Video Microscopy

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David G Grier - One of the best experts on this subject based on the ideXlab platform.

  • measuring boltzmann s constant through holographic Video Microscopy of a single colloidal sphere
    American Journal of Physics, 2014
    Co-Authors: Bhaskar Jyoti Krishnatreya, Arielle Colenlandy, Paige Hasebe, Breanna A Bell, Jasmine R Jones, Anderson Sundameya, David G Grier
    Abstract:

    The trajectory of a colloidal sphere diffusing in water records a history of the random forces exerted on the sphere by thermally driven fluctuations in the suspending fluid. The trajectory therefore can be used to characterize the spectrum of thermal fluctuations and thus to obtain an estimate for Boltzmann's constant. We demonstrate how to use holographic Video Microscopy to track a colloidal sphere's three-dimensional motions with nanometer precision while simultaneously measuring its radius to within a few nanometers. The combination of tracking and characterization data reliably yields Boltzmann's constant to within two percent and also provides the basis for many other useful and interesting measurements in statistical physics, physical chemistry, and materials science.

  • strategies for three dimensional particle tracking with holographic Video Microscopy
    Optics Express, 2010
    Co-Authors: Fook Chiong Cheong, Bhaskar Jyoti Krishnatreya, David G Grier
    Abstract:

    The Video stream captured by an in-line holographic microscope can be analyzed on a frame-by-frame basis to track individual colloidal particles’ three-dimensional motions with nanometer resolution. In this work, we compare the performance of two complementary analysis techniques, one based on fitting to the exact Lorenz-Mie theory and the other based on phenomenological interpretation of the scattered light field reconstructed with Rayleigh-Sommerfeld back-propagation. Although Lorenz-Mie tracking provides more information and is inherently more precise, Rayleigh-Sommerfeld reconstruction is faster and more general. The two techniques agree quantitatively on colloidal spheres’ in-plane positions. Their systematic differences in axial tracking can be explained in terms of the illuminated objects’ light scattering properties.

  • Flow visualization and flow cytometry with holographic Video Microscopy
    Optics express, 2009
    Co-Authors: Fook Chiong Cheong, Jesse Amato-grill, Lisa Dixon, Remi Dreyfus, Ke Xiao, Bo Sun, David G Grier
    Abstract:

    The Video stream captured by an in-line holographic microscope can be analyzed on a frame-by-frame basis to track individual colloidal particles' three-dimensional motions with nanometer resolution, and simultaneously to measure their sizes and refractive indexes. Through a combination of hardware acceleration and software optimization, this analysis can be carried out in near real time with off-the-shelf instrumentation. An efficient particle identification algorithm automates initial position estimation with sufficient accuracy to enable unattended holographic tracking and characterization. This technique's resolution for particle size is fine enough to detect molecular-scale coatings on the surfaces of colloidal spheres, without requiring staining or fluorescent labeling. We demonstrate this approach to label-free holographic flow cytometry by detecting the binding of avidin to biotinylated polystyrene spheres.

  • technical note characterizing individual milk fat globules with holographic Video Microscopy
    Journal of Dairy Science, 2009
    Co-Authors: F C Cheong, K Xiao, David G Grier
    Abstract:

    We used in-line holography to create images of individual milk fat globules in diluted samples of milk. Analyzing these images with the exact Lorenz-Mie light scattering theory then yields the droplets' radii with nanometer resolution and their refractive indexes to within one part in a thousand. This procedure rapidly and directly characterizes both the quantity and quality of fat in milk.

  • Methods of digital Video Microscopy for colloidal studies
    Journal of Colloid and Interface Science, 1996
    Co-Authors: John C Crocker, David G Grier
    Abstract:

    We describe a set of image processing algorithms for extracting quantitative data from digitized Video microscope images of colloidal suspensions. In a typical application, these direct imaging techniques can locate submicrometer spheres to within 10 nm in the focal plane and 150 nm in depth. Combining information from a sequence of Video images into single-particle trajectories makes possible measurements of quantities of fundamental and practical interest such as diffusion coefficients and pair-wise interaction potentials. The measurements we describe in detail combine the outstanding resolution of digital imaging with Video-synchronized optical trapping to obtain highly accurate and reproducible results very rapidly.

Sheila Macneil - One of the best experts on this subject based on the ideXlab platform.

