single molecule bleaching correction

[Jeff Spector]

*****
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...

[John Oreopoulos]

*****
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...

[Jeff Spector]

*****
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 <
[hidden email]> 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...
>

[Michelle Peckham]

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

You could try the approach described by Mashanov and Molloy in j biol chem 2003, effectively varying the laser power, which changes the rate of bleaching, but also tells you if there is unbinding. See paper for more explanation


Michelle

On 10 May 2013, at 16:19, "Jeff Spector" <[hidden email]> 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 <
> [hidden email]> 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...
>>

[John Oreopoulos]

*****
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 <
> [hidden email]> 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...
>>

[Kurt Thorn]

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

Jeff -

When I did this kind of work in graduate school, we assumed that the
glass-bound molecules never dissociated and used that as an estimate of
the bleaching rate.  Once you have the bleaching rate, the measured
dissocation rate is just the sum of the actual dissociation rate and the
bleach rate.  See the methods section here:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2174356/ for details.

Kurt

On 5/10/2013 8:18 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 <
> [hidden email]> 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...
>