Simple modification of a commercial flow cytometer to triple laser excitation. Simultaneous five-color fluorescence detection.

We describe a simple and inexpensive modification to the Coulter 753 flow cytometer which enables simultaneous triple laser excitation for advanced multi-color analysis and sorting applications. The salient feature of the modification was to split the rear laser operating in the all lines mode for use in pumping the rhodamine 6G dye laser in addition to its use as an independent 488 nm excitation beam. With the appropriate filters for fluorescence detection, and the measurement of two signals on one photomultiplier, this configuration allows for the simultaneous excitation and detection on single cells of fluorescein, phycoerythrin, Texas Red and allophycocyanin-conjugated antibodies together with certain UV-excited dyes.