UNIT 12.29 Total Internal Reflection Fluorescence (TIRF) Microscopy Illuminator for Improved Imaging of Cell Surface Events

  1. Daniel S. Johnson1,
  2. Jyoti K. Jaiswal2,3,
  3. Sanford Simon1

Published Online: 1 JUL 2012

DOI: 10.1002/0471142956.cy1229s61

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Johnson, D. S., Jaiswal, J. K. and Simon, S. 2012. Total Internal Reflection Fluorescence (TIRF) Microscopy Illuminator for Improved Imaging of Cell Surface Events. Current Protocols in Cytometry. 61:12.29:12.29.1–12.29.19.

Author Information

  1. 1

    The Rockefeller University, Laboratory of Cellular Biophysics, New York, New York

  2. 2

    Department of Integrative Systems Biology, The George Washington University Medical Center, Washington, D.C.

  3. 3

    Center for Genetic Medicine Research, Children's National Medical Center, Washington, D.C.

Publication History

  1. Published Online: 1 JUL 2012
  2. Published Print: JUL 2012


Total internal reflection fluorescence (TIRF) microscopy is a high-contrast imaging technique suitable for observing biological events that occur on or near the cell membrane. The improved contrast is accomplished by restricting the thickness of the excitation field to over an order of a magnitude narrower than the z-resolution of an epi-fluorescence microscope. This technique also increases signal-to-noise, making it a valuable tool for imaging cellular events such as vesicles undergoing exocytosis or endocytosis, viral particle formation, cell signaling, and dynamics of membrane proteins. This protocol describes the basic procedures for setting up a through-the-objective TIRF illuminator and a prism-based TIRF illuminator. In addition, an alternate protocol for incorporating an automated deflection system into through-the-objective TIRF is given. This system can be used to decrease aberrations in the illumination field, to quickly switch between epi- and TIRF illumination, and to adjust the penetration depth during multicolor TIRF applications. In the commentary, a description of the total internal reflection phenomenon is given, critical parameters of a TIRF microscope are discussed, and technical challenges and considerations are reviewed. Curr. Protoc. Cytom. 61:12.29.1-12.29.19. © 2012 by John Wiley & Sons, Inc.


  • total internal reflection fluorescence microcopy;
  • fluorescence;
  • cell imaging