Posted by
Emmanuel Gustin on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Researcher-postdoc-National-Imaging-Platform-Oslo-tp7000581p7004025.html
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It depends on what FLIM system they use. If they were able to acquire enough information to *reliably* fit the donor fluorescence data to a bi-exponential:
A exp(-(t-t0)/tau1) + B exp(-(t-t0)/tau2)
Where tau1 is the lifetime of the donor in a FRET couple and tau2 is the lifetime of the donor with "remote" acceptors, then the ratio of areas below the A and B curves would be a good measure for efficiency of dimerisation.
But in many FLIM experiments, as far as I know, the data is too rough to do that, and then the best option is to "fit" a single exponential. That gives you an "pragmatic" lifetime estimate tau that reflects a mixture of donor-acceptance distance and dimerisation efficiency. It actually depends on all of the four above parameters.
Best Regards,
Emmanuel
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Emmanuel Gustin, Tel. (+32) 14 64 1586, e-mail:
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-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Guy Cox
Sent: donderdag 17 november 2011 13:32
To:
[hidden email]
Subject: Re: FRET-FLIM question
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I'm a bit lost by this. As you say, donor lifetime reduction caused by FRET is independent of concentration (so long as there is no external constraint on dimer formation), but since dimer formation is typically what brings the FRET partners into close proximity, I cannot see how you can expect to have increased FRET efficiency with less dimerization. I may have misunderstood your point - if so, please clarify. My brain is probably slowing down with age!
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
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-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Straatman, Kees R. (Dr.)
Sent: Thursday, 17 November 2011 9:34 PM
To:
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Subject: FRET-FLIM question
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Dear list members,
We have a discussion about FRET-FLIM at the moment and somebody has published that they see an increase in FRET efficiency and argues that this is because there is more dimerisation between the two proteins they are interested in. Now, I thought that FRET-FLIM is independent from the concentration of the fluorochromes and that the increase in FRET efficiency is caused by a change in distance between the 2 reporter FP (CFP and YFP) and it would be possible to have less dimerisation of the proteins but an increase in FRET efficiency. Is this correct?
Thanks
Kees
Dr Ir K.R. Straatman
Senior Experimental Officer
Centre for Core Biotechnology Services
College of Medicine, Biological Sciences and Psychology
University of Leicester
http://www.le.ac.uk/biochem/microscopy/home.html-----
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