Optimization of FRET algorithms for sensitized-emission FRET detection

Sensitized-emission fluorescence resonance energy transfer（FRET） detection method based on threechannel fluorescence microscopy is widely used.Several FRET algorithms,such as N_FRET,FRETn,FR, FRET_R,and F^C/Df,are developed recently to quantitatively gauge and compare FRET signals between different experimental groups.However,the algorithms are difficult to choose and interpret.In this letter, we optimize the suitable yellow fluorescent protein（YFP） to cyan fluorescent protein（CFP） concentration ratio range for the above FRET algorithms.We also test the effect of YFP-to-CFP concentration ratio on the calculated energy transfer efficiency E and use the optimized FRET algorithms in the analysis of fas-associated protein with death domain（FADD） self-association directly in living cells.