Solution synchrotron x-ray diffraction reveals structural details of lipid domains in ternary mixtures

The influence of cholesterol on lipid bilayer structure is significant and the effect of cholesterol on lipid sorting and phase separation in lipid-raft-forming model membrane systems has been well investigated by microscopy methods on giant vesicles. An important consideration however is the influence of fluorescence illumination on the phase state of these lipids and this effect must be carefully minimized. In this paper, we show that synchrotron x-ray scattering on solution lipid mixtures is an effective alternative technique for the identification and characterization of the l_o (liquid ordered) and l_d (liquid disordered) phases. The high intensity of synchrotron x rays allows the observation of up to 5 orders of diffraction from the l_o phase, whereas only two are clearly visible when the l_d phase alone is present. This data can be collected in ∼1 min/sample, allowing rapid generation of phase data. In this paper, we measure the lamellar spacing in both the liquid-ordered and liquid-disordered phases simultaneously, as a function of cholesterol concentration in two different ternary mixtures. We also observe evidence of a third gel-phaselike population at 10–12 mol % cholesterol and determine the thickness of the bilayer for this phase. Importantly we are able to look at phase coexistence in the membrane independent of photoeffects.