Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dave,
I'm not sure I follow your question.
If you can see discrete photobleaching steps
in your spots intensity time-traces, then
I guess you're looking at single molecule.
In that case using the right buffer is
crucial.

Eli

---- Original message ----
>Date: Sun, 29 Jul 2007 14:05:08 -0400
>From: David Knecht-charter <[log in to unmask]>  
>Subject: Re: Single-molecule imaging with an Olympus Fluoview confocal microscope?  
>To: [log in to unmask]
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>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
>> Assoc Prof, Dept Neuroscience                 lab: (612) 624-2991
>> University of Minnesota             Preferred FAX: (612) 624-8118
>> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: (612) 626-5009
>> Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu