We too have noted colored, autofluorescent granules in frog parasympathetic
neurons.  These granules absorb strongly in the blue (488 nm) and fluoresce
in green and in orange-red.  But they are excited only weakly by 568 nm line
and even less so by 647 nm line.  So we can do double label fluorescence
with Cy3 and Cy5, for example, with a Kr/Ar laser.  The use of a Chroma
HQ598/40 emission filter for Cy3, which collects to within 10 nm of peak of
Cy3's emission (which coincides with 568 nm line, approximately) greately
improves things.  For more details, see a paper we published in NeuroImage
(1: 288-295).  This procedure is aa spin-off of a three channel procedure
originally described by Brelje, Wessendorf, and colleagues.
 
Peter Sargent
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 e-mail: [log in to unmask]    s-mail: Dr. Peter B. Sargent
 phone:  (415) 476-6156(7)                Div. Oral Biology, HSW-604
 fax:    (415) 502-7338                   University of California
      or (415) 476-4204                   San Francisco, CA  94143-0512
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