Lim James, Danuser Gaudenz
Department of Cell Biology, The Scripps Research Institute.
J Vis Exp. 2009 Aug 5(30):1325. doi: 10.3791/1325.
In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM). We describe the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.
在本实验方案中，我们描述了使用荧光斑点显微镜（FSM）来捕获PtK1细胞中肌动蛋白动力学的高分辨率图像。FSM的一个独特优势在于其能够捕获活细胞内F-肌动蛋白网络的运动和周转动力学（组装/拆卸）。当与计算机视觉软件（qFSM）配合使用时，该技术在获得F-肌动蛋白动力学的定量测量方面特别有用。我们描述了活细胞中荧光肌动蛋白探针的选择、显微注射和可视化。重要的是，类似的程序适用于可视化其他大分子组装体。FSM已被用于微管、中间丝和黏附复合体的研究。
