Color bias: Better visible than UV

Mario Roederer Roederer at Beadle.Stanford.EDU
Wed Jun 25 12:26:40 EST 1997


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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