Re: Acceptor Photobleaching vs. Sensitized Emission FRET results

Posted by yuansheng sun on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Acceptor-Photobleaching-vs-Sensitized-Emission-FRET-results-tp4828946p4830839.html

Pablo,

I forgot to ask if the FRET efficiency you mentioned refers to the
average of the whole cell. I think it is more appreciate to do
quantitative comparisons in details, such as comparing the FRET
efficiencies of different pairs for the same Donor : Acceptor ratios
or the same acceptor levels. If you can write me more details, we can
further discuss about the data analysis strategy.

sheng


On Tue, Mar 30, 2010 at 11:53 PM, Pablo German
<[hidden email]> wrote:

> Dear list members,
>
> I have been doing some FRET microscopy experiments on a
> 7-TransMembrane domain receptor tagged with either eCFP/eYFP at the
> different intra-cellular loops (ICL1, ICL2, and ICL3). I have tried
> the 9 different combinations (e.g ICL1-YFP + ICL1-CFP, ICL1-YFP +
> ICL2-CFP, etc) to see if I could detect any difference in FRET
> efficiency.
>
> I have anlyzed the images by both Sensitized Emission and Acceptor
> Photobleaching using the pFRET plugin on ImageJ developed at KCCI-UVa.
> The problem is the following: the results using Sensitized Emission
> give me significant differences between the different pairs but the
> results using APB give me no differences (all about 25% efficiency).
>
> I have the feeling that I should trust APB more than SE. I have
> noticed that, when using SE, the higher the difference in intensity
> between YFP and CFP, the higher the FRET efficiency.
>
> Has anyone had a similar experience? Which method of analysis should I trust?
>
> Regards,
> Pablo
>
> --
> Pablo German
> PhD Candidate
>
> Plant and Food Research
> Private Bag 92169
> Auckland Mail Centre
> Auckland 1142
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>