Use of TIRF microscopy to visualize actin and microtubules in migrating cells.

Total internal reflection fluorescence (TIRF) is the technique of choice to visualize and quantify cellular events localized at the basal plasma membrane of adherent cells. By selectively illuminating the first 200 nm above the basal membrane, it allows maximal resolution in the vertical z-axis. In this chapter, I describe a prism-based TIRF setup and the procedures to visualize the actin and microtubule cytoskeleton in migrating astrocytes. TIRF microscopy provides quantitative information on the organization of the cytoskeleton in both fixed and live migrating cells.