Pure Appl. Chem. 2014; 86(8): 1303–1320

IUPAC Technical Report

Alex E. Knight* Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

Abstract: Total internal reflection fluorescence (TIRF) is a popular illumination technique in microscopy, with many applications in and molecular and biophysics. The chief advantage of the technique is the high contrast that can be achieved by restricting fluorescent excitation to a thin layer. We summarise the optical theory needed to understand the technique and various aspects required for a practical imple- mentation of it, including the merits of different TIRF geometries. Finally, we discuss a variety of applications including super-resolution microscopy and high-throughput DNA sequencing technologies.

Keywords: DNA sequencing; evanescent ; fluorescence; fluorescence spectroscopy; IUPAC Analytical Chemistry Division; IUPAC Organic and Biomolecular Chemistry Division; IUPAC Physical and Biophysical Chemistry Division; polarisation; super-resolution microscopy; total internal reflection.

DOI 10.1515/pac-2012-0605 Received June 6, 2012; accepted April 17, 2014

Contents 1. INTRODUCTION ������������������������������������������������������������������������������������������������������������������������� 1304 2. THEORY ������������������������������������������������������������������������������������������������������������������������������������� 1305 2.1 Total internal reflection ��������������������������������������������������������������������������������������������������������������� 1305 2.2 Penetration depth ������������������������������������������������������������������������������������������������������������������������ 1307 2.3 Intensity and polarisation ������������������������������������������������������������������������������������������������������������1308 2.4 Emission characteristics �������������������������������������������������������������������������������������������������������������� 1310 3. IMPLEMENTING TIRF ����������������������������������������������������������������������������������������������������������������� 1311 3.1 Practical aspects ���������������������������������������������������������������������������������������������������������������������������1311 3.2 Lasers �������������������������������������������������������������������������������������������������������������������������������������������1311 3.3 Polarisation ����������������������������������������������������������������������������������������������������������������������������������1311 3.4 TIRF configurations ����������������������������������������������������������������������������������������������������������������������1311 3.4.1 Prism TIRF �������������������������������������������������������������������������������������������������������������������������1312 3.4.2 Objective TIRF ������������������������������������������������������������������������������������������������������������������ 1314 3.5 Optimising TIRF ����������������������������������������������������������������������������������������������������������������������������1315 3.6 Resolution ������������������������������������������������������������������������������������������������������������������������������������ 1316 3.7 Other matters ������������������������������������������������������������������������������������������������������������������������������� 1316

Article note: Sponsoring bodies: IUPAC Physical and Biophysical Chemistry Division; IUPAC Organic and Biomolecular Chemis- try Division; IUPAC Analytical Chemistry Division; see more details on p. 1318.

*Corresponding author: Alex E. Knight, Analytical Science Division, National Physical Laboratory, Hampton Road, Teddington, Middlesex, TW11 0LW, UK, e-mail: [email protected]

© 2014 IUPAC & De Gruyter 1304 A. E. Knight: Total internal reflection fluorescence microscopy

4. APPLICATIONS ���������������������������������������������������������������������������������������������������������������������������� 1317 5. CONCLUSION �������������������������������������������������������������������������������������������������������������������������������1318 6. MEMBERSHIP OF SPONSORING BODIES �������������������������������������������������������������������������������������1318 7. REFERENCES �������������������������������������������������������������������������������������������������������������������������������1319

Abbreviations FCS fluorescence correlation spectroscopy SNOM scanning near-field optical microscopy TEF tip-enhanced fluorescence TIRF total internal reflection fluorescence microscopy HILO highly inclined and laminated optical sheet microscopy SAF supercritical angle fluorescence TEM transverse electromagnetic mode NA numerical aperture PALM photo activated localisation microscopy STORM stochastic optical reconstruction microscopy GSDIM ground state depletion followed by individual molecule return CCD charge coupled device ICCD intensified CCD EMCCD electron multiplying CCD CMOS complementary metal oxide semiconductor FRET Förster resonance energy transfer AFM atomic force microscopy

1 Introduction

Recent years have seen a huge expansion of interest in single-molecule detection methods. These include electrical methods such as patch clamping, mechanical methods such as atomic force microscopy and optical tweezers, and a variety of optical methods, many of them based on fluorescence. The most widely used such method is fluorescence correlation spectroscopy (FCS) and its many variants; more than 1600 papers on FCS have been published in the last 5 years; see [1]. The common strategy in most such methods is to improve the selectivity for the molecules of interest by restricting the excitation volume. For example, in FCS and confocal approaches [2] excitation is effectively limited to a diffraction-limited focal volume, and a confocal pinhole or similar device is also used to restrict the emission volume. Similarly, in sheet microscopy, excitation is limited to a thin slice of the sample [3]. Probe-based methods, such as scanning near-field optical microscopy (SNOM) or tip-enhanced fluorescence (TEF) rely upon optical near-field effects to locally excite fluorescence. Total internal reflection fluorescence (TIRF) microscopy exploits a similar phenomenon, by using evanescent waves to restrict excitation to a very thin layer near a surface. The popularity of FCS is probably due to a number of factors: it probes molecules in solution, is versatile in application, and a number of commercial systems are available. It is also possible to use it to measure the properties of molecules in living cells. However, in many situations it is desirable to use a different method. Imaging approaches have the advan- tage that they can follow multiple discrete molecules over long periods of time; they can also provide spatial information such as position, and they track moving molecules with time. In contrast, while FCS exploits the molecular nature of the sample, it does not allow individual molecules to be identified and characterised, but only provides statistical information about populations of molecules. One of the most popular single- molecule imaging approaches is TIRF or evanescent microscopy. Like FCS, it is an optically rather simple technique and is therefore robust and relatively easy to set up. A number of commercial systems are available, although these are mostly targeted at cell biology applications, usually not involving single-molecule detection A. E. Knight: Total internal reflection fluorescence microscopy 1305

(although very recently TIRF-based super-resolution systems involving single-molecule imaging have been launched). Furthermore, a number of implementations of TIRF combined with other techniques have been developed, including with optical tweezers, scanning probe microscopy, and FCS. An advantage of TIRF is that it allows wide-field data collection and is, therefore, capable of continuous data collection in parallel on many hundreds or even thousands of molecules. TIRF is limited to viewing molecules at, or near, a surface; so in comparison to confocal microscopy, for example, it cannot be used for three-dimensional imaging of cells.1 However, its excellent depth discrimination is also one of its main advantages, as discussed below. In the following sections, the general theory of the technique is described, together with a discussion of some points of importance in experimental design and the interpretation of data. Although the scope of the article is restricted to TIRF, many of the observations will also apply to other single-molecule imaging modalities.

