Finite Phase-separation FRET II: Determination of domain size from simulated data

We have developed a new model to describe FRET efficiency (E FRET) between freely-diffusing membrane probes in phase-separated bilayers (Finite Phase-separation FRET, or FP-FRET), that in principle applies to any system where phase domain dimensions are larger than ~. Here we use Monte Carlo techniques to simulate E FRET for a range of probe partitioning behaviors and domain sizes, and then fit the simulated data to the FP-FRET model to recover simulation parameters. We find that FP-FRET can determine domain size to within 5% of simulated values for domain diameters up to ~ 5 , and to within 15% for diameters up to ~ 20. We also investigated the performance of the model in cases where specific model assumptions are not valid.