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Sample records for cell microscopy segmentation

  1. Segmentation of fluorescence microscopy cell images using unsupervised mining.

    Science.gov (United States)

    Du, Xian; Dua, Sumeet

    2010-05-28

    The accurate measurement of cell and nuclei contours are critical for the sensitive and specific detection of changes in normal cells in several medical informatics disciplines. Within microscopy, this task is facilitated using fluorescence cell stains, and segmentation is often the first step in such approaches. Due to the complex nature of cell issues and problems inherent to microscopy, unsupervised mining approaches of clustering can be incorporated in the segmentation of cells. In this study, we have developed and evaluated the performance of multiple unsupervised data mining techniques in cell image segmentation. We adapt four distinctive, yet complementary, methods for unsupervised learning, including those based on k-means clustering, EM, Otsu's threshold, and GMAC. Validation measures are defined, and the performance of the techniques is evaluated both quantitatively and qualitatively using synthetic and recently published real data. Experimental results demonstrate that k-means, Otsu's threshold, and GMAC perform similarly, and have more precise segmentation results than EM. We report that EM has higher recall values and lower precision results from under-segmentation due to its Gaussian model assumption. We also demonstrate that these methods need spatial information to segment complex real cell images with a high degree of efficacy, as expected in many medical informatics applications.

  2. 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.

  3. Towards automated segmentation of cells and cell nuclei in nonlinear optical microscopy.

    Science.gov (United States)

    Medyukhina, Anna; Meyer, Tobias; Schmitt, Michael; Romeike, Bernd F M; Dietzek, Benjamin; Popp, Jürgen

    2012-11-01

    Nonlinear optical (NLO) imaging techniques based e.g. on coherent anti-Stokes Raman scattering (CARS) or two photon excited fluorescence (TPEF) show great potential for biomedical imaging. In order to facilitate the diagnostic process based on NLO imaging, there is need for an automated calculation of quantitative values such as cell density, nucleus-to-cytoplasm ratio, average nuclear size. Extraction of these parameters is helpful for the histological assessment in general and specifically e.g. for the determination of tumor grades. This requires an accurate image segmentation and detection of locations and boundaries of cells and nuclei. Here we present an image processing approach for the detection of nuclei and cells in co-registered TPEF and CARS images. The algorithm developed utilizes the gray-scale information for the detection of the nuclei locations and the gradient information for the delineation of the nuclear and cellular boundaries. The approach reported is capable for an automated segmentation of cells and nuclei in multimodal TPEF-CARS images of human brain tumor samples. The results are important for the development of NLO microscopy into a clinically relevant diagnostic tool.

  4. Segmentation of vascular structures and hematopoietic cells in 3D microscopy images and quantitative analysis

    Science.gov (United States)

    Mu, Jian; Yang, Lin; Kamocka, Malgorzata M.; Zollman, Amy L.; Carlesso, Nadia; Chen, Danny Z.

    2015-03-01

    In this paper, we present image processing methods for quantitative study of how the bone marrow microenvironment changes (characterized by altered vascular structure and hematopoietic cell distribution) caused by diseases or various factors. We develop algorithms that automatically segment vascular structures and hematopoietic cells in 3-D microscopy images, perform quantitative analysis of the properties of the segmented vascular structures and cells, and examine how such properties change. In processing images, we apply local thresholding to segment vessels, and add post-processing steps to deal with imaging artifacts. We propose an improved watershed algorithm that relies on both intensity and shape information and can separate multiple overlapping cells better than common watershed methods. We then quantitatively compute various features of the vascular structures and hematopoietic cells, such as the branches and sizes of vessels and the distribution of cells. In analyzing vascular properties, we provide algorithms for pruning fake vessel segments and branches based on vessel skeletons. Our algorithms can segment vascular structures and hematopoietic cells with good quality. We use our methods to quantitatively examine the changes in the bone marrow microenvironment caused by the deletion of Notch pathway. Our quantitative analysis reveals property changes in samples with deleted Notch pathway. Our tool is useful for biologists to quantitatively measure changes in the bone marrow microenvironment, for developing possible therapeutic strategies to help the bone marrow microenvironment recovery.

  5. Statistical region based active contour using a fractional entropy descriptor: Application to nuclei cell segmentation in confocal \\ud microscopy images

    OpenAIRE

    Histace, A.; Meziou, B J; Matuszewski, Bogdan; Precioso, F.; Murphy, M F; Carreiras, F

    2013-01-01

    We propose an unsupervised statistical region based active contour approach integrating an original fractional entropy measure for image segmentation with a particular application to single channel actin tagged fluorescence confocal microscopy image segmentation. Following description of statistical based active contour segmentation and the mathematical definition of the proposed fractional entropy descriptor, we demonstrate comparative segmentation results between the proposed approach and s...

  6. A semi-Markov model for mitosis segmentation in time-lapse phase contrast microscopy image sequences of stem cell populations.

    Science.gov (United States)

    Liu, An-An; Li, Kang; Kanade, Takeo

    2012-02-01

    We propose a semi-Markov model trained in a max-margin learning framework for mitosis event segmentation in large-scale time-lapse phase contrast microscopy image sequences of stem cell populations. Our method consists of three steps. First, we apply a constrained optimization based microscopy image segmentation method that exploits phase contrast optics to extract candidate subsequences in the input image sequence that contains mitosis events. Then, we apply a max-margin hidden conditional random field (MM-HCRF) classifier learned from human-annotated mitotic and nonmitotic sequences to classify each candidate subsequence as a mitosis or not. Finally, a max-margin semi-Markov model (MM-SMM) trained on manually-segmented mitotic sequences is utilized to reinforce the mitosis classification results, and to further segment each mitosis into four predefined temporal stages. The proposed method outperforms the event-detection CRF model recently reported by Huh as well as several other competing methods in very challenging image sequences of multipolar-shaped C3H10T1/2 mesenchymal stem cells. For mitosis detection, an overall precision of 95.8% and a recall of 88.1% were achieved. For mitosis segmentation, the mean and standard deviation for the localization errors of the start and end points of all mitosis stages were well below 1 and 2 frames, respectively. In particular, an overall temporal location error of 0.73 ± 1.29 frames was achieved for locating daughter cell birth events.

  7. Neuron Segmentation in Electron Microscopy Images Using Partial Differential Equations.

    Science.gov (United States)

    Jones, Cory; Sayedhosseini, Mojtaba; Ellisman, Mark; Tasdizen, Tolga

    2013-01-01

    In connectomics, neuroscientists seek to identify the synaptic connections between neurons. Segmentation of cell membranes using supervised learning algorithms on electron microscopy images of brain tissue is often done to assist in this effort. Here we present a partial differential equation with a novel growth term to improve the results of a supervised learning algorithm. We also introduce a new method for representing the resulting image that allows for a more dynamic thresholding to further improve the result. Using these two processes we are able to close small to medium sized gaps in the cell membrane detection and improve the Rand error by as much as 9% over the initial supervised segmentation.

  8. 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...

  9. Automated seeding-based nuclei segmentation in nonlinear optical microscopy.

    Science.gov (United States)

    Medyukhina, Anna; Meyer, Tobias; Heuke, Sandro; Vogler, Nadine; Dietzek, Benjamin; Popp, Jürgen

    2013-10-01

    Nonlinear optical (NLO) microscopy based, e.g., on coherent anti-Stokes Raman scattering (CARS) or two-photon-excited fluorescence (TPEF) is a fast label-free imaging technique, with a great potential for biomedical applications. However, NLO microscopy as a diagnostic tool is still in its infancy; there is a lack of robust and durable nuclei segmentation methods capable of accurate image processing in cases of variable image contrast, nuclear density, and type of investigated tissue. Nonetheless, such algorithms specifically adapted to NLO microscopy present one prerequisite for the technology to be routinely used, e.g., in pathology or intraoperatively for surgical guidance. In this paper, we compare the applicability of different seeding and boundary detection methods to NLO microscopic images in order to develop an optimal seeding-based approach capable of accurate segmentation of both TPEF and CARS images. Among different methods, the Laplacian of Gaussian filter showed the best accuracy for the seeding of the image, while a modified seeded watershed segmentation was the most accurate in the task of boundary detection. The resulting combination of these methods followed by the verification of the detected nuclei performs high average sensitivity and specificity when applied to various types of NLO microscopy images.

  10. Advanced Microscopy of Microbial Cells

    DEFF Research Database (Denmark)

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

    2011-01-01

    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......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...

  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. 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...... for visualization of variation between cells in phenotypic traits such as gene expression....

  13. 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

  14. 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.

  15. A workflow for the automatic segmentation of organelles in electron microscopy image stacks.

    Science.gov (United States)

    Perez, Alex J; Seyedhosseini, Mojtaba; Deerinck, Thomas J; Bushong, Eric A; Panda, Satchidananda; Tasdizen, Tolga; Ellisman, Mark H

    2014-01-01

    Electron microscopy (EM) facilitates analysis of the form, distribution, and functional status of key organelle systems in various pathological processes, including those associated with neurodegenerative disease. Such EM data often provide important new insights into the underlying disease mechanisms. The development of more accurate and efficient methods to quantify changes in subcellular microanatomy has already proven key to understanding the pathogenesis of Parkinson's and Alzheimer's diseases, as well as glaucoma. While our ability to acquire large volumes of 3D EM data is progressing rapidly, more advanced analysis tools are needed to assist in measuring precise three-dimensional morphologies of organelles within data sets that can include hundreds to thousands of whole cells. Although new imaging instrument throughputs can exceed teravoxels of data per day, image segmentation and analysis remain significant bottlenecks to achieving quantitative descriptions of whole cell structural organellomes. Here, we present a novel method for the automatic segmentation of organelles in 3D EM image stacks. Segmentations are generated using only 2D image information, making the method suitable for anisotropic imaging techniques such as serial block-face scanning electron microscopy (SBEM). Additionally, no assumptions about 3D organelle morphology are made, ensuring the method can be easily expanded to any number of structurally and functionally diverse organelles. Following the presentation of our algorithm, we validate its performance by assessing the segmentation accuracy of different organelle targets in an example SBEM dataset and demonstrate that it can be efficiently parallelized on supercomputing resources, resulting in a dramatic reduction in runtime.

  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. Smart markers for watershed-based cell segmentation.

    Science.gov (United States)

    Koyuncu, Can Fahrettin; Arslan, Salim; Durmaz, Irem; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem

    2012-01-01

    Automated cell imaging systems facilitate fast and reliable analysis of biological events at the cellular level. In these systems, the first step is usually cell segmentation that greatly affects the success of the subsequent system steps. On the other hand, similar to other image segmentation problems, cell segmentation is an ill-posed problem that typically necessitates the use of domain-specific knowledge to obtain successful segmentations even by human subjects. The approaches that can incorporate this knowledge into their segmentation algorithms have potential to greatly improve segmentation results. In this work, we propose a new approach for the effective segmentation of live cells from phase contrast microscopy. This approach introduces a new set of "smart markers" for a marker-controlled watershed algorithm, for which the identification of its markers is critical. The proposed approach relies on using domain-specific knowledge, in the form of visual characteristics of the cells, to define the markers. We evaluate our approach on a total of 1,954 cells. The experimental results demonstrate that this approach, which uses the proposed definition of smart markers, is quite effective in identifying better markers compared to its counterparts. This will, in turn, be effective in improving the segmentation performance of a marker-controlled watershed algorithm.

  18. Smart markers for watershed-based cell segmentation.

    Directory of Open Access Journals (Sweden)

    Can Fahrettin Koyuncu

    Full Text Available Automated cell imaging systems facilitate fast and reliable analysis of biological events at the cellular level. In these systems, the first step is usually cell segmentation that greatly affects the success of the subsequent system steps. On the other hand, similar to other image segmentation problems, cell segmentation is an ill-posed problem that typically necessitates the use of domain-specific knowledge to obtain successful segmentations even by human subjects. The approaches that can incorporate this knowledge into their segmentation algorithms have potential to greatly improve segmentation results. In this work, we propose a new approach for the effective segmentation of live cells from phase contrast microscopy. This approach introduces a new set of "smart markers" for a marker-controlled watershed algorithm, for which the identification of its markers is critical. The proposed approach relies on using domain-specific knowledge, in the form of visual characteristics of the cells, to define the markers. We evaluate our approach on a total of 1,954 cells. The experimental results demonstrate that this approach, which uses the proposed definition of smart markers, is quite effective in identifying better markers compared to its counterparts. This will, in turn, be effective in improving the segmentation performance of a marker-controlled watershed algorithm.

  19. 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.

  20. A workflow for the automatic segmentation of organelles in electron microscopy image stacks

    Directory of Open Access Journals (Sweden)

    Alex Joseph Perez

    2014-11-01

    Full Text Available Electron microscopy (EM facilitates analysis of the form, distribution, and functional status of key organelle systems in various pathological processes, including those associated with neurodegenerative disease. Such EM data often provide important new insights into the underlying disease mechanisms. The development of more accurate and efficient methods to quantify changes in subcellular microanatomy has already proven key to understanding the pathogenesis of Parkinson’s and Alzheimer’s diseases, as well as glaucoma. While our ability to acquire large volumes of 3D EM data is progressing rapidly, more advanced analysis tools are needed to assist in measuring precise three-dimensional morphologies of organelles within data sets that can include hundreds to thousands of whole cells. Although new imaging instrument throughputs can exceed teravoxels of data per day, image segmentation and analysis remain significant bottlenecks to achieving quantitative descriptions of whole cell structural organellomes. Here, we present a novel method for the automatic segmentation of organelles in 3D EM image stacks. Segmentations are generated using only 2D image information, making the method suitable for anisotropic imaging techniques such as serial block-face scanning electron microscopy (SBEM. Additionally, no assumptions about 3D organelle morphology are made, ensuring the method can be easily expanded to any number of structurally and functionally diverse organelles. Following the presentation of our algorithm, we validate its performance by assessing the segmentation accuracy of different organelle targets in an example SBEM dataset and demonstrate that it can be efficiently parallelized on supercomputing resources, resulting in a dramatic reduction in runtime.

  1. 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.

  2. Segmentation of occluded hematopoietic stem cells from tracking.

    Science.gov (United States)

    Mankowski, Walter C; Winter, Mark R; Wait, Eric; Lodder, Mels; Schumacher, Ton; Naik, Shalin H; Cohen, Andrew R

    2014-01-01

    Image sequences of live proliferating cells often contain visual ambiguities that are difficult even for human domain experts to resolve. Here we present a new approach to analyzing image sequences that capture the development of clones of hematopoietic stem cells (HSCs) from live cell time lapse microscopy. The HSCs cannot survive long term imaging unless they are cultured together with a secondary cell type, OP9 stromal cells. The HSCs frequently disappear under the OP9 cell layer, making segmentation difficult or impossible from a single image frame, even for a human domain expert. We have developed a new approach to the segmentation of HSCs that captures these occluded cells. Starting with an a priori segmentation that uses a Monte Carlo technique to estimate the number of cells in a clump of touching cells, we proceed to track and lineage the image data. Following user validation of the lineage information, an a posteriori resegmentation step utilizing tracking results delineates the HSCs occluded by the OP9 layer. Resegmentation has been applied to 3031 occluded segmentations from 77 tracks, correctly recovering over 84% of the occluded segmentations.

  3. 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'.

  4. 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...

  5. 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.

  6. 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.

  7. 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

    Science.gov (United States)

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

    2016-10-01

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

  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. 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.

  10. 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.

  11. Segmentation Method of Time-Lapse Microscopy Images with the Focus on Biocompatibility Assessment.

    Science.gov (United States)

    Soukup, Jindřich; Císař, Petr; Šroubek, Filip

    2016-06-01

    Biocompatibility testing of new materials is often performed in vitro by measuring the growth rate of mammalian cancer cells in time-lapse images acquired by phase contrast microscopes. The growth rate is measured by tracking cell coverage, which requires an accurate automatic segmentation method. However, cancer cells have irregular shapes that change over time, the mottled background pattern is partially visible through the cells and the images contain artifacts such as halos. We developed a novel algorithm for cell segmentation that copes with the mentioned challenges. It is based on temporal differences of consecutive images and a combination of thresholding, blurring, and morphological operations. We tested the algorithm on images of four cell types acquired by two different microscopes, evaluated the precision of segmentation against manual segmentation performed by a human operator, and finally provided comparison with other freely available methods. We propose a new, fully automated method for measuring the cell growth rate based on fitting a coverage curve with the Verhulst population model. The algorithm is fast and shows accuracy comparable with manual segmentation. Most notably it can correctly separate live from dead cells.

  12. 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.

  13. 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%.

  14. 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.

  15. Telocytes and putative stem cells in the lungs: electron microscopy, electron tomography and laser scanning microscopy.

    Science.gov (United States)

    Popescu, Laurentiu M; Gherghiceanu, Mihaela; Suciu, Laura C; Manole, Catalin G; Hinescu, Mihail E

    2011-09-01

    This study describes a novel type of interstitial (stromal) cell - telocytes (TCs) - in the human and mouse respiratory tree (terminal and respiratory bronchioles, as well as alveolar ducts). TCs have recently been described in pleura, epicardium, myocardium, endocardium, intestine, uterus, pancreas, mammary gland, etc. (see www.telocytes.com ). TCs are cells with specific prolongations called telopodes (Tp), frequently two to three per cell. Tp are very long prolongations (tens up to hundreds of μm) built of alternating thin segments known as podomers (≤ 200 nm, below the resolving power of light microscope) and dilated segments called podoms, which accommodate mitochondria, rough endoplasmic reticulum and caveolae. Tp ramify dichotomously, making a 3-dimensional network with complex homo- and heterocellular junctions. Confocal microscopy reveals that TCs are c-kit- and CD34-positive. Tp release shed vesicles or exosomes, sending macromolecular signals to neighboring cells and eventually modifying their transcriptional activity. At bronchoalveolar junctions, TCs have been observed in close association with putative stem cells (SCs) in the subepithelial stroma. SCs are recognized by their ultrastructure and Sca-1 positivity. Tp surround SCs, forming complex TC-SC niches (TC-SCNs). Electron tomography allows the identification of bridging nanostructures, which connect Tp with SCs. In conclusion, this study shows the presence of TCs in lungs and identifies a TC-SC tandem in subepithelial niches of the bronchiolar tree. In TC-SCNs, the synergy of TCs and SCs may be based on nanocontacts and shed vesicles.

  16. From voxels to knowledge: a practical guide to the segmentation of complex electron microscopy 3D-data.

    Science.gov (United States)

    Tsai, Wen-Ting; Hassan, Ahmed; Sarkar, Purbasha; Correa, Joaquin; Metlagel, Zoltan; Jorgens, Danielle M; Auer, Manfred

    2014-08-13

    Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation. The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data

  17. Segmentation of scanning electron microscopy images from natural rubber samples with gold nanoparticles using starlet wavelets.

    Science.gov (United States)

    de Siqueira, Alexandre Fioravante; Cabrera, Flávio Camargo; Pagamisse, Aylton; Job, Aldo Eloizo

    2014-01-01

    Electronic microscopy has been used for morphology evaluation of different materials structures. However, microscopy results may be affected by several factors. Image processing methods can be used to correct and improve the quality of these results. In this article, we propose an algorithm based on starlets to perform the segmentation of scanning electron microscopy images. An application is presented in order to locate gold nanoparticles in natural rubber membranes. In this application, our method showed accuracy greater than 85% for all test images. Results given by this method will be used in future studies, to computationally estimate the density distribution of gold nanoparticles in natural rubber samples and to predict reduction kinetics of gold nanoparticles at different time periods.

  18. [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.

  19. Environmental scanning electron microscopy in cell biology.

    Science.gov (United States)

    McGregor, J E; Staniewicz, L T L; Guthrie Neé Kirk, S E; Donald, A M

    2013-01-01

    Environmental scanning electron microscopy (ESEM) (1) is an imaging technique which allows hydrated, insulating samples to be imaged under an electron beam. The resolution afforded by this technique is higher than conventional optical microscopy but lower than conventional scanning electron microscopy (CSEM). The major advantage of the technique is the minimal sample preparation needed, making ESEM quick to use and the images less susceptible to the artifacts that the extensive sample preparation usually required for CSEM may introduce. Careful manipulation of both the humidity in the microscope chamber and the beam energy are nevertheless essential to prevent dehydration and beam damage artifacts. In some circumstances it is possible to image live cells in the ESEM (2).In the following sections we introduce the fundamental principles of ESEM imaging before presenting imaging protocols for plant epidermis, mammalian cells, and bacteria. In the first two cases samples are imaged using the secondary electron (topographic) signal, whereas a transmission technique is employed to image bacteria.

  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. Electron Microscopy of Nanostructures in Cells

    DEFF Research Database (Denmark)

    Købler, Carsten

    with cells is therefore increasingly more relevant from both an engineering and a toxicological viewpoint. My work involves developing and exploring electron microscopy (EM) for imaging nanostructures in cells, for the purpose of understanding nanostructure-cell interactions in terms of their possibilities...... in science and concerns in toxicology. In the present work, EM methods for imaging nanostructure-cell interactions have been explored, and the complex interactions documented and ordered. In particular the usability of the focused ion beam scanning electron microscope (FIB-SEM) was explored. Using EM...

  2. 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.

  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. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. A fast and efficient segmentation scheme for cell microscopic image.

    Science.gov (United States)

    Lebrun, G; Charrier, C; Lezoray, O; Meurie, C; Cardot, H

    2007-04-27

    Microscopic cellular image segmentation schemes must be efficient for reliable analysis and fast to process huge quantity of images. Recent studies have focused on improving segmentation quality. Several segmentation schemes have good quality but processing time is too expensive to deal with a great number of images per day. For segmentation schemes based on pixel classification, the classifier design is crucial since it is the one which requires most of the processing time necessary to segment an image. The main contribution of this work is focused on how to reduce the complexity of decision functions produced by support vector machines (SVM) while preserving recognition rate. Vector quantization is used in order to reduce the inherent redundancy present in huge pixel databases (i.e. images with expert pixel segmentation). Hybrid color space design is also used in order to improve data set size reduction rate and recognition rate. A new decision function quality criterion is defined to select good trade-off between recognition rate and processing time of pixel decision function. The first results of this study show that fast and efficient pixel classification with SVM is possible. Moreover posterior class pixel probability estimation is easy to compute with Platt method. Then a new segmentation scheme using probabilistic pixel classification has been developed. This one has several free parameters and an automatic selection must dealt with, but criteria for evaluate segmentation quality are not well adapted for cell segmentation, especially when comparison with expert pixel segmentation must be achieved. Another important contribution in this paper is the definition of a new quality criterion for evaluation of cell segmentation. The results presented here show that the selection of free parameters of the segmentation scheme by optimisation of the new quality cell segmentation criterion produces efficient cell segmentation.

  10. 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.

  11. 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.

  12. 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.

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

    Science.gov (United States)

    Kreshuk, Anna; Straehle, Christoph N; Sommer, Christoph; Koethe, Ullrich; Cantoni, Marco; Knott, Graham; Hamprecht, Fred A

    2011-01-01

    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.

  14. 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.

  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. 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

  18. 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.

  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. 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.

  1. Cell wall extension results in the coordinate separation of parallel microfibrils: evidence from scanning electron microscopy and atomic force microscopy.

    Science.gov (United States)

    Marga, Francoise; Grandbois, Michel; Cosgrove, Daniel J; Baskin, Tobias I

    2005-07-01

    Enlargement of the cell wall requires separation of cellulose microfibrils, mediated by proteins such as expansin; according to the multi-net growth hypothesis, enlargement passively reorients microfibrils. However, at the molecular scale, little is known about the specific movement of microfibrils. To find out, we examined directly changes in microfibril orientation when walls were extended slowly in vitro under constant load (creep). Frozen-thawed cucumber hypocotyl segments were strained by 20-30% by incubation in pH 4.5 buffer or by incubation of heat-inactivated segments in alpha-expansin or a fungal endoglucanase (Cel12A). Subsequently, the innermost layer of the cell wall was imaged, with neither extraction nor homogenization, by field-emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). AFM images revealed that sample preparation for FESEM did not appreciably alter cell wall ultrastructure. In both FESEM and AFM, images from extended and non-extended samples appeared indistinguishable. To quantify orientational order, we used a novel algorithm to characterize the fast Fourier transform of the image as a function of spatial frequency. For both FESEM and AFM images, the transforms of non-extended samples were indistinguishable from those of samples extended by alpha-expansin or Cel12A, as were AFM images of samples extended by acidic buffer. We conclude that cell walls in vitro can extend slowly by a creep mechanism without passive reorientation of innermost microfibrils, implying that wall loosening agents act selectively on the cross-linking polymers between parallel microfibrils, rather than more generally on the wall matrix.

  2. SuperSegger: robust image segmentation, analysis and lineage tracking of bacterial cells.

    Science.gov (United States)

    Stylianidou, Stella; Brennan, Connor; Nissen, Silas B; Kuwada, Nathan J; Wiggins, Paul A

    2016-11-01

    Many quantitative cell biology questions require fast yet reliable automated image segmentation to identify and link cells from frame-to-frame, and characterize the cell morphology and fluorescence. We present SuperSegger, an automated MATLAB-based image processing package well-suited to quantitative analysis of high-throughput live-cell fluorescence microscopy of bacterial cells. SuperSegger incorporates machine-learning algorithms to optimize cellular boundaries and automated error resolution to reliably link cells from frame-to-frame. Unlike existing packages, it can reliably segment microcolonies with many cells, facilitating the analysis of cell-cycle dynamics in bacteria as well as cell-contact mediated phenomena. This package has a range of built-in capabilities for characterizing bacterial cells, including the identification of cell division events, mother, daughter and neighbouring cells, and computing statistics on cellular fluorescence, the location and intensity of fluorescent foci. SuperSegger provides a variety of postprocessing data visualization tools for single cell and population level analysis, such as histograms, kymographs, frame mosaics, movies and consensus images. Finally, we demonstrate the power of the package by analyzing lag phase growth with single cell resolution.

  3. 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.

  4. Optical Property Analyses of Plant Cells for Adaptive Optics Microscopy

    Science.gov (United States)

    Tamada, Yosuke; Murata, Takashi; Hattori, Masayuki; Oya, Shin; Hayano, Yutaka; Kamei, Yasuhiro; Hasebe, Mitsuyasu

    2014-04-01

    In astronomy, adaptive optics (AO) can be used to cancel aberrations caused by atmospheric turbulence and to perform diffraction-limited observation of astronomical objects from the ground. AO can also be applied to microscopy, to cancel aberrations caused by cellular structures and to perform high-resolution live imaging. As a step toward the application of AO to microscopy, here we analyzed the optical properties of plant cells. We used leaves of the moss Physcomitrella patens, which have a single layer of cells and are thus suitable for optical analysis. Observation of the cells with bright field and phase contrast microscopy, and image degradation analysis using fluorescent beads demonstrated that chloroplasts provide the main source of optical degradations. Unexpectedly, the cell wall, which was thought to be a major obstacle, has only a minor effect. Such information provides the basis for the application of AO to microscopy for the observation of plant cells.

  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. 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.

  7. 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.

  8. 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...

  9. Segmentation and Tracking of Lymphocytes Based on Modified Active Contour Models in Phase Contrast Microscopy Images

    Directory of Open Access Journals (Sweden)

    Yali Huang

    2015-01-01

    Full Text Available The paper proposes an improved active contour model for segmenting and tracking accurate boundaries of the single lymphocyte in phase-contrast microscopic images. Active contour models have been widely used in object segmentation and tracking. However, current external-force-inspired methods are weak at handling low-contrast edges and suffer from initialization sensitivity. In order to segment low-contrast boundaries, we combine the region information of the object, extracted by morphology gray-scale reconstruction, and the edge information, extracted by the Laplacian of Gaussian filter, to obtain an improved feature map to compute the external force field for the evolution of active contours. To alleviate initial location sensitivity, we set the initial contour close to the real boundaries by performing morphological image processing. The proposed method was tested on live lymphocyte images acquired through the phase-contrast microscope from the blood samples of mice, and comparative experimental results showed the advantages of the proposed method in terms of the accuracy and the speed. Tracking experiments showed that the proposed method can accurately segment and track lymphocyte boundaries in microscopic images over time even in the presence of low-contrast edges, which will provide a good prerequisite for the quantitative analysis of lymphocyte morphology and motility.

