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Hello David,
I get this kind of stuff all the time. I'm glad to hear that I'm not the
only one faced with unconventional, sometimes un-imageable, specimens on
a frequent basis. If you are getting serious reflections in the FITC
detection range then your SP-2 probably isn't set up right. I suspect
the wrong dichroic if you have a dichroic based model, also, check the
detection range to make sure it isn't overlapping with the laser output.
From the dawn of time the SP-2 factory pre-sets have been messed up. Set
up the laser, dichroic and detection range yourself and you'll be in
much better shape. If you have administrator priveledges you can fix all
of the default presets that users get when they log on. Another
possibility is that the detection sliders are stuck or mis-calibrated in
which case you'll need a service visit. You can test this by running a
lamda scan over the laser lines in reflected light mode. This happens
from time to time.
Some of these material people may want to look through the gold
substrate in order to detect fluorescence for one reason or another.
When this is the case I give them the "this is massively inefficient"
lecture, but often the gold deposition is thin enough to let some
fluorescence back through to the detector.
UV polymerized PMMA is basically plexiglass, we haven't had too much
trouble from background fluorescence, although sometimes the
"fluorescence" that is attributed to the bound protiens is orders of
magnitude below that which biologists are used to. Chemists and
materials people often have little concept of the fluorochrome
concentrations and quantum yield that biological specimens are prepared
with for microscopy. To some of them, if you can get it to fluoresce in
the IR with several WATTS of green laser light they think you can go
ahead and plop it on the 'scope and image it (I'm not kidding, I had a
guy come in with this a couple weeks ago). It's good to have a dilution
series of fluorescien on hand to demonstrate what our conception of
fluorescence is, and where the lower limits of detection for a
particular instrument are. Negative controls are great, but I always
recommend positive controls as well, this way I can prove that the
instrument works, and I can do this for known concentrations of
fluorochrome. In order to see the 'ol "protein on gold" experiment some
of our users are forced to take 30second -1minute exposures with a
cooled ccd camera on a widefield fluorescence rig. Patterns in the bound
protein help to distinguish fluorescence from stray light. The human eye
can't see this interpretation of fluorescence.
The materials people and chemists are fond of fluorescien conjugated to
protein. Fluorescien is far from the best fluorochrome for these
experiments; it bleaches very quickly, and anti-fade agents as well as
index-matching mountants are, more often than not, a no-no. You might
suggest Alexa 488 as a replacement.
Good Luck,
Karl

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 “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]
>

--
Karl Garsha
Light Microscopy Specialist
Imaging Technology Group
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
405 North Mathews Avenue
Urbana, IL 61801
Office: B650J
Phone: 217.244.6292
Fax: 217.244.6219
Mobile: 217.390.1874
www.itg.uiuc.edu