The fluorescence decay of membrane lipid bilayer probes are influenced by the environment of the probe and therefore by the properties of the bilayer in which it resides. Traditionally the fluorescence decay has been analyzed in the form of a multi-exponential. Analysis in the form of continuous distributions can provide a useful alternative to this approach since it introduces a new parameter, the distributional width. The physical basis underlying the distributional width, the fluorophore "environmental heterogeneity", arises from organizational aspects, compositional diversity and solvent effects. The "sampling" of environmental heterogeneity will depend on the intrinsic fluorescence lifetime, the lipid rate of motion and fluorophore charge and shape factors. We have found a good correlation of the distributional width with a number of bilayer properties. Thus as we increase the complexity of a bilayer the distributional width becomes broader, in order, from a single phospholipid molecular species, mixture of species, mixture of classes (i.e. PC+ PE etc) and species to intact natural membranes (i.e. including proteins and bilayer organizational factors). More recently we have been able to show that the protein-lipid interface itself can act as a source of fluorophore heterogeneity. Suggesting the possibility of spectroscopically isolating a region of the membrane of particular interest in the modulation of membrane processes. There are still a number of uncertainties in assigning a distributional width to a particular bilayer property. For example in comparing bilayers they could be compared at the same temperature or at the same temperature above the respective gel-liquid phase transition temperatures. In fact with regard to the relative degree of environmental heterogeneity sampled by the fluorophore it may be better to make such types of comparisons at the same respective rates of lipid motion since the rate of lipid motion is a key factor governing the sampling of environmental heterogeneity. Using this approach in the example of increasing levels of unsaturation our results suggest that unsaturation introduces structural diversity into lipid bilayers.
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