Vibrational Analysis With Scanning Probe Microscopy
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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 Scan may refer to: Acronyms * Schedules for Clinical Assessment in Neuropsychiatry (SCAN), a psychiatric diagnostic tool developed by WHO * Shared Check Authorization Network (SCAN), a database of bad check writers and collection agency for bad ...
(SPM) and vibrational spectroscopy (
Raman scattering Raman scattering or the Raman effect () is the inelastic scattering of photons by matter, meaning that there is both an exchange of energy and a change in the light's direction. Typically this effect involves vibrational energy being gained by a ...
or/and
Fourier transform infrared spectroscopy Fourier-transform infrared spectroscopy (FTIR) is a technique used to obtain an infrared spectrum of absorption or emission of a solid, liquid, or gas. An FTIR spectrometer simultaneously collects high-resolution spectral data over a wide spectr ...
, 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 Topography is the study of the forms and features of land surfaces. The topography of an area may refer to the land forms and features themselves, or a description or depiction in maps. Topography is a field of geoscience and planetary sci ...
images simultaneously.


History


Raman-NSOM

Near-field scanning optical microscopy Near-field scanning optical microscopy (NSOM) or scanning near-field optical microscopy (SNOM) is a microscopy technique for nanostructure investigation that breaks the far field resolution limit by exploiting the properties of evanescent waves ...
(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 Surface plasmon resonance (SPR) is the resonant oscillation of conduction electrons at the interface between negative and positive permittivity material in a particle stimulated by incident light. SPR is the basis of many standard tools for measu ...
(SPR) which is the precursor of tip-enhanced Raman scattering (TERS) and
surface-enhanced Raman scattering Surface-enhanced Raman spectroscopy or surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces or by nanostructures such as plasmonic-magnetic silica n ...
(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 Aluminium (aluminum in American and Canadian English) is a chemical element with the symbol Al and atomic number 13. Aluminium has a density lower than those of other common metals, at approximately one third that of steel. I ...
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 A scanning tunneling microscopy image of a single-walled carbon nanotube Rotating single-walled zigzag carbon nanotube A carbon nanotube (CNT) is a tube made of carbon with diameters typically measured in nanometers. ''Single-wall carbon na ...
(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 Brilliant cresyl blue is a supravital stain used for counting reticulocytes. It is classified as an oxazine dye Oxazines are heterocyclic compounds containing one oxygen and one nitrogen atom in a doubly unsaturated six-membered ring. Isome ...
) 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.Bruno Pettinger, ''Tip-Enhanced Raman Spectroscopy (TERS)'' 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 Brilliant cresyl blue is a supravital stain used for counting reticulocytes. It is classified as an oxazine dye Oxazines are heterocyclic compounds containing one oxygen and one nitrogen atom in a doubly unsaturated six-membered ring. Isome ...
, malachite green isothiocyanate and
rhodamine 6G Rhodamine 6G is a highly fluorescent rhodamine family dye. It is often used as a tracer dye within water to determine the rate and direction of flow and transport. Rhodamine dyes fluoresce and can thus be detected easily and inexpensively with ...
, 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 A solid immersion lens (SIL) has higher magnification and higher numerical aperture than common lenses by filling the object space with a high- refractive-index solid material. SIL was originally developed for enhancing the spatial resolution of o ...
with a
refractive index In optics, the refractive index (or refraction index) of an optical medium is a dimensionless number that gives the indication of the light bending ability of that medium. The refractive index determines how much the path of light is bent, or ...
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 Boron nitride is a thermally and chemically resistant refractory compound of boron and nitrogen with the chemical formula BN. It exists in various crystalline forms that are isoelectronic to a similarly structured carbon lattice. The hexagonal ...
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 Scanning thermal microscopy (SThM) is a type of scanning probe microscopy that maps the local temperature and thermal conductivity of an interface. The probe in a scanning thermal microscope is sensitive to local temperatures – providing a nano- ...
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 Urea, also known as carbamide, is an organic compound with chemical formula . This amide has two amino groups (–) joined by a carbonyl functional group (–C(=O)–). It is thus the simplest amide of carbamic acid. Urea serves an important r ...
in a
cyclohexane Cyclohexane is a cycloalkane with the molecular formula . Cyclohexane is non-polar. Cyclohexane is a colorless, flammable liquid with a distinctive detergent-like odor, reminiscent of cleaning products (in which it is sometimes used). Cyclohexan ...
/
butanol Butanol (also called butyl alcohol) is a four-carbon alcohol with a formula of C4 H9 O H, which occurs in five isomeric structures (four structural isomers), from a straight-chain primary alcohol to a branched-chain tertiary alcohol; all are a b ...
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 In optical engineering, the objective is the optical element that gathers light from the object being observed and Focus (optics), focuses the ray (optics), light rays to produce a real image. Objectives can be a single Lens (optics), lens or mirr ...
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 A parabolic (or paraboloid or paraboloidal) reflector (or dish or mirror) is a reflective surface used to collect or project energy such as light, sound, or radio waves. Its shape is part of a circular paraboloid, that is, the surface generated ...
.


