Introduction

The conventional optical microscope has been an important device for producing magnified images for rapid evaluation in research and routine studies, especially in the life science and medicine. Although the standard light microscope is easy to operate, one difficulty is generating a high-contrast image of specimens which are completely or almost transparent. To overcome this, fluorescence, dark field, and phase-contrast techniques have been developed; these result in microscopic images with sufficient contrast and high information content [1]. The lateral resolution of an optical microscope is, however, ultimately limited by diffraction. The best resolution for visible wavelengths is around 200 nm, according to the limit derived by Ernst Abbe [2]. After Abbe’s theory Rayleigh pointed out that objects are resolved when the maximum of one pattern coincides with the (first) minimum of the neighbouring feature and came up with the Rayleigh criterion, described as Eq. (1) [3–5]:

$$ d = 0.61\frac{\lambda } {{{\text{NA}}}} $$
(1)

Where d is the smallest distance distinguishable between two objects, λ is the wavelength of light applied and NA is the numerical aperture of the lens.

In the early 1980s scanning probe microscopy (SPM) techniques were introduced as a fundamentally different approach to obtaining information from a surface, with even better lateral resolution [6]. Different methods, for example atomic-force microscopy (AFM) and scanning tunnelling microscopy (STM), are categorised as SPM [7]. The common feature of these techniques is that a sharp tip is raster-scanned across a surface to map the topography. In AFM, for instance, the force between the tip and the sample surface is detected and kept constant throughout the scan, and hence yields the topography of the surface, whereas in STM the tunnelling current between the tip and the sample is exploited to generate a feedback signal and a contour of the electronic density of the states, which often reflects the topography of the surface.

The scanning near-field optical microscope (SNOM or NSOM) is a concept that combines scanning probe techniques and optical microscopy. A SNOM probe is scanned very close to the sample surface in the region called the optical near field. An overview of SNOM techniques will be presented in the next section. Instrumental and theoretical details are available in various books and review articles [8–16].

The basics of SNOM

The first concept of the optical near-field, which is to use a small aperture to image a surface with a resolution smaller than the wavelength of light, was proposed by Synge in 1928 [17]. The properties of light at apertures can be explained by diffraction theory. However, when the incident radiation passes through a sub-wavelength sized aperture, electromagnetic theory must be employed to describe the behaviour. If the distance between aperture and specimen is kept much smaller than the diameter of the aperture, the illumination area is then defined by the dimensions of the aperture. In this so called “optical near-field region” the light interacts with the sample before diffracting. Hence, the aperture dimensions and the distance from the sample determine the lateral resolution of such a system, as opposed to the wavelength of light and the collection efficiency in conventional optical instruments.

In 1972 Ash and Nichols first demonstrated near-field resolution using microwaves [18]. Twelve years later the first implementation of SNOM was reported by Pohl and co-workers [19] and by Lewis and co-workers [20]. Their experiments involved a sub-wavelength size aperture which they fabricated by coating the apex of a sharply tapered transparent glass tip with metal. In addition, they employed a feedback loop mechanism that maintained a constant distance of only a few nanometres. In 1991 Betzig et al. introduced the use of single-mode optical fibres as near-field optical probes; this is still one of the most popular set ups [21].

SPM techniques have the advantage that they are quite robust and can easily be used for routine investigations. In addition to the topography, under some conditions SPM techniques can yield material contrast, for instance lateral force detection or phase measurements. Although SNOM equipments are usually more complex and need a skilled person to operate the instrument [22], they provide the same topographic and contrast information. In addition, it is a particular advantage of SNOM that the optical properties of the specimen can be observed with a lateral resolution below the diffraction limit. In combination with spectroscopic techniques this enables a wide range of problems in chemistry, material science, and biology to be addressed.

Many spectroscopic methods can be coupled with SNOM. Fluorescence is the most popular method, especially in biological applications [9, 23–28]. Raman and infrared absorption (IR) spectroscopy are other examples [29–34]. Polarisation contrast SNOM can also be used to analyse a variety of phenomena, for example optical anisotropy, magneto-optical effects, and electron spin dynamics [35–37].

Figure 1 shows schematic illustrations of the major types of near-field optical set up, aperture (Figs. 1a–d) and scattering-type, often designated “apertureless” (Figs. 1e, f). In the following sections these two SNOM techniques are discussed and applications are presented.

