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In this course the general introduction to Raman spectroscopy and microscopy will be provided and practical tips as well as examples will be given. The capability of Raman spectroscopy for the analysis of real-life samples (paint components, clays, coating materials, etc.) taken from historical and archaeological objects will be discussed.

1. Principles of Raman spectroscopy

Raman spectroscopy is widely used in the investigation of cultural heritage materials due to its high spatial resolution (typically in the range of 1 to 10 µm), large amount of obtainable information, non-destructivity and ability to perform in-situ analysis.^1,2 With Raman spectroscopy it’s possible to analyse various materials: minerals, inorganic and organic pigments, binding media, varnishes, ceramics, plastics, textile fibres etc.²

The following video explains the principles and instrumentation of Raman spectroscopy.https://www.uttv.ee/embed?id=29055

Similarly to infrared spectroscopy, Raman spectroscopy is classified as vibrational spectroscopy.³ Raman spectroscopy is based on Raman scattering (or Raman effect) that reveals the vibrational, rotational and other low frequency modes of molecules⁴. In this technique, the sample is exposed to an intense beam of monochromatic light (typically a laser beam) in the frequency range of visible, near-infrared or near-ultraviolet region.⁵ The electromagnetic radiation, interacting with a substance, can be transmitted, absorbed, or scattered⁶. When the monochromatic radiation is scattered by molecules, the majority of the radiation undergoes the common Rayleigh scattering (radiation’s  frequency/wavelength is unchanged). However, a small fraction of the scattered radiation is observed to have a slightly different frequency from that of the incident radiation. This is known as the Raman effect⁷. The Raman lines show up pairwise. The dominant Stokes lines have a lower frequency (longer wavelength) than the initial radiation, whereas the weaker (often nondetectable) anti-Stokes lines have a higher frequency (shorter wavelength).^4,5 The frequency shifts are virtually independent of the excitation wavelength and are characteristic of the particular substance/molecule. Usually one only records the relatively strong Stokes lines, which therefore are attributed a positive frequency shift. Such spectral coordinate is called the Raman shift and measured in wavenumbers (in cm^-1).⁴ See scheme in Figure 1.

Figure 1. Scheme of Raman scattering.

In Raman spectroscopy, as it is a scattering technique, samples are simply placed in the laser beam and the scattered radiation is collected and analysed⁸. Raman spectrometer measures the wavelength-dependent intensity of the inelastically scattered light.

The obtained Raman spectra are essentially vibrational spectra. Hence, if presented in the Raman shift scale, they are directly comparable to corresponding infrared absorption spectra (see Figure 2). However, Raman spectrum arises in a different manner and the rules, which vibrations are Raman-active (and thus produce signals in the spectrum), are different. It turns out that a vibration is Raman-active (i.e. revealed as a spectral line in the Raman spectrum), if the polarizability of the molecule changes during the vibration.⁷ It often happens that vibrations that are active (or give high-intensity signals) in Raman scattering are inactive (or give low-intensity signals) in the infrared, and vice versa.⁷Therefore, Raman spectra often provide complementary information to IR spectra.

Figure 2. Raman (laser 514.5 nm) and IR spectra of benzene.

1.1. Instrumentation

There are two types of Raman spectrometers: dispersive spectrometers (based on the use of diffraction grating) and interferometer containing Fourier-transform Raman spectrometers (FT-Raman)⁹.

In general the main components of Raman spectrometers are presented on the following scheme:

In Raman spectroscopy, the choice of excitation wavelength and intensity is very important. Different wavelengths are suitable for the analysis of different types of material. The wavelength will affect the Raman intensity, spatial resolution, background fluorescence, and potential damage to the sample. Almost exclusively lasers are used as excitation sources, because they are highly monochromatic, give high-intensity radiation and can be efficiently focused due to their high coherence. Only continuous wave (CW) lasers are used, as pulsed lasers easily damage the sample. Some popular CW lasers are presented in Table 1. Traditionally, laser wavelengths up to 830 nm have been used in dispersive instruments while the 1064 nm laser line has been employed in FT-Raman setups. With the availability of sensitive InGaAs array detectors, it has become meaningful to use also the 1064 nm lasers with dispersive Raman instruments.

Table 1. Laser sources for Raman spectroscopy.

