Abstract
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
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Mattheyses AL, Simon SM, Rappoport JZ (2010) Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci 123:3621–3628
Axelrod D (2001) Total internal reflection fluorescence microscopy in cell biology. Traffic 2:764–774
Axelrod D (2008) Total internal reflection fluorescence microscopy. Methods Cell Biol 89:169–221
Rappoport JZ, Simon SM (2003) Real-time analysis of clathrin-mediated endocytosis during cell migration. J Cell Sci 116:847–855
Rappoport JZ, Benmerah A, Simon SM (2005) Analysis of the AP-2 adaptor complex and cargo during clathrin-mediated endocytosis. Traffic 6:539–547
Merrifield CJ, Perrais D, Zenisek D (2005) Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell 121:593–606
Merrifield CJ, Feldman ME, Wan L et al (2002) Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nat Cell Biol 4:691–698
Fix M, Melia TJ, Jaiswal JK et al (2004) Imaging single membrane fusion events mediated by SNARE proteins. Proc Natl Acad Sci U S A 101:7311–7316
Akopova I, Tatur S, Grygorczyk M et al (2011) Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells. Purinerg Signal 8:59–70
Grigoriev I, Splinter D, Keijzer N et al (2007) Rab6 regulates transport and targeting of exocytotic carriers. Dev Cell 13:305–314
Berginski ME, Vitriol EA, Hahn KM et al (2011) High-resolution quantification of focal adhesion spatiotemporal dynamics in living cells. PLoS One 6:e22025
Partridge MA, Marcantonio EE (2006) Initiation of attachment and generation of mature focal adhesions by integrin-containing filopodia in cell spreading. Mol Biol Cell 17:4237–4248
Fletcher SJ, Poulter NS, Haining EJ et al (2011) Clathrin‐mediated endocytosis regulates occludin, and not focal adhesion, distribution during epithelial wound healing. Biol Cell 104:238–256
Lock FE, Ryan KR, Poulter NS et al (2012) Differential regulation of adhesion complex turnover by ROCK1 and ROCK2. PLoS One 7:e31423
Manneville JB (2006) Use of TIRF microscopy to visualize actin and microtubules in migrating cells. Meth Enzymol 406:520–532
Webb RL, Rozov O, Watkins SC et al (2009) Using total internal reflection fluorescence (TIRF) microscopy to visualize cortical actin and microtubules in the drosophila syncytial embryo. Dev Dynam 238:2622–2632
Dixit R, Ross JL (2010) Studying plus-end tracking at single molecule resolution using TIRF microscopy. Method Cell Biol 95:543–554
Gardner MK, Charlebois BD, Jánosi IM et al (2011) Rapid microtubule self-assembly kinetics. Cell 146:582–592
Graham-Smith SF, King TA (2000) Optics and photonics: an introduction. Wiley, Chichester, UK
Millis BA (2012) Evanescent-wave field imaging: an introduction to total internal reflection fluorescence microscopy. Methods Mol Biol 823:295–309
Rappoport JZ, Simon SM (2009) Endocytic trafficking of activated EGFR is AP-2 dependent and occurs through preformed clathrin spots. J Cell Sci 122:1301–1305
Born M, Wolf E, Bhatia AB (1999) Principles of optics: electromagnetic theory of propagation, interference and diffraction of light. Cambridge Univ Press, Cambridge, UK
Anantharam A, Onoa B, Edwards RH et al (2010) Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM. J Cell Biol 188:415–428
Sund SE, Swanson JA, Axelrod D (1999) Cell membrane orientation visualized by polarized total internal reflection fluorescence. Biophys J 77:2266–2283
Axelrod D (1981) Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol 89:141–145
Stock K, Sailer R, Strauss W et al (2003) Variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM): realization and application of a compact illumination device. J Microsc 211:19–29
Fish KN (2009) Total internal reflection fluorescence (TIRF) microscopy. Curr Protoc Cytom Chapter 12, Unit 12.18
Olveczky BP, Periasamy N, Verkman A (1997) Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy. Biophys J 73:2836–2847
