It has been long recognized that the cell membrane is heterogeneous on scales ranging from a couple of molecules to micrometers in size and hence diffusion of receptors is length scale dependent. This heterogeneity modulates many cell-membrane-associated processes requiring transient spatiotemporal separation of components. The transient increase in local concentration of interacting signal components enables robust signaling in an otherwise thermally noisy system. Understanding how lipids and proteins self-organize and interact with the cell cortex requires quantifying the motion of the components. Multi-length scale diffusion measurements by single particle tracking, fluorescence correlation spectroscopy (FCS) or related techniques are able to identify components being transiently trapped in nanodomains, from freely moving one and from ones with reduced long-scale diffusion due to interaction with the cell cortex. One particular implementation of multi-length scale diffusion measurements is the combination of FCS with a spatially resolved detector, such as a camera and two-dimensional extended excitation profile. The main advantages of this approach are that all length scales are interrogated simultaneously, uniquely permits quantifying changes to the membrane structure caused by extrenal or internal perturbations. Here, we review how combining total internal reflection microscopy (TIRF) with FC resolves the membrane organization in living cells. We show how to implement the method, which requires only a few seconds of data acquisition to quantify membrane nanodomains, or the spacing of membrane fences caused by the actin cortex. The choice of diffusing fluorescent probe determines which membrane heterogeneity is detected. We review the instrument, sample preparation, experimental and computational requirements to perform such measurements, and discuss the potential and limitations. The discussion includes examples of spatial and temporal comparisons of the membrane structure in response to perturbations demonstrating the complex cell physiology.

中文翻译：

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通过 bimFCS 量化细胞膜超微结构的时空变化

人们早就认识到细胞膜在大小范围从几个分子到微米的尺度上是异质的，因此受体的扩散是长度尺度相关的。这种异质性调节了许多细胞膜相关过程，需要成分的瞬时时空分离。相互作用信号成分的局部浓度的瞬时增加能够在其他热噪声系统中实现稳健的信号传递。了解脂质和蛋白质如何自组织并与细胞皮层相互作用需要量化组件的运动。通过单粒子跟踪、荧光相关光谱 (FCS) 或相关技术进行的多长度尺度扩散测量能够识别被瞬时捕获在纳米域中的成分，由于与细胞皮层的相互作用，自由移动的一个和长期扩散减少的一个。多长度尺度扩散测量的一种特定实现是 FCS 与空间分辨检测器的组合，例如相机和二维扩展激发剖面。这种方法的主要优点是同时询问所有长度尺度，唯一地允许量化由外部或内部扰动引起的膜结构的变化。在这里，我们回顾了如何将全内反射显微镜 (TIRF) 与 FC 相结合来解决活细胞中的膜组织。我们展示了如何实施该方法，该方法只需要几秒钟的数据采集来量化膜纳米域，或由肌动蛋白皮层引起的膜栅栏的间距。扩散荧光探针的选择决定了检测到哪种膜异质性。我们回顾了执行此类测量的仪器、样品制备、实验和计算要求，并讨论了其潜力和局限性。讨论包括膜结构响应扰动的空间和时间比较的例子，证明了复杂的细胞生理学。