A linear measure of Förster resonant energy transfer (FRET) efficiency incorporating a shot noise uncertainty model for fluorescence microscopy intensity images

Abstract

We propose a linear measure of the Forster resonant energy transfer (FRET) efficiency derived in joint, donor and acceptor, intensity space and compare it quantitatively with the conventional ratiometric FRET measure normally used for FRET fluorescence microscopy images. Because of the low signal-to-noise characteristic of these images, particularly time-lapse images, we determine a quantitative model of shot noise and combine it with our measure. This provides a single measure of the certainty of FRET events that can be used as a pixel-wise classifier of FRET images. We evaluate our measures quantitatively by simulating photon emission under realistic conditions and demonstrate: linearity with FRET efficiency; less bias to shot noise than the ratio; and high correlation (r = 0.96) between our FRET certainty measure and SNR.