Total Internal Reflection Fluorescence Microscopy for High‐Resolution Imaging of Cell‐Surface Events

Jyoti K. Jaiswal1, Sanford M. Simon1

1 Rockefeller University, New York, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.12
DOI:  10.1002/0471143030.cb0412s20
Online Posting Date:  November, 2003
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Abstract

The wavelength of light imposes a physical limit of ∼400 nm on the maximum resolution that can be achieved using light microscopy. This unit will describe the use of total internal reflection fluorescence microscopy (TIR‐FM), or evanescent wave microscopy, an approach that partially overcomes this physical limit and permits one to selectively image just those fluorophores in the optical plane (along the z axis) within 50 nm of the cell surface. TIR‐FM works by means of limiting the depth of penetration of the excitation light within this narrow region. This narrow excitatory plane not only provides a high signal‐to‐noise ratio but also minimizes the photodamage to the cell.

Keywords: total internal reflection; evanescent wave; fluorescence microscopy

     
 
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Table of Contents

  • Basic Protocol 1: Setting Up a Through‐The‐Prism TIR‐FM System
  • Alternate Protocol 1: Setting Up a Through‐The‐Objective TIR‐FM System
  • Support Protocol 1: Measuring the Incident Angle of The Excitatory Beam
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Setting Up a Through‐The‐Prism TIR‐FM System

  Materials
  • 40‐ to 100‐nm fluorescent polystyrene beads (Molecular Probes)
  • 0.05× phosphate‐buffered saline (PBS; appendix 2A)
  • Immersion oil appropriate for objective (see Critical Parameters)
  • Wide‐field epifluorescence microscope, upright or inverted (unit 4.1) with appropriate excitation and emission filters
  • Prism (see Background Information; Edmund Scientific)
  • Laser (see Critical Parameters; Spectrum Physics, Coherent, Melles‐Griot, and others), with single‐mode laser optical fiber or system of mirrors and focusing lens
  • Sykes‐Moore chamber (Bellco Glass) or other coverslip mounting chamber
  • Laser‐safety goggles

Alternate Protocol 1: Setting Up a Through‐The‐Objective TIR‐FM System

  • Optical mounts including mirrors, optical fiber couplers, and fiber optics or lenses for coupling the laser beam expander to the microscope port (Olympus, TILL Photonics)
  • Plano‐convex lens of short radius of curvature or hemispherical or triangular prism and converging lens with focal length of several centimeters
  • High‐numerical‐aperture objective (see Critical Parameters)

Support Protocol 1: Measuring the Incident Angle of The Excitatory Beam

  • For materials, see protocol 2.
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Figures

Videos

Literature Cited

Literature Cited
   Amann, K.J. and Pollard, T.D. 2001. Direct real‐time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy. Proc. Natl. Acad. Sci. U.S.A. 98:15009‐15013.
   Axelrod, D. 1981. Cell‐substrate contacts illuminated by total internal reflection fluorescence. J. Cell Biol. 89:141‐145.
   Axelrod, D. 1983. Lateral motion of membrane proteins and biological function. J. Membrane Biol. 75:1‐10.
   Axelrod, D. 1989. Total internal reflection fluorescence microscopy. Methods Cell. Biol. 30:245‐270.
   Axelrod, D. 2001. Selective imaging of surface fluorescence with very high aperture microscope objectives. J. Biomed. Opt. 6:6‐13.
   Axelrod, D., Bauer, H.C., Stya, M., and Christian, C.N. 1981. A factor from neurons induces partial immobilization of nonclustered acetylcholine receptors on cultured muscle cells. J. Cell Biol. 88:459‐462.
   Gingell, D. 1981. The interpretation of interference‐reflection images of spread cells: Significant contributions from thin peripheral cytoplasm. J. Cell Sci. 49:237‐247.
   Greenslade, T.B. Jr. 1997. The Liquid Vein: Nineteenth Century Illustrations (LVII). Phys. Teach. 35:207.
   Kawano, Y., Abe, C., Kaneda, T., Aono, Y., Abe, K., Tamura, K., and Terakawa, S. 2000. High numerical aperature objective lenses and optical system improved objective type total internal reflection fluorescence microscopy. Proceedings SPIE 4098:142‐151.
   Pepper, J.H. 1860. The boy's playbook of science: Including the various manipulations and arrangements of chemical and philosophical apparatus required for the successful performance of scientific experiments. In Illustration of the Elementary Branches of Chemistry and Natural Philosophy. Routledge, Warne, and Routledge, London.
   Schmoranzer, J., Goulian, M., Axelrod, D., and Simon, S.M. 2000. Imaging constitutive exocytosis with total internal reflection fluorescence microscopy. J. Cell Biol. 149:23‐32.
   Steyer, J.A. and Almers, W. 2001. A real‐time view of life within 100 nm of the plasma membrane. Nat. Rev. Mol. Cell Biol. 2:268‐275.
   Sund, S.E. and Axelrod, D. 2000. Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching. Biophys. J. 79:1655‐1669.
   Terakawa, S., Sakurai, T., and Abe, K. 1997. Development of an objective lens with a high numerical aperature for light microscopy. Bioimages 5:24.
   Toomre, D. and Manstein, D.J. 2001. Lighting up the cell surface with evanescent wave microscopy. Trends Cell Biol. 11:298‐303.
   Wagner, M.L. and Tamm, L.K. 2000. Tethered polymer‐supported planar lipid bilayers for reconstitution of integral membrane proteins: Silane‐polyethyleneglycol‐lipid as a cushion and covalent linker. Biophys. J. 79:1400‐1414.
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