Fluorescence resonance energy transfer between DPH and Nile Red in a lipid bilayer

FRET between the well-known membrane binding probes DPH (donor) and Nile Red (acceptor) is investigated in a phosphatidylserine lipid bilayer. A ∼80% FRET efficiency was observed from the steady state experiments for a DPH:Nile Red:lipid molar ratio of 1:2:190, suggesting an efficient FRET process. Time resolved experiments however, provided a lower (∼50%) FRET efficiency. The emission of Nile Red was observed to rise with a time constant of 0.8–0.4 ns only when DPH is selectively excited. This time constant is ascribed due to FRET from which an average donor–acceptor distance of 27 Å is obtained for a DPH:Nile RED:lipid molar ratio of 1:1:190.