Dear John,

Though I cannot provide a reference, I think it should be well possible
to measure homo-FRET using FLIM; FRET will increase the probability to
de-populate the donor exited state and thus shorten its lifetime, so a
part of the GFP should show reduced lifetimes in case of homo-FRET. The
sensitivity of the approach will be reduced compared to hetero-FRET,
because you cannot spectrally restrict your observation to the
population of the donor molecules.

Just out of curiosity: How do you distinguish between the anisotropy due
to FRET from the anisotropy resulting from the proteins being
rotationally restricted by the membrane? 
Jahnig, F. (1979) Structural order of lipids and proteins in membranes:
Evaluation of fluorescence anisotropy data. Proc. Nati. Acad. Sci. USA
Vol. 76, No. 12, pp. 6361-6365. 

Best, jens.

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of John Oreopoulos
Sent: Wednesday, December 17, 2008 6:13 PM
To: [log in to unmask]
Subject: detection of homoFRET with FLIM?

I have a question for all of the FRET and/or FLIM experts out there  
in the world. I have read that FLIM can be used to spatially detect  
heteroFRET between CFP and YFP labeled proteins by looking for a drop  
in the donor fluorescence lifetime, but I'm wondering what happens to  
the fluorescence lifetime in the case of homoFRET between two YFP  
labeled proteins. We have a YFP labeled membrane protein that tends  
to form dimers with itself in some areas of the cell membrane. We  
imaged the polarization anisotropy of the fluorescence to confirm the  
presence of homoFRET with a spatially heterogeneous distribution on  
the membrane. I thought it would be great to use another sensitive  
FRET microscopy method like FLIM to verify this observation. Can  
anyone enlighten me on this topic or direct me to some relevant  
literature references? Thank you!

John Oreopoulos