Characterization of Lipid Bilayer Phases by Confocal Microscopy and Fluorescence Correlation Spectroscopy
Abstract
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× 10-8 cm2/s in the fluid phase, to ~ 2× 10-9 cm2/s in high-cholesterol-content phases, to ~ 2× 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.
- Publication:
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Proceedings of the National Academy of Science
- Pub Date:
- July 1999
- DOI:
- 10.1073/pnas.96.15.8461
- Bibcode:
- 1999PNAS...96.8461K