Raman scattering efficiency decreases with increasing excitation wavelength as λ⁻⁴. However, short-wavelength lasers more easily induce fluorescence, absorb in the sample or cause other undesirable effects due to their high photon energy. Hence, most common laser wavelengths in Raman spectroscopy are in the visible and NIR region (such as 633 or 785 nm) which offer low fluorescence whilst retaining relatively high Raman intensity. For samples which exhibit fluorescence even under red excitation (for example dyes), the 1064 nm laser may be needed. While near-infrared lasers have a smaller photon energy, compared to visible lasers, they are usually more powerful, in order to compensate for the reduced Raman scattering efficiency. Therefore, they may still damage the sample. It is especially important for strongly absorbing (black) samples, in which case the UV/visible lasers (operating at lower intensities) may yield a stronger Raman signal.

Dispersive Raman spectrometers

A dispersive spectrometer utilizes a diffraction grating to angularly disperse the light. As a result, at the detector plane, different wavelengths become spatially separated. Nevertheless, prior to entering the spectrometer, the incoming light should go through a special edge or notch filter to suppress the primary (Raman-scattered) light and thereby reduce the scattering inside the spectrometer. A matrix detector is used to record the dispersed spectrum. Typically, a silicon-based cooled CCD is used, which is very sensitive in the visible and NIR region (up to 1100 nm).

FT-Raman spectrometers

Commercial FT-Raman spectrometers were introduced in the late 1980s¹⁰. Their operating principle is similar to that of FTIR spectrometers and is based on an interferometer. As the Raman-scattered light enters the instrument, the interferometer selectively modulates the individual spectral components by systematically changing an optical path length difference. The resulting beam of light is recorded by a point detector. FT-Raman is superior to a dispersive instrument in the near-IR region beyond 1000 nm. Commonly, the 1064 nm laser excitation along with germanium or indium gallium arsenide (InGaAs) detector is used. They also offer excellent wavelength accuracy and can potentially combine IR absorption and Raman measurement capacity in single instrument. However, FT-Raman frequently needs to use high laser intensities due to the reduced Raman scattering efficiency at longer wavelengths, which may damage the sample.

Different types of Raman Spectroscopy

A variety of Raman instruments and special techniques are used for the analysis of cultural heritage materials. The choice of instrument determines the sensitivity, spectral range and resolution, spatial resolution, availability of different excitation sources, and convenience of operation. 

• Micro-Raman spectrometer (or Raman microscope) is the most common bench-top Raman instrument. A high-resolution spectrometer (either dispersive or FT) and one or several laser sources are coupled through an optical microscope. The excitation beam is focused and the secondary emission is collected simultaneously by the microscope objective in backscattering geometry. A high-numerical aperture (NA) objective yields both a high spatial resolution and a high collection efficiency.
• Surface-enhanced Raman spectroscopy (SERS) involves inelastic light scattering by molecules placed close to nanometal surfaces, which amplify the scattering by plasmonic resonance. One approach is to study molecules adsorbed onto corrugated metal surfaces such as silver or gold nanoparticles¹¹. Another approach is to stimulate the molecules by a sharp metal tip. Such tip-enhanced Raman spectroscopy is typically implemented by combining a confocal microscope and a scanning probe microscope. 
• In Resonance Raman spectroscopy (RRS) the incident photon energy is close in energy to an electronic transition of a compound or material under examination. 
• In a portable Raman spectrometer, a miniature dispersive spectrometer and a small laser source are integrated into a portable, hand-held device. Hence, the instrument can be used to perform in situ analysis in museums, archives, also outdoors on archaeological sites for the analysis of mural or cave paintings. Such portable devices frequently employ a fiber-optic probes. 

1.2. Problems with Raman spectroscopy

Compared to IR absorption, the primary disadvantage of Raman spectroscopy is the fluorescent background (see Figure 3). As Raman scattering is inherently weak, one has to use an intense laser beam for excitation, and for many materials, this results in a strong fluorescence – either due to the material itself of impurities. Sometimes even trace impurities – if they are strongly fluorescent – can lead to disturbing fluorescence background. Fortunately, Raman lines are spectrally close to the laser beam whereas fluorescence has typically a large Stokes shift. 

Figure 3. Example of the fluorescence in the Raman spectrum of red lead containing paint.