  • automated tracking of migrating cells in phase contrast Video Microscopy sequences using image registration
    Journal of Microscopy, 2009
    Co-Authors: Aj J Hand, Dc C Barber, D. Rodney Hose, Tao Sun, Sheila Macneil
    Abstract:

    Summary Analysis of in vitro cell motility is a useful tool for assessing cellular response to a range of factors. However, the majority of cell-tracking systems available are designed primarily for use with fluorescently labelled images. In this paper, five commonly used tracking systems are examined for their performance compared with the use of a novel in-house cell-tracking system based on the principles of image registration and optical flow. Image registration is a tool commonly used in medical imaging to correct for the effects of patient motion during imaging procedures and works well on low-contrast images, such as those found in bright-field and phase-contrast Microscopy. The five cell-tracking systems examined were Retrac, a manual tracking system used as the gold standard; CellTrack, a recently released freely downloadable software system that uses a combination of tracking methods; ImageJ, which is a freely available piece of software with a plug-in for automated tracking (MTrack2) and Imaris and Volocity, both commercially available automated tracking systems. All systems were used to track migration of human epithelial cells over ten frames of a phase-contrast time-lapse Microscopy sequence. This showed that the in-house image-registration system was the most effective of those tested when tracking non-dividing epithelial cells in low-contrast images, with a successful tracking rate of 95%. The performance of the tracking systems was also evaluated by tracking fluorescently labelled epithelial cells imaged with both phase-contrast and confocal Microscopy techniques. The results showed that using fluorescence Microscopy instead of phase contrast does improve the tracking efficiency for each of the tested systems. For the in-house software, this improvement was relatively small (<5% difference in tracking success rate), whereas much greater improvements in performance were seen when using fluorescence Microscopy with Volocity and ImageJ.

  • Automated tracking of migrating cells in phase contrast Video Microscopy sequences using image registration
    Journal of Microscopy, 2009
    Co-Authors: Aj J Hand, Dc C Barber, D. Rodney Hose, Tong Sun, Sheila Macneil
    Abstract:

    Analysis of in vitro cell motility is a useful tool for assessing cellular response to a range of factors. However, the majority of cell-tracking systems available are designed primarily for use with fluorescently labelled images. In this paper, five commonly used tracking systems are examined for their performance compared with the use of a novel in-house cell-tracking system based on the principles of image registration and optical flow. Image registration is a tool commonly used in medical imaging to correct for the effects of patient motion during imaging procedures and works well on low-contrast images, such as those found in bright-field and phase-contrast Microscopy. The five cell-tracking systems examined were Retrac, a manual tracking system used as the gold standard; CellTrack, a recently released freely downloadable software system that uses a combination of tracking methods; ImageJ, which is a freely available piece of software with a plug-in for automated tracking (MTrack2) and Imaris and Volocity, both commercially available automated tracking systems. All systems were used to track migration of human epithelial cells over ten frames of a phase-contrast time-lapse Microscopy sequence. This showed that the in-house image-registration system was the most effective of those tested when tracking non-dividing epithelial cells in low-contrast images, with a successful tracking rate of 95%. The performance of the tracking systems was also evaluated by tracking fluorescently labelled epithelial cells imaged with both phase-contrast and confocal Microscopy techniques. The results showed that using fluorescence Microscopy instead of phase contrast does improve the tracking efficiency for each of the tested systems. For the in-house software, this improvement was relatively small (

Fook Chiong Cheong - One of the best experts on this subject based on the ideXlab platform.

  • rapid high throughput tracking of bacterial motility in 3d via phase contrast holographic Video Microscopy
    Biophysical Journal, 2015
    Co-Authors: Fook Chiong Cheong, Chui Ching Wong, Yunfeng Gao, Mui Hoon Nai, Yidan Cui, Sungsu Park, Linda J Kenney, Chwee Teck Lim
    Abstract:

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

  • strategies for three dimensional particle tracking with holographic Video Microscopy
    Optics Express, 2010
    Co-Authors: Fook Chiong Cheong, Bhaskar Jyoti Krishnatreya, David G Grier
    Abstract:

    The Video stream captured by an in-line holographic microscope can be analyzed on a frame-by-frame basis to track individual colloidal particles’ three-dimensional motions with nanometer resolution. In this work, we compare the performance of two complementary analysis techniques, one based on fitting to the exact Lorenz-Mie theory and the other based on phenomenological interpretation of the scattered light field reconstructed with Rayleigh-Sommerfeld back-propagation. Although Lorenz-Mie tracking provides more information and is inherently more precise, Rayleigh-Sommerfeld reconstruction is faster and more general. The two techniques agree quantitatively on colloidal spheres’ in-plane positions. Their systematic differences in axial tracking can be explained in terms of the illuminated objects’ light scattering properties.

