Can anyone help about using confocal imaging in connection with FRET with CFP and TFP fluorophores? Briefly, we've got a confocal microscope with Leica software. I'm transfecting or cotransfecting cells with constructs encoding nuclear receptors fused with either CFP or YFP. I harvest the transfected cells, and excite them with 458 nm (CFP excitation) and want to detect FRET by either a reduction in CFP emission or by an increase in YFP emission. When I excite them at 458, I can scan the emission images of the individual cells and then use the LEICA software to quantify how intense is the emission of interest. With the software, I can scan the emission across 465 to 600, to capture the effect of FRET on either the CFP emission max or the YFP emission max. Now, how can I use this set up to tell, on a cell by cell analysis basis, whether FRET is occurring? I've tried a number of approaches, and want to run my ideas by someone with experience in this area. Can anyone help? Thanks Dr. Grace Jones School of Biological Sciences University of Kentucky Lexington, KY 40506
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