  10. Model-based segmentation and quantification of subcellular structures in 2D and 3D fluorescent microscopy images

    Science.gov (United States)

    Wörz, Stefan; Heinzer, Stephan; Weiss, Matthias; Rohr, Karl

    2008-03-01

    We introduce a model-based approach for segmenting and quantifying GFP-tagged subcellular structures of the Golgi apparatus in 2D and 3D microscopy images. The approach is based on 2D and 3D intensity models, which are directly fitted to an image within 2D circular or 3D spherical regions-of-interest (ROIs). We also propose automatic approaches for the detection of candidates, for the initialization of the model parameters, and for adapting the size of the ROI used for model fitting. Based on the fitting results, we determine statistical information about the spatial distribution and the total amount of intensity (fluorescence) of the subcellular structures. We demonstrate the applicability of our new approach based on 2D and 3D microscopy images.

  11. 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.

  12. Hierarchical partial matching and segmentation of interacting cells.

    Science.gov (United States)

    Wu, Zheng; Gurari, Danna; Wong, Joyce Y; Betke, Margrit

    2012-01-01

    We propose a method that automatically tracks and segments living cells in phase-contrast image sequences, especially for cells that deform and interact with each other or clutter. We formulate the problem as a many-to-one elastic partial matching problem between closed curves. We introduce Double Cyclic Dynamic Time Warping for the scenario where a collision event yields a single boundary that encloses multiple touching cells and that needs to be cut into separate cell boundaries. The resulting individual boundaries may consist of segments to be connected to produce closed curves that match well with the individual cell boundaries before the collision event. We show how to convert this partial-curve matching problem into a shortest path problem that we then solve efficiently by reusing the computed shortest path tree. We also use our shortest path algorithm to fill the gaps between the segments of the target curves. Quantitative results demonstrate the benefit of our method by showing maintained accurate recognition of individual cell boundaries across 8068 images containing multiple cell interactions.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. Unstained viable cell recognition in phase-contrast microscopy

    Science.gov (United States)

    Skoczylas, M.; Rakowski, W.; Cherubini, R.; Gerardi, S.

    2011-09-01

    Individual cell recognition is a relevant task to be accomplished when single-ion microbeam irradiations are performed. At INFN-LNL facility cell visualization system is based on a phase-contrast optical microscope, without the use of any cell dye. Unstained cells are seeded in the special designed Petri dish, between two mylar foils, and at present the cell recognition is achieved manually by an expert operator. Nevertheless, this procedure is time consuming and sometimes it could be not practical if the amount of living cells to be irradiated is large. To reduce the time needed to recognize unstained cells on the Petri dish, it has been designed and implemented an automated, parallel algorithm. Overlapping ROIs sliding in steps over the captured grayscale image are firstly pre-classified and potential cell markers for the segmentation are obtained. Segmented objects are additionally classified to categorize cell bodies from other structures considered as sample dirt or background. As a result, cell coordinates are passed to the dedicated CELLView program that controls all the LNL single-ion microbeam irradiation protocol, including the positioning of individual cells in front of the ion beam. Unstained cell recognition system was successfully tested in experimental conditions with two different mylar surfaces. The recognition time and accuracy was acceptable, however, improvements in speed would be useful.

  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. 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.

  20. 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.

  1. 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

  2. Changing cell behaviours during beetle embryogenesis correlates with slowing of segmentation.

    Science.gov (United States)

    Nakamoto, A; Hester, S D; Constantinou, S J; Blaine, W G; Tewksbury, A B; Matei, M T; Nagy, L M; Williams, T A

    2015-04-10

    Segmented animals are found in major clades as phylogenetically distant as vertebrates and arthropods. Typically, segments form sequentially in what has been thought to be a regular process, relying on a segmentation clock to pattern budding segments and posterior mitosis to generate axial elongation. Here we show that segmentation in Tribolium has phases of variable periodicity during which segments are added at different rates. Furthermore, elongation during a period of rapid posterior segment addition is driven by high rates of cell rearrangement, demonstrated by differential fates of marked anterior and posterior blastoderm cells. A computational model of this period successfully reproduces elongation through cell rearrangement in the absence of cell division. Unlike current models of steady-state sequential segmentation and elongation from a proliferative growth zone, our results indicate that cell behaviours are dynamic and variable, corresponding to differences in segmentation rate and giving rise to morphologically distinct regions of the embryo.

  3. Automated counting of morphologically normal red blood cells by using digital holographic microscopy and statistical methods

    Science.gov (United States)

    Moon, Inkyu; Yi, Faliu

    2015-09-01

    In this paper we overview a method to automatically count morphologically normal red blood cells (RBCs) by using off-axis digital holographic microscopy and statistical methods. Three kinds of RBC are used as training and testing data. All of the RBC phase images are obtained with digital holographic microscopy (DHM) that is robust to transparent or semitransparent biological cells. For the determination of morphologically normal RBCs, the RBC's phase images are first segmented with marker-controlled watershed transform algorithm. Multiple features are extracted from the segmented cells. Moreover, the statistical method of Hotelling's T-square test is conducted to show that the 3D features from 3D imaging method can improve the discrimination performance for counting of normal shapes of RBCs. Finally, the classifier is designed by using statistical Bayesian algorithm and the misclassification rates are measured with leave-one-out technique. Experimental results show the feasibility of the classification method for calculating the percentage of each typical normal RBC shape.

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

    Science.gov (United States)

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

    2009-11-26

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

  5. Atomic force microscopy for the examination of single cell rheology.

    Science.gov (United States)

    Okajima, Takaharu

    2012-11-01

    Rheological properties of living cells play important roles in regulating their various biological functions. Therefore, measuring cell rheology is crucial for not only elucidating the relationship between the cell mechanics and functions, but also mechanical diagnosis of single cells. Atomic force microscopy (AFM) is becoming a useful technique for single cell diagnosis because it allows us to measure the rheological properties of adherent cells at any region on the surface without any modifications. In this review, we summarize AFM techniques for examining single cell rheology in frequency and time domains. Recent applications of AFM for investigating the statistical analysis of single cell rheology in comparison to other micro-rheological techniques are reviewed, and we discuss what specificity and universality of cell rheology are extracted using AFM.

  6. 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.

  7. Electron microscopy in cell biology: integrating structure and function

    NARCIS (Netherlands)

    Koster, A.J.; Klumperman, J.

    2003-01-01

    Electron microscopy (EM) is at the highest-resolution limit of a spectrum of complementary morphological techniques. When combined with molecular detection methods, EM is the only technique with sufficient resolution to localize proteins to small membrane subdomains in the context of the cell. Recen

  8. 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

  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. Single molecule microscopy in 3D cell cultures and tissues.

    Science.gov (United States)

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy.

  12. Bright field microscopy as an alternative to whole cell fluorescence in automated analysis of macrophage images.

    Directory of Open Access Journals (Sweden)

    Jyrki Selinummi

    Full Text Available BACKGROUND: Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes, overlapping excitation and emission spectra of the stains, and phototoxicity. METHODOLOGY: We here present and validate a method to automatically detect cell population outlines directly from bright field images. By imaging samples with several focus levels forming a bright field -stack, and by measuring the intensity variations of this stack over the -dimension, we construct a new two dimensional projection image of increased contrast. With additional information for locations of each cell, such as stained nuclei, this bright field projection image can be used instead of whole cell fluorescence to locate borders of individual cells, separating touching cells, and enabling single cell analysis. Using the popular CellProfiler freeware cell image analysis software mainly targeted for fluorescence microscopy, we validate our method by automatically segmenting low contrast and rather complex shaped murine macrophage cells. SIGNIFICANCE: The proposed approach frees up a fluorescence channel, which can be used for subcellular studies. It also facilitates cell shape measurement in experiments where whole cell fluorescent staining is either not available, or is dependent on a particular experimental condition. We show that whole cell area detection results using our projected bright field images match closely to the standard approach where cell areas are localized using fluorescence, and conclude that the high contrast bright field projection image can directly replace one fluorescent channel in whole cell quantification. Matlab code for calculating the projections can be downloaded from the supplementary site: http://sites.google.com/site/brightfieldorstaining.

  13. 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.

  14. 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...

  15. Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy.

    Science.gov (United States)

    Chen, Weili; Long, Kenneth D; Yu, Hojeong; Tan, Yafang; Choi, Ji Sun; Harley, Brendan A; Cunningham, Brian T

    2014-11-21

    We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives.

  16. 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.

  17. 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.

  18. 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.

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

    DEFF Research Database (Denmark)

    Liesche, Johannes; Schulz, Alexander

    2015-01-01

    cell wall interface. Transport through plasmodesmata, the cell wall channels that directly connect plant cells, is regulated not only by a fixed size exclusion limit, but also by physiological and pathological adaptation. The noninvasive approach described here offers the possibility of precisely......Movement of nutrients and signaling compounds from cell to cell is an essential process for plant growth and development. To understand processes such as carbon allocation, cell communication, and reaction to pathogen attack it is important to know a specific molecule’s capacity to pass a specific...... determining the plasmodesmata-mediated cell wall permeability for small molecules in living cells. The method is based on photoactivation of the fluorescent tracer caged fluorescein. Non-fluorescent caged fluorescein is applied to a target tissue, where it is taken up passively into all cells. Imaged...

  20. 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.

  1. 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.

  2. Preparation of Drosophila S2 cells for Light Microscopy

    Science.gov (United States)

    Buster, Daniel W.; Nye, Jonathan; Klebba, Joseph E.; Rogers, Gregory C.

    2010-01-01

    The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells. S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24°C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks. Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is

  3. STED microscopy of living cells--new frontiers in membrane and neurobiology.

    Science.gov (United States)

    Eggeling, Christian; Willig, Katrin I; Barrantes, Francisco J

    2013-07-01

    Recent developments in fluorescence far-field microscopy such as STED microscopy have accomplished observation of the living cell with a spatial resolution far below the diffraction limit. Here, we briefly review the current approaches to super-resolution optical microscopy and present the implementation of STED microscopy for novel insights into live cell mechanisms, with a focus on neurobiology and plasma membrane dynamics.

  4. 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

  5. Correlating intravital multi-photon microscopy to 3D electron microscopy of invading tumor cells using anatomical reference points.

    Directory of Open Access Journals (Sweden)

    Matthia A Karreman

    Full Text Available Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis.

  6. Poroelasticity of cell nuclei revealed through atomic force microscopy characterization

    Science.gov (United States)

    Wei, Fanan; Lan, Fei; Liu, Bin; Liu, Lianqing; Li, Guangyong

    2016-11-01

    With great potential in precision medical application, cell biomechanics is rising as a hot topic in biology. Cell nucleus, as the largest component within cell, not only contributes greatly to the cell's mechanical behavior, but also serves as the most vital component within cell. However, cell nucleus' mechanics is still far from unambiguous up to now. In this paper, we attempted to characterize and evaluate the mechanical property of isolated cell nuclei using Atomic Force Microscopy with a tipless probe. As indicated from typical indentation, changing loading rate and stress relaxation experiment results, cell nuclei showed significant dynamically mechanical property, i.e., time-dependent mechanics. Furthermore, through theoretical analysis, finite element simulation and stress relaxation experiment, the nature of nucleus' mechanics was better described by poroelasticity, rather than viscoelasticity. Therefore, the essence of nucleus' mechanics was clarified to be poroelastic through a sophisticated analysis. Finally, we estimated the poroelastic parameters for nuclei of two types of cells through a combination of experimental data and finite element simulation.

  7. 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.

  8. 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.

  9. Segmentation of the Clustered Cells with Optimized Boundary Detection in Negative Phase Contrast Images.

    Directory of Open Access Journals (Sweden)

    Yuliang Wang

    Full Text Available Cell image segmentation plays a central role in numerous biology studies and clinical applications. As a result, the development of cell image segmentation algorithms with high robustness and accuracy is attracting more and more attention. In this study, an automated cell image segmentation algorithm is developed to get improved cell image segmentation with respect to cell boundary detection and segmentation of the clustered cells for all cells in the field of view in negative phase contrast images. A new method which combines the thresholding method and edge based active contour method was proposed to optimize cell boundary detection. In order to segment clustered cells, the geographic peaks of cell light intensity were utilized to detect numbers and locations of the clustered cells. In this paper, the working principles of the algorithms are described. The influence of parameters in cell boundary detection and the selection of the threshold value on the final segmentation results are investigated. At last, the proposed algorithm is applied to the negative phase contrast images from different experiments. The performance of the proposed method is evaluated. Results show that the proposed method can achieve optimized cell boundary detection and highly accurate segmentation for clustered cells.

  10. Segmentation of the Clustered Cells with Optimized Boundary Detection in Negative Phase Contrast Images.

    Science.gov (United States)

    Wang, Yuliang; Zhang, Zaicheng; Wang, Huimin; Bi, Shusheng

    2015-01-01

    Cell image segmentation plays a central role in numerous biology studies and clinical applications. As a result, the development of cell image segmentation algorithms with high robustness and accuracy is attracting more and more attention. In this study, an automated cell image segmentation algorithm is developed to get improved cell image segmentation with respect to cell boundary detection and segmentation of the clustered cells for all cells in the field of view in negative phase contrast images. A new method which combines the thresholding method and edge based active contour method was proposed to optimize cell boundary detection. In order to segment clustered cells, the geographic peaks of cell light intensity were utilized to detect numbers and locations of the clustered cells. In this paper, the working principles of the algorithms are described. The influence of parameters in cell boundary detection and the selection of the threshold value on the final segmentation results are investigated. At last, the proposed algorithm is applied to the negative phase contrast images from different experiments. The performance of the proposed method is evaluated. Results show that the proposed method can achieve optimized cell boundary detection and highly accurate segmentation for clustered cells.

  11. 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.

  12. Segmented polymer electrolyte membrane fuel cells - A review

    Energy Technology Data Exchange (ETDEWEB)

    Perez, Luis C.; Brandao, Lucia; Mendes, Adelio [LEPAE, Chemical Engineering Department, Faculty of Engineering, University of Porto, Rua Roberto Frias, 4200-465 Porto (Portugal); Sousa, Jose M. [LEPAE, Chemical Engineering Department, Faculty of Engineering, University of Porto, Rua Roberto Frias, 4200-465 Porto (Portugal); Chemistry Department, University of Tras-os-Montes e Alto Douro, Apartado 202, 5001-911 Vila-Real Codex (Portugal)

    2011-01-15

    A complex interaction of many design, assembling and operating parameters as well as the properties of the materials used in the construction of polymer electrolyte membrane fuel cells (PEMFC) result in an uneven electrochemical performance over the MEA active area. For more than one decade, segmented PEMFC (SFC) have been used to study the factors responsible for that uneven performance. This paper reviews relevant literature related to SFC published since 1998 focusing on the three most important SFC design techniques: (1) printed circuit board, (2) resistors network and (3) Hall effect sensors. First, the three techniques are described and fundamental considerations for its design, construction and electrochemical characterization are provided. After that, the effect of most important parameters on the current density distribution is highlighted. Finally, representative results combining current density distribution measurements with other analytical techniques for distributed analysis are presented. (author)

  13. A Marked Poisson Process Driven Latent Shape Model for 3D Segmentation of Reflectance Confocal Microscopy Image Stacks of Human Skin.

    Science.gov (United States)

    Ghanta, Sindhu; Jordan, Michael I; Kose, Kivanc; Brooks, Dana H; Rajadhyaksha, Milind; Dy, Jennifer G

    2017-01-01

    Segmenting objects of interest from 3D data sets is a common problem encountered in biological data. Small field of view and intrinsic biological variability combined with optically subtle changes of intensity, resolution, and low contrast in images make the task of segmentation difficult, especially for microscopy of unstained living or freshly excised thick tissues. Incorporating shape information in addition to the appearance of the object of interest can often help improve segmentation performance. However, the shapes of objects in tissue can be highly variable and design of a flexible shape model that encompasses these variations is challenging. To address such complex segmentation problems, we propose a unified probabilistic framework that can incorporate the uncertainty associated with complex shapes, variable appearance, and unknown locations. The driving application that inspired the development of this framework is a biologically important segmentation problem: the task of automatically detecting and segmenting the dermal-epidermal junction (DEJ) in 3D reflectance confocal microscopy (RCM) images of human skin. RCM imaging allows noninvasive observation of cellular, nuclear, and morphological detail. The DEJ is an important morphological feature as it is where disorder, disease, and cancer usually start. Detecting the DEJ is challenging, because it is a 2D surface in a 3D volume which has strong but highly variable number of irregularly spaced and variably shaped "peaks and valleys." In addition, RCM imaging resolution, contrast, and intensity vary with depth. Thus, a prior model needs to incorporate the intrinsic structure while allowing variability in essentially all its parameters. We propose a model which can incorporate objects of interest with complex shapes and variable appearance in an unsupervised setting by utilizing domain knowledge to build appropriate priors of the model. Our novel strategy to model this structure combines a spatial Poisson

  14. 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.

  15. Bioluminescence microscopy: application to ATP measurements in single living cells

    Science.gov (United States)

    Brau, Frederic; Helle, Pierre; Bernengo, Jean C.

    1997-12-01

    Bioluminescence microscopy can be used to measure intracellular cofactors and ionic concentrations (Ca2+, K+, ATP, NADH), as an alternative to micro- spectrophotometry and micro-fluorimetry, due to the development of sensitive detectors (cooled photomultipliers tubes and CCD). The main limitation comes from the very small and brief intensity of the emitted light. Our instrumentation based on an inverted microscope, equipped with high aperture immersion lenses is presented. Light intensity measurements are carried out through a photomultiplier sorted for low dark current and cooled at -5 degree(s)C to reduce thermal noise. Our first aim is to quantify ATP on single living cells using the firefly luciferin-luciferase couple. Experimental and kinetic aspects are presented to emphasize the potentialities of the technique.

  16. 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.

  17. 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.

  18. 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

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

    Science.gov (United States)

    Hatipoglu, Nuh; Bilgin, Gokhan

    2017-02-28

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

  20. Integrating Real-Time Analysis With The Dendritic Cell Algorithm Through Segmentation

    CERN Document Server

    Gu, Feng; Aickelin, Uwe

    2010-01-01

    As an immune inspired algorithm, the Dendritic Cell Algorithm (DCA) has been applied to a range of problems, particularly in the area of intrusion detection. Ideally, the intrusion detection should be performed in real-time, to continuously detect misuses as soon as they occur. Consequently, the analysis process performed by an intrusion detection system must operate in real-time or near-to real-time. The analysis process of the DCA is currently performed offline, therefore to improve the algorithm's performance we suggest the development of a real-time analysis component. The initial step of the development is to apply segmentation to the DCA. This involves segmenting the current output of the DCA into slices and performing the analysis in various ways. Two segmentation approaches are introduced and tested in this paper, namely antigen based segmentation (ABS) and time based segmentation (TBS). The results of the corresponding experiments suggest that applying segmentation produces different and significantl...

  1. 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.

  2. Segmented filamentous bacteria antigens presented by intestinal dendritic cells drive mucosal Th17 cell differentiation.

    Science.gov (United States)

    Goto, Yoshiyuki; Panea, Casandra; Nakato, Gaku; Cebula, Anna; Lee, Carolyn; Diez, Marta Galan; Laufer, Terri M; Ignatowicz, Leszek; Ivanov, Ivaylo I

    2014-04-17

    How commensal microbiota contributes to immune cell homeostasis at barrier surfaces is poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by commensal segmented filamentous bacteria (SFB). Here we show that MHCII-dependent antigen presentation of SFB antigens by intestinal dendritic cells (DCs) is crucial for Th17 cell induction. Expression of MHCII on CD11c(+) cells was necessary and sufficient for SFB-induced Th17 cell differentiation. Most SFB-induced Th17 cells recognized SFB in an MHCII-dependent manner. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. The importance of other innate cells was unveiled by the finding that MHCII deficiency in group 3 innate lymphoid cells (ILCs) resulted in an increase in SFB-independent Th17 cell differentiation. Our results outline the complex role of DCs and ILCs in the regulation of intestinal Th17 cell homeostasis.

  3. Synchronizing atomic force microscopy force mode and fluorescence microscopy in real time for immune cell stimulation and activation studies

    Energy Technology Data Exchange (ETDEWEB)

    Cazaux, Séverine; Sadoun, Anaïs; Biarnes-Pelicot, Martine; Martinez, Manuel; Obeid, Sameh [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France); Bongrand, Pierre [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France); APHM, Hôpital de la Conception, Laboratoire d’Immunologie, Marseille F-13385 (France); Limozin, Laurent [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France); Puech, Pierre-Henri, E-mail: pierre-henri.puech@inserm.fr [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France)

    2016-01-15

    A method is presented for combining atomic force microscopy (AFM) force mode and fluorescence microscopy in order to (a) mechanically stimulate immune cells while recording the subsequent activation under the form of calcium pulses, and (b) observe the mechanical response of a cell upon photoactivation of a small G protein, namely Rac. Using commercial set-ups and a robust signal coupling the fluorescence excitation light and the cantilever bending, the applied force and activation signals were very easily synchronized. This approach allows to control the entire mechanical history of a single cell up to its activation and response down to a few hundreds of milliseconds, and can be extended with very minimal adaptations to other cellular systems where mechanotransduction is studied, using either purely mechanical stimuli or via a surface bound specific ligand. - Highlights: • A signal coupling AFM and fluorescence microscopy was characterized for soft cantilevers. • It can be used as an intrinsic timer to synchronize images and forces. • Mechanical stimulation of single immune cells while recording calcium fluxes was detailed. • Light-induced mechanical modifications of lymphocytes using a PA-Rac protein were demonstrated. • The precautions and limitations of use of this effect were presented.

  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. 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.

  6. 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.

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

    NARCIS (Netherlands)

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

    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

  8. Blood Cell Segmentation Based on Improved Pulse Coupled Neural Network and Fuzzy Entropy

    Directory of Open Access Journals (Sweden)

    Zhanbo Liu

    2016-12-01

    Full Text Available In the field of biomedical image processing, because of the low intensity and brightness of the cell image, and the complex structure of the cell image, the segmentation of cell images is very difficult. A large number of studies have shown that the Pulse Coupled Neural Networks (PCNN is suitable for image segmentation. However, the traditional PCNN must set a large number of parameters in image segmentation, and the optimal number of iterations cannot be automatically determined. In this paper, a new improved PCNN model is proposed. The work of improved PCNN includes the acceptance portion of the PCNN model being simplified and the connection portion of PCNN being improved. In addition, the maximum fuzzy entropy is used as the criterion to determine the optimal number of iterations. Experimental results on blood cell image segmentation show that this proposed method can automatically determine the number of loop iterations and automatically select the best threshold. It also has the characteristics of fast convergence, high accuracy and good segmentation effect in blood cell image segmentation processing.

  9. Cell wall modification in grapevine cells in response to UV stress investigated by atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lesniewska, E.; Adrian, M.; Klinguer, A.; Pugin, A

    2004-08-15

    Despite cell wall reinforcement being a well-known defence mechanism of plants, it remains poorly characterized from a physical point of view. The objective of this work was to further describe this mechanism. Vitis vinifera cv Gamay cells were treated with UV-light (254 nm), a well-known elicitor of defence mechanisms in grapevines, and physical cell wall modifications were observed using the atomic force microscopy (AFM) under native conditions. The grapevine cell suspensions were continuously observed in their culture medium from 30 min to 24 h after elicitation. In the beginning, cellulose fibrils covered by a matrix surrounded the control and treated cells. After 3 h, the elicited cells displayed sprouted expansions around the cell wall that correspond to pectin chains. These expansions were not observed on untreated grapevine cells. The AFM tip was used to determine the average surface elastic modulus of cell wall that account for cell wall mechanical properties. The elasticity is diminished in UV-treated cells. In a comparative study, grapevine cells showed the same decrease in cell wall elasticity when treated with a fungal biotic elicitor of defence response. These results demonstrate cell wall strengthening by UV stress.

  10. Serial block face scanning electron microscopy--the future of cell ultrastructure imaging.

    Science.gov (United States)

    Hughes, Louise; Hawes, Chris; Monteith, Sandy; Vaughan, Sue

    2014-03-01

    One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

  11. Induction of morphological changes in death-induced cancer cells monitored by holographic microscopy.

    Science.gov (United States)

    El-Schich, Zahra; Mölder, Anna; Tassidis, Helena; Härkönen, Pirkko; Falck Miniotis, Maria; Gjörloff Wingren, Anette

    2015-03-01

    We are using the label-free technique of holographic microscopy to analyze cellular parameters including cell number, confluence, cellular volume and area directly in the cell culture environment. We show that death-induced cells can be distinguished from untreated counterparts by the use of holographic microscopy, and we demonstrate its capability for cell death assessment. Morphological analysis of two representative cell lines (L929 and DU145) was performed in the culture flasks without any prior cell detachment. The two cell lines were treated with the anti-tumour agent etoposide for 1-3days. Measurements by holographic microscopy showed significant differences in average cell number, confluence, volume and area when comparing etoposide-treated with untreated cells. The cell volume of the treated cell lines was initially increased at early time-points. By time, cells decreased in volume, especially when treated with high doses of etoposide. In conclusion, we have shown that holographic microscopy allows label-free and completely non-invasive morphological measurements of cell growth, viability and death. Future applications could include real-time monitoring of these holographic microscopy parameters in cells in response to clinically relevant compounds.

  12. 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.

  13. 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.

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

    Science.gov (United States)

    Millaku, Agron; Drobne, Damjana; Torkar, Matjaz; Novak, Sara; Remškar, Maja; Pipan-Tkalec, Živa

    2013-09-15

    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.

  15. Attached segment has higher CD34+ cells and CFU-GM than the main bag after thawing.

    Science.gov (United States)

    Lee, Hye Ryun; Shin, Sue; Yoon, Jong Hyun; Roh, Eun Youn; Song, Eun Young; Han, Kyou Sup; Kim, Byoung Jae

    2015-01-01

    A contiguous segment attached to the cord blood unit (CBU) is required for verifying HLA types, cell viability, and, possibly, potency before transplantation since such a segment is considered to be representative of the CBU. However, little is known regarding the characteristics of contiguous segments in comparison to main bag units due to the difficulty experienced in accessing a large number of cryopreserved CBUs. In this study, we used 245 nonconforming CBUs for allogeneic transplantation. After thawing the cryopreserved CBU, the number of total nucleated cells (TNCs), CD34(+) cells, and CFUs in CB from main bags and segments, as well as cell viability and apoptosis, were examined. The comparative analysis showed that the number of TNCs was significantly higher in CB from main bags, whereas the numbers of CD34(+) cells and CFU-GM were significantly higher in CB from segments. While the cell viability of TNCs in segments was higher, the proportion of apoptotic TNCs was also higher. In contrast, no difference was observed between the proportion of apoptotic CD34(+) cells in main bags and segments. In the correlation analysis, the numbers of TNCs, CD34(+) cells, and CFU-GM in main bags were highly correlated with those in segments, indicating that CB from segments is indeed representative of CB in main bags. Taken together, we conclude that segments have higher CD34(+) cells and CFU-GM and lower TNCs than the main cryopreserved bag, although the two compartments are highly correlated with each other.

