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Farid Jalali wrote:
>  We are seeing what seems to be the Alexa 555 
> signal in the GFP channel. We are illuminating with an EXFO Hg lamp,  
> and using the Semrock Quad-band Sedat filter set. The excitation, 
> emission and dichroic mirror specs from the Semrock website are provided 
> below. A small amount of Alexa 555 is excited by the GFP excitation 
> indicated below, but the cut-off on the GFP EM should preclude any Alexa 
> 555 EM getting through. I am not sure if I am interpreting the Mol 
> Probes Spectraviewer correctly and therefore I am asking the group for 
> thoughts. We are obtaining what appears to be Alexa 555 signal in the 
> GFP channel at very long exposure times (400ms) and high gain (>3800 on 
> a scale max'ing at 4095) on a Photometrics Cascade II EM-CCD, imaging in 
> widefield.

When you excite a longer wavelength fluorophore with a short wavelength, you can 
get emission from the short wavelength "tail" of the emission spectrum. The 
various spectrum viewer show the spectra on a linear scale, so it looks like 
there is no emission, but that is not necessarily the case. In many cases, the 
spectral data are not provided so far from the peaks, but again, that does not 
mean that there is absolutely nothing. You are detecting at very high gain, and 
perhaps you have strong staining with the alexa555.

If you want to test this, you can run spectra of alexa555 in a high quality 
spectrofluorometer, and acquire the emission spectrum with 485nm excitation.

--aryeh
-- 
Aryeh Weiss
School of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: 972-3-7384050