Fluorescence Detection of Signs of Sterol Superlattice Formation in Lipid Membranes

There is a significant amount of experimental data, obtained predominantly from fluorescence studies, showing that sterol-containing liposomes can exhibit multiple biphasic changes in membrane properties at specific critical mole fractions of sterol such as 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol%. This can be understood in terms of the sterol regular distribution (e.g., superlattice) model. Here, the authors use excitation generalized polarization of 6-lauroyl-2-dimethylamino-naphthalene fluorescence in fluid 1-palmitoyl-2-oleoyl-sn -glycero-3-phosphocholine/cholesterol unilamellar vesicles to illustrate the experimental procedures and conditions that are required to detect multiple biphasic changes at predicted sterol mole fraction values in liposomal membranes. For this detection, the use of small sterol increments over a wide sterol mole fraction range is essential. Lipid concentration, incubation time, thermal history, and degree of sterol oxidation of liposomal membranes are critical factors. The principles and methodologies described here can be extended to other probes or bioactive molecules, such as enzymes, and can be applied to study sterol lateral organization in multicomponent lipid membranes.