  16. 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

  17. 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.

  18. Active contour-based cell segmentation during freezing and its application in cryopreservation.

    Science.gov (United States)

    Wu, Pengxiang; Yi, Jingru; Zhao, Gang; Huang, Zhangjin; Qiu, Bensheng; Gao, Dayong

    2015-01-01

    Water permeability of the plasma membrane plays an important role in making optimal cryopreservation protocols for different types of cells. To quantify water permeability effectively, automated cell volume segmentation during freezing is necessary. Unfortunately, there exists so far no efficient and accurate segmentation method to handle this kind of image processing task gracefully. The existence of extracellular ice and variable background present significant challenges for most traditional segmentation algorithms. In this paper, we propose a novel approach to reliably extract cells from the extracellular ice, which attaches to or surrounds cells. Our method operates on temporal image sequences and is composed of two steps. First, for each image from the sequence, a greedy search strategy is employed to track approximate locations of cells in motion. Second, we utilize a localized competitive active contour model to obtain the contour of each cell. Based on the first step's result, the initial contour for level set evolution can be determined appropriately, thus considerably easing the pain of initialization for an active contour model. Experimental results demonstrate that the proposed method is efficient and effective in segmenting cells during freezing.

  19. Low impact to fixed cell processing aiming transmission electron microscopy

    Science.gov (United States)

    Barth, Ortrud Monika; da Silva, Marcos Alexandre Nunes; Barreto-Vieira, Debora Ferreira

    2016-01-01

    In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible. PMID:27276186

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

    Science.gov (United States)

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

    2014-11-01

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

  1. 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

  2. Confocal supercritical angle fluorescence microscopy for cell membrane imaging

    CERN Document Server

    Sivankutty, Siddharth; Mayet, Céline; Dupuis, Guillaume; Fort, Emmanuel; Lévêque-Fort, Sandrine

    2013-01-01

    We demonstrate sub-wavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fuorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modi?cation on the detection channel. Confocal-SAF microscopy would be a powerful tool to achieve high resolution surface imaging, especially for membrane imaging in biological samples

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 5D imaging via light sheet microscopy reveals cell dynamics during the eye-antenna disc primordium formation in Drosophila

    Science.gov (United States)

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

    2017-03-01

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

  10. 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.

  11. 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).

  12. Segmentation of White Blood Cells through Nucleus Mark Watershed Operations and Mean Shift Clustering

    Directory of Open Access Journals (Sweden)

    Zhi Liu

    2015-09-01

    Full Text Available This paper presents a novel method for segmentation of white blood cells (WBCs in peripheral blood and bone marrow images under different lights through mean shift clustering, color space conversion and nucleus mark watershed operation (NMWO. The proposed method focuses on obtaining seed points. First, color space transformation and image enhancement techniques are used to obtain nucleus groups as inside seeds. Second, mean shift clustering, selection of the C channel component in the CMYK model, and illumination intensity adjustment are employed to acquire WBCs as outside seeds. Third, the seeds and NMWO are employed to precisely determine WBCs and solve the cell adhesion problem. Morphological operations are further used to improve segmentation accuracy. Experimental results demonstrate that the algorithm exhibits higher segmentation accuracy and robustness compared with traditional methods.

  13. 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...

  14. 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.

  15. 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

    Exposure to small size particulates is regarded as a risk factor for cardiovascular disease. We investigated effects of exposure to nanosized carbon black (CB) in human umbilical vein endothelial cells (HUVECs) and segments of arteries from rodents. The CB exposure was associated with increased...

  16. 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

    Schwann cell proliferation was studied in cultured segments of the rat sciatic nerve by measurement of [3H] thymidine incorporation or through bromodeoxyuridine-(BrdU)-labelling and immunocytochemistry. The aim was to delineate mechanisms involved in the injury-induced proliferative response...

  17. 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

  18. 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+.

  19. 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.

  20. 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.

  1. Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy.

    Science.gov (United States)

    Jiang, Huaidong; Song, Changyong; Chen, Chien-Chun; Xu, Rui; Raines, Kevin S; Fahimian, Benjamin P; Lu, Chien-Hung; Lee, Ting-Kuo; Nakashima, Akio; Urano, Jun; Ishikawa, Tetsuya; Tamanoi, Fuyuhiko; Miao, Jianwei

    2010-06-22

    Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3-5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50-60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5-10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.

  2. Three-dimensional counting of morphologically normal human red blood cells via digital holographic microscopy

    Science.gov (United States)

    Yi, Faliu; Moon, Inkyu; Lee, Yeon H.

    2015-01-01

    Counting morphologically normal cells in human red blood cells (RBCs) is extremely beneficial in the health care field. We propose a three-dimensional (3-D) classification method of automatically determining the morphologically normal RBCs in the phase image of multiple human RBCs that are obtained by off-axis digital holographic microscopy (DHM). The RBC holograms are first recorded by DHM, and then the phase images of multiple RBCs are reconstructed by a computational numerical algorithm. To design the classifier, the three typical RBC shapes, which are stomatocyte, discocyte, and echinocyte, are used for training and testing. Nonmain or abnormal RBC shapes different from the three normal shapes are defined as the fourth category. Ten features, including projected surface area, average phase value, mean corpuscular hemoglobin, perimeter, mean corpuscular hemoglobin surface density, circularity, mean phase of center part, sphericity coefficient, elongation, and pallor, are extracted from each RBC after segmenting the reconstructed phase images by using a watershed transform algorithm. Moreover, four additional properties, such as projected surface area, perimeter, average phase value, and elongation, are measured from the inner part of each cell, which can give significant information beyond the previous 10 features for the separation of the RBC groups; these are verified in the experiment by the statistical method of Hotelling's T-square test. We also apply the principal component analysis algorithm to reduce the dimension number of variables and establish the Gaussian mixture densities using the projected data with the first eight principal components. Consequently, the Gaussian mixtures are used to design the discriminant functions based on Bayesian decision theory. To improve the performance of the Bayes classifier and the accuracy of estimation of its error rate, the leaving-one-out technique is applied. Experimental results show that the proposed method can

  3. In SITU Transmission Electron Microscopy on Operating Electrochemical CELLS

    DEFF Research Database (Denmark)

    Gualandris, Fabrizio; Simonsen, Søren Bredmose; Mogensen, Mogens Bjerg

    2016-01-01

    Solid oxide cells (SOC) have the potential of playing a significant role in the future efficient energy system scenario. In order to become widely commercially available, an improved performance and durability of the cells has to be achieved [1]. Conventional scanning and transmission SEM and TEM...... have been often used for ex-situ post mortem characterization of SOFCs and SOECs [2,3]. However, in order to get fundamental insight of the microstructural development of SOFC/SOEC during operation conditions in situ studies are necessary [4]....

  4. 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.

  5. An automated approach to the segmentation of HEp-2 cells for the indirect immunofluorescence ANA test.

    Science.gov (United States)

    Tonti, Simone; Di Cataldo, Santa; Bottino, Andrea; Ficarra, Elisa

    2015-03-01

    The automatization of the analysis of Indirect Immunofluorescence (IIF) images is of paramount importance for the diagnosis of autoimmune diseases. This paper proposes a solution to one of the most challenging steps of this process, the segmentation of HEp-2 cells, through an adaptive marker-controlled watershed approach. Our algorithm automatically conforms the marker selection pipeline to the peculiar characteristics of the input image, hence it is able to cope with different fluorescent intensities and staining patterns without any a priori knowledge. Furthermore, it shows a reduced sensitivity to over-segmentation errors and uneven illumination, that are typical issues of IIF imaging.

  6. Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy.

    Science.gov (United States)

    Puech, Pierre-Henri; Taubenberger, Anna; Ulrich, Florian; Krieg, Michael; Muller, Daniel J; Heisenberg, Carl-Philipp

    2005-09-15

    During vertebrate gastrulation, progenitor cells of different germ layers acquire specific adhesive properties that contribute to germ layer formation and separation. Wnt signals have been suggested to function in this process by modulating the different levels of adhesion between the germ layers, however, direct evidence for this is still lacking. Here we show that Wnt11, a key signal regulating gastrulation movements, is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin, an abundant extracellular matrix component during gastrulation. To measure this effect, we developed an assay to quantify the adhesion of single zebrafish primary mesendodermal progenitors using atomic-force microscopy (AFM). We observed significant differences in detachment force and work between cultured mesendodermal progenitors from wild-type embryos and from slb/wnt11 mutant embryos, which carry a loss-of-function mutation in the wnt11 gene, when tested on fibronectin-coated substrates. These differences were probably due to reduced adhesion to the fibronectin substrate as neither the overall cell morphology nor the cell elasticity grossly differed between wild-type and mutant cells. Furthermore, in the presence of inhibitors of fibronectin-integrin binding, such as RGD peptides, the adhesion force and work were strongly decreased, indicating that integrins are involved in the binding of mesendodermal progenitors in our assay. These findings demonstrate that AFM can be used to quantitatively determine the substrate-adhesion of cultured primary gastrulating cells and provide insight into the role of Wnt11 signalling in modulating cell adhesion at the single cell scale.

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

    Science.gov (United States)

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

    2008-03-01

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

  8. 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.

  9. 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.

  10. Fast Cell Segmentation Using Scalable Sparse Manifold Learning and Affine Transform-approximated Active Contour.

    Science.gov (United States)

    Xing, Fuyong; Yang, Lin

    2015-10-01

    Efficient and effective cell segmentation of neuroendocrine tumor (NET) in whole slide scanned images is a difficult task due to a large number of cells. The weak or misleading cell boundaries also present significant challenges. In this paper, we propose a fast, high throughput cell segmentation algorithm by combining top-down shape models and bottom-up image appearance information. A scalable sparse manifold learning method is proposed to model multiple subpopulations of different cell shape priors. Followed by a shape clustering on the manifold, a novel affine transform-approximated active contour model is derived to deform contours without solving a large amount of computationally-expensive Euler-Lagrange equations, and thus dramatically reduces the computational time. To the best of our knowledge, this is the first report of a high throughput cell segmentation algorithm for whole slide scanned pathology specimens using manifold learning to accelerate active contour models. The proposed approach is tested using 12 NET images, and the comparative experiments with the state of the arts demonstrate its superior performance in terms of both efficiency and effectiveness.

  11. 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

  12. High-Resolution Transmission Electron Microscopy Observation of Colloidal Nanocrystal Growth Mechanisms using Graphene Liquid Cells

    Energy Technology Data Exchange (ETDEWEB)

    Yuk, Jong Min; Park, Jungwon; Ercius, Peter; Kim, Kwanpyo; Hellebusch, Danny J.; Crommie, Michael F.; Lee, Jeong Yong; Zettl, A.; Alivisatos, A. Paul

    2011-12-12

    We introduce a new type of liquid cell for in-situ electron microscopy based upon entrapment of a liquid film between layers of graphene. We employ this cell to achieve high-resolution imaging of colloidal platinum nanocrystal growth. The ability to directly image and resolve critical steps at atomic resolution provides new insights into nanocrystal coalescence and reshaping during growth.

  13. 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.

  14. 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.

  15. Electron Microscopy to Correlate Cell Structure and Biochemical Activity

    Science.gov (United States)

    1993-03-10

    Plasmodium coatneyi-infected rhesus monkeys: a primate model for human cerebral malaria. Memorias do Instituto Osvald Cruz (In Press). 24. Sim, K.L...P. coatney1 and found th.’t cvtoadherence of PRBC to ei.dothelial cells is a consistent feature of infections with this primate parasite. Cerebral ...Malaria; iaraunoe loct ror.-micros.’opy ; Chemotherapy; DFO; Cerebral malaria; HA 1 17 SECURITY CLASSIFICATION Of REPORT Unclass1 fled 1

  16. 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.

  17. 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.

  18. Monitoring the elasticity changes of HeLa cells during mitosis by atomic force microscopy

    Science.gov (United States)

    Jiang, Ningcheng; Wang, Yuhua; Zeng, Jinshu; Ding, Xuemei; Xie, Shusen; Yang, Hongqin

    2016-10-01

    Cell mitosis plays a crucial role in cell life activity, which is one of the important phases in cell division cycle. During the mitosis, the cytoskeleton micro-structure of the cell changed and the biomechanical properties of the cell may vary depending upon different mitosis stages. In this study, the elasticity property of HeLa cells during mitosis was monitored by atomic force microscopy. Also, the actin filaments in different mitosis stages of the cells were observed by confocal imaging. Our results show that the cell in anaphase is stiffer than that in metaphase and telophase. Furthermore, lots of actin filaments gathered in cells' center area in anaphase, which contributes to the rigidity of the cell in this phase. Our findings demonstrate that the nano-biomechanics of living cells could provide a new index for characterizing cell physiological states.

  19. In situ visualization of intracellular morphology of epidermal cells using stimulated Raman scattering microscopy

    Science.gov (United States)

    Egawa, Mariko; Tokunaga, Kyoya; Hosoi, Junichi; Iwanaga, Shinya; Ozeki, Yasuyuki

    2016-08-01

    Visualization of epidermal cells is important because the differentiation patterns of keratinocytes (KCs) are considered to be related to the functions and condition of skin. Optical microscopy has been widely used to investigate epidermal cells, but its applicability is still limited because of the need for sample fixation and staining. Here, we report our staining-free observation of epidermal cells in both tissue and culture by stimulated Raman scattering (SRS) microscopy that provides molecular vibrational contrast. SRS allowed us to observe a variety of cellular morphologies in skin tissue, including ladder-like structures in the spinous layer, enucleation of KCs in the granular layer, and three-dimensional cell column structures in the stratum corneum. We noticed that some cells in the spinous layer had a brighter signal in the cytoplasm than KCs. To examine the relevance of the observation of epidermal layers, we also observed cultured epidermal cells, including KCs at various differentiation stages, melanocytes, and Langerhans cell-like cells. Their SRS images also demonstrated various morphologies, suggesting that the morphological differences observed in tissue corresponded to the cell lineage. These results indicate the possible application of SRS microscopy to dermatological investigation of cell lineages and types in the epidermis by cellular-level analysis.

  20. 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

  1. 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.

  2. 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.

  3. STED super-resolution microscopy in Drosophila tissue and in mammalian cells

    Science.gov (United States)

    Lau, Lana; Lee, Yin Loon; Matis, Maja; Axelrod, Jeff; Stearns, Tim; Moerner, W. E.

    2011-03-01

    Far-field super-resolution microscopy is a rapidly emerging method that is opening up opportunities for biological imaging beyond the optical diffraction limit. We have implemented a Stimulated Emission Depletion (STED) microscope to image single dye, cell, and tissue samples with 50-80 nm resolution. First, we compare the STED performance imaging single molecules of several common dyes and report a novel STED dye. Then we apply STED to image planar cell polarity protein complexes in intact fixed Drosophila tissue for the first time. Finally, we present a preliminary study of the centrosomal protein Cep164 in mammalian cells. Our images suggest that Cep164 is arranged in a nine-fold symmetric pattern around the centriole, consistent with findings suggested by cryoelectron tomography. Our work demonstrates that STED microscopy can be used for superresolution imaging in intact tissue and provides ultrastructural information in biological samples as an alternative to immuno-electron microscopy.

  4. Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Koh, Ai Leen [Department of Materials Science and Engineering, Stanford University, Durand Building Room 139, 496 Lomita Mall, Stanford, CA 94305 (United States); Stanford Nanocharacterization Laboratory, Stanford University, Stanford, CA 94305 (United States); Department of Mechanical Engineering, Stanford University, Stanford, CA 94305 (United States)], E-mail: alkoh@stanford.edu; Shachaf, Catherine M.; Elchuri, Sailaja; Nolan, Garry P. [Baxter Laboratory in Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305 (United States); Sinclair, Robert [Department of Materials Science and Engineering, Stanford University, Durand Building Room 139, 496 Lomita Mall, Stanford, CA 94305 (United States); Stanford Nanocharacterization Laboratory, Stanford University, Stanford, CA 94305 (United States)

    2008-12-15

    We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.

  5. 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.

  6. Multi-resolution cell orientation congruence descriptors for epithelium segmentation in endometrial histology images.

    Science.gov (United States)

    Li, Guannan; Raza, Shan E Ahmed; Rajpoot, Nasir M

    2017-04-01

    It has been recently shown that recurrent miscarriage can be caused by abnormally high ratio of number of uterine natural killer (UNK) cells to the number of stromal cells in human female uterus lining. Due to high workload, the counting of UNK and stromal cells needs to be automated using computer algorithms. However, stromal cells are very similar in appearance to epithelial cells which must be excluded in the counting process. To exclude the epithelial cells from the counting process it is necessary to identify epithelial regions. There are two types of epithelial layers that can be encountered in the endometrium: luminal epithelium and glandular epithelium. To the best of our knowledge, there is no existing method that addresses the segmentation of both types of epithelium simultaneously in endometrial histology images. In this paper, we propose a multi-resolution Cell Orientation Congruence (COCo) descriptor which exploits the fact that neighbouring epithelial cells exhibit similarity in terms of their orientations. Our experimental results show that the proposed descriptors yield accurate results in simultaneously segmenting both luminal and glandular epithelium.

  7. Spectral and lifetime fluorescence imaging microscopies: new modalities of multiphoton microscopy applied to tissue or cell engineering.

    Science.gov (United States)

    Dumas, D; Gaborit, N; Grossin, L; Riquelme, B; Gigant-Huselstein, C; De Isla, N; Gillet, P; Netter, P; Stoltz, J F

    2004-01-01

    Spectral and multiphoton imaging is the preferred approach for non-invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross-talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM-FRET).

  8. Functional screening of intracardiac cell transplants using two-photon fluorescence microscopy.

    Science.gov (United States)

    Tao, Wen; Soonpaa, Mark H; Field, Loren J; Chen, Peng-Sheng; Firulli, Anthony B; Shou, Weinian; Rubart, Michael

    2012-08-01

    Although the adult mammalian myocardium exhibits a limited ability to undergo regenerative growth, its intrinsic renewal rate is insufficient to compensate for myocyte loss during cardiac disease. Transplantation of donor cardiomyocytes or cardiomyogenic stem cells is considered a promising strategy for reconstitution of cardiac mass, provided the engrafted cells functionally integrate with host myocardium and actively contribute to its contractile force. The authors previously developed a two-photon fluorescence microscopy-based assay that allows in situ screening of donor cell function after intracardiac delivery of the cells. This report reviews the techniques of two-photon fluorescence microscopy and summarizes its application for quantifying the extent to which a variety of donor cell types stably and functionally couple with the recipient myocardium.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. Cytogenetic Characterization of the TM4 Mouse Sertoli Cell Line. II. Chromosome Microdissection, FISH, Scanning Electron Microscopy, and Confocal Laser Scanning Microscopy.

    Science.gov (United States)

    Schmid, Michael; Guttenbach, Martina; Steinlein, Claus; Wanner, Gerhard; Houben, Andreas

    2015-01-01

    The chromosomes and interphase cell nuclei of the permanent mouse Sertoli cell line TM4 were examined by chromosome microdissection, FISH, scanning electron microscopy, and confocal laser scanning microscopy. The already known marker chromosomes m1-m5 were confirmed, and 2 new large marker chromosomes m6 and m7 were characterized. The minute heterochromatic marker chromosomes m4 and m5 were microdissected and their DNA amplified by DOP-PCR. FISH of this DNA probe on TM4 metaphase chromosomes demonstrated that the m4 and m5 marker chromosomes have derived from the centromeric regions of normal telocentric mouse chromosomes. Ectopic pairing of the m4 and m5 marker chromosomes with the centromeric region of any of the other chromosomes (centromeric associations) was apparent in ∼60% of the metaphases. Scanning electron microscopy revealed DNA-protein bridges connecting the centromeric regions of normal chromosomes and the associated m4 and m5 marker chromosomes. Interphase cell nuclei of TM4 Sertoli cells did not exhibit the characteristic morphology of Sertoli cells in the testes of adult mice as shown by fluorescence microscopy and confocal laser scanning microscopy.

  14. 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

  15. 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.

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

    Institute of Scientific and Technical Information of China (English)

    SHAO; Manjun(邵曼君); JIANG; Lei(姜蕾); CONG; Wei(丛威); OUYANG; Fan(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.

  17. Cell lineage analysis of the mandibular segment of the amphipod Orchestia cavimana reveals that the crustacean paragnaths are sternal outgrowths and not limbs

    Directory of Open Access Journals (Sweden)

    Scholtz Gerhard

    2006-12-01

    Full Text Available Abstract The question of arthropod head segmentation has become one of the central issues in Evolutionary Developmental Biology. The number of theories pertaining to head segments progressively enlarges, old concepts have been revitalized, and nearly every conceivable composition of the arthropod head has at some point received discussion. One contentious issue involves a characteristic mouthpart in crustaceans – the lower lips or the so-called paragnaths. The paragnaths build the posterior border of the mouth region antagonistic to the upper lip – the labrum. We show here the development of the appendage-like structures in the mandibular region of the amphipod crustacean Orchestia cavimana at a high level of cellular resolution. The embryos are examined during development of the mouthparts using in vivo labeling. An invariant cell division pattern of the mandibular segment was detected by 4D-microscopy and a preliminary model for pattern of the first cleavages in the mandibular region created. With this indispensable precondition single ectodermal cells of the grid-like pattern were labeled with DiI – a lipophilic fluorescent dye – to trace cell lineages and determine the clonal composition of the developing mouthparts, especially the mandibular segment. From our data it is evident that the paragnaths are sternal outgrowths of the mandible segment. The assumption of the limb nature of paragnaths and the presence of an additional head segment between the mandibular and the second antennal segments are clearly refuted by our data. Our results show the power of cell lineage and clonal analyses for inferences on the nature, origin and thus homology of morphological structures. With this kind of investigation morphological and gene expression data can be complemented. We discuss notable similarities of paragnath anlagen to those of the hypopharynx complex in myriapods and hexapods. The fact that both structures grow out as two lateral buds

  18. 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.

  19. Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

    NARCIS (Netherlands)

    Manen, van Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; Berg, van den Timo K.; Roos, Dirk; Otto, Cees

    2008-01-01

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase

  20. Developments in application of light and scanning electron microscopy techniques for cell wall degradation studies.

    NARCIS (Netherlands)

    Engels, F.M.

    1996-01-01

    The results of recent technological developments in light and scanning electron microscopy closely used for research on forage cell wall degradation in ruminants, are reviewed. The indigestibility of forages by rumen microorganisms used to be ascribed mainly to an overall presence of lignin in the p

  1. 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.

  2. 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.

  3. Mapping nanomechanical properties of live cells using multi-harmonic atomic force microscopy

    Science.gov (United States)

    Raman, A.; Trigueros, S.; Cartagena, A.; Stevenson, A. P. Z.; Susilo, M.; Nauman, E.; Contera, S. Antoranz

    2011-12-01

    The nanomechanical properties of living cells, such as their surface elastic response and adhesion, have important roles in cellular processes such as morphogenesis, mechano-transduction, focal adhesion, motility, metastasis and drug delivery. Techniques based on quasi-static atomic force microscopy techniques can map these properties, but they lack the spatial and temporal resolution that is needed to observe many of the relevant details. Here, we present a dynamic atomic force microscopy method to map quantitatively the nanomechanical properties of live cells with a throughput (measured in pixels/minute) that is ~10-1,000 times higher than that achieved with quasi-static atomic force microscopy techniques. The local properties of a cell are derived from the 0th, 1st and 2nd harmonic components of the Fourier spectrum of the AFM cantilevers interacting with the cell surface. Local stiffness, stiffness gradient and the viscoelastic dissipation of live Escherichia coli bacteria, rat fibroblasts and human red blood cells were all mapped in buffer solutions. Our method is compatible with commercial atomic force microscopes and could be used to analyse mechanical changes in tumours, cells and biofilm formation with sub-10 nm detail.

  4. Segmentation and abnormality detection of cervical cancer cells using fast elm with particle swarm optimization

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    Sukumar P.

    2015-01-01

    Full Text Available Cervical cancer arises when the anomalous cells on the cervix mature unmanageable obviously in the renovation sector. The most probably used methods to detect abnormal cervical cells are the routine and there is no difference between the abnormal and normal nuclei. So that the abnormal nuclei found are brown in color while normal nuclei are blue in color. The spread or cells are examined and the image denoising is performed based on the Iterative Decision Based Algorithm. Image Segmentation is the method of paneling a digital image into compound sections. The major utilize of segmentation is to abridge or modify the demonstration of an image. The images are segmented by applying anisotropic diffusion on the Denoised image. Image can be enhanced using dark stretching to increase the quality of the image. It separates the cells into all nuclei region and abnormal nuclei region. The abnormal nuclei regions are further classified into touching and non-touching regions and touching regions undergoes feature selection process. The existing Support Vector Machines (SVM is classified few nuclei regions but the time to taken for execution is high. The abnormality detected from the image is calculated as 45% from the total abnormal nuclei. Thus the proposed method of Fast Particle Swarm Optimization with Extreme Learning Machines (Fast PSO-ELM to classify all nuclei regions further into touching region and separated region. The iterative method for to training the ELM and make it more efficient than the SVM method. In experimental result, the proposed method of Fast PSO-ELM may shows the accuracy as above 90% and execution time is calculated based on the abnormality (ratio of abnormal nuclei regions to all nuclei regions image. Therefore, Fast PSO-ELM helps to detect the cervical cancer cells with maximum accuracy.

  5. Two-photon microscopy for imaging germinal centers and T follicular helper cells.

    Science.gov (United States)

    Clatworthy, Menna R

    2015-01-01

    One of the principle features of immune cells is their dynamic nature. Lymphocytes circulate in the blood between secondary lymphoid organs and tissues in an effort to maximize the likelihood of a rapid and appropriate immune response to invading pathogens and tissue damage. Conventional experimental techniques such as histology and flow cytometry have greatly increased our understanding of immune cells, but in the last decade, two-photon microscopy has revolutionized our ability to interrogate the dynamic behavior of immune cells, a facet so critical to their function. Two-photon microscopy relies on the excitation of fluorophores by simultaneous application of two photons of longer wavelength light. This allows a greater depth of imaging with minimal photodamage. Thus, living tissues can be imaged, including immune cells in lymph nodes. This technique has been used to interrogate the events occurring in a germinal center response and the interactions between cells in the germinal center, including T follicular helper cells (Tfh), germinal center B cells, and follicular dendritic cells (FDC). Herein, a method is described by which the interactions between Tfh and B cells within a germinal center in a popliteal lymph node can be imaged in a live mouse.

  6. Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy.

    Science.gov (United States)

    Andolfi, Laura; Bourkoula, Eugenia; Migliorini, Elisa; Palma, Anita; Pucer, Anja; Skrap, Miran; Scoles, Giacinto; Beltrami, Antonio Paolo; Cesselli, Daniela; Lazzarino, Marco

    2014-01-01

    Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.

  7. Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy.

    Directory of Open Access Journals (Sweden)

    Laura Andolfi

    Full Text Available Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.

  8. Automatic measurement of compression wood cell attributes in fluorescence microscopy images.

    Science.gov (United States)

    Selig, B; Luengo Hendriks, C L; Bardage, S; Daniel, G; Borgefors, G

    2012-06-01

    This paper presents a new automated method for analyzing compression wood fibers in fluorescence microscopy. Abnormal wood known as compression wood is present in almost every softwood tree harvested. Compression wood fibers show a different cell wall morphology and chemistry compared to normal wood fibers, and their mechanical and physical characteristics are considered detrimental for both construction wood and pulp and paper purposes. Currently there is the need for improved methodologies for characterization of lignin distribution in wood cell walls, such as from compression wood fibers, that will allow for a better understanding of fiber mechanical properties. Traditionally, analysis of fluorescence microscopy images of fiber cross-sections has been done manually, which is time consuming and subjective. Here, we present an automatic method, using digital image analysis, that detects and delineates softwood fibers in fluorescence microscopy images, dividing them into cell lumen, normal and highly lignified areas. It also quantifies the different areas, as well as measures cell wall thickness. The method is evaluated by comparing the automatic with a manual delineation. While the boundaries between the various fiber wall regions are detected using the automatic method with precision similar to inter and intra expert variability, the position of the boundary between lumen and the cell wall has a systematic shift that can be corrected. Our method allows for transverse structural characterization of compression wood fibers, which may allow for improved understanding of the micro-mechanical modeling of wood and pulp fibers.