2 Theory

The essence of total internal reflection (TIR) is that it generates an exponentially decaying wave (or evanescent wave) at the interface between two media, such as and air, or glass and (e.g. the surface of a micro- scope slide). It therefore provides a highly localised fluorescence excitation. In this section, I briefly outline some of the theory necessary to understand some of the properties of the evanescent wave that are relevant to its practical applications. For a more detailed introduction to the theory, consult the comprehensive review by Axelrod and colleagues [5]. Figure 1 schematically illustrates the scenario discussed in the following sections.

2.1 Total internal reflection

Internal reflection occurs when light propagating in a medium (such as glass) encounters an interface with a more rarefied medium (such as water or air) – that is, a medium with a lower (defined in


x-z plane

θi θr

y θt x x-y plane z n3 n1

Fig. 1 Geometry of total internal reflection, showing the coordinate system used in this report. The x-y plane is the interface between the denser medium (refractive index n3) – typically a slide or cover slip – and the less dense medium

(refractive index n1) – typically water or a buffer solution. The x-z plane is the plane of incidence, within which the incident and reflected rays (solid lines) and the transmitted ray (dotted line) lie. The angle between the incident ray and the normal (dash-dot line) is the angle of incidence, θi; the angle of the reflected ray is θr, and of the transmitted (refracted) ray is θt. Incident light that is s-polarised has its vectors perpendicular to the plane of incidence (i.e. purely in the y direction) and the evanescent wave has a corresponding polarisation. Incident light that is p-polarised has its electric field vectors parallel to the plane of incidence, with components in the x and z directions – as does the resulting evanescent wave (see also Fig. 5).

1 However, at subcritical angles, greater depths can be imaged whilst retaining some of the advantages of TIRF illumination. This is sometimes referred to as highly inclined illumination or HILO [4]. 1306 A. E. Knight: Total internal reflection fluorescence microscopy the “Green Book” [6]). (The opposite case – where the light is propagating in the more rarefied medium – is correspondingly termed external reflection [7].) At the interface, the light may be reflected or transmitted. The angle of the transmitted ray can be found from the familiar “Snell’s law”:

nnsinsθθ= in (1) 3i1t where n3 is the refractive index of the first medium, and θi is the angle of incidence; and n1 is the refractive 2 index of the medium and θt is the angle of the transmitted ray. The angle of the reflected ray, θr, is equal to θi. These relationships apply for both internal and external reflection.

Let us consider the internal reflection case in more detail. Figure 2 shows the values of θt and θr for angles of incidence from 0° (normal incidence) to 90° (π/2 rad; glancing incidence). While the angle of the reflected ray is always equal to the angle of incidence, the angle of the transmitted ray is greater (except at normal inci- dence); this corresponds to the transmitted ray bending away from the normal as one would expect. However, the angle of the transmitted ray obviously cannot exceed 90° as this would mean that the ray would re-enter the first medium. The angle of incidence at which θt = 90° is known as the critical angle and, since sin 90° = 1, can be calculated by rearranging eq. 1 as

n == −1 1 θθcisin  (2) n 3

For example, for the quartz–water interface shown in Fig. 2, the critical angle is 65.6° or 1.14 rad.

θi/rad 0 0.5 1.0 1.5

90 θt 1.6 θr 80 θc 1.4 70 1.2 60 1.0 50 θ /rad θ /deg 0.8 40

0.6 30

0.4 20

10 0.2

0 0 010 20 30 40 50 60 70 80 90


Fig. 2 Internal reflection and refraction. Calculated using water (n1 = 1.33) and quartz (n3 = 1.46) as an example. The angle of the reflected ray, θr, is always equal to the angle of incidence at the interface, θi; contrastingly, the angle of the transmitted ray

θt is always greater than the angle of the incident ray (corresponding to the ray being bent away from the normal as it enters the water), except at normal incidence. The difference between the two angles increases until θt reaches 90° (π/2 rad). This occurs when θi reaches a value, θc, known as the critical angle, which is determined by the refractive indices of the two media (see eq. 2) – in this example, 65.6° (1.14 rad). Since the angle of the transmitted ray cannot exceed 90°, mathematically at this point

θt becomes complex (real component plotted in the figure) and physically, total internal reflection occurs.

2 The use of the subscripts 1 and 3 for the refractive indices accommodates the possibility of an intermediate layer at the interface of refractive index n2 [5], although this is beyond the scope of this report. A. E. Knight: Total internal reflection fluorescence microscopy 1307

4.0 532 nm, 66° (1.15 rad) 1064 nm, 66° (1.15 rad) 3.5 532 nm, 70° (1.22 rad)



I 2.0




0 0 100 200 300 400 500 600 700 800 900 1000 z/nm

Fig. 3 Exponential decay of the evanescent intensity away from the surface. The dotted lines indicate the 1/e intensities and the corresponding values of d. For a 532-nm laser at θi = 66° (1.15 rad, just above the critical angle) the penetration depth is 422 nm, and for a 1064-nm laser at the same angle of incidence, the penetration depth is 844 nm. The plots show the decay of the s-polarised intensity with increasing z, with both lasers having an s-polarised incident intensity of 1. The intensity at z = 0 is 3 3.89 for both, as this is not influenced by the wavelength. In contrast, for the 532-nm laser with a larger θi = 70° (or 1.22 rad), the intensity is not only smaller at z = 0, but also decays more rapidly (d = 126 nm).

