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Abstract
We use (2)H-NMR, (1)H-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 (>160 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 (2)H-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 (2)H-NMR observations and a previously published ternary phase diagram. (2)H-
and (1)H-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 ( approximately 80 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 (2)H-NMR experiments in similar membranes.