Fig. 1
figure 1

Six common types of scanning near-field optical microscopy (SNOM) configurations. (a) Illumination and collection mode. Illumination and collection through the same aperture. (b) Transmission mode. Illumination through aperture, transmitted signal is collected by external optics. (c) Reflection mode. Illumination through aperture, reflected signal is collected by external optics. (d) Photon scanning tunnelling mode (PSTM). An evanescent field is generated at a prism surface, and collection is through a tapered fibre tip. (e) Scattering evanescent excitation mode. (f) Scattering-type SNOM. In (b) and (c), excitation and detection can be exchanged. In (e) and (f), the illuminated tip gives an enhancement and serves as a subwavelength light source

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Aperture SNOM

Principle

In aperture SNOM an optical fibre probe with an aperture is scanned across a sample at a short distance from the surface. As has been mentioned, the distance between the probe and sample must be sufficiently small that the specimen is in the optical near-field and diffraction effects can be neglected. In addition, the aperture size must be considerably smaller than the diffraction limit, otherwise the information can be obtained much more easily by conventional microscopy. For these reasons, the optical fibre probe is the most important part of the aperture SNOM equipment and essential for obtaining high-quality images. The fibre must be highly transmissive, light should not leak through the coating film, and a small circular aperture is essential. The quality of the fibre material itself is also crucial. For instance, impurities in the core or cladding might cause luminescence, which results in an undesired background.

Instrumentation

The general procedure used to make tips from glass fibres for aperture SNOM consists of three steps. Initially, a taper structure with a sharp apex on the edge of the fibre must be fabricated. Typical approaches are either the “heating-and-pulling” method using a CO2 laser, or chemical etching using hydrofluoric acid solution [38, 39]. The second step is to coat the tapered tip with metal (Al, Au, or Ag) to obtain a completely opaque cover on the probe. Because of the small skin depth in the optical region, aluminium is usually the metal of choice. However, aluminium is oxidised easily, which affects the properties of the coating. The final step is aperture formation at the apex. A popular method of forming an aperture is based on geometrical shadowing [15, 40]. The metal is evaporated from the angle slightly from behind, such that the very end of the probe points slightly away from the metal source. This geometry automatically creates an aperture at the point of the fibre tip which can be adjusted by the angle of evaporation. A more sophisticated procedure is milling and polishing a fully coated fibre using a focussed ion beam [41, 42]. With both techniques it is important to obtain a flat end and a well-defined circular aperture. If the aperture plane is not parallel to the sample surface, or rough, the distance between aperture and surface increases, resulting in lower optical resolution. Bouhelier et al. reported an alternative method of forming nano-apertures using amorphous silver metaphosphate-iodide (AgPO3.AgI) [43]. In their procedure, called “controlled all solid state electrolysis (CASSE)”, the tip is initially completely coated with silver. The probe is then brought into feedback above a solid electrolyte. Because only the silver coating in contact with the electrolyte is dissolved, a nano-aperture with a flat rim is obtained.

Another approach for manufacturing SNOM probes is fabrication of micromachined aperture probes [40, 44–47]. These tips are similar to AFM tips and are produced by similar micromechanical techniques that enable batch production of the tips, and the SNOM apparatus can be conveniently combined with an AFM system. When these tips are generally available they have clear advantages as industrial manufacturing improves the quality and reproducibility.

The precise distance control of the probes is also a crucial issue in SNOM experiments, because the lateral resolution and the intensity of the evanescent fields generated at the aperture are very dependent on the distance between the near-field aperture and sample. In general scanning-force microscopic techniques two different mechanisms of maintaining a close tip-to-sample distance are commonly used—contact mode and non-contact mode, including intermittent and shear-force feedback mode. In contact mode the tip applies a constant force to the sample surface. In non-contact mode the tip is vibrating at its resonance frequency above the surface and the feedback signal here is the damping of the free oscillation amplitude. In general, both techniques could be destructive to the sample. With the non-contact mode, however, very gentle scanning can be achieved. Optical detection of the tip oscillation with broad-band diode lasers can cause problems in optical measurements because of stray light interference. To avoid this, tuning fork-based shear-force feedback was introduced [48]. This approach has already been employed in SNOM experiments by many research groups. The near-field optical fibre tips are glued alongside the prongs of a quartz tuning fork and the whole system is mounted perpendicular to the surface of a specimen. The feedback is obtained in a manner similar to that used for the intermittent mode discussed earlier. The device is excited to vibrate at its resonance frequency. As the tip approaches the sample surface the amplitude of the tip vibration decreases; this can be utilised as a feed-back signal.

