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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  •   FigureFigure 12.29.1 (A) Schematic of through‐the‐objective TIRF illuminator. Lasers are combined with longpass mirror(s) (LP) and then the beam diameter is increased with a telescope beam expander (BE). The periscope mirror system (PM) facilitates alignment by allowing translation of the beam on the TIR mirror (TIR M). Following the reflection of light from the TIR mirror, the collimated light is focused with a TIR lens (TIR L) onto the back‐focal‐plane (BFP) of the objective. Between the TIR lens and the objective is a dichroic mirror (DM), which allows emitted light from fluorophores to pass to an imaging camera (C). Light focused to the BFP becomes collimated in the sample‐plane (SP), where the biological sample is on the surface of a glass dish. This figure illustrates two potential configuration scenarios of the TIR mirror. One laser is focused to the radial center of the BFP, which results in the excitation light propagating vertically through the sample. The other laser demonstrates a different tilt of the TIR mirror that results in the beam being focused radially away from the center of the BFP. In this scenario, the light incident on the glass sample chamber is above the critical angle, resulting in TIR. NOTE: The is realized by replacing the TIR mirror with an electro‐mechanical mirror(s). (B) Schematic of through‐the‐prism TIRF illuminator. Separate beams are combined with longpass filters and then reflected off of a TIR mirror into a weakly focusing TIR lens. Light passes through a prism (P) where TIR occurs on a glass slide above the sample. Changing the tilt of the TIR mirror translates the position of the focused field, while translating the mirror position changes the angle of incidence.
  •   FigureFigure 12.29.2 Illustration of refraction of light between media of different refractive index. Three different incident angles are shown: (A) below the critical angle, (B) at the critical angle, and (C) above the critical angle. The incident medium has a refractive index, n i, which is higher than the refractive index of the transmitted medium, n t. Incident light is illustrated as an infinite plane wave, with the distance between the wavefronts (solid lines) being the wavelength (λ). The speed of light is faster in the transmitted medium; therefore, as illustrated by the larger distance between wavefronts, the wavelength (λ t) is longer in the transmitted medium. At the boundary, wavefronts must remain continuous. In order for (1) the wavefronts to remain continuous and (2) the wavelength to increase, the light must bend further from normal at the interface. (B) At the critical angle (θ d), the transmitted light propagates along the surface of the interface. (C) Beyond the critical angle, light does not propagate into the transmitted medium. TIR results in an exponentially decaying evanescent field with a depth constant of d.
  •   FigureFigure 12.29.3 Illustration of evanescent field penetration depth, d, versus incident angle. Depth is given for two different types of microscope glass coverslips; conventional ( n t = 1.52) and high refractive index ( n t = 1.78). For each incident glass, two transmitted media are given: water ( n i = 1.33) and cell cytoplasm ( n i = 1.38). The maximum potential incident angle is given for three objectives: a 1.45 NA objective (using 1.52 glass), a 1.49 NA objective (using 1.52 glass), and a 1.65 NA objective (using 1.78 glass).
  •   FigureFigure 12.29.4 TIRF microscopy image of acridine orange stuck to glass coverslip (A) with the azimuthal incident angle of the light fixed during imaging or (B) with the azimuthal incident angle rotating during imaging. Presence of patterned interference fringes decrease significantly when the excitation field is rotating.


Literature Cited

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