At angles greater than the critical angle, the transmitted ray ceases to exist, and all of the incident light is reflected. This is the condition known as total internal reflection. At first glance, if no light is transmitted into the sample, one might expect that no fluorescence could be excited. In fact, some energy does penetrate into the sample, in the form of an evanescent wave. One could visualise this as the light “dipping its toe” into the second medium before being reflected. It is the particular properties of this evanescent wave that make it so useful for single-molecule fluorescence imaging. If there is no absorption or scattering at the interface, all the energy passes into the reflected light. This helps to greatly reduce the background from excitation light. However, if energy is absorbed from the evanescent wave it can excite fluorescence in the normal way.

2.2 Penetration depth

The chief feature of the evanescent wave that makes it so useful is the fact that it decays exponentially with depth into the second medium (see Fig. 3)

− I()zI= (0)e zd/ (3)

where z is the distance into the second medium, I(0) is the intensity4 at the interface, and I(z) is the intensity at depth z. The parameter d is referred to as the penetration depth and is the depth at which the intensity falls to 1/e times the intensity at the interface. The penetration depth depends on the refractive indices of the media, the angle of incidence, and the wavelength of the light.

3 This analysis assumes for simplicity that the refractive indices are not wavelength dependent. In practice, of course, there will be small differences due to the dispersion of the media involved. 4 Intensity here refers to the intensity or , which is usually measured in W m-2. In this context, all intensities are expressed relative to an initial intensity, and are therefore dimensionless. See the “Green Book”, Section 2.7 [6]. 1308 A. E. Knight: Total internal reflection fluorescence microscopy

λ dn=−0 (s22in θ n21)− /2 (4) π 3i1 4 where λ0 is the wavelength of the light in a vacuum. The effects of angle of incidence and laser wavelength are illustrated in Fig. 3. As can be seen from Fig. 4, d is a fraction of the wavelength for most angles greater than the critical angle. This extreme z-sectioning of excitation is the chief advantage of the evanescent wave method.

2.3 Intensity and polarisation

What is the intensity of the evanescent wave – is it enough to excite sufficiently bright fluorescence? To answer this fully, we must also introduce a consideration of the effect of polarisation in both the incident light and the evanescent wave. The coordinate system for the discussion that follows is depicted in Fig. 1. The two orthogonal linear polarisations of the incident light are s (perpendicular to the plane of incidence) and p (parallel to the plane of incidence). For s-polarised light, the electric field vectors are oriented solely along the y-axis, whereas for p-polarisation, there will be components along both the z and x axes. Correspondingly, for s-polarised inci- I dent light of intensity s, the intensity of the evanescent wave in the y direction is [5]

4cos2 θ = I i Iy (0) s 2 (5) 1− n

where Iy(0) indicates the intensity at the interface (where z = 0), and n is the ratio of the two refractive indices, I n1/n3. Similarly, the x and y components are related to p, the incident p-polarised intensity, as follows [5]: 4cos22θθ(sin −n2 ) I (0) = I ii (6) x p nn42cossθθ+−in22 ii

θi/deg 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1000 532 nm 635 nm 900 1064 nm




500 d /nm





0 70 75 80 85 90


Fig. 4 Penetration depth, d, as a function of angle of incidence, for a quartz–water interface and for λ0 corresponding to three common laser wavelengths (532, 635, and 1064 nm). Note the greater penetration depth of the longer wavelength lasers. A. E. Knight: Total internal reflection fluorescence microscopy 1309

4cos22θθsin I ii Iz (0) = p (7) 42θθ22− nncossii+ in

The x, y, and z intensities are plotted versus the angle of incidence in Fig. 5a for incident intensities of 1. The most striking feature of this plot is that near the critical angle, the z and y evanescent intensities are four or more times higher than the incident intensities! This effect further enhances the signal-to-noise ratio of the technique. Away from the critical angle, the intensities fall off rapidly; the x intensity is always much weaker

θ /rad a i 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 5.0 Iy (0) 4.5 Ix (0) Iz (0) 4.0



I 2.5





0 70 75 80 85 90 θi/deg

θi/rad b 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 5.0 Is (0) 4.5 Ip (0)




I 2.5





0 70 75 80 85 90 θi/deg

Fig. 5 Intensity of the evanescent wave as a function of angle of incidence and polarisation: (a) x, y, and z components; (b) s and p components. Examples shown are for a quartz–water interface as before. 1310 A. E. Knight: Total internal reflection fluorescence microscopy than the others. For incident light where the s and p intensities are equal, the x, y, and z components will be as shown (e.g. where the incident light is linearly polarised at 45° (π/4 rad) or circularly polarised); for pure s-polarised input there will only be the y component, and for p-polarised input there will only be the x and z components. Fig. 5b shows the evanescent intensities expressed in terms of s and p components, where the s compo- nent is the same as the y component [5]

4cos2 θ ==I i Iss(0)(I y 0) 2 (8) 1 − n and the p component is the sum of the x and y components [5] I (0)(=+II0) (0) (9) p xz which gives [5] 4cos22θθ(2sin)−n2 I ii I pp(0) = (10) 42θθ22− nncossii+ in

The exponential decay and the penetration depth are unaffected by polarisation; the effect of polarisa- tion is simply to change the value of I(0) in eq. 3. For some cell-imaging applications, angles just below the critical angle are used – this technique is referred to as highly inclined illumination or HILO [4]. At the barely subcritical angles used, the intensities near the interface are similar to those seen in the evanescent wave. In all of the preceding discussion, we have implicitly considered the simplest possible case of a single ray of light, representing an infinite plane wave and having a single angle of incidence. In the real-world imple- mentation of the technique, of course, we are dealing with a finite-width beam, with a particular intensity profile, and with the possibility of more than one angle of incidence. Some of the theoretical aspects of this are discussed in [5], but for our purposes we will discuss the practical implications in Section 3.

