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Hello Group,
Thanks very much to you all for the helpful thoughts and comments. Much
appreciated. This user was un-necessarily using a long exposure time and
gain to pick up very very weak GFP signal. This bleedthrough is not
something that I have ever seen or documented on my system. Good thing to be
aware of as well a point to reinforce to users.

Thanks again group.
Cheers
Farid


On Sat, Aug 2, 2008 at 1:54 PM, David Knecht ATT <[log in to unmask]>wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> It looks to me like you are getting the early tail of the emission of Alexa
> 555 through your GFP filer.  You have a 525/30, which cuts off around 550,
> but the Alexa emission starts up around 538nm..  You could do as others
> suggested, but if you already have samples stained, you could switch to a
> narrower GFP filter, such as the Chroma 41020 which cuts off around 520 and
> should eliminate the problem.  Dave
> On Jul 31, 2008, at 10:47 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
>
>
> Dr. David Knecht
> Department of Molecular and Cell Biology
> Co-head Flow Cytometry and Confocal Microscopy Facility
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)
>
>
>


-- 
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 Suite