Applications

As already mentioned, the spectroscopic technique most frequently combined with SNOM is fluorescence, especially in biological applications. A standard procedure for imaging cells is to introduce fluorescent molecules, for example green fluorescent proteins, which are cloned into the cells [49–51]. The huge number of publications showing images of living cells obtained by use of confocal fluorescence techniques have recently been reviewed [52–54]. Similar investigations have also been made using SNOM. The confocal fluorescence imaging technique also enables study of the inner sections of the cell while scanning vertical to the surface [50]. SNOM, on the other hand, can be used to map the distribution of fluorescent proteins on the membrane simultaneously with the topography. In the following section we will discuss several examples of aperture SNOM.

Clancy et al. measured single human retinal lipofuscin granules using fluorescence SNOM [55]. In their experiment, lipofuscin granules were deposited on a mica substrate and the orange fluorophores in the sample were detected. Figure 2 shows the simultaneous SNOM and AFM images of two different acquisitions of lipofuscin granules. It is interesting that the combination of topography and fluorescence images enables investigation of the distribution of orange fluorophores in the samples. For example, in the top pair of images in Fig. 2, only a small portion of the granules’ surface is emissive, and in the bottom image the emission is only observed at the junction of the two granules and the granules themselves are non-emissive.

Fig. 2
figure 2

Simultaneously collected aperture scanning near-field optical images, topography (left) and fluorescence (right) of two different collections of lipofuscin granules demonstrating the range of structures observed. The 458 nm line from a continuous argon ion laser is used as the excitation source. Courtesy of Professors J. Simon and R.C. Dunn [55]

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Although the confocal method for fluorescence imaging is limited by diffraction, it can be improved by techniques like two-photon microscopy [56] or the 4-pi confocal imaging technique [57–60]. Hell and co-workers demonstrated that in multi-photon 4-pi confocal microscopy the combination of wavefronts by two high numerical aperture lenses enables three-dimensional imaging of biological molecules with “super-high axial resolution”, which they claim is approximately 100–140 nm [59]. They also demonstrated stimulated emission depletion (STED), which obtained the lowest resolution using propagating waves [61–63]. Presently, fluorescence SNOM gives higher resolution, going down to 30–50 nm, irrespective of the wavelength used, and enables observation of a few hundred fluorescent molecules per μm2, which is an order of magnitude more molecules than that distinguishable in the confocal fluorescent technique [64]. Dunn’s group studied the formation of lipid bilayers using confocal fluorescence, AFM, and SNOM and concluded that SNOM is advantageous in imaging smaller domains compared with confocal imaging [9, 65]. A disadvantage of SNOM is that it involves use of a sharp tip and close tip–sample distance separation, which might damage fragile biological samples. Feedback settings must be precisely controlled to maintain a constant tip–sample distance and to avoid destruction or manipulation of the specimen with the sharp optical fibre tip.

An attractive example of the combination of aperture SNOM and spectroscopy is the adoption of Raman scattering. This combination was first demonstrated simultaneously by Jahncke et al. [66] and Smith et al. [67]. Interesting studies on highly resolved stress mapping of silicon have been performed by Webster and co-workers [68–70]. An application of aperture SNOM/Raman that demonstrates the high spatial resolution in the aperture direction is investigation of liquid–liquid interfaces [71, 72]. The experiment was done by approaching the interfacial region of two immiscible liquids with a near-field optical fibre probe. The purpose of this research is to investigate the behaviour and structure of molecules at the actual interface. Figure 3 shows the colour coded intensity plot of the Raman signal during the approach of an aperture SNOM probe to a p-xylene/ethylene glycol interface [71]. The intensities of the p-xylene Raman band remain constant until the interface is reached. Then, a rapid decrease of the intensity is observed. This step-like intensity change was only observed with aperture SNOM tips. Uncoated tips resulted in far-field behaviour, as expected. Recent studies with a water/carbon tetrachloride interface revealed that the water molecules undergo structural rearrangement near the interface, corresponding to weakening of the hydrogen bonds between neighbouring water molecules [72].