  9. A microfluidic platform for correlative live-cell and super-resolution microscopy.

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    Johnny Tam

    Full Text Available Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images.

  10. A microfluidic platform for correlative live-cell and super-resolution microscopy.

    Science.gov (United States)

    Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

    2014-01-01

    Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images.

  11. Analysis of cancer cell morphology in fluorescence microscopy image exploiting shape descriptor

    Science.gov (United States)

    Kang, Mi-Sun; Kim, Hye-Ryun; Kim, Sudong; Ryu, Gyu Ha; Kim, Myoung-Hee

    2016-04-01

    Cancer cell morphology is closely related to their phenotype and activity. These characteristics are important in drug-response prediction for personalized cancer therapeutics. We used multi-channel fluorescence microscopy images to analyze the morphology of highly cohesive cancer cells. First, we detected individual nuclei regions in single-channel images using advanced simple linear iterative clustering. The center points of the nuclei regions were used as seeds for the Voronoi diagram method to extract spatial arrangement features from cell images. Human cancer cell populations form irregularly shaped aggregates, making their detection more difficult. We overcame this problem by identifying individual cells using an image-based shape descriptor. Finally, we analyzed the correlation between cell agglutination and cell shape.

  12. Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.

    Science.gov (United States)

    Cahalan, Michael D; Parker, Ian

    2008-01-01

    The immune system is the most diffuse cellular system in the body. Accordingly, long-range migration of cells and short-range communication by local chemical signaling and by cell-cell contacts are vital to the control of an immune response. Cellular homing and migration within lymphoid organs, antigen recognition, and cell signaling and activation are clearly vital during an immune response, but these events had not been directly observed in vivo until recently. Introduced to the field of immunology in 2002, two-photon microscopy is the method of choice for visualizing living cells deep within native tissue environments, and it is now revealing an elegant cellular choreography that underlies the adaptive immune response to antigen challenge. We review cellular dynamics and molecular factors that contribute to basal motility of lymphocytes in the lymph node and cellular interactions leading to antigen capture and recognition, T cell activation, B cell activation, cytolytic effector function, and antibody production.

  13. Induction of Th17 cells by segmented filamentous bacteria in the murine intestine.

    Science.gov (United States)

    Farkas, Adam M; Panea, Casandra; Goto, Yoshiyuki; Nakato, Gaku; Galan-Diez, Marta; Narushima, Seiko; Honda, Kenya; Ivanov, Ivaylo I

    2015-06-01

    Segmented filamentous bacteria (SFB) are Gram-positive, anaerobic, spore-forming commensals that reside in the gut of many animal species. Described more than forty years ago, SFB have recently gained interest due to their unique ability to modulate the host immune system through induction of IgA and Th17 cells. Here, we describe a collection of methods to detect and quantify SFB and SFB adhesion in intestinal mucosa, as well as SFB-specific CD4 T cells in the lamina propria. In addition, we describe methods for purification of SFB from fecal material of SFB-monoassociated gnotobiotic mice. Using these methods we examine the kinetics of SFB colonization and Th17 cell induction. We also show that SFB colonize unevenly the intestinal mucosa and that SFB adherence occurs predominantly in the terminal ileum and correlates with an increased proportion of SFB-specific Th17 cells.

  14. 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

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

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    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.

  16. 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.

  17. 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.

  18. Binding Strength Between Cell Adhesion Proteoglycans Measured by Atomic Force Microscopy

    Science.gov (United States)

    Dammer, Ulrich; Popescu, Octavian; Wagner, Peter; Anselmetti, Dario; Guntherodt, Hans-Joachim; Misevic, Gradimir N.

    1995-02-01

    Measurement of binding forces intrinsic to adhesion molecules is necessary to assess their contribution to the maintenance of the anatomical integrity of multicellular organisms. Atomic force microscopy was used to measure the binding strength between cell adhesion proteoglycans from a marine sponge. Under physiological conditions, the adhesive force between two cell adhesion molecules was found to be up to 400 piconewtons. Thus a single pair of molecules could hold the weight of 1600 cells. High intermolecular binding forces are likely to form the basis for the integrity of the multicellular sponge organism.

  19. Differences in Nuclear DNA Organization Between Lymphocytes, Hodgkin and Reed–Sternberg Cells Revealed by Structured Illumination Microscopy

    NARCIS (Netherlands)

    Righolt, C.H.; Guffei, A.; Knecht, H.; Young, I.T.; Stallinga, S.; Van Vliet, L.J.; Mai, S.

    2014-01-01

    Advances in light microscopy have enabled the visualization of DNA in the interphase nucleus with more detail than is visible with conventional light microscopy. The nuclear architecture is assumed to be different in cancer cells compared to normal cells. In this paper we have studied, for the first

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

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    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.

  1. Calculation of Intracellular Pressure of Red Blood Cells at Jaundice According to Atomic Force Microscopy Data

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    Yu.S. Nagornov

    2016-03-01

    Full Text Available The present work is devoted to the analysis of three-dimensional data of atomic force microscopy for research of the morphology of red blood cells. In this paper we built a biomechanical model of the erythrocyte, which allowed calculating the intracellular pressure of erythrocyte based on data of atomic force microscopy. As a result, we obtained the dependence intracellular pressure on the morphology of red blood cell. We have proposed a method of estimating of intracellular pressure of erythrocytes based on numerical modeling and data of atomic force microscopy of erythrocytes scan, which involves a comparison of the experimental data with the results of numerical calculation. The method is applied to the data of atomic force microscopy of erythrocytes of experimental animals - dwarf domestic pigs with different degrees of obstructive jaundice and normal. It is shown that with increasing severity of the disease and the concentration of bilirubin in the blood there is an infringement erythrocyte membranes, by an average increasing their volume and intracellular pressure.

  2. Near-field scanning optical microscopy in cell biology and cytogenetics.

    Science.gov (United States)

    Hausmann, Michael; Perner, Birgit; Rapp, Alexander; Wollweber, Leo; Scherthan, Harry; Greulich, Karl-Otto

    2006-01-01

    Light microscopy has proven to be one of the most versatile analytical tools in cell biology and cytogenetics. The growing spectrum of scientific knowledge demands a continuous improvement of the optical resolution of the instruments. In far-field light microscopy, the attainable resolution is dictated by the limit of diffraction, which, in practice, is about 250 nm for high-numerical-aperture objective lenses. Near-field scanning optical microscopy (NSOM) was the first technique that has overcome this limit up to about one order of magnitude. Typically, the resolution range below 100 nm is accessed for biological applications. Using appropriately designed scanning probes allows for obtaining an extremely small near-field light excitation volume (some tens of nanometers in diameter). Because of the reduction of background illumination, high contrast imaging becomes feasible for light transmission and fluorescence microscopy. The height of the scanning probe is controlled by atomic force interactions between the specimen surface and the probe tip. The control signal can be used for the production of a topographic (nonoptical) image that can be acquired simultaneously. In this chapter, the principle of NSOM is described with respect to biological applications. A brief overview of some requirements in biology and applications described in the literature are given. Practical advice is focused on instruments with aperture-type illumination probes. Preparation protocols focussing on NSOM of cell surfaces and chromosomes are presented.

  3. The crocidolite fibres interaction with human mesothelial cells as investigated by combining electron microscopy, atomic force and scanning near-field optical microscopy.

    Science.gov (United States)

    Andolfi, Laura; Trevisan, Elisa; Zweyer, Marina; Prato, Stefano; Troian, Barbara; Vita, Francesca; Borelli, Violetta; Soranzo, Maria Rosa; Melato, Mauro; Zabucchi, Giuliano

    2013-03-01

    In this study, we have performed a morphological analysis of crocidolite fibres interaction with mesothelial cells (MET5A) by combining conventional electron microscopy with atomic force (AFM) and scanning near-field optical microscopy (SNOM). After 6-h exposure at a crocidolite dose of 5 μg cm(-2), 90% of MET5A cells interact with fibres that under these conditions have a low cytotoxic effect. SEM images point out that fibres can be either engulfed by the cells that lose their typical morphology or they can accumulate over or partially inside the cells, which preserve their typical spread morphology. By using AFM we are able to directly visualize the entry-site of nanometric-sized fibres at the plasma membrane of the spread mesothelial cells. More importantly, the crocidolite fibres that are observed to penetrate the plasma membrane in SNOM topography can be simultaneously followed beneath the cell surface in the SNOM optical images. The analysis of SNOM data demonstrates the entrance of crocidolite fibres in proximity of nuclear compartment, as observed also in the TEM images. Our findings indicate that the combination of conventional electron microscopy with novel nanoscopic techniques can be considered a promising approach to achieve a comprehensive morphological description of the interaction between asbestos fibres and mesothelial cells that represents the early event in fibre pathogenesis.

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

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    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

  5. 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

  6. Quantitative index imaging of coculture cells by scanning focused refractive index microscopy

    Science.gov (United States)

    Sun, Teng-Qian; Ye, Qing; Hu, Fen; Liu, Shi-ke; Wang, Xiao-Wan; Wang, Jin; Deng, Zhi-Chao; Mei, Jian-Chun; Zhou, Wen-Yuan; Zhang, Chun-Ping; Wang, Xin-Yu; Pan, Lei-Ting; Tian, Jian-Guo

    2016-08-01

    We report the quantitative refractive index (RI) imaging of cocultured cells in their living environment by scanning focused refractive index microscopy (SFRIM). Mouse microglial cells and synovial cells are cocultured on the top surface of a trapezoid prism. The RI imaging of living cells is obtained in a reflection-type method. The RI information is deduced with the simple derivative total internal reflection method, where a complex retrieval algorithm or reconstruction process is unnecessary. The outline of each cell is determined according to the RI value compared with that of the immersion liquid. The cocultured cells can be discriminated in the RI image. The measurement is nondestructive and label-free. The experimental results prove that SFRIM is a promising tool in the field of biological optics.

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

    Science.gov (United States)

    Hartsuiker, L; VAN Es, P; Petersen, W; VAN Leeuwen, T G; Terstappen, L W M M; Otto, C

    2011-11-01

    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 preparation protocol was developed to enable imaging of cells and gold nanoparticles with a conventional below lens scanning electron microscopes. The negative influence of 'charging' on the quality of scanning electron microscopes' images could be limited by deposition of biological cells on a conductive (gold) surface. The novel protocol enabled high-resolution scanning electron microscopes' imaging of small clusters and individual gold nanoparticles on uncoated cell surfaces. Gold nanoparticles could be counted on cancer cells with automated routines.

  8. Imaging of apoptotic HeLa cells by using scanning near-field optical microscopy

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    By using scanning near-field optical microscopy (SNOM), HeLa cells in apoptosis process are imaged with a higher optical resolution beyond the diffraction limit. Since SNOM provides both topographic and transmitted light intensity information of a cell, it can correlate the structural characteristics and optical properties with the spatial position of the apoptotic cells. Wavelength imaging by using near-field spectroscopy shows that there is a great difference in light propagation and absorption in the cell. This unique technique can be applied to the super high resolution imaging of different components in the cell. The observations by near-field optical imaging and near-field spectroscopy indicate an inhomogeneous aggregation of the inner structure in the apoptotic HeLa cells and the change of transmission intensity of light with the apoptosis status.

  9. 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.

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

    Science.gov (United States)

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

    2015-06-01

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

  11. 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.

  12. 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.

  13. 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.

  14. Investigating the use of in situ liquid cell scanning transmission electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Nguy, Amanda [Iowa State Univ., Ames, IA (United States)

    2016-02-19

    Engineering nanoparticles with desired shape-dependent properties is the key to many applications in nanotechnology. Although many synthetic procedures exist to produce anisotropic gold nanoparticles, the dynamics of growth are typically unknown or hypothetical. In the case of seed-mediated growth in the presence of DNA into anisotropic nanoparticles, it is not known exactly how DNA directs growth into specific morphologies. A series of preliminary experiments were carried out to contribute to the investigation of the possible mechanism of DNA-mediated growth of gold nanoprisms into gold nanostars using liquid cell scanning transmission electron microscopy (STEM). Imaging in the liquid phase was achieved through the use of a liquid cell platform and liquid cell holder that allow the sample to be contained within a “chip sandwich” between two electron transparent windows. Ex situ growth experiments were performed using Au-T30 NPrisms (30-base thymine oligonucleotide-coated gold nanoprisms) that are expected to grow into gold nanostars. Growth to form these nanostars were imaged using TEM (transmission electron microscopy) and liquid cell STEM (scanning transmission electron microscopy). An attempt to perform in situ growth experiments with the same Au-T30 nanoprisms revealed challenges in obtaining desired morphology results due to the environmental differences within the liquid cell compared to the ex situ environment. Different parameters in the experimental method were explored including fluid line set up, simultaneous and alternating reagent addition, and the effect of different liquid cell volumes to ensure adequate flow of reagents into the liquid cell. Lastly, the binding affinities were compared for T30 and A30 DNA incubated with gold nanoparticles using zeta potential measurements, absorption spectroscopy, and isothermal titration calorimetry (ITC). It was previously reported thymine bases have a lower binding affinity to gold surfaces than adenine

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

    Science.gov (United States)

    Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

    2014-01-01

    Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment.

  16. 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.

  17. High-resolution fast ion microscopy of single whole biological cells

    Science.gov (United States)

    Bettiol, Andrew A.; Mi, Zhaohong; Watt, Frank

    2016-12-01

    High-resolution microscopy techniques have become an essential tool in both biological and biomedical sciences, enabling the visualization of biological processes at cellular and subcellular levels. For many years, these imaging techniques utilized conventional optical microscopes including those with confocal facilities. However, the spatial resolutions achieved were largely limited to around 200 nm, as determined by the diffraction of light. To overcome this diffraction barrier, considerable scientific and technological effort has resulted in the development of super-resolution optical-based techniques, scanning probe microscopies, and also techniques utilizing charged particles (e.g., energetic electrons and ions) or high-energy photons (e.g., X-ray), which exhibit much shorter de Broglie wavelengths. Among the charged particle techniques, those utilizing mega-electron-volt (MeV) ion beams appear to have unique advantages primarily because MeV ions can penetrate through several microns of biological tissue (e.g., whole cells) with little deflection in their trajectories, and hence spatial resolutions are maintained while traversing the sample. Recently, we have witnessed the significant development of MeV ion beam focusing systems in reducing beam dimensions well below 100 nm, such that single whole cell imaging at 20 nm spatial resolutions is now possible. In this review, two super resolution imaging modalities that utilize MeV highly focused ion beams are discussed: Scanning Transmission Ion Microscopy (STIM), which images the areal density of cells and gives an insight into the cellular structure, and Proton/Helium-ion Induced Fluorescence Microcopy (P/HeIFM), which images the fluorescence emission of fluorescent markers and probes used as labels within the cells. This review hopes to demonstrate the potential of MeV ion microscopy, which is still in its infancy, and describe the simultaneous use of STIM and P/HeIFM as a new and powerful multifaceted

  18. Breast cancer cell movement: imaging invadopodia by TIRF and IRM microscopy.

    Science.gov (United States)

    Xu, Xuehua; Johnson, Peter; Mueller, Susette C

    2009-01-01

    Invadopodia are hair-like membrane protrusions projecting from the ventral side of the plasma membrane of tumor cells invading into an extracellular matrix (ECM). Formation of invadopodia and phagocytosis of partially degraded ECM is correlated with invasiveness of cancer cells. Many proteins associated with actin-rich punctae associated with invadopodia have been identified. However, the dynamic temporal and spatial relationship of invadopodia-related proteins and the mechanisms required for invadopodia formation remain largely unknown. Total Internal Reflection Fluorescence (TIRF) microscopy provides a powerful tool to directly visualize the dynamic membrane transportation of invadopodia-related, GFP-tagged proteins and to simultaneously monitor invadopodia formation by observation of localized degradation and phagocytosis of fluorescently labeled gelatin. Cell-substratum contacts can be visualized using a related technique, Interference Reflection Microscopy (IRM). In this chapter, we provide detailed methodologies to monitor the dynamic localizations of c-Src-eGFP using two-color TIRF microscopy along with IRM to simultaneously visualize translocation of c-Src-eGFP and invadopodia formation by degradation of AlexaFluor 568-labeled gelatin.

  19. 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.

  20. 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.

  1. 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.

  2. Investigating Effects of Proteasome Inhibitor on Multiple Myeloma Cells Using Confocal Raman Microscopy

    Directory of Open Access Journals (Sweden)

    Jeon Woong Kang

    2016-12-01

    Full Text Available Due to its label-free and non-destructive nature, applications of Raman spectroscopic imaging in monitoring therapeutic responses at the cellular level are growing. We have recently developed a high-speed confocal Raman microscopy system to image living biological specimens with high spatial resolution and sensitivity. In the present study, we have applied this system to monitor the effects of Bortezomib, a proteasome inhibitor drug, on multiple myeloma cells. Cluster imaging followed by spectral profiling suggest major differences in the nuclear and cytoplasmic contents of cells due to drug treatment that can be monitored with Raman spectroscopy. Spectra were also acquired from group of cells and feasibility of discrimination among treated and untreated cells using principal component analysis (PCA was accessed. Findings support the feasibility of Raman technologies as an alternate, novel method for monitoring live cell dynamics with minimal external perturbation.

  3. 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.

  4. 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......, inoculation of the flow cells, running of the system, confocal laser scanning microscopy and image analysis, and disassembly and cleaning of the system.......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...

  5. Lab-On-Chip Clinorotation System for Live-Cell Microscopy Under Simulated Microgravity

    Science.gov (United States)

    Yew, Alvin G.; Atencia, Javier; Chinn, Ben; Hsieh, Adam H.

    2013-01-01

    Cells in microgravity are subject to mechanical unloading and changes to the surrounding chemical environment. How these factors jointly influence cellular function is not well understood. We can investigate their role using ground-based analogues to spaceflight, where mechanical unloading is simulated through the time-averaged nullification of gravity. The prevailing method for cellular microgravity simulation is to use fluid-filled containers called clinostats. However, conventional clinostats are not designed for temporally tracking cell response, nor are they able to establish dynamic fluid environments. To address these needs, we developed a Clinorotation Time-lapse Microscopy (CTM) system that accommodates lab-on- chip cell culture devices for visualizing time-dependent alterations to cellular behavior. For the purpose of demonstrating CTM, we present preliminary results showing time-dependent differences in cell area between human mesenchymal stem cells (hMSCs) under modeled microgravity and normal gravity.

  6. Performance evaluation of multi-junction solar cells by spatially resolved electroluminescence microscopy.

    Science.gov (United States)

    Kong, Lijing; Wu, Zhiming; Chen, Shanshan; Cao, Yiyan; Zhang, Yong; Li, Heng; Kang, Junyong

    2015-01-01

    An electroluminescence microscopy combined with a spectroscopy was developed to visually analyze multi-junction solar cells. Triple-junction solar cells with different conversion efficiencies were characterized by using this system. The results showed that the mechanical damages and material defects in solar cells can be clearly distinguished, indicating a high-resolution imaging. The external quantum efficiency (EQE) measurements demonstrated that different types of defects or damages impacted cell performance in various degrees and the electric leakage mostly degraded the EQE. Meanwhile, we analyzed the relationship between electroluminescence intensity and short-circuit current density J SC. The results indicated that the gray value of the electroluminescence image corresponding to the intensity was almost proportional to J SC. This technology provides a potential way to evaluate the current matching status of multi-junction solar cells.

  7. Comet assay, cloning assay, and light and electron microscopy on one preselected cell

    Science.gov (United States)

    Koenig, Karsten; Oehring, Hartmut; Halbhuber, Karl-Juergen; Fiedler, Ursula; Bauer, Eckhard; Greulich, Karl-Otto

    1998-01-01

    In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular

  8. 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.

  9. 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.

  10. Characterization of Combinatorial Effects of Toxic Substances by Cell Cultivation in Micro Segmented Flow

    Science.gov (United States)

    Cao, J.; Kürsten, D.; Funfak, A.; Schneider, S.; Köhler, J. M.

    This chapter reviews the application of micro segmented flow for the screening of toxic effects on bacteria, eukaryotic microorganisms, human cells and multicellular systems. Besides, the determination of complete dose/response functions of toxic substances with a minimum of cells and chemicals, it is reviewed how two- and multi-dimensional concentration spaces can be screened in order to evaluate combinatorial effects of chemicals on cells. The challenge for the development of new and miniaturized methods is derived from the increase of the number of different used substances in technique, agriculture and medicine, from the increasing release of new substances and nanomaterials into our environment and from the improvement of the insight of toxicity of natural substances and the interferences between different substances resulting in toxic effects on different organisms, cells and tissues. The application of two-dimensional toxicological screenings on selected examples of effector combinations is described. Examples for the detection of an independent, an additive and a synergistic interference between two substances are given. In addition, it is shown that the screening for toxicological effects in complete two-dimensional concentration spaces allows the detection of complex response behaviour—for example, the formation of tolerances and stimulation peaks—which thereby can be characterized. The characterization of interference of toxic organic substances with silver nanoparticles is reported as an example for the potential of micro segmented-flow technique for evaluating the toxicological impact of new materials. Finally, it is demonstrated that the technique can be applied for different organisms like simple bacteria, single cell alga such as Chlorella vulgaris and multicellular systems up to the development of complete organisms beginning from eggs.

  11. 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).

  12. New data-driven method from 3D confocal microscopy for calculating phytoplankton cell biovolume.

    Science.gov (United States)

    Roselli, L; Paparella, F; Stanca, E; Basset, A

    2015-06-01

    Confocal laser scanner microscopy coupled with an image analysis system was used to directly determine the shape and calculate the biovolume of phytoplankton organisms by constructing 3D models of cells. The study was performed on Biceratium furca (Ehrenberg) Vanhoeffen, which is one of the most complex-shaped phytoplankton. Traditionally, biovolume is obtained from a standardized set of geometric models based on linear dimensions measured by light microscopy. However, especially in the case of complex-shaped cells, biovolume is affected by very large errors associated with the numerous manual measurements that this entails. We evaluate the accuracy of these traditional methods by comparing the results obtained using geometric models with direct biovolume measurement by image analysis. Our results show cell biovolume measurement based on decomposition into simple geometrical shapes can be highly inaccurate. Although we assume that the most accurate cell shape is obtained by 3D direct biovolume measurement, which is based on voxel counting, the intrinsic uncertainty of this method is explored and assessed. Finally, we implement a data-driven formula-based approach to the calculation of biovolume of this complex-shaped organism. On one hand, the model is obtained from 3D direct calculation. On the other hand, it is based on just two linear dimensions which can easily be measured by hand. This approach has already been used for investigating the complexities of morphology and for determining the 3D structure of cells. It could also represent a novel way to generalize scaling laws for biovolume calculation.

  13. A novel supervised trajectory segmentation algorithm identifies distinct types of human adenovirus motion in host cells.

    Science.gov (United States)

    Helmuth, Jo A; Burckhardt, Christoph J; Koumoutsakos, Petros; Greber, Urs F; Sbalzarini, Ivo F

    2007-09-01

    Biological trajectories can be characterized by transient patterns that may provide insight into the interactions of the moving object with its immediate environment. The accurate and automated identification of trajectory motifs is important for the understanding of the underlying mechanisms. In this work, we develop a novel trajectory segmentation algorithm based on supervised support vector classification. The algorithm is validated on synthetic data and applied to the identification of trajectory fingerprints of fluorescently tagged human adenovirus particles in live cells. In virus trajectories on the cell surface, periods of confined motion, slow drift, and fast drift are efficiently detected. Additionally, directed motion is found for viruses in the cytoplasm. The algorithm enables the linking of microscopic observations to molecular phenomena that are critical in many biological processes, including infectious pathogen entry and signal transduction.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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...

  20. Investigation of osteoblast cells behavior in polymeric 3D micropatterned scaffolds using digital holographic microscopy.

    Science.gov (United States)

    Mihailescu, M; Popescu, R C; Matei, A; Acasandrei, A; Paun, I A; Dinescu, M

    2014-08-01

    The effect of micropatterned polymeric scaffolds on the features of the cultured cells at different time intervals after seeding was investigated by digital holographic microscopy. Both parallel and perpendicular walls, with different heights, were fabricated using two-photon lithography on photopolymers. The walls were subsequently coated with polypyrrole-based thin films using the matrix assisted pulsed laser evaporation technique. Osteoblast-like cells, MG-63 line, were cultured on these polymeric 3D micropatterned scaffolds. To analyze these scaffolds with/without cultured cells, an inverted digital holographic microscope, which provides 3D images, was used. Information about the samples' refractive indices and heights was obtained from the phase shift introduced in the optical path. Characteristics of cell adhesion, alignment, orientation, and morphology as a function of the wall heights and time from seeding were highlighted.

  1. 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.

    2016-05-03

    Abstract

    Liquid cell transmission electron microscopy (LCTEM) provides a unique insight into the dynamics of nanomaterials in solution. Controlling the addition of multiple solutions to the liquid cell remains a key hurdle in our ability to increase throughput and to study processes dependent on solution mixing including chemical reactions. Here, we report that a piezo dispensing technique allows for mixing of multiple solutions directly within the viewing area. This technique permits deposition of 50 pL droplets of various aqueous solutions onto the liquid cell window, before assembly of the cell in a fully controlled manner. This proof-of-concept study highlights the great potential of picoliter dispensing in combination with LCTEM for observing nanoparticle mixing in the solution phase and the creation of chemical gradients.

  2. 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.

  3. 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.

  4. Fluorescence microscopy methods for determining the viability of bacteria in association with mammalian cells.

    Science.gov (United States)

    Johnson, M Brittany; Criss, Alison K

    2013-09-05

    Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

  5. Fluorescence fluctuation microscopy: a diversified arsenal of methods to investigate molecular dynamics inside cells.

    Science.gov (United States)

    Weidemann, Thomas; Mücksch, Jonas; Schwille, Petra

    2014-10-01

    Fluorescence microscopy provides insight into the subcellular organization of biological functions. However, images are snap shots averaging over a highly dynamic molecular system. Fluorescence fluctuation microscopy, employing similar detection technology, encompasses a powerful arsenal of analysis tools that investigate the molecular heterogeneity in space and time. Analyzing signal fluctuations from small ensembles (several hundred particles) reveals their concentration, the stoichiometry, the stochastic motion, as well as superimposed signatures of the environment such as spatial confinement and binding events. Thus, fluctuation analysis provides access to dynamic molecular properties that can be used to build physical models of cellular processes. In the last decade these methods experienced a remarkable diversification, which we revisit here with a particular focus on live cell applications.

  6. 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.

  7. Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K; Roos, Dirk; Otto, Cees

    2008-04-15

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91(phox), which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91(phox) are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91(phox). By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91(phox) are approximately 1.38 and approximately 1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane.

  8. Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy.