=Comparison of TERS and SERS

= Both TERS and SERS rely on a localized surface plasmon for increasing the ought-to-be weak Raman signal. hat TERS microscopy can do, SERS cannot What TERS microscopy can do, SERS cannotSatoshi Kawata Department of Applied Physics, Osaka University, Suita, Osaka and RIKEN, Wako, Saitama, Japan 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.Pettinger B.''Topics Appl. Phys.'', 103, 217–240 (2006) 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 Rayleigh scattering ( ), named after the 19th-century British physicist Lord Rayleigh (John William Strutt), is the predominantly elastic scattering of light or other electromagnetic radiation by particles much smaller than the wavelength of the ...
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,L. Rayleigh, ''Phil. Mag.''. 8, 261–274 (1879) 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 Tem or TEM may refer to: Acronyms * Threat and error management, an aviation safety management model. * Telecom Expense Management * Telecom Equipment Manufacturer * TEM (currency), local to Volos, Greece * TEM (nuclear propulsion), a Russian ...
) 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.R. M. Silverstein, G. C. Bassler, T. C. Morill, ''Spectrometric Identification of Organic Compounds, 5th edition'', Wiley, New York (1991) By combining SPM and vibrational spectroscopy, AFM/IR-NSOM and
AFM-IR AFM-IR (atomic force microscope-infrared spectroscopy) or infrared nanospectroscopy is one of a family of techniques (published online, Feb 2008) with erratum, 19(5), 14 May 2004 that are derived from a combination of two parent instrumental techn ...
have emerged as useful characterization tools that integrate the high spatial resolution abilities of AFM with IR spectroscopy.Craig Prater, Kevin Kjoller, Debra Cook, Roshan Shetty, Gregory Meyers, Carl Reinhardt, Jonathan Felts, William King, Konstantin Vodopyanov and Alexandre Dazzi
Nanoscale Infrared Spectroscopy of Materials by Atomic Force Microscopy
''Microscopy and Analysis'', 24, 5–8 (2010)
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 AFM-IR (atomic force microscope-infrared spectroscopy) or infrared nanospectroscopy is one of a family of techniques (published online, Feb 2008) with erratum, 19(5), 14 May 2004 that are derived from a combination of two parent instrumental techn ...
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 can be obtained through the measurement of the amplitude of the cantilever, which is a function of the IR source wavelength. For example, when the radiation laser wavelength is tuned at the resonance frequency with the vibrational absorption frequency of the sample, the displacement intensity of the cantilever will increase until the laser wavelength reaches the maximum of sample absorption. The displacement of the cantilever will then be reduced as the laser wavelength is tuned past the absorption maximum. This approach can map chemical composition beyond the diffraction-limit resolution and can also provide three-dimensional topographic, thermal and mechanical information at the nanoscale. Overall, it overcomes the resolution limit of traditional IR spectroscopy and adds chemical and mechanical mapping to the AFM and NSOM.


