Intramolecular FRET Efficiency Measures for Time-Lapse Fluorescence Microscopy Images

Here we investigate quantitative measures of Forster resonance energy transfer (FRET) efficiency that can be used to quantify protein-protein interactions using fluorescence microscopy images of living cells. We adopt a joint intensity space approach and develop a parametric shot noise model to estimate the uncertainty of FRET efficiency on a per pixel basis. We evaluate our metrics rigorously by simulating photon emission events corresponding to typical conditions and demonstrate advantages of our metrics over the conventional ratiometric one. In particular, our measure is linear, normalised and has greater tolerance to low SNR characteristic of FRET fluorescence microscopy images.