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 (l(d)), solid-ordered (s(o)) and liquid-ordered (l(o)) 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 approximately 27 and approximately 22 mol% for l(d)-l(o) and s(o)-l(o) 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.