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I would think that the FITC wide setting uses too much of the
spectral range.  Try adjusting the width of the detector spectrum by
using the sliders in the "beam window".  You might be able to remove
much of the reflection by careful selection of the wavelengths.

Date sent:              Fri, 13 Aug 2004 14:16:53 -0500
Send reply to:          Confocal Microscopy List <[log in to unmask]>
From:                   David Burk <[log in to unmask]>
Subject:                Problems scanning FITC labeled proteins on gold monolayer with Leica
                TCS SP2
To:                     [log in to unmask]

> 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 "reflection" 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]
>
>

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [log in to unmask]