MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, U.K.
Essays Biochem. 2015;57:69-80. doi: 10.1042/bse0570069.
Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid-protein interactions and the traditional lipid 'raft' theory.
质膜动力学是细胞活动的重要调节者,特别是通过膜嵌入蛋白和脂质的相互作用和扩散动力学。在光学(共焦)显微镜上进行的 FCS(荧光相关光谱)是研究此类动力学的常用工具。不幸的是,其完全适用性受到传统光学显微镜空间分辨率的限制。本章描述了光学超分辨率 STED(受激辐射损耗)显微镜与 FCS 的结合,以及为什么它是研究活细胞中分子膜动力学的重要工具。与传统的 FCS 相比,STED-FCS 方法具有更好的可能性来区分自由扩散和异常扩散,从而深入了解脂质-蛋白相互作用和传统的脂质“筏”理论。