Relative to the Raman signal, the fluorescent background can be highly intense and even the tail of the fluorescence band may obscure the Raman spectrum. Although the problem can be partially resolved by careful sample preparation, time resolved spectroscopy or coherent anti-Stokes Raman spectroscopy (CARS), there will always be experiments that remain difficult to perform.⁷

In addition to fluorescence, intense focused laser irradiation can cause heating and degradation of the sample. The problems are typical for organic, soft, photosensitive or dark/coloured materials whereas transparent inorganic materials have usually quite high damage threshold.

2. Analysis with Raman spectroscopy

In the following video Senior Research Fellow Dr. Valter Kiisk demonstrates and explains how to perform measurements with a typical micro-Raman spectrometer.https://www.uttv.ee/embed?id=29396

Identification of the composition of the studied material is often based on the comparison of its Raman spectrum with a spectral library of reference materials.¹² Different papers and books have been published from where Raman spectra or information about excitation wavelengths and list of wavenumbers in the Raman spectra are available ^5,13,14. Also a very valuable on-line database is made available by the Infrared & Raman Users Group (IRUG)– http://irug.org/ – from where different Raman (and also IR) spectra of cultural heritage materials can be obtained free of charge.

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The Scanning Electron Microscope (SEM) produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen (raster scanning).

There are two families of electron guns:

• Conventional thermionic emitters such as Tungsten (W) or Lanthanum hexaboride (LaB6) tipped filaments.
• Tungsten field emission gun (FEG) , warm or Cold FEG. A pointed emitter is held at several kilovolts (2000-7000 V) so that there is sufficient potential at the emitter surface to cause field electron emission.

Field emission gun (FEG) is used to produce an electron beam that is smaller in diameter, more coherent and up to three orders of magnitude greater current density or brightness.

scrollable

Energy of electrons is depending of Voltage: 1 Kev to 50KeV

Current (A): Number of electrons /unit of time

1 amp = 1 coulomb/sec 1 coulomb ~ 6 x10¹⁸ electrons

Example if the current measured at sample is around 10^-9A to 10^-12 A then the number of electrons is around 6X10⁶ to 6X10⁹ electrons/sec.

Environmental Scanning Electron Microscope (ESEM)

ESEM is a variety of SEM called environmental scanning electron microscope. It can produce images of sufficient quality and resolution with the samples being wet or contained in low vacuum or gas. This greatly facilitates imaging biological samples that are unstable in the high vacuum of conventional electron microscopes. The major disadvantage of transmission electron microscope is the need for extremely thin sections of the specimens, typically about 100 nanometers. Biological specimens are typically required to be chemically fixed, dehydrated and embedded in a polymer resin to stabilize them sufficiently to allow ultrathin sectioning. Sections of biological specimens, organic polymers and similar materials may require special treatment with heavy atom labels in order to achieve the required image contrast.

ESEM is especially useful for non-metallic, uncoated and biological materials. The presence of gas, mainly Argon, around a sample permits to work with pressure greater than 500 Pa compared to conventional SEM requirements samples under vacuum about 10-3 to 10-4 Pa. This vacuum level creates the possibility to operate on non-conductive samples without any preparation or hydrated specimens without charging.

Transmission Electron Microscope (TEM)

In a Transmission Electron Microscope (TEM), the electron beam is accelerated by an anode typically at +100 keV (40 to 400 keV) with respect to the cathode, focused by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope.

The spatial variation in this information (the “image”) may be viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide. Alternatively, the image can be photographically recorded by exposing a photographic film or plate directly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a fiber optic light-guide to the sensor of a digital camera. The image detected by the digital camera may be displayed on a monitor or computer.

A transmission electron microscope can achieve better than 50 pm resolution and magnifications of up to about 10,000,000x whereas most light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x. Generally, the image resolution of an SEM is at least an order of magnitude poorer than that of a TEM. However, because the SEM image relies on surface processes rather than transmission, it is able to image bulk samples up to many centimeters in size and (depending on instrument design and settings) has a great depth of field, and so can produce images that are good representations of the three dimensional shape of the sample.