Oheim M, Loerke D, Chow RH et al (1999) Evanescent-wave microscopy: a new tool to gain insight into the control of transmitter release. Philos T Roy Soc B 354:307–318
Rohrbach A (2000) Observing secretory granules with a multiangle evanescent wave microscope. Biophys J 78:2641–2654
Loerke D, Stühmer W, Oheim M (2002) Quantifying axial secretory-granule motion with variable-angle evanescent-field excitation. J Neurosci Methods 119:65–73
Weisswange I, Bretschneider T, Anderson KI (2005) The leading edge is a lipid diffusion barrier. J Cell Sci 118:4375–4380
Millán J, Hewlett L, Glyn M et al (2006) Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola-and F-actin-rich domains. Nat Cell Biol 8:113–123
Saffarian S, Kirchhausen T (2008) Differential evanescence nanometry: live-cell fluorescence measurements with 10-nm axial resolution on the plasma membrane. Biophys J 94:2333–2342
Pitkeathly WT, Poulter NS, Claridge E et al (2011) Auto-align – multi-modality fluorescence microscopy image co-registration. Traffic 13:204–217
Mattheyses AL, Axelrod D (2006) Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence. J Biomed Opt 11:014006
Doherty GJ, McMahon HT (2009) Mechanisms of endocytosis. Annu Rev Biochem 78:857–902
Damke H (1996) Dynamin and receptor-mediated endocytosis. FEBS Lett 389:48–51
Roth MG (2005) Clathrin-mediated endocytosis before fluorescent proteins. Nat Rev Mol Cell Biol 7:63–68
Gaidarov I, Santini F, Warren RA et al (1999) Spatial control of coated-pit dynamics in living cells. Nat Cell Biol 1:1–7
Cao H, Garcia F, McNiven MA (1998) Differential distribution of dynamin isoforms in mammalian cells. Mol Biol Cell 9:2595–2609
Rappoport JZ, Taha BW, Lemeer S et al (2003) The AP-2 complex is excluded from the dynamic population of plasma membrane-associated clathrin. J Biol Chem 278:47357–47360
Yarar D, Waterman-Storer CM, Schmid SL (2005) A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis. Mol Biol Cell 16:964–975
Rappoport JZ (2008) Focusing on clathrin-mediated endocytosis. Biochem J 412:415–423
Soulet F, Yarar D, Leonard M et al (2005) SNX9 regulates dynamin assembly and is required for efficient clathrin-mediated endocytosis. Mol Biol Cell 16:2058–2067
Lee D, Wu X, Eisenberg E et al (2006) Recruitment dynamics of GAK and auxilin to clathrin-coated pits during endocytosis. J Cell Sci 119:3502–3512
Jahn R, Südhof TC (1999) Membrane fusion and exocytosis. Annu Rev Biochem 68:863–911
Simon SM (2009) Partial internal reflections on total internal reflection fluorescent microscopy. Trends Cell Biol 19:661–668
Jaiswal JK, Fix M, Takano T et al (2007) Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes. Proc Natl Acad Sci U S A 104:14151–14156
Schmoranzer J, Goulian M, Axelrod D et al (2000) Imaging constitutive exocytosis with total internal reflection fluorescence microscopy. J Cell Biol 149:23–32
Burridge K, Fath K, Kelly T et al (1988) Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol 4:487–525
Burridge K, Chrzanowska-Wodnicka M (1996) Focal adhesions, contractility, and signaling. Annu Rev Cell Dev Biol 12:463–519
Webb DJ, Parsons JT, Horwitz AF (2002) Adhesion assembly, disassembly and turnover in migrating cells-over and over and over again. Nat Cell Biol 4:E97–E100
Ezratty EJ, Bertaux C, Marcantonio EE et al (2009) Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells. J Cell Biol 187:733–747
Acknowledgments
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Poulter, N.S., Pitkeathly, W.T.E., Smith, P.J., Rappoport, J.Z. (2015). The Physical Basis of Total Internal Reflection Fluorescence (TIRF) Microscopy and Its Cellular Applications. In: Verveer, P. (eds) Advanced Fluorescence Microscopy. Methods in Molecular Biology, vol 1251. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2080-8_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2080-8_1
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2079-2
Online ISBN: 978-1-4939-2080-8
eBook Packages: Springer Protocols