2.4 Emission characteristics

The foregoing discussion covers the theoretical aspects relating to the excitation of fluorescent molecules in TIRF geometry, as these are the primary consideration for the implementer or user of TIRF imaging. However, it should be noted that in addition to the near-field effects in excitation, there are also near-field effects in emission. Where a fluorophore is very close to the cover slip, its emitted near-field can interact with the surface and be converted into propagating light [8, 9]. This near-field component is predominantly seen as “supercriti- cal” emission (i.e. emission above the critical angle) and therefore appears at the periphery of the objective back aperture. This component also contains information about the orientation of the fluorophore (although in many applications the fluorophores are rotating rapidly and randomly such that this information is lost). These effects have a number of interesting implications: –– Collecting just supercritical emission allows selective detection of molecules near a surface (even when excitation is not by TIRF). This is known as supercritical angle fluorescence or SAF [10, 11]. –– The point spread function of a single molecule with a fixed orientation will differ significantly from the classical Airy disc (see Section 3.6 and Fig. 7). Where molecules are rapidly tumbling, the Airy disc remains a reasonable approximation [8]. –– The information contained in the supercritical emission can be used to measure molecular orientations, distance from the surface, and thickness of intermediate layers [8, 11]. –– Near the surface, the majority of the emitted light is emitted into the glass, further enhancing the effi- ciency of detection of molecules in an objective TIRF configuration (see Section 3.4) [9].

For more information about these effects, please refer to the articles referenced above. A. E. Knight: Total internal reflection fluorescence microscopy 1311

3 Implementing TIRF

3.1 Practical aspects

As has been previously noted, the intensity of the evanescent wave falls off exponentially away from the surface, and this is illustrated in Fig. 3. As shown in eq. 4 and Fig. 4, the penetration depth is strongly influ- enced by the wavelength of the laser used, but the intensity of the evanescent wave is not (see eqs. 5, 6, and 7, and Fig. 3). On the other hand, both the intensity and the penetration depth are strongly influenced by the angle of incidence, falling off rapidly away from the critical angle (Figs. 4 and 5, respectively). Therefore, in many cases, where signal is limiting, it will be desirable to work close to the critical angle. Alternatively, where there is a stronger signal, it may be possible to use greater angles of incidence to reduce the penetra- tion depth if required.

3.2 Lasers

Because of the strong angle-dependence of the properties of the evanescent wave, particularly around the critical angle, and to avoid a mixture of glancing illumination and TIRF, it is desirable to use excitation light that is collimated, or at least converging at a narrow angle. This makes lasers ideal as the light source for TIRF, which is why they are almost universally used. Other beneficial aspects include the monochromatic light, which is easier to filter out, reducing the background from excitation light. Modern solid-state lasers are also quiet, stable, and produce relatively little heat; furthermore, they can readily be controlled by computer, facilitating the automation of experiments.

3.3 Polarisation

Another feature of lasers is that most produce strongly linearly polarised light, which is often beneficial where the polarisation of the evanescent wave needs to be controlled. Many workers, however, attempt to remove the polarisation by inserting a quarter-wave plate into the beam. This results in a circularly polarised beam, which will contain s and p components of equal intensity. However, as we have shown, this will not result in equal intensities in the evanescent wave (Fig. 5) which will contain x, y, and z components of differ- ing intensities [12]. The importance of these polarisation effects depends on the sample under observation. In some cases polarisation will not be a concern; in others it will be a nuisance which must be eliminated, and in yet others it can be exploited to obtain useful information about the orientation of molecules [13–15]. For example, where the fluorophores under observation are in fixed (but random) orientations, polarised excitation will selectively excite a subset of the molecules, with the intensity of fluorescence related to the orientation of the molecules. Where molecules are free to rotate rapidly (with respect to the integration time of observation) this effect will not be apparent. However, where the molecules undergo slower rotations, this may cause fluctuations of the observed intensities of the molecules. Where this is a problem, one solution is to create an isotropic excitation in the x-y plane by the use of two beams intersecting at 90° [12], although this is likely to be difficult to align. In principle, this approach could be extended to obtain isotropic excitation in all three axes [12].

3.4 TIRF configurations

There are two principal configurations for TIRF microscopy, usually called “prism” and “objective” styles (Fig. 6). Both these implementations have particular advantages and limitations, and the choice of approach will typically be guided by the type of sample to be visualised and other experimental requirements, for 1312 A. E. Knight: Total internal reflection fluorescence microscopy

1 a





f g




Fig. 6 Optical arrangements for TIRF. This diagram shows the two most common optical arrangements for TIRF: objective and prism-based. In prism-based TIRF, a collimated (or nearly collimated) laser beam (1) is coupled via a prism (a) and a coupling medium (b) such as glycerol into a microscope slide (c). Total internal reflection occurs at the interface with a sample (f), gen- erating an evanescent wave. The fluorescence is imaged with a microscope objective (h) through the thickness of the sample, which is determined by spacers (d), and through a microscope cover slip (e). The objective is typically coupled to the cover slip with immersion oil (g). In objective TIRF, the laser beam (2) is brought to a focus near the edge of the aperture at the back focal plane (i) of the objective lens. The lens collimates the beam and it passes through the immersion oil and the cover slip, under- going total internal reflection at the interface with the sample. example, whether the TIRF observation is to be combined with another technique. The pros and cons of the two configurations are summarised in Table 1. In all these discussions, we assume that an inverted micro- scope is being used (which is the preference of most researchers). In most cases, similar arguments apply to upright instruments, with the obvious inversion of the frame of reference.

3.4.1 Prism TIRF

In prism-based TIRF, the evanescent wave is typically generated at the surface of a microscope slide, or pos- sibly at the surface of a cover slip or directly on the prism [5, 16]. The function of the prism is to efficiently couple the laser beam (Fig. 6, beam 1) into the microscope slide by providing a surface at close to normal incidence. However, the beam is unlikely to strike the prism at exactly normal incidence, which means that

Table 1 Comparison of the objective and prism configurations of TIRF.

