Topic Introduction

Principles of Total Internal Reflection Microscopy (TIRFM)

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

Total internal reflection fluorescence microscopy (TIRFM) is a powerful technique for studying events that 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. TIRFM has been used to study (1) exocytosis of single synaptic vesicles stained with FM1-43 in living goldfish retinal bipolar neurons and (2) exocytosis of single dense core granules stained with neuropeptide Y-enhanced green fluorescent protein (NPY-EGFP) in living bovine chromaffin cells. This article describes the basic theory behind TIRFM and provides an overview for setting up a TIRFM system.