Microtubule dynamics at the cell cortex probed by TIRF microscopy

Abstract

Total internal reflection fluorescence (TIRF) microscopy is a technique that allows selective excitation of fluorescence at a liquid/solid interface within a short distance from the boundary. The penetration depth of TIRF microscopy depends on the angle of illumination resulting in a range of depths, which typically vary from approximately similar 70-200 nm up to reverse approximately 500 nm. The advantages of TIRF microscopy include excellent signal-to-noise ratio, high sensitivity, low photobleaching, and low photodamage. TIRF microscopy is widely used for studying cell adhesion, exo- and endocytosis, and the dynamics of plasma membrane-associated molecules. TIRF microscopy can also be applied for selective visualization of any other cellular processes that occur near the basal membrane even if their localization is not restricted to this part of the cell. For example, microtubules are distributed throughout the cytoplasm, but the use of TIRF microscopy makes it possible to visualize specifically the microtubule subpopulation in the vicinity of the basal cortex and thus study cortical microtubule attachment and stabilization, interactions between microtubules and matrix adhesion structures, and the behavior of specific molecules involved in these processes. In this chapter we describe the application of a commercially available setup to analyze microtubule behavior in live mammalian cells using TIRF microscopy.