Membrane structural domains. Resolution limits using diphenylhexatriene fluorescence decay.

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.