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| Date: | Fri, 13 Aug 2004 15:40:39 -0400 |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi David,
I've seen the reflection from several metal surfaces also. Others here
have solved the problem by coating with carbon. For example,
someone wanted to see EPS-stained bacteria on stainless steel
"coupons". The reflectance was particularly bad on the SS.
Your sample may not allow coating while maintaining the data.
John Shields
On 13 Aug 2004 at 14:16, David Burk wrote:
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Leica
TCS
> SP2 and other confocal experts,
>
> I have been having a devil of a time recently trying to visualize FITC
> labeled proteins attached to a gold monolayer deposited on glass
> slides. I can™t go into too much detail as I am not privy to all of
> the gory steps involved in prepping these samples but I can explain
> what sort of results I™m getting.
>
> With the Leica confocal set to detect FITCwide fluorescence (488
> excitation) I get what looks like bands of reflectance off of the gold
> surface that renders any attempt at detecting weak FITC signal
> useless. I have no trouble seeing the fluorescence with my eyes
using
> an I3 (blue excitation; long pass 515nm) but can™t get a good
image
> with the TCS SP2 due to extremely bright bands of background
signal
> (this background banding pattern appears on the negative controls
> also). Any ideas what causes this śreflectionť and how to eliminate
> it?
>
> I know the system is operating as we have no problems with typical
> biological specimens but as our facility gets more business from the
> material scientists I™d like to be able to provide them with some
> results or at least a knowledgeable explanation as to why I can™t
help
> them.
>
> Thanks for any advice!
>
> P.S. If any of you also have experience looking at proteins attached
> to UV-modified PMMA sheets please contact me off-list (or on, if you
> like). I™d like to know what excitation wavelengths you use that
don™t
> lead to auto-fluorescence from the PMMA.
>
> David Burk
> Socolofsky Microscopy Center
> Department of Biological Sciences
> Louisiana State University
> (225)578-8246
> [log in to unmask]
>
--
John Shields
E.M. Lab, 151 Barrow Hall
University of Georgia
Athens, GA 30602
706-542-4080
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