Wide-Field Time Resolved Anisotropy for In-Situ Lipid Phase Dynamics

Abstract

We have developed a new time-resolved fluorescence platform which enables us to follow the molecular orientation and dynamics of a lipid monolayer at the air - water interface. Confocal microscopy is limited in its ability to characterize dynamic orientation changes within cellular membranes. By implementing an all reflective Cassegrain objective we minimize dispersion while eliminating the restriction of collinear excitation. This enables us to unambiguously identify fluorescence probe orientation and dynamic freedom within our membrane model, with the highest available temporal resolution, and without the restriction imposed by a supporting substrate. We investigate miscibility transition of ternary lipid mixture, DPPC / DOPC/ Cholesterol, using a combination of fluorescence imaging and time-resolved fluorescence anisotropy. The technique affords unprecedented dynamic characterization for lipid orientation, self-assembly, and dynamic freedom as the monolayer is forced from the liquid to the gel phase. We demonstrate the novelty and applicability of this device by contrasting the time-resolved fluorescence signal of three different lipid probes: 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC), 5-butyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-nonanoic acid (BIODIPY), and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) which show dramatically different orientation and dynamic freedom when bound to the lipid layer, over a range of lipid phases. Using this technique we can resolve highly dynamic processes such as the insertion of peptide and proteins into the lipid membrane.