Phasor based analysis of FRET images recorded using spectrally resolved lifetime imaging

The combined analysis of spectral and lifetime images has the potential to provide more accurate and more detailed information about Förster resonance energy transfer (FRET). We have developed a novel FRET analysis method to analyze images recorded by multispectral lifetime imaging. The new method is based on a phasor approach and facilitates the simultaneous analysis of decay kinetics of donor and acceptor molecules. The method is applicable to both molecules that exhibit a monoexponential decay and a biexponential decay. As an example we show the possibility of extracting the energy transfer efficiency and the fraction of interacting molecules even in the presence of noninteracting molecules. The reliability of the method is investigated by comparing it with conventional FRETFLIM analyses. We show that, with the same number of detected photons, the spectrally resolved phasor approach provides higher accuracy than other analysis methods; the confidence interval is improved and the FRET efficiency is closer to the real value.