Lux-FRET method for quantitive analysis of FRET signal
A novel method for spectral analysis of FRET-signals in living cells will be presented. The method allows for the determination of exact stoichiometry of interacting molecules as well as apparent FRET effi ciency from two-wavelength measurement. Further considerations allow us to predict the infl uence of incomplete labeling of interacting partners. The method is based on information obtained from the full fl uorescence emission spectra. In addition our analysis explicitly takes into account the contributions from simultaneously present free donors, acceptors and FRET-pairs, as well as the effects of non-functional fl uorophores. The determination of apparent FRET effi ciency in our method does not require, as in some other methods, acceptor photobleaching experiments, fl uorescence lifetime measurements nor cell fi xation. The method presented here can be directly applied to individual pixels of a fl uorescence image in fl uorescence microscopy image analysis.