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Is that the only way to know for sure whether or not you're looking  
at single-molecules? You only know after the fact that what you saw  
was a single fluorescing molecule if you saw only one discrete  
photobleaching step in your intensity vs. time trace?
So for ten molecules I should see hopefully ten discrete steps into  
the background signal?

John


On 30-Jul-07, at 3:36 PM, Eli Rothenberg wrote:

> 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