The Scanning Transmission Electron Microscope (STEM) rasters a focused incident probe across a specimen that (as with the TEM) has been thinned to facilitate detection of electrons scattered through the specimen. The high resolution of the TEM is thus possible in STEM. The focusing action (and aberrations) occurs before the electrons hit the specimen in the STEM, but afterward in the TEM.

Focused ion beam (FIB)

Focused ion beam, also known as FIB, is a technique used particularly in the semiconductor industry, materials science and increasingly in the biological field for site-specific analysis, deposition, and ablation of materials. A FIB setup is a scientific instrument that resembles a scanning electron microscope (SEM). However, while the SEM uses a focused beam of electrons to image the sample in the chamber, a FIB setup uses a focused beam of ions instead. Unlike an electron microscope, FIB is inherently destructive to the specimen.

When the high-energy gallium ions strike the sample, they will sputter atoms from the surface. Gallium atoms will also be implanted into the top few nanometers of the surface, and the surface will be made amorphous. A FIB-SEM consists in a system with both electron and ion beam columns, allowing the same feature to be investigated using either of the beams. A FIB-SEM system uses a beam of Ga+ ion to mill into the surface to locate a feature or defect of interest. The integrated SEM then uses a focused beam of electrons to image the sample in the chamber.

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Atomic force microscopy (AFM) is a technique with multiple applications in biology. This method is a member of the broad family of scanning probe microscopy and was initially developed in 1986 by Binnig et al to overcome the disadvantages of the scanning tunneling microscopy (STM) [1]. In the case of STM, only conductive materials can be studied as the resolution is obtained by using a tunneling current between a sharp probe and the sample surface[1]. In contrast, AFM uses small forces on the surface by a probe, thus do not damage samples and can provide information of surface topography of biological materials. AFM soon attracted the attention of the biophysical scientists in biomembrane as well as synthetic membrane research due to its capability of observing biological molecular system with resolution on nanometer scale and its possibility of three dimensional imaging [2].

Fundamental Elements of AFM

An atomic force microscope consists of a flexible cantilever containing a sharp probe, laser, photodiode detector, piezoelectric scanner and feedback electronics [3]. The microscope obtains the surface topography by scanning the tip in gentle touch with the sample. The tip motion is monitored by the piezoelectric scanner. As the tip scans the sample, the forces between the tip and the sample surface cause the cantilever to bend. A photodiode detector detects the deflection of a laser beam reflected off the back of the cantilever onto a two- segment photodiode. In most operating modes, a feedback circuit connected to the cantilever deflection sensor keeps the interaction between the tip and the sample at a fixed value and controls the tip-sample distance. The feedback signal is recorded by a computer to reconstruct a 3D image of the surface topography.

The force between the probe and the sample surface depends on the spring constant (stiffness of the cantilever) and the tip-sample distance). The amount of force is calculated based on Hooke’s law:F=−kx(6.1.1)(6.1.1)F=−kx

• FF: Force
• kk: spring constant
• xx: cantilever deflection.

Imaging modes

There are three primary imaging modes in AFM: contact, non-contact and intermittent (tapping) mode [4]. During contact mode, the probe is in contact with the sample and repulsive Van der Waals forces prevail, whilst attractive Van der Waals forces are dominant when the tip moves further away from the sample surface [5].

Contact Mode

In contact mode, the tip is constantly in touch with the sample surface. The applied force is kept constant while the tip scans the surface, creating the surface image [4]. This imaging mode is good for samples with rough and rigid surfaces as it provides fast scanning with high resolution. One disadvantage of this imaging mode is that soft samples like tissues can be deformed or damaged due to the applied force. This drawback can be solved by measuring the sample in aqueous environments to reduce the interaction force between the tip and the sample.

Tapping Mode

In tapping mode, the tip is not in constant contact with the sample surface. Instead, the cantilever is oscillated at its resonant frequency, which makes the tip lightly tap on the surface during scanning. A constant tip-sample interaction is maintained by monitoring the oscillation amplitude and an image is obtained [4]. Several parameters that affect the image contrast are the height, phase signals and amplitude [6]. Phase signals are influenced by material properties of the sample, for example viscoelasticity [6]. The force during scanning is greatly reduced, therefore this mode is useful for biological samples, where the samples are easily damageable or loosely bound to their surface. However, this imaging mode requires a slower scanning speed and is more challenging to measure in liquids.