Prism Objective

Wide range of angles of incidence. Limited range of angles of incidence. Limited to thin specimens (unless using water immersion objective) Specimens may be of any thickness (e.g. cells). by spherical aberration, e.g. 10 μm. Difficult to exchange solutions. Specimens typically immobilised on microscope slides. Specimens typically immobilised on cover slips. Limited access to upper surface. Free access to upper surface of specimen. Often incompatible with bright-field illumination. Compatible with bright-field illumination. Frequent realignment may be necessary. No need for realignment when changing specimens. Exposed laser beams above microscope stage may cause safety Laser beam typically confined. concerns. Usually custom-built. Commercial systems available. A. E. Knight: Total internal reflection fluorescence microscopy 1313 some refraction will occur, changing the actual angle of incidence, and this must be allowed for. Various prism arrangements have been used [5, 16], although the most common arrangement is a triangular or cubic prism on the optical axis, as shown in Fig. 6. This arrangement allows a single internal reflection to occur on the optical axis of the system. Cubic prisms may have the advantage of allowing optical components to be used above the sample. The choice of prism will often depend on the desired angle of incidence and the space available for guiding the beams above the microscope stage. For example, for typical TIRF angles in excess of 60° (π/3 rad), the incoming beam will need to strike a 60° (equilateral) prism at a greater angle, as it will be bent towards the normal. This can cause fouling of the beam when space is limited above the microscope stage. In contrast, for a cubic prism the sides are at 90° (π/4 rad) and the beam will be refracted in the oppo- site direction. Another possibility is the use of a hemi-cylindrical prism, which ensures normal incidence (if correctly positioned), so that the angle of incidence can be adjusted without changing the alignment of the beams. This is convenient, as some realignment is typically necessary with cubic or triangular prisms to ensure the evanescent wave is centred in the field of view. It is often desirable to adjust the angle of incidence without changing the position of the evanescent wave. This can be achieved, for example, by translating the laser beam across a lens that is located with the desired TIRF position at one of its focal points [17]. Alternatively, a combination of linear and rotation stages under computer control may be useful, and permits precise control of the angle of incidence, provided that allowance is made for the refractions that occur at the interfaces between media [18]. An alternative arrangement [16] uses two prisms either side of the observation , one to couple the beam into the slide and one to couple it out again. Multiple internal reflections occur at the top and bottom of the slide. The advantage of this arrangement is that the top (back) of the slide is readily accessible, which is therefore more conveniently compatible with other techniques such as bright field imaging using a con- denser. The disadvantage, however, is the more complex mounting requirements for the prisms, more dif- ficult alignment, and the possibility of more light being scattered within the slide at the multiple reflections. Recently, a variant of prism TIRF has been developed, known as “variable-angle TIRF” [19, 20], which takes the form of a substitute for a conventional microscope condenser. The within the condenser permit the control of the angle of incidence. The prism itself is usually optically coupled to the slide in some way. One possibility is to glue the prism to the slide using some form of optical cement. Apart from rendering the slide nondisposable, this also means that a beam realignment is required whenever the microscope stage is moved. Most workers have instead chosen to use a film of liquid (such as glycerol or immersion oil) between prism and slide. This allows rela- tive motion of the prism and slide. For example, the slide can be moved using an x-y stage whilst keeping the prism (and hence the evanescent wave) in a fixed position. This requires that the prism be mounted in some way to the microscope body or optical table in this situation, the prism is typically removed and replaced every time a slide is exchanged, which can mean that fiddly realignment is required for each fresh sample. It is not necessary that the prism, slide, and liquid be index-matched, as long as they are similar enough to avoid strong reflections. However, differences in refractive index between slide and prism, in particular, will cause small shifts in beam alignment, which may need to be allowed for in more critical work. Typically, the beam used in prism-style TIRF is either collimated or nearly collimated; that is, converging at a very small angle. This is to allow a defined angle of incidence and avoid a mixture of supercritical and subcritical illumination, which would degrade the background reduction advantage of the TIRF illumination. The “footprint” or illuminated area where the evanescent wave is generated is obviously a critical param- eter for imaging experiments. Typically, the lasers used have a TEM00, or Gaussian, emission profile, and the intensity profile of the evanescent wave reflects this. In a prism-based TIRF experiment, the width of the “footprint” (in the y direction) reflects the 1/e2 diameter of the incident laser beam. In principle, one would expect the length of the footprint to correspond to a diagonal “slice” through the beam at angle θi. In practice, the shape of the footprint may become distorted by multiple reflections at interfaces (particularly between the prism and the slide) and by other aberrations, typically resulting in an elongated footprint [21]. In imaging applications, it is usually desirable to have the most uniform possible illumination across the field of view, and most workers in practice achieve this by arranging for the y-dimension of the TIR footprint to 1314 A. E. Knight: Total internal reflection fluorescence microscopy be rather larger than the imaging area. However, the intensity will still be non-uniform, and this needs to be taken into account during data analysis. Since the 1/e2 diameters of most lasers are in the millimetre range, and the field of view with a typical camera and a high-magnification objective is likely to be of the order of 100 μm or less, it is often useful to reduce the size of the footprint by focusing the incident beam with a lens. Ideally, the use of a longer focal length lens will give the minimum convergence angle of the beam. For non-imaging applications such as TIR- FCS, where uniformity of illumination is not so important, the beam can be focused down to a smaller area to achieve a higher intensity. Both prism and slide should be made of low-fluorescence material, as the high intensity of the laser light can excite a significant level of background fluorescence (although this is dependent on the excitation wavelength). For example, many inexpensive microscope slides exhibit high levels of fluorescence. An ideal material is quartz, particularly highly purified synthetic fused silica, such as Spectrosil 2000 (Saint-Gobain Quartz, Wallsend UK). However, conventional optical-grade quartz is usually sufficient for the prism. For the slide, quartz is rather expensive, and in practice a low-fluorescence glass (“extra-white” glass, available from a variety of suppliers) will usually suffice. Imperfections and dirt on the surface of the microscope slide will cause scattering of the evanescent wave, which can lead to background problems. However, good-quality microscope slides are usually smooth and clean enough to achieve good results. A major problem with prism TIRF is that the evanescent wave is formed on the opposite side of the chamber from the microscope objective. Most high-numerical aperture objectives are designed to image specimens through the thickness of a No. 1½ cover slip (0.17 mm), and greater thicknesses lead to spherical aberrations, which severely degrade image quality. Typically, the specimen chambers need to be no more than ∼10 μm thick. Achieving thin enough sample chambers for prism TIRF can therefore be difficult. One approach is to include silica microspheres to act as spacers [17]; also, strips of 10-μm-thick PTFE (Goodfel- low, Cambridge UK) work well. Alternatively, microfluidic flow cells can be used provided that they meet the optical constraints and are made of low-fluorescence materials. This restriction on the thickness of the sample limits the applications of prism-based TIRF. For example, in in vitro experiments it is often difficult to exchange solutions; and most cell types are too thick to be visu- alised in this type of chamber. In some situations, however, this restriction may be relaxed by the use of water immersion objectives. The prism-based method is preferable where it is desirable to use a range of angles of incidence, and to control the angle (and therefore the penetration depth). It may also achieve better signal-to-noise ratios, perhaps because it avoids the risk of scattering of excitation light within the microscope objective [16, 17].

