Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Elisabeth Bugnard wrote:

> We used a Cy5 antibody + an anti tubulin on mRFP and GFP co-transfected cell. I was extremely surprised to be able to see the Cy5 staining in Cy5, red (Rho or TRed) and green channels with the eyepieces of the microscope. I didn't need to turn the camera on to see the Cy5 staining. I can understand that I can see a fluorochrome in other channels if it's too concentrated or the filters are not very "selective". But in the case of Cy5 that one can normaly see only with the camera....Is it also possible for a fluorochrome of the far red?

Dear Elizabeth--

When you looked at the Cy5 through the rhodamine and fluorescein
filters, what color did it appear to be?

If it looked red in all 3 cases, then you simply have long-pass emission
filters on your FITC and rhodamine filter cubes.  (You probably also can
see rhodamine through your FITC filter, if that's the case.)  If it
doesn't look red through all the different filters, I'd expect that the
Cy5 has become contaminated with other fluorophores somewhere along the
line.

The emission spectrum of Cy5 peaks at about 670 nm--quite far out in the
red, but not THAT much "more red" than the 635 nm line of a
laser-pointer.  The bottom line is that its emission is quite visible
to the naked eye, if it's bright enough.  It will be much harder to see
than Cy3 or FITC, but if it's bright enough (e.g. an intracellularly
recorded and filled cell), you'll see it.

Good luck!

Martin Wessendorf
-- 
Martin Wessendorf, Ph.D.                   office: (612) 626-0145
Assoc Prof, Dept Neuroscience                 lab: (612) 624-2991
University of Minnesota             Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: (612) 626-5009
Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu