Ananthanarayanan Vaishnavi, Tolić Iva M
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany; Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia.
Methods Cell Biol. 2015;125:1-12. doi: 10.1016/bs.mcb.2014.10.001. Epub 2015 Jan 7.
While early fluorescence microscopy experiments employing fluorescent probes afforded snapshots of the cell, the power of live-cell microscopy is required to understand complex dynamics in biological processes. The first successful cloning of green fluorescent protein in the 1990s paved the way for development of approaches that we now utilize for visualization in a living cell. In this chapter, we discuss a technique to observe fluorescently tagged single molecules in fission yeast. With a few simple modifications to the established total internal reflection fluorescence microscopy, cytoplasmic dynein molecules in the cytoplasm and on the microtubules can be visualized and their intracellular dynamics can be studied. We illustrate a technique to study motor behavior, which is not apparent in conventional ensemble studies of motors. In general, this technique can be employed to study single-molecule dynamics of fluorescently tagged proteins in the cell interior.
虽然早期使用荧光探针的荧光显微镜实验能够提供细胞的瞬间图像，但要理解生物过程中的复杂动态，还需要活细胞显微镜技术。20世纪90年代绿色荧光蛋白的首次成功克隆为我们现在用于活细胞可视化的方法的发展铺平了道路。在本章中，我们将讨论一种在裂殖酵母中观察荧光标记单分子的技术。对已有的全内反射荧光显微镜进行一些简单的改进后，细胞质和微管上的细胞质动力蛋白分子就可以被可视化，并且可以研究它们在细胞内的动态变化。我们阐述了一种研究运动行为的技术，这种行为在传统的动力蛋白整体研究中并不明显。一般来说，这种技术可用于研究细胞内部荧光标记蛋白的单分子动态。
