Live-Cell Total Internal Reflection Fluorescence (TIRF) Microscopy to Investigate Protein Internalization Dynamics.
Live-Cell Total Internal Reflection Fluorescence (TIRF) Microscopy to Investigate Protein Internalization Dynamics.
复制标题
活细胞全内反射荧光 (TIRF) 显微镜研究蛋白质内化动力学。
DOI：
10.1007/978-1-0716-2035-9_3
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Mattheyses,AlexaL
中科院分区：
文献类型：
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作者：
Rao,TejeshwarC;Nawara,TomaszJ;Mattheyses,AlexaL
顶端基底或平面细胞极性的建立涉及在质膜处或附近发生的许多事件，包括粘着斑动力学、胞吞作用、胞吐作用和细胞骨架重组。希望在不受细胞内其他更深处区域干扰的情况下可视化这些事件。全内反射荧光 (TIRF) 显微镜利用优雅的光学切片方法来可视化样品-盖玻片界面附近的荧光团。 TIRF 提供背景有限且几乎没有失焦光的高对比度荧光图像，非常适合可视化和跟踪质膜附近的动态。在本章中，我们提出了用于研究细胞表面受体内吞作用的通用实验和分析 TIRF 流程。所提出的方法可以很容易地应用于使用 TIRF 显微镜研究质膜处或附近的其他动态生物过程。
The establishment of apicobasal or planar cell polarity involves many events that occur at or near the plasma membrane including focal adhesion dynamics, endocytosis, exocytosis, and cytoskeletal reorganization. It is desirable to visualize these events without interference from other regions deeper within the cell. Total internal reflection fluorescence (TIRF) microscopy utilizes an elegant optical sectioning approach to visualize fluorophores near the sample–coverslip interface. TIRF provides high-contrast fluorescence images with limited background and virtually no out-of-focus light, ideal for visualizing and tracking dynamics near the plasma membrane. In this chapter, we present a general experimental and analysis TIRF pipeline for studying cell surface receptor endocytosis. The approach presented can be easily applied to study other dynamic biological processes at or near the plasma membrane using TIRF microscopy.