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

Daniel S. Johnson1, Jyoti K. Jaiswal2, Sanford Simon1

1 The Rockefeller University, Laboratory of Cellular Biophysics, New York, New York, 2 Center for Genetic Medicine Research, Children's National Medical Center, Washington, D.C.
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.29
DOI:  10.1002/0471142956.cy1229s61
Online Posting Date:  July, 2012
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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.

Keywords: total internal reflection fluorescence microcopy; fluorescence; cell imaging

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Table of Contents

  • Introduction
  • Basic Protocol 1: Through‐the‐Objective TIRF Protocol
  • Alternate Protocol 1: Improved Uniformity in the Excitation Field Protocol
  • Basic Protocol 2: Through‐the‐Prism TIRF Protocol
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Through‐the‐Objective TIRF Protocol

  • 40‐ to 100‐nm fluorescent microspheres (e.g., Invitrogen FluoSpheres)
  • Biological sample
  • Upright or inverted infinity‐corrected fluorescence microscope including appropriate emission filters and dichroics
  • High numerical aperture (≥1.45) objective lens (see Critical Parameters)
  • Laser(s) at desired excitation wavelength(s), with system of lenses and mirrors to combine lasers into similar beam diameter
  • TIR focusing lens and TIR steering mirror; appropriate mounts with ability to translate optical components (if exciting with multiple wavelengths, select a lens with minimal chromatic aberrations)
  • Periscope mirror systems, optional
  • Beam expander
  • Glass sample chamber [e.g. glass bottom dish (MakTek) or Sykes‐Moore chamber (Bellco Glass)]
NOTE: For all of these studies, the excitation source will be a laser. However, it is also possible to use other light sources.

Alternate Protocol 1: Improved Uniformity in the Excitation Field Protocol

  • Steerable mirror, such as a 2‐axis Galvo scan head (Nutfield Technology, Cambridge Technology), a fast steering mirror (Newport, Optics in Motion, Thorlabs), or a tip‐tilt piezomirror (PhysikInstrumente, MadCity Labs, Piezosystem Jena)
  • Two‐channel function generator or a function‐generating computer card (such as a National Instruments multifunction DAQ card with analog output channels); if using a computer card, control software will also be necessary

Basic Protocol 2: Through‐the‐Prism TIRF Protocol

  • Immersion oil
  • Biological sample
  • Upright or inverted microscope with appropriate emission filters
  • Triangular‐ or hemispherical‐shaped glass prism (such as right angle BK7 or fused silica prism) and mount to hold the prism on the appropriate side of the sample (to ease adjustment, the mount should be able to translate easily away from the sample)
  • Appropriate microscope objective for imaging (in order to minimize spherical aberrations over long working distances, it is advisable to use a water‐dipping or water‐immersion objective)
  • Focusing lens (∼100‐ to 200‐mm focal length) in a mount that can be easily translated; select a weakly focusing lens so that laser light is focused to a diffraction limited spot roughly the size of the viewable imaging plane
  • Beam steering mirror in a mount so that it can be easily translated and rotated around small angles
  • Laser (and beam combining optics if using multiple lasers)
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Literature Cited

Literature Cited
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   Axelrod, D. 1979. Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization. Biophys. J. 26:557‐574.
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   Axelrod, D., Burghardt, T.P., and Thompson, N.L., 1984. Total internal reflection fluorescence. Ann. Rev. Biophys. Bioeng. 13:247‐268.
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