Non-contact mode

There is no contact between the sample surface and the probe in non-contact mode. The probe oscillates above the sample surface, forming a weak attractive force between the tip apex atom and the sample surface atom. Feedback signals are obtained by measuring a frequency shift in the mechanical oscillation of the cantilever [6].

The force exerted on the surface sample is very low in this case. Moreover, as there is no contact between the probe and the surface, the probe lifetime can be extended. Another advantage of this operating mode is the possibility to observe an atomic defect if the very weak attractive force can be detected. The drawback of this mode is the reduction of resolution, and the oscillation of the cantilever is affected in case there are contaminants on the sample surface. Usually this operation mode requires a careful control of the environment (in UHV) to carry out [7].

Cantilever and Tips

The scanning probe is an important component of the AFM. The dimensions of the cantilever are in micrometer range, while its tip has a radius of a few nanometers [4]. Different cantilever lengths, shapes and materials lead to various spring constants and resonant frequencies. The most common materials of the probes are silicon nitride (Si₃N₄) or silicon (Si) [5]. Figure 6.1.46.1.4 shows a scanning electron microscope (SEM) image of a silicon nitride (4a, 4b) and silicon (4c, 4d) cantilever chip, where a tiny tip having a pyramid shape is integrated at the end. The silicon nitride probe is often used in static contact mode, where the stiffness of the cantilever should be as low as possible [4]. The silicon probe is usually used in dynamic operation mode, which requires higher values for the spring constant to reduce noise and instabilities [4].

Typically, spring constants of AFM cantilevers vary between 0.01 Nm^-1 and 100 Nm^-1, enabling a force sensitivity of 10-11N [4]. The force sensitivity is influenced by thermal, electrical and optical noise. [5] For biological samples, cantilevers in contact mode often have resonance frequencies between 5 and 50 kHz in vacuum [8]. Figure 6.1.56.1.5 reports an AFM tip made of carbon nanotubes (CNTs), which was a breakthrough in terms of resolution. CNTs tips have a high aspect ratio, small diameter, and a well-defined surface chemistry, therefore appearing to be the ideal probe for biological applications [4].

AFM on membranes

Native membranes studied by AFM

Biomembrane sample preparation

In order to study membranes using AFM, the membranes need to be fixed on a flat solid support [8]. Several solid supports such as mica, highly ordered pyrolitic graphite (HOPG), template stripped gold and molybdenum disulfide have proved to give high resolution images [6, 8]. While mica is an insulator and exposes a hydrophilic surface, HOPG is a good conductor and hydrophobic [8]. The similarity between these two substrates is an atomically flat surface [8]. Based on chemical adsorption mechanism, membranes are attached onto the solid support by adjusting pH and ionic strength. As the gap between membrane and the support is very small (0.5-2nm), this may cause impaired mobility for membrane proteins that are directly attached to the support [6]. Furthermore, the adsorption force may affect the conformation of the membrane proteins. To sum up, current sample preparation methods still need further consideration in order to study dynamics and structure of native membrane proteins.

AFM images of native membrane

AFM can obtain the images of native membranes at submolecular resolution, which is a great advantage comparing to other methods [6]. It is effective in providing information of the native organization of membrane proteins and their complexes. Figure 6.1.66.1.6 shows an example of the study of native membrane using AFM. Disk membranes were prepared from mouse retina and then attached on mica support [6]. The AFM image revealed tight rows of dimers packed in the structure arrangement of rhodopsin [6]. This provides a platform for interaction with arrestin and transducin.

Model lipid membranes studied by AFM

Preparation of supported lipid bilayers.

Supported lipid bilayers (SLBs) have been used as a biomimetic model for biomembranes in numerous studies [3, 9]. This system consists of two lipid leaflets spread on a solid support [3]. Although this model lacks some features of the real membranes, it still provides an insight of the structural organization and characteristics of cell membranes. The first method to prepare SLBs is the fusion of lipid vesicles on solid supports [9]. The vesicles are prepared via sonication or extrusion, then adsorbed on the surface of the solid support. The adsorbed vesicles either form larger vesicles by fusing together, or directly rupture and form SLBs.

