The Physical Basis of Total Internal Reflection Fluorescence (TIRF) Microscopy and Its Cellular Applications

  • Natalie S. Poulter
  • William T. E. Pitkeathly
  • Philip J. Smith
  • Joshua Z. RappoportEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1251)


Total internal reflection fluorescence (TIRF) microscopy has gained popularity in recent years among cell biologists due to its ability to clearly visualize events that occur at the adherent plasma membrane of cells. TIRF microscopy systems are now commercially available from nearly all microscope suppliers. This review aims to give the reader an introduction to the physical basis of TIRF and considerations that need to be made when purchasing a commercial system. We explain how TIRF can be combined with other microscopy modalities and describe how to use TIRF to study processes such as endocytosis, exocytosis, and focal adhesion dynamics. Finally, we provide a step-by-step guide to imaging and analyzing focal adhesion dynamics in a migrating cell using TIRF microscopy.

Key words

Total internal reflection fluorescence (TIRF) Endocytosis Exocytosis Focal adhesions 



The authors would like to acknowledge funding through BBSRC Project grant BB/H002308/1. WTEP and PJS are funded through the Physical Sciences of Imaging for the Biomedical Sciences (PSIBS) Doctoral Training Centre, and NSP is funded through British Heart Foundation New Horizons grant NH/11/6/29061. The TIRF microscope used in this research was obtained through Birmingham Science City Translational Medicine Clinical Research and Infrastructure Trials Platform, with support from Advantage West Midlands (AWM).


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Natalie S. Poulter
    • 4
  • William T. E. Pitkeathly
    • 2
  • Philip J. Smith
    • 2
  • Joshua Z. Rappoport
    • 1
    • 3
    Email author
  1. 1.School of BiosciencesUniversity of BirminghamBirminghamUK
  2. 2.Physical Sciences of Imaging for the Biomedical Sciences (PSIBS) Doctoral Training CentreUniversity of BirminghamBirminghamUK
  3. 3.Center for Advanced Microscopy and the Nikon Imaging Center at Northwestern UniversityNorthwestern University Feinberg School of MedicineChicagoUSA
  4. 4.Centre for Cardiovascular Sciences, Institute of Biomedical Research, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUK

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