Dear Grace, I am in the process to figure out which "GFP" pairs are the best to perform energy transfer experiments. I cannot make a decision yet, but right now I prefer the CFP/YFP pair. Let me comment your letter using ALL CAPITAL WITHIN YOUR TEXT. At 08:10 PM 1/28/00 -0800, you wrote: > >Does anyone have experience doing BFP/GFP or CFP/GFP FRET in transfected >cells using a confocal microscope with LEICA software? I HAVE NO EXPERIENCE WITH THIS SETUP. We'd like to get >that geared up, but in our naivity have a basic conceptual problem. If we >excite the BFP with its UV excitation, then we imagine its emission ought >excite GFP that is close enough by, and then we can monitor emission of the >GFP as the indicator of energy transfer. (Why not alternatively monitor >quenching of the BFP emission by GFP as the indicator of FRET?). BECAUSE IF YOU WANT TO DETERMINE THE FRET EFFICIENCY ON A CELL-BY-CELL OR IN THE MICROSCOPE ON A PIXEL-BY-PIXEL BASIS YOU CANNOT USE THIS APPROACH. HOW CAN YOU TELL FROM THE FLUORESCENCE INTENSITY OF ONE PIXEL (CELL) THAT THIS INTENSITY IS SMALL BECAUSE OF THE FRET OR BECAUSE AMOUNT OF THE DYE SMALLER IN THE PIXEL (CELL) DUE TO BIOLOGICAL VARIATION IN EXPRESSION, OR IN TRANSFECTION. However, >the BFP emission itself does have an overlap into the GFP emission, so that >has to be subtracted out of the 'GFP emission.' THAT IS RIGHT. The problem is how to >estimate the amount of BFP emission that overlaps into the GFP emission >range. We can just use some BFP-only transfected cells, since there is >great variation from one cell to the next on the intensity of the BFP >emission in BFP-only transfected cells. Can we do something alternative, >such as scan the shape of the BFP emissionin BFP-only transfected cells, >establish an empirical distribution of BFP emission vs. wavelength, and >then derive a statistic on, given a certain peak value of BFP emission in >BFP/GFP cotransfected cells, we know that x-amount of BFP emission value >must be happening in the region of emission overlap between BFP and GFP? THAT IS THE WAY TO DO. USE BFP ONLY SAMPLE TAKE TWO IMAGES, ONE IN THE BFP EMISSION ONE IN THE GFP EMISSION. AFTER BACKGROUND AND AUTOFLUORESCENCE CORRECTION THE RATIOS OF TWO INTENSITIES AT VARIOUS PIXELS SHOULD BE THE SAME. THIS "CONSTANT" CAN BE USED TO MAKE THE CORRECTION ON A PIXEL BY PIXEL BASIS. >That calculated x-amount can then be substrated from the observed 'GFP >(+BFP tail) emission'? Help! > YOU FORGOT TO MENTION THE FLUORESCENCE INTENSITY OF THE GFP, WHICH CAN BE EXCITED AT THE UV EXCITATION. THIS CAN BE CORRECTED SIMILARLY TO THE BFP CONTRIBUTION. SO, DO NOT FORGET THE "FRET SIGNNAL" WILL HAVE SOME INTENSITY COMING FROM THE BFP EMISSION OVERLAP, AND SOME INTENSITY COMING FROM THE GFP EXCITATION OVERLAP. >Is it feasible to use a CFP/GFP set? > I PREFER THE CPF/YFP SET. IF YOU ARE REALLY INTO THE INTENSITY BASED FRET MEASUREMENTS IN DIGITAL IMAGING MICROSCOPY, YOU CAN CONSULT OUR PAPER: NAGY, P., VAMOSI, G., BODNAR, A., LOCKETT, S.J., SZOLLOSI, J.: INTENSITY-BASED ENERGY TRANSFER MEASUREMENTS IN DIGITAL IMAGING MICROSCOPY. EUR. BIOPHYS. J. 27:377-389, 1998. >Grace Jones > IF YOU HAVE QUESTIONS, DO NOT HESITATE TO CONTACT ME! REGARDS JANOS SZOLLOSI Janos Szollosi Department of Biophysics and Cell Biology Faculty of Medicine University of Debrecen Nagyerdei krt. 98, P.O.Box 39, H-4012 Debrecen, HUNGARY Fax/Phone: (36) (52) 412-623 e-mail: szollo@jaguar.dote.hu
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