人工膜と生体膜の接点を探る 最近の膜研究の方法 環境感受性色素によるリポソームと細胞の膜物性のイメージング

Abstract

The environment-sensitive fluorescent probes, such as prodan, acrylodan and laurdan, change their emission wavelength depending on the polarity of their environment. For example, laurdan changes its emission peak from 440 nm in the gel phase of lipid bilayer to 490 nm in the liquid-crystalline phase. Time-resolved fluorescence spectra of laurdan in liposomes suggested that the 490 nm component of fluorescence was a emission from dipolar-relaxed states of excited fluorophores and that the dipolar relaxation was due to water molecules penetrated into the hydrophobic region of membranes. The generalized polarization (GP), defined by two fluorescence intensities at these characteristic emission wavelengths, is a measure of membrane fluidity and polarity. We have constructed an imaging system which enables us to obtain spatial distribution of the GP. The system is composed of a fluorescence microscope, monochromatic filters, a cooled-CCD camera, and an image-analysis system. It was found that the average GP value in a liposome and its temperature dependence were almost the same as those obtained by a conventional fluorescence spectrometer, by appending only one instrumental constant which described the difference of relative sensitivity between the two wavelength channels. By using this apparatus to the study of membrane heterogeneity of CHO (chinese hamster ovary) cells, it was found that plasma membranes were less polar than the membranes of organelles.