28 May 2002 Tracking tissue section surfaces for automated 3D confocal cytometry
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Proceedings Volume 4622, Optical Diagnostics of Living Cells V; (2002) https://doi.org/10.1117/12.468353
Event: International Symposium on Biomedical Optics, 2002, San Jose, CA, United States
Abstract
Three-dimensional cytometry, whereby large volumes of tissue would be measured automatically, requires a computerized method for detecting the upper and lower tissue boundaries. In conventional confocal microscopy, the user interactively sets limits for axial scanning for each field-of-view. Biological specimens vary in section thickness, thereby driving the requirement for setting vertical scan limits. Limits could be set arbitrarily large to ensure the entire tissue is scanned, but automatic surface identification would eliminate storing undue numbers of empty optical sections and forms the basis for incorporating lateral microscope stage motion to collect unlimited numbers of stacks. This walk-away automation of 3D confocal scanning for biological imaging is the first sep towards practical, computerized statistical sampling from arbitrarily large tissue volumes. Preliminary results for automatic tissue surface tracking were obtained for phase-contrast microscopy by measuring focus sharpness (previously used for high-speed autofocus by our group). Measurements were taken from 5X5 fields-of-view from hamster liver sections, varying from five to twenty microns in thickness, then smoothed to lessen variations of in-focus information at each axial position. Because image sharpness (as the power of high spatial frequency components) drops across the axial boundaries of a tissue section, mathematical quantities including the full-width at half-maximum, extrema in the first derivative, and second derivative were used to locate the proximal and distal surfaces of a tissue. Results from these tests were evaluated against manual (i.e., visual) determination of section boundaries.
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Ramses Agustin, Ramses Agustin, Jeffrey H. Price, Jeffrey H. Price, "Tracking tissue section surfaces for automated 3D confocal cytometry", Proc. SPIE 4622, Optical Diagnostics of Living Cells V, (28 May 2002); doi: 10.1117/12.468353; https://doi.org/10.1117/12.468353
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