Infrared light source

The ideal IR source should be monochromatic and tunable within a wide range of wavelength. According to ''T'' ∝''d''4/''λ''4, where ''T'' is the transmission coefficient, ''d'' the aperture diameter and ''λ'' is wavelength, the aperture-based NSOM/FTIR transmission is even more limited due to the long infrared wavelength;C. J. Bouwkamp, ''Philips Res. Rep.'', 5, 321–332 (1950) therefore, an intense IR source is needed to offset the low transmission through the optical fiber. The common bright IR light sources are the
free-electron laser A free-electron laser (FEL) is a (fourth generation) light source producing extremely brilliant and short pulses of radiation. An FEL functions and behaves in many ways like a laser, but instead of using stimulated emission from atomic or molecula ...
(FEL), color-center lasers, CO2 lasers and
laser diode file:Laser diode chip.jpg, The laser diode chip removed and placed on the eye of a needle for scale A laser diode (LD, also injection laser diode or ILD, or diode laser) is a semiconductor device similar to a light-emitting diode in which a di ...
s. FEL is an excellent IR source, with 2–20 μm spectral range, short pulses (picosecond) and high average power (0.1-1 W). Alternately, a tabletop picosecond
optical parametric oscillator An optical parametric oscillator (OPO) is a parametric oscillator that oscillates at optical frequencies. It converts an input laser wave (called "pump") with frequency \omega_p into two output waves of lower frequency (\omega_s, \omega_i) by means ...
(OPO) can be used which is less expensive, but has a limited tunability and a lower power-output.


NSOM/FTIR experimental setup

The essence of NSOM/FTIR is that it allows the detection of non-propagating evanescent waves in the near-field (less than one wavelength from the sample), thus yielding high spatial resolution. Depending on the detection modes of these non-propagating evanescent waves, two NSOM/FTIR instrumentations are available: apertureless NSOM/FTIR and aperture-based NSOM/FTIR. ;Aperture-based NSOM/FTIR In aperture-based NSOM/FTIR, the probe is a waveguide with a tapered tip with a very small, sub-wavelength size aperture. When the aperture is brought into the near-field, it collects the non-propagating light and guides it to the detector. In general, there are two modes when the aperture is scanned over the sample: illumination mode and collection mode (Figure 7). The high-quality infrared fiber tip is very important in realizing NSOM/FTIR technique. There are several types of fibers, such as
sapphire Sapphire is a precious gemstone, a variety of the mineral corundum, consisting of aluminium oxide () with trace amounts of elements such as iron, titanium, chromium, vanadium, or magnesium. The name sapphire is derived via the Latin "sapphir ...
,
chalcogenide glass Chalcogenide glass (pronounced hard ''ch'' as in ''chemistry'') is a glass containing one or more chalcogens (sulfur, selenium and tellurium, but excluding oxygen). Such glasses are covalently bonded materials and may be classified as covalent netw ...
,
fluoride glass Fluoride glass is a class of non-oxide optical glasses composed of fluorides of various metals. They can contain heavy metals such as zirconium, or be combined with lighter elements like aluminum and beryllium. These heavier elements cause the gla ...
and hollow silica guides.Sanghera, J. S., and Aggarwal, I. D.,'' Infrared Fiber Optics'' (Boca Raton; Florida: CRC) 1998 Chalcogenide glasses are widely used because of their high transmittance in the broad IR range of 2–12 μm. The fluoride fibers also exhibit low transmitting losses beyond 3.0 μm. ;Apertureless NSOM/FTIR The probe is a sharp metal tip ending with a single or a few atoms. The sample is illuminated from
far-field The near field and far field are regions of the electromagnetic (EM) field around an object, such as a transmitting antenna, or the result of radiation scattering off an object. Non-radiative ''near-field'' behaviors dominate close to the ante ...
and the radiation is focused at the contact area between probe and sample. When this tip approaches the sample, usually within 10 nm, the incident electromagnetic field is enhanced due to the resonant surface plasma excitation as well as due to hot-spots in the sharp tip. The dipole interaction between the tip and sample change the non-propagating waves into propagating waves by scattering, and a detector collects the signal in the far-field. An apertureless NSOM/FTIR usually has better resolution (~5–30 nm) compared with aperture-based NSOM/FTIR (~50–150 nm). One main challenge in apertureless NSOM/FTIR is a strong background signal because the scattering is obtained from both near-field and remote area of the probe. Thus, the small near-field contribution to the signal has to be extracted from the background. One solution is to use a very flat sample with only optical spatial fluctuation. Another solution is to apply constant-height mode scanning or pseudo-constant-height mode scanning. ;Experimental scheme of aperture-based NSOM/FTIR Figure 8 shows the experimental setup used in NSOM/FTIR in the external reflection mode. FEL source is focused on the sample from the far-field using a mirror. The distance between the probe and a sample is kept at a few nanometers during scanning. Figure 9 is the cross-section of a NSOM/FTIR instrument. As shown below, sample is placed on a piezo-electric tube scanner, in which the x-y tube has four parts, namely x+, x-, y+ and y-. Lateral (x-y plane) oscillation of the fiber tip is induced by applying an AC voltage to a
dither Dither is an intentionally applied form of image noise, noise used to randomize quantization error, preventing large-scale patterns such as color banding in images. Dither is routinely used in processing of both digital audio and digital vide ...
piezo-scanner. Also, the fiber tip is fixed to a bimorph piezo-scanner so that the amplitude of the oscillation of the tip can be monitored through the scanner.


