Cell Cycle Analysis With The Laser Scanning Cytometer I: High Resolution Analysis Using A Single Detector.
E. Luther* and L.A. Kamentsky, CompuCyte Corporation, Cambridge MA.
Laser scanning cytometry generates data that is similar to flow cytometry in the sense that a list of extracted parameters is generated for each of thousands of cells in a sample. The way cells are identified and parameters are calculated is similar to image processing, with measurements of the slide being made on a pixel by pixel basis, and about a hundred measurements are used to calculate parameters for each cell.
When stained with a DNA specific fluorochrome, in addition to the amount of DNA per cell, the computationally intensive segmenting provides non-stoichiometric sub parameters which give additional information about the nuclei being analyzed. One of these parameters is very sensitive to the condensation of the chromatin of the nuclei, and allows the discrimination of mitotic from interphase cells. In bivariate histograms, this can be seen as a long extension away from the G2 cluster, corresponding to the progression from prophase to metaphase. Similarly, an extension from the G1 cluster represents newly formed G1a cells. This was demonstrated by colchicine blocking, as well as by relocation and visualization of cells using either epifluorescence illumination or restaining the cells with chromatic dyes and using bright field illumination.
The technique is demonstrated on cells fixed in suspension, cytocentrifuge preparations of suspension cells, adherent cells fixed on slides in situ, viable cells staired with the vital stain SYT016, and a frozen section of pathological material.