Total Internal Reflection Fluorescence (TIRF) Microscopy

Kenneth N. Fish1

1 Department of Psychiatry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.18
DOI:  10.1002/0471142956.cy1218s50
Online Posting Date:  October, 2009
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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.

Keywords: axial resolution; fluorescence microscopy; live cell imaging; receptor trafficking; neurons

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Table of Contents

  • Introduction
  • The Theory Behind the Technique
  • TIRF Objectives
  • Empirically Determining Incident Angle/Penetration Depth
  • TIRF Imaging of Plasma Membrane Receptors in Neurons
  • Multi‐Wavelength TIRFM
  • Final Experimental Suggestions
  • Concluding Remarks
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
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