Fig. 3
figure 3

Top Colour coded contour plot of the Raman signal during the approach of a metal coated near-field tip to a p-xylene/ethylene glycol interface. The Y-axis corresponds to the piezo positioning. Bottom Near-field Raman spectrum of p-xylene. Adapted from [71]

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A new concept, use of aperture SNOM probes in combination with analytical tools, is laser ablation. Laser pulses are delivered through the nanometre-sized probe and subsequently the ablated material can be analysed with mass spectrometry [73–75]. In this method, the ablation resolution is 70 nm at full width at half maximum, which enables highly localised “nano-sampling” [74]. This technique is still in the early development stage and improvement of ionisation efficiency and sample transport must be achieved.

A general problem of metal-coated fibre probes is the quality of tips, which strongly influences the resolution of the image. Reproducibility and lifetime of the aperture are the factors to be aware of. As micromachined tips become widely available this problem could be resolved. Aperture tips also have relatively low light throughput. The damage threshold must also be considered when increasing the laser power.

The alternative to aperture SNOM is the so-called scattering-type SNOM or apertureless SNOM, which is discussed in the next section.

Scattering-type SNOM

Principle

The major difference between aperture and scattering-type SNOM is the shape of the probes. Instead of metal-coated fibre optic probes, standard AFM tips can be used; the geometries of these leads to strongly localised fields. The aim is to obtain super-resolution without involving an aperture. Wessel first suggested in 1985 [76], and Zenhausern and co-workers first reported the experiments [77], that an externally illuminated sharp tip can be used as a highly localised light source. As has already been discussed, the resolution of aperture SNOM is limited by the aperture dimensions. The potential resolution of scattering-type SNOM is determined by the dimension of the tip, where strongly confined fields are created if the tip is illuminated externally. This, in principle, yields much higher spatial resolution.

A disadvantage of the scattering-type SNOM technique is that in addition to the tip a large area of the sample is also irradiated. This results in an intense background. Because the signal has to be observed externally, it is difficult to avoid this unwanted signal contribution. In aperture SNOM, in contrast, the light is shining only at a specific part of the sample, which can be controlled by the diameter of the aperture. In other words, the aperture probe provides a localised light source with very low background; the intensity of the illumination, however, is smaller than for scattering-type SNOM. Frey et al. [78] recently combined scattering-type and aperture-type SNOM by using a metal tip mounted on an aperture probe. The purpose of this approach is to overcome the background problem; it can improve results under certain conditions.

Basic theory

The basic principle of the physical phenomena in scattering-type SNOM is optical resonance by the excitation of collective electron oscillations, so-called surface plasmons, at a metal nanostructure on the surface. These create strong electromagnetic fields confined to the surface that result in substantial enhancement in the small tip region. Such highly localised fields play a central role in scattering-type SNOM. A number of theoretical calculations of field enhancement have been published [13, 79–82]. Many properties, for instance, tip shape, material, roughness, excitation wavelength, will affect not only the size of the enhancement region but also the enhancement factors.

Applications

IR spectroscopy in combination with scattering-type SNOM has recently been used to obtain molecular information [33, 83–88]. Because silica based fibres do not transmit wavelengths longer than 1.6 μm, because of absorption effects, the combination of aperture SNOM with IR spectroscopy would require special fibre materials. By employing the scattering technique, this prerequisite is avoided.

Keilmann and co-workers have studied two immiscible polymers, PS and PMMA [85], by scattering-type SNOM/IR. The Au-coated AFM tip was irradiated from the side with a CO2laser. The contrast of vibrational resonances was plotted to obtain images of the surfaces. In this way, direct comparison of sample materials can be visualised. Their experiments revealed the IR signal can be enhanced by a factor of about 200 by use of a gold-coated tip [83].

Raman spectroscopy, which is a complementary method to IR absorption spectroscopy, has also been combined with scattering-type SNOM [31, 89–93]. In this specific set up Raman has a particular sensitivity advantage over IR spectroscopy. If a sharp metal tip, typically made of silver or gold, is used, external illumination gives rise to a huge field enhancement known from surface-enhanced Raman scattering (SERS). SERS can increase the signal by up to a factor of 1015, theoretically enabling detection of even single molecules [94]. Therefore, even weakly fluorescent substances can be characterised by this method as long as the SERS signal intensity is above the fluorescent background. This further increases the sensitivity of Raman scattering, and is the basic concept of the scattering-type SNOM/Raman technique.