Another method is to use a hydrophilic substrate on which two consecutive lipid monolayers are deposited by Langmuir-Blodgett transfer [3]. A Teflon-coated trough contains the aqueous solution, with two movable Teflon barriers used to control the area for lipid spread and form a monolayer at the air-water interface. There is also a balance to measure the surface pressure, controlling lipid packing. The solid support is then pulled vertically through the lipid monolayer, depositing the first lipid layer on the substrate. The transference of the second lipid layer can be completed by dipping the lipid support either horizontally or vertically. This technique can be applied to fabricate SLBs having two different lipid composition.

AFM studies of SLBs formation.

SLBs formation process by fusing lipid vesicles on solid supports can be studied in situ by AFM technique, as illustrated in Figure 6.1.76.1.7. The vesicles spread from the edge towards the center, then stacked on top of each other. The edges of the top and bottom bilayers are joined together to form bigger patches.

Different protocols to prepare multi-component and phase-separated SLBs can affect the asymmetry of the resulting SLBs. In Figure 6.1.86.1.8, the extruded small unilamellar vesicles (SUVs) composed of DLPC/DSPC are heated at 65⁰C, then fused on mica at 20⁰C. The resulting SLB are fully symmetric fluid/gel membranes. On the other hand, using sonicated SUVs heated at 20⁰C prior to fusion results in a completely asymmetric SLBs.

Summary

AFM is a powerful technique for scientists to have an insight in membrane biophysics. Advantages of this method including:

• The ability to image cell surfaces, molecular assemblies in their native aqueous environment at a very high resolution.
• No requirement of a conductive sample.
• Provide 3-D surface profiles.
• The ability to operate in liquid environment and ambient air.

Disadvantages of AFM are limited scanning area and the scanning speed.

Recently there have been some progress in improving AFM scanning speed and resolution, for example high speed AFM (HS-AFM) or high resolution AFM (HR-AFM) [10, 11]. HF-AFM consists of modified components in order to maximize scanning seed, for example soft small cantilevers having high resonance frequencies, high resonance frequency scanners and fast data acquisition devices [10]. AFM is also combined with several techniques such as fluorescence microscopy to acquire more efficient data [9]. The potential improvement of AFM will enhance the possibility to apply this technique in a wider range of biological fields.

References

1. Binnig, G., C. F. Quate, and C. Gerber, Atomic force microscope. Phys. Rev. Lett, 1986(56): p. 930–933.
2. D.J.Muller, Y.F.D., Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology. Nat.Nanotechnol, 2008. 3: p. 261-269.
3. Morandat, S., et al., Atomic force microscopy of model lipid membranes. Anal Bioanal Chem, 2013. 405(5): p. 1445-61.
4. Alessandrini, A. and P. Facci, AFM: a versatile tool in biophysics. Measurement Science and Technology, 2005. 16(6): p. R65- R92.
5. Bullen, R.A.W.a.H.A., Lecture notes: Introduction to Scanning Probe Microscopy
6. Frederix, P.L., P.D. Bosshart, and A. Engel, Atomic force microscopy of biological membranes. Biophys J, 2009. 96(2): p. ​329- ​38.
7. S. Morita, R.W., E. Meyer, Noncontact Atomic Force Microscopy. Springer, 2002. 1.
8. Muller, D.J. and A. Engel, Atomic force microscopy and spectroscopy of native membrane proteins. Nat Protoc, 2007. 2(9): p. ​2191-7.
9. Goksu, E.I., et al., AFM for structure and dynamics of biomembranes. Biochim Biophys Acta, 2009. 1788(1): p. 254-66.
10. Ando, T., T. Uchihashi, and N. Kodera, High-speed AFM and applications to biomolecular systems. Annu Rev Biophys, 2013. ​42: p. 393-414.
11. Bippes, C.A. and D.J. Muller, High-resolution atomic force microscopy and spectroscopy of native membrane proteins. Reports on Progress in Physics, 2011. 74(8): p. 086601.

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Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very-high-resolution type of scanning probe microscopy (SPM), with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction-limit.

Atomic force microscopy is a type of scanning probe microscopy (SPM), with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit. The information is gathered by “feeling” or “touching” the surface with a mechanical probe. Piezoelectric elements that facilitate tiny but accurate and precise movements on (electronic) command enable precise scanning.

Abilities

Atomic Force Microscope

The AFM has three major abilities: force measurement, topographic imaging, and manipulation.

