The technique of vibrational analysis with scanning probe microscopy allows probing vibrational properties of materials at the submicrometer scale, and even of individual molecules. This is accomplished by integrating scanning probe microscopy (SPM) and vibrational spectroscopy (Raman scattering or/and Fourier transform infrared spectroscopy, FTIR). This combination allows for much higher spatial resolution than can be achieved with conventional Raman/FTIR instrumentation. The technique is also nondestructive, requires non-extensive sample preparation, and provides more contrast such as intensity contrast, polarization contrast and wavelength contrast, as well as providing specific chemical information and topography images simultaneously.
History.
Raman-NSOM.
Near-field scanning optical microscopy (NSOM) was described in 1984, and used in many applications since then. The combination of Raman scattering and NSOM techniques was first realized in 1995, when it was used for imaging a Rb-doped KTP crystal at a spatial resolution of 250 nm.
NSOM employs two different methods for data collection and analysis: the fiber tip aperture approach and the apertureless metal tip approach. NSOM with aperture probes has a smaller aperture that can increase the spatial resolution of NSOM; however, the transmission of light to the sample and the collection efficiency of the scattered/emitted light is also diminished. The apertureless near-field scanning microscopy (ANSOM) was developed in the 1990s. ANSOM employs a metalized tip instead of an optical fiber probe. The performance of the ANSOM strongly depends on the electric field enhancement factor of the metalized tip. This technique is based on surface plasmon resonance (SPR) which is the precursor of tip-enhanced Raman scattering (TERS) and surface-enhanced Raman scattering (SERS).
In 1997, Martin and Girard demonstrated theoretically that electric field under a metallic or dielectric tip (belonging to NSOM apertureless technique) can be strongly enhanced if the incident field is along the tip axis. Since then a few groups have reported Raman or fluorescence enhancement in near field optical spectroscopy by apertureless microscopy. In 2000, T. Kalkbrenner "et al." used a single gold particle as a probe for apertureless scanning and presented images of an aluminium film with 3 μm holes on a glass substrate. The resolution of this apertureless method was 100 nm, that is comparable to that of fiber-based systems Recently, a carbon nanotube (CNT) having a conical end, tagged with gold nanoparticles, was applied as a nanometer-resolution optical probe tip for NSOM. NSOM images were obtained with a spatial resolution of ~5 nm, demonstrating the potential of a composite CNT probe tip for nanoscale-resolution optical imaging.
Tip-enhanced Raman scattering.
There are two options for realizing apertureless NSOM-Raman technique: TERS and SERS. TERS is frequently used for apertureless NSOM-Raman and can significantly enhance the spatial resolution. This technique requires a metal tip to enhance the signal of the sample. That is why an AFM metal tip is usually used for enhancing the electric field for molecule excitation. Raman spectroscopy was combined with AFM in 1999. A very narrow aperture of the tip was required to obtain a relatively high spatial resolution; such aperture reduced the signal and was difficult to prepare. In 2000, Stȍckle "et al." first designed a setup combining apertureless NSOM, Raman and AFM techniques, in which the tip had a 20 nm thick granular silver film on it. They reported a large gain in the Raman scattering intensity of a dye film (brilliant cresyl blue) deposited on a glass substrate if a metal-coated AFM tip was brought very close to the sample. About 2000-fold enhancement of Raman scattering and a spatial resolution of ~55 nm were achieved.
Similarly, Nieman "et al". used an illuminated AFM tip coated with a 100 nm thick film of gold to enhance Raman scattering from polymers samples and achieved a resolution of 100 nm. In the early research of TERS, the most commonly used coating materials for the tip probe were silver and gold. High-resolution spatial maps of Raman signals were obtained with this technique from molecular films of such compounds as brilliant cresyl blue, malachite green isothiocyanate and rhodamine 6G, as well as individual carbon nanotubes.
IR-NSOM and AFM.
IR near-field scanning optical microscopy (IR-NSOM) is a powerful spectroscopic tool because it allows subwavelength resolution in IR spectroscopy. Previously, IR-NSOM was realized by applying a solid immersion lens with a refractive index of "n", which shortens wavelength ("λ") to ("λ/n"), compared to FTIR-based IR microscopy. In 2004, an IR-SNOM achieved a spatial resolution ~"λ"/7 that is less than 1 μm. This resolution was further improved to about "λ"/60 that is 50–150 nm for a boron nitride thin film sample.
IR-NSOM uses an AFM to detect the absorption response of a material to the modulated infrared radiation from an FTIR spectrometer and therefore is also referred to as AFM/FTIR spectroscopy. Two approaches have been used to measure the response of polymer systems to infrared absorption. The first mode relies on the AFM contact mode, and the second mode of operation employs a scanning thermal microscopy probe (invented in 1986) to measure the polymer's temperature increase. In 2007, AFM was combined with infrared attenuated total reflection (IR-ATR) spectroscopy to study the dissolution process of urea in a cyclohexane/butanol solution with a high spatial resolution.
Theory and instrumentation.
Raman-NSOM.
There are two modes for the operation of NSOM technique, with and without an aperture. These two mode have also been combined with the near-field Raman spectroscopy. The near-field aperture must be nanosized that complicates the probe manufacturing process. Also, the aperture method usually has a very weak signal due to weak excitation and Raman scattering signal. Overall, these factors lower the signal-to-noise ratio in aperature based NSOM/Raman technique. Apertureless probes are based on a metal-coated tip and provide a stronger signal.
Aperture-based detection.
