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

Author:

Carter Ramirez Daniel M.12,Ding Jason12,Guan Jack12,Vobornik Dusan12,Carnini Anna12,Ogilvie William W.12,Jakubek Zygmunt J.12,Johnston Linda J.12

Affiliation:

1. Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, K1A 0R6 ON, Canada.

2. Department of Chemistry, University of Ottawa, Ottawa, ON K1N 6N5, Canada

Abstract

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 Förster 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.

Publisher

Canadian Science Publishing

Subject

Organic Chemistry,General Chemistry,Catalysis

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