TIRF (Total Internal Reflection Fluorescence)

Abstract

Total internal reflection fluorescence (TIRF) stands out as a technique allowing wide‐field near‐membrane imaging by selectively exciting molecules located close to the substrate/water boundary onto which cells are grown. TIRF probes a ∼200‐nm thin layer close to the basal plasma membrane of the cell. This optical sectioning of TIRF is key for individual‐organelle and single‐molecule detection and it is the basis for a growing number of superresolution microscopies, including PALM, STORM and variants of STED and SIM. TIRF microscopy has matured from an expert technique to a routine method in cell‐biological imaging. Implemented with an external prism more than 50 years ago, TIRF has been popularised through the advent of ‘through‐the‐objective’ TIRF, in which a laser beam is focused in the extreme periphery of a high‐numerical aperture objective to direct a highly inclined collimated beam to the reflecting interface. Inexpensive and conceptually simple, the technique is not without flaws particularly when it comes to image quantification. After a short introduction, this tutorial recalls the principles and main geometries for setting up TIRF, gives an overview of experiments relying on evanescent‐wave excitation and also discusses some problems and remedies when TIRF images shall be used as measurements.

Key Concepts

  • Spatially confined fluorescence can be excited in close proximity of a dielectric interface by the evanescent wave set up by total internal reflection.
  • 100% pure evanescent fields are impractical; there is always a bit of contaminating epifluorescence.
  • The selective detection of supercritical angle fluorescence (SAF) is the optical equivalent to TIRF on the emission site, with similar optical sectioning.
  • Combining TIRF and SAF improves optical sectioning and facilitates image quantification.
  • Apart from the z‐localisation of excitation light, evanescent fields have other properties interesting for biological imaging, including an unusual polarisation behaviour.

Keywords: membrane dynamics; lipid rafts; superresolution; single‐vesicle; background reduction

Figure 1. Principle of total internal reflection (TIR). (a) Refraction and reflection of light at the dielectric interface between a glass coverslip (n3 = 1.52, shaded) and an aqueous medium (n1 = 1.33, clear). A possible intermediate layer was neglected (n2 = 0, t2 = 0). The relative intensities of the transmitted and reflected beams, T(θ) and R(θ), respectively, are given by the Fresnel equation and are schematised for two incoming beams (through and dashed line). θ is the polar beam angle measured against the surface normal (optical axis). (b) TIR occurs for θ > θc. Inset shows the evanescent wave (EW) set up in the optically rarer medium through TIR. The EW is an inhomogeneous surface wave propagating in +x‐direction (arrow). Its intensity decays exponentially along z with a length constant (penetration depth) δ(θ, λ). (c) Evolution of δ as a function of θ, for n3 = 1.52 and different sample indices n1 = 1.33, 1.35 and 1.37. Note the shift that occurs in θc from 61.2° to 62.7° and 64.4°. δ asymptotically approaches λ/8 and only slowly depends on θ for very large beam angles. In practice, the maximal beam angle is eventually limited by the objective NA, of the order of 74° for an NA 1.45 objective.
Figure 2. Popular TIR configurations. (a) Upright prism configuration. A laser beam (turquoise) is directed to the reflecting interface via an external glass prism, depicted here as a hemisphere. Scattered light or EW‐excited fluorescence is detected through a water‐immersion microscope objective (MO), that is, excitation and collection optics are totally separated. This arrangement facilitates beam angle (θ) scans. (b) Prismless ‘through‐the‐objective’ TIR. Schematic representation of how a high‐NA objective can be used to guide light at a supercritical angle to the reflecting interface. Fluorescence is collected in through the same objective. Inset: focusing a laser beam in the objective back‐focal plane (bfp) generates a collimated beam emerging from the objective. Moving it from the optical axis (dash‐dotted) to the periphery inclines the beam until it suffers TIR.
Figure 3. Problems of and remedies for better TIRF images. (a) Uneven illumination affects the interpretation of biological TIRF images. Unidirectional and azimuthal beam spinning (spTIRF) images of fluorescent lysosomes in a cultured mouse cortical astrocyte labelled with FM2–10 at θ = 73°. Symbols indicate position of the focused spot in the objective bfp. Note the flare of intracellular fluorescence collinear with EW propagation direction, absent on the spTIRF image. Scale bar, 20 µm. Reproduced from Brunstein et al. (2014a) © Elsevier. (b) Nonlinear EW excitation is one way to suppress unwanted scattered light. 860‐nm 2PEF (top) and 488‐nm 1PEF (bottom) images of acridine orange (AO) labelled chromaffin granules at equal probe depth of θ ≈ 110 nm. Scale bar, 5 µm; integration time 100 ms; images are subsequent frames, with <3 ms interframe interval. (Reproduced with permission from Schapper et al. (2003) © Springer Science + Business Media.) (c) Pseudocolour overlay of an EW‐excited virtual supercritical angle fluorescence (vSAF) image (red) and the corresponding undercritical‐angle fluorescence (UAF, red) Scale bar, 5 µm. Note the movement of small vesicles during the successive acquisition of the UAF and (UAF + SAF) component images (arrowheads). Inset shows pseudocoloured BFP image of collected SAF and UAF‐iris stop. (d) schematic optical layout of an improved SAF‐emission path, which is, in principle, a dual‐view (image splitter) device for the simultaneous detection of SAF and UAF images. Abbreviations: obj – objective, BFP – back‐focal plane, (EM)CCD – (electron‐multiplying) charge‐coupled device camera, dic – dichroic mirror, TL – tube lens, BS – beam splitter, BL – Bertrand lens, FL – focusing lens. Brunstein et al. ().
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Further Reading

Axelrod D (2013) Evanescent excitation and emission in fluorescence microscopy. Biophysical Journal 104:1014–1019.

Barocca T, Balaa K, Lévêque‐Fort S and Fort S (2012) Full‐field near‐field optical microscope for cell imaging. Physical Reviews Letters 21: 218101.

Brunstein M, Teremetz M, Hérault K, Tourain C and Oheim M (2015a) Eliminating unwanted far‐field excitation in objective‐type TIRF. Part I. Identifying sources of nonevanescent excitation light. Biophysical Journal 106 (5):1020–1032.

Brunstein M, Hérault K and Oheim M (2015b) Eliminating unwanted far‐field excitation in objective‐type TIRF. Part II. Combined evanescent‐wave excitation and supercritical‐angle fluorescence detection improves optical sectioning. Biophysical Journal 106 (5):1044–1056.

Oheim M and Schapper F (2006) Non‐linear evanescent field imaging. Journal of Physics D: Applied Physics 38: R185‐R197

Rohrbach A (2000b) Observing secretory granules with a multiangle evanescent wave microscope. Biophysical Journal 78: 2641–2654.

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How to Cite close
Oheim, Martin(Mar 2016) TIRF (Total Internal Reflection Fluorescence). In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0022505]