    Science.gov (United States)

    Takahashi, Yasufumi; Shevchuk, Andrew I; Novak, Pavel; Babakinejad, Babak; Macpherson, Julie; Unwin, Patrick R; Shiku, Hitoshi; Gorelik, Julia; Klenerman, David; Korchev, Yuri E; Matsue, Tomokazu

    2012-07-17

    We describe voltage-switching mode scanning electrochemical microscopy (VSM-SECM), in which a single SECM tip electrode was used to acquire high-quality topographical and electrochemical images of living cells simultaneously. This was achieved by switching the applied voltage so as to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest. This imaging method is robust, and a single nanoscale SECM electrode, which is simple to produce, is used for both topography and activity measurements. In order to minimize the delay at voltage switching, we used pyrolytic carbon nanoelectrodes with 6.5-100 nm radii that rapidly reached a steady-state current, typically in less than 20 ms for the largest electrodes and faster for smaller electrodes. In addition, these carbon nanoelectrodes are suitable for convoluted cell topography imaging because the RG value (ratio of overall probe diameter to active electrode diameter) is typically in the range of 1.5-3.0. We first evaluated the resolution of constant-current mode topography imaging using carbon nanoelectrodes. Next, we performed VSM-SECM measurements to visualize membrane proteins on A431 cells and to detect neurotransmitters from a PC12 cells. We also combined VSM-SECM with surface confocal microscopy to allow simultaneous fluorescence and topographical imaging. VSM-SECM opens up new opportunities in nanoscale chemical mapping at interfaces, and should find wide application in the physical and biological sciences.

  9. Measurement of intracellular calcium gradients in single living cells using optical sectioning microscopy

    Science.gov (United States)

    Yelamarty, Rao V.; Cheung, Joseph Y.

    1992-06-01

    Intracellular free calcium has been recognized as a regulator of many cellular processes and plays a key role in mediating actions of many drugs. To elucidate subcellular spatial calcium changes throughout the cell in three dimensions (3-D), optical sectioning microscopy was applied using digital imaging coupled fluorescence microscopy. The cell was loaded with a fluorescent indicator, fura-2, and a stack of sectional fluorescent images were acquired, digitized and finally stored on-line for post image analysis. Each sectional image was then deconvolved, to remove contaminating light signals from adjacent planes, using the Nearest Neighboring Deconvolution Algorithm (NNDA) and the overall imaging system's empirical Point Spread Function (PSF) that is measured with a 0.25 micrometers fluorescent bead. Using this technique, we measured that the addition of growth factors caused a 2 - 3 fold increase (1) in nuclear calcium compared to cytosolic calcium in blood cells and (2) in both nuclear and cytosolic calcium in liver cells. Such spatial information, which is important in understanding subcellular processes, would not be possible to measure with other methods.

  10. 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.

  11. 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.

  12. Fast, accurate reconstruction of cell lineages from large-scale fluorescence microscopy data.

    Science.gov (United States)

    Amat, Fernando; Lemon, William; Mossing, Daniel P; McDole, Katie; Wan, Yinan; Branson, Kristin; Myers, Eugene W; Keller, Philipp J

    2014-09-01

    The comprehensive reconstruction of cell lineages in complex multicellular organisms is a central goal of developmental biology. We present an open-source computational framework for the segmentation and tracking of cell nuclei with high accuracy and speed. We demonstrate its (i) generality by reconstructing cell lineages in four-dimensional, terabyte-sized image data sets of fruit fly, zebrafish and mouse embryos acquired with three types of fluorescence microscopes, (ii) scalability by analyzing advanced stages of development with up to 20,000 cells per time point at 26,000 cells min(-1) on a single computer workstation and (iii) ease of use by adjusting only two parameters across all data sets and providing visualization and editing tools for efficient data curation. Our approach achieves on average 97.0% linkage accuracy across all species and imaging modalities. Using our system, we performed the first cell lineage reconstruction of early Drosophila melanogaster nervous system development, revealing neuroblast dynamics throughout an entire embryo.

  13. 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.

  14. Mean cell size and collagen orientation from 2D Fourier analysis on confocal laser scanning microscopy and two-photon fluorescence microscopy on human skin in vivo

    Science.gov (United States)

    Lucassen, Gerald W.; Bakker, Bernard L.; Neerken, Sieglinde; Hendriks, Rob F. M.

    2003-07-01

    We present results from 2D Fourier analysis on 3D stacks of images obtained by confocal laser scanning reflectance microscopy (CLSM) and two-photon fluorescence microscopy (2PM) on human skin in vivo. CLSM images were obtained with a modified commercial system (Vivascope1000, Lucid Inc, excitation wavelength 830 nm) equipped with a piezo-focusing element (350 μm range) for depth positioning of the objective lens. 2PM was performed with a specially designed set-up with excitation wavelength 730 nm. Mean cell size in the epidermal layer and structural orientation in the dermal layer have been determined as a function of depth by 2D Fourier analysis. Fourier analysis on microscopic images enables automatic non-invasive quantitative structural analysis (mean cell size and orientation) of living human skin.

  15. Refractometry of melanocyte cell nuclei using optical scatter images recorded by digital Fourier microscopy.

    Science.gov (United States)

    Seet, Katrina Y T; Nieminen, Timo A; Zvyagin, Andrei V

    2009-01-01

    The cell nucleus is the dominant optical scatterer in the cell. Neoplastic cells are characterized by cell nucleus polymorphism and polychromism-i.e., the nuclei exhibits an increase in the distribution of both size and refractive index. The relative size parameter, and its distribution, is proportional to the product of the nucleus size and its relative refractive index and is a useful discriminant between normal and abnormal (cancerous) cells. We demonstrate a recently introduced holographic technique, digital Fourier microscopy (DFM), to provide a sensitive measure of this relative size parameter. Fourier holograms were recorded and optical scatter of individual scatterers were extracted and modeled with Mie theory to determine the relative size parameter. The relative size parameter of individual melanocyte cell nuclei were found to be 16.5+/-0.2, which gives a cell nucleus refractive index of 1.38+/-0.01 and is in good agreement with previously reported data. The relative size parameters of individual malignant melanocyte cell nuclei are expected to be greater than 16.5.

  16. 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...

  17. 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.

  18. Looking at cell mechanics with atomic force microscopy: experiment and theory.

    Science.gov (United States)

    Benitez, Rafael; Toca-Herrera, José L

    2014-11-01

    This review reports on the use of the atomic force microscopy in the investigation of the mechanical properties of cells. It is shown that the technique is able to deliver information about the cell surface properties (e.g., topography), the Young modulus, the viscosity, and the cell the relaxation times. Another aspect that this short review points out is the utilization of the atomic force microscope to investigate basic questions related to materials physics, biology, and medicine. The review is written in a chronological way to offer an overview of phenomenological facts and quantitative results to the reader. The final section discusses in detail the advantages and disadvantages of the Hertz and JKR models. A new implementation of the JKR model derived by Dufresne is presented.

  19. 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.

  20. Just Look! Intravital Microscopy as the Best Means to Study Kidney Cell Death Dynamics.

    Science.gov (United States)

    Schießl, Ina Maria; Hammer, Anna; Riquier-Brison, Anne; Peti-Peterdi, Janos

    2016-05-01

    Kidney cell death plays a key role in the progression of life-threatening renal diseases, such as acute kidney injury and chronic kidney disease. Injured and dying epithelial and endothelial cells take part in complex communication with the innate immune system, which drives the progression of cell death and the decrease in renal function. To improve our understanding of kidney cell death dynamics and its impact on renal disease, a study approach is needed that facilitates the visualization of renal function and morphology in real time. Intravital multiphoton microscopy of the kidney has been used for more than a decade and made substantial contributions to our understanding of kidney physiology and pathophysiology. It is a unique tool that relates renal structure and function in a time- and spatial-dependent manner. Basic renal function, such as microvascular blood flow regulation and glomerular filtration, can be determined in real time and homeostatic alterations, which are linked inevitably to cell death and can be depicted down to the subcellular level. This review provides an overview of the available techniques to study kidney dysfunction and inflammation in terms of cell death in vivo, and addresses how this novel approach can be used to improve our understanding of cell death dynamics in renal disease.

  1. Superresolution microscopy reveals a dynamic picture of cell polarity maintenance during directional growth.

    Science.gov (United States)

    Ishitsuka, Yuji; Savage, Natasha; Li, Yiming; Bergs, Anna; Grün, Nathalie; Kohler, Daria; Donnelly, Rebecca; Nienhaus, G Ulrich; Fischer, Reinhard; Takeshita, Norio

    2015-11-01

    Polar (directional) cell growth, a key cellular mechanism shared among a wide range of species, relies on targeted insertion of new material at specific locations of the plasma membrane. How these cell polarity sites are stably maintained during massive membrane insertion has remained elusive. Conventional live-cell optical microscopy fails to visualize polarity site formation in the crowded cell membrane environment because of its limited resolution. We have used advanced live-cell imaging techniques to directly observe the localization, assembly, and disassembly processes of cell polarity sites with high spatiotemporal resolution in a rapidly growing filamentous fungus, Aspergillus nidulans. We show that the membrane-associated polarity site marker TeaR is transported on microtubules along with secretory vesicles and forms a protein cluster at that point of the apical membrane where the plus end of the microtubule touches. There, a small patch of membrane is added through exocytosis, and the TeaR cluster gets quickly dispersed over the membrane. There is an incessant disassembly and reassembly of polarity sites at the growth zone, and each new polarity site locus is slightly offset from preceding ones. On the basis of our imaging results and computational modeling, we propose a transient polarity model that explains how cell polarity is stably maintained during highly active directional growth.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. Identification of tumor cells infiltrating into connective tissue in esophageal cancer by multiphoton microscopy

    Science.gov (United States)

    Xu, Jian; Jiang, Liwei; Kang, Deyong; Wu, Xuejing; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Lin, Jiangbo; Chen, Jianxin

    2016-10-01

    Esophageal cancer is one of the most common malignancies of the gastrointestinal cancers and carries poorer prognosis than other gastrointestinal cancers. In general practice, the depth of tumor infiltration in esophageal wall is crucial to establishing appropriate treatment plan which is established by detecting the tumor infiltration depth. Connective tissue is one of the main structures that form the esophageal wall. So, identification of tumor cells infiltrating into connective tissue is helping for detecting the tumor infiltration depth. Our aim is to evaluate whether multiphoton microscopy (MPM) can be used to detect tumor cells infiltrating into connective tissue in the esophageal cancer. MPM is well-suited for real-time detecting morphologic and cellular changes in fresh tissues since many endogenous fluorophores of fresh tissues are excited through two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). In this work, microstructure of tumor cells and connective tissue are first studied. Then, morphological changes of collagen fibers after the infiltration of tumor cells are shown. These results show that MPM has the ability to detect tumor cells infiltrating into connective tissue in the esophageal cancer. In the future, MPM may be a promising imaging technique for detecting tumor cells in esophageal cancer.

  9. Time-stretch microscopy on a DVD for high-throughput imaging cell-based assay.

    Science.gov (United States)

    Tang, Anson H L; Yeung, P; Chan, Godfrey C F; Chan, Barbara P; Wong, Kenneth K Y; Tsia, Kevin K

    2017-02-01

    Cell-based assay based on time-stretch imaging is recognized to be well-suited for high-throughput phenotypic screening. However, this ultrafast imaging technique has primarily been limited to suspension-cell assay, leaving a wide range of solid-substrate assay formats uncharted. Moreover, time-stretch imaging is generally restricted to intrinsic biophysical phenotyping, but lacks the biomolecular signatures of the cells. To address these challenges, we develop a spinning time-stretch imaging assay platform based on the functionalized digital versatile disc (DVD). We demonstrate that adherent cell culture and biochemically-specific cell-capture can now be assayed with time-stretch microscopy, thanks to the high-speed DVD spinning motion that naturally enables on-the-fly cellular imaging at an ultrafast line-scan rate of >10MHz. As scanning the whole DVD at such a high speed enables ultra-large field-of-view imaging, it could be favorable for scaling both the assay throughput and content as demanded in many applications, e.g. drug discovery, and rare cancer cell screening.

  10. Active intracellular transport in metastatic cells studied by spatial light interference microscopy.

    Science.gov (United States)

    Ceballos, Silvia; Kandel, Mikhail; Sridharan, Shamira; Majeed, Hassaan; Monroy, Freddy; Popescu, Gabriel

    2015-01-01

    Spatiotemporal patterns of intracellular transport are very difficult to quantify and, consequently, continue to be insufficiently understood. While it is well documented that mass trafficking inside living cells consists of both random and deterministic motions, quantitative data over broad spatiotemporal scales are lacking. We studied the intracellular transport in live cells using spatial light interference microscopy, a high spatiotemporal resolution quantitative phase imaging tool. The results indicate that in the cytoplasm, the intracellular transport is mainly active (directed, deterministic), while inside the nucleus it is both active and passive (diffusive, random). Furthermore, we studied the behavior of the two-dimensional mass density over 30 h in HeLa cells and focused on the active component. We determined the standard deviation of the velocity distribution at the point of cell division for each cell and compared the standard deviation velocity inside the cytoplasm and the nucleus. We found that the velocity distribution in the cytoplasm is consistently broader than in the nucleus, suggesting mechanisms for faster transport in the cytosol versus the nucleus. Future studies will focus on improving phase measurements by applying a fluorescent tag to understand how particular proteins are transported inside the cell.

  11. 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.

  12. Hyperspectral microscopy for characterization of gold nanoparticles in biological media and cells for toxicity assessment.

    Science.gov (United States)

    Grabinski, Christin; Schlager, John; Hussain, Saber

    2013-01-01

    Nanoparticles (NPs) are being implemented in a wide range of applications, and it is critical to proactively investigate their toxicity. Due to the extensive range of NPs being produced, in vitro studies are a valuable approach for toxicity screening. Key information required to support in vitro toxicity assessments include NP stability in biologically relevant media and fate once exposed to cells. Hyperspectral microscopy is a sensitive, real-time technique that combines the use of microscopy and spectroscopy for the measurement of the reflectance spectrum at individual pixels in a micrograph. This method has been used extensively for molecular imaging with plasmonic NPs as contrast agents (Aaron et al., Opt Express 16:2153-2167, 2008; Kumar et al., Nano Lett 7:1338-1343, 2007; Wax and Sokolov, Laser Photon Rev 3:146-158, 2009; Curry et al., Opt Express 14:6535-6542, 2006; Curry et al., J Biomed Opt 13:014022, 2008; Cognet et al., Proc Natl Acad Sci U S A 100:11350-11355, 2003; Sokolov et al., Cancer Res 63:1999-2004, 2003; Sönnichsen et al., Nat Biotechnol 23:741-745, 2005; Nusz et al., Anal Chem 80:984-989, 2008) and/or sensors (Nusz et al., Anal Chem 80:984-989, 2008; Ungureanu et al., Sens Actuators B 150:529-536, 2010; McFarland and Van Duyne, Nano Lett 3:1057-1062, 2003; Galush et al., Nano Lett 9:2077-2082, 2009; El-Sayed et al., Nano Lett 5:829-834, 2005). Here we describe an approach for using hyperspectral microscopy to characterize the agglomeration and stability of plasmonic NPs in biological media and their interactions with cells.

  13. 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.

  14. Investigating Ceria Nanocrystals Uptake by Glioblastoma Multiforme Cells and its Related Effects: An Electron Microscopy Study

    KAUST Repository

    Aloufi, Bader

    2017-01-22

    Cerium oxide nanoparticles have been utilized widely nowadays in cancer research. It has been suggested by many studies that these nanoparticles are capable of having dual antioxidant behavior in healthy and cancer microenvironment; where in physiological condition, they act as antioxidant and do not affect the healthy cells, while in tumor-like condition; they act as an oxidase, and result in a selective killing for the cancer cells. In this experiment, the interaction of nanoceria with glioblastoma and healthy astrocyte cells was examined, and further correlated with the in vitro cytotoxic effects of various nanoceria concentrations (100 and 300 µg/ml) and exposure times (12, 24, and 48 hours). Electron microscopes were used to investigate the cellular-NPs interactions, and to examine the related cytotoxic effects in combination with trypan blue and propidium iodide viability assays. Our data suggest the following results. First, the two cell lines demonstrated capability of taken up the ceria through endocytosis pathway, where the NPs were recognized engulfed by double membrane vesicles at various regions over the cellular cytoplasm. Secondly, cerium oxide nanoparticles were found to affect the glioblastoma cells, but not so severely the corresponding healthy astrocytes at the various concentrations and incubation times, as revealed by the viability assays and the electron microscopy analysis. Thirdly, the viability of the glioblastoma cells after the treatment displayed a declined trend when increasing the ceria concentrations, but did not show such dependency with regard to the different time points. In all cases, the healthy astrocyte cells showed slight alterations in mitochondrial shape which did not influence their viability. Among the various nanoceria concentrations and exposure times, the most efficient dose of treatment was found to be with a concentration of 300 µg/ml at a time point of 24-hour, where higher reduction on the viability of

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

    Directory of Open Access Journals (Sweden)

    Adam E Lynch

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2017-02-24

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

  18. Fourier ptychographic microscopy for filtration-based circulating tumor cell enumeration and analysis

    Science.gov (United States)

    Williams, Anthony; Chung, Jaebum; Ou, Xiaoze; Zheng, Guoan; Rawal, Siddarth; Ao, Zheng; Datar, Ram; Yang, Changhuei; Cote, Richard

    2014-06-01

    Circulating tumor cells (CTCs) are recognized as a candidate biomarker with strong prognostic and predictive potential in metastatic disease. Filtration-based enrichment technologies have been used for CTC characterization, and our group has previously developed a membrane microfilter device that demonstrates efficacy in model systems and clinical blood samples. However, uneven filtration surfaces make the use of standard microscopic techniques a difficult task, limiting the performance of automated imaging using commercially available technologies. Here, we report the use of Fourier ptychographic microscopy (FPM) to tackle this challenge. Employing this method, we were able to obtain high-resolution color images, including amplitude and phase, of the microfilter samples over large areas. FPM's ability to perform digital refocusing on complex images is particularly useful in this setting as, in contrast to other imaging platforms, we can focus samples on multiple focal planes within the same frame despite surface unevenness. In model systems, FPM demonstrates high image quality, efficiency, and consistency in detection of tumor cells when comparing corresponding microfilter samples to standard microscopy with high correlation (R2=0.99932). Based on these results, we believe that FPM will have important implications for improved, high throughput, filtration-based CTC analysis, and, more generally, image analysis of uneven surfaces.

  19. Optical coherence tomography segmentation reveals ganglion cell layer pathology after optic neuritis.

    Science.gov (United States)

    Syc, Stephanie B; Saidha, Shiv; Newsome, Scott D; Ratchford, John N; Levy, Michael; Ford, E'tona; Crainiceanu, Ciprian M; Durbin, Mary K; Oakley, Jonathan D; Meyer, Scott A; Frohman, Elliot M; Calabresi, Peter A

    2012-02-01

    Post-mortem ganglion cell dropout has been observed in multiple sclerosis; however, longitudinal in vivo assessment of retinal neuronal layers following acute optic neuritis remains largely unexplored. Peripapillary retinal nerve fibre layer thickness, measured by optical coherence tomography, has been proposed as an outcome measure in studies of neuroprotective agents in multiple sclerosis, yet potential swelling during the acute stages of optic neuritis may confound baseline measurements. The objective of this study was to ascertain whether patients with multiple sclerosis or neuromyelitis optica develop retinal neuronal layer pathology following acute optic neuritis, and to systematically characterize such changes in vivo over time. Spectral domain optical coherence tomography imaging, including automated retinal layer segmentation, was performed serially in 20 participants during the acute phase of optic neuritis, and again 3 and 6 months later. Imaging was performed cross-sectionally in 98 multiple sclerosis participants, 22 neuromyelitis optica participants and 72 healthy controls. Neuronal thinning was observed in the ganglion cell layer of eyes affected by acute optic neuritis 3 and 6 months after onset (P optica, with and without a history of optic neuritis, when compared with healthy controls (P optica and a history of optic neuritis exhibited the greatest reduction in ganglion cell layer thickness. Results from our in vivo longitudinal study demonstrate retinal neuronal layer thinning following acute optic neuritis, corroborating the hypothesis that axonal injury may cause neuronal pathology in multiple sclerosis. Further, these data provide evidence of subclinical disease activity, in both participants with multiple sclerosis and with neuromyelitis optica without a history of optic neuritis, a disease in which subclinical disease activity has not been widely appreciated. No pathology was seen in the inner or outer nuclear layers of eyes with optic

  20. 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.

  1. 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.

  2. Quantitative microscopy and nanoscopy of sickle red blood cells performed by wide field digital interferometry

    Science.gov (United States)

    Shaked, Natan T.; Satterwhite, Lisa L.; Telen, Marilyn J.; Truskey, George A.; Wax, Adam

    2011-03-01

    We have applied wide-field digital interferometry (WFDI) to examine the morphology and dynamics of live red blood cells (RBCs) from individuals who suffer from sickle cell anemia (SCA), a genetic disorder that affects the structure and mechanical properties of RBCs. WFDI is a noncontact, label-free optical microscopy approach that can yield quantitative thickness profiles of RBCs and measurements of their membrane fluctuations at the nanometer scale reflecting their stiffness. We find that RBCs from individuals with SCA are significantly stiffer than those from a healthy control. Moreover, we show that the technique is sensitive enough to distinguish classes of RBCs in SCA, including sickle RBCs with apparently normal morphology, compared to the stiffer crescent-shaped sickle RBCs. We expect that this approach will be useful for diagnosis of SCA and for determining efficacy of therapeutic agents.

  3. An approach to estimate spatial distribution of analyte within cells using spectrally-resolved fluorescence microscopy

    Science.gov (United States)

    Sharma, Dharmendar Kumar; Irfanullah, Mir; Basu, Santanu Kumar; Madhu, Sheri; De, Suman; Jadhav, Sameer; Ravikanth, Mangalampalli; Chowdhury, Arindam

    2017-03-01

    While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in

  4. 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.

  5. Nanoparticle interactions with live cells: Quantitative fluorescence microscopy of nanoparticle size effects

    Directory of Open Access Journals (Sweden)

    Li Shang

    2014-12-01

    Full Text Available Engineered nanomaterials are known to enter human cells, often via active endocytosis. Mechanistic details of the interactions between nanoparticles (NPs with cells are still not well enough understood. NP size is a key parameter that controls the endocytic mechanism and affects the cellular uptake yield. Therefore, we have systematically analyzed the cellular uptake of fluorescent NPs in the size range of 3.3–100 nm (diameter by live cells. By using spinning disk confocal microscopy in combination with quantitative image analysis, we studied the time courses of NP association with the cell membrane and subsequent internalization. NPs with diameters of less than 10 nm were observed to accumulate at the plasma membrane before being internalized by the cells. In contrast, larger NPs (100 nm were directly internalized without prior accumulation at the plasma membrane, regardless of their surface charges. We attribute this distinct size dependence to the requirement of a sufficiently strong local interaction of the NPs with the endocytic machinery in order to trigger the subsequent internalization.

  6. 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.

  7. Monitoring exocytosis and release from individual mast cells by capillary electrophoresis and UV imaging microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Yeung, E.S. [Ames Lab., IA (United States)]|[Iowa State Univ., Ames, IA (United States); Lillard, S.J. [Stanford Univ., CA (United States); McCloskey, M.A. [Iowa State Univ., Ames, IA (United States)

    1997-12-31

    The complex temporal evolution of on-column exocytotic release of serotonin from individual peritoneal mast cells (RPMCs) was monitored by using capillary electrophoresis and UV imaging microscopy. Laser-induced native fluorescence detection with 275-nm excitation was used, and a detection limit of 1.7 amol (S/N = 3; rms) was obtained for serotonin. A physiological running buffer was used to ensure that the cell remained viable throughout. The secretagogue was polymyxin B sulfate (Pmx). Following the injection of a single mast cell into the capillary, electromigration of Pmx toward and past the cell induced degranulation and release of serotonin. The time course of release was registered in the electropherograms with subsecond resolution. Subsequent introduction of SDS caused the cell to lyse completely and allowed the residual serotonin to be quantified. The average amount of serotonin observed per RPMC was 1.6 {+-} 0.6 fmol; the average percentage of serotonin released was 28 {+-} 14%. Events that are consistent with released serontonin from single submicron granules (250 aL each) were evident, each of which contained an average amount of 5.9 {+-} 3 amol. Alternatively, UV movies can be taken of the entire event to provide temporal and spatial information.

  8. 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

  9. 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.

  10. 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

  11. Detection of activated parietal epithelial cells on the glomerular tuft distinguishes early focal segmental glomerulosclerosis from minimal change disease

    NARCIS (Netherlands)

    Smeets, B.; Stucker, F.; Wetzels, J.; Brocheriou, I.; Ronco, P.; Grone, H.J.; D'Agati, V.; Fogo, A.B.; Kuppevelt, T.H. van; Fischer, H.P.; Boor, P.; Floege, J.; Ostendorf, T.; Moeller, M.J.

    2014-01-01

    In rodents, parietal epithelial cells (PECs) migrating onto the glomerular tuft participate in the formation of focal segmental glomerulosclerosis (FSGS) lesions. We investigated whether immunohistologic detection of PEC markers in the initial biopsies of human patients with first manifestation of i

  12. Human mesenchymal stem cell behavior on segmented polyurethanes prepared with biologically active chain extenders.

    Science.gov (United States)

    Kavanaugh, Taylor E; Clark, Amy Y; Chan-Chan, Lerma H; Ramírez-Saldaña, Maricela; Vargas-Coronado, Rossana F; Cervantes-Uc, José M; Hernández-Sánchez, Fernando; García, Andrés J; Cauich-Rodríguez, Juan V

    2016-02-01

    The development of elastomeric, bioresorbable and biocompatible segmented polyurethanes (SPUs) for use in tissue-engineering applications has attracted considerable interest because of the existing need of mechanically tunable scaffolds for regeneration of different tissues, but the incorporation of osteoinductive molecules into SPUs has been limited. In this study, SPUs were synthesized from poly (ε-caprolactone)diol, 4,4'-methylene bis(cyclohexyl isocyanate) using biologically active compounds such as ascorbic acid, L-glutamine, β-glycerol phosphate, and dexamethasone as chain extenders. Fourier transform infrared spectroscopy (FTIR) revealed the formation of both urethanes and urea linkages while differential scanning calorimetry, dynamic mechanical analysis, X-ray diffraction and mechanical testing showed that these polyurethanes were semi-crystalline polymers exhibiting high deformations. Cytocompatibility studies showed that only SPUs containing β-glycerol phosphate supported human mesenchymal stem cell adhesion, growth, and osteogenic differentiation, rendering them potentially suitable for bone tissue regeneration, whereas other SPUs failed to support either cell growth or osteogenic differentiation, or both. This study demonstrates that modification of SPUs with osteogenic compounds can lead to new cytocompatible polymers for regenerative medicine applications.

  13. 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.

  14. 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).

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Towards quantitative molecular mapping of cells by Raman microscopy: using AFM for decoupling molecular concentration and cell topography.

