Lipid domains in giant unilamellar vesicles and their correspondence with equilibrium thermodynamic phases: a quantitative fluorescence microscopy imaging approach.

We report a novel analytical procedure to measure the surface areas of coexisting lipid domains in giant unilamellar vesicles (GUVs) based on image processing of 3D fluorescence microscopy data. The procedure involves the segmentation of lipid domains from fluorescent image stacks and reconstruction of 3D domain morphology using active surface models. This method permits the reconstruction of the spherical surface of GUVs and determination of the area fractions of coexisting lipid domains at the level of single vesicles. Obtaining area fractions enables the scrutiny of the lever rule along lipid phase diagram's tie lines and to test whether or not the coexistence of lipid domains in GUVs correspond to equilibrium thermodynamic phases. The analysis was applied to DLPC/DPPC GUVs displaying coexistence of lipid domains. Our results confirm the lever rule, demonstrating that the observed membrane domains correspond to equilibrium thermodynamic phases (i.e., solid ordered and liquid disordered phases). In addition, the fact that the lever rule is validated from 11 to 14 randomly selected GUVs per molar fraction indicates homogeneity in the lipid composition among the explored GUV populations. In conclusion, our study shows that GUVs are reliable model systems to perform equilibrium thermodynamic studies of membranes.

Melting of individual lipid components in binary lipid mixtures studied by FTIR spectroscopy, DSC and Monte Carlo simulations.

Monte Carlo (MC) simulations, Differential Scanning Calorimetry (DSC) and Fourier Transform InfraRed (FTIR) spectroscopy were used to study the melting behavior of individual lipid components in two-component membranes made of DMPC and DSPC. We employed Monte Carlo simulations based on parameters obtained from DSC profiles to simulate the melting of the different lipids as a function of temperature. The simulations show good agreement with the FTIR data recorded for deuterated and non-deuterated lipids, which demonstrates that the information on the differential melting of the individual components is already contained in the calorimetric profiles. In mixtures, both lipids melt over a wide temperature range. As expected, the lipid melting events of the lipid with the lower melting temperature occur on average at lower temperatures. The simulations also yield information on the lateral distribution of the lipids that is neither directly contained in the DSC nor in the FTIR data. In the phase coexistence region, liquid disordered domains are typically richer in the lower-melting-temperature lipid species.

Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures containing cholesterol.

The effect of temperature on the lateral structure of lipid bilayers composed of porcine brain ceramide and 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC), with and without addition of cholesterol, were studied using differential scanning calorimetry, Fourier transformed infrared spectroscopy, atomic force microscopy, and confocal/two-photon excitation fluorescence microscopy (which included LAURDAN generalized polarization function images). A broad gel/fluid phase coexistence temperature regime, characterized by the presence of micrometer-sized gel-phase domains with stripe and flowerlike shapes, was observed for different POPC/ceramide mixtures (up to approximately 25 mol % ceramide). This observed phase coexistence scenario is in contrast to that reported previously for this mixture, where absence of gel/fluid phase coexistence was claimed using bulk LAURDAN generalized polarization (GP) measurements. We demonstrate that this apparent discrepancy (based on the direct comparison between the LAURDAN GP data obtained in the microscope and the fluorometer) disappears when the additive property of the LAURDAN GP function is taken into account to examine the data obtained using bulk fluorescence measurements. Addition of cholesterol to the POPC/ceramide mixtures shows a gradual transition from a gel/fluid to gel/liquid-ordered phase coexistence scenario as indicated by the different experimental techniques used in our experiments. This last result suggests the absence of fluid-ordered/fluid-disordered phase coexistence in the ternary mixtures studied in contrast to that observed at similar molar concentrations with other ceramide-base-containing lipid mixtures (such as POPC/sphingomyelin/cholesterol, which is used as a canonical raft model membrane). Additionally, we observe a critical cholesterol concentration in the ternary mixtures that generates a peculiar lateral pattern characterized by the observation of three distinct regions in the membrane.