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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


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

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Principle of Operation
  • Practical Guidelines
  • Advantages of TIRF
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

   Adams, M.C., Matov, A., Yarar, D., Gupton, S.L., Danuser, G., and Waterman‐Storer, C.M. 2004. Signal analysis of total internal reflection fluorescent speckle microscopy (TIR‐FSM) and wide‐field epifluorescence FSM of the actin cytoskeleton and focal adhesions in living cells. J. Microsc. 216:138‐152.
   Axelrod, D. 2001a. Total internal reflection fluorescence microscopy. In Methods in Cellular Imaging (A. Periasamy, ed.) pp. 362‐380. American Physiological Society, Bethesda, Md.
   Axelrod, D. 2001b. Total internal reflection fluorescence microscopy in cell biology. Traffic 2:764‐774.
   Axelrod, D. 2001c. Selective imaging of surface fluorescence with very high aperture microscope objectives. J. Biomed. Opt. 6:6‐13.
   Axelrod, D., Thompson, N.L., and Burghardt, T.P. 1983. Total internal reflection fluorescent microscopy. J Microsc. 129:19‐28.
   Axelrod, D., Burghardt, T.P., and Thompson, N.L. 1984. Total internal reflection fluorescence. Annu. Rev. Biophys. Bioeng. 13:247‐268.
   Axelrod, D., Fulbright, R.M., and Hellen, E.H. 1986. Adsorption kinetics on biological membranes: Measurements by total internal reflection fluorescence. In Applications of Fluorescence in Biomedical Sciences (L. Taylor, A.S. Waggoner, R.F. Murphy, F. Lanni, and R.R. Birge, eds.) pp. 461‐476. A.R. Liss, New York.
   Bonifacino, J.S. and Traub, L.M. 2003. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72:395‐447.
   Burmeister, J.S., Truskey, G.A., Yarbrough, J.L., and Reichert, W.M. 1994. Imaging of cell/substrate contacts on polymers by total internal reflection fluorescence microscopy. Biotechnol. Prog. 10:26‐31.
   Burmeister, J.S., Olivier, L.A., Truskey, G.A., and Reichert, W.M. 1998. Application of total internal reflection fluorescence microscopy to study cell adhesion to biomaterials. Biomaterials 19:307‐325.
   Burtey, A., Rappoport, J.Z., Bouchet, J., Basmaciogullari, S., Guatelli, J., Simin, S.M., Benichou, S., and Benmerah, A. 2007. Dynamic interaction of HIV‐1 with the clathrin‐mediated endocytic pathway at the plasma membrane. Traffic 8:61‐76.
   Conner, S.F. and Schmid, S.L. 2003. Regulated portals of entry into the cell. Nature 422:37‐44.
   Demuro, A. and Parker, I. 2004. Imaging the activity and localization of single voltage‐gated Ca2+ channels by total internal reflection fluorescence microscopy. Biophys. J. 86:3250‐3259.
   Ewers, H., Smith, A.E., Sbalzarini, I.F., Lilie, H., Koumouysakos, P., and Helenius, A. 2005. Single‐particle tracking of murine polyoma virus–like particles on live cells and artificial membranes. Proc. Natl. Acad. Sci. U.S.A. 102:15110‐15115.
   Fulbright, R.M. and Axelrod, D. 1993. Dynamics of nonspecific adsorption of insulin to erythrocyte membranes. J. Fluoresc. 3:1‐16.
   Gilbert, J. and Benjamin, T. 2004. Uptake pathway of polyomavirus via ganglioside GD1a. J. Virol. 78:12259‐12267.
   Gingell, D., Heavens, O.S., and Mellor, J.S. 1987. General electromagnetic theory of total internal reflection fluorescence: The quantitative basis for mapping cell‐substratum topography. Cell Sci. 87:677‐693.
   Gleiter, S. and Lilie, H. 2001. Coupling of antibodies via protein Z on modified polyoma virus–like particles. Protein Sci. 10:434‐444.
   Hirschfeld, T. 1965. Total reflection fluorescence (TRF). Can. Spectrosc. 10:128.
   Lang, M.J., Fordyce, P.M., Engh, A.M., Neuman, K.C., and Block, S.M. 2004. Simultaneous, coincident optical trapping and single‐molecule fluorescence. Nat. Methods 1:133‐139.
   Lieto, A.M., Randall, C.C., and Thompson, N.L. 2003. Ligand‐receptor kinetics by total internal reflection with fluorescence correlation spectroscopy. Biophys. J. 85:3294‐3302.
   Mathur, A.B., Truskey, G.A. and Reichert, W.M. 2000. Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells. Biophys. J. 78:1725‐1735.
