Multiple immunofluorescence analysis of cells using flow cytometry

Prior to 1982, the measurement of more than one antigen simultaneously by flow cytometry required two lasers-an argon-ion laser to excite fluorescein (at 488 nm) and a krypton or a dye laser to excite rhodamine or one of its derivatives. The discovery of a naturally occurring fluorochrome, phycoerythrin (PE), changed this (1). PE is a phycobiloprotein found in red algae. It can be excited efficiently at 488 nm (simultaneously with fluorescein) and has a peak fluorescence at 578 nm, sufficiently removed from the peak of 520 nm from fluorescein. There is some overlap in the emission spectra from the two dyes (in particular, there is still some emission from fluorescein above 580 nm) and this must be corrected, either electronically or by the computer software.