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 models for biomembranes. Giant unilamellar vesicles of dilauroyl phosphatidylcholine/dipalmitoyl phosphatidylcholine (DLPC/ DPPC)/cholesterol were imaged by confocal fluorescence microscopy with two fluorescent probes, 1,1'-dieicosanyl-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-hexadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC), Phase separation was visualized by differential probe partition into the coexisting phases. Three-dimensional image reconstructions of confocal z-scans through giant unilamellar vesicles reveal the anisotropic morphology of coexisting phase domains on the surface of these vesicles with full two-dimensional resolution. This method demonstrates by direct visualization the exact superposition of like phase domains in apposing monolayers, thus answering a long-standing open question. Cholesterol was found to induce a marked change in the phase boundary shapes of the coexisting phase domains. To further characterize the phases, the translational diffusion coefficient, D-T, of the DiI-C-20 was measured 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-phase coexistence on a single vesicle.