The fluorescence lifetime, τf, of merocyanine 540 (MC540) in small unilamellar vesicles was measured as a function of temperature and cholesterol content by using phase modulation fluorometry. These vesicles were formed by probe sonication of aqueous suspensions of egg phosphatidylcholine (PC) and cholesterol. The fluorescence lifetime of MC540 in these vesicles decreased with increasing temperature, but was independent of cholesterol. The decrease in τf with temperature is attributed to trans-cis photoisomerization. At low temperatures, the inverse of τf, or the fluorescence rate constant, kf, approaches a constant value of 0.45 ± 0.02 ns-1, which corresponds to the value of the radiative rate constant, kr, of the dye. The photoisomerization rate constant, kiso, was obtained by subtracting kr from kf. The temperature dependence of kiso is well described by an Arrhenius equation, with an activation energy of 31.5 ± 0.9 kJ mol-1. This Arrhenius behavior is rationalized in terms of the Smoluchowski limit for the Kramers theory for activated barrier crossing. The electronic spectra and kiso for MC540 in these vesicles are consistent with the dye being located in the polar headgroup region of the lipid bilayer.
- Merocyanine 540