3.4.2 Objective TIRF

Objective TIRF is the most popular form of the technique. This is probably because of the ready availability of commercial systems from the major microscope manufacturers, the applicability of the technique to visualis- ing cells, and the greater simplicity of use (see Table 1 and Fig. 6). The limitations of the technique include the relatively narrow range of angles of incidence that can be accommodated. There is also a concern that scattering of laser light in the objective will lead to a higher background [16, 17]. The geometry of objective TIRF is shown in Fig. 6 (beam 2). The incident laser beam is focused at a point in the back focal plane of the objective lens. This results in a collimated beam exiting the front element of the microscope objective. Various optical arrangements are possible to achieve objective TIRF (see, e.g. [5, 17] or the commercial systems available). The absolutely critical component in this technique is the objective lens itself. The numerical aperture (or NA) of an objective lens is given by NAn= sinθ (11) where n is the refractive index of the medium between the lens and the sample and θ is the half-cone angle of the light emerging from the objective lens. At the critical angle, the NA will therefore be given by NA = n3 sin θc, A. E. Knight: Total internal reflection fluorescence microscopy 1315

where n3 is the refractive index of the immersion oil. The critical angle will be given by eq. 2, so that the NA corresponding to the critical angle will be given by nn NAn==sinθ 31= n (12) 3cn 1 3 In other words, for an objective lens to be usable for TIRF, the numerical aperture must be greater than the refractive index of water, which is 1.33. Commercially available objective lenses with NA = 1.45 should be suit- able, but it is important to ensure that the lens chosen meets the stated specification [16]. Special TIRF lenses with NA as high as 1.65 are now commercially available. The angle of incidence for objective TIRF is adjusted by translating the focused beam across the back focal plane of the objective. The actual angle at which the beam emerges can be determined by coupling a glass block onto the objective lens with immersion oil, and measuring the angle of the beam in the block. A facility for translating the focus across the back aperture provides a convenient means of switching between epi-illumination (with the beam on the optical axis), highly inclined illumination (with the beam just inside the radius corresponding to the critical angle), and TIRF (outside this critical radius). The penetration depth in TIRF mode can also be adjusted by changing the position outside the critical radius. When in TIRF mode, the reflected beam is coupled out of the specimen and emerges at the back focal plane opposite to the incident beam. Typically, the optics are arranged so as to guide this beam back out of the microscope, so as to avoid stray light problems; however, this beam can also be used for checking the status and alignment of the illumination, or as part of an autofocus mechanism [17]. Typically, the excitation beam is separated from the emitted light by using small silvered mirrors or a dichroic mirror (as in a normal fluorescence filter cube) mounted at a 45° (π/4 rad) angle below the objective lens. It is important to note that when not in TIRF mode, which is when the angle of incidence is subcritical, a collimated laser beam is emitted from the objective lens. This can be a serious eye hazard, and therefore it is vital to avoid exposure of the operator to the beam. For example, an interlocked enclosure can be placed over the stage. The size of the TIR footprint depends on the cone angle of the incident beam at the back focal plane. It may be desirable to expand the beam to increase the area of illumination; the size of the illuminated area can then be adjusted by using an iris diaphragm in the expanded beam [17]. As with prism TIRF, the illumination intensity across the beam will be non-uniform. In summary, objective TIRF has a number of attractive features: –– slides or specimens can be exchanged rapidly without the need for realignment or refocusing; –– fluorescence excitation occurs at the near side of the flow cell, minimising spherical aberration of the objects being imaged; –– thick specimens such as cells can therefore be accommodated; and –– the same illumination arrangement can be used for epi-illumination, highly inclined illumination, and TIRF.

3.5 Optimising TIRF

One of the difficulties in adjusting any optical system, particularly a custom-built one, is the correct align- ment and adjustment of all the components to give the best-quality data. This can often be helped by using some kind of standard sample. Fluorescent beads or quantum dots are particularly useful in this regard, as they are typically much brighter and more photostable than single molecules. Large beads (with diameters of microns or larger) typi- cally display unexpected behaviour in a TIRF system because of a phenomenon known as frustrated TIR [7], where the evanescent wave “jumps” across a narrow gap between two media of high refractive index, leading to a high degree of scattering. Instead, it is better to use fluorescent beads of a diameter less than the resolu- tion of the imaging system. Such beads are often used as “point sources” for calibrating confocal and similar systems, and are available from a number of suppliers. 1316 A. E. Knight: Total internal reflection fluorescence microscopy

A suspension of these beads can be used to empirically adjust the system for optimal TIR conditions. Ideally, the beads should be suspended in a medium of similar refractive index to the experimental buffer, to ensure that the critical angle remains the same. By focusing the microscope at the interface and adjusting the angle of incidence, the transition between the subcritical and supercritical illumination can be observed. At subcritical angles, there will typically be a high background from beads suspended in the liquid. When TIRF has been achieved, only beads at, or very close to, the surface will be observed, against a dark background. Often their fluorescence will “flicker” or “blink” as they diffuse up and down within the evanescent wave. For single-molecule applications it is not normally necessary to precisely control the penetration depth, and the angle of incidence can simply be set empirically to achieve the best contrast. In any case, it is difficult to measure the penetration depth directly, and it is better to control it via the angle of incidence and refractive indices of the media.

