Fig. 2. Sensitized acceptor emission FRET imaging confirms the distinct FRET levels on the plasma membrane. Left to right: FRET-channel images, FRET-efficiency images and dependencies of the FRET efficiency of indicated FRET pairs on the acceptor intensity, corresponding to approximately 500-2000 acceptors/µm². Each datapoint was calculated on one region of interest on plasma membranes with donor mole fractions of x_D=0.5±0.17 were analysed. Note that for mCFP-tH/mCit-tH the FRET originating from putative internal membranes show similar values as that originating from the plasma membrane. Consistent with the negligible FRET from soluble fluorophores at these concentrations (supplementary material Fig. S2E), FRET from the nucleus is close to zero for mCFP-tR/mCit-tR. Bars, 5 µm.