Entering the Portal: Understanding the Digital Image Recorded Through a Microscope

Hazelwood, Kristin L.; Olenych, Scott G.; Griffin, John D.; Cathcart, Judith A.; Davidson, Michael W.

doi:10.1007/978-3-540-71331-9_1

Cited by 17 publications

(4 citation statements)

References 35 publications

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“…Dyes that fluoresce at longer wavelengths are preferred due to lower phototoxicity and autofluorescence contributions. 59 Both sensing and normalizing dyes should be spectrally well separated and placed spatially at least 10 nm apart to avoid cross-talk. 60 Further substantial spatial separation between sensing and normalizing dyes would enable better binding between sensor and its protein targets as well as preserve the photophysical properties of both dyes.…”

Section: Discussionmentioning

confidence: 99%

Rational design of a quantitative, pH-insensitive, nucleic acid based fluorescent chloride reporter

et al. 2016

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Section: Discussionmentioning

confidence: 99%

Rational design of a quantitative, pH-insensitive, nucleic acid based fluorescent chloride reporter

et al. 2016

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“…Compared with the fluorescence-based microscopy which is known for photodamage to the functions of live cells, brightfield intravital microscopy is more physiological and less harmful to the cells and tissues (therefore with less artifacts) when long-time imaging for observing dynamic cellular behaviors in live animals is necessary 7 . It is also more convenient, less costly and no fluorescent labeling is needed.…”

Section: Discussionmentioning

confidence: 99%

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

Liu

2011

JoVE

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The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation 1,2 . In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling 3 . Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis) 4 . Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment 2,5 . In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm 3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo. Video LinkThe video component of this article can be found at http://www.jove.com/details.php?id=3296 Protocol Preparation of Chemoattractant in Agarose Gel Preparation of Cremaster Muscle for Intravital Microscopy (Figure 1)Note: All procedures from 2.6 to 2.14 must be performed very gently 5 .

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“…For example, one can start by segmenting the cells in the first frame of a video and evolve their contours in consecutive frames [13], [14]. However, often biological experiment does not allow the high temporal resolution to minimize unnecessarily oversampling time point or exposing the cells to the excessive level of illumination to measure these processes of individual cells over days [15]. Thus, a scenario of low temporal resolution and/or high cell speed (or large movement) is a more practical and challenging problem since this setting complicates the identification of the correspondence between cells.…”

Section: Introductionmentioning

confidence: 99%

Single cell tracking based on Voronoi partition via stable matching

Chang

Linsley

Lamstein

et al. 2020

Preprint

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Live-cell imaging is an important technique to study cell migration and proliferation as well as image-based profiling of drug perturbations over time. To gain biological insights from live-cell imaging data, it is necessary to identify individual cells, follow them over time and extract quantitative information. However, since often biological experiment does not allow the high temporal resolution to reduce excessive levels of illumination or minimize unnecessary oversampling to monitor long-term dynamics, it is still a challenging task to obtain good tracking results with coarsely sampled imaging data. To address this problem, we consider cell tracking problem as "stable matching problem" and propose a robust tracking method based on Voronoi partition which adapts parameters that need to be set according to the spatio-temporal characteristics of live cell imaging data such as cell population and migration. We demonstrate the performance improvement provided by the proposed method using numerical simulations and compare its performance with proximity-based tracking and nearest neighbor-based tracking.

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Entering the Portal: Understanding the Digital Image Recorded Through a Microscope

Cited by 17 publications

References 35 publications

Rational design of a quantitative, pH-insensitive, nucleic acid based fluorescent chloride reporter

Rational design of a quantitative, pH-insensitive, nucleic acid based fluorescent chloride reporter

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

Single cell tracking based on Voronoi partition via stable matching

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