Acceptor Photobleaching vs. Sensitized Emission FRET results

Posted by Pablo German on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Acceptor-Photobleaching-vs-Sensitized-Emission-FRET-results-tp4828946.html

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
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