study of Listeria monocytogenes serotype 1/2a cells exposed to sublethal heat stress and carvacrol

    Science.gov (United States)

    The objective of this study was to investigate the morphological changes that occurred in Listeria monocytogenes serotype 1/2a cells as visualized by transmission electron microscopy (TEM) after exposure to sublethal heat stress at 48°C for 60 min and in combination with lethal concentration of carv...

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

    NARCIS (Netherlands)

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

    2011-01-01

    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

  11. In vivo confocal microscopy of basal cell carcinoma: a systematic review of diagnostic accuracy.

    Science.gov (United States)

    Kadouch, D J; Schram, M E; Leeflang, M M; Limpens, J; Spuls, P I; de Rie, M A

    2015-10-01

    Basal cell carcinoma (BCC) is the most prevalent type of skin cancer. Histologic analysis of punch biopsy or direct excision specimen is used to confirm clinical diagnosis. In vivo reflectance confocal microscopy (RCM) is a non-invasive imaging modality that could facilitate early diagnosis and minimize unnecessary invasive procedures. We systematically reviewed diagnostic accuracy (sensitivity and specificity) of RCM in diagnosing primary BCCs to judge its usefulness. Eligible studies were reviewed for methodological quality using the QUADAS-2 tool. We used the bivariate random-effects model to calculate summary estimates of sensitivity and specificity. Six studies met the selection criteria and were included for analysis. The meta-analysis showed a summary estimate of sensitivity 0.97 (95% CI, 0.90-0.99) and specificity 0.93 (95% CI, 0.88-0.96). All but one of the QUADAS-2 items showed a high or unclear risk of bias with regards to patient selection. RCM may be a promising diagnostic tool, but the limited number of available studies and potential risk of bias of included studies do not allow us to draw firm conclusions. Future accuracy studies should take these limitations into account. PMID:26290493

  12. Electrodeposition and Screening of Photoelectrochemical Activity in Conjugated Polymers Using Scanning Electrochemical Cell Microscopy.

    Science.gov (United States)

    Aaronson, Barak D B; Garoz-Ruiz, Jesus; Byers, Joshua C; Colina, Alvaro; Unwin, Patrick R

    2015-11-24

    A number of renewable energy systems require an understanding and correlation of material properties and photoelectrochemical activity on the micro to nanoscale. Among these, conducting polymer electrodes continue to be important materials. In this contribution, an ultrasensitive scanning electrochemical cell microscopy (SECCM) platform is used to electrodeposit microscale thin films of poly(3-hexylthiophene) (P3HT) on an optically transparent gold electrode and to correlate the morphology (film thickness and structural order) with photoactivity. The electrochemical growth of P3HT begins with a thin ordered film up to 10 nm thick, after which a second more disordered film is deposited, as revealed by micro-Raman spectroscopy. A decrease in photoactivity for the thicker films, measured in situ immediately following film deposition, is attributed to an increase in bulk film disorder that limits charge transport. Higher resolution ex situ SECCM phototransient measurements, using a smaller diameter probe, show local variations in photoactivity within a given deposit. Even after aging, thinner, more ordered regions within a deposit exhibit sustained enhanced photocurrent densities compared to areas where the film is thicker and more disordered. The platform opens up new possibilities for high-throughput combinatorial correlation studies, by allowing materials fabrication and high spatial resolution probing of processes in photoelectrochemical materials. PMID:26502089

  13. Characterization of carrier concentration in CIGS solar cells by scanning capacitance microscopy

    International Nuclear Information System (INIS)

    Thin films of copper indium gallium selenide (CIGS) designed for highly efficient solar cell material were investigated to characterize the two-dimensional carrier distribution using scanning capacitance microscopy (SCM). We optimized a preparation method of the cross-section samples and concluded that bevel polishing by 25° to 30° was effective for crumbly polycrystalline materials such as CIGS, so as to provide not the surface property of cracked crystalline grains but the cross-section property of individual cut grains. Because of improvement in this preparation procedure, changes in carrier distribution have been observed directly in the active CIGS layer before and after turning on a 100 W halogen lamp irradiation. A calibration curve between carrier concentration N and SCM's dC/dV signals was applied for qualitatively calculating relative values of N in CIGS. Increased carrier concentration peaks on the grains were estimated to become about three times as high as those with the light on. (paper)

  14. Combined Optical Coherence and Fluorescence Microscopy to assess dynamics and specificity of pancreatic beta-cell tracers

    OpenAIRE

    Corinne Berclaz; Christophe Pache; Arno Bouwens; Daniel Szlag; Antonio Lopez; Lieke Joosten; Selen Ekim; Maarten Brom; Martin Gotthardt; Anne Grapin-Botton; Theo Lasser

    2015-01-01

    The identification of a beta-cell tracer is a major quest in diabetes research. However, since MRI, PET and SPECT cannot resolve individual islets, optical techniques are required to assess the specificity of these tracers. We propose to combine Optical Coherence Microscopy (OCM) with fluorescence detection in a single optical platform to facilitate these initial screening steps from cell culture up to living rodents. OCM can image islets and vascularization without any labeling. Thereby, it ...

  15. Accurate segmentation of leukocyte in blood cell images using Atanassov's intuitionistic fuzzy and interval Type II fuzzy set theory.

    Science.gov (United States)

    Chaira, Tamalika

    2014-06-01

    In this paper automatic leukocyte segmentation in pathological blood cell images is proposed using intuitionistic fuzzy and interval Type II fuzzy set theory. This is done to count different types of leukocytes for disease detection. Also, the segmentation should be accurate so that the shape of the leukocytes is preserved. So, intuitionistic fuzzy set and interval Type II fuzzy set that consider either more number of uncertainties or a different type of uncertainty as compared to fuzzy set theory are used in this work. As the images are considered fuzzy due to imprecise gray levels, advanced fuzzy set theories may be expected to give better result. A modified Cauchy distribution is used to find the membership function. In intuitionistic fuzzy method, non-membership values are obtained using Yager's intuitionistic fuzzy generator. Optimal threshold is obtained by minimizing intuitionistic fuzzy divergence. In interval type II fuzzy set, a new membership function is generated that takes into account the two levels in Type II fuzzy set using probabilistic T co norm. Optimal threshold is selected by minimizing a proposed Type II fuzzy divergence. Though fuzzy techniques were applied earlier but these methods failed to threshold multiple leukocytes in images. Experimental results show that both interval Type II fuzzy and intuitionistic fuzzy methods perform better than the existing non-fuzzy/fuzzy methods but interval Type II fuzzy thresholding method performs little bit better than intuitionistic fuzzy method. Segmented leukocytes in the proposed interval Type II fuzzy method are observed to be distinct and clear.

  16. Comparison of the viscoelastic properties of cells from different kidney cancer phenotypes measured with atomic force microscopy

    International Nuclear Information System (INIS)

    The viscoelastic properties of human kidney cell lines from different tumor types (carcinoma (A-498) and adenocarcinoma (ACHN)) are compared to a non-tumorigenic cell line (RC-124). Our methodology is based on the mapping of viscoelastic properties (elasticity modulus E and apparent viscosity η) over the surface of tens of individual cells with atomic force microscopy (AFM). The viscoelastic properties are averaged over datasets as large as 15000 data points per cell line. We also propose a model to estimate the apparent viscosity of soft materials using the hysteresis observed in conventional AFM deflection–displacement curves, without any modification to the standard AFM apparatus. The comparison of the three cell lines show that the non-tumorigenic cells are less deformable and more viscous than cancerous cells, and that cancer cell lines have distinctive viscoelastic properties. In particular, we obtained that ERC−124 > EA−498 > EACHN and ηRC−124 > ηA−498 > ηACHN. (paper)

  17. The New Electron Microscopy: Cells and Molecules in Three Dimensions | Poster

    Science.gov (United States)

    NCI recently announced the launch of the new National Cryo-Electron Microscopy Facility (NCEF) at the Frederick National Laboratory for Cancer Research (FNLCR). The launch comes while cryo-electron microscopy (cryo-EM) is enjoying the spotlight as a newly emerging, rapidly evolving technology with the potential to revolutionize the field of structural biology. Read more...

  18. Use of CMEIAS Image Analysis Software to Accurately Compute Attributes of Cell Size, Morphology, Spatial Aggregation and Color Segmentation that Signify in Situ Ecophysiological Adaptations in Microbial Biofilm Communities

    Directory of Open Access Journals (Sweden)

    Frank B. Dazzo

    2015-03-01

    Full Text Available In this review, we describe computational features of computer-assisted microscopy that are unique to the Center for Microbial Ecology Image Analysis System (CMEIAS software, and examples illustrating how they can be used to gain ecophysiological insights into microbial adaptations occurring at micrometer spatial scales directly relevant to individual cells occupying their ecological niches in situ. These features include algorithms that accurately measure (1 microbial cell length relevant to avoidance of protozoan bacteriovory; (2 microbial biovolume body mass relevant to allometric scaling and local apportionment of growth-supporting nutrient resources; (3 pattern recognition rules for morphotype classification of diverse microbial communities relevant to their enhanced fitness for success in the particular habitat; (4 spatial patterns of coaggregation that reveal the local intensity of cooperative vs. competitive adaptations in colonization behavior relevant to microbial biofilm ecology; and (5 object segmentation of complex color images to differentiate target microbes reporting successful cell-cell communication. These unique computational features contribute to the CMEIAS mission of developing accurate and freely accessible tools of image bioinformatics that strengthen microscopy-based approaches for understanding microbial ecology at single-cell resolution.

  19. Identification of the Genome Segments of Bluetongue Virus Serotype 26 (Isolate KUW2010/02) that Restrict Replication in a Culicoides sonorensis Cell Line (KC Cells).

    Science.gov (United States)

    Pullinger, Gillian D; Guimerà Busquets, Marc; Nomikou, Kyriaki; Boyce, Mark; Attoui, Houssam; Mertens, Peter P

    2016-01-01

    Bluetongue virus (BTV) can infect most ruminant species and is usually transmitted by adult, vector-competent biting midges (Culicoides spp.). Infection with BTV can cause severe clinical signs and can be fatal, particularly in naïve sheep and some deer species. Although 24 distinct BTV serotypes were recognized for several decades, additional 'types' have recently been identified, including BTV-25 (from Switzerland), BTV-26 (from Kuwait) and BTV-27 from France (Corsica). Although BTV-25 has failed to grow in either insect or mammalian cell cultures, BTV-26 (isolate KUW2010/02), which can be transmitted horizontally between goats in the absence of vector insects, does not replicate in a Culicoides sonorensis cell line (KC cells) but can be propagated in mammalian cells (BSR cells). The BTV genome consists of ten segments of linear dsRNA. Mono-reassortant viruses were generated by reverse-genetics, each one containing a single BTV-26 genome segment in a BTV-1 genetic-background. However, attempts to recover a mono-reassortant containing genome-segment 2 (Seg-2) of BTV-26 (encoding VP2), were unsuccessful but a triple-reassortant was successfully generated containing Seg-2, Seg-6 and Seg-7 (encoding VP5 and VP7 respectively) of BTV-26. Reassortants were recovered and most replicated well in mammalian cells (BSR cells). However, mono-reassortants containing Seg-1 or Seg-3 of BTV-26 (encoding VP1, or VP3 respectively) and the triple reassortant failed to replicate, while a mono-reassortant containing Seg-7 of BTV-26 only replicated slowly in KC cells. PMID:26890863

  20. Characterizing the intracellular distribution of metabolites in intact Chlamydia-infected cells by Raman and two-photon microscopy.

    Science.gov (United States)

    Szaszák, Márta; Chang, Jiun Chiun; Leng, Weinan; Rupp, Jan; Ojcius, David M; Kelley, Anne Myers

    2013-06-01

    Chlamydia species are obligate intracellular pathogens that proliferate only within infected cells. Currently, there are no known techniques or systems that can probe the spatial distribution of metabolites of interest within intact Chlamydia-infected cells. Here we investigate the ability of Raman microscopy to probe the chemical composition of different compartments (nucleus, inclusion, and cytoplasm) of Chlamydia trachomatis-infected epithelial cells. The overall intensity of the Raman spectrum is greatest in the inclusions and lowest in the cytoplasm in fixed cells. Difference spectra generated by normalizing to the intensity of the strong 1004 cm(-1) phenylalanine line show distinct differences among the three compartments. Most notably, the concentrations of adenine are greater in both the inclusions and the nucleus than in the cytoplasm, as seen by Raman microscopy. The source of the adenine was explored through a complementary approach, using two-photon microscopy imaging. Autofluorescence measurements of living, infected cells show that the adenine-containing molecules, NAD(P)H and FAD, are present mainly in the cytoplasm, suggesting that these molecules are not the source of the additional adenine signal in the nucleus and inclusions. Experiments of infected cells stained with a DNA-binding dye, Hoechst 33258, reveal that most of the DNA is present in the nucleus and the inclusions, suggesting that DNA/RNA is the main source of the additional Raman adenine signal in the nucleus and inclusions. Thus, Raman and two-photon microscopy are among the few non-invasive methods available to investigate cells infected with Chlamydia and, together, should also be useful for studying infection by other intracellular pathogens that survive within intracellular vacuoles.

  1. A phase-contrast microscopy-based method for modeling the mechanical behavior of mesenchymal stem cells.

    Science.gov (United States)

    Saeed, Mayssam; Sharabani-Yosef, Orna; Weihs, Daphne; Gefen, Amit

    2016-10-01

    We present three-dimensional (3D) finite element (FE) models of single, mesenchymal stem cells (MSCs), generated from images obtained by optical phase-contrast microscopy and used to quantify the structural responses of the studied cells to externally applied mechanical loads. Mechanical loading has been shown to affect cell morphology and structure, phenotype, motility and other biological functions. Cells experience mechanical loads naturally, yet under prolonged or sizable loading, damage and cell death may occur, which motivates research regarding the structural behavior of loaded cells. For example, near the weight-bearing boney prominences of the buttocks of immobile persons, tissues may become highly loaded, eventually leading to massive cell death that manifests as pressure ulcers. Cell-specific computational models have previously been developed by our group, allowing simulations of cell deformations under compressive or stretching loads. These models were obtained by reconstructing specific cell structures from series of 2D fluorescence, confocal image-slices, requiring cell-specific fluorescent-staining protocols and costly (confocal) microscopy equipment. Alternative modeling approaches represent cells simply as half-spheres or half-ellipsoids (i.e. idealized geometries), which neglects the curvature details of the cell surfaces associated with changes in concentrations of strains and stresses. Thus, we introduce here for the first time an optical image-based FE modeling, where loads are simulated on reconstructed 3D geometrical cell models from a single 2D, phase-contrast image. Our novel modeling method eliminates the need for confocal imaging and fluorescent staining preparations (both expensive), and makes cell-specific FE modeling affordable and accessible to the biomechanics community. We demonstrate the utility of this cost-effective modeling method by performing simulations of compression of MSCs embedded in a gel. PMID:26856632

  2. Biological cell morphology studies by scanning electrochemical microscopy imagery at constant height: Contrast enhancement using biocompatible conductive substrates

    International Nuclear Information System (INIS)

    Scanning ElectroChemical Microscopy (SECM) has emerged as a very attractive method to image living cells activity due to its non invasive character and to the possibility of concomitant electro- and physico-chemical measurements. One of the difficulties when studying morphology of living cells in real time by SECM, using classical constant height mode, is the low contrast of the obtained images due to the insulating character of both the cells and of the underlying substrates. We propose here a technical approach to improve the contrast of SECM imagery obtained at constant height in the feedback mode without the need of Faraday cage. To this aim, a piece of biocompatible transparent conductive substrate (indium tin oxide, ITO coated PET) was attached into the bottom of cell culture well over which the cells were cultured. The transparency of ITO is intended to perform simultaneously SECM and optical microscopy measurements. The concept was applied to the study of endothelial cells, EA.hy926, whose morphology may be altered via an antivascular treatment. Our results show that the differences in the conductivity of the substrate and of the cells enhance the contrast of SECM image in feedback mode at constant height using highly charged redox mediator. In addition, differences in cell morphology are significantly observed by SECM after cell treatment with Combretastatin A4 antivascular agent

  3. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    CERN Document Server

    Kim, Kyoohyun; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mouse were also investigated.

  4. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    Science.gov (United States)

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, Yongkeun

    2016-09-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated.

  5. Cross-Sectional Investigations on Epitaxial Silicon Solar Cells by Kelvin and Conducting Probe Atomic Force Microscopy: Effect of Illumination.

