Liquid domains in vesicles investigated by NMR and fluorescence microscopy

We use H-2-NMR, H-1-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (much greater than160 nm) phase separation into liquid-ordered (L-o) and liquid-crystalline (L-alpha) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed H-2-NMR spectra, we quantitatively determine that the L-o phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both H-2-NMR observations and a previously published ternary phase diagram. H-2- and H-1-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer- scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller (similar to80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, T-low. A transition at T-low is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting H-2-NMR experiments in similar membranes.