Direct measurement of phase coexistence in DPPC/cholesterol vesicles using Raman spectroscopy

The phase behavior of bilayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol has been studied using Raman spectroscopy. It is observed that the shape of the cholesterol vibrational spectrum in lipid–cholesterol binary mixtures does not vary significantly with either the cholesterol concentration or the temperature. This permits determination of the lipid vibrational signatures of the liquid-disordered (ld), solid-ordered (so) and liquid-ordered (lo) phases. Within the phase coexistence region, the measured spectra are described very well by a linear combination of the different spectral components, which permits a quantitative analysis of the phase diagram. In contrast to earlier findings, our experiments provide no indication of a phase boundary at low cholesterol concentration. The upper boundary of the phase coexistence region is found at ∼27 and ∼22 mol% for ld–lo and so–lo coexistence region, respectively. Within these phase coexistence regions, the partitioning of cholesterol between the cholesterol-poor and the cholesterol-rich phases is in close agreement with the lever rule.