    Science.gov (United States)

    Narchi, Paul; Alvarez, Jose; Chrétien, Pascal; Picardi, Gennaro; Cariou, Romain; Foldyna, Martin; Prod'homme, Patricia; Kleider, Jean-Paul; I Cabarrocas, Pere Roca

    2016-12-01

    Both surface photovoltage and photocurrent enable to assess the effect of visible light illumination on the electrical behavior of a solar cell. We report on photovoltage and photocurrent measurements with nanometer scale resolution performed on the cross section of an epitaxial crystalline silicon solar cell, using respectively Kelvin probe force microscopy and conducting probe atomic force microscopy. Even though two different setups are used, the scans were performed on locations within 100-μm distance in order to compare data from the same area and provide a consistent interpretation. In both measurements, modifications under illumination are observed in accordance with the theory of PIN junctions. Moreover, an unintentional doping during the deposition of the epitaxial silicon intrinsic layer in the solar cell is suggested from the comparison between photovoltage and photocurrent measurements. PMID:26831693

  6. Separation of Normal and Premalignant Cervical Epithelial Cells Using Confocal Light Absorption and Scattering Spectroscopic Microscopy Ex Vivo

    Directory of Open Access Journals (Sweden)

    Ling Yang

    2011-01-01

    Full Text Available Confocal light absorption and scattering spectroscopic (CLASS microscopy can detect changes in biochemicals and the morphology of cells. It is therefore used to detect high-grade cervical squamous intraepithelial lesion (HSIL cells in the diagnosis of premalignant cervical lesions. Forty cervical samples from women with abnormal Pap smear test results were collected, and twenty cases were diagnosed as HSIL; the rest were normal or low-grade cervical squamous intraepithelial lesion (LSIL. The enlarged and condensed nuclei of HSIL cells as viewed under CLASS microscopy were much brighter and bigger than those of non-HSIL cells. Cytological elastic scattered light data was then collected at wavelengths between 400 and 1000 nm. Between 600 nm to 800 nm, the relative elastic scattered light intensity of HSIL cells was higher than that of the non-HSIL. Relative intensity peaks occurred at 700 nm and 800 nm. CLASS sensitivity and specificity results for HSIL and non-HSIL compared to cytology diagnoses were 80% and 90%, respectively. This study demonstrated that CLASS microscopy could effectively detect cervical precancerous lesions. Further study will verify this conclusion before the method is used in clinic for early detection of cervical cancer.

  7. Development of an automated fluorescence microscopy system for photomanipulation of genetically encoded photoactivatable proteins (optogenetics) in live cells.

    Science.gov (United States)

    Araki, Nobukazu; Ikeda, Yuka; Kato, Takuma; Kawai, Katsuhisa; Egami, Youhei; Miyake, Katsuya; Tsurumaki, Nobuhide; Yamaguchi, Mitsunari

    2014-06-01

    Photomanipulation of genetically encoded light-sensitive protein activity, also known as optogenetics, is one of the most innovative recent microscopy techniques in the fields of cell biology and neurobiology. Although photomanipulation is usually performed by diverting the photobleaching mode of a confocal laser microscope, photobleaching by the laser scanning unit is not always suitable for photoactivation. We have developed a simple automated wide-field fluorescence microscopy system for the photomanipulation of genetically encoded photoactivatable proteins in live cells. An electrically automated fluorescence microscope can be controlled through MetaMorph imaging software, making it possible to acquire time-lapse, multiwavelength images of live cells. Using the journal (macro recording) function of MetaMorph, we wrote a macro program to change the excitation filter for photoactivation and illumination area during the intervals of image acquisition. When this program was run on the wide-field fluorescence microscope, cells expressing genetically encoded photoactivatable Rac1, which is activated under blue light, showed morphological changes such as lamellipodial extension and cell surface ruffling in the illuminated region. Using software-based development, we successfully constructed a fully automated photoactivation microscopy system for a mercury lamp-based fluorescence microscope.

  8. Clinical efficacy and safety of autologous stem cell transplantation for patients with ST-segment elevation myocardial infarction

    Directory of Open Access Journals (Sweden)

    Li R

    2016-08-01

    Full Text Available Rong Li,1,* Xiao-Ming Li,2,* Jun-Rong Chen,3 1Department of Intensive Care Unit, The People’s Hospital of Baoji City, 2Department of Cardiovascular Medicine, 3Department of Function, Baoji Central Hospital, Baoji, Shaanxi, People’s Republic of China *These authors contributed equally to this work Purpose: The purpose of this study is to evaluate the therapeutic efficacy and safety of stem cells for the treatment of patients with ST-segment elevation myocardial infarction (STEMI.Materials and methods: We performed a systematic review and meta-analysis of relevant published clinical studies. A computerized search was conducted for randomized controlled trials of stem cell therapy for STEMI.Results: Twenty-eight randomized controlled trials with a total of 1,938 STEMI patients were included in the present meta-analysis. Stem cell therapy resulted in an improvement in long-term (12 months left ventricular ejection fraction of 3.15% (95% confidence interval 1.01–5.29, P<0.01. The 3-month to 4-month, 6-month, and 12-month left ventricular end-systolic volume showed favorable results in the stem cell therapy group compared with the control group (P≤0.05. Significant decrease was also observed in left ventricular end-diastolic volume after 3-month to 4-month and 12-month follow-up compared with controls (P<0.05. Wall mean score index was reduced significantly in stem cell therapy group when compared with the control group at 6-month and 12-month follow-up (P=0.01. Moreover, our analysis showed a significant change of 12-month infarct size decrease in STEMI patients treated with stem cells compared with controls (P<0.01. In addition, no significant difference was found between treatment group and control in adverse reactions (P>0.05.Conclusion: Overall, stem cell therapy is efficacious in the treatment of patients with STEMI, with low rates of adverse events compared with control group patients. Keywords: ST-segment elevation myocardial

  9. Characterization of microbially Fe(III)-reduced nontronite: Environmental cell-transmission electron microscopy study

    Science.gov (United States)

    Kim, J.-W.; Furukawa, Y.; Daulton, T.L.; Lavoie, D.; Newell, S.W.

    2003-01-01

    Microstructural changes induced by the microbial reduction of Fe(III) in nontronite by Shewanella oneidensis were studied using environmental cell (EC)-transmission electron microscopy (TEM), conventional TEM, and X-ray powder diffraction (XRD). Direct observations of clays by EC-TEM in their hydrated state allowed for the first time an accurate and unambiguous TEM measurement of basal layer spacings and the contraction of layer spacing caused by microbial effects, most likely those of Fe(III) reduction. Non-reduced and Fe(III)-reduced nontronite, observed by EC-TEM, exhibited fringes with mean d001 spacings of 1.50 nm (standard deviation, ?? = 0.08 nm) and 1.26 nm (?? = 0.10 nm), respectively. In comparison, the same samples embedded with Nanoplast resin, sectioned by microtome, and observed using conventional TEM, displayed layer spacings of 1.0-1.1 nm (non-reduced) and 1.0 nm (reduced). The results from Nanoplast-embedded samples are typical of conventional TEM studies, which have measured nearly identical layer spacings regardless of Fe oxidation state. Following Fe(III) reduction, both EC- and conventional TEM showed an increase in the order of nontronite selected area electron diffraction patterns while the images exhibited fewer wavy fringes and fewer layer terminations. An increase in stacking order in reduced nontronite was also suggested by XRD measurements. In particular, the ratio of the valley to peak intensity (v/p) of the 1.7 nm basal 001 peak of ethylene glycolated nontronite was measured at 0.65 (non-reduced) and 0.85 (microbially reduced).

  10. Correlating confocal microscopy and atomic force indentation reveals metastatic cancer cells stiffen during invasion into collagen I matrices

    Science.gov (United States)

    Staunton, Jack R.; Doss, Bryant L.; Lindsay, Stuart; Ros, Robert

    2016-01-01

    Mechanical interactions between cells and their microenvironment dictate cell phenotype and behavior, calling for cell mechanics measurements in three-dimensional (3D) extracellular matrices (ECM). Here we describe a novel technique for quantitative mechanical characterization of soft, heterogeneous samples in 3D. The technique is based on the integration of atomic force microscopy (AFM) based deep indentation, confocal fluorescence microscopy, finite element (FE) simulations and analytical modeling. With this method, the force response of a cell embedded in 3D ECM can be decoupled from that of its surroundings, enabling quantitative determination of the elastic properties of both the cell and the matrix. We applied the technique to the quantification of the elastic properties of metastatic breast adenocarcinoma cells invading into collagen hydrogels. We found that actively invading and fully embedded cells are significantly stiffer than cells remaining on top of the collagen, a clear example of phenotypical change in response to the 3D environment. Treatment with Rho-associated protein kinase (ROCK) inhibitor significantly reduces this stiffening, indicating that actomyosin contractility plays a major role in the initial steps of metastatic invasion. PMID:26813872

  11. Correlating confocal microscopy and atomic force indentation reveals metastatic cancer cells stiffen during invasion into collagen I matrices.

    Science.gov (United States)

    Staunton, Jack R; Doss, Bryant L; Lindsay, Stuart; Ros, Robert

    2016-01-01

    Mechanical interactions between cells and their microenvironment dictate cell phenotype and behavior, calling for cell mechanics measurements in three-dimensional (3D) extracellular matrices (ECM). Here we describe a novel technique for quantitative mechanical characterization of soft, heterogeneous samples in 3D. The technique is based on the integration of atomic force microscopy (AFM) based deep indentation, confocal fluorescence microscopy, finite element (FE) simulations and analytical modeling. With this method, the force response of a cell embedded in 3D ECM can be decoupled from that of its surroundings, enabling quantitative determination of the elastic properties of both the cell and the matrix. We applied the technique to the quantification of the elastic properties of metastatic breast adenocarcinoma cells invading into collagen hydrogels. We found that actively invading and fully embedded cells are significantly stiffer than cells remaining on top of the collagen, a clear example of phenotypical change in response to the 3D environment. Treatment with Rho-associated protein kinase (ROCK) inhibitor significantly reduces this stiffening, indicating that actomyosin contractility plays a major role in the initial steps of metastatic invasion. PMID:26813872

  12. Resolving protein interactions and organization downstream the T cell antigen receptor using single-molecule localization microscopy: a review

    Science.gov (United States)

    Sherman, Eilon

    2016-06-01

    Signal transduction is mediated by heterogeneous and dynamic protein complexes. Such complexes play a critical role in diverse cell functions, with the important example of T cell activation. Biochemical studies of signalling complexes and their imaging by diffraction limited microscopy have resulted in an intricate network of interactions downstream the T cell antigen receptor (TCR). However, in spite of their crucial roles in T cell activation, much remains to be learned about these signalling complexes, including their heterogeneous contents and size distribution, their complex arrangements in the PM, and the molecular requirements for their formation. Here, we review how recent advancements in single molecule localization microscopy have helped to shed new light on the organization of signalling complexes in single molecule detail in intact T cells. From these studies emerges a picture where cells extensively employ hierarchical and dynamic patterns of nano-scale organization to control the local concentration of interacting molecular species. These patterns are suggested to play a critical role in cell decision making. The combination of SMLM with more traditional techniques is expected to continue and critically contribute to our understanding of multimolecular protein complexes and their significance to cell function.

  13. Correlating confocal microscopy and atomic force indentation reveals metastatic cancer cells stiffen during invasion into collagen I matrices

    Science.gov (United States)

    Staunton, Jack R.; Doss, Bryant L.; Lindsay, Stuart; Ros, Robert

    2016-01-01

    Mechanical interactions between cells and their microenvironment dictate cell phenotype and behavior, calling for cell mechanics measurements in three-dimensional (3D) extracellular matrices (ECM). Here we describe a novel technique for quantitative mechanical characterization of soft, heterogeneous samples in 3D. The technique is based on the integration of atomic force microscopy (AFM) based deep indentation, confocal fluorescence microscopy, finite element (FE) simulations and analytical modeling. With this method, the force response of a cell embedded in 3D ECM can be decoupled from that of its surroundings, enabling quantitative determination of the elastic properties of both the cell and the matrix. We applied the technique to the quantification of the elastic properties of metastatic breast adenocarcinoma cells invading into collagen hydrogels. We found that actively invading and fully embedded cells are significantly stiffer than cells remaining on top of the collagen, a clear example of phenotypical change in response to the 3D environment. Treatment with Rho-associated protein kinase (ROCK) inhibitor significantly reduces this stiffening, indicating that actomyosin contractility plays a major role in the initial steps of metastatic invasion.

  14. CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells.

    Science.gov (United States)

    Ratz, Michael; Testa, Ilaria; Hell, Stefan W; Jakobs, Stefan

    2015-04-20

    Overexpression is a notorious concern in conventional and especially in super-resolution fluorescence light microscopy studies because it may cause numerous artifacts including ectopic sub-cellular localizations, erroneous formation of protein complexes, and others. Nonetheless, current live cell super-resolution microscopy studies generally rely on the overexpression of a host protein fused to a fluorescent protein. Here, we establish CRISPR/Cas9-mediated generation of heterozygous and homozygous human knockin cell lines expressing fluorescently tagged proteins from their respective native genomic loci at close to endogenous levels. We tagged three different proteins, exhibiting various localizations and expression levels, with the reversibly switchable fluorescent protein rsEGFP2. We demonstrate the benefit of endogenous expression levels compared to overexpression and show that typical overexpression-induced artefacts were avoided in genome-edited cells. Fluorescence activated cell sorting analysis revealed a narrow distribution of fusion protein expression levels in genome-edited cells, compared to a pronounced variability in transiently transfected cells. Using low light intensity RESOLFT (reversible saturable optical fluorescence transitions) nanoscopy we show sub-diffraction resolution imaging of living human knockin cells. Our strategy to generate human cell lines expressing fluorescent fusion proteins at endogenous levels for RESOLFT nanoscopy can be extended to other fluorescent tags and super-resolution approaches.

  15. The Potential Impact of Biofield Treatment on Human Brain Tumor Cells: A Time-Lapse Video Microscopy

    OpenAIRE

    Trivedi, Mahendra Kumar

    2015-01-01

    Study background: Glioblastoma (GBM) is the most common subtype of primary brain tumor in adults. The aim was to evaluate the impact of biofield treatment potential on human GBM and non-GBM brain cells using two time-lapse video microscopy technique. Methods: The human brain tumor, GBM cultured cells were divided into two groups viz. GBM control and GBM treatment. Similarly, human normal brain cultured cells (non-GBM) were taken and divided into two groups viz. non- GBM control ...

  16. Compact diffraction phase microscopy for quantitative visualization of cells in biomedical applications

    Science.gov (United States)

    Talaikova, N. A.; Ryabukho, V. P.

    2016-08-01

    We consider a simplified and compact scheme of interference phase microscopy using a diffraction grating and spatial filtering of the diffracted field, i.e., diffraction phase microscopy. The scheme and the parameters of the device with the possibility of using the optical system of a smartphone and its software are analysed. The results of experimental determination of the spatial structure parameters of erythrocytes are presented.

  17. SIM and PALM: high-resolution microscopy methods and their consequences for cell biology

    Science.gov (United States)

    Krampert, Gerhard; Kleppe, Ingo; Kalkbrenner, Thomas; Weisshart, Klaus; Wolleschensky, Ralf; Kempe, Michael

    2010-04-01

    The diffraction limit in traditional fluorescence microscopy (approximately 200 and 600 nanometers in lateral and axial directions, respectively) has restricted the applications in bio-medical research. However, over the last 10 years various techniques have emerged to overcome this limit. Each of these techniques has its own characteristics that influence its application in biology. This paper will show how two of the techniques, Structured Illumination Microscopy (SIM) and PhotoActivated Localization Microscopy (PALM), complement each other in imaging of biological samples beyond the resolution of classical widefield fluorescence microscopy. As a reference the properties of two well known standard imaging techniques in this field, confocal Laser Scanning Microscopy (LSM) and Total Internal Reflection (TIRF) microscopy, are compared to the properties of the two high resolution techniques. Combined SIM/PALM imaging allows the extremely accurate localization of individual molecules within the context of various fluorescent structures already resolved in 3D with a resolution of up to 100nm using SIM. Such a combined system provides the biologist with an unprecedented view of the sub-cellular organization of life.

  18. Visualizing Cell Architecture and Molecular Location Using Soft X-Ray Tomography and Correlated Cryo-Light Microscopy

    Science.gov (United States)

    McDermott, Gerry; Le Gros, Mark A.; Larabell, Carolyn A.

    2012-05-01

    Living cells are structured to create a range of microenvironments that support specific chemical reactions and processes. Understanding how cells function therefore requires detailed knowledge of both the subcellular architecture and the location of specific molecules within this framework. Here we review the development of two correlated cellular imaging techniques that fulfill this need. Cells are first imaged using cryogenic fluorescence microscopy to determine the location of molecules of interest that have been labeled with fluorescent tags. The same specimen is then imaged using soft X-ray tomography to generate a high-contrast, 3D reconstruction of the cells. Data from the two modalities are then combined to produce a composite, information-rich view of the cell. This correlated imaging approach can be applied across the spectrum of problems encountered in cell biology, from basic research to biotechnological and biomedical applications such as the optimization of biofuels and the development of new pharmaceuticals.

