Mario Roederer has remarked: "We routinely run four colors off of 488 nm, and 3 off of 600 nm, with outstanding results. With apologies to Howard Shapiro, it would be far more complex (technologically) to consider 7 different excitation beams--or even 4. Perhaps future technology development, however, will prove me wrong." Apologies are unnecessary, now that Mario and so many people are running three beams. When we didn't have dyes with a variety of Stokes shifts, like the phycobiliproteins and tandems, multiple excitation beams provided the only practical approach to multicolor immunofluorescence measurement. We can now use fewer beams, but, as the number of colors per beam increases, the problem of compensation becomes harder to solve with either hardware or software. It's hard to build systems deriving 4 or more separated beams when you get the beams from big lasers, but is considerably easier when you use diode or solid state lasers, now eminently suitable for green, red, and infrared excitation, and probably available for UV through blue within a few years. If our appetites for more and more immunofluorescence colors keep increasing, we'll have to consider both approaches. Of course, the original Block system (1974) derived five separated beams from one arc lamp, but, then again, we weren't doing immunofluorescence. -Howard
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