    Science.gov (United States)

    Boitor, Radu; Sinjab, Faris; Strohbuecker, Stephanie; Sottile, Virginie; Notingher, Ioan

    2016-06-23

    Raman micro-spectroscopy (RMS) is a non-invasive technique for imaging live cells in vitro. However, obtaining quantitative molecular information from Raman spectra is difficult because the intensity of a Raman band is proportional to the number of molecules in the sampled volume, which depends on the local molecular concentration and the thickness of the cell. In order to understand these effects, we combined RMS with atomic force microscopy (AFM), a technique that can measure accurately the thickness profile of the cells. Solution-based calibration models for RNA and albumin were developed to create quantitative maps of RNA and proteins in individual fixed cells. The maps were built by applying the solution-based calibration models, based on partial least squares fitting (PLS), on raster-scan Raman maps, after accounting for the local cell height obtained from the AFM. We found that concentrations of RNA in the cytoplasm of mouse neuroprogenitor stem cells (NSCs) were as high as 25 ± 6 mg ml(-1), while proteins were distributed more uniformly and reached concentrations as high as ∼50 ± 12 mg ml(-1). The combined AFM-Raman datasets from fixed cells were also used to investigate potential improvements for normalization of Raman spectral maps. For all Raman maps of fixed cells (n = 10), we found a linear relationship between the scores corresponding to the first component (PC1) and the cell height profile obtained by AFM. We used PC1 scores to reconstruct the relative height profiles of independent cells (n = 10), and obtained correlation coefficients with AFM maps higher than 0.99. Using this normalization method, qualitative maps of RNA and protein were used to obtain concentrations for live NSCs. While this study demonstrates the potential of using AFM and RMS for measuring concentration maps for individual NSCs in vitro, further studies are required to establish the robustness of the normalization method based on principal component analysis when comparing

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

    Science.gov (United States)

    2013-06-01

    samples   with   inertial   microfluidics  can  reduce  false  positive  rates  by  excluding  cell...HDLS       22   The   microfluidic  device  was  fabricated  in  thermoset  polyester  (TPE).  We  used   samples ...conventional microscopy are incapable of statistically relevant evaluation and screening of large populations with high accuracy due to its

  1. Polarization Force Microscopy of the Cell-Mineral Interface: Insights Into the Bioelectric Signature

    Science.gov (United States)

    Bartosik, E. M.; Kendall, T. A.

    2007-12-01

    The success of bioremediation strategies is dependent upon effective monitoring of microorganisms in the subsurface. Induced polarization (IP) may represent a cost-effective, complementary technique to existing borehole-based microbe detection schemes. Recent studies show a significant, yet poorly understood IP effect associated with the presence of bacteria in aqueous and porous media. This effect is believed to be rooted in the physicochemical surface interactions between cells and minerals which we probe using polarization and electric force microscopy. Dispersions of the local permittivity inferred from polarization force data that was collected over a hydrated mineral surface correspond to dispersions modeled for a bacterium. In each case, absolute permittivities and frequency cut-off values increase with surface potential and ion mobility, respectively. Potentially similar polarization mechanisms between the inorganic and organic condition are inferred. Further polarization force microscopy measurements of the mineral-microbe interface will provide molecular-level insight that complements column and field-scale IP observations. Anticipated is a more comprehensive mechanisitic description of the bioelectric IP response that facilitates application of IP to bioremediation.

  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. 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.

  4. 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.

  5. An introduction to the wound healing assay using live-cell microscopy.

    Science.gov (United States)

    Jonkman, James E N; Cathcart, Judith A; Xu, Feng; Bartolini, Miria E; Amon, Jennifer E; Stevens, Katarzyna M; Colarusso, Pina

    2014-01-01

    The wound healing assay is used in a range of disciplines to study the coordinated movement of a cell population. In this technical review, we describe the workflow of the wound healing assay as monitored by optical microscopy. Although the assay is straightforward, a lack of standardization in its application makes it difficult to compare results and reproduce experiments among researchers. We recommend general guidelines for consistency, including: (1) sample preparation including the creation of the gap, (2) microscope equipment requirements, (3) image acquisition, and (4) the use of image analysis to measure the gap size and its rate of closure over time. We also describe parameters that are specific to the particular research question, such as seeding density and matrix coatings. All of these parameters must be carefully controlled within a given set of experiments in order to achieve accurate and reproducible results.

  6. Atomic Force Microscopy Investigation of Morphological and Nanomechanical Properties of Pseudomonas aeruginosa Cells

    DEFF Research Database (Denmark)

    Mortensen, Ninell Pollas

    2008-01-01

    Atomic Force Microscopy (AFM) is unique in the aspect of studying living biological sample under physiological conditions. AFM was invented in 1986 by Binnig and Gerber and began in the early 1990’s to be implemented in life science. AFM can give a detailed three dimensional image of an intact cell...... caused by the dehydration. When visualizing bacteria in liquid the image resolution is reduced, but the bacteria are kept in the natural environment and therefore not subject to the same degree of artifact formation as observed for dehydrated bacteria. However, when imaging rode-shape Gram...... spectrum diagnostic tool originally visualized. Low antibody-antigen affinity and inefficient exposure of the antibody recognition sites could be an explanation for the lack of success. Work presented in this thesis proves what powerful tool AFM is in bacteriology. AFM of bacteria in liquid can be used...

  7. A simple and stable auto focusing protocol for long multidimensional live cell microscopy.

    Science.gov (United States)

    Wolf, F; Geley, S

    2006-01-01

    Focus maintenance is a challenging problem in multidimensional wide-field microscopy. Most automated microscopes use software algorithms, which are applied to z-sections of the object, to select for the plane with the best signal to noise ratio. When applied automatically in multidimensional imaging applications, auto focus routines significantly increase light exposure and can become cytotoxic if applied too frequently. In addition, automated focusing procedures can readily focus on unwanted high contrast objects. By labelling a defined position with a fluorescent marker, we were able to separate the focusing procedure from the actual image acquisition positions and therefore overcome some of the major drawbacks of routine auto focus procedures. To implement this method in a multidimensional acquisition experiment, we created a visual basic-based program, which is run prior to each image acquisition. This technique allows tight control of focus whilst keeping light toxicity in live cell imaging experiments to a minimum.

  8. 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.

  9. 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.

  10. 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.

  11. Charge transport in CdTe solar cells revealed by conductive tomographic atomic force microscopy

    Science.gov (United States)

    Luria, Justin; Kutes, Yasemin; Moore, Andrew; Zhang, Lihua; Stach, Eric A.; Huey, Bryan D.

    2016-11-01

    The influence of microstructural defects on the device properties in CdTe remains largely unknown. This is partly because characterization techniques have been unable to image electrical pathways throughout three-dimensional grains and grain boundaries with nanoscale resolution. Here, we employ a conductive and tomographic variation of atomic force microscopy to study charge transport at the nanoscale in a functioning thin-film solar cell with 12.3% efficiency. Images of electric current collected through the device thickness reveal spatially dependent short-circuit and open-circuit performance, and confirm that grain boundaries are preferential pathways for electron transport. Results on samples with and without cadmium chloride treatment reveal little difference in grain structure at the microscale, with samples without treatment showing almost no photocurrent either at planar defects or at grain boundaries. Our results supports an energetically orthogonal transport system of grain boundaries and interconnected planar defects as contributing to optimal solar cell performance, contrary to the conventional wisdom of the deleterious role of planar defects on polycrystalline thin-film solar cells.

  12. 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...

  13. Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

    Energy Technology Data Exchange (ETDEWEB)

    Estrela-Lopis, I; Donath, E [Institute of Medical Physics and Biophysics, Leipzig University, Haertelstrasse 16, 04107 Leipzig (Germany); Romero, G; Rojas, E; Moya, S E, E-mail: Irina.Estrela-Lopis@medizin.uni-leipzig.de [CIC biomaGUNE, Paseo Miramon 182 Edificio Empresarial C, 20009 San Sebastian, Gipuzkoa (Spain)

    2011-07-06

    Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al{sub 2}O{sub 3} NPs were found in the cytoplasm. CeO{sub 2} NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

  14. Neuroblastoma after Childhood: Prognostic Relevance of Segmental Chromosome Aberrations, ATRX Protein Status, and Immune Cell Infiltration

    Directory of Open Access Journals (Sweden)

    Ana P. Berbegall

    2014-06-01

    Full Text Available Neuroblastoma (NB is a common malignancy in children but rarely occurs during adolescence or adulthood. This subgroup is characterized by an indolent disease course, almost uniformly fatal, yet little is known about the biologic characteristics. The aim of this study was to identify differential features regarding DNA copy number alterations, α-thalassemia/mental retardation syndrome X-linked (ATRX protein expression, and the presence of tumor-associated inflammatory cells. Thirty-one NB patients older than 10 years who were included in the Spanish NB Registry were considered for the current study; seven young and middle-aged adult patients (range 18-60 years formed part of the cohort. We performed single nucleotide polymorphism arrays, immunohistochemistry for immune markers (CD4, CD8, CD20, CD11b, CD11c, and CD68, and ATRX protein expression. Assorted genetic profiles were found with a predominant presence of a segmental chromosome aberration (SCA profile. Preadolescent and adolescent NB tumors showed a higher number of SCA, including 17q gain and 11q deletion. There was also a marked infiltration of immune cells, mainly high and heterogeneous, in young and middle-aged adult tumors. ATRX negative expression was present in the tumors. The characteristics of preadolescent, adolescent, young adult, and middle-aged adult NB tumors are different, not only from childhood NB tumors but also from each other. Similar examinations of a larger number of such tumor tissues from cooperative groups should lead to a better older age–dependent tumor pattern and to innovative, individual risk-adapted therapeutic approaches for these patients.

  15. Dynamic quantitative microscopy and nanoscopy of red blood cells in sickle cell disease

    Science.gov (United States)

    Shaked, Natan T.; Satterwhite, Lisa L.; Telen, Marilyn J.; Truskey, George A.; Wax, Adam

    2012-03-01

    We have applied wide-field digital interferometric techniques to quantitatively image sickle red blood cells (RBCs) [1] in a noncontact label-free manner, and measure the nanometer-scale fluctuations in their thickness as an indication of their stiffness. The technique can simultaneously measure the fluctuations for multiple spatial points on the RBC and thus yields a map describing the stiffness of each RBC in the field of view. Using this map, the local rigidity regions of the RBC are evaluated quantitatively. Since wide-field digital interferometry is a quantitative holographic imaging technique rather than one-point measurement, it can be used to simultaneously evaluate cell transverse morphology plus thickness in addition to its stiffness profile. Using this technique, we examine the morphology and dynamics of RBCs from individuals who suffer from sickle cell disease, and find that the sickle RBCs are significantly stiffer than healthy RBCs. Furthermore, we show that the technique is sensitive enough to distinguish various classes of sickle RBCs, including sickle RBCs with visibly-normal morphology, compared to the stiffer crescent-shaped sickle RBCs.

  16. Visualization of Mesenchymal Stromal Cells in 2Dand 3D-Cultures by Scanning Electron Microscopy with Lanthanide Contrasting.

    Science.gov (United States)

    Novikov, I A; Vakhrushev, I V; Antonov, E N; Yarygin, K N; Subbot, A M

    2017-02-01

    Mesenchymal stromal cells from deciduous teeth in 2D- and 3D-cultures on culture plastic, silicate glass, porous polystyrene, and experimental polylactoglycolide matrices were visualized by scanning electron microscopy with lanthanide contrasting. Supravital staining of cell cultures with a lanthanide-based dye (neodymium chloride) preserved normal cell morphology and allowed assessment of the matrix properties of the carriers. The developed approach can be used for the development of biomaterials for tissue engineering.

  17. Study of Collagen Birefringence in Different Grades of Oral Squamous Cell Carcinoma Using Picrosirius Red and Polarized Light Microscopy

    OpenAIRE

    2015-01-01

    Objectives. The present study was done to evaluate birefringence pattern of collagen fibres in different grades of oral squamous cell carcinoma using Picrosirius red stain and polarization microscopy and to determine if there is a change in collagen fibres between different grades of oral squamous cell carcinoma. Materials and Methods. Picrosirius red stained 5 μm thick sections of previously diagnosed different grades of squamous cell carcinoma and normal oral mucosa were studied under polar...

  18. 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

  19. Total internal reflection fluorescence (TIRF) microscopy for real-time imaging of nanoparticle-cell plasma membrane interaction

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Moghimi, Seyed Moien

    2012-01-01

    Nanoparticulate systems are widely used for site-specific drug and gene delivery as well as for medical imaging. The mode of nanoparticle-cell interaction may have a significant effect on the pathway of nanoparticle internalization and subsequent intracellular trafficking. Total internal reflection...... fluorescence (TIRF) microscopy allows for real-time monitoring of nanoparticle-membrane interaction events, which can provide vital information in relation to design and surface engineering of therapeutic nanoparticles for cell-specific targeting. In contrast to other microscopy techniques, the bleaching...

  20. 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

    2013-01-01

    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

  1. 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.

  2. Exploring the Dynamics of Cell Processes through Simulations of Fluorescence Microscopy Experiments

    Science.gov (United States)

    Angiolini, Juan; Plachta, Nicolas; Mocskos, Esteban; Levi, Valeria

    2015-01-01

    Fluorescence correlation spectroscopy (FCS) methods are powerful tools for unveiling the dynamical organization of cells. For simple cases, such as molecules passively moving in a homogeneous media, FCS analysis yields analytical functions that can be fitted to the experimental data to recover the phenomenological rate parameters. Unfortunately, many dynamical processes in cells do not follow these simple models, and in many instances it is not possible to obtain an analytical function through a theoretical analysis of a more complex model. In such cases, experimental analysis can be combined with Monte Carlo simulations to aid in interpretation of the data. In response to this need, we developed a method called FERNET (Fluorescence Emission Recipes and Numerical routines Toolkit) based on Monte Carlo simulations and the MCell-Blender platform, which was designed to treat the reaction-diffusion problem under realistic scenarios. This method enables us to set complex geometries of the simulation space, distribute molecules among different compartments, and define interspecies reactions with selected kinetic constants, diffusion coefficients, and species brightness. We apply this method to simulate single- and multiple-point FCS, photon-counting histogram analysis, raster image correlation spectroscopy, and two-color fluorescence cross-correlation spectroscopy. We believe that this new program could be very useful for predicting and understanding the output of fluorescence microscopy experiments. PMID:26039162

  3. Multiplexed high-content analysis of mitochondrial morphofunction using live-cell microscopy.

    Science.gov (United States)

    Iannetti, Eligio F; Smeitink, Jan A M; Beyrath, Julien; Willems, Peter H G M; Koopman, Werner J H

    2016-09-01

    Mitochondria have a central role in cellular (patho)physiology, and they display a highly variable morphology that is probably coupled to their functional state. Here we present a protocol that allows unbiased and automated quantification of mitochondrial 'morphofunction' (i.e., morphology and membrane potential), cellular parameters (size, confluence) and nuclear parameters (number, morphology) in intact living primary human skin fibroblasts (PHSFs). Cells are cultured in 96-well plates and stained with tetramethyl rhodamine methyl ester (TMRM), calcein-AM (acetoxy-methyl ester) and Hoechst 33258. Next, multispectral fluorescence images are acquired using automated microscopy and processed to extract 44 descriptors. Subsequently, the descriptor data are subjected to a quality control (QC) algorithm based upon principal component analysis (PCA) and interpreted using univariate, bivariate and multivariate analysis. The protocol requires a time investment of ∼4 h distributed over 2 d. Although it is specifically developed for PHSFs, which are widely used in preclinical research, the protocol is portable to other cell types and can be scaled up for implementation in high-content screening.

  4. 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.

  5. 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.

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

    Science.gov (United States)

    Hashim, Fatimah; Amin, Nakisah Mat

    2017-02-01

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

  7. 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.

  8. Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry

    Energy Technology Data Exchange (ETDEWEB)

    Pham, Son [Deakin University, Victoria 3216 (Australia); CSIRO Australian Animal Health Laboratory, Victoria 3220 (Australia); Tabarin, Thibault [ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, New South Wales 3220 (Australia); Garvey, Megan; Pade, Corinna [Deakin University, Victoria 3216 (Australia); CSIRO Australian Animal Health Laboratory, Victoria 3220 (Australia); Rossy, Jérémie [ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, New South Wales 3220 (Australia); Monaghan, Paul; Hyatt, Alex [CSIRO Australian Animal Health Laboratory, Victoria 3220 (Australia); Böcking, Till [ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, New South Wales 3220 (Australia); Leis, Andrew [CSIRO Australian Animal Health Laboratory, Victoria 3220 (Australia); Gaus, Katharina, E-mail: k.gaus@unsw.edu.au [ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, New South Wales 3220 (Australia); Mak, Johnson, E-mail: j.mak@deakin.edu.au [Deakin University, Victoria 3216 (Australia); CSIRO Australian Animal Health Laboratory, Victoria 3220 (Australia)

    2015-12-15

    Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the ‘lynchpin’ for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection. - Highlights: • Cell free viruses are able to receive external trigger that leads to apparent size expansion. • Virus envelope and CD4 receptor engagement is the lynchpin of virus size expansion. • Internal capsid organisation can influence receptor mediated virus size expansion. • Pre-existing virus-associated lipid membrane in cell free virus can accommodate the receptor mediated virus size expansion.

  9. 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.

  10. Enhanced multi-spectral imaging of live breast cancer cells using immunotargeted gold nanoshells and two-photon excitation microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Bickford, Lissett; Sun Jiantang; Fu, Kun; Lewinski, Nastassja; Nammalvar, Vengadesan; Chang, Joseph; Drezek, Rebekah [Department of Bioengineering, Rice University, Houston, TX 77005 (United States)], E-mail: drezek@rice.edu

    2008-08-06

    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.

  11. Enhanced multi-spectral imaging of live breast cancer cells using immunotargeted gold nanoshells and two-photon excitation microscopy

    Science.gov (United States)

    Bickford, Lissett; Sun, Jiantang; Fu, Kun; Lewinski, Nastassja; Nammalvar, Vengadesan; Chang, Joseph; Drezek, Rebekah

    2008-08-01

    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.

  12. Macrophages and dendritic cells in the rat meninges and choroid plexus: three-dimensional localisation by environmental scanning electron microscopy and confocal microscopy.

    Science.gov (United States)

    McMenamin, Paul G; Wealthall, Rosamund J; Deverall, Marie; Cooper, Stephanie J; Griffin, Brendan

    2003-09-01

    The present investigation provides novel information on the topographical distribution of macrophages and dendritic cells (DCs) in normal meninges and choroid plexus of the rat central nervous system (CNS). Whole-mounts of meninges and choroid plexus of Lewis rats were incubated with various anti-leucocyte monoclonal antibodies and either visualised with gold-conjugated secondary antibody followed by silver enhancement and subsequent examination by environmental scanning electron microscopy or by the use of fluorochromes and confocal microscopy. Large numbers of MHC class II(+) putative DCs were identified on the internal or subarachnoid aspect of dural whole-mounts, on the surface of the cortex (pia/arachnoid) and on the surface of the choroid plexus. Occupation of these sites would allow DCs access to cerebrospinal fluid (CSF) and therefore allow antigens into the subarachnoid space and ventricles. By contrast, macrophages were less evident at sites exposed to CSF and were more frequently located within the connective tissue of the dura/arachnoid and choroid plexus stroma and also in a sub-pial location. The present data suggest that DC may be strategically located within the CNS to sample CSF-borne antigens. Furthermore, the data suggest that CNS tissue samples collected without careful removal of the meninges may inadvertently be contaminated by DCs and meningeal macrophages.

  13. 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.

  14. 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.

  15. 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.

  16. Differences in nuclear DNA organization between lymphocytes, Hodgkin and Reed-Sternberg cells revealed by structured illumination microscopy.

    Science.gov (United States)

    Righolt, Christiaan H; Guffei, Amanda; Knecht, Hans; Young, Ian T; Stallinga, Sjoerd; van Vliet, Lucas J; Mai, Sabine

    2014-08-01

    Advances in light microscopy have enabled the visualization of DNA in the interphase nucleus with more detail than is visible with conventional light microscopy. The nuclear architecture is assumed to be different in cancer cells compared to normal cells. In this paper we have studied, for the first time, the organization of nuclear DNA and that of DNA-free space in control lymphocytes, Hodgkin cells and Reed-Sternberg cells using 3D structured illumination microscopy (SIM). We have observed detail in these SIM images that was not observed in conventional widefield images. We have measured the size distribution of the DNA structure using granulometry and noted a significant, progressive increase in the amount of sub-micron structures from control lymphocytes to Hodgkin cells to Reed-Sternberg cells. The DNA-free space changes as well; "holes" in the DNA distribution start to appear in the malignant cells. We have studied whether these "holes" are nucleoli by staining for upstream binding factor (UBF), a protein associated with the nucleolus. We have found that the relative UBF content progressively and significantly decreases-or is absent-in the DNA-free space when measured as either the Pearson correlation coefficient with the DNA-free space or as the number of "holes" that contain UBF. Similar differences exist within the population of Reed-Sternberg cells between binucleated and multinucleated cells with four or more subnuclei. To our knowledge, this is the first study that investigates the changes of the nuclear DNA structure in any disease with superresolution light microscopy.

  17. 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.

  18. 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...

  19. 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

  20. Nanoscale Spatial Organization of Prokaryotic Cells Studied by Super-Resolution Optical Microscopy

    Science.gov (United States)

    McEvoy, Andrea Lynn

    All cells spatially organize their interiors, and this arrangement is necessary for cell viability. Until recently, it was believed that only eukaryotic cells spatially segregate their components. However, it is becoming increasingly clear that bacteria also assemble their proteins into complex patterns. In eukaryotic cells, spatial organization arises from membrane bound organelles as well as motor transport proteins which can move cargos within the cell. To date, there are no known motor transport proteins in bacteria and most microbes lack membrane bound organelles, so it remains a mystery how bacterial spatial organization emerges. In hind-sight it is not surprising that bacteria also exhibit complex spatial organization considering much of what we have learned about the basic processes that take place in all cells, such as transcription and translation was first discovered in prokaryotic cells. Perhaps the fundamental principles that govern spatial organization in prokaryotic cells may be applicable in eukaryotic cells as well. In addition, bacteria are attractive model organism for spatial organization studies because they are genetically tractable, grow quickly and much biochemical and structural data is known about them. A powerful tool for observing spatial organization in cells is the fluorescence microscope. By specifically tagging a protein of interest with a fluorescent probe, it is possible to examine how proteins organize and dynamically assemble inside cells. A significant disadvantage of this technology is its spatial resolution (approximately 250 nm laterally and 500 nm axially). This limitation on resolution causes closely spaced proteins to look blurred making it difficult to observe the fine structure within the complexes. This resolution limit is especially problematic within small cells such as bacteria. With the recent invention of new optical microscopies, we now can surpass the existing limits of fluorescence imaging. In some cases, we can

  1. Convective heat transfer in a measurement cell for scanning electrochemical microscopy.

    Science.gov (United States)

    Novev, Javor K; Compton, Richard G

    2016-11-21

    Electrochemical experiments, especially those performed with scanning electrochemical microscopy (SECM), are often carried out without taking special care to thermostat the solution; it is usually assumed that its temperature is homogeneous and equal to the ambient. The present study aims to test this assumption via numerical simulations of the heat transfer in a particular system - the typical measurement cell for SECM. It is assumed that the temperature of the solution is initially homogeneous but different from that of its surroundings; convective heat transfer in the solution and the surrounding air is taken into account within the framework of the Boussinesq approximation. The hereby presented theoretical treatment indicates that an initial temperature difference of the order of 1 K dissipates with a characteristic time scale of ∼1000 s; the thermal equilibration is accompanied by convective flows with a maximum velocity of ∼10(-4) m s(-1); furthermore, the temporal evolution of the temperature profile is influenced by the sign of the initial difference. These results suggest that, unless the temperature of the solution is rigorously controlled, convection may significantly compromise the interpretation of data from SECM and other electrochemical techniques, which is usually done on the basis of diffusion-only models.

  2. Conductive Atomic Force Microscopy Applied to CdTe/CdS Solar Cells

    Energy Technology Data Exchange (ETDEWEB)

    Moutinho, H. R.; Dhere, R. G.; Jiang, C. -S.; Al-Jassim, M. M.; Kazmerski, L. L.

    2004-08-01

    In this work we describe for the first time the use of conductive atomic force microscopy (C-AFM) in the study of CdTe/CdS solar cells, before and after the etching processes used in device fabrication. C-AFM is a new technique that provides information on the electrical properties of the sample in conjunction with topographic images with high lateral resolution. At the same time, this technique allows for the generation of I-V curves at very well-defined locations. A potential is applied between the sample and a very sharp tip, which scans the sample in contact mode. The current images showed that different CdTe grains produce different contrast. Etching the CdTe with a bromine/methanol solution enhanced the current along grains boundaries when compared to the intragrain material. Etching with a solution of nitric and phosphoric acids did not show this effect. Instead, it increased the current through the whole sample surface.

  3. Electrical properties of CdTe/CdS solar cells investigated with conductive atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Moutinho, H.R. [National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, CO 80401 (United States)]. E-mail: helio_moutinho@nrel.gov; Dhere, R.G. [National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, CO 80401 (United States); Jiang, C.-S. [National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, CO 80401 (United States); Al-Jassim, M.M. [National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, CO 80401 (United States); Kazmerski, L.L. [National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, CO 80401 (United States)

    2006-08-30

    We report on the application of conductive atomic force microscopy (C-AFM) for studying the electrical properties of CdTe/CdS solar cells, and discuss the advantages and limitations of this technique. C-AFM is a new technique that uses the tip of an AFM to apply a potential between the tip and the sample, resulting in high spatial-resolution current images, as well as current versus voltage curves. The analyses were made before and after the standard vapor CdCl{sub 2} treatment, as well as two etching processes, using solutions of bromine/methanol and nitric-phosphoric acids. The current images from the untreated and CdCl{sub 2}-treated samples showed grains with different contrasts, due to differences in electrical conductivity or a nonuniform surface. The bromine/methanol etch resulted in more conductive grain boundaries as compared to intragrain material, while the nitric/phosphoric etch increased the conductivity of the whole film close to the surface and resulted in films with significant photocurrent.

  4. Correlated Light Microscopy and Electron Microscopy

    NARCIS (Netherlands)

    Sjollema, Klaas A.; Schnell, Ulrike; Kuipers, Jeroen; Kalicharan, Ruby; Giepmans, Ben N. G.; MullerReichert, T; Verkade, P

    2012-01-01

    Understanding where, when, and how biomolecules (inter)act is crucial to uncover fundamental mechanisms in cell biology. Recent developments in fluorescence light microscopy (FLM) allow protein imaging in living cells and at the near molecular level. However, fluorescence microscopy only reveals sel

  5. Confocal microscopy, a major tool for the cell and gene therapy of the muscle and nervous central system.

    OpenAIRE

    2012-01-01

    The specific research areas of the UMR 703 are focused on cell and gene therapies ofDuchenne Muscular Dystrophy (DMD) and Pompe disease (glycogenosis type II),a lysosomal storage disease with both CNS and muscle involvement. The unit has alsodeveloped a pathology coreAPEX. Histopathological expertise is the main mission ofAPEX by providing pathology and phenotypic analysis to support both academic andprivate research teams. Confocal microscopy is a major tool to explore cells andtissues in bi...

  6. 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...

  7. Visualizing viral protein structures in cells using genetic probes for correlated light and electron microscopy.

    Science.gov (United States)

    Ou, Horng D; Deerinck, Thomas J; Bushong, Eric; Ellisman, Mark H; O'Shea, Clodagh C

    2015-11-15

    Structural studies of viral proteins most often use high-resolution techniques such as X-ray crystallography, nuclear magnetic resonance, single particle negative stain, or cryo-electron microscopy (EM) to reveal atomic interactions of soluble, homogeneous viral proteins or viral protein complexes. Once viral proteins or complexes are separated from their host's cellular environment, their natural in situ structure and details of how they interact with other cellular components may be lost. EM has been an invaluable tool in virology since its introduction in the late 1940's and subsequent application to cells in the 1950's. EM studies have expanded our knowledge of viral entry, viral replication, alteration of cellular components, and viral lysis. Most of these early studies were focused on conspicuous morphological cellular changes, because classic EM metal stains were designed to highlight classes of cellular structures rather than specific molecular structures. Much later, to identify viral proteins inducing specific structural configurations at the cellular level, immunostaining with a primary antibody followed by colloidal gold secondary antibody was employed to mark the location of specific viral proteins. This technique can suffer from artifacts in cellular ultrastructure due to compromises required to provide access to the immuno-reagents. Immunolocalization methods also require the generation of highly specific antibodies, which may not be available for every viral protein. Here we discuss new methods to visualize viral proteins and structures at high resolutions in situ using correlated light and electron microscopy (CLEM). We discuss the use of genetically encoded protein fusions that oxidize diaminobenzidine (DAB) into an osmiophilic polymer that can be visualized by EM. Detailed protocols for applying the genetically encoded photo-oxidizing protein MiniSOG to a viral protein, photo-oxidation of the fusion protein to yield DAB polymer staining, and

  8. Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.

