The problems with UV/violet excitation of fluorescent antibodies have been: 1. UV/violet sources are not ideal - Argon and krypton UV/violet lasers are expensive and power hungry, and burn out plasma tubes much faster than when run at 488 (although this may have improved; He-Cd lasers are noisy and temperamental, and arc lamps put out relatively low power in the UV and violet, although they work quite well in systems designed for them, such as Bio-Rad's, Partec's, and the old B-D FACS Analyzer and Cytomutt I. UV does, of course, excite at least some phycobiliproteins and other relatively long wavelength dyes, such as Texas red, but that only makes for compensation problems when you use these with longer wavelength excitation... 2. Pyridine and flavin nucleotides, which are major sources of tissue and cell autofluorescence, are, respectively, UV-excited/blue fluorescent and blue-violet excited/yellow-green fluorescent. Autofluorescence of flavins is even a problem with 488 excitation; that and the PE absorption maximum at about 545 accounts for the fact that the old FACS Analyzer with 546 nm Hg arc excitation gave ratios of fluorescence from PE-stained and unstained cells about 10x higher than a FACS with any level of laser power at 488. UV-excited tandems might fix problem 2, but not problem 1. However, there is still problem 3... 3. There are a few dyes which are only useful with UV or violet excitation; if you want to stain live cells for DNA, you need Hoechst 33342, and indo-1 is still the easiest calcium probe to use, and anybody who wants to combine either of these with antibody measurements more or less has to use the longer wavelengths to excite antibody labels. Chromomycin dyes and many GFP variants also require violet excitation. Within a few years, we'll probably have tractable solid state lasers running from the violet to the infrared, and diodes from the red (or maybe the yellow-green) to the infrared, leveling the playing field in that respect, but I suspect that it will still make sense in most cases to excite antibody labels at longer rather than shorter wavelengths. -Howard
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