In force measurement, AFMs can be used to measure the forces between the probe and the sample as a function of their mutual separation. This can be applied to perform force spectroscopy, to measure the mechanical properties of the sample, such as the sample’s Young’s modulus, a measure of stiffness.

For imaging, the reaction of the probe to the forces that the sample imposes on it can be used to form an image of the three-dimensional shape (topography) of a sample surface at a high resolution. This is achieved by raster scanning the position of the sample with respect to the tip and recording the height of the probe that corresponds to a constant probe-sample interaction (see section topographic imaging in AFM for more details). The surface topography is commonly displayed as a pseudocolor plot. Although the initial publication about the atomic force microscopy by Binnig, Quate and Gerber in 1986 speculated about the possibility of achieving atomic resolution, profound experimental challenges needed to be overcome before atomic resolution of defects and step edges in ambient (liquid) conditions was demonstrated in 1993 by Ohnesorge and Binnig. True atomic resolution of the silicon 7×7 surface – the atomic images of this surface obtained by STM had convinced the scientific community of the spectacular spatial resolution of scanning tunneling microscopy – had to wait a little longer before it was shown by Giessibl,

In manipulation, the forces between tip and sample can also be used to change the properties of the sample in a controlled way. Examples of this include atomic manipulation, scanning probe lithography and local stimulation of cells.

Simultaneous with the acquisition of topographical images, other properties of the sample can be measured locally and displayed as an image, often with similarly high resolution. Examples of such properties are mechanical properties like stiffness or adhesion strength and electrical properties such as conductivity or surface potential. In fact, the majority of SPM techniques are extensions of AFM that use this modality.

Other microscopy technologies

The major difference between atomic force microscopy and competing technologies such as optical microscopy and electron microscopy is that AFM does not use lenses or beam irradiation. Therefore, it does not suffer from a limitation in spatial resolution due to diffraction and aberration, and preparing a space for guiding the beam (by creating a vacuum) and staining the sample are not necessary.

There are several types of scanning microscopy including scanning probe microscopy (which includes AFM, scanning tunneling microscopy (STM) and near-field scanning optical microscope (SNOM/NSOM), STED microscopy (STED), and scanning electron microscopy and electrochemical AFM, EC-AFM). Although SNOM and STED use visible, infrared or even terahertz light to illuminate the sample, their resolution is not constrained by the diffraction limit.

Configuration

Fig. 3 shows an AFM, which typically consists of the following features. Numbers in parentheses correspond to numbered features in Fig. 3. Coordinate directions are defined by the coordinate system (0).Fig. 3: Typical configuration of an AFM.
(1): Cantilever, (2): Support for cantilever, (3): Piezoelectric element (to oscillate cantilever at its eigen frequency), (4): Tip (Fixed to open end of a cantilever, acts as the probe), (5): Detector of deflection and motion of the cantilever, (6): Sample to be measured by AFM, (7): xyz drive, (moves sample (6) and stage (8) in x, y, and z directions with respect to a tip apex (4)), and (8): Stage.

The small spring-like cantilever (1) is carried by the support (2). Optionally, a piezoelectric element (typically made of a ceramic material) (3) oscillates the cantilever (1). The sharp tip (4) is fixed to the free end of the cantilever (1). The detector (5) records the deflection and motion of the cantilever (1). The sample (6) is mounted on the sample stage (8). An xyz drive (7) permits to displace the sample (6) and the sample stage (8) in x, y, and z directions with respect to the tip apex (4). Although Fig. 3 shows the drive attached to the sample, the drive can also be attached to the tip, or independent drives can be attached to both, since it is the relative displacement of the sample and tip that needs to be controlled. Controllers and plotter are not shown in Fig. 3.

According to the configuration described above, the interaction between tip and sample, which can be an atomic scale phenomenon, is transduced into changes of the motion of cantilever which is a macro scale phenomenon. Several different aspects of the cantilever motion can be used to quantify the interaction between the tip and sample, most commonly the value of the deflection, the amplitude of an imposed oscillation of the cantilever, or the shift in resonance frequency of the cantilever (see section Imaging Modes).