    Directory of Open Access Journals (Sweden)

    Alice C N Brown

    2011-09-01

    Full Text Available Natural Killer (NK cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.

  9. Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.

    Science.gov (United States)

    Brown, Alice C N; Oddos, Stephane; Dobbie, Ian M; Alakoskela, Juha-Matti; Parton, Richard M; Eissmann, Philipp; Neil, Mark A A; Dunsby, Christopher; French, Paul M W; Davis, Ilan; Davis, Daniel M

    2011-09-01

    Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.

  10. 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.

  11. 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

  12. Prevalence and Distribution of Segmentation Errors in Macular Ganglion Cell Analysis of Healthy Eyes Using Cirrus HD-OCT.

    Directory of Open Access Journals (Sweden)

    Rayan A Alshareef

    Full Text Available To determine the frequency of different types of spectral domain optical coherence tomography (SD-OCT scan artifacts and errors in ganglion cell algorithm (GCA in healthy eyes.Infrared image, color-coded map and each of the 128 horizontal b-scans acquired in the macular ganglion cell-inner plexiform layer scans using the Cirrus HD-OCT (Carl Zeiss Meditec, Dublin, CA macular cube 512 × 128 protocol in 30 healthy normal eyes were evaluated. The frequency and pattern of each artifact was determined. Deviation of the segmentation line was classified into mild (less than 10 microns, moderate (10-50 microns and severe (more than 50 microns. Each deviation, if present, was noted as upward or downward deviation. Each artifact was further described as per location on the scan and zones in the total scan area.A total of 1029 (26.8% out of total 3840 scans had scan errors. The most common scan error was segmentation error (100%, followed by degraded images (6.70%, blink artifacts (0.09% and out of register artifacts (3.3%. Misidentification of the inner retinal layers was most frequent (62%. Upward Deviation of the segmentation line (47.91% and severe deviation (40.3% were more often noted. Artifacts were mostly located in the central scan area (16.8%. The average number of scans with artifacts per eye was 34.3% and was not related to signal strength on Spearman correlation (p = 0.36.This study reveals that image artifacts and scan errors in SD-OCT GCA analysis are common and frequently involve segmentation errors. These errors may affect inner retinal thickness measurements in a clinically significant manner. Careful review of scans for artifacts is important when using this feature of SD-OCT device.

  13. 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.

  14. 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.

  15. 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.

  16. 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).

  17. Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

    Science.gov (United States)

    Höhn, K; Fuchs, J; Fröber, A; Kirmse, R; Glass, B; Anders-Össwein, M; Walther, P; Kräusslich, H-G; Dietrich, C

    2015-08-01

    In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells.

  18. 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

    content in the catalyst layer. Transmission electron microscopy (TEM) was carried out on selective electrodes to provide additional information and confirmed with the AFM results. Cyclic voltammetry (CV) showed that the electrode containing 30 wt.% ionomer has maximum catalyst utilization....

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. Confocal microscopy-based three-dimensional cell-specific modeling for large deformation analyses in cellular mechanics.

    Science.gov (United States)

    Slomka, Noa; Gefen, Amit

    2010-06-18

    This study introduces a new confocal microscopy-based three-dimensional cell-specific finite element (FE) modeling methodology for simulating cellular mechanics experiments involving large cell deformations. Three-dimensional FE models of undifferentiated skeletal muscle cells were developed by scanning C2C12 myoblasts using a confocal microscope, and then building FE model geometries from the z-stack images. Strain magnitudes and distributions in two cells were studied when the cells were subjected to compression and stretching, which are used in pressure ulcer and deep tissue injury research to induce large cell deformations. Localized plasma membrane and nuclear surface area (NSA) stretches were observed for both the cell compression and stretching simulation configurations. It was found that in order to induce large tensile strains (>5%) in the plasma membrane and NSA, one needs to apply more than approximately 15% of global cell deformation in cell compression tests, or more than approximately 3% of tensile strains in the elastic plate substrate in cell stretching experiments. Utilization of our modeling can substantially enrich experimental cellular mechanics studies in classic cell loading designs that typically involve large cell deformations, such as static and cyclic stretching, cell compression, micropipette aspiration, shear flow and hydrostatic pressure, by providing magnitudes and distributions of the localized cellular strains specific to each setup and cell type, which could then be associated with the applied stimuli.

  4. 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.

  5. 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.

  6. Mass transfer in fuel cells. [electron microscopy of components, thermal decomposition of Teflon, water transport, and surface tension of KOH solutions

    Science.gov (United States)

    Walker, R. D., Jr.

    1973-01-01

    Results of experiments on electron microscopy of fuel cell components, thermal decomposition of Teflon by thermogravimetry, surface area and pore size distribution measurements, water transport in fuel cells, and surface tension of KOH solutions are described.

  7. 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.

  8. 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.

  9. 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.

  10. The structure and function of cell membranes examined by atomic force microscopy and single-molecule force spectroscopy.

    Science.gov (United States)

    Shan, Yuping; Wang, Hongda

    2015-06-07

    The cell membrane is one of the most complicated biological complexes, and long-term fierce debates regarding the cell membrane persist because of technical hurdles. With the rapid development of nanotechnology and single-molecule techniques, our understanding of cell membranes has substantially increased. Atomic force microscopy (AFM) has provided several unprecedented advances (e.g., high resolution, three-dimensional and in situ measurements) in the study of cell membranes and has been used to systematically dissect the membrane structure in situ from both sides of membranes; as a result, novel models of cell membranes have recently been proposed. This review summarizes the new progress regarding membrane structure using in situ AFM and single-molecule force spectroscopy (SMFS), which may shed light on the study of the structure and functions of cell membranes.

  11. 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.

  12. 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.

  13. [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.

  14. Cell enumeration and visualisation by transmission electron microscopy of Lactobacillus rhamnosus treated with cinnamon (Cinnamomum zeylanicum B.) essential oil.

    Science.gov (United States)

    Feniman, C M; Rall, V L M; Doyama, J T; Júnior, A Fernandes

    2012-01-01

    The use of essential oils (EOs) in functional foods containing probiotic microorganisms must consider the antimicrobial activity of these oils against beneficial bacteria such as Lactobacillus rhamnosus. This study aimed to evaluate the sensitivity of L. rhamnosus cultures treated with cinnamon EO through viable cell counts and visualisation by transmission electron microscopy. Cinnamon EO at a concentration of 0.04% had a bacteriostatic activity after 2 h of incubation. Although slight alterations were detected in the cell structure, this concentration was considered to be bactericidal, since it led to a significant reduction in cell numbers after 24 h. On the other hand, cinnamon EO at a 1.00% concentration decreased cell counts by 3 log units after 2 h incubation and no viable cell count was detected after 24 h. Transmission electron microscopy indicated that cells treated with 1.00% cinnamon EO were severely damaged and presented cell membrane disruption and cytoplasmic leakage.

  15. A multi-model approach to simultaneous segmentation and classification of heterogeneous populations of cell nuclei in 3D confocal microscope images.

    Science.gov (United States)

    Lin, Gang; Chawla, Monica K; Olson, Kathy; Barnes, Carol A; Guzowski, John F; Bjornsson, Christopher; Shain, William; Roysam, Badrinath

    2007-09-01

    Automated segmentation and morphometry of fluorescently labeled cell nuclei in batches of 3D confocal stacks is essential for quantitative studies. Model-based segmentation algorithms are attractive due to their robustness. Previous methods incorporated a single nuclear model. This is a limitation for tissues containing multiple cell types with different nuclear features. Improved segmentation for such tissues requires algorithms that permit multiple models to be used simultaneously. This requires a tight integration of classification and segmentation algorithms. Two or more nuclear models are constructed semiautomatically from user-provided training examples. Starting with an initial over-segmentation produced by a gradient-weighted watershed algorithm, a hierarchical fragment merging tree rooted at each object is built. Linear discriminant analysis is used to classify each candidate using multiple object models. On the basis of the selected class, a Bayesian score is computed. Fragment merging decisions are made by comparing the score with that of other candidates, and the scores of constituent fragments of each candidate. The overall segmentation accuracy was 93.7% and classification accuracy was 93.5%, respectively, on a diverse collection of images drawn from five different regions of the rat brain. The multi-model method was found to achieve high accuracy on nuclear segmentation and classification by correctly resolving ambiguities in clustered regions containing heterogeneous cell populations.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. β-d-Glucan Antibodies Inhibit Auxin-Induced Cell Elongation and Changes in the Cell Wall of Zea Coleoptile Segments 1

    Science.gov (United States)

    Hoson, Takayuki; Nevins, Donald J.

    1989-01-01

    Antiserum was raised against the Avena sativa L. caryopsis β-d-glucan fraction with an average molecular weight of 1.5 × 104. Polyclonal antibodies recovered from the serum after Protein A-Sepharose column chromatography precipitated when cross-reacted with high molecular weight (1→3), (1→4)-β-d-glucans. These antibodies were effective in suppression of cell wall autohydrolytic reactions and auxin-induced decreases in noncellulosic glucose content of the cell wall of maize (Zea mays L.) coleoptiles. The results indicate antibody-mediated interference with in situ β-d-glucan degradation. The antibodies at a concentration of 200 micrograms per milliliter also suppress auxin-induced elongation by about 40% and cell wall loosening (measured by the minimum stress-relaxation time of the segments) of Zea coleoptiles. The suppression of elongation by antibodies was imposed without a lag period. Auxin-induced elongation, cell wall loosening, and chemical changes in the cell walls were near the levels of control tissues when segments were subjected to antibody preparation precipitated by a pretreatment with Avena caryopsis β-d-glucans. These results support the idea that the degradation of (1→3), (1→4)-β-d-glucans by cell wall enzymes is associated with the cell wall loosening responsible for auxin-induced elongation. PMID:16666935

  4. beta-d-Glucan Antibodies Inhibit Auxin-Induced Cell Elongation and Changes in the Cell Wall of Zea Coleoptile Segments.

    Science.gov (United States)

    Hoson, T; Nevins, D J

    1989-08-01

    Antiserum was raised against the Avena sativa L. caryopsis beta-d-glucan fraction with an average molecular weight of 1.5 x 10(4). Polyclonal antibodies recovered from the serum after Protein A-Sepharose column chromatography precipitated when cross-reacted with high molecular weight (1-->3), (1-->4)-beta-d-glucans. These antibodies were effective in suppression of cell wall autohydrolytic reactions and auxin-induced decreases in noncellulosic glucose content of the cell wall of maize (Zea mays L.) coleoptiles. The results indicate antibody-mediated interference with in situ beta-d-glucan degradation. The antibodies at a concentration of 200 micrograms per milliliter also suppress auxin-induced elongation by about 40% and cell wall loosening (measured by the minimum stress-relaxation time of the segments) of Zea coleoptiles. The suppression of elongation by antibodies was imposed without a lag period. Auxin-induced elongation, cell wall loosening, and chemical changes in the cell walls were near the levels of control tissues when segments were subjected to antibody preparation precipitated by a pretreatment with Avena caryopsis beta-d-glucans. These results support the idea that the degradation of (1-->3), (1-->4)-beta-d-glucans by cell wall enzymes is associated with the cell wall loosening responsible for auxin-induced elongation.

  5. A comparative study of metabolic state of stem cells during osteogenic and adipogenic differentiations via fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Chakraborty, Sandeep; Ou, Meng-Hsin; Kuo, Jean-Cheng; Chiou, Arthur

    2016-10-01

    Cellular metabolic state can serve as a biomarker to indicate the differentiation potential of stem cells into other specialized cell lineages. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was applied to determine the fluorescence lifetime and the amounts of the auto-fluorescent metabolic co-factor reduced nicotinamide adenine dinucleotide (NADH) to elucidate the cellular metabolism of human mesenchymal stem cells (hMSCs) in osteogenic and adipogenic differentiation processes. 2P-FLIM provides the free to protein-bound NADH ratio which can serve as the indicator of cellular metabolic state. We measured NADH fluorescence lifetime at 0, 7, and 14 days after hMSCs were induced for either osteogenesis or adipogenesis. In both cases, the average fluorescence lifetime increased significantly at day 14 (P stem cells into other specialized cell lineages.

  6. 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.

  7. 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.

  8. Investigation of quercetin-induced HepG2 cell apoptosis-associated cellular biophysical alterations by atomic force microscopy.

    Science.gov (United States)

    Pi, Jiang; Li, Baole; Tu, Lvying; Zhu, Haiyan; Jin, Hua; Yang, Fen; Bai, Haihua; Cai, Huaihong; Cai, Jiye

    2016-01-01

    Quercetin, a wildly distributed bioflavonoid, has been proved to possess excellent antitumor activity on hepatocellular carcinoma (HCC). In the present study, the biophysical properties of HepG2 cells were qualitatively and quantitatively determined using high resolution atomic force microscopy (AFM) to understand the anticancer effects of quercetin on HCC cells at nanoscale. The results showed that quercetin could induce severe apoptosis in HepG2 cells through arrest of cell cycle and disruption of mitochondria membrane potential. Additionally, the nuclei and F-actin structures of HepG2 cells were destroyed by quercetin treatment as well. AFM morphological data showed some typical apoptotic characterization of HepG2 cells with increased particle size and roughness in the ultrastructure of cell surface upon quercetin treatment. As an important biophysical property of cells, the membrane stiffness of HepG2 cells was further quantified by AFM force measurements, which indicated that HepG2 cells became much stiffer after quercetin treatment. These results collectively suggest that quercetin can be served as a potential therapeutic agent for HCC, which not only extends our understanding of the anticancer effects of quercetin against HCC cells into nanoscale, but also highlights the applications of AFM for the investigation of anticancer drugs.

  9. 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

  10. 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.

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

    Science.gov (United States)

    2012-08-01

    microfilter platform captures CTC from the cancer patients’ blood cost effectively, where the larger CTC are preferentially retained on the membrane ...capture through parylene-based microfiltration technology for breast cancer with high sensitivity (Aim 1), defining imaging parameters for CTC analysis...finally, we have combined the microfiltration and WFOV microscopy components to create an integrated CTC analysis system (Aim 4). 15

  12. Segmentation and Classification of Bone Marrow Cells Images Using Contextual Information for Medical Diagnosis of Acute Leukemias

    Science.gov (United States)

    Reta, Carolina; Altamirano, Leopoldo; Gonzalez, Jesus A.; Diaz-Hernandez, Raquel; Peregrina, Hayde; Olmos, Ivan; Alonso, Jose E.; Lobato, Ruben

    2015-01-01

    Morphological identification of acute leukemia is a powerful tool used by hematologists to determine the family of such a disease. In some cases, experienced physicians are even able to determine the leukemia subtype of the sample. However, the identification process may have error rates up to 40% (when classifying acute leukemia subtypes) depending on the physician’s experience and the sample quality. This problem raises the need to create automatic tools that provide hematologists with a second opinion during the classification process. Our research presents a contextual analysis methodology for the detection of acute leukemia subtypes from bone marrow cells images. We propose a cells separation algorithm to break up overlapped regions. In this phase, we achieved an average accuracy of 95% in the evaluation of the segmentation process. In a second phase, we extract descriptive features to the nucleus and cytoplasm obtained in the segmentation phase in order to classify leukemia families and subtypes. We finally created a decision algorithm that provides an automatic diagnosis for a patient. In our experiments, we achieved an overall accuracy of 92% in the supervised classification of acute leukemia families, 84% for the lymphoblastic subtypes, and 92% for the myeloblastic subtypes. Finally, we achieved accuracies of 95% in the diagnosis of leukemia families and 90% in the diagnosis of leukemia subtypes. PMID:26107374

  13. Segmentation and Classification of Bone Marrow Cells Images Using Contextual Information for Medical Diagnosis of Acute Leukemias.

    Science.gov (United States)

    Reta, Carolina; Altamirano, Leopoldo; Gonzalez, Jesus A; Diaz-Hernandez, Raquel; Peregrina, Hayde; Olmos, Ivan; Alonso, Jose E; Lobato, Ruben

    2015-01-01

    Morphological identification of acute leukemia is a powerful tool used by hematologists to determine the family of such a disease. In some cases, experienced physicians are even able to determine the leukemia subtype of the sample. However, the identification process may have error rates up to 40% (when classifying acute leukemia subtypes) depending on the physician's experience and the sample quality. This problem raises the need to create automatic tools that provide hematologists with a second opinion during the classification process. Our research presents a contextual analysis methodology for the detection of acute leukemia subtypes from bone marrow cells images. We propose a cells separation algorithm to break up overlapped regions. In this phase, we achieved an average accuracy of 95% in the evaluation of the segmentation process. In a second phase, we extract descriptive features to the nucleus and cytoplasm obtained in the segmentation phase in order to classify leukemia families and subtypes. We finally created a decision algorithm that provides an automatic diagnosis for a patient. In our experiments, we achieved an overall accuracy of 92% in the supervised classification of acute leukemia families, 84% for the lymphoblastic subtypes, and 92% for the myeloblastic subtypes. Finally, we achieved accuracies of 95% in the diagnosis of leukemia families and 90% in the diagnosis of leukemia subtypes.

  14. Segmentation and Shape Classification of Nuclei in DAPI Images

    OpenAIRE

    Snell, V; Kittler, J.; Christmas, W

    2011-01-01

    This paper addresses issues of analysis of DAPI-stained microscopy images of cell samples, particularly classification of objects as single nuclei, nuclei clusters or nonnuclear material. First, segmentation is significantly improved compared to Otsu’s method[5] by choosing a more appropriate threshold, using a cost-function that explicitly relates to the quality of resulting boundary, rather than image histogram. This method applies ideas from active contour models to threshold-based segment...

  15. The CS1 segment of fibronectin is involved in human OSCC pathogenesis by mediating OSCC cell spreading, migration, and invasion

    Directory of Open Access Journals (Sweden)

    D'Silva Nisha J

    2010-06-01

    Full Text Available Abstract Background The alternatively spliced V region or type III connecting segment III (IIICS of fibronectin is important in early development, wound healing, and tumorigenesis, however, its role in oral cancer has not been fully investigated. Thus, we investigated the role of CS-1, a key site within the CSIII region of fibronectin, in human oral squamous cell carcinoma (OSCC. Methods To determine the expression of CS-1 in human normal and oral SCC tissue specimens immunohistochemical analyses were performed. The expression of CS1 was then associated with clinicopathological factors. To investigate the role of CS-1 in regulating OSCC cell spreading, migration and invasion, OSCC cells were assayed for spreading and migration in the presence of a CS-1 peptide or a CS-1 blocking peptide, and for invasion using Matrigel supplemented with these peptides. In addition, integrin α4siRNA or a focal adhesion kinase (FAK anti-sense oligonucleotide was transfected into OSCC cells to examine the mechanistic role of integrin α4 or FAK in CS1-mediated cell spreading and migration, respectively. Results CS-1 expression levels were significantly higher in OSCC tissues compared to normal tissues (p Conclusion These data indicate that the CS-1 site of fibronectin is involved in oral cancer pathogenesis and in regulating OSCC cell spreading, migration and invasion.

  16. 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}

  17. A confocal microscopy image analysis method to measure adhesion and internalization of Pseudomonas aeruginosa multicellular structures into epithelial cells.

    Science.gov (United States)

    Lepanto, Paola; Lecumberry, Federico; Rossello, Jéssica; Kierbel, Arlinet

    2014-02-01

    Formation of multicellular structures such as biofilms is an important feature in the physiopathology of many disease-causing bacteria. We recently reported that Pseudomonas aeruginosa adheres to epithelial cells rapidly forming early biofilm-like aggregates, which can then be internalized into cells. Conventional methods to measure adhesion/internalization, such as dilution plating for total cell-associated or antibiotic protected bacteria, do not distinguish between single and aggregated bacteria. We report a procedure that combining double bacteria labeling, confocal microscopy and image analysis allows identification and quantification of the number of adhered and internalized bacteria distinguishing between single and aggregated bacterial cells. A plugin for Fiji to automatically perform these procedures has been generated.

  18. Multichannel fluorescence spinning disk microscopy reveals early endogenous CD4 T cell recruitment in contact sensitivity via complement.

    Science.gov (United States)

    Norman, M Ursula; Hulliger, Sara; Colarusso, Pina; Kubes, Paul

    2008-01-01

    Contact sensitivity (CS) is one of the primary in vivo models of T cell-mediated inflammation. The presence of CS-initiating CD4 T lymphocytes at the time of challenge is essential for transfer and full development of the late phase CS inflammatory response. From this observation investigators have speculated that early recruitment of CD4 T cells to the site of challenge must occur. Moreover, there must be rapid synthesis/release and disappearance of an important mediator during the first hours after hapten challenge. Using spinning disk confocal microscopy, we observed the very early effector events of the immune response. Simultaneous, real-time visualization of predominant neutrophil and extremely rare CD4 T cell trafficking in the challenged skin vasculature was noted (one rolling CD4 T cell for every 10-18 rolling and adherent neutrophils). We demonstrate that neutrophil adhesion during the early CS response was reduced in C5a receptor-deficient (C5aR-/-) mice or leukotriene B4 receptor antagonist-treated mice, whereas CD4 T cell recruitment was only inhibited in C5aR-/- mice. In line with these observations, leukocyte infiltration and the associated tissue damage were significantly reduced in C5aR-/- mice but not in leukotriene B4 receptor antagonist-treated wild-type mice 24 h after challenge. C5a receptor expression on T cells and not on tissue resident cells was important for the development of a CS response. Thus, by using spinning disk confocal microscopy we visualized the early events of an adaptive immune response and identified the rare but essential recruitment of CD4 T cells via the complement pathway.

  19. 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.

  20. 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.

  1. 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.

  2. Segmentation: Identification of consumer segments

    DEFF Research Database (Denmark)

    Høg, Esben

    2005-01-01

    origin in other sciences as for example biology, anthropology etc. From an economic point of view, it is called segmentation when specific scientific techniques are used to classify consumers to different characteristic groupings. What is the purpose of segmentation? For example, to be able to obtain...... 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...... and analysed possible segments in the market. Results show that the statistical model used identified two segments - a segment of so-called "fish lovers" and another segment called "traditionalists". The "fish lovers" are very fond of eating fish and they actually prefer fish to other dishes...

  3. A tubular segmented-flow bioreactor for the infection of insect cells with recombinant baculovirus

    OpenAIRE

    Hu, Yu-Chen; Wang, Ming-Ying; Bentley, William E.

    1997-01-01

    A continuous process of insect cell (S f9) growth and baculovirus infection is tested with the sequential combination of a CSTR and a tubular reactor. A tubular infection reactor enables continuous introduction of baculovirus and therefore avoids the ‘passage effect’ observed in two-stage CSTR systems. Moreover, a tubular reactor can be used to test cell infection kinetics and the subsequent metabolism of infected insect cells. Unlike batch and CSTR culture, cells in a horizontally positioned...

  4. 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...

  5. Study of Collagen Birefringence in Different Grades of Oral Squamous Cell Carcinoma Using Picrosirius Red and Polarized Light Microscopy

    Directory of Open Access Journals (Sweden)

    Pillai Arun Gopinathan

    2015-01-01

    Full Text Available Objectives. The present study was done to evaluate birefringence pattern of collagen fibres in different grades of oral squamous cell carcinoma using Picrosirius red stain and polarization microscopy and to determine if there is a change in collagen fibres between different grades of oral squamous cell carcinoma. Materials and Methods. Picrosirius red stained 5 μm thick sections of previously diagnosed different grades of squamous cell carcinoma and normal oral mucosa were studied under polarization microscopy for arrangement as well as birefringence of collagen fibres around tumour islands. Results. It was found that thin collagen fibres increased and thick collagen fibres decreased with dedifferentiation of OSCC (P<0.0001 . It was observed that there was change in polarization colours of thick fibres from yellowish orange to greenish yellow with dedifferentiation of OSCC indicating loosely packed fibres (P<0.0001. Conclusion. There was a gradual change of birefringence of collagen from yellowish orange to greenish yellow from well to poorly differentiated squamous cell carcinoma, indicating that there is a change from mature form of collagen to immature form as tumour progresses. Studying collagen fibres with Picrosirius red for stromal changes around tumour islands along with routine staining may help in predicting the prognosis of tumour.

  6. Nanomechanical analysis of insulinoma cells after glucose and capsaicin stimulation using atomic force microscopy

    Institute of Scientific and Technical Information of China (English)

    Rui-guo YANG; Ning XI; King Wai-chiu LAI; Bei-hua ZHONG; Carmen Kar-man Fung; Chen-geng QU; Donna H Wang

    2011-01-01

    Aim: Glucose stimulates insulin secretion from pancreatic islet β cells by altering ion channel activity and membrane potential in the β cells. TRPV1 channel is expressed in the β cells and capsaicin induces insulin secretion similarly to glucose. This study aims to investigate the biophysical properties of the β ceils upon stimulation of membrane channels using an atomic force microscopic (AFM)nanoindentation system.Methods: ATCC insulinoma cell line was used. Cell stiffness, a marker of reorganization of cell membrane and cytoskeleton due to ion channel activation, was measured in real time using an integrated AFM nanoindentation system. Cell height that represented structural changes was simultaneously recorded along with cell stiffness.Results: After administration of glucose (16,20,and 40 mmol/L), the cell stiffness was markedly increased in a dose-dependent manner, whereas cell height was changed in an opposite way. Lower concentrations of capsaicin (1.67×10-9 and 1.67×10-8 mol/L)increased the cell stiffness without altering cell height. In contrast, higher concentrations of capsaicin (1.67×10-6 and 1.67×10-7mol/L) had no effect on the cell physical properties.Conclusion: A unique bio-nanomechanical signature was identified for characterizing biophysical properties of insulinoma cells upon general or specific activation of membrane channels. This study may deepen our understanding of stimulus-secretion coupling of pancreatic islet cells that leads to insulin secretion.

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

    Science.gov (United States)

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

    2005-04-01

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

  8. Real-time in vivo confocal laser scanning microscopy of melanin-containing cells: A promising diagnostic intervention.

    Science.gov (United States)

    Xiang, Wenzhong; Song, Xiuzu; Peng, Jianzhong; Xu, Aie; Bi, Zhigang

    2015-12-01

    The use of noninvasive imaging techniques to evaluate different types of skin lesions is increasing popular. In vivo confocal laser scanning microscopy (CLSM) is a new method for high resolution non-invasive imaging of intact skin in situ and in vivo. Although many studies have investigated melanin-containing cells in lesions by in vivo CLSM, few studies have systematically characterized melanin-containing cells based on their morphology, size, arrangement, density, borders, and brightness. In this study, the characteristics of melanin-containing cells were further investigated by in vivo CLSM. A total of 130 lesions, including common nevi, giant congenital pigmented nevi, vitiligo, melasma, melanoma, and chronic eczema, were imaged by in vivo CLSM. This research helps dermatologists understand the characteristics of melanin-containing cells and facilitate the clinical application of melanin-containing cells in the investigation of dermatological disease. In summary, melanin-containing cells include keratinocytes, melanocytes, macrophages, and melanocytic skin tumor cells. Our study presents the CLSM characteristics of melanin-containing cells to potentially facilitate in vivo diagnosis based on shape, size, arrangement, density, borders, and brightness.

  9. 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.

