Quantitative characterization of coexisting phases in DOPC/DPPC/cholesterol mixtures: Comparing confocal fluorescence microscopy and deuterium nuclear magnetic resonance

The differential miscibility of membrane lipids is thought to be the basis for the formation of dynamic microdomain assemblies in cell membranes known as membrane rafts. Because of their relevance to the existence of rafts, there has been much interest in recent years in model membrane systems that display coexisting liquid ordered (lo) and liquid disordered phases (ld), such as the ternary mixture composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol. Carefully equilibrating the samples at well controlled temperatures allows us to use a quantitative confocal fluorescence microscopy approach to measure the area fractions of coexisting fluid phases in DOPC/DPPC/cholesterol mixtures. We can then compare the behaviour of a large population of unilamellar vesicles with the domain fractions deduced from 2H NMR experiments. The fluorescence results are established for the first time to be in quantitative agreement with those obtained using 2H NMR spectroscopy within the two phase region of the phase diagram. We are also able to describe fine details of the phase separation and the approach to equilibrium not previously reported, in particular the existence of small spots of lo phase at temperatures higher than that at which the samples display domain fluctuations. A better understanding of coexisting fluid phases in model systems will assist in interpreting the behaviour of rafts in more complex biological membranes.