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The partitioning of the dye-labeled lipid probe, NBD-DHPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-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 fluorescence 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 F\uf6rster resonance energy transfer between the NBD donor and TR acceptor in the fluid phase, which has a TR concentration that is approximately 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 behaviour 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.Faisant appel \ue0 la microscopie de fluorescence, on a \ue9tudi\ue9 la partition de la sonde de lipide marqu\ue9e d'un colorant, la 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl, NBD-DHPE, dans des bicouches de lipides s\ue9par\ue9s par phases coexistentes de fluide ou de liquide ordonn\ue9. Cette sonde pr\ue9sente une intensit\ue9 de fluorescence qui n'est que l\ue9g\ue8rement sup\ue9rieure dans le domaine ordonn\ue9, elle subit un renversement de contraste qui conduit \ue0 une phase fluide beaucoup plus fluorescente en pr\ue9sence de plus de 0,2 % de Texas Rouge-DHPE (TR-DHPE). On a d\ue9montr\ue9 que le changement de contraste r\ue9sulte d'un transfert d'\ue9nergie de r\ue9sonance de F\uf6rster entre le donneur NBD et l'accepteur TR dans la phase fluide qui comporte une concentration de TR qui est approximativement 3 fois plus \ue9lev\ue9e que dans les autres domaines. Dans le but d'obtenir une augmentation du contraste, on a fait appel \ue0 une autre approche impliquant un lipide substitu\ue9 par un groupe nitroxyde qui se partitionne entre la phase fluide comme d\ue9sactivant; toutefois, le d\ue9sactivant a modifi\ue9 le comportement de la bicouche. La m\ue9thode d'\ue9nergie de transfert pour augmenter le contraste entre les phases fluides et ordonn\ue9es a donc \ue9t\ue9 utilis\ue9e pour examiner la morphologie des bicouches trait\ue9es par un enzyme.Peer reviewed: YesNRC publication: Ye

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