Although the apertureless mode is more promising than the aperture mode, the latter is more widely used because of easier instrumental setup and operation. To obtain a high resolution Raman micrograph/spectrum, the following conditions should be met: (1) the size of the aperture must be on the order of the wavelength of the excitation light. (2) The distance from the tip of the probe to the sample must be smaller than excitation wavelength. (3) The instrument must remain stable over a long time. An important AFM feature is the ability to accurately control the distance between the sample and probe tip, which is the reason why the AFM-Raman combination is preferred for realizing Raman-NSOM.
Apertureless mode.
The main drawback of the aperture mode is that the small aperture size reduces the signal intensity and is difficult to fabricate. Recently, researchers have focused on the apertureless mode, which utilizes SPR theory to produce stronger signals. There are two techniques supporting this mode: SERS and TERS.
TERS technique.
Theory and instrumentation of Raman/AFM and IR/AFM combine the theory of SPR (AFM and NSOM) and Raman scattering, and this combination is based on TERS. In TERS, the electric field of excitation source induces an SPR in the tip of the probe. If the electric field vector of the incidence light is perpendicular (s-polarized) to the metal tip axis, the free electrons are driven to the sides lateral of the tip. If it is parallel (p-polarized) to the tip axis, the free electrons on the surface of the metal are confined to the end of the apex of tip. As a consequence, there is a very large electric-field enhancement which is sensed by the molecules close to it leading to a stronger signal. 
A typical approach in a TERS experiment is to focus the laser beam on a metal tip with the light polarized along the tip axis, followed by collection of the surface-enhanced Raman scattered light from the sample in the enhancement zone of the tip using optics.
Depending on the sample and experiment, different illumination geometries have been applied in TERS experiments, as shown in figure 4. With p-polarized (parallel to the surface normal) incidence light, the plasmon excitation at the tip is most efficient. If the focusing objective lens is also used for collecting the scattered photons (backscattering geometry), the optimum angle is around 55° with respect to the surface normal. This is because the scattering lobe is maximum with this configuration and it provides a much enhanced signal. The setup of figure 4(A) is usually used for the large thick samples. Setup (B) handles semi-transparent or transparent samples, such as single cells, tissue samples and biopolymers. The setup of figure 4(C) is preferred for opaque samples because all the light would be focused by the parabolic mirror.
Comparison of TERS and SERS.
Both TERS and SERS rely on a localized surface plasmon for increasing the ought-to-be weak Raman signal. The only difference between them is that the sample in SERS has a rough surface that hinders application of a sharp AFM-like tip. TERS, on the other hand, uses a metal-coated tip having some roughness at nanoscale. The “hot spot” theory is very popular in explaining the large enhancement in the signal. That is, the signal from “hot spots” on the surface of the sample dominates the total signal from the sample. This is also reinforced by the fact that the distance between nanoparticles and sample is an important factor in obtaining high Raman signal.
Raman/AFM instrumentation.
The Raman/AFM technique has two approaches: aperture and apertureless, and the apertureless mode is realized with SERS and TERS. Figure 5 is the example of an integrated TERS system. It shows that there are five main components for a whole integrated TERS (apertureless) system. These components are: microscope, one objective lens, one integrated AFM head, a Raman spectrometer and a CCD. The laser is focused on the sample, on piezo-stage and the AFM tip by the moving the laser beam along the tip. The movement of the laser beam is achieved by the mirror in the top left corner. The XYZ piezo-stage in the left bottom holds the sample. In this design, the laser beam is focused on the sample through an objective lens, and the scattered light is collected by the same lens.
This setup utilizes a low contact-pressure to reduce the damage to the AFM tip and sample. The laser power is typically below 1 mW. The notch filter can filter Rayleigh scattering from the excitation laser light from the back of the cantilever. The laser beam is focused on the apex of the gold-coated AFM tip and the sample. The laser scanning is completed by the moving the mirror across the approaching tip. A small enhance in background occurs when the laser spot focuses on the tip area. The movement of the XYZ piezo-stage finishes the sample scanning. The wide red signal is Raman signal which is collected through the objective lens. The same lens is also used for excitation of the sample and collecting the Raman signal.
NSOM/FTIR, AFM/FTIR and AFM-IR.
Because of the diffraction limit in the resolution of conventional lens-based microscopes, namely D = 0.61"λ"/nsinθ, the maximum resolution obtainable with an optical microscope is ~200 nm. A new type of lens using multiple scattering of light allowed to improve the resolution to about 100 nm. Several new microscopy techniques with a sub-nanometer resolution have been developed in the last several decades, such as electron microscopy (SEM and TEM) and scanning probe microscopy (NSOM, STM and AFM). SPM differs from other techniques in that the excitation and signal collection are very close (less than diffraction limit distance) to the sample. Instead of using a conventional lens to obtain magnified images of samples, an SPM scans across the sample with a very sharp probe. Whereas SEM and TEM usually require vacuum and an extensive sample preparation, SPM measurements can be performed in atmospheric or liquid conditions.
Despite the achievable resolution of atomic scale for AFM and NSOM techniques, it does not provide chemical information of the sample. The infrared part of the electromagnetic spectrum covers molecular vibrations which can characterize chemical bonding within the sample.
By combining SPM and vibrational spectroscopy, AFM/IR-NSOM and AFM-IR have emerged as useful characterization tools that integrate the high spatial resolution abilities of AFM with IR spectroscopy. This new technique can be referred to as AFM-FTIR, AFM-IR and NSOM/FTIR. AFM and NSOM can be used to detect the response when a modulated infrared radiation generated by an FTIR spectrometer is absorbed by a material. In the AFM-IR technique the absorption of the radiation by sample will cause a rapid thermal expansion wave which will be transferred to the vibrational modes of the AFM cantilever. Specifically, thermal expansion wave induces a vertical displacement of the ATM tip (Figure 6). A local IR absorption spectrum then