3.6 Resolution

As TIRF is a far-field imaging technique, its resolution is limited by diffraction. A fluorescent molecule behaves as a , which gives a diffraction pattern (or point spread function) known as an Airy disc5 in the imaging system; the diameter of this disc limits the resolution of the system. This is conventionally expressed as the Rayleigh criterion6 for resolution where NA refers to the numerical aperture 1.22 λ r = 0 (13) 2NA For example, for light at 532 nm the resolution, r, is 232 nm. Figure 7 shows how, as the separation between two molecules is decreased, the Airy discs overlap until they cannot be distinguished. However, a common error is to confuse this resolution criterion with the ability to (a) identify overlapping fluorophores and (b) obtain positional information with high resolution. In the first case, if the intensities of the molecules are known, one molecule can be distinguished from two by the intensity of the peak, at least in principle (see Fig. 7). In practice, this can be achieved more readily by comparing the images before and after the pho- tobleaching of one of the molecules [22, 23]. In the second case, very high positional accuracies have been reported (down to 1 nm) [24, 25]. This can be achieved by imaging the point-spread function across several pixels of the camera. Then the accuracy of localisation of the molecule can be smaller than the pixel reso- lution of the camera.7 This principle has recently been extended by the development of localisation-based super-resolution microscopy techniques such as PALM [27, 28] and STORM/GSDIM [29, 30].

3.7 Other matters

Imaging with the low levels of light found in single-molecule applications is challenging. One of the key requirements is obviously an extremely sensitive, low-noise detector. In the past, most workers in the field used image-intensified CCD cameras (ICCDs) as this was the best available technology. The state of the art at present is the electron-multiplication CCD technology, or EMCCD, where the signal is amplified on the chip, giving extremely low noise and high resolution. However, scientific CMOS is a promising new technology. A further critical component is the emission filter used to select the fluorescence light and exclude the excita- tion light and other extraneous light sources. Due to the small Stokes shift of most fluorescent dyes, such

5 In practice, the Airy disc is only an approximation of the true point-spread function, but is adequate for practical purposes where the fluorophores are tumbling rapidly, such that their emission is effectively unpolarised (see Section 2.4). 6 Other criteria are encountered – such as the Abbe and Sparrow criteria – but the Rayleigh criterion is the one most frequently encountered in microscopy. 7 The accuracy depends on the number of , the background noise, and the pixel size [26]. A. E. Knight: Total internal reflection fluorescence microscopy 1317

Separation 0 nm Separation 232 nm -500 -500 2.0 2.0

1.5 1.5

0 0 1.0 1.0 y /nm y /nm 0.5 0.5

500 500 0 0 -500 0 500 -500 0 500 x/nm x/nm Separation 116 nm Separation 463 nm -500 -500 2.0 2.0

1.5 1.5

0 0 1.0 1.0 y /nm y /nm 0.5 0.5

500 500 0 0 -500 0 500 -500 0 500 x/nm x/nm

Fig. 7 Point-spread function and resolution. A single molecule acts as a point source of light and is imaged as a diffraction pattern known as an Airy disc. The size of the disc limits the resolution of the imaging system. In these simulated plots, the intensity of the diffraction pattern is shown in false colour; the white circle indicates the radius, r, of the Airy disc (the first minimum in the diffraction pattern), which is given by eq. 13. These diffraction patterns were calculated for light at 532 nm and an objective with NA 1.4. When the separation between two molecules is zero, the two diffraction patterns are perfectly super- imposed, and the molecules cannot be resolved. When the separation is r/2, the shape of the combined diffraction patterns is no longer circular, but there is no dip in intensity between the peaks. When the separation is exactly r, there is a clear dip in intensity between the peaks and the molecules are considered to be resolved; at greater separations still the molecules are more readily separated. Although in the first two cases the molecules are considered not to be resolvable, the presence of more than one molecule may be inferred, under ideal circumstances, from the intensity of the pattern.

filters require extremely good blocking of the laser line, sharp cut-on and high transmission of the fluores- cence emission. Very high quality interference filters are therefore required. Band-pass filters are preferable as they exclude more sources of stray light, and can also be used for FRET experiments. A critical component of a single-molecule imaging system that is often overlooked is focusing. It can be very difficult to focus accurately, especially where there is no bright-field illumination available, as is often the case in prism TIRF. High NA objectives tend to have an extremely narrow depth of field, < 1 μm for a 1.4 NA lens, and the specimen may be difficult to visualise until it is brought into sharp focus. Focus drift, which is present in many microscopes, can become a major problem, particularly if the acquisition of long sequences of images is required. In this case, it is extremely useful to use a motorised focusing system under computer control; the mechanical stability of the specimen chamber is also a significant concern.

