Spectral bleedthrough (SBT) ratio is dependent on the level of fluorescence intensity in confocal imaging. Precision Forster resonance energy transfer (FRET) algorithm corrects SBT ratio according to fluorescence intensity and avoids over- or under-estimation of SBT ratio. In this letter, we propose a new method to accurately measure the FRET efficiency of FRET plasmid in single living cells by combining the calculation of SBT in precision FRET algorithm with E-FRET formulae. We also use this me
