论文标题
成像动态微管和相关的蛋白质通过同时干扰 - 反射和总内反转荧光显微镜
Imaging Dynamic Microtubules and Associated Proteins by Simultaneous Interference-Reflection and Total-Internal-Reflection-Fluorescence Microscopy
论文作者
论文摘要
已经采用了几种技术来直接可视化细胞骨骼细丝及其相关蛋白。总内部反射荧光（TIRF）显微镜具有较高的信噪比，但荧光蛋白的光漂白和光损伤遭受了损坏。无标签技术，例如干扰反射显微镜（IRM）和干扰散射显微镜（ISCAT），避免了光漂白的问题，但不能容易地可视化单分子。在这里，我们提出了一种将IRM与商用TIRF显微镜相结合的方案，用于同时在体外成像微管相关蛋白（MAP）和动态微管。我们的协议允许高速观察与动态微管相互作用的地图。通过消除对微管标记的需求和对几种其他光学组件（例如第二个激发激光器）的需求，这可以改善现有的两色TIRF设置。我们在同一相机芯片上图像两个通道，以避免图像注册和框架同步问题。我们通过可视化在动态微管上行走的单个驱动蛋白分子来展示我们的设置。
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 interference scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. Here, we present a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. Our 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. We image both channels on the same camera chip to avoid image-registration and frame-synchronization problems. We demonstrate our setup by visualizing single kinesin molecules walking on dynamic microtubules.
