Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy
AUTOR(ES)
Korlach, Jonas
FONTE
The National Academy of Sciences
RESUMO
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°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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=17538Documentos Relacionados
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