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Laser Scanning Microscopy Applied to Studies of the Cell Cycle.

Published online by Cambridge University Press:  02 July 2020

Ed Luther
Affiliation:
CompuCyte Corporation, 12 Emily St, Cambridge, MA
Louis A. Kamentsky
Affiliation:
CompuCyte Corporation, 12 Emily St, Cambridge, MA
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Non-confocal Laser Scanning Microscopy (LSCM) was developed to allow applying the tenents of flow cytometery to specimens attached to a microscope slide (1). Areas of the slide are scanned, fluorescently labled cells are automatically segmented, and a list of features for each cell is calculated and stored in a list mode data file. Figure 1 shows a block diagram of the LSCM. A collimated laser beam scans in the y direction, and values from photomultiplier and photodiode detectors are digitized to .25 micron resolution. The automated stage advances in .5 micron steps in the x direction. When cascading memory banks are filled, the “image” is segmented. Figure 2 shows contours drawn as part of the segmentation process. The innermost contour is drawn around pixels that exceed a predetermined threshold, and is used to define events. Other contours are used to define integration areas, background calculation and correction, and integrating nuclear vs. peripheral fluorescence areas.

Type
Imaging Cells and Organelles
Copyright
Copyright © Microscopy Society of America 1997

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References

1.Kamentsky, L. A. and Kamentsky, L. D., Cytometry, 12:381387, 1991 10.1002/cyto.990120502CrossRefGoogle ScholarPubMed
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3.Pines, J. and Hunter, T., The Journal of Cell Biology, 115,1:117, 1991 10.1083/jcb.115.1.1CrossRefGoogle Scholar