Gaus Katharina, Zech Tobias, Harder Thomas
Centre for Vascular Research at the School of Medical Sciences, University of New South Wales and The Department of Haematology, Prince of Wales Hospital, Sydney, Australia.
Mol Membr Biol. 2006 Jan-Feb;23(1):41-8. doi: 10.1080/09687860500466857.
Lateral segregation of cell membrane components gives rise to microdomains with a different structure within the membrane. Most prominently, lipid rafts are defined as domains in liquid ordered phase whereas surrounding membranes are more fluid. Here we review a 2-photon fluorescence microscopy approach, which allows the visualization of membrane fluidity. The fluorescent probe Laurdan exhibits a blue shift in emission with increasing membrane condensation caused by an alteration in the dipole moment of the probe as a consequence of exclusion of water molecules from the lipid bilayer. The quantification of membrane order is achieved by the Generalized Polarization (GP) values, which are defined as normalized intensity ratios of two emission channels. GP images are therefore not biased by probe concentrations and membrane ruffles. Furthermore, Laurdan reports membrane structure independently from the lipid and protein cargo of the membrane domains. We give examples where Laurdan microscopy was instrumental in quantifying the formation of condensed membrane domains and their cellular requirements. Moreover we discuss how microdomains identified by Laurdan microscopy are consistent with domains identified by other methodologies and put GP images in the context of current raft hypotheses.
细胞膜成分的侧向分离会在膜内产生具有不同结构的微区。最显著的是，脂筏被定义为处于液晶有序相的区域，而周围的膜则流动性更强。在此，我们综述一种双光子荧光显微镜方法，该方法可实现对膜流动性的可视化。荧光探针劳丹（Laurdan）随着膜凝聚程度的增加，其发射光会发生蓝移，这是由于脂质双分子层中水分子的排除导致探针偶极矩发生变化所致。膜有序性的量化通过广义极化（GP）值来实现，GP值定义为两个发射通道的归一化强度比。因此，GP图像不受探针浓度和膜褶皱的影响。此外，劳丹能够独立于膜区的脂质和蛋白质成分来反映膜结构。我们给出了一些例子，说明劳丹显微镜在量化凝聚膜区的形成及其细胞条件方面发挥了重要作用。此外，我们还讨论了通过劳丹显微镜鉴定的微区如何与通过其他方法鉴定的区域相一致，并将GP图像置于当前脂筏假说的背景下进行探讨。
