We have investigated the phase behavior of dipalmitoylphosphatidylcholine-cholesterol bilayers using both the fluorescence of bilayer-associated 1,6-diphenyl-1,3,5-hexatriene (DPH) and freeze-fracture electron microscopy to elucidate specimen structure. Arrhenius analysis of the fluorescence-derived "microviscosity" parameter reveals temperature-induced structural changes in these membranes. In addition, isotherms of DPH fluorescence anisotropy and total intensity are used to detect alterations in membrane structure with varying cholesterol content. Freeze-fracture electron microscopic studies, utilizing rapid "jet-freezing" techniques, show strikingly different fracture-face morphologies for different combinations of sample cholesterol content and temperature. A phase diagram is proposed that offers a unifying interpretation of the fluorescence and freeze-fracture results. In this interpretation, inflections in temperature-scanning and isothermal fluorescence measurements reveal phase lines in the dipalmitoylphosphatidylcholine-cholesterol membranes Two-phase regions of the proposed phase diagram correspond to samples showing two coexisting fracture-face morphologies, while single-phase regions produce membranes having only one clearly identifiable structure. The proposed phase diagram provides an explanation for several conflicting literature proposals of stoichiometries for phosphatidylcholine-cholesterol complexes in membranes. These stoichiometric complexes correspond to the boundaries of two-phase areas in the gel region of the phase diagram. To better approximate the effect of cholesterol on natural membranes, the structure of egg phosphatidylcholine-cholesterol multilamellar vesicles was also investigated by using DPH fluorescence. The results for this complex natural phospholipid system are interpreted by comparison with the synthetic phospholipid results.

Data from several membrane systems are presented to confirm an empirical means of correcting diphenylhexatriene fluorescence for depolarization caused by sample turbidity. The depolarization proportionally constants obtained are not equal, but are shown to vary with (a) the physical state of the membrane, (b) the cholesterol content of the membrane, (c) the protein content of the membrane, and (d) the method of membrane preparation or isolation. It is concluded that depolarization corrections must always be considered when diphenylhexatriene fluorescence anisotropy is used to compare the fluidities within different membrane bilayers.

Measurement of multiple fluorescence decay times of 1,6-diphenyl-1,3,5-hexatriene (DPH) in membranes can in principle be used to investigate structural domains of lipid bilayers. To assess the feasibility of this approach using phase and modulation techniques, we reduced experimental errors specifically associated with performing these measurements on membrane suspensions (probe self-quenching, background fluorescence, turbidity-induced artifacts) and determined empirically the level of precision thereby obtainable. Next we used these precision limits in theoretical calculations to conclude that the ratio of two coexisting decay times must exceed 1.3 if they are to be resolved with reliable accuracy. To demonstrate that such resolutions could be accomplished experimentally in membrane suspensions, three approaches were taken. First, the fluorescence decay of aqueous quinine sulfate quenched by chloride ion was resolved from that of membrane-associated DPH as long as the lifetime ratios of these two fluorophores exceeded the predicted value. Second, populations of DPH-containing lipid vesicles with single (or nearly single) decay times were mixed together, and when there were only two major lifetime components that differed by more than 30%, the resulting heterogeneous fluorescence could be resolved into the two expected lifetime components. Finally, DPH fluorescence decay measurements were correlated with phase behavior in well-characterized lipid systems, revealing a short lifetime component of DPH fluorescence associated with gel-phase lipid vesicles. From these studies, we conclude that only in special cases can co-existing gel and fluid phases be resolved by means of DPH lifetime heterogeneity, within the limits of precision defined herein.

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