Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
You're basically right, though you must have a control, that
you know it to have a single fluorophore labeling.
If you're working with a multiple labeled sample,
and if you're S/N is good enough for single molecule
detection, than photobleaching steps are expected, since
photobleaching events are not correlated. When
you look at a few traces that show a step like behavior,
the average Intensity count of a step (the steps should yield
a similar counts difference, since each results from a single fluorophore) would be the count of a single molecule
fluorescence.
If your background is too high and your doing epi, consider switching to TIR.
Eli
---- Original message ----
>Date: Mon, 30 Jul 2007 15:44:34 -0400
>From: John Oreopoulos <[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
>
>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
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