Membrane fluidity expressed as the regiospecific anisotropy of fluorescence polarization.

Artificial membranes were prepared via hydration of lipide film (2.5 mg, 0.0034 mmol DPPC + MitoTam 10 mol.% + 0.1 mol% DPH or 0.2 mol % TMA-DPH) with physiological saline (2 mL). The lipide solution was extruded through polycarbonate membrane with pore size 0.4 μm. An 100 μL aliquot of liposomal solution was diluted with additional 900 μL of physiological saline to give clear solution of subvisible particles (≈ 0.125 mg/mL). Fluorescence anisotropy was measured over range of temperatures (22 – 49°C) in 3°C increments while the slides in the spectrometer FLS1000 were set to 1 to 3 nm. In between the individual temperature measurements was the sample stirred with magnetic stir bar. Each measurement was average of three subsequent scans and the whole data set was collected as triplicate (DPH excitation 360 nm, emission 435 nm; TMA-DPH excitation 365 nm, emission 425 nm). Fluorescence anisotropy (r) is calculated automatically by software provided with the instrument, according to r = (Ivv−IvhG)/(Ivv + 2IvhG), where Ivv and Ivh are the intensities of the vertically and horizontally polarized components of the fluorescent light, respectively, after excitation with vertically polarized light. G = Ihv/Ihh is a grating correction factor for the optical system.