期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1999
卷号：96
期号：15
页码：8461-8466
DOI：10.1073/pnas.96.15.8461
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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-C20) 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, DT, of the DiI-C20 was measured by FCS. DT values at [~]25{degrees}C ranged from [~] 3 x 10-8 cm2/s in the fluid phase, to [~] 2 x 10-9 cm2/s in high-cholesterol-content phases, to [~] 2 x 10-10 cm2/s in the spatially ordered phases that coexist with fluid phases. In favorable cases, FCS could distinguish two different values of DT in a region of two-phase coexistence on a single vesicle.
