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 -phosphoc holine (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 FCS. D(T) values at approximately 25 degrees C ranged from approximately 3 x 10(-8) cm(2)/s in the fluid phase, to approximately 2 x 10(-9) cm(2)/s in high-cholesterol-content phases, to approximately 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.
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.
J
J
Korlach
P
P
Schwille
W W
WW
Webb
G W
GW
Feigenson
D002232:Carbocyanines; D002784:Cholesterol / Q000737:chemistry; D004058:Diffusion; D005456:Fluorescent Dyes; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D018613:Microscopy, Confocal; D010743:Phospholipids / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D002232:Carbocyanines; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008081:Liposomes; D010743:Phospholipids; D002784:Cholesterol