UNIT 12.18 Total Internal Reflection Fluorescence (TIRF) Microscopy

  1. Kenneth N. Fish

Published Online: 1 OCT 2009

DOI: 10.1002/0471142956.cy1218s50

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Fish, K. N. 2009. Total Internal Reflection Fluorescence (TIRF) Microscopy. Current Protocols in Cytometry. 50:12.18:12.18.1–12.18.13.

Author Information

  1. Department of Psychiatry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Publication History

  1. Published Online: 1 OCT 2009
  2. Published Print: OCT 2009


Total internal reflection fluorescence (TIRF) microscopy (TIRFM) is an elegant optical technique that provides for the excitation of fluorophores in an extremely thin axial region (“optical section”). The method is based on the principle that when excitation light is totally internally reflected in a transparent solid (e.g., coverglass) at its interface with liquid, an electromagnetic field, called the evanescent wave, is generated in the liquid at the solid-liquid interface and is the same frequency as the excitation light. Since the intensity of the evanescent wave exponentially decays with distance from the surface of the solid, only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. This unit will briefly review the history, optical theory, and different hardware configurations used in TIRFM. In addition, it will provide experimental details and methodological considerations for studying receptors at the plasma membrane in neurons. Curr. Protoc. Cytom. 50:12.18.1-12.18.13. © 2009 by John Wiley & Sons, Inc.


  • axial resolution;
  • fluorescence microscopy;
  • live cell imaging;
  • receptor trafficking;
  • neurons