Biophysics Institute, Johannes Kepler University Linz, Linz, Austria.
Biophys J. 2011 Jun 8;100(11):2839-45. doi: 10.1016/j.bpj.2011.04.035.
Resolving the dynamical interplay of proteins and lipids in the live-cell plasma membrane represents a central goal in current cell biology. Superresolution concepts have introduced a means of capturing spatial heterogeneity at a nanoscopic length scale. Similar concepts for detecting dynamical transitions (superresolution chronoscopy) are still lacking. Here, we show that recently introduced spot-variation fluorescence correlation spectroscopy allows for sensing transient confinement times of membrane constituents at dramatically improved resolution. Using standard diffraction-limited optics, spot-variation fluorescence correlation spectroscopy captures signatures of single retardation events far below the transit time of the tracer through the focal spot. We provide an analytical description of special cases of transient binding of a tracer to pointlike traps, or association of a tracer with nanodomains. The influence of trap mobility and the underlying binding kinetics are quantified. Experimental approaches are suggested that allow for gaining quantitative mechanistic insights into the interaction processes of membrane constituents.
解析活细胞质膜中蛋白质和脂质的动态相互作用是当前细胞生物学的主要目标。超分辨率概念为捕捉纳米级空间异质性提供了一种手段。用于检测动态转变（超分辨率实时显微镜）的类似概念仍然缺乏。在这里，我们表明，最近引入的点变异荧光相关光谱法允许以显著提高的分辨率来检测膜成分的瞬态限制时间。使用标准的衍射极限光学，点变异荧光相关光谱法可以捕获示踪剂通过焦点远远低于通过时间的单个延迟事件的特征。我们提供了一种分析描述，用于描述示踪剂与点状陷阱的瞬时结合，或示踪剂与纳米域的结合的特殊情况。量化了陷阱迁移率和基础结合动力学的影响。提出了实验方法，可以深入了解膜成分相互作用过程的定量机制。
