Spectra of fluorescent dyes used in flow cytometry.

Flow cytometry uses a relatively small set of dyes for immunochemistry and nucleic acid detection, most of which have been known and used reliably in flow cytometry for several years. These can usually be combined to make simultaneous two-color measurements of multiple cell-surface antigens and nucleic acid content. Because of the overlap of dye spectra and difficulties in finding dyes with substantial Stokes shifts that can be excited by the argon laser, simultaneous three-color or more detection can be more difficult. A basic knowledge of the factors that go into producing the fluorescent signal, including the spectra of dyes and their overlap, is necessary in planning multicolor experiments. By contrast, there have been a number of new fluorescent probes developed for detecting ions, membrane potential, metabolism, organelles, and other properties of living cells as well as for determining cell viability, proliferation, and cell tracking. So far most of these physiological probes are being used only for fundamental research rather than for cell classification. However, as research activity expands in this area, its diagnostic potential is likely to be increasingly appreciated.