Assessment of Merocyanine Subpopulations in DPPC Vesicles using Ansitropy and Lifetime Measurements

Abstract

The purpose of this study was to further investigate the properties of the fluorescent probe merocyanine 540, which has been used frequently for over two decades to assess membrane phase properties under various conditions. Differences in emission spectrum shape at temperatures above and below the thermotropic phase transition of model membranes have been hypothesized to represent changes in the position and orientation of the probe in the bilayer. This hypothesis suggests specific predictions concerning probe mobility in the membrane as a function of temperature and emission wavelength. We tested the hypothesis using measurements of steady state anisotropy and fluorescence lifetimes in dipalmitoylphosphatidylcholine vesicles. Below the lipid phase transition temperature, steady state anisotropy decreased by 0.2 units across the emission spectrum from short to long wavelength. In contrast, anisotropy was more stable as a function of emission wavelength when measured above the transition temperature. Fluorescence lifetimes showed minimal wavelength dependence at either temperature. Anisotropy experiments were repeated at a variety of probe-to-lipid ratios to assess the role of probe aggregation on the observations. The data supported previous findings from measurements of the quantum yield of merocyanine 540: in the gel phase, two separate populations of the probe (monomers and dimers) fluoresce. The monomers, which emit at short wavelengths, are oriented perpendicular to the bilayer surface, and are limited in mobility by neighboring phospholipids. The dimmers, which emit at long wavelengths, are oriented parallel to the bilayer surface, and are localized in a membrane region where motion is less restricted, perhaps in the region between the membrane leaflets. At higher temperatures, only monomers fluoresce but exhibit higher mobility due to the lower order of the membrane phase.