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Given Martin's comment, what is a good simple and conclusive test for  
determining sensitivity in the 1-10 fluorophore/molecule range if  
testing cameras/systems?  Dave

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)


On Jul 27, 2007, at 10:00 AM, Martin Wessendorf wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> John Oreopoulos wrote:
>
>> I'm wondering if anyone has had experience imaging single  
>> fluorescing molecules immobilized on a surface using a standard  
>> laser-scanning Olympus confocal microscope (Fluoview - any of the  
>> models). Someone came to me today with a sample of  a dilute  
>> solution of proteins that had been labeled with 1-10 Oregon Green  
>> and Texas Red fluorochormes per protein. Using TIRF and an EMCCD  
>> camera, it was very easy to see bright little spots spread around  
>> the surface.
>
>> I tried imaging once, and I was able to see bright spots, but only  
>> after driving the gains of the PMTs very high. It was a very noisy  
>> image, and the spots bleached fairly quickly unfortunately, and so  
>> I can't be sure if what I was seeing with the confocal was the  
>> protein or fluorescent junk in the sample.
>
> --This is a bit off your question, but you may need to consider  
> whether you're really seeing single protein molecules.  The higher  
> the fluorochrome-to-protein ratio, the more the solubility of their  
> protein will have changed.  (They're substituting large, planar  
> lipophilic moieties for small hydrophilic amino groups.)  My guess  
> is that unless they've done something like ion-exchange  
> chromatography after conjugation, they'll probably have at least  
> some cases where highly conjugated proteins have glommed together  
> into multimers.
>
> Good luck!
>
> Martin Wessendorf
> -- 
> Martin Wessendorf, Ph.D.                   office: (612) 626-0145
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