A Förster resonance energy transfer (FRET) approach for enhancing fluorescence contrast in phase-separated membranes

The partitioning of the dye-labeled lipid probe, NBD-DHPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanol- amine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt)), was examined by fluorescence microscopy in phase- separated lipid bilayers with mixtures of coexisting liquid-ordered and fluid phases. This probe shows slightly higher fluo- rescence intensity in the ordered domains but undergoes a contrast reversal to give a more strongly fluorescent fluid phase in the presence of >0.2% Texas red-DHPE (TR-DHPE). The change in contrast is shown to result from Forster resonance energy transfer between the NBD donor and TR acceptor in the fluid phase, which has a TR concentration that is approxi- mately 3 times higher than in the domains. An alternate approach using a nitroxide-substituted lipid that partitions into the fluid phase as a quencher, was also examined as a means to enhance the contrast; however, the quencher modified the be- haviour of the bilayer. The energy transfer method for enhancing the contrast between ordered and fluid phases was used to examine the morphology of enzyme-treated bilayers.