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-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.

Figure 1

The principle of lipid phase identification, showing confocal images at the equator of GUVs at a phospholipid/cholesterol composition yielding (1A) a single fluid phase (DLPC/DPPC = 1/0), and (1B) ordered-fluid two-phase coexistence (DLPC/DPPC = 0.60/0.40). In each figure, DiI-C₂₀ fluorescence is shown in the upper left small image and Bodipy-PC fluorescence in the upper right. Thus, the merged large lower image shows spatially ordered phase regions in red (DiI-C₂₀ fluorescence) and fluid phase in green (Bodipy-PC fluorescence). The asphericity of these GUVs indicates lack of osmotic stress. A small adherent vesicle is visible in 1B and in several subsequent images. (Bars = 10 μm.) (2A–2E) Visualization of phase separation in the binary lipid mixture of DLPC/DPPC. The images show a progression of increasing DPPC concentration relative to DLPC at DLPC/DPPC values: 1/0 (2A), 0.80/0.20 (2B), 0.60/0.40 (2C), 0.40/0.60 (2D), and 0.20/0.80 (2E). Note that the vesicle shown in 2D is not unilamellar, but instead consists of two bilayers which are very close to each other. Image 2D shows two concentric GUVs, chosen to demonstrate the principle of superposition of phase domains in apposing monolayers (see text). No GUVs were formed in pure DPPC; apparently some fluid phase must be present for successful preparations of GUVs by this method. The circular rings of contrast in these and subsequent images are due to nonuniform axial stepping between confocal images and do not indicate compositional inhomogeneities. (Bars = 10 μm.) (3A and 3B) Influence of cholesterol on the two-phase region. GUVs at a constant ratio of DLPC/DPPC = 0.50/0.50 were prepared with increasing cholesterol concentrations of 0 (3A) and 5 mol % (3B). For cholesterol ≥10 mol %, images were identical in appearance to 2A and 2B. For explanation, see text. (Bars = 10 μm.)

Figure 2

Diffusion properties in laterally ordered and fluid phases in the absence of cholesterol, corresponding to Fig. 2. FCS autocorrelation curves were obtained for samples of composition DLPC/DPPC (□ = 1/0, ○ = 0.60/0.40, and ▵ = 0.20/0.80). Solid curves are data-fitting curves for diffusion theory from which D_T values were determined. In this and all subsequent figures, correlation amplitudes are normalized to 1 by multiplying G(τ) with the average number of fluorescent molecules in the focal area 〈N〉 to compare the shapes of the curves for different compositions.

Figure 3

The binary lipid mixture DLPC/cholesterol exhibits a continuous change in diffusion coefficient. (A) Autocorrelation curves at increasing cholesterol concentration are shown (from left to right) for the compositions of DLPC with 0, 15, 30, 45, and 60 mol % cholesterol. Each autocorrelation curve represents the average of five separate vesicles measured. (B) Average diffusion coefficients determined from the autocorrelation curves in A. The error bars correspond to the entire range of diffusion coefficients obtained from the individual FCS measurements.

Figure 4

FCS autocorrelation curves corresponding to the cholesterol compositions shown in Fig. 3, at constant DLPC/DPPC = 0.50/0.50. Cholesterol concentrations: red indicates 0, green indicates 5, blue indicates 10, and black indicates 15 mol %. For explanation, see text.