Fluorescent Probe: Diphenylhexatriene

This chapter discusses the fluorescent probe—diphenylhexatriene. Depolarization of fluorescence has proved to be a very reliable technique with which to characterize the thermotropic and dynamic aspects of the hydrophobic regions of lipid bilayers and lipoproteins. Fluorescence analysis of dynamic systems can be performed either with time-dependent or steady-state type anisotropy measurements. The former approach can yield information concerning the heterogeneity of the lifetime of the fluorophore in the system, allowing in some cases a resolution of fluorophore subpopulations. This technique also allows an evaluation of the hindrance of the probe motion because of the degree of order of the phospholipid acyl side chains. Fluorescence polarization is measured by exciting the fluorophore with monochromatic light through a polarizer whose polarization axis is oriented vertically to the light path and the emission intensity is detected through an analyzer, whose polarization axis is oriented sequentially parallel to and perpendicular to the polarization axis of the exciting light.