  10. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Carriere, Marie [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France)]. E-mail: carriere@drecam.cea.fr; Gouget, Barbara [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France); Gallien, Jean-Paul [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France); Avoscan, Laure [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France); Gobin, Renee [Laboratoire d' imagerie cellulaire et moleculaire, DBJC/SBFM/LTMD, CEA/Saclay, 91191 Gif sur Yvette (France); Verbavatz, Jean-Marc [Laboratoire d' imagerie cellulaire et moleculaire, DBJC/SBFM/LTMD, CEA/Saclay, 91191 Gif sur Yvette (France); Khodja, Hicham [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France)

    2005-04-01

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

  12. Ependymal cell reactions in spinal cord segments after compression injury in adult rat.

    Science.gov (United States)

    Takahashi, Masaki; Arai, Yasuhisa; Kurosawa, Hisashi; Sueyoshi, Noriyoshi; Shirai, Shunichi

    2003-02-01

    Recently, it has been suggested that neural stem cells and neural progenitor cells exist in the ependyma that forms the central canal of the spinal cord. In this study, we produced various degrees of thoracic cord injury in adult rats using an NYU-weight-drop device, assessed the degree of recovery of lower limb motor function based on a locomotor rating scale, and analyzed the kinetics of ependymal cell proliferation and differentiation by proliferating cell nuclear antigen (PCNA), nestin, glial fibrillary acidic protein (GFAP), or GAP-43 immunostaining. The results showed that the time course of the ependymal cell proliferation and differentiation reactions differed according to the severity of injury, and that the responses occurred not only in the neighborhood of the injury but in the entire spinal cord. An increase in the locomotor rating score was related to an increase in the number of PCNA-positive cells, and the differentiation of ependymal cells into reactive astrocytes was involved in injury repair. No apoptotic cells in the ependyma were detectable by the TUNEL method. These results indicate that the ependymal cells of the spinal central canal are themselves multipotent, can divide and proliferate according to the severity of injury, and differentiate into reactive astrocytes within the ependyma without undergoing apoptosis or cell death.

  13. Automatic segmentation of closed-contour features in ophthalmic images using graph theory and dynamic programming.

    Science.gov (United States)

    Chiu, Stephanie J; Toth, Cynthia A; Bowes Rickman, Catherine; Izatt, Joseph A; Farsiu, Sina

    2012-05-01

    This paper presents a generalized framework for segmenting closed-contour anatomical and pathological features using graph theory and dynamic programming (GTDP). More specifically, the GTDP method previously developed for quantifying retinal and corneal layer thicknesses is extended to segment objects such as cells and cysts. The presented technique relies on a transform that maps closed-contour features in the Cartesian domain into lines in the quasi-polar domain. The features of interest are then segmented as layers via GTDP. Application of this method to segment closed-contour features in several ophthalmic image types is shown. Quantitative validation experiments for retinal pigmented epithelium cell segmentation in confocal fluorescence microscopy images attests to the accuracy of the presented technique.

  14. 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.

  15. Robust demarcation of basal cell carcinoma by dependent component analysis-based segmentation of multi-spectral fluorescence images.

    Science.gov (United States)

    Kopriva, Ivica; Persin, Antun; Puizina-Ivić, Neira; Mirić, Lina

    2010-07-02

    This study was designed to demonstrate robust performance of the novel dependent component analysis (DCA)-based approach to demarcation of the basal cell carcinoma (BCC) through unsupervised decomposition of the red-green-blue (RGB) fluorescent image of the BCC. Robustness to intensity fluctuation is due to the scale invariance property of DCA algorithms, which exploit spectral and spatial diversities between the BCC and the surrounding tissue. Used filtering-based DCA approach represents an extension of the independent component analysis (ICA) and is necessary in order to account for statistical dependence that is induced by spectral similarity between the BCC and surrounding tissue. This generates weak edges what represents a challenge for other segmentation methods as well. By comparative performance analysis with state-of-the-art image segmentation methods such as active contours (level set), K-means clustering, non-negative matrix factorization, ICA and ratio imaging we experimentally demonstrate good performance of DCA-based BCC demarcation in two demanding scenarios where intensity of the fluorescent image has been varied almost two orders of magnitude.

  16. Repair of Rat Segmental Defects with Mineralized Collagen Grafts Combined with or without Mesenchymal Stem Cells and BMP-2

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The aim of the present study was to investigate and compare the bone formation capacity with three different grafts. Four millimeter segmental defects were created in adult rat tibias and were either left empty(control defects) or implanted with (1) nano-hydroxyapatite/collagen/PLA (nHAC/PLA) composite, (2)nHAC/ PLA composite added with bone marrow mesenchymal stem cells ( BMSCs ), (3) nHAC/PLA composite added with bone morphogenetic protein 2 ( BMP- 2). Radiographs of the defects were taken weekly post-surgery.After 1 or 2 months, the rats were euthanized. Histologic analyses were performed on the harvested tissue.nHAC/ PLA composite could enhance the repair of rat tibia segmental defects. Addition of BMSCs or BMP- 2 to nHAC/ PLA led to an increase in osteogenesis. nHAC/ PLA composite could be an ideal alternative bone-graft material and it could also be used as an ideal carrier of BMSCs or BMP- 2.

  17. Multimodal discrimination of immune cells using a combination of Raman spectroscopy and digital holographic microscopy

    Science.gov (United States)

    McReynolds, Naomi; Cooke, Fiona G. M.; Chen, Mingzhou; Powis, Simon J.; Dholakia, Kishan

    2017-03-01

    The ability to identify and characterise individual cells of the immune system under label-free conditions would be a significant advantage in biomedical and clinical studies where untouched and unmodified cells are required. We present a multi-modal system capable of simultaneously acquiring both single point Raman spectra and digital holographic images of single cells. We use this combined approach to identify and discriminate between immune cell populations CD4+ T cells, B cells and monocytes. We investigate several approaches to interpret the phase images including signal intensity histograms and texture analysis. Both modalities are independently able to discriminate between cell subsets and dual-modality may therefore be used a means for validation. We demonstrate here sensitivities achieved in the range of 86.8% to 100%, and specificities in the range of 85.4% to 100%. Additionally each modality provides information not available from the other providing both a molecular and a morphological signature of each cell.

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

    Science.gov (United States)

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

    2008-04-01

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

  19. 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.

  20. In situ transmission electron microscopy of ionic conductivity and reaction mechanisms in ultrathin solid oxide fuel cells.

    Science.gov (United States)

    Tavabi, Amir H; Arai, Shigeo; Muto, Shunsuke; Tanji, Takayoshi; Dunin-Borkowski, Rafal E

    2014-12-01

    Solid oxide fuel cells (SOFCs) are promising candidates for use in alternative energy technologies. A full understanding of the reaction mechanisms in these dynamic material systems is required to optimize device performance and overcome present limitations. Here, we show that in situ transmission electron microscopy (TEM) can be used to study redox reactions and ionic conductivity in SOFCs in a gas environment at elevated temperature. We examine model ultrathin half and complete cells in two environmental TEMs using off-axis electron holography and electron energy-loss spectroscopy. Our results from the model cells provide insight into the essential phenomena that are important for the operation of commercial devices. Changes in the activities of dopant cations in the solid electrolyte are detected during oxygen anion conduction, demonstrating the key role of dopants in electrolyte architecture in SOFCs.

  1. Non-invasive discrimination between pancreatic islets and exocrine cells using multiphoton microscopy

    Science.gov (United States)

    Wu, Binlin; Li, Ge; Hao, Mingming; Mukherjee, Sushmita

    2015-03-01

    In this study, we propose a non-invasive method to distinguish pancreatic islet cells from exocrine cell clusters using multiphoton (MP) imaging. We demonstrate the principle of distinguishing them based on autofluorescence. The results show that MP imaging has a potential to distinguish pancreatic islets from exocrine cells. This ability to distinguish the two cell types could have many applications, such as the examination of fresh pancreatic biopsies when staining is not possible or desirable.

  2. Enabling user-guided segmentation and tracking of surface-labeled cells in time-lapse image sets of living tissues.

    Science.gov (United States)

    Mashburn, David N; Lynch, Holley E; Ma, Xiaoyan; Hutson, M Shane

    2012-05-01

    To study the process of morphogenesis, one often needs to collect and segment time-lapse images of living tissues to accurately track changing cellular morphology. This task typically involves segmenting and tracking tens to hundreds of individual cells over hundreds of image frames, a scale that would certainly benefit from automated routines; however, any automated routine would need to reliably handle a large number of sporadic, and yet typical problems (e.g., illumination inconsistency, photobleaching, rapid cell motions, and drift of focus or of cells moving through the imaging plane). Here, we present a segmentation and cell tracking approach based on the premise that users know their data best-interpreting and using image features that are not accounted for in any a priori algorithm design. We have developed a program, SeedWater Segmenter, that combines a parameter-less and fast automated watershed algorithm with a suite of manual intervention tools that enables users with little to no specialized knowledge of image processing to efficiently segment images with near-perfect accuracy based on simple user interactions.

  3. Quantification of GPCR internalization by single-molecule microscopy in living cells.

    NARCIS (Netherlands)

    Serge, A.; Keijzer, S. de; Hemert, F. Van; Hickman, M.R.; Hereld, D.; Spaink, H.P.; Schmidt, T.; Snaar-Jagalska, B.E.

    2011-01-01

    Receptor internalization upon ligand stimulation is a key component of a cell's response and allows a cell to correctly sense its environment. Novel fluorescent methods have enabled the direct visualization of the agonist-stimulated G-protein-coupled receptors (GPCR) trafficking in living cells. How

  4. A simple optical fiber device for quantitative fluorescence microscopy of single living cells

    NARCIS (Netherlands)

    Graft, van Marja; Oosterhuis, Bernard; Werf, van der Kees O.; Grooth, de Bart G.; Greve, Jan

    1993-01-01

    simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical fibe

  5. 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

  6. 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.

  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. Imaging the uptake of gold nanoshells in live cells using plasmon resonance enhanced four wave mixing microscopy.

    Science.gov (United States)

    Garrett, Natalie; Whiteman, Matt; Moger, Julian

    2011-08-29

    Gold nanoshells (GNS) are novel metal nanoparticles exhibiting attractive optical properties which make them highly suitable for biophotonics applications. We present a novel investigation using plasmon-enhanced four wave mixing microscopy combined with coherent anti-Stokes Raman scattering (CARS) microscopy to visualize the distribution of 75 nm radius GNS within live cells. During a laser tolerance study we found that cells containing nanoshells could be exposed to nanoshell uptake using two donor molecules, NaHS and GYY4137. As GYY4137 concentration was increased from 10 µM to 1 mM, the nanoshell pixel percentage as a function of cell volume (PPCV) increased from 2.15% to 3.77%. As NaHS concentration was increased over the same range, the nanoshell PPCV decreased from 12.67% to 11.47%. The most important factor affecting uptake in this study was found to be the rate of H2S release, with rapid-release from NaHS resulting in significantly greater uptake.

  9. Calibrated complex impedance of CHO cells and E. coli bacteria at GHz frequencies using scanning microwave microscopy

    Science.gov (United States)

    Tuca, Silviu-Sorin; Badino, Giorgio; Gramse, Georg; Brinciotti, Enrico; Kasper, Manuel; Oh, Yoo Jin; Zhu, Rong; Rankl, Christian; Hinterdorfer, Peter; Kienberger, Ferry

    2016-04-01

    The application of scanning microwave microscopy (SMM) to extract calibrated electrical properties of cells and bacteria in air is presented. From the S 11 images, after calibration, complex impedance and admittance images of Chinese hamster ovary cells and E. coli bacteria deposited on a silicon substrate have been obtained. The broadband capabilities of SMM have been used to characterize the bio-samples between 2 GHz and 20 GHz. The resulting calibrated cell and bacteria admittance at 19 GHz were Y cell = 185 μS + j285 μS and Y bacteria = 3 μS + j20 μS, respectively. A combined circuitry-3D finite element method EMPro model has been developed and used to investigate the frequency response of the complex impedance and admittance of the SMM setup. Based on a proposed parallel resistance-capacitance model, the equivalent conductance and parallel capacitance of the cells and bacteria were obtained from the SMM images. The influence of humidity and frequency on the cell conductance was experimentally studied. To compare the cell conductance with bulk water properties, we measured the imaginary part of the bulk water loss with a dielectric probe kit in the same frequency range resulting in a high level of agreement.

  10. 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.

  11. 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.

  12. Automated single-cell motility analysis on a chip using lensfree microscopy

    Science.gov (United States)

    Pushkarsky, Ivan; Lyb, Yunbo; Weaver, Westbrook; Su, Ting-Wei; Mudanyali, Onur; Ozcan, Aydogan; di Carlo, Dino

    2014-04-01

    Quantitative cell motility studies are necessary for understanding biophysical processes, developing models for cell locomotion and for drug discovery. Such studies are typically performed by controlling environmental conditions around a lens-based microscope, requiring costly instruments while still remaining limited in field-of-view. Here we present a compact cell monitoring platform utilizing a wide-field (24 mm2) lensless holographic microscope that enables automated single-cell tracking of large populations that is compatible with a standard laboratory incubator. We used this platform to track NIH 3T3 cells on polyacrylamide gels over 20 hrs. We report that, over an order of magnitude of stiffness values, collagen IV surfaces lead to enhanced motility compared to fibronectin, in agreement with biological uses of these structural proteins. The increased throughput associated with lensfree on-chip imaging enables higher statistical significance in observed cell behavior and may facilitate rapid screening of drugs and genes that affect cell motility.

  13. Simple and fast spectral domain algorithm for quantitative phase imaging of living cells with digital holographic microscopy.

    Science.gov (United States)

    Min, Junwei; Yao, Baoli; Ketelhut, Steffi; Engwer, Christian; Greve, Burkhard; Kemper, Björn

    2017-01-15

    We present a simple and fast phase aberration compensation method in digital holographic microscopy (DHM) for quantitative phase imaging of living cells. By analyzing the frequency spectrum of an off-axis hologram, phase aberrations can be compensated for automatically without fitting or pre-knowledge of the setup and/or the object. Simple and effective computation makes the method suitable for quantitative online monitoring with highly variable DHM systems. Results from automated quantitative phase imaging of living NIH-3T3 mouse fibroblasts demonstrate the effectiveness and the feasibility of the method.

  14. 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.

  15. Segmentation of leukocytes and erythrocytes in blood smear images.

    Science.gov (United States)

    Bergen, Tobias; Steckhan, Dirk; Wittenberg, Thomas; Zerfass, Thorsten

    2008-01-01

    Differential blood count is a standard method in hematological laboratory diagnosis. In the course of developing a computer-assisted microscopy system for the generation of differential blood counts, the detection and segmentation of white and red blood cells forms an essential step and its exactness is a fundamental prerequisite for the effectiveness of the subsequent classification step. We propose a method for the exact segmentation of leukocytes and erythrocytes in a simultaneous and cooperative way. We combine pixel-wise classification with template matching to locate erythrocytes and use a level-set approach in order to get the exact cell contours of leukocyte nucleus and plasma regions as well as erythrocyte regions. An evaluation comparing the performance of the algorithm to the manual segmentation performed by several persons yielded good results.

  16. Comparative electron microscopy of basophils and mast cells, in vivo and in vitro.

    Science.gov (United States)

    Eguchi, M

    1991-01-01

    We compared the fine structure and electron microscopic cytochemical findings of basophils and mast cells from humans, guinea pigs, rabbits, mice and rats. The particulate structure was the most frequently observed and most typical structure of human and rabbit basophil granules and of guinea pig mast cell granules. The most prominent feature of guinea pig basophils and murine mast cells was that the fine structure of the granules was homogeneous. The fine structure of the granules in guinea pig basophils resembled that in murine mast cells, while the fine structure of the granules of guinea pig mast cells resembled those in human and rabbit mast cells. In mouse mast cells in culture, the majority of the granules contained small vesicles, which were also observed in human basophils in culture and in mouse basophils in vivo. The degrees of cytochemical reactivity of acid mucopolysaccharides among the species were different. Peroxidase activity was positive in most basophils and in human mast cells. Among mammals, the granules of basophils and mast cells present heterogeneous fine structure. It is of interest that the basophil granules of some species resemble the mast cell granules rather than the basophil granules of other species.

  17. 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.

  18. Conductive Atomic Free Microscopy of CdTe/CdS Solar Cells

    Energy Technology Data Exchange (ETDEWEB)

    Moutinho, H. R.; Dhere, R. G.; Jiang, C. S.; Al-Jassim, M. M.; Kazmerski, L. L.

    2005-01-01

    Conductive atomic force microscopy (C-AFM) is a recently developed technique that applies an electric voltage between a very sharp tip and the sample, permitting the study of the electrical properties of the sample with very high spatial resolution. It also provides current-voltage measurements at well-defined spots. C-AFM is applied simultaneously with atomic force microscopy, providing topographic and current images of the same region. In this work, we analyze CdTe/CdS samples, before and after CdCl2 treatment, and after bromine/methanol and nitric/phosphoric etches. The as-deposited samples show grains with different contrasts, indicating that the material is not electrically uniform. The CdCl2 treatment resulted in less conductive grain boundaries, suggesting a relative decrease in the conductivity at these locations. After the bromine/methanol etch, the conductivity at grains boundaries was higher than inside the grains, whereas for the nitric/phosphoric etch the conductivity increased over the entire surface.

  19. Discrimination of bladder cancer cells from normal urothelial cells with high specificity and sensitivity: combined application of atomic force microscopy and modulated Raman spectroscopy.

    Science.gov (United States)

    Canetta, Elisabetta; Riches, Andrew; Borger, Eva; Herrington, Simon; Dholakia, Kishan; Adya, Ashok K

    2014-05-01

    Atomic force microscopy (AFM) and modulated Raman spectroscopy (MRS) were used to discriminate between living normal human urothelial cells (SV-HUC-1) and bladder tumour cells (MGH-U1) with high specificity and sensitivity. MGH-U1 cells were 1.5-fold smaller, 1.7-fold thicker and 1.4-fold rougher than normal SV-HUC-1 cells. The adhesion energy was 2.6-fold higher in the MGH-U1 cells compared to normal SV-HUC-1 cells, which possibly indicates that bladder tumour cells are more deformable than normal cells. The elastic modulus of MGH-U1 cells was 12-fold lower than SV-HUC-1 cells, suggesting a higher elasticity of the bladder cancer cell membranes. The biochemical fingerprints of cancer cells displayed a higher DNA and lipid content, probably due to an increase in the nuclear to cytoplasm ratio. Normal cells were characterized by higher protein contents. AFM studies revealed a decrease in the lateral dimensions and an increase in thickness of cancer cells compared to normal cells; these studies authenticate the observations from MRS. Nanostructural, nanomechanical and biochemical profiles of bladder cells provide qualitative and quantitative markers to differentiate between normal and cancerous cells at the single cellular level. AFM and MRS allow discrimination between adhesion energy, elasticity and Raman spectra of SV-HUC-1 and MGH-U1 cells with high specificity (83, 98 and 95%) and sensitivity (97, 93 and 98%). Such single-cell-level studies could have a pivotal impact on the development of AFM-Raman combined methodologies for cancer profiling and screening with translational significance.

  20. Spatial organization of cellulose microfibrils and matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic force microscopy.

    Science.gov (United States)

    Zhang, Tian; Zheng, Yunzhen; Cosgrove, Daniel J

    2016-01-01

    We used atomic force microscopy (AFM), complemented with electron microscopy, to characterize the nanoscale and mesoscale structure of the outer (periclinal) cell wall of onion scale epidermis - a model system for relating wall structure to cell wall mechanics. The epidermal wall contains ~100 lamellae, each ~40 nm thick, containing 3.5-nm wide cellulose microfibrils oriented in a common direction within a lamella but varying by ~30 to 90° between adjacent lamellae. The wall thus has a crossed polylamellate, not helicoidal, wall structure. Montages of high-resolution AFM images of the newly deposited wall surface showed that single microfibrils merge into and out of short regions of microfibril bundles, thereby forming a reticulated network. Microfibril direction within a lamella did not change gradually or abruptly across the whole face of the cell, indicating continuity of the lamella across the outer wall. A layer of pectin at the wall surface obscured the underlying cellulose microfibrils when imaged by FESEM, but not by AFM. The AFM thus preferentially detects cellulose microfibrils by probing through the soft matrix in these hydrated walls. AFM-based nanomechanical maps revealed significant heterogeneity in cell wall stiffness and adhesiveness at the nm scale. By color coding and merging these maps, the spatial distribution of soft and rigid matrix polymers could be visualized in the context of the stiffer microfibrils. Without chemical extraction and dehydration, our results provide multiscale structural details of the primary cell wall in its near-native state, with implications for microfibrils motions in different lamellae during uniaxial and biaxial extensions.

  1. 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.

  2. Time-dependent traction force microscopy for cancer cells as a measure of invasiveness.

    Science.gov (United States)

    Peschetola, Valentina; Laurent, Valérie M; Duperray, Alain; Michel, Richard; Ambrosi, Davide; Preziosi, Luigi; Verdier, Claude

    2013-04-01

    The migration of tumor cells of different degrees of invasivity is studied, on the basis of the traction forces exerted in time on soft substrates (Young modulus∼10 kPa). It is found that the outliers of the traction stresses can be an effective indicator to distinguish cancer cell lines of different invasiveness. Here, we test two different epithelial bladder cancer cell lines, one invasive (T24), and a less invasive one (RT112). Invasive cancer cells move in a nearly periodic motion, with peaks in velocity corresponding to higher traction forces exerted on the substrate, whereas less invasive cells develop traction stresses almost constant in time. The dynamics of focal adhesions (FAs) as well as cytoskeleton features reveals that different mechanisms are activated to migrate: T24 cells show an interconnected cytoskeleton linked to mature adhesion sites, leading to small traction stresses, whereas less invasive cells (RT112) show a less-structured cytoskeleton and unmature adhesions corresponding to higher traction stresses. Migration velocities are smaller in the case of less invasive cells. The mean squared displacement shows super-diffusive motion in both cases with higher exponent for the more invasive cancer cells. Further correlations between traction forces and the actin cytoskeleton reveal an unexpected pattern of a large actin rim at the RT112 cell edge where higher forces are colocalized, whereas a more usual cytoskeleton structure with stress fibers and FAs are found for T24 cancer cells. We conjecture that this kind of analysis can be useful to classify cancer cell invasiveness.

  3. Fourier-ring descriptor to characterize rare circulating cells from images generated using immunofluorescence microscopy.

    Science.gov (United States)

    Emerson, Tegan; Kirby, Michael; Bethel, Kelly; Kolatkar, Anand; Luttgen, Madelyn; O'Hara, Stephen; Newton, Paul; Kuhn, Peter

    2015-03-01

    We address the problem of subclassification of rare circulating cells using data driven feature selection from images of candidate circulating tumor cells from patients diagnosed with breast, prostate, or lung cancer. We determine a set of low level features which can differentiate among candidate cell types. We have implemented an image representation based on concentric Fourier rings (FRDs) which allow us to exploit size variations and morphological differences among cells while being rotationally invariant. We discuss potential clinical use in the context of treatment monitoring for cancer patients with metastatic disease.

  4. Electron Microscopy.

    Science.gov (United States)

    Beer, Michael

    1980-01-01

    Reviews technical aspects of structure determination in biological electron microscopy (EM). Discusses low dose EM, low temperature microscopy, electron energy loss spectra, determination of mass or molecular weight, and EM of labeled systems. Cites 34 references. (CS)

  5. 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.

  6. Measuring bacterial adaptation dynamics at the single-cell level using a microfluidic chemostat and time-lapse fluorescence microscopy.

    Science.gov (United States)

    Long, Zhicheng; Olliver, Anne; Brambilla, Elisa; Sclavi, Bianca; Lagomarsino, Marco Cosentino; Dorfman, Kevin D

    2014-10-21

    We monitored the dynamics of cell dimensions and reporter GFP expression in individual E. coli cells growing in a microfluidic chemostat using time-lapse fluorescence microscopy. This combination of techniques allows us to study the dynamical responses of single bacterial cells to nutritional shift-down or shift-up for longer times and with more precision over the chemical environment than similar experiments performed on conventional agar pads. We observed two E. coli strains containing different promoter-reporter gene constructs and measured how both their cell dimensions and the GFP expression change after nutritional upshift and downshift. As expected, both strains have similar adaptation dynamics for cell size rearrangement. However, the strain with a ribosomal RNA promoter dependent reporter has a faster GFP production rate than the strain with a constitutive promoter reporter. As a result, the mean GFP concentration in the former strain changes rapidly with the nutritional shift, while that in the latter strain remains relatively stable. These findings characterize the present microfluidic chemostat as a versatile platform for measuring single-cell bacterial dynamics and physiological transitions.

  7. 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

  8. 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...

  9. Noninvasive Imaging of Protein Metabolic Labeling in Single Human Cells Using Stable Isotopes and Raman Microscopy

    NARCIS (Netherlands)

    Manen, van Henk-Jan; Lenferink, Aufried; Otto, Cees

    2008-01-01

    We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C−D stret

  10. 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

  11. Insight into the Microbial Multicellular Lifestyle via Flow-Cell Technology and Confocal Microscopy

    DEFF Research Database (Denmark)

    Pamp, Sünje Johanna; Sternberg, Claus; Tolker-Nielsen, Tim

    2009-01-01

    formed in flow-chamber experimental systems by genetically color-coded bacteria have provided detailed knowledge about biofilm developmental processes, cell differentiations, spatial organization, and function of laboratory-grown biofilms, in some cases down to the single cell level. In addition...

  12. 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

  13. Consistency and distribution of reflectance confocal microscopy features for diagnosis of cutaneous T cell lymphoma

    Science.gov (United States)

    Lange-Asschenfeldt, Susanne; Babilli, Jasmin; Beyer, Marc; Ríus-Diaz, Francisca; González, Salvador; Stockfleth, Eggert; Ulrich, Martina

    2012-01-01

    Reflectance confocal microscopy (RCM) represents a noninvasive imaging technique that has previously been used for characterization of mycosis fungoides (MF) in a pilot study. We aimed to test the applicability of RCM for diagnosis and differential diagnosis of MF in a clinical study. A total of 39 test sites of 15 patients with a biopsy-proven diagnosis of either MF, parapsoriasis, Sézary syndrome, or lymphomatoid papulosis were analyzed for presence and absence of RCM features of MF. Cochran and Chi2 analysis were applied to test the concordance between investigators and the distribution of RCM features, respectively. For selected parameters, the Cochran analysis showed good concordance between investigators. Inter-observer reproducibility was highest for junctional atypical lymphocytes, architectural disarray, and spongiosis. Similarly, Chi2 analysis demonstrated that selected features were present at particularly high frequency in individual skin diseases, with values ranging from 73% to 100% of all examined cases.

  14. Imaging immune and metabolic cells of visceral adipose tissues with multimodal nonlinear optical microscopy.

    Directory of Open Access Journals (Sweden)

    Yasuyo Urasaki

    Full Text Available Visceral adipose tissue (VAT inflammation is recognized as a mechanism by which obesity is associated with metabolic diseases. The communication between adipose tissue macrophages (ATMs and adipocytes is important to understanding the interaction between immunity and energy metabolism and its roles in obesity-induced diseases. Yet visualizing adipocytes and macrophages in complex tissues is challenging to standard imaging methods. Here, we describe the use of a multimodal nonlinear optical (NLO microscope to characterize the composition of VATs of lean and obese mice including adipocytes, macrophages, and collagen fibrils in a label-free manner. We show that lipid metabolism processes such as lipid droplet formation, lipid droplet microvesiculation, and free fatty acids trafficking can be dynamically monitored in macrophages and adipocytes. With its versatility, NLO microscopy should be a powerful imaging tool to complement molecular characterization of the immunity-metabolism interface.

  15. 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