*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

This sounds like a very common type of measurement that people in the single-molecule imaging field would be doing. I don't have much experience with that kind of analysis, but as I recall, Nancy Thompson at UNC has been doing these kinds of experiments for quite some time (maybe even from the beginning). Here's the link to her contact information:

http://www.chem.unc.edu/people/faculty/thompson/

Perhaps she might be give you some more details?

John Oreopoulos


On 2013-05-10, at 11:18 AM, Jeff Spector wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> Thanks for the references. I've looked at a lot of that type of literature.
> The issue I am having is that we have a nice TIRF image of single molecules
> landing on our substrate and then disappearing. I need to know if they are
> dissociating from the substrate or bleaching.  I see a lot of molecule on
> glass in my samples, and I guess I could measure their bleaching curves,
> but how do I know they aren't dissociating from the glass? I guess I could
> get a distribution of glass bound molecules and measure their bleaching
> curves to get an average bleaching time, and then compare that with the
> dwell times I measure on the sample, and as long as the dwell times are
> shorter than the average on-glass bleaching time then I can use this
> measurement, and if not then I need to adjust my framerate or laser power.
> Is this the correct way to do this sort of  'dwell time' analysis?  We are
> not doing FRET and we don't really need to count the number of molecules in
> a given fluorescent spot, we really just want to measure the "on" and "off"
> rates. Anyone else have some literature I can look at, I seem to be having
> a hard time finding anything..
> thanks...
> -jeff
> 
> 
> On Thu, May 9, 2013 at 11:44 PM, John Oreopoulos <
> [log in to unmask]> wrote:
> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> Jeff,
>> 
>> Take a look at these articles. Might be some help here:
>> 
>> Coffman, V.C. and J.Q. Wu, Counting protein molecules using quantitative
>> fluorescence microscopy. Trends in Biochemical Sciences, 2012. 37(11): p.
>> 499-506.
>> 
>> McGuire, H., et al., Automating single subunit counting of membrane
>> proteins in mammalian cells. Journal of Biological Chemistry, 2012.
>> 287(43): p. 35912-35921.
>> 
>> John Oreopoulos
>> 
>> 
>> On 2013-05-09, at 10:07 PM, Jeff Spector wrote:
>> 
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>> 
>>> Greetings,
>>> this may be somewhat out of place in this listserv but I know a lot of
>> good
>>> microscopists are on this list.
>>> I'm using a single molecule assay to try and measure the binding
>> kinetics of an
>>> enzyme. Essentially, I can see them land on my substrate and then
>> disappear. I
>>> would like to measure the on and off rates but am unsure as to how to
>> include
>>> the effects of photobleaching. I have seen a lot of papers where people
>> "correct"
>>> for this but don't explain how. Could someone on this list point me
>> towards some
>>> literature that might help me out?
>>> thanks...
>>