Molecular collisions or resonance energy transfer in lipid vesicles? A methodology to tackle this question

•Collisional quenching in membrane depends on fluorophore lifetime and its diffusion.•Even visually small absorption/emission spectral overlap can reveal significant FRET.•FRET and not collisional quenching occurs between Laurdan and 2-TSC in DMPC membrane.•FRET results confirm localization of Laurdan and 2-TSC close to the bilayer interface.•Distance distribution indicates membrane FRET pairs at the same and opposite layers.