Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy

Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy

^*Section of Biochemistry, Molecular and Cell Biology and ^†School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853

Contributed by Watt W. Webb

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, D _T, of the DiI-C₂₀ was measured by FCS. D _T 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 D _T in a region of two-phase coexistence on a single vesicle.

Footnotes

↵ ‡ To whom reprint requests should be addressed at: Section of Biochemistry, Molecular, and Cell Biology, 201 Biotechnology Building, Cornell University, Ithaca, NY 14853. e-mail: gwf3{at}cornell.edu.
↵ § The term “spatially ordered phases” (often called “solid” or “gel”) denotes two-dimensional spatial order in lipid bilayers that corresponds to the long-range order of three-dimensional smectic liquid crystal hydrated phases of smectic B type, and the two-dimensional fluid phases (often called “liquid-crystalline” and “liquid-ordered”) that correspond to hydrated smectic A type, in which short-range order may also occur (20, 21).
ABBREVIATIONS:

Bodipy-PC,

2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine;

DiI-C20,

1,1′-dieicosanyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate;

DLPC,

dilauroyl phosphatidylcholine (1,2-dilauroyl-sn-glycero-3-phosphocholine);

DPPC,

dipalmitoyl phosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine);

FCS,

fluorescence correlation spectroscopy;

GUV,

giant unilamellar vesicle;

POPS,

1-palmitoyl 2-oleoyl phosphatidylserine [1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-l-serine)] (sodium salt)