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We do not have any experience with the 590nm HeNe line except to note
that it is weaker than the primary line.
We have had extensive experience with Argon/dye setup, and find it
works quite well for TexRed/APC combination. It is expensive, though,
and requires dye changing depending on use. We use several hundred mw
output from the dye laser for TR excitation, so I would doubt a yellow
HeNe would have much success with a jet-in-air configuration.
FL/PE/TR/APC has been a very reliable 4 color setup for us for quite a
while. The addition of Cy5-PE makes this a solid 5-color setup.
We are starting to use some of the Cy7 tandem dyes (Cy7-PE and Cy7-APC)
and find them to work quite well,too (see our poster on 7 and 8 color
immunofluorescence at ISAC). We use TR, APC, and Cy7-APC with the
Argon/dye lasers, giving three colors. Cy7-PE gives a 4th color off of
the 488nm argon line.
Cy7-APC should work with a red HeNe, thus giving 2 colors (with APC),
but we do not have any experience with this configuration.
Becasue of the far red emission of the Cy7 dye, it is necessary to have
PMTs optimized for red detection. The same will be true to a lesser
extent for TR and APC. These PMTs will cost more. Even better would be
to get the GaAs cathode type PMTs which have (in our hands) a five-fold
increase in sensitivity for Cy7, but cost even more. Also these tubes
have lower overall gain so higher gain preamps are required.
Hope this helps.
-Marty Bigos
Stanford Shared FACS Facility
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