Light scattered at two wavelengths can discriminate viable lymphoid cell populations on a fluorescence-activated cell sorter.

Abstract

A fluorescence-activated cell sorter was modified to simultaneously detect 2 different colors of light scattered by cells as they passed through the instrument. A single Ar ion laser with output from 351-488 nm was used as the light source. The signals from 2 light scattering detectors (using the same collection optics but having different optical filters) were compared. In the analysis of glutaraldehyde-fixed chicken erythrocytes the histograms collected for UV and 488 nm scattered light were similar in shape. The correlation plot between the 2 signals indicated that a simple linear transformation could not be made between UV and 488 nm light scattering signals. The analysis of mouse bone marrow indicated that 2 distinct nucleated cell populations could be identified by 488 nm light scattering; only 1 population was identified by UV light scattering. Erythrocytes could be distinguished from nucleated cells by UV and by 488 nm light scattering. By correlating the 2 colors of light scattering a 3rd population of bone marrow cells, predominantly large lymphocytes, was identified. Among splenic lymphocytes, T cells could be distinguished from B cells based on the differences in scattering of these 2 wavelengths. Light scattering patterns change drastically when different colors of light are used to analyze the population. The correlation of light scattered at 2 different wavelengths can aid in distinguishing populations of cells that could not be identified by a single wavelength of scattered light.