In Vitro Reconstitution of Microtubule Dynamics and Severing Imaged by Label-Free Interference-Reflection Microscopy.
In Vitro Reconstitution of Microtubule Dynamics and Severing Imaged by Label-Free Interference-Reflection Microscopy.
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DOI：
10.1007/978-1-0716-1983-4_5
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发表时间：
2022
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微管细胞骨架的动态结构对于细胞分裂、运动和形态发生至关重要。微管的动态特性-生长、收缩、成核和切断-由微管相关蛋白（MAP）调节。许多这些MAP的活性已经在体外使用显微镜测定法重建。作为荧光显微镜的替代方法，干涉反射显微镜（Interference-Reflection Microscopy，缩写为EFT）作为一种易于使用的宽视场成像技术，可以以高对比度和速度对微管进行无标记可视化。荧光显微镜避免了与荧光显微镜相关的几个问题，包括荧光标记所需的高浓度微管蛋白，荧光团引起的功能的潜在扰动，以及光损伤的风险。荧光显微镜可以在标准落射荧光显微镜上以低成本实现，并且可以与荧光技术如全内反射荧光（TIRF）显微镜相结合。在这里，我们描述了实验过程中的图像微管动力学和切断使用的ESTA，提供实用的提示和指导方针，以解决可能的实验障碍。
The dynamic architecture of the microtubule cytoskeleton is crucial for cell division, motility and morphogenesis. The dynamic properties of microtubules – growth, shrinkage, nucleation and severing - are regulated by an arsenal of microtubule-associated proteins (MAPs). The activities of many of these MAPs have been reconstituted in vitro using microscope assays. As an alternative to fluorescence microscopy, interference-reflection microscopy (IRM) has been introduced as an easy-to-use, wide-field imaging technique that allows label-free visualization of microtubules with high contrast and speed. IRM circumvents several problems associated with fluorescence microscopy including the high concentrations of tubulin required for fluorescent labeling, the potential perturbation of function caused by the fluorophores, and the risks of photodamage. IRM can be implemented on a standard epifluorescence microscope at low cost and can be combined with fluorescence techniques like total-internal-reflection-fluorescence (TIRF) microscopy. Here we describe the experimental procedure to image microtubule dynamics and severing using IRM, providing practical tips and guidelines to resolve possible experimental hurdles.