An improved dual-laser flow cytometric system for quantitative analysis and sorting of mammalian cells has been developed using a low-power argon and high-power krypton laser as illumination sources, thus permitting the excitation of fluorescent dyes having absorption regions ranging from the ultraviolet to infrared. Cells stained in liquid suspension with fluorescent dyes enter a flow chamber where they intersect two spatially separated laser beams. Separate pairs of quartz beam-shaping optics focus each beam onto the cell stream. Electro-optical sensors measure fluorescence and light scatter from cells that are processed electronically and displayed as frequency distribution histograms. Cells also can be electronically separated and microscopically identified. The ease and versatility of operation designed into this system represent a marked technological improvement for dual-laser excited flow systems. Details of this instrument are described along with illustrative examples of cells stained with mithramycin and rhodamine and analyzed for DNA content, total protein, and nuclear and cytoplasmic diameter.

For optimum performance in cell sorting, it is critical to assure proper timing in the charging of droplets to be deflected. A method for determining the transit delay time in cell sorters has been devised and applied to daily operation in the Los Alamos sorter systems. This delay monitor relies on detection of either scattered or absorbed light from cells in the fluid stream near the point of droplet breakoff.