Protocol

Imaging Exocytosis with Total Internal Reflection Microscopy (TIRFM)

This protocol was adapted from "A Practical Guide: Imaging Exocytosis with Total Internal Reflection Microscopy," Chapter 64, in Imaging in Neuroscience and Development, (eds. Yuste and Konnerth). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

Although electrophysiological techniques such as membrane capacitance measurements, electrochemical detection, and post-synaptic recordings are powerful ways of studying exocytosis, information concerning any steps prior to vesicle fusion must be inferred indirectly. Total internal reflection fluorescence microscopy (TIRFM) is a powerful technique for studying events that, like exocytosis, occur near a cell surface. The technique allows selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass. In this protocol, TIRFM is used to investigate the steps leading up to vesicle fusion in both retinal bipolar neurons and chromaffin cells by directly imaging synaptic vesicles and dense core granules prior to and including exocytosis.