Live-Cell Total Internal Reflection Fluorescence (TIRF) Microscopy to Investigate Protein Internalization Dynamics.

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.