Ashdown George, Pandžić Elvis, Cope Andrew, Wiseman Paul, Owen Dylan
Department of Physics and Randall Division of Cell and Molecular Biophysics, King's College London.
ARC Centre for Advanced Molecular Imaging, Australian Centre for NanoMedicine, University of New South Wales Australia.
J Vis Exp. 2015 Dec 17(106):e53749. doi: 10.3791/53749.
Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.
丝状肌动蛋白在大多数细胞过程中发挥着关键作用，包括细胞运动，在免疫细胞中，它还参与一种关键的细胞间相互作用，即免疫突触的形成。据推测，F-肌动蛋白在调节细胞膜上的分子分布方面也发挥作用，包括膜下囊泡动力学和蛋白质聚集。虽然标准光学显微镜技术能够进行广义的、受衍射限制的观察，但许多细胞和分子事件，包括聚集和分子流动，发生在远低于标准光学显微镜分辨率的长度尺度上的群体中。通过将全内反射荧光与超分辨率成像方法结构照明显微镜相结合，记录了T细胞免疫突触处F-肌动蛋白的二维分子流动。然后应用时空图像相关光谱法（STICS），该方法以速度直方图和代表流动方向性和大小的矢量图的形式生成可量化的结果。本方案描述了超分辨率成像和STICS技术的结合，以在亚衍射水平的细节上生成流动矢量。该技术用于证实突触形成时T细胞外周存在对称逆行和向心的肌动蛋白流。
