Department of Molecular Biophysics & Biochemistry, Yale University.
Department of Molecular Biophysics & Biochemistry, Yale University; Department of Physics, Yale University;
J Vis Exp. 2022 May 3(183). doi: 10.3791/63730.
Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.
已经采用了几种技术来直接可视化细胞骨架丝及其相关蛋白。全内反射荧光（TIRF）显微镜具有高的信号背景比，但它受到荧光蛋白的光漂白和光损伤的影响。无标记技术，如干涉反射显微镜（IRM）和干涉散射显微镜（iSCAT），规避了光漂白的问题，但不能轻易地可视化单个分子。本文提出了一种将 IRM 与商业 TIRF 显微镜结合使用的方案，用于在体外同时成像微管相关蛋白（MAPs）和动态微管。该方案允许高速观察与动态微管相互作用的 MAPs。这通过消除微管标记的需要和几个额外的光学组件（如第二激发激光）的需要，改进了现有的双色 TIRF 装置。两个通道都在同一个相机芯片上成像，以避免图像注册和帧同步问题。通过可视化单个驱动蛋白分子在动态微管上的行走，展示了该设置。
