Total Internal Reflection Fluorescence (TIRF) Microscopy

Andreea Trache1, Gerald A. Meininger2

1 Department of Systems Biology and Translational Medicine, College of Medicine, Texas A&M Health Science Center, College Station, Texas, 2 Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 2A.2
DOI:  10.1002/9780471729259.mc02a02s10
Online Posting Date:  August, 2008
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Abstract

Total internal reflection fluorescence (TIRF) microscopy represents a method of exciting and visualizing fluorophores present in the near‐membrane region of live or fixed cells grown on coverslips. TIRF microscopy is based on the total internal reflection phenomenon that occurs when light passes from a high‐refractive medium (e.g., glass) into a low‐refractive medium (e.g., cell, water). The evanescent field produced by total internally reflected light excites the fluorescent molecules at the cell‐substrate interface and is accompanied by minimal exposure of the remaining cell volume. This technique provides high‐contrast fluorescence images, with very low background and virtually no out‐of‐focus light, ideal for visualization and spectroscopy of single‐molecule fluorescence near a surface. This unit presents, in a concise manner, the principle of operation, instrument diversity, and TIRF microscopy applications for the study of biological samples. Curr. Protoc. Microbiol. 10:2A.2.1‐2A.2.22. © 2008 by John Wiley & Sons, Inc.

Keywords: total internal reflection fluorescence microscopy

     
 
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Table of Contents

  • Introduction
  • Principle of Operation
  • Practical Guidelines
  • Advantages of TIRF
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Figures

Videos

Literature Cited

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Key References
   Axelrod, D. 2003. Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol. 31:1‐33.
  Good review of TIRF theory, applications, and practical protocols to set up a TIRF system.
   Gingell et al., 1987. See above.
  Quantitative theoretical approach of TIRF.
   Oheim, M. 2001. Imaging transmitter release. II. A practical guide to evanescent‐wave imaging. Laser Med. Sci. 16:159‐170.
  A how‐to guide for constructing a TIRF system.
   Wazawa, T. and Ueda, M. 2005. Total internal reflection fluorescence microscopy in single molecule nanobioscience. Adv. Biochem. Eng. Biotechnol. 95:77‐106.
  Good review of TIRF applied to single molecule studies.
Internet Resources
   http://www.micro.magnet.fsu.edu/primer/index.html
  Molecular Expressions Microscopy Primer. Excellent presentation of optical microscopy.
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