An example of scattering-type SNOM combined with Raman spectroscopy is shown in Figure 4 [31]. In this study, the silver deposited AFM tip was brought into contact with a thin brilliant cresyl blue layer. The area of Raman enhancement was 55 nm in diameter, which is in good agreement with the size of the tip (50 nm) determined by SEM. This indicates that, although the laser is illuminating a larger area, the Raman signal originates only from the tip apex, and gives very high lateral resolution.

Fig. 4
figure 4

Ultraflat homogeneous BCB sample measured by use of the tip-scan mode of AFM combined with tip-enhanced Raman scattering. (a) Colour-coded intensity plot of the Raman band between 1630 and 1680 cm−1. (b) Two selected Raman spectra measured at tip locations No. 8 and No. 15. Adapted from [31]

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Kawata’s group has also studied the SNOM/Raman combination [95–98]. In their studies, the central section of the excitation laser was blocked. In this way, only the evanescent field (p-polarised) is generated and interacts with the silver-coated cantilevers. The enhancement is very sensitive to polarisation, because only p-polarised excitation yields enhancement, not side illumination (s-polarised) [97].

A very elegant scattering-type SNOM experiment has recently been reported by Novotony’s group [32, 89]. They studied single-walled carbon nanotubes (SWNTs) prepared by chemical vapour deposition and by arc discharge. The Raman spectra of SWNT samples consist of two characteristics bands—the sharp “G-band” at 1596 cm−1, due to the tangential stretching mode, and the broader “G′-band” at 2615 cm−1, which originates from a double resonance Raman scattering process. Figure 5 shows the simultaneous optical near-field Raman image (a) and the topographic image (b) of SWNTs grown by chemical vapour deposition. The authors stated that although far-field confocal Raman imaging of this sample is not possible, because of weak scattering, scattering-type SNOM can be used to study such a sample. A very interesting fact in this example is that the lateral resolution of the Raman image is better than the resolution of the simultaneously recorded topographic image.

Fig. 5
figure 5

Simultaneous (a) (scattering-type) near-field optical Raman image and (b) topographic image of single-walled carbon nanotubes (SWNTs) grown by chemical vapour deposition on glass. Scan area 1×1 μm2. The “G′-band” (2615 cm−1) is used for Raman imaging. Excitation wavelength: 633 nm. (c) Cross-section taken along the dashed line indicated in the Raman image. Vertical units are photon counts per second. Courtesy of Professor A. Hartschuh [89].

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Application of the scattering-type SNOM technique to fluorescence is not as straightforward as for IR and Raman spectroscopies. The tip can cause quenching instead of signal enhancement. A very interesting approach is two-photon fluorescence, that makes use of second-harmonic generation (SHG) at the tip [26, 99–101]. Because the SHG source has the dimension of the tip, high lateral resolution can be achieved without the above mentioned background problems.

Conclusions

Scanning near-field optical microscopy techniques are powerful tools for investigation of a specimen whenever the resolution of a standard optical microscope is insufficient, or whenever the combination of topography and spectroscopic information is necessary to solve a problem. In fluorescence SNOM, in particular, well established techniques for biological applications can be adopted, and higher resolution can be achieved compared with standard optical microscopy. Currently all SNOM types are restricted to the surface of a sample, which is clearly a disadvantage compared with the three dimensional possibilities of a confocal set up. This situation might change when optical force microscopy [102, 103] becomes available—in this technique a 3D light source can be controlled and positioned from outside.

Several aspects of aperture SNOM need improvement. By far the most important is the quality and reproducibility of the tips. This situation should improve when micromachined apertures become generally available.

The scattering-type SNOM approach avoids all the issues concerned with nanometre-sized apertures and can achieve even better lateral resolution. Although the problem of high background signal levels has been solved by different approaches, the main concerns are still the irradiation of large parts of the sample which do not contribute to the desired signal. Furthermore, probe geometries in terms of resolution and enhancement are still not fully optimised. Improvements based on theoretical modelling of scattering probes are in progress and will help to advance the preparation of tips for particular experiments.