A ternary phase diagram is proposed for the hydrated lamellar lipid mixture dipalmitoylphosphatidylcholine/dilauroylphosphatidylcholine/cholesterol (DPPC/DLPC/cholesterol) at room temperature. The entire composition space has been thoroughly mapped by complementary experimental techniques, revealing interesting phase behavior that has not been previously described. Confocal fluorescence microscopy shows a regime of coexisting DPPC-rich ordered and DLPC-rich fluid lamellar phases, having an upper boundary at apparently constant cholesterol mole fraction chi(chol) approximately 0.16. Fluorescence resonance energy transfer experiments confirm the identification and extent of this two-phase regime and, furthermore, reveal a 1-phase regime between chi(chol) approximately 0.16 and 0.25, consisting of ordered and fluid nanoscopic domains. Dipyrene-PC excimer/monomer measurements confirm the new regime between chi(chol) approximately 0.16 and 0.25 and also show that rigidly ordered phases seem to disappear around chi(chol) approximately 0.25. This study should be considered as a step toward a more complete understanding of lateral heterogeneity within biomembranes. Cholesterol may play a role in domain separation on the nanometer scale.

In any lipid bilayer membrane, there is an upper limit on the cholesterol concentration that can be accommodated within the bilayer structure; excess cholesterol will precipitate as crystals of pure cholesterol monohydrate. This cholesterol solubility limit is a well-defined quantity. It is a first-order phase boundary in the phospholipid/cholesterol phase diagram. There are many different solubility limits in the literature, but no clear picture has emerged that can unify the disparate results. We have studied the effects that different sample preparation methods can have on the apparent experimental solubility limit. We find that artifactual demixing of cholesterol can occur during conventional sample preparation and that this demixed cholesterol may produce artifactual cholesterol crystals. Therefore, phospholipid/cholesterol suspensions which are prepared by conventional methods may manifest variable, falsely low cholesterol solubility limits. We have developed two novel preparative methods which are specifically designed to prevent demixing during sample preparation. For detection of the cholesterol crystals, X-ray diffraction has proven to be quantitative and highly sensitive. Experiments based on these methods yield reproducible and precise cholesterol solubility limits: 66 mol% for phosphatidylcholine (PC) bilayers and 51 mol% for phosphatidylethanolamine (PE) bilayers. We present evidence that these are true, equilibrium values. In contrast to the dramatic headgroup effect (PC vs. PE), acyl chain variations had no effect on the cholesterol solubility limit in four different PC/cholesterol mixtures.

During the preparation of multi-component model membranes, a primary consideration is that compositional homogeneity should prevail throughout the suspension. Some conventional sample preparation methods pass the lipid mixture through an intermediary, solvent-free state. This is an ordered, solid state and may favor the demixing of membrane components. A new preparative method has been developed which is specifically designed to avoid this intermediary state. This novel strategy is called rapid solvent exchange (RSE) and entails the direct transfer of lipid mixtures between organic solvent and aqueous buffer. RSE liposomes require no more than a minute to prepare and manifest considerable entrapment volumes with a high fraction of external surface area. In phospholipid/cholesterol mixtures of high cholesterol content, suspensions prepared by more conventional methods reveal evidence of artifactual demixing, whereas samples prepared by rapid solvent exchange do not. The principles which may lead to artifactual demixing during conventional sample preparation are discussed.

Phase diagrams of lipid mixtures can show several different regions of phase coexistence, which include liquid-disordered, liquid-ordered, and gel phases. Some phase regions are small, and some have sharp boundaries. The identity of the phases, their location in composition space, and the nature of the transitions between the phases are important for understanding the behavior of lipid mixtures. High fidelity phase boundary detection requires high compositional resolution, on the order of 2% compositional increments. Sample artifacts, especially the precipitation of crystals of anhydrous cholesterol, can occur at higher cholesterol concentrations unless precautions are taken. Fluorescence resonance energy transfer (FRET) can be used quantitatively to find the phase boundaries and even partition coefficients of the dyes between coexisting phases, but only if data are properly corrected for non-FRET contributions. Self-quenching of the dye fluorescence can be significant, distorting the data at dye concentrations that intuitively might be considered acceptable. Even more simple than FRET experiments, measurements of single-dye fluorescence can be used to find phase boundaries. Both FRET and single-dye fluorescence readily detect the formation of phase domains that are much smaller than the wavelength of light, i.e. "nanoscopic" domains.

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We modified the original design for a rapid solvent exchange (RSE) device with the intent of making the RSE method (i) more efficient and (ii) easier to adopt and implement. Our modifications improved solvent-removal kinetics by a factor of 2, while reducing sample-prep time by a factor of 3. In this paper, we develop the kinetic model that informed the device revision and we address several RSE parameters that have not yet been discussed in the literature. We also provide detailed mechanical drawings and present solvent-removal efficiency data that confirm the improved performance of our device.

For some time now, we have been using a fluorescence resonance energy transfer (FRET)-based strategy to conduct high-resolution studies of phase behavior in ternary lipid-raft membrane mixtures. Our FRET experiments can be carried out on ordinary, polydisperse multilamellar vesicle suspensions, so we are able to prepare our samples according to a procedure that was designed specifically to guard against artifactual phase separation. In some respects (i.e., the number and nature of two-phase regions observed), our phase diagrams are consistent with those in previously published reports. However, in other respects (i.e., overall size of miscibility gaps, phase boundary locations and their dependence on temperature), there are clear differences. Here, we present FRET data taken in dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine/cholesterol (DOPC/DPPC/Chol) mixtures at 25.0, 35.0, and 45.0 degrees C. Comparisons between our results and previously reported phase boundaries suggest that lipid-raft mixtures may be particularly susceptible to demixing effects during sample preparation.

A general strategy is proposed for determining the very low aqueous solubility limits of bilayer-forming phospholipids. The strategy exploits the inherent surface activity of phospholipids and has been termed EDSB, which stands for Equilibrium Distribution between Surface and Bulk phases. In this report, EDSB has been used to determine the critical bilayer concentration of dilauroylphosphatidylycholine (DLPC), a short-chain bilayer-forming phospholipid. At room temperature in neutral pH buffer, CBC(DLPC) = 2.5 x 10(-)(8) M. Using a mole fraction concentration scale, this corresponds to a standard-state free energy change of -12.8 kcal/mol for DLPC bilayer membrane formation.