AFM-IR setup

;Spatial resolution The spatial resolution of an
AFM-IR AFM-IR (atomic force microscope-infrared spectroscopy) or infrared nanospectroscopy is one of a family of techniques (published online, Feb 2008) with erratum, 19(5), 14 May 2004 that are derived from a combination of two parent instrumental techn ...
instrument is related to the contact area between the probe and sample. The contact area is given by ''a''3 = 3''PR''/4''E''* and 1/''E''* = (1-''n''12)/ ''E''1+ (1-''n''22)/ ''E''2, where ''P'' is the force employed to the probe, ''n''1 and ''n''2 represent the Poisson ratios of the sample and probe, respectively, and ''E''1 and ''E''2 are the elastic moduli of the sample and probe materials respectively. Typically, an AFM-IR has a lateral spatial resolution of 10–400 nm, for example, 100 nm, ''λ''/150, and ''λ''/400. Recently, Ruggeri et al. have demonstrated the acquisition of infrared absorption spectra and chemical maps at the single molecule level in the case of protein molecules with ca. 10 nm diameter and a molecular weight of 400 kDa. ;Instrumentation In AFM-IR, an AFM probe is used to measure the absorption response of the sample to infrared radiation. The general approach for AFM/FTIR is shown in Figure 10. There are a few different experimental setups when the infrared radiation is projected onto the sample as shown below: top, side, and bottom illumination setups (Figure 11). In the first developed setup of
AFM-IR AFM-IR (atomic force microscope-infrared spectroscopy) or infrared nanospectroscopy is one of a family of techniques (published online, Feb 2008) with erratum, 19(5), 14 May 2004 that are derived from a combination of two parent instrumental techn ...
, a sample is mounted onto an infrared-transparent
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prism for excitation purposes (Figure 12), then an optical parametric oscillator (OPO)-based tunable IR lased is radiated on the molecules to be probed by the instrument. Similar to conventional ATR spectroscopy, IR beam illuminates the sample through total internal reflection mechanism (Figure 12). The sample will heat up while absorbing radiation which causes a rapid thermal expansion of the sample surface. This expansion will increase the resonant oscillations of the AFM cantilever in a characteristic ringdown pattern (ringdown patterns means the decay of cantilever oscillation exponential in nature). Through Fourier transformation analysis, the signal could be isolated to obtain the amplitudes and frequencies of the oscillations. The amplitudes of the cantilever provide information of local absorption spectra, whereas the oscillation frequencies depend on the mechanical stiffness of the sample (Figure 12).


