Dear Grace! How do you want to measure FRET? Using the photobleaching FRET approach or the intensity based approach? For the intensity based approach you need an additional excitation where preferentially only the YFP is excited (514 nm). Then you can monitor three intensitites for each cell: 458 475 donor intensity (CFP) 458 540 energy transfer intensity (mostly but some CFP and YFP intensity as well) 514 540 acceptor intensity (YFP) You have to measure these three intensities on unlabeled cell, cell labeled only with donor, cells labeled only with acceptor and on cells labeled both with donor and acceptor. From this experimental setup you can estimate the transfer efficiency using various equations for correcting the overlapping fluorescence intensities. You have to see how much is the autofluorescence and how fast is the photobleaching. If you can correct for these two problems the FRET measurement would be easy. If you need specific help please do not hesitate to contact me. For references I can recommend: Nagy, P., Vamosi, G., Bodnar, A., Lockett, S. J. and Szollosi, J.: Intensity-based energy transfer measurements in digital imaging microscopy. Eur. Biophys. J., 1998. 27, 377-389 Szollosi, J., Damjanovich, S. and Mátyus, L.: Application of fluorescence resonance energy transfer in the clinical laboratory: Routine and research. Cytometry, 1998. 34, 159-179. Szollosi, J., Damjanovich, S. and Mátyus, L.: Principles of resonance energy transfer. In: Current Protocols in Cytometry (eds. J. Paul Robinson et al.) John Wiley & Sons, Inc. New York, 1999. pp. 1.12.1-1.12.13 Best regards Janos At 05:13 PM 7/14/00 -0700, you wrote: >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 Janos Szollosi University of Debrecen Medical and Health Science Center Faculty of Medicine Department of Biophysics and Cell Biology P.O.Box 39, Nagyerdei krt. 98 H-4012 Debrecen Hungary Phone/Fax: (36) (52) 412-623 E-mail: szollo@jaguar.dote.hu
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