Mueller Veronika, Honigmann Alf, Ringemann Christian, Medda Rebecca, Schwarzmann Günter, Eggeling Christian
Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Methods Enzymol. 2013;519:1-38. doi: 10.1016/B978-0-12-405539-1.00001-4.
Details of molecular membrane dynamics in living cells such as lipid-protein interactions or the incorporation of molecules into lipid "rafts" are often hidden to the observer because of the limited spatial resolution of conventional far-field optical microscopy. Fortunately, the superior spatial resolution of far-field stimulated-emission-depletion (STED) nanoscopy allows gaining new insights. Applying fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes free from anomalous molecular diffusion due to transient binding, as for the diffusion of fluorescent phosphoglycero- and sphingolipid analogs in the plasma membrane of living cells. STED-FCS data recorded at different environmental conditions and on different lipid analogs reveal molecular details of the observed nanoscale trapping. Dependencies on the molecular structure of the lipids point to the distinct connectivity of the various lipids to initiate or assist cellular signaling events, but also outline strong differences to the characteristics of liquid-ordered and disordered phase separation in model membranes. STED-FCS is a highly sensitive and exceptional tool to study the membrane organization by introducing a new class of nanoscale biomolecular studies.
由于传统远场光学显微镜的空间分辨率有限，活细胞中分子膜动力学的细节，如脂质 - 蛋白质相互作用或分子纳入脂质“筏”的过程，常常难以被观察者察觉。幸运的是，远场受激发射损耗（STED）纳米显微镜卓越的空间分辨率使得人们能够获得新的见解。在直径持续调降至30纳米的焦斑中应用荧光相关光谱（FCS），可以区分自由分子扩散与由于瞬时结合导致的异常分子扩散，就像活细胞质膜中荧光磷酸甘油和鞘脂类似物的扩散情况一样。在不同环境条件下以及针对不同脂质类似物记录的STED - FCS数据揭示了所观察到的纳米级捕获的分子细节。对脂质分子结构的依赖性表明，各种脂质具有独特的连接性，可启动或协助细胞信号转导事件，同时也勾勒出与模型膜中液相有序和无序相分离特征的显著差异。通过引入一类新的纳米级生物分子研究，STED - FCS是研究膜组织的一种高度灵敏且独特的工具。