  19. Immunogold electron microscopy and confocal analyses reveal distinctive patterns of histone H3 phosphorylation during mitosis in MCF-7 cells.

    Science.gov (United States)

    Yan, Yitang; Cummings, Connie A; Sutton, Deloris; Yu, Linda; Castro, Lysandra; Moore, Alicia B; Gao, Xiaohua; Dixon, Darlene

    2016-04-01

    Histone phosphorylation has a profound impact on epigenetic regulation of gene expression, chromosome condensation and segregation, and maintenance of genome integrity. Histone H3 Serine 10 is evolutionally conserved and heavily phosphorylated during mitosis. To examine Histone H3 Serine 10 phosphorylation (H3S10ph) dynamics in mitosis, we applied immunogold labeling and confocal microscopy to visualize H3S10ph expression in MCF-7 cells. Confocal observations showed that MCF-7 cells had abundant H3S10ph expression in prophase and metaphase. In anaphase, the H3S10ph expression was significantly decreased and displayed only sparsely localized staining that mainly associated with the chromatid tips. We showed that immunogold bead density distribution followed the H3S10ph expression patterns observed in confocal analysis. At a higher magnification in metaphase, the immunogold beads were readily visible and the bead distribution along the condensed chromosomes was distinctive, indicating the specificity and reliability of the immunogold staining procedure. In anaphase, the beads were found to distribute focally in specific regions of chromatids, reinforcing the confocal observations of differential H3 phosphorylation. To our knowledge, this is the first report to show the specific H3S10ph expression with an immunogold technique and transmission electron microscopy. Additionally, with confocal microscopy, we analyzed H3S10ph expression in an immortalized cell line derived from benign uterine smooth muscle tumor cells. H3S10ph epitope was expressed more abundantly during anaphase in the benign tumor cells, and there was no dramatic differential expression within the condensed chromatid clusters as observed in MCF-7 cells. The differences in H3S10ph expression pattern and dynamics may contribute to the differential proliferative potential between benign tumor cells and MCF-7 cells.

  20. High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy.

    Science.gov (United States)

    Reymann, Jürgen; Baddeley, David; Gunkel, Manuel; Lemmer, Paul; Stadter, Werner; Jegou, Thibaud; Rippe, Karsten; Cremer, Christoph; Birk, Udo

    2008-01-01

    Spatially modulated illumination (SMI) microscopy is a method of wide field fluorescence microscopy featuring interferometric illumination, which delivers structural information about nanoscale architecture in fluorescently labelled cells. The first prototype of the SMI microscope proved its applicability to a wide range of biological questions. For the SMI live cell imaging this system was enhanced in terms of the development of a completely new upright configuration. This so called Vertico-SMI transfers the advantages of SMI nanoscaling to vital biological systems, and is shown to work consistently at different temperatures using both oil- and water-immersion objective lenses. Furthermore, we increased the speed of data acquisition to minimize errors in the detection signal resulting from cellular or object movement. By performing accurate characterization, the present Vertico-SMI now offers a fully-fledged microscope enabling a complete three-dimensional (3D) SMI data stack to be acquired in less than 2 seconds. We have performed live cell measurements of a tet-operator repeat insert in U2OS cells, which provided the first in vivo signatures of subnuclear complexes. Furthermore, we have successfully implemented an optional optical configuration allowing the generation of high-resolution localization microscopy images of a nuclear pore complex distribution.

  1. Correlations between Photovoltaic Characteristics, Adsorption Number, and Regeneration Kinetics in Dye-Sensitized Solar Cells Revealed by Scanning Photocurrent Microscopy.

    Science.gov (United States)

    Mitsui, Masaaki; Kawano, Yuya; Mori, Kyosuke; Wakabayashi, Naoto

    2015-06-30

    Newly developed simultaneous scanning photocurrent and luminescence microscopy was applied to ruthenium-based dye-sensitized solar cells (DSCs) comprising a cover glass photoanode with a 100 nm thick TiO2 layer. Using this, we have investigated the lateral variations of several parameters of these DSCs under short-circuit conditions. Simultaneous measurement of photocurrent and luminescence images for the same area of the DSC demonstrated submicrometric lateral resolution of our photocurrent microscopy, which is approximately 10 times better than the resolution of photocurrent microscopy used in past studies. The photovoltaic parameters, such as short-circuit current density, open-circuit voltage, and charge-collection efficiency, were thus evaluated for local (or submicrometric) regions of the DSCs. Furthermore, the photocurrent saturation behavior of the DSCs was examined as a function of the excitation rate and analyzed on the basis of a three-state kinetic model. This protocol allowed for quantification of the dye-adsorption number and dye-regeneration rate constant for any local area of the DSCs. Consequently, the correlations between the dye adsorption number, photovoltaic parameters, and regeneration rate constant, which are difficult to address through examination of the entire cell, were revealed by the "zoom-in" approach utilizing this high-resolution photocurrent microscopy.

  2. Chromosome condensation and segmentation

    International Nuclear Information System (INIS)

    Some aspects of chromosome condensation in mammalians -humans especially- were studied by means of cytogenetic techniques of chromosome banding. Two further approaches were adopted: a study of normal condensation as early as prophase, and an analysis of chromosome segmentation induced by physical (temperature and γ-rays) or chemical agents (base analogues, antibiotics, ...) in order to show out the factors liable to affect condensation. Here 'segmentation' means an abnormal chromosome condensation appearing systematically and being reproducible. The study of normal condensation was made possible by the development of a technique based on cell synchronization by thymidine and giving prophasic and prometaphasic cells. Besides, the possibility of inducing R-banding segmentations on these cells by BrdU (5-bromodeoxyuridine) allowed a much finer analysis of karyotypes. Another technique was developed using 5-ACR (5-azacytidine), it allowed to induce a segmentation similar to the one obtained using BrdU and identify heterochromatic areas rich in G-C bases pairs

  3. The consequences of reconfiguring the ambisense S genome segment of Rift Valley fever virus on viral replication in mammalian and mosquito cells and for genome packaging.

    Directory of Open Access Journals (Sweden)

    Benjamin Brennan

    2014-02-01

    Full Text Available Rift Valley fever virus (RVFV, family Bunyaviridae is a mosquito-borne pathogen of both livestock and humans, found primarily in Sub-Saharan Africa and the Arabian Peninsula. The viral genome comprises two negative-sense (L and M segments and one ambisense (S segment RNAs that encode seven proteins. The S segment encodes the nucleocapsid (N protein in the negative-sense and a nonstructural (NSs protein in the positive-sense, though NSs cannot be translated directly from the S segment but rather from a specific subgenomic mRNA. Using reverse genetics we generated a virus, designated rMP12:S-Swap, in which the N protein is expressed from the NSs locus and NSs from the N locus within the genomic S RNA. In cells infected with rMP12:S-Swap NSs is expressed at higher levels with respect to N than in cells infected with the parental rMP12 virus. Despite NSs being the main interferon antagonist and determinant of virulence, growth of rMP12:S-Swap was attenuated in mammalian cells and gave a small plaque phenotype. The increased abundance of the NSs protein did not lead to faster inhibition of host cell protein synthesis or host cell transcription in infected mammalian cells. In cultured mosquito cells, however, infection with rMP12:S-Swap resulted in cell death rather than establishment of persistence as seen with rMP12. Finally, altering the composition of the S segment led to a differential packaging ratio of genomic to antigenomic RNA into rMP12:S-Swap virions. Our results highlight the plasticity of the RVFV genome and provide a useful experimental tool to investigate further the packaging mechanism of the segmented genome.

  4. [Artefacts of confocal microscopy].

    Science.gov (United States)

    Vekshin, N L; Frolov, M S

    2014-01-01

    Typical artefacts caused by using confocal fluorescent microscopy while studying living cells are considered. The role of light scattering, mobility, staining, local concentrations, etc. is discussed.

  5. Systematic Characterization of Cell Cycle Phase-dependent Protein Dynamics and Pathway Activities by High-content Microscopy-assisted Cell Cycle Phenotyping

    Institute of Scientific and Technical Information of China (English)

    Christopher Bruhn; Torsten Kroll; Zhao-Qi Wang

    2014-01-01

    Cell cycle progression is coordinated with metabolism, signaling and other complex cel-lular functions. The investigation of cellular processes in a cell cycle stage-dependent manner is often the subject of modern molecular and cell biological research. Cell cycle synchronization and immunostaining of cell cycle markers facilitate such analysis, but are limited in use due to unphysiological experimental stress, cell type dependence and often low flexibility. Here, we describe high-content microscopy-assisted cell cycle phenotyping (hiMAC), which integrates high-resolution cell cycle profiling of asynchronous cell populations with immunofluorescence microscopy. hiMAC is compatible with cell types from any species and allows for statistically pow-erful, unbiased, simultaneous analysis of protein interactions, modifications and subcellular locali-zation at all cell cycle stages within a single sample. For illustration, we provide a hiMAC analysis pipeline tailored to study DNA damage response and genomic instability using a 3–4-day protocol, which can be adjusted to any other cell cycle stage-dependent analysis.

  6. Segmental neurofibromatosis

    OpenAIRE

    Galhotra, Virat; Sheikh, Soheyl; Jindal, Sanjeev; Singla, Anshu

    2014-01-01

    Segmental neurofibromatosis is a rare disorder, characterized by neurofibromas or cafι-au-lait macules limited to one region of the body. Its occurrence on the face is extremely rare and only few cases of segmental neurofibromatosis over the face have been described so far. We present a case of segmental neurofibromatosis involving the buccal mucosa, tongue, cheek, ear, and neck on the right side of the face.

  7. 3D Plant Cell Architecture of Arabidopsis thaliana (Brassicaceae Using Focused Ion Beam–Scanning Electron Microscopy

    Directory of Open Access Journals (Sweden)

    Bhawana

    2014-06-01

    Full Text Available Premise of the study: Focused ion beam–scanning electron microscopy (FIB-SEM combines the ability to sequentially mill the sample surface and obtain SEM images that can be used to create 3D renderings with micron-level resolution. We have applied FIB-SEM to study Arabidopsis cell architecture. The goal was to determine the efficacy of this technique in plant tissue and cellular studies and to demonstrate its usefulness in studying cell and organelle architecture and distribution. Methods: Seed aleurone, leaf mesophyll, stem cortex, root cortex, and petal lamina from Arabidopsis were fixed and embedded for electron microscopy using protocols developed for animal tissues and modified for use with plant cells. Each sample was sectioned using the FIB and imaged with SEM. These serial images were assembled to produce 3D renderings of each cell type. Results: Organelles such as nuclei and chloroplasts were easily identifiable, and other structures such as endoplasmic reticula, lipid bodies, and starch grains were distinguishable in each tissue. Discussion: The application of FIB-SEM produced 3D renderings of five plant cell types and offered unique views of their shapes and internal content. These results demonstrate the usefulness of FIB-SEM for organelle distribution and cell architecture studies.

  8. WHOLE CELL TOMOGRAPHY/MOLECULAR BIOLOGY/STRUCTURAL BIOLOGY: Affordable x-ray microscopy with nanoscale resolution

    Energy Technology Data Exchange (ETDEWEB)

    Evans, James E.; Blackborow, Paul; Horne, Stephen J.; Gelb, Jeff

    2013-03-01

    Biological research spans 10 orders of magnitude from angstroms to meters. While electron microscopy can reveal structural details at most of these spatial length scales, transmission electron tomography only reliably reconstructs three-dimensional (3-D) volumes of cellular material with a spatial resolution between 1-5 nm from samples less than 500 nm thick1. Most biological cells are 2-30 times thicker than this threshold, which means that a cell must be cut into consecutive slices with each slice reconstructed individually in order to approximate the contextual information of the entire cell. Fortunately, due to a larger penetration depth2, X-ray computed tomography bypasses the need to physically section a cell and enables imaging of intact cells and tissues on the micrometer or larger scale with tens to hundreds of nanometer spatial resolution. While the technique of soft x-ray microscopy has been extensively developed in synchrotron facilities, advancements in laboratory x-ray source designs now increase its accessibility by supporting commercial systems suitable for a standard laboratory. In this paper, we highlight a new commercial compact cryogenic soft x-ray microscope designed for a standard laboratory setting and explore its capabilities for mesoscopic investigations of intact prokaryotic and eukaryotic cells.

  9. Nanoscale imaging of untreated mammalian cells in a medium with low radiation damage using scanning electron-assisted dielectric microscopy

    Science.gov (United States)

    Okada, Tomoko; Ogura, Toshihiko

    2016-07-01

    Imaging of untreated living cells in a medium at a nanometre-scale resolution under physiological conditions is a significant challenge. Scanning electron microscopy (SEM) is widely used to observe cells in various atmospheric holders or special equipment. However, untreated biological specimens in aqueous solution generally incur heavy radiation damage from the direct electron beam (EB); and these images exhibit very poor contrast. Therefore, a new method for generating high-contrast images of living cells under physiological conditions without radiation damage has been strongly desired. Here, we demonstrate the first nanoscale observation of living cultured mammalian cells using our newly developed scanning-electron assisted dielectric microscopy (SE-ADM) method with a culture dish holder. Using the difference in relative permittivity between water and specimens, our SE-ADM system aids in the visualisation of untreated biological samples in aqueous solution. In addition, specimens incurred only a low level of radiation damage because the tungsten (W)-coated silicon nitride (SiN) film absorbs irradiated electrons. Untreated cells and organelles are clearly visible in high-contrast and high-resolution images without staining and fixation. Furthermore, our method enables the detection of changes in organelle structures within cells via time-lapse imaging with minimal radiation damage.

  10. Nanoscale imaging of untreated mammalian cells in a medium with low radiation damage using scanning electron-assisted dielectric microscopy

    Science.gov (United States)

    Okada, Tomoko; Ogura, Toshihiko

    2016-01-01

    Imaging of untreated living cells in a medium at a nanometre-scale resolution under physiological conditions is a significant challenge. Scanning electron microscopy (SEM) is widely used to observe cells in various atmospheric holders or special equipment. However, untreated biological specimens in aqueous solution generally incur heavy radiation damage from the direct electron beam (EB); and these images exhibit very poor contrast. Therefore, a new method for generating high-contrast images of living cells under physiological conditions without radiation damage has been strongly desired. Here, we demonstrate the first nanoscale observation of living cultured mammalian cells using our newly developed scanning-electron assisted dielectric microscopy (SE-ADM) method with a culture dish holder. Using the difference in relative permittivity between water and specimens, our SE-ADM system aids in the visualisation of untreated biological samples in aqueous solution. In addition, specimens incurred only a low level of radiation damage because the tungsten (W)-coated silicon nitride (SiN) film absorbs irradiated electrons. Untreated cells and organelles are clearly visible in high-contrast and high-resolution images without staining and fixation. Furthermore, our method enables the detection of changes in organelle structures within cells via time-lapse imaging with minimal radiation damage. PMID:27375121

  11. Investigations on the change of texture of plant cells due to preservative treatments by digital holographic microscopy

    Science.gov (United States)

    Vora, Priyanka; Anand, Arun

    2014-10-01

    Texture change is observed in preserved fruits and vegetables. Responsible factors for texture change during preservative treatments are cell morphology, cell wall structure, cell turger, water content and some biochemical components, and also the environmental conditions. Digital Holographic microscopy (DHM) is a quantitative phase contrast imaging technique, which provides three dimensional optical thickness profiles of transparent specimen. Using DHM the morphology of plant cells preserved by refrigeration or stored in vinegar or in sodium chloride can be obtained. This information about the spatio-temporal evolution of optical volume and thickness can be an important tool in area of food processing. Also from the three dimensional images, the texture of the cell can be retrieved and can be investigated under varying conditions.

  12. Large-scale time-lapse microscopy of Oct4 expression in human embryonic stem cell colonies.

    Science.gov (United States)

    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.

  13. Sipunculans and segmentation

    DEFF Research Database (Denmark)

    Wanninger, Andreas; Kristof, Alen; Brinkmann, Nora

    2009-01-01

    Comparative molecular, developmental and morphogenetic analyses show that the three major segmented animal groups- Lophotrochozoa, Ecdysozoa and Vertebrata-use a wide range of ontogenetic pathways to establish metameric body organization. Even in the life history of a single specimen, different...... mechanisms may act on the level of gene expression, cell proliferation, tissue differentiation and organ system formation in individual segments. Accordingly, in some polychaete annelids the first three pairs of segmental peripheral neurons arise synchronously, while the metameric commissures of the ventral...... nervous system form in anterior-posterior progression. Contrary to traditional belief, loss of segmentation may have occurred more often than commonly assumed, as exemplified in the sipunculans, which show remnants of segmentation in larval stages but are unsegmented as adults. The developmental...