  • flow visualization and flow cytometry with holographic Video Microscopy
    Proceedings of SPIE the International Society for Optical Engineering, 2010
    Co-Authors: Fook Chiong Cheong, Remi Dreyfus, Ke Xiao, Jesse Amatogrill, Lisa Dixo, David G Grie
    Abstract:

    The Video stream captured by an in-line holographic microscope can be analyzed on a frame-by-frame basis to track individual colloidal particles' three-dimensional motions with nanometer resolution, and simultaneously to measure their sizes and refractive indexes. An efficient particle-tracking algorithm automates initial position estimation with sufficient accuracy to enable unattended holographic particle tracking and characterization. In this work, we demonstrated this approach to flow visualization in a microfluidic channel and also to flow cytometry of micrometer-scale colloidal spheres.

  • Flow visualization and flow cytometry with holographic Video Microscopy
    Optics express, 2009
    Co-Authors: Fook Chiong Cheong, Jesse Amato-grill, Lisa Dixon, Remi Dreyfus, Ke Xiao, Bo Sun, David G Grier
    Abstract:

    The Video stream captured by an in-line holographic microscope can be analyzed on a frame-by-frame basis to track individual colloidal particles' three-dimensional motions with nanometer resolution, and simultaneously to measure their sizes and refractive indexes. Through a combination of hardware acceleration and software optimization, this analysis can be carried out in near real time with off-the-shelf instrumentation. An efficient particle identification algorithm automates initial position estimation with sufficient accuracy to enable unattended holographic tracking and characterization. This technique's resolution for particle size is fine enough to detect molecular-scale coatings on the surfaces of colloidal spheres, without requiring staining or fluorescent labeling. We demonstrate this approach to label-free holographic flow cytometry by detecting the binding of avidin to biotinylated polystyrene spheres.

Aj J Hand - One of the best experts on this subject based on the ideXlab platform.

  • automated tracking of migrating cells in phase contrast Video Microscopy sequences using image registration
    Journal of Microscopy, 2009
    Co-Authors: Aj J Hand, Dc C Barber, D. Rodney Hose, Tao Sun, Sheila Macneil
    Abstract:

    Summary Analysis of in vitro cell motility is a useful tool for assessing cellular response to a range of factors. However, the majority of cell-tracking systems available are designed primarily for use with fluorescently labelled images. In this paper, five commonly used tracking systems are examined for their performance compared with the use of a novel in-house cell-tracking system based on the principles of image registration and optical flow. Image registration is a tool commonly used in medical imaging to correct for the effects of patient motion during imaging procedures and works well on low-contrast images, such as those found in bright-field and phase-contrast Microscopy. The five cell-tracking systems examined were Retrac, a manual tracking system used as the gold standard; CellTrack, a recently released freely downloadable software system that uses a combination of tracking methods; ImageJ, which is a freely available piece of software with a plug-in for automated tracking (MTrack2) and Imaris and Volocity, both commercially available automated tracking systems. All systems were used to track migration of human epithelial cells over ten frames of a phase-contrast time-lapse Microscopy sequence. This showed that the in-house image-registration system was the most effective of those tested when tracking non-dividing epithelial cells in low-contrast images, with a successful tracking rate of 95%. The performance of the tracking systems was also evaluated by tracking fluorescently labelled epithelial cells imaged with both phase-contrast and confocal Microscopy techniques. The results showed that using fluorescence Microscopy instead of phase contrast does improve the tracking efficiency for each of the tested systems. For the in-house software, this improvement was relatively small (<5% difference in tracking success rate), whereas much greater improvements in performance were seen when using fluorescence Microscopy with Volocity and ImageJ.

  • Automated tracking of migrating cells in phase contrast Video Microscopy sequences using image registration
    Journal of Microscopy, 2009
    Co-Authors: Aj J Hand, Dc C Barber, D. Rodney Hose, Tong Sun, Sheila Macneil
    Abstract:

    Analysis of in vitro cell motility is a useful tool for assessing cellular response to a range of factors. However, the majority of cell-tracking systems available are designed primarily for use with fluorescently labelled images. In this paper, five commonly used tracking systems are examined for their performance compared with the use of a novel in-house cell-tracking system based on the principles of image registration and optical flow. Image registration is a tool commonly used in medical imaging to correct for the effects of patient motion during imaging procedures and works well on low-contrast images, such as those found in bright-field and phase-contrast Microscopy. The five cell-tracking systems examined were Retrac, a manual tracking system used as the gold standard; CellTrack, a recently released freely downloadable software system that uses a combination of tracking methods; ImageJ, which is a freely available piece of software with a plug-in for automated tracking (MTrack2) and Imaris and Volocity, both commercially available automated tracking systems. All systems were used to track migration of human epithelial cells over ten frames of a phase-contrast time-lapse Microscopy sequence. This showed that the in-house image-registration system was the most effective of those tested when tracking non-dividing epithelial cells in low-contrast images, with a successful tracking rate of 95%. The performance of the tracking systems was also evaluated by tracking fluorescently labelled epithelial cells imaged with both phase-contrast and confocal Microscopy techniques. The results showed that using fluorescence Microscopy instead of phase contrast does improve the tracking efficiency for each of the tested systems. For the in-house software, this improvement was relatively small (

Cédric Allier - One of the best experts on this subject based on the ideXlab platform.

  • Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture
    Journal of Visualized Experiments, 2018
    Co-Authors: Cédric Allier, Romaric Vincent, Fabrice Navarro, Mathilde Menneteau, Lamya Ghenim, Xavier Gidrol, Thomas Bordy, Lionel Hervé, Olivier Cioni, Sabine Bardin
    Abstract:

    Here, we demonstrate that lens-free Video Microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free Video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

  • imaging of dense cell cultures by multiwavelength lens free Video Microscopy
    Cytometry Part A, 2017
    Co-Authors: Cédric Allier, Fabrice Navarro, Mathilde Menneteau, Lamya Ghenim, Thomas Bordy, Lionel Hervé, Olivier Cioni, S Morel, R Vincent, Xavier Gidrol
    Abstract:

    They present results for lens-free Microscopy for the imaging of dense cell culture. With this aim, they use a multiwavelength LED illumination with well separated wavelengths, together with the implementation of an appropriate holographic reconstruction algorithm. This allows for a fast and efficient reconstruction of the phase image of densely packed cells (up to 700 cells/mm2) over a large field of view of 29.4 mm2. Combined with the compactness of the system which fits altogether inside an incubator, lens-free Microscopy becomes a unique tool to monitor cell cultures over several days. The high contrast phase shift images provide robust cell segmentation and tracking, and enable high throughput monitoring of individual cell dimensions, dry mass, and motility. They tested the multiwavelength lens-free Video-microscope over a broad range of cell lines, including mesenchymal, endothelial, and epithelial cells. © 2017 International Society for Advancement of Cytometry

  • Video lensfree Microscopy of 2d and 3d culture of cells
    Proceedings of SPIE, 2014
    Co-Authors: Cédric Allier, Fabrice Navarro, Mathilde Menneteau, Olivier Cioni, Vinjimore S Kesavan, Jeanguillaume Coutard, Fabien Momey, Bernard Chalmond, Patricia Obeid, Vincent Haguet
    Abstract:

    Innovative imaging methods are continuously developed to investigate the function of biological systems at the microscopic scale. As an alternative to advanced cell Microscopy techniques, we are developing lensfree Video Microscopy that opens new ranges of capabilities, in particular at the mesoscopic level. Lensfree Video Microscopy allows the observation of a cell culture in an incubator over a very large field of view (24 mm 2 ) for extended periods of time. As a result, a large set of comprehensive data can be gathered with strong statistics, both in space and time. Video lensfree Microscopy can capture images of cells cultured in various physical environments. We emphasize on two different case studies: the quantitative analysis of the spontaneous network formation of HUVEC endothelial cells, and by coupling lensfree Microscopy with 3D cell culture in the study of epithelial tissue morphogenesis. In summary, we demonstrate that lensfree Video Microscopy is a powerful tool to conduct cell assays in 2D and 3D culture experiments. The applications are in the realms of fundamental biology, tissue regeneration, drug development and toxicology studies.

  • real time label free detection of dividing cells by means of lensfree Video Microscopy
    Journal of Biomedical Optics, 2014
    Co-Authors: Srikanth Vinjimore Kesavan, Fabrice Navarro, Mathilde Menneteau, Bernard Chalmond, Frederique Mittler, Brigitte Davidwatine, Nelly Dubrulle, Spencer Shorte, Jeanmarc Dinten, Cédric Allier
    Abstract:

    Quantification of cell proliferation and monitoring its kinetics are essential in fields of research such as developmental biology, oncology, etc. Although several proliferation assays exist, monitoring cell proliferation kinetics remains challenging. We present a novel cell proliferation assay based on real-time monitoring of cell culture inside a standard incubator using a lensfree Video-microscope, combined with automated detection of single cell divisions over a population of several thousand cells. Since the method is based on direct visualization of dividing cells, it is label-free, continuous, and not sample destructive. Kinetics of cell proliferation can be monitored from a few hours to several days. We compare our method to a standard assay, the EdU proliferation assay, and as proof of principle, we demonstrate concentration-dependent and time-dependent effect of actinomycin D—a cell proliferation inhibitor.