Fluorescence Anisotropy

Membrane fluidity at various bilayer depths was measured by means of fluorescence anisotropy that provides information on membrane microviscosity (inversely related to fluidity). The changes in the local environment of the fluorophores within a membrane system can be marked by monitoring the orientation and rotational correlation time, which would be indicated by fluorescence anisotropy.^50,51 Fluorescence anisotropy of DPH/TMA–DPH-labeled lipid membranes, in the presence and absence of the drug, was measured using a temperature-controlled Varian Cary Eclipse fluorescence spectrophotometer attached with a polarizer (Varian Cary Eclipse Manual Polarizer), considering the range of temperature from 5 to 90 °C giving a period of 3 min for equilibration with an accuracy of ±0.1 °C. The samples were excited with vertically and horizontally polarized lights, and respective polarized emission intensities were recorded. The degree of fluorescence steady-state anisotropy (r) was calculated from the following equation.⁵²⁻⁵⁴

where I_VV and I_VH are the parallel and perpendicular emission intensities of the vertically polarized excitation beams, respectively, is the correction factor to determine the sensitivity of the instrument (G should be ∼1), and I_HV and I_HH are the parallel and perpendicular emission intensities of the horizontally polarized excitation beams, respectively.