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Farid,
As Aryeh says, check the spectra. You can't rely on the stated maxima
to judge bleedthrough or cross talk. If you check EGFP and Alexa555
on a spectra viewer, like at Molecular Probes, you will see a small
overlap from the blue side of the A555 excitation curve and your
exciter filter. Small, but enough to generate enough photons to be a
problem when sufficiently amplified by long exposures with a cranked
up EMCCD. You are amplifying everything, regardless of the source.
I'd suggest switching to Alexa568, unless you can either cut
exposure time or gain to minimize the problem, or move to a blue-
shifted excitation for the GFP.
Regards,
Glen
On Jul 31, 2008, at 7:47 PM, Farid Jalali wrote:
> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Hello Group,
> In January there was a post and comments regarding Cy3
> photobleaching of DAPI and I have re-read them trying to come up
> with an explanation for something I was shown today on my system. A
> user has fixed cells stained with DAPI, stably expressing GFP, and
> targets stained indirectly with Alexa 555 and Alexa 647. 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.
> Apologies if this seems to be an amateurish question, but I can't
> seem to figure it out.
>
> Thanks very much all.
> Cheers
> Farid
>
> Filter
> Average Transmission
> Center Wavelengths
> Bandwidths *
> DAPI EX
> > 85%
> 387 nm
> 11 nm
> GFP EX
> > 90%
> 485 nm
> 20 nm
> Alexa555 EX
> > 90%
> 560 nm
> 25 nm
> Alexa647 EX
> > 90%
> 650 nm
> 13 nm
> DAPI EM
> > 90%
> 440 nm
> 40 nm
> GFP EM
> > 90%
> 525 nm
> 30 nm
> Alexa555 EM
> > 90%
> 607 nm
> 36 nm
> Alexa647 EM
> > 90%
>
> 684 nm
> 24 nm
>
> Average Reflection
> Reflection Bands
> Transmission Bands
> Dichroic
> > 95%
> > 95%
> > 95%
> > 95%
> 381 - 392 nm
> 475 - 495 nm
> 547 - 572 nm
> 643 - 656 nm
> 420 - 460 nm
> 510 - 531 nm
> 589 - 623 nm
> 677 - 722 nm
> --
> Farid Jalali MSc
> Senior Research Technician- Lab Manager
> Applied Molecular Oncology/ Princess Margaret Hospital
> STTARR Innovation Facility/ Radiation Medicine Program
> Toronto, Canada
> 416-946-4501 X4351 (Princess Margaret Hospital)
> 416-581-7754 STTARR at MaRS Building
> 416-581-7791 STTARR Microscopy Suit
Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923 USA
(206) 616-4156
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