2004 年 29 巻 3 号 p. 169-179
Laurdan, one of the environment-sensitive fluorescent probes, is useful for monitoring membrane fluidity and is widely used for spectroscopic and imaging applications. In the imaging applications, the generalized polarization parameter (GP), defined by two fluorescent intensities at characteristic wavelengths, is used as a measure of membrane fluidity and polarity. However, time-resolved fluorescent spectra of laurdan revealed that the three excited states concern with the emission spectra. We have measured the steady-state fluorescence spectra and the fluorescence anisotropy of laurdan, prodan and NBD-PE in the DPPC and DLPC bilayers at various temperatures. The spectral dependence on the excitation wavelength is interpreted as the red edge effects that are related to the existence of excited-state distribution of fluorophores on their interaction energy with the environment and the slow dielectric relaxation of this environment. The emission peak of laurdan in the liquid-crystalline phase is independent of the red edge excitation, while prodan and NBD-PE show significant red edge effects. This results strongly suggest that laurdan changes its electronic excited state by the specific interaction with a few water molecules in the bilayers and that in addition the non-specific solvent relaxation causes further spectral shifts in the case of prodan in the bilayers and laurdan in the micella. A new three state model for the absorption and emission of laurdan is proposed. The availability and limitation of the GP method are disscussed.