  14. Serial block face-scanning electron microscopy: a tool for studying embryonic development at the cell-matrix interface.

    Science.gov (United States)

    Starborg, Tobias; Kadler, Karl E

    2015-03-01

    Studies of gene regulation, signaling pathways, and stem cell biology are contributing greatly to our understanding of early embryonic vertebrate development. However, much less is known about the events during the latter half of embryonic development, when tissues comprising mostly extracellular matrix (ECM) are formed. The matrix extends far beyond the boundaries of individual cells and is refractory to study by conventional biochemical and molecular techniques; thus major gaps exist in our knowledge of the formation and three-dimensional (3D) organization of the dense tissues that form the bulk of adult vertebrates. Serial block face-scanning electron microscopy (SBF-SEM) has the ability to image volumes of tissue containing numerous cells at a resolution sufficient to study the organization of the ECM. Furthermore, whereas light microscopy was once relatively straightforward and electron microscopy was performed in specialist laboratories, the tables are turned; SBF-SEM is relatively straightforward and is becoming routine in high-end resolution studies of embryonic structures in vivo. In this review, we discuss the emergence of SBF-SEM as a tool for studying embryonic vertebrate development.

  15. Detection of Lipid-Rich Prostate Circulating Tumour Cells with Coherent Anti-Stokes Raman Scattering Microscopy

    Directory of Open Access Journals (Sweden)

    Mitra Ranjana

    2012-11-01

    Full Text Available Abstract Background Circulating tumour cells (CTC are an important indicator of metastasis and associated with a poor prognosis. Detection sensitivity and specificity of CTC in the peripheral blood of metastatic cancer patient remain a technical challenge. Methods Coherent anti-Stokes Raman scattering (CARS microscopy was employed to examine the lipid content of CTC isolated from the peripheral blood of metastatic prostate cancer patients. CARS microscopy was also employed to evaluate lipid uptake and mobilization kinetics of a metastatic human prostate cancer cell line. Results One hundred CTC from eight metastatic prostate cancer patients exhibited strong CARS signal which arose from intracellular lipid. In contrast, leukocytes exhibited weak CARS signal which arose mostly from cellular membrane. On average, CARS signal intensity of prostate CTC was 7-fold higher than that of leukocytes (P Conclusions Intracellular lipid could serve as a biomarker for prostate CTC which could be sensitively detected with CARS microscopy in a label-free manner. Strong affinity for lipid by metastatic prostate cancer cells could be used to improve detection sensitivity and therapeutic targeting of prostate CTC.

  16. Local variations of HER2 dimerization in breast cancer cells discovered by correlative fluorescence and liquid electron microscopy.

    Science.gov (United States)

    Peckys, Diana B; Korf, Ulrike; de Jonge, Niels

    2015-07-01

    The formation of HER2 homodimers plays an important role in breast cancer aggressiveness and progression; however, little is known about its localization. We have studied the intra- and intercellular variation of HER2 at the single-molecule level in intact SKBR3 breast cancer cells. Whole cells were visualized in hydrated state with correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM). The locations of individual HER2 receptors were detected using an anti-HER2 affibody in combination with a quantum dot (QD), a fluorescent nanoparticle. Fluorescence microscopy revealed considerable differences of HER2 membrane expression between individual cells and between different membrane regions of the same cell (that is, membrane ruffles and flat areas). Subsequent ESEM of the corresponding cellular regions provided images of individually labeled HER2 receptors. The high spatial resolution of 3 nm and the close proximity between the QD and the receptor allowed quantifying the stoichiometry of HER2 complexes, distinguishing between monomers, dimers, and higher-order clusters. Downstream data analysis based on calculating the pair correlation function from receptor positions showed that cellular regions exhibiting membrane ruffles contained a substantial fraction of HER2 in homodimeric state. Larger-order clusters were also present. Membrane areas with homogeneous membrane topography, on the contrary, displayed HER2 in random distribution. Second, HER2 homodimers appeared to be absent from a small subpopulation of cells exhibiting a flat membrane topography, possibly resting cells. Local differences in homodimer presence may point toward functional differences with possible relevance for studying metastasis and drug response. PMID:26601217

  17. Electron microscopy of the germ cells and the ovarian wall in Xiphinema (Nematoda).

    Science.gov (United States)

    Van de Velde, M C; Coomans, A

    1988-01-01

    The ovary of Xiphinema theresiae is studied ultrastructurally. It consists of two cell types, the ovarian epithelial cells and the germ cells. The ovarian epithelial cells form a thin layer around the germ cells. Their nuclei are located in between the germ cells. At some sites, processes of the ovarian epithelial cells migrate inward and form a central cytoplasmic mass. The germ cells have a large lobated nucleus, with an eccentric nucleolus, and are considered to represent young previtellogenic oocytes. In contact with the central cytoplasmic mass, the germ cells develop two membrane derived features, the villi and the small coated bulges, which most probably play a role in transport.

  18. Carbon black nanoparticles and vascular dysfunction in cultured endothelial cells and artery segments

    DEFF Research Database (Denmark)

    Vesterdal, Lise K; Mikkelsen, Lone; Folkmann, Janne K;

    2012-01-01

    surface expression of intercellular cell adhesion molecule 1 (ICAM-1) and vascular adhesion molecule 1 (VCAM-1) in HUVECs at 100µg/ml. CB exposure was also associated with increased reactive oxygen species production and damage to the cell membranes in the form of increased lactate dehydrogenase leakage...

  19. Rhodopsin gene expression determines rod outer segment size and rod cell resistance to a dominant-negative neurodegeneration mutant.

    Directory of Open Access Journals (Sweden)

    Brandee A Price

    Full Text Available Two outstanding unknowns in the biology of photoreceptors are the molecular determinants of cell size, which is remarkably uniform among mammalian species, and the mechanisms of rod cell death associated with inherited neurodegenerative blinding diseases such as retinitis pigmentosa. We have addressed both questions by performing an in vivo titration with rhodopsin gene copies in genetically engineered mice that express only normal rhodopsin or an autosomal dominant allele, encoding rhodopsin with a disease-causing P23H substitution. The results reveal that the volume of the rod outer segment is proportional to rhodopsin gene expression; that P23H-rhodopsin, the most common rhodopsin gene disease allele, causes cell death via a dominant-negative mechanism; and that long term survival of rod cells carrying P23H-rhodopsin can be achieved by increasing the levels of wild type rhodopsin. These results point to promising directions in gene therapy for autosomal dominant neurodegenerative diseases caused by dominant-negative mutations.

  20. Segmentation: Identification of consumer segments

    DEFF Research Database (Denmark)

    Høg, Esben

    2005-01-01

    It is very common to categorise people, especially in the advertising business. Also traditional marketing theory has taken in consumer segments as a favorite topic. Segmentation is closely related to the broader concept of classification. From a historical point of view, classification has its...... a basic understanding of grouping people. Advertising agencies may use segmentation totarget advertisements, while food companies may usesegmentation to develop products to various groups of consumers. MAPP has for example investigated the positioning of fish in relation to other food products...... their evaluation of fish - for example by focusing on quality. Therefore, taking heterogeneity between segments into account makes a lot of difference, quantitatively as well as qualitatively....

  1. Complete staining of human spermatozoa and immature germ cells combined with phase contrast microscopy

    DEFF Research Database (Denmark)

    Michael, A Y; Drejer, J O; Bagger, P V;

    1987-01-01

    A method combining Janus green B and Thymol blue stains the anterior part of the head, the nuclear membrane, middle piece, and tail of spermatozoa light green and the nucleus deep purple. The method provides excellent stained preparations for the evaluation of sperm morphology by phase contrast...... microscopy. It produces significantly less abnormal spermatozoa compared with the Papanicolaou stain....

  2. Hybrid Rayleigh, Raman and two-photon excited fluorescence spectral confocal microscopy of living cells

    NARCIS (Netherlands)

    Pully, Vishnu Vardhan; Lenferink, Aufried; Otto, Cees

    2010-01-01

    A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low-wavenumber-resolution Raman imaging, Rayleigh scatter imaging and two-photon fluorescence (TPE) spectral imaging, fast ‘amplitude-only’ TPE-fluorescence imaging and high-spectral-resolution Raman imaging. Thi

  3. Direct visualization of secretion from single bovine adrenal chromaffin cells by laser-induced native fluorescence imaging microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tong, W.; Yeung, E.S. [Ames Laboratory---USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 (United States)

    1998-03-01

    Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved with laser-induced native fluorescence imaging microscopy. By monitoring the native fluorescence of catecholamines excited by the 275 nm laser line with an intensified charge-coupled-device (CCD) camera, we obtained good temporal and spatial resolution simultaneously without using additional fluorescent probes. Large variations were found among individual cells in terms of the amounts of catecholamines secreted and the rates of secretion. Different regions of a cell also behave differently during the secretion process. However, the degree of this local heterogeneity is smaller than in neurons and neuralgia. The influence of deep-ultraviolet (UV) laser excitation on cells is also discussed. This quantitative imaging technique provides a useful noninvasive approach for the study of dynamic cellular changes and the understanding of the molecular mechanisms of secretory processes. {copyright} {ital 1998} {ital Society for Applied Spectroscopy}

  4. Mapping Cd²⁺-induced membrane permeability changes of single live cells by means of scanning electrochemical microscopy.

    Science.gov (United States)

    Filice, Fraser P; Li, Michelle S M; Henderson, Jeffrey D; Ding, Zhifeng

    2016-02-18

    Scanning Electrochemical Microscopy (SECM) is a powerful, non-invasive, analytical methodology that can be used to investigate live cell membrane permeability. Depth scan SECM imaging allowed for the generation of 2D current maps of live cells relative to electrode position in the x-z or y-z plane. Depending on resolution, one depth scan image can contain hundreds of probe approach curves (PACs). Individual PACs were obtained by simply extracting vertical cross-sections from the 2D image. These experimental PACs were overlaid onto theoretically generated PACs simulated at specific geometry conditions. Simulations were carried out using 3D models in COMSOL Multiphysics to determine the cell membrane permeability coefficients at different locations on the surface of the cells. Common in literature, theoretical PACs are generated using a 2D axially symmetric geometry. This saves on both compute time and memory utilization. However, due to symmetry limitations of the model, only one experimental PAC right above the cell can be matched with simulated PAC data. Full 3D models in this article were developed for the SECM system of live cells, allowing all experimental PACs over the entire cell to become usable. Cd(2+)-induced membrane permeability changes of single human bladder (T24) cells were investigated at several positions above the cell, displaced from the central axis. The experimental T24 cells under study were incubated with Cd(2+) in varying concentrations. It is experimentally observed that 50 and 100 μM Cd(2+) caused a decrease in membrane permeability, which was uniform across all locations over the cell regardless of Cd(2+) concentration. The Cd(2+) was found to have detrimental effects on the cell, with cells shrinking in size and volume, and the membrane permeability decreasing. A mapping technique for the analysis of the cell membrane permeability under the Cd(2+) stress is realized by the methodology presented. PMID:26826690

  5. Third Harmonic Generation microscopy as a diagnostic tool for the investigation of microglia BV-2 and breast cancer cells activation

    Science.gov (United States)

    Gavgiotaki, E.; Filippidis, G.; Psilodimitrakopoulos, S.; Markomanolaki, H.; Kalognomou, M.; Agelaki, S.; Georgoulias, V.; Athanassakis, I.

    2015-07-01

    Nonlinear optical imaging techniques have created new opportunities of research in the biomedical field. Specifically, Third Harmonic Generation (THG) seems to be a suitable noninvasive imaging tool for the delineation and quantification of biological structures at the microscopic level. The aim of this study was to extract information as to the activation state of different cell types by using the THG imaging microscopy as a diagnostic tool. BV-2 microglia cell line was used as a representative biological model enabling the study of resting and activated state of the cells linked to various pathological conditions. Third Harmonic Generation (THG) and Two Photon Excitation Fluorescence (TPEF) measurements were simultaneously collected from stained breast cancer cells, by employing a single homemade experimental apparatus and it was shown that high THG signals mostly arise from lipid bodies. Continuously, BV-2 microglia cells were examined with or without activation by lipopolysaccharide (LPS) in order to discriminate between control and activated cells based on the quantification of THG signals. Statistically quantification was accomplished in both mean area and mean intensity values of THG. The values for mean total area and mean THG intensity values have been increased in activated versus the non-activated cells. Similar studies of quantification are underway in breast cancer cells for the exact discrimination on different cell lines. Furthermore, laser polarization dependence of SHG and THG signal in unstained biological samples is investigated.

  6. A fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells

    Directory of Open Access Journals (Sweden)

    Franks Douglas

    2001-01-01

    Full Text Available In platelets, PGHS-1-dependant formation of thromboxane A2 is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin. Since platelets are a-nucleated cells, we have used the immortalized human megakaryoblastic cell line MEG-01 which can be induced to differentiate into platelet-like structures upon addition of TPA as a model system to study PGHS-1 gene expression. Using a specific antibody to PGHS-1 we have developed a technique utilizing immunofluorescence microscopy and analysis of multiple digital images to monitor PGHS-1 protein levels as MEG-01 cells were induced to differentiate by a single addition of TPA (1.6 x 10-8 M over a period of 8 days. The method represents a rapid and economical alternative to flow cytometry. Using this technique we observed that TPA induced adherence of MEG-01 cells, and only the non-adherent TPA-stimulated cells demonstrated compromised viability. The differentiation of MEG-01 cells was evaluated by the expression of the platelet-specific cell surface antigen, CD-41. The latter was expressed in MEG-01 cells at the later stages of differentiation. We demonstrated a good correlation between PGHS-1 levels and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein level, which shows a consistent increase over the entire course of differentiation, can be used as an additional and better index by which to monitor megakaryocyte differentiation.

  7. Segmental Neurofibromatosis: Atypical Localisation

    OpenAIRE

    Filiz Topaloğlu Demir; Burçe Can; Berkant Oman; İlkin Zindancı; Tülay Zenginkinet; Mukaddes Kavala

    2015-01-01

    Neurofibromatosis (NF) is a genetic disease leading pathological findings in skin, soft tissue, bone and nervous system by affecting neural crest cells. Due to its heterogeneity neurofibromatosis was divided into eight different subgroups (NF-I NF-VIII) by Riccardi. Segmental neurofibromatosis (NF type V) is characterized by cutaneous neurofibromas and Café-au-lait spots limited with a segment of dermatome. Here we report this case with numerous, painless cutaneous nodules showing extension f...

  8. CARS and SHG microscopy to follow the collagen production in living human corneal fibroblasts and mesenchymal stem cells in fibrin gel 3D cultures

    CERN Document Server

    Mortati, Leonardo; Sassi, Maria Paola

    2011-01-01

    Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with second harmonic generation (SHG) technique in order to follow the early stage of stem cell differentiation within a 3D scaffold. CARS microscopy can detect lipid membranes and droplet compartments in living cells and SHG microscopy enables a strong imaging contrast for molecules with a non-centrosymmetric ordered structure like collagen. One of the first evidence of hMSCs differentiation is the formation of an extracellular matrix (ECM) where the collagen protein is its main component. This work demonstrated the multimodal CARS and SHG microscopy as a powerful non-invasive label free technique to investigate the collagen production dynamic in living cell 3D cultures. Its ability to image the cell morphology and the produced collagen distribution on a long term (4 weeks) experiment allowed to obtain important information about the cell-scaffold interaction and the ECM production. The very low limit reached in detecting collagen has permit...

  9. Functional imaging of a single cell: far-field infrared super-resolution microscopy using autofluorescence detection

    Science.gov (United States)

    Ohmori, Tsutomu; Inoue, Keiichi; Sakai, Makoto; Fujii, Masaaki; Ishihara, Miya; Kikuchi, Makoto

    2009-02-01

    We demonstrated cell imaging without any stain by far-field 2-color infrared (IR) super-resolution microscopy, combining laser fluorescence microscope and picosecond transient fluorescence detected IR (TFD-IR) spectroscopy. TFD-IR spectroscopy detects IR absorption by monitoring fluorescence due to an electronic transition from a vibrational excited level by an additional visible light. By using the IR microscopy based on TFD-IR spectroscopy, the spatial resolution of the image can be increased to the visible diffraction limit of sub-μm, i.e., the IR is super-resolved. Cell auto-fluorescence due to flavin molecules was monitored for label-free detection of the cellular components. The fluorescence image of an A549 cell was obtained by introducing both an IR light at 3300 nm and a visible light at 560 nm. The spatial resolution of the image was estimated to be 1.6 μm. This is about 2.5-times higher resolution than the diffraction limit of IR light. The fluorescence intensity of the images at 3448 nm was smaller than that at 3300 nm, corresponding to the smaller IR absorption. Therefore, IR spectral imaging of a single cell was achieved with superresolution.