4 Applications

As a robust technique for single-molecule detection and imaging, TIRF has been applied to many different types of measurement, particularly within the life sciences. In this section, I aim to provide a flavour of the range of applications in the literature. FRET is a familiar technique for measuring the distance between two fluorophores, which in recent years has been extended to single-molecule measurements. The distance is inferred from the efficiency of the energy transfer, although there are some uncertainties in this method, which mean that results are often interpreted qualitatively [31]. This approach can be extended to an enormous variety of applications, such as 1318 A. E. Knight: Total internal reflection fluorescence microscopy investigating conformational changes in proteins or nucleic acids, protein folding, or protein–protein inter- actions [31]. As mentioned above, positional or distance information beyond the normal resolution limit may be obtained by single-molecule approaches. Individual fluorophores can be tracked with a resolution approach- ing 1 nm [25]; this has been applied, for example, to measure the motions of individual myosin V motor domains to distinguish between alternative mechanisms of processive motility [24]. Distances between mol- ecules separated by sub-resolution spacings can be measured by comparing the diffraction patterns as the molecules photobleach. For example, the distances between fluorophores bound to DNA and separated by 10 nm have been measured [22]. Photobleaching (and subsequent recovery) can also be used to investigate the stoichiometry of multi-protein complexes and the turnover of subunits, as was recently done for the bac- terial flagellar motor [32]. TIRF is also a popular route to single-molecule detection in cells [17, 33, 34]. Further- more, as mentioned above, TIRF excitation may be used in a number of forms of super-resolution microscopy. TIRF is highly amenable to being combined with a variety of other techniques, enabling a host of pow- erful new measurement methodologies. For example, TIRF can be combined with fluorescence correlation spectroscopy to measure binding at, and diffusion near surfaces, a technique known as TIR-FCS [35, 36]. It can also be combined with single-molecule mechanical measurements [37] by optical tweezers [38] or a variant of AFM [39]. Finally, a single-molecule approach to DNA microarrays has been developed [40], as have high-through- put [41] and single-molecule [42–44] DNA sequencing methods. These examples are only a few of the many ways in which TIRF has been applied, and new uses for this simple yet versatile technique continue to emerge.

5 Conclusion

The intention of this report has been to present the main technical aspects of performing successful single- molecule observations using TIRF microscopy. We have discussed the theory governing the evanescent wave, and how TIRF can be implemented for single-molecule detection. As the previous section shows, this is a highly robust and yet versatile methodology, and is likely to continue to be applied more and more widely in the life sciences and beyond.

6 Membership of sponsoring bodies

Membership of the IUPAC Physical and Biophysical Chemistry Division Committee for the period 2014–2015 is as follows: President: R. Marquardt (France); Vice President: A. K. Wilson (USA); Secretary: A. Friedler (Israel); Past President: K. Yamanouchi (Japan); Titular Members: K. Bartik (Belgium); A. Goodwin (USA); A. E. Russell (UK); J. Stohner (Switzerland); Y. H. Taufiq-Yap (Malaysia); F. van Veggel (Canada); Associate Members: K. Battacharyya (India); A. G. Császár (Hungary); J. de Faria (Portugal); V. Kukushkin (Russia); Á. W. Mombrú (Uruguay); X. S. Zhao (China/Beijing); National Representatives: Md. Abu bin Hassan Susan (Bangladesh); H. R. Corti (Argentina); J. Cejka (Czech Republic); S. Hannongbua (Thailand); S. J. Kim (Korea); E. Klein (Bulgaria); M. Koper (Netherlands); M. Korenko (Slovakia); K. E. Laasonen (Sweden); J. E. G. Mdoe (Tanzania); V. Tomišić (Croatia). Membership of the IUPAC Organic and Biomolecular Chemistry Division Committee for the period 2014– 2015 is as follows: President: M. J. Garson (Australia); Vice President: M. A. Brimble (New Zealand); Secretary: A. ­Griesbeck (Germany); Past President: K. N. Ganesh (India); Titular Members: G. Blackburn (UK); A. Brandi (Italy); T. Carell (Germany); B. Han (China); F. Nicotra (Italy) N. E. Nifantiev (Russia); Associate Members: A. ­Al-Aboudi (Jordan); V. Dimitrov (Bulgaria); J. F. Honek (Canada); P. Mátyus (Hungary); A. P. A. E. Knight: Total internal reflection fluorescence microscopy 1319

Rauter (Portugal); Z. Xi (China/Beijing); National Representatives: Y.-M. Choo (Malaysia); O. M. Demchuk (Poland); M. Ludwig (Czech Republic); V. Milata (Slovakia); M. Olire Edema (Nigeria); P. M. Pihko (Finland); N. Sultana (Bangladesh); H. Vančik (Croatia); B.-J. Uang (China/Taipei); T. Vilaivan (Thailand). Membership of the IUPAC Analytical Chemistry Division Committee for the period 2014–2015 is as follows: President: D. Hibbert (Australia); Vice President: J. Labuda (Slovakia); Secretary: Z. Mester (Canada); Past President: M. F. Camões (Portugal); Titular Members: C. Balarew (Bulgaria); Y. Chen (China/Beijing); A. Felinger (Hungary); H. Kim (Korea); M. C. Magalhães (Portugal); H. M. M. Sirén (Finland); Associate Members: R. Apak (Turkey); P. Bode (Netherlands); D. Craston (UK); Y. H. Lee (Malaysia); T. Maryutina (Russia); N. Torto (South Africa); National Representatives: L. Charles (France); O. C. Othman (Tanzania); P. DeBièvre (Belgium); M. N. Eberlin (Brazil); A. Fajgelj (Slovenia); K. Grudpan (Thailand); J. Hanif (Pakistan); D. Mandler (Israel); P. Novak (Croatia); D. G. Shaw (USA). This document was prepared in the frame of IUPAC Project 3#2004-021-1-300, Reference Methods, Stand- ards and Applications of Photoluminescence. Chairs: Fred Brouwer, Enrique San Román; Members: Ulises Acuña, Marcel Ameloot, Noël Boens, Cornelia Bohne, Paul DeRose, Jörg Enderlein, Nikolaus Ernsting, Thomas Gustavsson, Niels Harrit, Johan Hofkens, Alex E. Knight, Helge Lemmetyinen, Hiroshi Miyasaka, Ute Resch-Genger, Alan Ryder, Trevor Smith, Mark Thompson, Bernard Valeur, Hiroyuki Yoshikawa.


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