Bob, your questions deserve far more complex answers that I am giving below, but such a discussion is not apropos for this mailing list! I apologize to the uninterested readers for how long this answer is already! I will give some brief "thoughts" on colors, based on our extensive experience doing multi-color applications (we currently do 8-color FACS using excitations from 400 to 600 nm, and emission ranging to 800 nm). There are two principle problems with UV excitation: (1) the availability of "good" dyes, and (2) background. (1:) The advantages of phycobiliproteins is their enormous epsilon and a quantum efficiency of nearly 1. Comparatively, UV dyes in general have very small absorption coefficients and quantum efficiencies. Thus, the amount of signal is considerably less. Some UV dyes can come close to FITC in terms of brightness, but most cannot. (2) Background, being mostly autofluorescence (AF). AF has excitation peaks around 360 and 490, with 360 particularly bad. Thus, the UV dyes suffer in that the cellular contribution to background is much greater than for other excitations. Recently, Coherent has come out with a 405 nm (Krypton) laser. This laser line is good because there is a relative "hole" in the AF excitation spectrum at 405, and is still good for exciting dyes like Cascade Blue. Using this line, Cascade Blue becomes about one-half as bright (useful brightness) as FITC. Obviously, it still isn't a "great" reagent, but that's the best you can do with UV dyes. There are other UV dyes that can be used independently and simultaneously as Cascade Blue with emissions further to the red, but they aren't even as bright as CB. Using CB with typical (351) UV excitation makes it difficult to detect directly conjugated antibodies (with the exception of very bright ones like CD8 or CD45). Avidin or indirect stains are somewhat better. so: "IS CONJUGATE-SPACE SYMMETRIC, OR IS IT BETTER RED THAN DEAD?" The answer is that is is not symmetric for a variety of reasons. The red dyes (even nonphycobiliproteins like Cy5) are outstanding because they combine high absorbance and high quantum efficieny in a spectral region with very low AF. and: "Is the He-Ne path of higher wavelength excitation superior to the He-Cd (or Hg arc) path of shorter wavelength excitation?" Yes, HeNe is superior. Finally: "In the quest for extra colours, we can add tandems for 488-excitation, or we can add light sources which move to higher or to lower wavelengths of excitation. But which way is better?" Based on the high degree of success we have had with tandems, I would say that this is the better way to go. 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. mr
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