Total Internal Reflection Fluorescence Microscopy

Total internal reflection fluorescence microscopy, often abbreviated as TIRF microcopy, is best suited to image and probe a cellular environment within a

Total Internal Reflection Microscopy

distance of 100nm from a solid substrate. It relies on excitation of fluorescence in a thin zone of 100nm from a solid substrate of refractive index higher than that of the cellular environment being imaged, by using the electromagnetic energy in the form of an evanescent wave. The concept of an evanescent wave can be understood by using the propagation of light through a prism of refractive index n1 to the cellular environment of a lower refractive index n2. At the interface, a refraction would occur at a small incidence angle. But when the angle of incidence exceeds a value 8c, called the critical angle, the light beam is reflected from the interface as shown in Figure 7.16. This process is called total internal reflection (TIR). The critical angle 8c is given by the equation

As shown in the figure, for incidence angle >8c, the light is totally internally reflected back to the prism from the prism/cellular environment interface. The refractive index n1 of a standard glass prism is about 1.52, while the refractive index n2 of an intact cell interior can be as high as 1.38. The critical angle for these n1 and n2 parameters is 65°. For permeabilized, hemolyzed, or fixed cells, the n2 value is that of an aqueous buffer which is 1.33, yielding a critical angle of 61°.

Even under the condition of TIR, a portion of the incident energy penetrates the prism surface and enters the cellular environment in contact with the prism surface. This penetrating light energy (or wave) is called an evanescent wave or an evanescent field (Figure 7.17). In contrast to a propagating mode (oscillating electromagnetic field with the propagation

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Standing wave n1 n2

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Figure 7.17. Evanescent wave extending beyond the guiding region and decaying exponentially. For waveguiding, n1 > n2, where n2 is the refractive index of surrounding medium and n1 is the refractive index of guiding region.

constant k, defined in Chapter 2, as a real quantity), an evanescent wave has a rapidly decaying electric field amplitude, with an imaginary propagation constant k. Therefore, its electric field amplitude Ez decays exponentially with distance z into the surrounding cellular medium of lower refractive index n2 as where E0 is the electric field at the surface of the prism (solid substrate of higher refractive index). The parameter dp, also called the penetration depth, is defined as the distance at which the electric field amplitude reduces to 1/e of E0. The term dp can be shown to be given as (Sutherland et al., 1984; Boisde and Harmer, 1996).

Typically, the penetration depths dp for the visible light are 50-100 nm. The evanescent wave energy can be absorbed by a fluorophore to generate fluorescence which can be used to image fluorescently labeled biological targets. However, because of the rapidly (exponentially) decaying nature of the evanescent field, only the fluorescently labeled biological specimen near the substrate (prism) surface generates fluorescence and can thus be imaged. The fluorophores that are further away in the bulk of the cellular medium are not excited. This feature allows one to obtain a high-quality image of the flu-

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Figure 7.18. Inverted microscope TIP configuration. (Reproduced with permission from tion.html.)

Figure 7.18. Inverted microscope TIP configuration. (Reproduced with permission from tion.html.)

orescently labeled biologic near the surface, with the following advantages (Axelrod, 2001):

• Very low background fluorescence

• No out-of-focus fluorescence

• Minimal exposure of cells to light in any other planes in the sample, except near the interface

The TIRF imaging offers a number of relative merits compared to the con-focal microscopy. TIRF allows one to achieve a narrower depth of optical section (0.1 mm) compared to a typical value of 0.5 mm achieved in confocal microscopy. The illumination and hence the excitation are confined to a thin section (near the interface) in the case of TIRF, thus limiting any light-induced damage to cell viability. The TIRF microscopy is also much less expensive than the confocal microscopy, because one can use a standard microcope with TIRF attachment (or TIRF microscopy kits) available from a number of commercial sources.

Figure 7.18 shows two different prism based TIRF setups utilizing an inverted microscope. In Figure 7.18a, a prism is employed to achieve total internal reflection; the maximum incidence angle is obtained by introducing the laser beam from the horizontal direction. This arrangement is not compatible with conventional transmission imaging techniques. In Figure 7.18b, a trapezoidal prism is used and the incoming laser beam is vertical, so the total internal reflection area does not shift laterally when the prism is raised and lowered during specimen changes. In addition, transmission imaging techniques are compatible with this experimental design. Another approach is to utilize a hemispherical prism which permits continuous variation of the incidence angle over a wide range.

TIRF microscopy has been used for numerous applications that take advantage of the surface selectivity. Some of these are:

• Single-molecule fluorescence detection near a surface (Dickson et al., 1996; Vale et al., 1996; Ha et al., 1999; Sako et al., 2000)

• Study of binding of extracellular and intracellular proteins to cell surface receptors and artificial membranes (McKiernan et al., 1997; Sand et al., 1999; Lagerholm et al., 2000)

TIRF microscopy can be used with other optical imaging techniques such as fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM), fluorescence recovery after photobleaching (FRAP), and nonlinear optical imaging. FRET and FLIM are discussed in Sections 7.13 and 7.14. The TIRF microscopy can also utilize two-photon or multiphoton excitation of the fluorophores, similar to what was discussed above under two-photon laser scanning microscopy. Lakowicz and co-workers (Gryczynski et al., 1997) demonstrated two-photon excitation of a calcium probe Indo-1 using an evanescent wave.

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