  10. Development of confocal immunofluorescence FRET microscopy to Investigate eNOS and GSNOR localization and interaction in pulmonary endothelial cells

    Science.gov (United States)

    Rehman, Shagufta; Brown-Steinke, Kathleen; Palmer, Lisa; Periasamy, Ammasi

    2015-03-01

    Confocal FRET microscopy is a widely used technique for studying protein-protein interactions in live or fixed cells. Endothelial nitric oxide synthase (eNOS) and S-nitrosoglutathione reductase (GSNOR) are enzymes involved in regulating the bioavailability of S-nitrosothiols (SNOs) in the pulmonary endothelium and have roles in the development of pulmonary arterial hypertension. Labeling of endogenous proteins to better understand a disease process can be challenging. We have used immunofluorescence to detect endogenous eNOS and GSNOR in primary pulmonary endothelial cells to co-localize these proteins as well as to study their interaction by FRET. The challenge has been in selecting the right immunofluorescence labeling condition, right antibody, the right blocking reagent, the right FRET pair and eliminating cross-reactivity of secondary antibodies. We have used Alexa488 and Alexa568 as a FRET pair. After a series of optimizations, the data from Confocal Laser Scanning Microscopy (CLSM) demonstrate co-localization of eNOS and GSNOR in the perinuclear region of the pulmonary endothelial cell primarily within the cis-Golgi with lower levels of co-localization seen within the trans-Golgi. FRET studies demonstrate, for the first time, interaction between eNOS and GSNOR in both murine and bovine pulmonary endothelial cells. Further characterization of eNOSGSNOR interaction and the subcellular location of this interaction will provide mechanistic insight into the importance of S-nitrosothiol signaling in pulmonary biology, physiology and pathology.

  11. Glucose and Lactate Biosensors for Scanning Electrochemical Microscopy Imaging of Single Live Cells

    OpenAIRE

    Ciobanu, Madalina; Taylor, Dale E.; Wilburn, Jeremy P.; Cliffel, David E

    2008-01-01

    We have developed glucose and lactate ultramicroelectrode (UME) biosensors based on glucose oxidase and lactate oxidase (with enzymes immobilized onto Pt UMEs by either electropolymerization or casting) for scanning electrochemical microscopy (SECM), and have determined their sensitivity to glucose and lactate, respectively. The results of our evaluations reveal different advantages for sensors constructed by each method: improved sensitivity and shorter manufacturing time for hand-casting, a...

  12. Sonic hedgehog enhances somite cell viability and formation of primary slow muscle fibers in avian segmented mesoderm.

    Science.gov (United States)

    Cann, G M; Lee, J W; Stockdale, F E

    1999-09-01

    Primary skeletal muscle fibers first form in the segmented portions of paraxial mesoderm called somites. Although the neural tube and notochord are recognized as crucial in patterning myogenic cell lineages during avian and mammalian somitic myogenesis, the source, identities, and actions of the signals governing this process remain controversial. It has been shown that signals emanating from the ventral neural tube and/or notochord alone or Shh alone serve to activate MyoD expression in somites. However, beyond a role in initiating MyoD expression, little is known about the effects of Shh on primary muscle fiber formation in somites of higher vertebrates. The studies reported here investigate how the ventral neural tube promotes myogenesis and compare the effects of the ventral neural tube with those of purified Shh protein on fiber formation in somites. We show that purified Shh protein mimics actions of the ventral neural tube on somites including initiation of muscle fiber formation, enhancement of numbers of primary muscle fibers, and particularly, the formation of primary fibers that express slow myosin. There is a marked increase in slow myosin expression in fibers in response to Shh as somites mature. The effects of ventral neural tube on fiber formation can be blocked by disrupting the Shh signaling pathway by increasing the activity of somitic cyclic AMP-dependent protein kinase A. Furthermore, it was demonstrated that apoptosis is a dominant fate of somite cells, but not somitic muscle fibers, when cultured in the absence of the neural tube, and that application of Shh protein to somites reduced apoptosis. The block to apoptosis by Shh is a manifestation of the maturity of the somite with a progressive increase in the block as somites are displaced rostrally from somite III forward. We conclude that purified Shh protein in mimicking the effects of the ventral neural tube on segmented mesoderm can exert pleiotropic effects during primary myogenesis

  13. Differences in elasticity of vinculin-deficient F9 cells measured by magnetometry and atomic force microscopy

    Science.gov (United States)

    Goldmann, W. H.; Galneder, R.; Ludwig, M.; Xu, W.; Adamson, E. D.; Wang, N.; Ezzell, R. M.; Ingber, D. E. (Principal Investigator)

    1998-01-01

    We have investigated a mouse F9 embryonic carcinoma cell line, in which both vinculin genes were inactivated by homologous recombination, that exhibits defective adhesion and spreading [Coll et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9161-9165]. Using a magnetometer and RGD-coated magnetic microbeads, we measured the local effect of loss and replacement of vinculin on mechanical force transfer across integrins. Vinculin-deficient F9Vin(-/-) cells showed a 21% difference in relative stiffness compared to wild-type cells. This was restored to near wild-type levels after transfection and constitutive expression of increasing amounts of vinculin into F9Vin(-/-) cells. In contrast, the transfection of vinculin constructs deficient in amino acids 1-288 (containing the talin- and alpha-actinin-binding site) or substituting tyrosine for phenylalanine (phosphorylation site, amino acid 822) in F9Vin(-/-) cells resulted in partial restoration of stiffness. Using atomic force microscopy to map the relative elasticity of entire F9 cells by 128 x 128 (n = 16,384) force scans, we observed a correlation with magnetometer measurements. These findings suggest that vinculin may promote cell adhesions and spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, thereby affecting the elastic properties of the cell.

  14. Imaging and measuring the rituximab-induced changes of mechanical properties in B-lymphoma cells using atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Li, Mi [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Liu, Lianqing, E-mail: lqliu@sia.cn [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Xi, Ning, E-mail: xin@egr.msu.edu [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI 48824 (United States); Wang, Yuechao; Dong, Zaili [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Tabata, Osamu [Department of Micro Engineering, Kyoto University, Kyoto 606-8501 (Japan); Xiao, Xiubin [Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071 (China); Zhang, Weijing, E-mail: zhangwj3072@163.com [Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071 (China)

    2011-01-14

    Research highlights: {yields} Single B-lymphoma living cells were imaged by AFM with the assistance of microfabricated pillars. {yields} The apoptosis of B-lymphoma cells triggered by rituximab without cross-linking was observed by AO/EB double fluorescent staining. {yields} The B-lymphoma cells became dramatically softer after adding rituximab. -- Abstract: The topography and mechanical properties of single B-lymphoma cells have been investigated by atomic force microscopy (AFM). With the assistance of microfabricated patterned pillars, the surface topography and ultrastructure of single living B-lymphoma cell were visualized by AFM. The apoptosis of B-lymphoma cells induced by rituximab alone was observed by acridine orange/ethidium bromide (AO/EB) double fluorescent staining. The rituximab-induced changes of mechanical properties in B-lymphoma cells were measured dynamically and the results showed that B-lymphoma cells became dramatically softer after incubation with rituximab. These results can improve our understanding of rituximab'effect and will facilitate the further investigation of the underlying mechanisms.

  15. White Blood Cell Segmentation by Circle Detection Using Electromagnetism-Like Optimization

    Directory of Open Access Journals (Sweden)

    Erik Cuevas

    2013-01-01

    Full Text Available Medical imaging is a relevant field of application of image processing algorithms. In particular, the analysis of white blood cell (WBC images has engaged researchers from fields of medicine and computer vision alike. Since WBCs can be approximated by a quasicircular form, a circular detector algorithm may be successfully applied. This paper presents an algorithm for the automatic detection of white blood cells embedded into complicated and cluttered smear images that considers the complete process as a circle detection problem. The approach is based on a nature-inspired technique called the electromagnetism-like optimization (EMO algorithm which is a heuristic method that follows electromagnetism principles for solving complex optimization problems. The proposed approach uses an objective function which measures the resemblance of a candidate circle to an actual WBC. Guided by the values of such objective function, the set of encoded candidate circles are evolved by using EMO, so that they can fit into the actual blood cells contained in the edge map of the image. Experimental results from blood cell images with a varying range of complexity are included to validate the efficiency of the proposed technique regarding detection, robustness, and stability.

  16. White Blood Cell Segmentation by Circle Detection Using Electromagnetism-Like Optimization

    Science.gov (United States)

    Oliva, Diego; Díaz, Margarita; Zaldivar, Daniel; Pérez-Cisneros, Marco; Pajares, Gonzalo

    2013-01-01

    Medical imaging is a relevant field of application of image processing algorithms. In particular, the analysis of white blood cell (WBC) images has engaged researchers from fields of medicine and computer vision alike. Since WBCs can be approximated by a quasicircular form, a circular detector algorithm may be successfully applied. This paper presents an algorithm for the automatic detection of white blood cells embedded into complicated and cluttered smear images that considers the complete process as a circle detection problem. The approach is based on a nature-inspired technique called the electromagnetism-like optimization (EMO) algorithm which is a heuristic method that follows electromagnetism principles for solving complex optimization problems. The proposed approach uses an objective function which measures the resemblance of a candidate circle to an actual WBC. Guided by the values of such objective function, the set of encoded candidate circles are evolved by using EMO, so that they can fit into the actual blood cells contained in the edge map of the image. Experimental results from blood cell images with a varying range of complexity are included to validate the efficiency of the proposed technique regarding detection, robustness, and stability. PMID:23476713

  17. Regulation of Schwann cell proliferation in cultured segments of the adult rat sciatic nerve

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Kanje, M

    1998-01-01

    of Schwann cells. Removal of extracellular Ca2+ by addition of EGTA to the culture medium suppressed [3H] thymidine incorporation as did the calmodulin inhibitor 48/80. The Ca2+ ionophore A23187 increased incorporation. Staurosporin, an inhibitor of protein kinase C (PKC), suppressed [3H] thymidine...

  18. Quantification of Mesenchymal Stem Cell (MSC delivery to a target site using in vivo confocal microscopy.

    Directory of Open Access Journals (Sweden)

    Luke J Mortensen

    Full Text Available The ability to deliver cells to appropriate target tissues is a prerequisite for successful cell-based therapy. To optimize cell therapy it is therefore necessary to develop a robust method of in vivo cell delivery quantification. Here we examine Mesenchymal Stem Cells (MSCs labeled with a series of 4 membrane dyes from which we select the optimal dye combination for pair-wise comparisons of delivery to inflamed tissue in the mouse ear using confocal fluorescence imaging. The use of an optimized dye pair for simultaneous tracking of two cell populations in the same animal enables quantification of a test population that is referenced to an internal control population, thereby eliminating intra-subject variations and variations in injected cell numbers. Consistent results were obtained even when the administered cell number varied by more than an order of magnitude, demonstrating an ability to neutralize one of the largest sources of in vivo experimental error and to greatly reduce the number of cells required to evaluate cell delivery. With this method, we are able to show a small but significant increase in the delivery of cytokine pre-treated MSCs (TNF-α & IFN-γ compared to control MSCs. Our results suggest future directions for screening cell strategies using our in vivo cell delivery assay, which may be useful to develop methods to maximize cell therapeutic potential.

  19. Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry.

    Science.gov (United States)

    Kurki, P; Ogata, K; Tan, E M

    1988-04-22

    Proliferating cell nuclear antigen (PCNA)/cyclin is an intranuclear polypeptide antigen that is found in both normal and transformed proliferating cells. We have recently described two mouse monoclonal antibodies reacting with PCNA. In this report we describe the application of these antibodies to the study of proliferating human cells by indirect immunofluorescence microscopy and by flow cytometry. A fixation/permeation procedure was developed in order to obtain satisfactory binding of monoclonal PCNA-specific antibodies to proliferating cells. This method involved fixation with 1% paraformaldehyde followed by methanol treatment. For the staining of cells in suspension with the IgM type monoclonal antibodies lysolecithin was added to the paraformaldehyde solution to achieve a better permeation by the antibody molecules. This procedure gave a good ratio of specific staining relative to the background staining. It also preserved the shape and normal architecture of the cells as judged by visual microscopic observation and by light scatter measurements using a flow cytometer. Furthermore, this fixation technique permits simultaneous labeling of DNA by propidium iodide and PCNA by monoclonal antibodies. PCNA was detected in various types of normal and transformed proliferating cells by indirect immunofluorescence. Quiescent peripheral blood mononuclear cells were PCNA-negative whereas a fraction of lectin-stimulated lymphocytes became PCNA-positive. Similarly, early passages of fetal skin fibroblasts were PCNA-positive but non-proliferating senescent fibroblasts of later passages were PCNA-negative. The association of PCNA-staining by monoclonal antibodies with cell proliferation was confirmed by flow cytometry. Simultaneous labeling of PCNA and DNA showed that the PCNA signal increased during the G1 phase of the cell cycle, reached its maximum in the S-phase, and declined during the G2/M phase. Using cell sorting we demonstrated that mitotic cells had a very low PCNA

  20. Comparison between power-law rheological parameters of living cells in frequency and time domains measured by atomic force microscopy

    Science.gov (United States)

    Takahashi, Ryosuke; Okajima, Takaharu

    2016-08-01

    We investigated how stress relaxation mapping is quantified compared with the force modulation mapping of confluent epithelial cells using atomic force microscopy (AFM). Using a multi-frequency AFM technique, we estimated the power-law rheological behaviors of cells simultaneously in time and frequency domains. When the power-law exponent α was low ( 0.1), α in the time domain was underestimated relative to that in the frequency domain, and the difference increased with α, whereas the cell modulus was overestimated in the time domain. These results indicate that power-law rheological parameters estimated by stress relaxation are sensitive to lag time during initial indentation, which is inevitable in time-domain AFM experiments.

  1. Mapping power-law rheology of living cells using multi-frequency force modulation atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Ryosuke; Okajima, Takaharu, E-mail: okajima@ist.hokudai.ac.jp [Graduate School of Information Science and Technology, Hokkaido University, Kita-ku N14 W9, Sapporo 060-0814 (Japan)

    2015-10-26

    We present multi-frequency force modulation atomic force microscopy (AFM) for mapping the complex shear modulus G* of living cells as a function of frequency over the range of 50–500 Hz in the same measurement time as the single-frequency force modulation measurement. The AFM technique enables us to reconstruct image maps of rheological parameters, which exhibit a frequency-dependent power-law behavior with respect to G{sup *}. These quantitative rheological measurements reveal a large spatial variation in G* in this frequency range for single cells. Moreover, we find that the reconstructed images of the power-law rheological parameters are much different from those obtained in force-curve or single-frequency force modulation measurements. This indicates that the former provide information about intracellular mechanical structures of the cells that are usually not resolved with the conventional force measurement methods.

  2. Mapping power-law rheology of living cells using multi-frequency force modulation atomic force microscopy

    International Nuclear Information System (INIS)

    We present multi-frequency force modulation atomic force microscopy (AFM) for mapping the complex shear modulus G* of living cells as a function of frequency over the range of 50–500 Hz in the same measurement time as the single-frequency force modulation measurement. The AFM technique enables us to reconstruct image maps of rheological parameters, which exhibit a frequency-dependent power-law behavior with respect to G*. These quantitative rheological measurements reveal a large spatial variation in G* in this frequency range for single cells. Moreover, we find that the reconstructed images of the power-law rheological parameters are much different from those obtained in force-curve or single-frequency force modulation measurements. This indicates that the former provide information about intracellular mechanical structures of the cells that are usually not resolved with the conventional force measurement methods

  3. MARKET SEGMENTATION

    OpenAIRE

    Munaga Ramakrishna Mohan Rao

    2015-01-01

    Market segmentation is a marketing strategy that involves dividing a broad target market into subsets of consumers, businesses, or countries who have common needs and priorities, and then designing and implementing strategies to target them. Market segmentation strategies may be used to identify the target customers, and provide supporting data for positioning to achieve a marketing plan objective. Businesses may develop product differentiation strategies, or an undifferentiated approach, inv...

  4. Fingerprint Segmentation

    OpenAIRE

    Jomaa, Diala

    2009-01-01

    In this thesis, a new algorithm has been proposed to segment the foreground of the fingerprint from the image under consideration. The algorithm uses three features, mean, variance and coherence. Based on these features, a rule system is built to help the algorithm to efficiently segment the image. In addition, the proposed algorithm combine split and merge with modified Otsu. Both enhancements techniques such as Gaussian filter and histogram equalization are applied to enhance and improve th...

