Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy

We report the application of confocal imaging and fluorescence correlation spectroscopy (FCS) to characterize chemically well-defined lipid bilayer mo dels for biomembranes. Giant unilamellar vesicles of dilauroyl phosphatidyl choline/dipalmitoyl phosphatidylcholine (DLPC/ DPPC)/cholesterol were image d by confocal fluorescence microscopy with two fluorescent probes, 1,1'-die icosanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C-20) and 2 -(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-he xadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC), Phase separation was vi sualized by differential probe partition into the coexisting phases. Three- dimensional image reconstructions of confocal z-scans through giant unilame llar vesicles reveal the anisotropic morphology of coexisting phase domains on the surface of these vesicles with full two-dimensional resolution. Thi s method demonstrates by direct visualization the exact superposition of li ke phase domains in apposing monolayers, thus answering a long-standing ope n question. Cholesterol was found to induce a marked change in the phase bo undary shapes of the coexisting phase domains. To further characterize the phases, the translational diffusion coefficient, D-T, of the DiI-C-20 was m easured by BCS. DT values at similar to 25 degrees C ranged from similar to 3 x 10(-8) cm(2)/s in the fluid phase, to similar to 2 x 10(-9) cm(2)/s in high-cholesterol-content phases, to similar to 2 x 10(-10) cm(2)/s in the spatially ordered phases that coexist with fluid phases, In favorable cases , FCS could distinguish two different values of D-T in a region of two-phas e coexistence on a single vesicle.