An empirical quantitative fluorescence resonance energy transfer method for multiple acceptors based on partial acceptor photobleaching

We present an empirical quantitative fluorescence resonance energy transfer (FRET) method based on partial acceptor photobleaching, termed emp-PbFRET. This method can be used to determine the FRET efficiency of construct with multiple acceptors by measuring the degree of acceptor photobleaching (x) and the donor intensity before and after photobleaching. We validated the emp-PbFRET method using the constructs consisting one Cerulean and two (VCV) or three (VCVV) Venus. The FRET efficiencies of VCV and VCVV in living cells obtained by emp-PbFRET method under different x (10%-60%) were consistent with those obtained by fluorescence lifetime imaging and spectral imaging methods