  5. Long segmental hyperplasia of interstitial cells of Cajal with giant diverticulum formation

    OpenAIRE

    Xue, Liyan; Qiu, Tian; Song, Ying; Shan, Ling; Liu, Xiuyun; Guo, Lei; Ying, Jianming; Zou, Shuangmei; Shi, Susheng; Polydorides, Alexandros D.; Zhao, Xinming; Lu, Ning; Lin, Dongmei

    2013-01-01

    Sporadic gastrointestinal stromal tumors (GISTs) usually form a well-circumscribed mass. In contrast, diffuse interstitial cell of Cajal (ICC) hyperplasia along the Auerbach plexus without a discrete mass may occur in patients with germline mutations in the NF1, c-KIT or PDGFRA genes. However, sporadic, diffuse ICC hyperplasia without c-KIT or PDGFRA mutations has not been reported. We describe herein one such case, forming a giant diverticulum. A 63-year-old woman with no features of Neurofi...

  6. Multidendritic sensory neurons in the adult Drosophila abdomen: origins, dendritic morphology, and segment- and age-dependent programmed cell death

    Directory of Open Access Journals (Sweden)

    Sugimura Kaoru

    2009-10-01

    Full Text Available Abstract Background For the establishment of functional neural circuits that support a wide range of animal behaviors, initial circuits formed in early development have to be reorganized. One way to achieve this is local remodeling of the circuitry hardwiring. To genetically investigate the underlying mechanisms of this remodeling, one model system employs a major group of Drosophila multidendritic sensory neurons - the dendritic arborization (da neurons - which exhibit dramatic dendritic pruning and subsequent growth during metamorphosis. The 15 da neurons are identified in each larval abdominal hemisegment and are classified into four categories - classes I to IV - in order of increasing size of their receptive fields and/or arbor complexity at the mature larval stage. Our knowledge regarding the anatomy and developmental basis of adult da neurons is still fragmentary. Results We identified multidendritic neurons in the adult Drosophila abdomen, visualized the dendritic arbors of the individual neurons, and traced the origins of those cells back to the larval stage. There were six da neurons in abdominal hemisegment 3 or 4 (A3/4 of the pharate adult and the adult just after eclosion, five of which were persistent larval da neurons. We quantitatively analyzed dendritic arbors of three of the six adult neurons and examined expression in the pharate adult of key transcription factors that result in the larval class-selective dendritic morphologies. The 'baseline design' of A3/4 in the adult was further modified in a segment-dependent and age-dependent manner. One of our notable findings is that a larval class I neuron, ddaE, completed dendritic remodeling in A2 to A4 and then underwent caspase-dependent cell death within 1 week after eclosion, while homologous neurons in A5 and in more posterior segments degenerated at pupal stages. Another finding is that the dendritic arbor of a class IV neuron, v'ada, was immediately reshaped during post

  7. Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Peddie, Christopher J.; Blight, Ken; Wilson, Emma [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Melia, Charlotte [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Department of Molecular Cell Biology, Leiden University Medical Centre, 2300 RC Leiden (Netherlands); Marrison, Jo [Department of Biology, The University of York, Heslington, York (United Kingdom); Carzaniga, Raffaella [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Domart, Marie-Charlotte [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); O' Toole, Peter [Department of Biology, The University of York, Heslington, York (United Kingdom); Larijani, Banafshe [Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, Unidad de Biofísica (CSIC-UPV/EHU),Sarriena s/n, 48940 Leioa (Spain); IKERBASQUE, Basque Foundation for Science, Bilbao (Spain); Collinson, Lucy M. [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom)

    2014-08-01

    Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure. - Highlights: • GFP and mCherry fluorescence are preserved in heavy-metal stained mammalian cells embedded in resin • Fluorophores are stable and intensity is sufficient for detection in ultrathin sections • Overlay of separate LM and EM images from the same ultrathin section improves CLEM protein localisation precision • GFP is stable and active in the vacuum of an integrated light and scanning EM • Integrated light and electron microscopy shows new subcellular locations of the lipid diacylglycerol.

  8. In situ liquid-cell electron microscopy of silver-palladium galvanic replacement reactions on silver nanoparticles

    Science.gov (United States)

    Sutter, E.; Jungjohann, K.; Bliznakov, S.; Courty, A.; Maisonhaute, E.; Tenney, S.; Sutter, P.

    2014-09-01

    Galvanic replacement reactions provide an elegant way of transforming solid nanoparticles into complex hollow morphologies. Conventionally, galvanic replacement is studied by stopping the reaction at different stages and characterizing the products ex situ. In situ observations by liquid-cell electron microscopy can provide insight into mechanisms, rates and possible modifications of galvanic replacement reactions in the native solution environment. Here we use liquid-cell electron microscopy to investigate galvanic replacement reactions between silver nanoparticle templates and aqueous palladium salt solutions. Our in situ observations follow the transformation of the silver nanoparticles into hollow silver-palladium nanostructures. While the silver-palladium nanocages have morphologies similar to those obtained in ex situ control experiments the reaction rates are much higher, indicating that the electron beam strongly affects the galvanic-type process in the liquid-cell. By using scavengers added to the aqueous solution we identify the role of radicals generated via radiolysis by high-energy electrons in modifying galvanic reactions.

  9. Reflectance confocal microscopy of red blood cells: simulation and experiment (Conference Presentation)

    Science.gov (United States)

    Zeidan, Adel; Yeheskely-Hayon, Daniella; Minai, Limor; Yelin, Dvir

    2016-03-01

    The properties of red blood cells are a remarkable indicator of the body's physiological condition; their density could indicate anemia or polycythemia, their absorption spectrum correlates with blood oxygenation, and their morphology is highly sensitive to various pathologic states including iron deficiency, ovalocytosis, and sickle cell disease. Therefore, measuring the morphology of red blood cells is important for clinical diagnosis, providing valuable indications on a patient's health. In this work, we simulated the appearance of normal red blood cells under a reflectance confocal microscope and discovered unique relations between the cells' morphological parameters and the resulting characteristic interference patterns. The simulation results showed good agreement with in vitro reflectance confocal images of red blood cells, acquired using spectrally encoded flow cytometry (SEFC) that imaged the cells during linear flow and without artificial staining. By matching the simulated patterns to the SEFC images of the cells, the cells' three-dimensional shapes were evaluated and their volumes were calculated. Potential applications include measurement of the mean corpuscular volume, cell morphological abnormalities, cell stiffness under mechanical stimuli, and the detection of various hematological diseases.

  10. Combined reflectance confocal microscopy-optical coherence tomography for delineation of basal cell carcinoma margins: an ex vivo study

    Science.gov (United States)

    Iftimia, Nicusor; Peterson, Gary; Chang, Ernest W.; Maguluri, Gopi; Fox, William; Rajadhyaksha, Milind

    2016-01-01

    We present a combined reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) approach, integrated within a single optical layout, for diagnosis of basal cell carcinomas (BCCs) and delineation of margins. While RCM imaging detects BCC presence (diagnoses) and its lateral spreading (margins) with measured resolution of ˜1 μm, OCT imaging delineates BCC depth spreading (margins) with resolution of ˜7 μm. When delineating margins in 20 specimens of superficial and nodular BCCs, depth could be reliably determined down to ˜600 μm, and agreement with histology was within about ±50 μm.

  11. Confocal reflectance quantitative phase microscopy system for cell biology studies (Conference Presentation)

    Science.gov (United States)

    Singh, Vijay Raj; So, Peter T. C.

    2016-03-01

    Quantitative phase microscopy (QPM), used to measure the refractive index, provides the optical path delay measurement at each point of the specimen under study and becomes an active field in biological science. In this work we present development of confocal reflection phase microscopy system to provide depth resolved quantitative phase information for investigation of intracellular structures and other biological specimen. The system hardware development is mainly divided into two major parts. First, creates a pinhole array for parallel confocal imaging of specimen at multiple locations simultaneously. Here a digital micro mirror device (DMD) is used to generate pinhole array by turning on a subset micro-mirrors arranged on a grid. Second is the detection of phase information of confocal imaging foci by using a common path interferometer. With this novel approach, it is possible to measure the nuclei membrane fluctuations and distinguish them from the plasma membrane fluctuations. Further, depth resolved quantitative phase can be correlated to the intracellular contents and 3D map of refractive index measurements.

  12. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells.

    Science.gov (United States)

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. PMID:27537197

  13. Fluorescence lifetime imaging microscopy (FLIM) to quantify protein-protein interactions inside cells.

    Science.gov (United States)

    Duncan, R R

    2006-11-01

    Recent developments in cellular imaging spectroscopy now permit the minimally invasive study of protein dynamics inside living cells. These advances are of interest to cell biologists, as proteins rarely act in isolation, but rather in concert with others in forming cellular machinery. Until recently, all protein interactions had to be determined in vitro using biochemical approaches: this biochemical legacy has provided cell biologists with the basis to test defined protein-protein interactions not only inside cells, but now also with high spatial resolution. These techniques can detect and quantify protein behaviours down to the single-molecule level, all inside living cells. More recent developments in TCSPC (time-correlated single-photon counting) imaging are now also driving towards being able to determine protein interaction rates with similar spatial resolution, and together, these experimental advances allow investigators to perform biochemical experiments inside living cells. PMID:17052173

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

    Science.gov (United States)

    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.

  15. In Situ Confocal Raman Microscopy of Hydrated Early Stages of Bacterial Biofilm Formation on Various Surfaces in a Flow Cell.

    Science.gov (United States)

    Smith-Palmer, Truis; Lin, Sicheng; Oguejiofor, Ikenna; Leng, Tianyang; Pustam, Amanda; Yang, Jin; Graham, Lori L; Wyeth, Russell C; Bishop, Cory D; DeMont, M Edwin; Pink, David

    2016-02-01

    Bacterial biofilms are precursors to biofouling by other microorganisms. Understanding their initiation may allow us to design better ways to inhibit them, and thus to inhibit subsequent biofouling. In this study, the ability of confocal Raman microscopy to follow the initiation of biofouling by a marine bacterium, Pseudoalteromonas sp. NCIMB 2021 (NCIMB 2021), in a flow cell, using optical and confocal Raman microscopy, was investigated. The base of the flow cell comprised a cover glass. The cell was inoculated and the bacteria attached to, and grew on, the cover glass. Bright field images and Raman spectra were collected directly from the hydrated biofilms over several days. Although macroscopically the laser had no effect on the biofilm, within the first 24 h cells migrated away from the position of the laser beam. In the absence of flow, a buildup of extracellular substances occurred at the base of the biofilm. When different coatings were applied to cover glasses before they were assembled into the flow cells, the growth rate, structure, and composition of the resulting biofilm was affected. In particular, the ratio of Resonance Raman peaks from cytochrome c (CC) in the extracellular polymeric substances, to the Raman phenylalanine (Phe) peak from protein in the bacteria, depended on both the nature of the surface and the age of the biofilm. The ratios were highest for 24 h colonies on a hydrophobic surface. Absorption of a surfactant with an ethyleneoxy chain into the hydrophobic coating created a surface similar to that given with a simple PEG coating, where bacteria grew in colonies away from the surface rather than along the surface, and CC:Phe ratios were initially low but increased at least fivefold in the first 48 h.

  16. Effects of cholesterol on plasma membrane lipid order in MCF-7 cells by two-photon microscopy

    Science.gov (United States)

    Zeng, Yixiu; Chen, Jianling; Yang, Hongqin; Wang, Yuhua; Li, Hui; Xie, Shusen

    2014-09-01

    Lipid rafts are cholesterol- and glycosphingolipids- enriched microdomains on plasma membrane surface of mammal cells, involved in a variety of cellular processes. Depleting cholesterol from the plasma membrane by drugs influences the trafficking of lipid raft markers. Optical imaging techniques are powerful tools to study lipid rafts in live cells due to its noninvasive feature. In this study, breast cancer cells MCF-7 were treated with different concentrations of MβCD to deplete cholesterol and an environmentally sensitive fluorescence probe, Laurdan was loaded to image lipid order by two-photon microscopy. The generalized polarization (GP) values were calculated to distinguish the lipid order and disorder phase. GP images and GP distributions of native and cholesterol-depleted MCF-7 cells were obtained. Our results suggest that even at low concentration (0.5 mM) of MβCD, the morphology of the MCF-7 cells changes. Small high GP areas (lipid order phase) decrease more rapidly than low GP areas (lipid disorder phase), indicating that lipid raft structure was altered more severely than nonraft domains. The data demonstrates that cholesterol dramatically affect raft coverage and plasma membrane fluidity in living cells.

  17. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles

    Science.gov (United States)

    Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

    2016-01-01

    Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs. PMID:27022410

  18. Analyzing the effect of absorption and refractive index on image formation in high numerical aperture transmission microscopy of single cells

    Science.gov (United States)

    Coe, Ryan L.; Seibel, Eric J.

    2013-02-01

    Transmission bright-field microscopy is the clinical mainstay for disease diagnosis where image contrast is provided by absorptive and refractive index differences between tissue and the surrounding media. Different microscopy techniques often assume one of the two contrast mechanisms is negligible as a means to better understand the tissue scattering processes. This particular work provides better understanding of the role of refractive index and absorption within Optical Projection Tomographic Microscopy (OPTM) through the development of a generalized computational model based upon the Finite-Difference Time-Domain method. The model mimics OPTM by simulating axial scanning of the objective focal plane through the cell to produce projection images. These projection images, acquired from circumferential positions around the cell, are reconstructed into isometric three-dimensional images using the filtered backprojection normally employed in Computed Tomography (CT). The model provides a platform to analyze all aspects of bright-field microscopes, such as the degree of refractive index matching and the numerical aperture, which can be varied from air-immersion to high NA oil-immersion. In this preliminary work, the model is used to understand the effects of absorption and refraction on image formation using micro-shells and idealized nuclei. Analysis of absorption and refractive index separately provides the opportunity to better assess their role together as a complex refractive index for improved interpretation of bright-field scattering, essential for OPTM image reconstruction. This simulation, as well as ones in the future looking at other effects, will be used to optimize OPTM imaging parameters and triage efforts to further improve the overall system design.

  19. A self-contained, programmable microfluidic cell culture system with real-time microscopy access

    DEFF Research Database (Denmark)

    Skafte-Pedersen, Peder; Hemmingsen, Mette; Sabourin, David;

    2011-01-01

    Utilizing microfluidics is a promising way for increasing the throughput and automation of cell biology research. We present a complete self-contained system for automated cell culture and experiments with real-time optical read-out. The system offers a high degree of user-friendliness, stability...

  20. A New cell design for Potentiostatically Controlled In Situ Atomic Force Microscopy

    DEFF Research Database (Denmark)

    Madsen, Lars Lithen; Friis, Esben P.; Andersen, Jens Enevold Thaulov;

    1998-01-01

    We describe the design and construction of a new type of AFM cell for in situ imaging under potentiostatic control. The cell is specifically designed for a Rasterscope 4000TM AFM instrument with no need for instrumental modification, but can easily be adapted to other commercial instruments...

  1. Methods for studying biofilm formation: flow cells and confocal laser scanning microscopy

    DEFF Research Database (Denmark)

    Tolker-Nielsen, Tim; Sternberg, Claus

    2014-01-01

    In this chapter methods for growing and analyzing biofilms under hydrodynamic conditions in flow cells are described. Use of flow cells allows for direct microscopic investigation of biofilm formation. The flow in these chambers is essentially laminar, which means that the biofilms can be grown u...

  2. Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

    NARCIS (Netherlands)

    Jong, Imke G. de; Beilharz, Katrin; Kuipers, Oscar P.; Veening, Jan-Willem

    2011-01-01

    During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously. However, ma

  3. Deformable Graph Model for Tracking Epithelial Cell Sheets in Fluorescence Microscopy.

    Science.gov (United States)

    Zou, Roger S; Tomasi, Carlo

    2016-07-01

    We propose a novel method for tracking cells that are connected through a visible network of membrane junctions. Tissues of this form are common in epithelial cell sheets and resemble planar graphs where each face corresponds to a cell. We leverage this structure and develop a method to track the entire tissue as a deformable graph. This coupled model in which vertices inform the optimal placement of edges and vice versa captures global relationships between tissue components and leads to accurate and robust cell tracking. We compare the performance of our method with that of four reference tracking algorithms on four data sets that present unique tracking challenges. Our method exhibits consistently superior performance in tracking all cells accurately over all image frames, and is robust over a wide range of image intensity and cell shape profiles. This may be an important tool for characterizing tissues of this type especially in the field of developmental biology where automated cell analysis can help elucidate the mechanisms behind controlled cell-shape changes. PMID:26829784

  4. Measurements of light and pH dependence of single-cell photosynthesis by fluorescence microscopy

    NARCIS (Netherlands)

    Snel, J.F.H.; Dassen, J.H.A.

