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Hello Elizabeth (and Martin!)

Martin's explanation seems pretty likely, but occasionally we have seen 
emission from Cy5 down to green wavelengths, especially when the dye is 
in very high concentrations (eg in a dye-filled neuron). I seem to 
remember that there was some discussion on the list about this sort of 
phenomenon a while back??

hope that helps

IAN

On Friday, June 22, 2007, at 12:37  AM, Martin Wessendorf wrote:

> 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
>
>

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