Three‐color fluorescence measurements on single cells excited at three laser wavelengths

A three-laser flow cytometer for quantitative analysis and sorting of cells and microscopic particles has been developed and evaluated using argon- and krypton-ion lasers as excitation sources. Cells stained with three fluorescent dyes having different excitation spectra enter a flow chamber where they first pass through an electronic volume sensor and then intersect three spatially separated laser beams for fluorescence excitation of bound dyes and light scatter measurements. Separate pairs of beam-shaping optics independently position and focus each beam onto the cell stream thus permitting cells to be sequentially illuminated. Fluorescence is measured by a detector that employs a single collecting lens for single or multilaser excitation experiments. Fluorescence signals are processed and displayed as frequency distribution histograms using an LSI-11 computer. This instrument is described in detail with illustrative examples using cells stained for DNA content, protein, and cell surface antigens and cells that have phagocytized fluorescent microspheres.