   Presley, J.F., Cole, N.B., Schroer, T.A., Hirschberg, K., Zaal, K.J., and Lippincott‐Schwartz, J. 1997. ER‐to‐Golgi transport visiualized in living cells. Nature 389:81‐85.
   Reichert, W.M. 1989. Evanscent detection of absorbed films: Assessment of optical considerations for absorbance and fluorescence spectroscopy at the crystal/solution and polymer/solution interfaces. Crit. Rev. Biocompat. 5:173‐205.
   Reichert, W.M. and Truskey, G.A. 1990. Total internal reflection fluorescence (TIRF) microscopy. I. Modeling cell contact region fluorescence. J. Cell Sci. 96:219‐230.
   Ried, T., Liyanage, M., du Manoir, S., Heselmeyer, K., Auer, G., Macville, M., and Schrock, E. 1997. Tumor cytogenetics revisited: Comparative genomic hybridization and spectral karyotyping. J. Mol. Med. 75:801‐814.
   Sanger, F. and Coulson, A.R. 1975. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol. 94:441‐448.
   Sbalzarini, I.F. and Koumoutsakos, P. 2005. Feature point tracking and trajectory analysis for video imaging in cell biology. J. Struct. Biol. 151:182‐195.
   Schmoranzer, J., Goulian, M., Axelrod, D., and Simon, S.M. 2000. Imaging constitutive exocytosis with total internal reflection fluorescence microscopy. J. Cell Biol. 149:23‐31.
   Seong, J.A. and Almers, W. 2004. Tracking SNARE complex formation in live endocrine cells. Science 306:1042‐1046.
   Stehle, T., Yan, Y., Benjamin, T.L., and Harrison, S.C. 1994. Structure of murine polyomavirus complexed with an oligosaccharide receptor fragment. Nature 369:160‐163.
   Stout, A.L. and Axelrod, D. 1989. Evanescent field excitation of fluorescence by epi‐illumination microscopy. Appl. Optics 28:5237‐5242.
   Sund, S.E., Swanson, J., and Axelrod, D. 1999. Cell membrane orientation visualized by polarized total internal reflection fluorescence. Biophys. J. 77:2266‐2283.
   Temple, P. 1981. Total internal reflection microscopy: A surface inspection technique. Appl. Optics 20:2656‐2664.
   Toomre, D., Keller, P., White, J., Olivo, J.C., and Simons, K. 1999. Dual‐color vistubles in living cells. J. Cell Biol. 112:21‐33.
   Toomre, D., Steyer, J.A., Keller, P., Almers, W., and Simons, K. 2000. Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy. J. Cell Biol. 149:33‐40.
   Trache, A. and Meininger, G.A. 2005. An atomic force–multi optical imaging integrated microscope for monitoring molecular dynamics in live cells. J. Biomed. Opt. 10:064023:1‐17.
   Truskey, G.A., Burmeister, J.S., Grapa, E., and Reichert, W.M. 1992. Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances. J. Cell Sci. 103:491‐499.
   Tsai, B., Gilbert, J.M., Stehle, T., Lencer, W., Benjamin, T.L., and Rapoport, T.A. 2003. Gangliosides are receptors for murine polyoma virus and SV40. EMBO J. 22:4346‐4355.
   van Belkum, A. 1994. DNA fingerprinting of medically important microorganisms by use of PCR. Clin. Microbiol. Rev. 7:174‐184.
   Wallace, M.I., Molloy, J.E., and Trentham, D.R. 2003. Combined single‐molecule force and fluorescence measurements for biology. J. Biol. 2(1):4.
   Wang, M.D. and Axelrod, D. 1994. Time‐lapse total internal reflection fluorescence video of acetylcholine receptor cluster formation on myotubes. Dev. Dynam. 201:29‐40.
   Wehland, J., Willingham, M.C., Gallo, M.G., and Pastan, I. 1982. The morphologic pathway of exocytosis of the vesicular somatitis virus G protein in cultured fibrolasts. Cell 28:831‐841.
   Wong, G.K., Yu, J., Thayer, E.C., and Olson, M.V. 1997. Multiple‐complete‐digest restriction fragment mapping: Generating sequence‐ready maps for large scale DNA sequencing. Proc. Natl. Acad. Sci. U.S.A. 94:5225‐5230.
   Xiao, M., Phong, A., Ha, C., Chan, T., Cai, D., Leung, L., Wan, E., Kistler, A.L., DeRisi, J.L., Selvin, P.R., and Kwok, P. 2007. Rapid DNA mapping by fluorescent single molecule detection. Nucleic Acids Res. 35:e16.
   Yildiz, A., Forkey, J.N., McKinney, S.A., Ha, T., Goldman, Y.E., and Selvin, P.R. 2003. Myosin V walks hand‐over‐hand: Single fluorophore imaging with 1.5 nm localization. Science 300:2061‐2065.
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
  Molecular Expressions Microscopy Primer. Excellent presentation of optical microscopy.
PDF or HTML at Wiley Online Library