    2000-01-01

    Measurement of algal photosynthetic performance with conventional methods requires thousands of cells obtained by isolation and subsequent cultivation. This is a time-consuming process for many species. We describe a new method to study photosynthetic performance of single algal cells under various

  5. Fluorescence microscopy imaging of cells with a plasmonic dish integrally molded

    Science.gov (United States)

    Tawa, Keiko; Sasakawa, Chisato; Fujita, Tsuyoshi; Kiyosue, Kazuyuki; Hosokawa, Chie; Nishii, Junji; Oike, Makoto; Kakinuma, Norihiro

    2016-03-01

    A plastic dish with a wavelength-scale periodic structure at a bottom panel was integrally molded and coated with thin metal films. The integrally molded dish called plasmonic dish was applied to bioimaging under a fluorescence microscope. On the plasmonic substrate, the enhanced electric field based on a grating-coupled surface plasmon resonance (GC-SPR) can provide an enhanced fluorescence. In this study, two kinds of cells, human embryonic kidney (HEK) cells and neuronal cells, were observed in our plasmonic dish. Fluorescence images of HEK cells were above 10 times brighter than those obtained on a conventional glass-bottomed dish. Neuronal cells were successfully cultured for 10 d on the plasmonic dish integrally molded, and in fluorescence images with transmitted light, a higher contrast was obtained than in epifluorescence images. The plasmonic dish integrally molded, as well as that fabricated by the UV nanoimprint method, was also found to be useful for sensitive bioimaging.

  6. CELL TRACKING USING PARTICLE FILTERS WITH IMPLICIT CONVEX SHAPE MODEL IN 4D CONFOCAL MICROSCOPY IMAGES

    Science.gov (United States)

    Ramesh, Nisha; Tasdizen, Tolga

    2016-01-01

    Bayesian frameworks are commonly used in tracking algorithms. An important example is the particle filter, where a stochastic motion model describes the evolution of the state, and the observation model relates the noisy measurements to the state. Particle filters have been used to track the lineage of cells. Propagating the shape model of the cell through the particle filter is beneficial for tracking. We approximate arbitrary shapes of cells with a novel implicit convex function. The importance sampling step of the particle filter is defined using the cost associated with fitting our implicit convex shape model to the observations. Our technique is capable of tracking the lineage of cells for nonmitotic stages. We validate our algorithm by tracking the lineage of retinal and lens cells in zebrafish embryos. PMID:27403085

  7. Scalable, incremental learning with MapReduce parallelization for cell detection in high-resolution 3D microscopy data

    KAUST Repository

    Sung, Chul

    2013-08-01

    Accurate estimation of neuronal count and distribution is central to the understanding of the organization and layout of cortical maps in the brain, and changes in the cell population induced by brain disorders. High-throughput 3D microscopy techniques such as Knife-Edge Scanning Microscopy (KESM) are enabling whole-brain survey of neuronal distributions. Data from such techniques pose serious challenges to quantitative analysis due to the massive, growing, and sparsely labeled nature of the data. In this paper, we present a scalable, incremental learning algorithm for cell body detection that can address these issues. Our algorithm is computationally efficient (linear mapping, non-iterative) and does not require retraining (unlike gradient-based approaches) or retention of old raw data (unlike instance-based learning). We tested our algorithm on our rat brain Nissl data set, showing superior performance compared to an artificial neural network-based benchmark, and also demonstrated robust performance in a scenario where the data set is rapidly growing in size. Our algorithm is also highly parallelizable due to its incremental nature, and we demonstrated this empirically using a MapReduce-based implementation of the algorithm. We expect our scalable, incremental learning approach to be widely applicable to medical imaging domains where there is a constant flux of new data. © 2013 IEEE.

  8. Cell and brain tissue imaging of the flavonoid fisetin using label-free two-photon microscopy.

    Science.gov (United States)

    Krasieva, Tatiana B; Ehren, Jennifer; O'Sullivan, Thomas; Tromberg, Bruce J; Maher, Pamela

    2015-10-01

    Over the last few years, we have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity against microglia and astrocytes and inhibits the activity of lipoxygenases, thereby reducing the production of pro-inflammatory eicosanoids and their by-products. However, key questions about its targets and brain penetration remain. In this study, we used label-free two-photon microscopy of intrinsic fisetin fluorescence to examine the localization of fisetin in living nerve cells and the brains of living mice. In cells, fisetin but not structurally related flavonols with different numbers of hydroxyl groups, localized to the nucleoli suggesting that key targets of fisetin may reside in this organelle. In the mouse brain, following intraperitoneal injection and oral administration, fisetin rapidly distributed to the blood vessels of the brain followed by a slower dispersion into the brain parenchyma. Thus, these results provide further support for the effects of fisetin on brain function. In addition, they suggest that label-free two-photon microscopy may prove useful for studying the intracellular and tissue distribution of other intrinsically-fluorescent flavonoids.

  9. Simultaneous multi-parameter observation of Harring-tonine-treating HL-60 cells with both two-photon and confo-cal laser scanning microscopy

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluores-cent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simulta-neously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.

  10. 3D exploration of light scattering from live cells in the presence of gold nanomarkers using holographic microscopy

    CERN Document Server

    Joud, Fadwa; Bun, P; Verpillat, Frédéric; Suck, Sarah Y; Tessier, G; Atlan, Michael; Desbiolles, Pierre; Coppey-Moisan, Maite; Abboud, Marie; Gross, Michel

    2011-01-01

    In this paper, we explore the 3D structure of light scattering from dark-field illuminated live 3T3 cells in the presence of 40 nm gold nanomarkers. For this purpose, we use a high resolution holographic microscope combining the off-axis heterodyne geometry and the phase-shifting acquisition of the digital holograms. A comparative study of the 3D reconstructions of the scattered fields allows us to locate the gold markers which yield, contrarily to the cell structures, well defined bright scattering patterns that are not angularly titled and clearly located along the optical axis (z). This characterization is an unambiguous signature of the presence of gold biological nanomarkers, and validates the capability of digital holographic microscopy to discriminate them from background signals in live ce

  11. Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells.

    Science.gov (United States)

    Romero-Brey, Inés; Bartenschlager, Ralf

    2015-12-01

    As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications.

  12. Mechanical properties of single cells by high-frequency time-resolved acoustic microscopy.

    Science.gov (United States)

    Weiss, Eike C; Anastasiadis, Pavlos; Pilarczyk, Götz; Lemor, Robert M; Zinin, Pavel V

    2007-11-01

    In this paper, we describe a new, high-frequency, time-resolved scanning acoustic microscope developed for studying dynamical processes in biological cells. The new acoustic microscope operates in a time-resolved mode. The center frequency is 0.86 GHz, and the pulse duration is 5 ns. With such a short pulse, layers thicker than 3 microm can be resolved. For a cell thicker than 3 microm, the front echo and the echo from the substrate can be distinguished in the signal. Positions of the first and second pulses are used to determine the local impedance of the cell modeled as a thin liquid layer that has spatial variations in its elastic properties. The low signal-to-noise ratio in the acoustical images is increased for image generation by averaging the detected radio frequency signal over 10 measurements at each scanning point. In conducting quantitative measurements of the acoustic parameters of cells, the signal can be averaged over 2000 measurements. This approach enables us to measure acoustical properties of a single HeLa cell in vivo and to derive elastic parameters of subcellular structures. The value of the sound velocity inside the cell (1534.5 +/- 33.6 m/s) appears to be only slightly higher than that of the cell medium (1501 m/s). PMID:18051160

  13. The measurement of red blood cell volume change induced by Ca2+ based on full field quantitative phase microscopy

    Science.gov (United States)

    Lee, Seungrag; Lee, Ji Yong; Yang, Wenzhong; Kim, Dug Young

    2009-02-01

    We present the measurement of red blood cell (RBC) volume change induced by Ca2+ for a live cell imaging with full field quantitative phase microscopy (FFQPM). FFQPM is based on the Mach-Zehnder interferometer combined with an inverted microscopy system. We present the effective method to obtain a clear image and an accurate volume of the cells. An edge detection technique is used to accurately resolve the boundary between the cell line and the suspension medium. The measurement of the polystyrene bead diameter and volume has been demonstrated the validity of our proposed method. The measured phase profile can be easily converted into thickness profile. The measured polystyrene bead volume and the simulated result are about 14.74 μm3 and 14.14 μm3, respectively. The experimental results of our proposed method agree well with the simulated results within less than 4 %. We have also measured the volume variation of a single RBC on a millisecond time scale. Its mean volume is 54.02 μm3 and its standard deviation is 0.52 μm3. With the proposed system, the shape and volume changes of RBC induced by the increased intracellular Ca2+ are measured after adding ionophore A23187. A discocyte RBC is deformed to a spherocyte due to the increased intracellular Ca2+ in RBC. The volume of the spherocyte is 47.88 μm3 and its standard deviation is 0.19 μm3. We have demonstrated that the volume measurement technique is easy, accurate, and robust method with high volume sensitivity (<0.0000452 μm3) and this provides the ability to study a biological phenomenon in Hematology.

  14. Scanning transmission and computer-aided volumic electron microscopy: 3-D modeling of entire cells by electronic imaging

    Science.gov (United States)

    Bron, Christophe; Gremillet, Philip; Launay, D.; Jourlin, Michel; Gautschi, H. P.; Baechi, Thomas; Schuepbach, Joerg

    1990-05-01

    The digital processing of electron microscopic images from serial sections containing laser-induced topographical references allows a 3-D reconstruction at a depth resolution of 30 to 40 nm of entire cells by the use of image analysis methods, as already demonstrated for Transmission Electron Microscopy (TEM) coupled with a video camera. We decided to use a Scanning Transmission Electron Microscope (STEM) to get higher contrast and better resolution at medium magnification. The scanning of our specimens at video frequencies is an attractive and easy way to link a STEM with an image processing system but the hysteresis of the electronic spools responsible for the magnetic deviation of the scanning electron beam induces deformations of images which have to be modelized and corrected before registration. Computer algorithms developed for image analysis and treatment correct the artifacts caused by the use of STEM and by serial sectioning to automatically reconstruct the third dimension of the cells. They permit the normalization of the images through logarithmic processing of the original grey level infonnation. The automatic extraction of cell limits allows to link the image analysis and treatments with image synthesis methods by minimal human intervention. The surface representation and the registered images provide an ultrastructural data base from which quantitative 3-D morphological parameters, as well as otherwise impossible visualizations, can be computed. This 3-D image processing named C.A.V.U.M. for Computer Aided Volumic Ultra-Microscopy offers a new tool for the documentation and analysis of cell ultrastructure and for 3-D morphometric studies at EM magnifications. Further, a virtual observer can be computed in such a way as to simulate a visit of the reconstructed object.

  15. Characterizations of individual mouse red blood cells parasitized by Babesia microti using 3-D holographic microscopy

    CERN Document Server

    Park, HyunJoo; Kim, Kyoohyun; Cho, Shin-Hyeong; Lee, Won-Ja; Kim, Youngchan; Lee, SangEun; Park, YongKeun

    2015-01-01

    Babesia microti causes emergency human babesiosis. However, little is known about the alterations in B. microti invaded red blood cells (Bm-RBCs) at the individual cell level. Through quantitative phase imaging techniques based on laser interferometry, we present the simultaneous measurements of structural, chemical, and mechanical modifications in individual mouse Bm-RBCs. 3-D refractive index maps of individual RBCs and in situ parasite vacuoles are imaged, from which total contents and concentration of dry mass are also precisely quantified. In addition, we examine the dynamic membrane fluctuation of Bm-RBCs, which provide information on cell membrane deformability.

  16. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    International Nuclear Information System (INIS)

    The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals

  17. Vibrational imaging of glucose uptake activity in live cells and tissues by stimulated Raman scattering microscopy (Conference Presentation)

    Science.gov (United States)

    Hu, Fanghao; Chen, Zhixing; Zhang, Luyuan; Shen, Yihui; Wei, Lu; Min, Wei

    2016-03-01

    Glucose is consumed as an energy source by virtually all living organisms, from bacteria to humans. Its uptake activity closely reflects the cellular metabolic status in various pathophysiological transformations, such as diabetes and cancer. Extensive efforts such as positron emission tomography, magnetic resonance imaging and fluorescence microscopy have been made to specifically image glucose uptake activity but all with technical limitations. Here, we report a new platform to visualize glucose uptake activity in live cells and tissues with subcellular resolution and minimal perturbation. A novel glucose analogue with a small alkyne tag (carbon-carbon triple bond) is developed to mimic natural glucose for cellular uptake, which can be imaged with high sensitivity and specificity by targeting the strong and characteristic alkyne vibration on stimulated Raman scattering (SRS) microscope to generate a quantitative three dimensional concentration map. Cancer cells with differing metabolic characteristics can be distinguished. Heterogeneous uptake patterns are observed in tumor xenograft tissues, neuronal culture and mouse brain tissues with clear cell-cell variations. Therefore, by offering the distinct advantage of optical resolution but without the undesirable influence of bulky fluorophores, our method of coupling SRS with alkyne labeled glucose will be an attractive tool to study energy demands of living systems at the single cell level.

  18. Continuous live cell imaging of cellulose attachment by microbes under anaerobic and thermophilic conditions using confocal microscopy

    Institute of Scientific and Technical Information of China (English)

    Zhi-Wu Wang; Seung-Hwan Lee; James G.Elkins; Yongchao Li; Scott Hamilton-Brehm; Jennifer L.Morrell-Falvey

    2013-01-01

    Live cell imaging methods provide important insights into the dynamics of cellular processes that cannot be derived easily from population-averaged datasets.In the bioenergy field,much research is focused on fermentation of cellulosic biomass by thermophilic microbes to produce biofuels; however,little effort is dedicated to the development of imaging tools to monitor this dynamic biological process.This is,in part,due to the experimental challenges of imaging ceils under both anaerobic and thermophilic conditions.Here an imaging system is described that integrates confocal microscopy,a flow cell device,and a lipophilic dye to visualize cells.Solutions to technical obstacles regarding suitable fluorescent markers,photodamage during imaging,and maintenance of environmental conditions during imaging are presented.This system was utilized to observe cellulose colonization by Clostridium thermocellum under anaerobic conditions at 60℃.This method enables live cell imaging of bacterial growth under anaerobic and thermophilic conditions and should be widely applicable to visualizing different cell types or processes in real time.

  19. In situ observation of C60(C(COOH)2)2 interacting with living cells using fluorescence microscopy

    Institute of Scientific and Technical Information of China (English)

    YE Chang; ZHAO Yuliang; CHAI Zhifang; FANG Xiaohong; HAN Dong; CHEN Long; WANG Chen; WEI Taotao; CHEN Chunying; CHEN Zhen; MENG Huan; XING Li; JIANG Yaxin; YUAN Hui; XING Gengmei; ZHAO Feng

    2006-01-01

    The interactions between nanoparticles and living cells were investigated by an imaging technique of fluorescence microscopy. For this purpose, the C60 derivative C60(C(COOH)2)2, a therapeutic agent for degeneration diseases of central nervous system, was synthesized, purified and characterized. Its interaction with the living cell and penetration of the cellular membrane were in situ studied using the real time imaging technique, and its potential cytotoxicity was also examined by flow cytometry. The results indicate that C60(C(COOH)2)2 can easily enter cells, and is mainly located in cytoplasm by fluorescein labeling. Furthermore, C60(C(COOH)2)2 can carry the molecule that cannot cross cellular membranes into cells, because fluorescein compound itself cannot enter the cell or adhere to membrane. At concentrations ranging from 1×10?2 to 1×102 mg/L, C60(C(COOH)2)2 does not show any detectable cytotoxicity.

  20. Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells

    Science.gov (United States)

    Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

    2016-01-01

    Background Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. Results We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50–60 nm on a time scale of 2.3 s. Conclusion Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level.

  1. Cell volume increase in murine MC3T3-E1 pre-osteoblasts attaching onto biocompatible Tantalum observed by magnetic AC mode Atomic Force Microscopy

    Directory of Open Access Journals (Sweden)

    Klembt Andersen L.

    2005-12-01

    Full Text Available Magnetic AC mode (MACmode atomic force microscopy (AFM was used to study murine (mouse MC3T3-E1 preosteoblastic cells attached to biocompatible tantalum substrates. Cell volumes of attached cells derived from AFM images were compared to volumes of detached cells in suspension measured by the Coulter sizing technique. An increase of similar 50 % in cell volume was observed when the cells attached to planar tantalum substrates and developed a flattened structure including lamellipodia. We address thoroughly the issues general to the AFM determination of absolute cell volumes, and compare our magnetic AC mode AFM measurements to hitherto reported cell volume determinations by contact mode AFM.