Pros and cons

NSOM combined with FTIR/Raman techniques can provide local chemical information together with topographical details. This technique is non-destructive and can work in a variety of environments (liquids), for example, when detecting single biomolecules. The illuminated area of sample is relatively big at around 1 μm. However, the sampling area is only ~10 nm. This means that a strong background from an unclean tip contributes to the overall signal, hindering the signal analysis. The Raman spectroscopy in general could be time-consuming due to the low scattering efficiency (<1 in 107 photons). It usually takes several minutes to accumulate a conventional Raman spectrum, and this time could be much longer in Raman-NSOM; for example, 9 hours for a 32×32-pixel image. As to near-field IR/AFM, high optical losses in aqueous environments (water is strongly absorbing in the IR range) reduces the signal-to-noise ratio.


Applications

Improving the resolution and enhancing the instrumentation with user-friendly hardware and software will make AFM/NSOM coupled with IR/Raman a useful characterization tool in many areas including biomedical, materials and life sciences. For example, this technique was used in detecting the spin-cast thin film of poly(dimethylsiloxane) with polystyrene on it by scanning the tip over the sample. The shape and size of polystyrene fragments was detected at a high spatial resolution due to its high absorption at specific resonance frequencies. Other examples include inorganic boron nitride thin films characterization with IR-NSOM. The images of single molecule
rhodamine 6G Rhodamine 6G is a highly fluorescent rhodamine family dye. It is often used as a tracer dye within water to determine the rate and direction of flow and transport. Rhodamine dyes fluoresce and can thus be detected easily and inexpensively with ...
(Rh-6G) was obtained with a spatial resolution of 50 nm. These techniques can be also used in numerous biological related applications including the analysis of plant materials, bone, and single cells. Biological application was demonstrated by detecting details of conformation changes of cholesteryl-oleate caused by FEL irradiation with a spatial resolution below the diffraction limit. Researchers also used Raman/NSOM in tracking the formation of energy-storing polymer
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in bacteria ''
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capsulatus''. This characterization tool may also help in the kinetic studies on physical and chemical processes at a wide variety of surfaces giving chemical specificity via IR spectroscopy as well as high-resolution imaging via AFM. For example, the study of the hydrogen termination of Si (100) surface was performed by studying the absorbance of Si-O bond to characterize the reaction between silicon surface and atmospheric oxygen.E. Romano, S. Trabattoni, M. Campione, E. Merati, A. Sassella and D. Narducc
''Combined use of AFM and FTIR in the analysis of the hydrogen termination of Si(100) surfaces''
Microscopy: Science, Technology, Applications and Education, A. Méndez-Vilas and J. Diaz (Eds.), Vol. 3, pp. 1984–1992 (2010)
Studies were also conducted where the reactivity of a polymer, a 1000-nm-thick poly-(tert-butylmethacrylate) (PTBMA) combined with a photochemically modified 500-nm-thick poly(methacrylic acid) (PMAA), with water vapor depicted the different absorption bands before and after water uptake by the polymer. Not only the increased swell of PMAA (280 nm) was observed but also the different absorption ability of water was shown by the different transmission of IR light at a much smaller dimension (<500 nm). These results are related to polymer, chemical and biological sensors, and tissue engineering and artificial organ studies. Because of their high spatial resolution, NSOM/AFM-Raman/IR techniques can be used for measuring the width of multilayer films, including layers which are too small (in the x and y directions) to be probed with conventional IR or Raman spectroscopy.


References

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