Re: FRET-FLIM question

Posted by Deepak nair on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Researcher-postdoc-National-Imaging-Platform-Oslo-tp7000581p7004906.html

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Hi All

I am sorry a bit of understanding/misunderstanding from my part. I thought
that the concentration dependence of life times depend on how you do your
analysis (Like Emmanuel suggested). If the analysis is based on mean or
average lifetimes, it is concentration dependent since it averages
fractional contributions of multiple lifetimes in the decay.On the
otherhand If you can find out the individual components (wether bi (for
GFP) or multiexpoential (for CFP)) of the decay, it is concentration
independent since it gives out the unaveraged lifetimes of individual
species.

Can someone correct me if I got it wrong...

Best
Deepak

On Thu, Nov 17, 2011 at 1:58 PM, Gustin, Emmanuel [JRDBE] <
[hidden email]> wrote:

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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
>
>
> --
>  Emmanuel Gustin,    Tel. [phone],    e-mail: [hidden email]
>
>
> -----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,
> Madsen Building F09, University of Sydney, NSW 2006
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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: [hidden email]
> 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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