  2. Incubator embedded cell culture imaging system (EmSight) based on Fourier ptychographic microscopy

    OpenAIRE

    Kim, Jinho; Henley, Beverley M.; Kim, Charlene H.; Lester, Henry A.; Yang, Changhuei

    2016-01-01

    Multi-day tracking of cells in culture systems can provide valuable information in bioscience experiments. We report the development of a cell culture imaging system, named EmSight, which incorporates multiple compact Fourier ptychographic microscopes with a standard multiwell imaging plate. The system is housed in an incubator and presently incorporates six microscopes. By using the same low magnification objective lenses as the objective and the tube lens, the EmSight is configured as a 1:1...

  3. Counting White Blood Cells from a Blood Smear Using Fourier Ptychographic Microscopy

    OpenAIRE

    Chung, Jaebum; Ou, Xiaoze; Kulkarni, Rajan P.; Yang, Changhuei

    2015-01-01

    White blood cell (WBC) count is a valuable metric for assisting with diagnosis or prognosis of various diseases such as coronary heart disease, type 2 diabetes, or infection. Counting WBCs can be done either manually or automatically. Automatic methods are capable of counting a large number of cells to give a statistically more accurate reading of the WBC count of a sample, but the specialized equipment tends to be expensive. Manual methods are inexpensive since they only involve a convention...

  4. Rapid telomere motions in live human cells analyzed by highly time-resolved microscopy

    Directory of Open Access Journals (Sweden)

    Wang Xueying

    2008-10-01

    Full Text Available Abstract Background Telomeres cap chromosome ends and protect the genome. We studied individual telomeres in live human cancer cells. In capturing telomere motions using quantitative imaging to acquire complete high-resolution three-dimensional datasets every second for 200 seconds, telomere dynamics were systematically analyzed. Results The motility of individual telomeres within the same cancer cell nucleus was widely heterogeneous. One class of internal heterochromatic regions of chromosomes analyzed moved more uniformly and showed less motion and heterogeneity than telomeres. The single telomere analyses in cancer cells revealed that shorter telomeres showed more motion, and the more rapid telomere motions were energy dependent. Experimentally increasing bulk telomere length dampened telomere motion. In contrast, telomere uncapping, but not a DNA damaging agent, methyl methanesulfonate, significantly increased telomere motion. Conclusion New methods for seconds-scale, four-dimensional, live cell microscopic imaging and data analysis, allowing systematic tracking of individual telomeres in live cells, have defined a previously undescribed form of telomere behavior in human cells, in which the degree of telomere motion was dependent upon telomere length and functionality.

  5. Dissecting eukaryotic cells by coherent phase microscopy: quantitative analysis of quiescent and activated T lymphocytes

    Science.gov (United States)

    Tychinsky, Vladimir P.; Kretushev, Alexander V.; Vyshenskaya, Tatiana V.; Shtil, Alexander A.

    2012-07-01

    We present a concept for quantitative characterization of a functional state of an individual eukaryotic cell based on interference imaging. The informative parameters of the phase images of quiescent and mitogen-activated T lymphocytes included the phase thickness, phase volume, the area, and the size of organelles. These parameters were obtained without a special hypothesis about cell structure. Combinations of these parameters generated a ``phase portrait'' of the cell. A simplified spherical multilayer optic model of a T lymphocyte was used to calculate the refractivity profile, to identify structural elements of the image with the organelles, and to interpret the parameters of the phase portrait. The values of phase image parameters underwent characteristic changes in the course of mitogenic stimulation of T cells; thereby, the functional state of individual cells can be described using these parameters. Because the values of the components of the phase portrait are measured in absolute units, it is possible to compare the parameters of images obtained with different interference microscopes. Thus, the analysis of phase portraits provides a new and perspective approach for quantitative, real-time analysis of subcellular structure and physiologic state of an individual cell.

  6. Segmental neurofibromatosis and malignancy.

    Science.gov (United States)

    Dang, Julie D; Cohen, Philip R

    2010-01-01

    Segmental neurofibromatosis is an uncommon variant of neurofibromatosis type I characterized by neurofibromas and/or café-au-lait macules localized to one sector of the body. Although patients with neurofibromatosis type I have an associated increased risk of certain malignancies, malignancy has only occasionally been reported in patients with segmental neurofibromatosis. The published reports of patients with segmental neurofibromatosis who developed malignancy were reviewed and the characteristics of these patients and their cancers were summarized. Ten individuals (6 women and 4 men) with segmental neurofibromatosis and malignancy have been reported. The malignancies include malignant peripheral nerve sheath tumor (3), malignant melanoma (2), breast cancer (1), colon cancer (1), gastric cancer (1), lung cancer (1), and Hodgkin lymphoma (1). The most common malignancies in patients with segmental neurofibromatosis are derived from neural crest cells: malignant peripheral nerve sheath tumor and malignant melanoma. The incidence of malignancy in patients with segmental neurofibromatosis may approach that of patients with neurofibromatosis type I. PMID:21137621

  7. Automatic segmentation and classification of tendon nuclei from IHC stained images

    Science.gov (United States)

    Kuok, Chan-Pang; Wu, Po-Ting; Jou, I.-Ming; Su, Fong-Chin; Sun, Yung-Nien

    2015-12-01

    Immunohistochemical (IHC) staining is commonly used for detecting cells in microscopy. It is used for analyzing many types of diseases, e.g. breast cancer. Dispersion problem often exist at cell staining which will affect the accuracy of automatic counting. In this paper, we introduce a new method to overcome this problem. Otsu's thresholding method is first applied to exclude the background, so that only cells with dispersed staining are left at foreground, and then refinement will be applied by local adaptive thresholding method according to the irregularity index of the segmented shape at foreground. The segmentation results are also compared to the refinement results using Otsu's thresholding method. Cell classification based on the shape and color indices obtained from the segmentation result is applied to determine the cell condition into normal, abnormal and suspected abnormal cases.

  8. Investigation of prostate cancer cells using NADH and Tryptophan as biomarker: multiphoton FLIM-FRET microscopy

    Science.gov (United States)

    Rehman, Shagufta; O'Melia, Meghan J.; Wallrabe, Horst; Svindrych, Zdenek; Chandra, Dhyan; Periasamy, Ammasi

    2016-03-01

    Fluorescence Lifetime Imaging (FLIM) can be used to understand the metabolic activity in cancer. Prostate cancer is one of the leading cancers in men in the USA. This research focuses on FLIM measurements of NAD(P)H and Tryptophan, used as biomarkers to understand the metabolic activity in prostate cancer cells. Two prostate cancers and one normal cell line were used for live-cell FLIM measurements on Zeiss780 2P confocal microscope with SPCM FLIM board. Glucose uptake and glycolysis proceeds about ten times faster in cancer than in non-cancerous tissues. Therefore, we assessed the glycolytic activity in the prostate cancer in comparison to the normal cells upon glucose stimulation by analyzing the NAD(P)H and Trp lifetime distribution and efficiency of energy transfer (E%). Furthermore, we treated the prostate cancer cells with 1μM Doxorubicin, a commonly used anti-cancer chemotherapeutic. Increase in NADH a2%, an indicator of increased glycolysis and increased E% between Trp and NAD(P)H were seen upon glucose stimulation for 30min. The magnitude of shift to the right for NAD(P)H a2% and E% distribution was higher in prostate cancer versus the normal cells. Upon treatment with Doxorubicin decrease in cellular metabolism was seen at 15 and 30 minutes. The histogram for NAD(P)H a2% post-treatment for prostate cancer cells showed a left shift compared to the untreated control suggesting decrease in glycolysis and metabolic activity opposite to what was observed after glucose stimulation. Hence, NAD(P)H and Trp lifetimes can be used biomarkers to understand metabolic activity in prostate cancer and upon chemotherapeutic interventions.

  9. Atomic force microscopy combined with human pluripotent stem cell derived cardiomyocytes for biomechanical sensing.

    Science.gov (United States)

    Pesl, Martin; Pribyl, Jan; Acimovic, Ivana; Vilotic, Aleksandra; Jelinkova, Sarka; Salykin, Anton; Lacampagne, Alain; Dvorak, Petr; Meli, Albano C; Skladal, Petr; Rotrekl, Vladimir

    2016-11-15

    Cardiomyocyte contraction and relaxation are important parameters of cardiac function altered in many heart pathologies. Biosensing of these parameters represents an important tool in drug development and disease modeling. Human embryonic stem cells and especially patient specific induced pluripotent stem cell-derived cardiomyocytes are well established as cardiac disease model.. Here, a live stem cell derived embryoid body (EB) based cardiac cell syncytium served as a biorecognition element coupled to the microcantilever probe from atomic force microscope thus providing reliable micromechanical cellular biosensor suitable for whole-day testing. The biosensor was optimized regarding the type of cantilever, temperature and exchange of media; in combination with standardized protocol, it allowed testing of compounds and conditions affecting the biomechanical properties of EB. The studied effectors included calcium , drugs modulating the catecholaminergic fight-or-flight stress response such as the beta-adrenergic blocker metoprolol and the beta-adrenergic agonist isoproterenol. Arrhythmogenic effects were studied using caffeine. Furthermore, with EBs originating from patient's stem cells, this biosensor can help to characterize heart diseases such as dystrophies. PMID:27266660

  10. Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

    Science.gov (United States)

    Itano, Michelle S; Bleck, Marina; Johnson, Daniel S; Simon, Sanford M

    2016-02-01

    Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

  11. Atomic force microscopy observation on nuclear reassembly in a cell-free system

    Institute of Scientific and Technical Information of China (English)

    YANG Ning; CHEN Zhongcai; ZHANG Zhaohui; ZHU Xing; ZHAI Zhonghe; TANG Xiaowei

    2003-01-01

    Cell-free system is interesting and useful for studying nuclear assembly in mitosis. Atomic force micro- scopy (AFM), which is a simple way for imaging fixed reassemble nuclei with high resolution, has not been used in the cell-free system. In this paper, we put forward an air-drying sample preparation for AFM. Using AFM, we observed nuclear reassembly process within 100 nm resolution ina cell-free system. As a result, we found that the images were artifact-free, and with higher resolution compared with fluorescent optical microscope images. Furthermore, the morphology of membrane vesicles was obtained clearly, and a dynamic change of morphology during the vesicles' approaching to nuclear envelope was also observed, which is enlightened to understand the mechanism of nuclear envelope assembly.

  12. Single cells and intracellular processes studied by a plasmonic-based electrochemical impedance microscopy

    Science.gov (United States)

    Wang, Wei; Foley, Kyle; Shan, Xiaonan; Wang, Shaopeng; Eaton, Seron; Nagaraj, Vinay J; Wiktor, Peter; Patel, Urmez; Tao, Nongjian

    2012-01-01

    We report an electrochemical impedance microscope (EIM) based on surface plasmon resonance. The new EIM can resolve local impedance with sub-micron spatial resolution, and monitor dynamics of various processes, such as apoptosis and electroporation of individual cells with millisecond time resolution. The high spatial and temporal resolution images make it possible to not only study individual cells, but also resolve the sub-cellular structures and processes without labels. The detection sensitivity achieved with the current setup is ~2 pS, which is excellent considering the conductance of a single ion channel is in the range of 5–400 pS. We describe also a model that simulates the EIM images of cells based on local dielectric constant and conductivity. PMID:21336333

  13. Solving the mysteries of the bacterial cell – application of novel techniques in fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Magdalena Donczew

    2011-01-01

    Full Text Available We have reviewed how the development of fluorescent markers, triggered by the discovery of green fluorescence protein and its other color variants leading to the establishment of methods for studies of protein interactions with application of fluorescent proteins, affected the view of bacterial cell organization. Application of the new microscopic methods allowed localization of proteins and chromosomal regions, and observation of their migration in real time. These studies revealed the spatial organization of bacterial cells which includes specific subcellular localization of proteins, the presence of dynamic cytoskeletal structures, orchestrated and active segregation of chromosomes, and spatiotemporal gene regulation.

  14. Label-free assessment of adipose-derived stem cell differentiation using coherent anti-Stokes Raman scattering and multiphoton microscopy

    OpenAIRE

    Mouras, Rabah; Bagnaninchi, Pierre O.; Downes, Andrew R; Elfick, Alistair P D

    2012-01-01

    ABSTRACT. Adult stem cells (SCs) hold great potential as likely candidates for disease therapy but also as sources of differentiated human cells in vitro models of disease. In both cases, the label-free assessment of SC differentiation state is highly desirable, either as a quality-control technology ensuring cells to be used clinically are of the desired lineage or to facilitate in vitro time-course studies of cell differentiation. We investigate the potential of nonlinear optical microscopy...

  15. Dual-wavelength optical-resolution photoacoustic microscopy for cells with gold nanoparticle bioconjugates in three-dimensional cultures

    Science.gov (United States)

    Lee, Po-Yi; Liu, Wei-Wen; Chen, Shu-Ching; Li, Pai-Chi

    2016-03-01

    Three-dimensional (3D) in vitro models bridge the gap between typical two-dimensional cultures and in vivo conditions. However, conventional optical imaging methods such as confocal microscopy and two-photon microscopy cannot accurately depict cellular processing in 3D models due to limited penetration of photons. We developed a dualwavelength optical-resolution photoacoustic microscopy (OR-PAM), which provides sufficient penetration depth and spatial resolution, for studying CD8+ cytotoxic T lymphocytes (CTLs) trafficking in an in vitro 3D tumor microenvironment. CTLs play a cardinal role in host defense against tumor. Efficient trafficking of CTLs to the tumor microenvironment is a critical step for cancer immunotherapy. For the proposed system, gold nanospheres and indocyanine green (ICG) have been remarkable choices for contrast agents for photoacoustic signals due to their excellent biocompatibility and high optical absorption. With distinct absorption spectrums, targeted cells with gold nanospheres and ICG respectively can be identified by switching 523-nm and 800-nm laser irradiation. Moreover, we use an x-y galvanometer scanner to obtain high scanning rate. In the developed system, lateral and axial resolutions were designed at 1.6 μm and 5 μm, respectively. We successfully showed that dual-spectral OR-PAM can map either the distribution of CTLs with gold nanospheres at a visible wavelength of 523 nm or the 3D structure of tumor spheres with ICG in an in vitro 3D microenvironment. Our OR-PAM can provide better biological relevant information in cellular interaction and is potential for preclinical screening of anti-cancer drugs.

  16. Analysis of energy metabolism of HeLa cancer cells in vitro and in vivo using fluorescence lifetime microscopy

    Science.gov (United States)

    Lukina, Maria; Shirmanova, Marina; Dudenkova, Varvara; Druzhkova, Irina; Shumilova, Anastasia; Zagaynova, Elena

    2016-04-01

    The aim of the present work was to study energy metabolism in human cervical carcinoma (HeLa) cells in vitro and in vivo using two-photon FLIM. Cellular metabolism was examined by monitoring of the fluorescence lifetimes of free and protein-bound forms of NAD(P)H and FAD and their relative contributions. Two-photon fluorescence and second harmonic generation microscopy as well as standard histopathology with hematoxylin and eosin were used to characterize tissue structure. Cellular metabolism was analyzed in cancer cells co-cultured with human fibroblasts and in tumor xenografts transplanted to nude mice. In the HeLa-huFB co-culture we observed a metabolic shift from OXPHOS toward glycolysis in cancer cells, and from glycolysis to OXPHOS in fibroblasts, starting from Day 2 of co-culturing. In the tumor tissue we detected metabolic heterogeneity with more glycolytic metabolism of cancer cells in the stroma-rich zones. The results of the study are of a great importance for understanding metabolic behavior of tumors and for development of anticancer drugs targeted to metabolic pathways.

  17. Architectural Analysis of Picrosirius Red Stained Collagen in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma using Polarization Microscopy

    Science.gov (United States)

    Sharma, Rashi; Rehani, Shweta; Mehendiratta, Monica; Kumra, Madhumani; Mathias, Yulia; Yadav, Jyoti; Sahay, Khushboo

    2015-01-01

    Introduction Collagen degradation is important both for carcinogenesis and in its progression. Research regarding the co-relation of collagen with Oral Epithelial Dysplasia (OED) and Oral Squamous Cell Carcinoma (OSCC) is less explored. Aim To elucidate the nature of collagen in Oral Epithelial Dysplasia (OED) and Oral Squamous Cell Carcinoma (OSCC) using Picrosirius Red Stain (PSR) under polarizing microscopy. Materials and Methods The study consisted of a total 40 samples which were divided into three groups. Group I included buccal mucosa as negative and irritation fibroma as positive control, group II consisted of OED and group III consisted of Oral Squamous