Recently we upgraded our Zeiss cell observer D1 for the real time imaging experiment with ratiometric probes and FRET probes. And for the calculation of the "real" FRET signal, the Axiovision provided several formulas which of course are based on different principles, but all of them need an input of "correction factor" from calibration. I learned that for the calibration of FRET probes, in principle I have to measure, for example with the CFP/YFP pair, the overlapping part of CFP signal to the YFP channel and the false signal from the YFP excitation, and also the signal from a paired CFP/YFP without FRET. But I did not find any detailed protocol for this calibration, unfortunately. The question is, of course, how to do it? And do I need some special equipment for this purpose?
Thanks for your attention!
Hello,
If understand your question, you need to measure a couple of signals to do this correction.
1-You need to measure the emission of the acceptor in the absence of the donor (you can use a molecular construct that is labeled with the aceptor only). And measure the emission using the exact laser and detection arrangement that you will use in your fret measurements. This intensity needs to be subtracted from any acceptor emission prior to calculation of the FRET values.
2-You need to know the collection efficiencies of the two channels
3-You need to measure the dark signal, some times called backgound intensity on each channel.
You need to know the quantum yield of the donor and that for the acceptor.
Once you have these parameters you can fairly well correct the data. Please take a look at equations 1-6 in the following article to see if this is of any help:
J. L. Fiore, Hodak, J. H. , Piestert, O. , Downey, C. D. , and Nesbitt, D. J. , “Monovalent and Divalent Promoted GAAA Tetraloop-Receptor Tertiary Interactions from Freely Diffusing Single-Molecule Studies”, Biophysical Journal, vol. 95, pp. 3892 - 3905, 2008.
Jose Hodak
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