Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
Methods Mol Biol. 2022;2438:45-58. doi: 10.1007/978-1-0716-2035-9_3.
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.
细胞的顶端-基底或平面细胞极性的建立涉及许多发生在质膜或其附近的事件，包括黏着斑动力学、内吞作用、胞吐作用和细胞骨架重组。人们希望在不干扰细胞内更深区域的情况下对这些事件进行可视化。全内反射荧光（TIRF）显微镜利用一种巧妙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 提供具有有限背景和几乎没有离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力学。在本章中，我们提出了一个用于研究细胞表面受体内吞作用的通用实验和分析 TIRF 工作流程。所提出的方法可以很容易地应用于使用 TIRF 显微镜研究质膜或其附近的其他动态生物学过程。
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