Detector

The detector (5) of AFM measures the deflection (displacement with respect to the equilibrium position) of the cantilever and converts it into an electrical signal. The intensity of this signal will be proportional to the displacement of the cantilever.

Various methods of detection can be used, e.g. interferometry, optical levers, the piezoresistive method, the piezoelectric method, and STM-based detectors (see section “AFM cantilever deflection measurement”.).

Image formation

Note: The following paragraphs assume that ‘contact mode’ is used (see section Imaging Modes). For other imaging modes, the process is similar, except that ‘deflection’ should be replaced by the appropriate feedback variable.

When using the AFM to image a sample, the tip is brought into contact with the sample, and the sample is raster scanned along an x–y grid (fig 4). Most commonly, an electronic feedback loop is employed to keep the probe-sample force constant during scanning. This feedback loop has the cantilever deflection as input, and its output controls the distance along the z axis between the probe support (2 in fig. 3) and the sample support (8 in fig 3). As long as the tip remains in contact with the sample, and the sample is scanned in the x–y plane, height variations in the sample will change the deflection of the cantilever. The feedback then adjusts the height of the probe support so that the deflection is restored to a user-defined value (the setpoint). A properly adjusted feedback loop adjusts the support-sample separation continuously during the scanning motion, such that the deflection remains approximately constant. In this situation, the feedback output equals the sample surface topography to within a small error.

Historically, a different operation method has been used, in which the sample-probe support distance is kept constant and not controlled by a feedback (servo mechanism). In this mode, usually referred to as ‘constant height mode’, the deflection of the cantilever is recorded as a function of the sample x–y position. As long as the tip is in contact with the sample, the deflection then corresponds to surface topography. The main reason this method is not very popular anymore, is that the forces between tip and sample are not controlled, which can lead to forces high enough to damage the tip or the sample. It is however common practice to record the deflection even when scanning in ‘constant force mode’, with feedback. This reveals the small tracking error of the feedback, and can sometimes reveal features that the feedback was not able to adjust for.

The AFM signals, such as sample height or cantilever deflection, are recorded on a computer during the x–y scan. They are plotted in a pseudocolor image, in which each pixel represents an x–y position on the sample, and the color represents the recorded signal.Fig. 5: Topographic image forming by AFM.
(1): Tip apex, (2): Sample surface, (3): Z-orbit of Tip apex, (4): Cantilever.

History

The AFM was invented by IBM scientists in 1985, The precursor to the AFM, the scanning tunneling microscope (STM), was developed by Gerd Binnig and Heinrich Rohrer in the early 1980s at IBM Research – Zurich, a development that earned them the 1986 Nobel Prize for Physics. Binnig invented the atomic force microscope and the first experimental implementation was made by Binnig, Quate and Gerber in 1986.

The first commercially available atomic force microscope was introduced in 1989. The AFM is one of the foremost tools for imaging, measuring, and manipulating matter at the nanoscale.

Applications

The AFM has been applied to problems in a wide range of disciplines of the natural sciences, including solid-state physics, semiconductor science and technology, molecular engineering, polymer chemistry and physics, surface chemistry, molecular biology, cell biology, and medicine.

Applications in the field of solid state physics include (a) the identification of atoms at a surface, (b) the evaluation of interactions between a specific atom and its neighboring atoms, and (c) the study of changes in physical properties arising from changes in an atomic arrangement through atomic manipulation.

In molecular biology, AFM can be used to study the structure and mechanical properties of protein complexes and assemblies. For example, AFM has been used to image microtubules and measure their stiffness.

In cellular biology, AFM can be used to attempt to distinguish cancer cells and normal cells based on a hardness of cells, and to evaluate interactions between a specific cell and its neighboring cells in a competitive culture system. AFM can also be used to indent cells, to study how they regulate the stiffness or shape of the cell membrane or wall.

In some variations, electric potentials can also be scanned using conducting cantilevers. In more advanced versions, currents can be passed through the tip to probe the electrical conductivity or transport of the underlying surface, but this is a challenging task with few research groups reporting consistent data (as of 2004).

Principles

Electron micrograph of a used AFM cantilever. Image width ~100 micrometers

Electron micrograph of a used AFM cantilever. Image width ~30 micrometers

The AFM consists of a cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface. The cantilever is typically silicon or silicon nitridewith a tip radius of curvature on the order of nanometers. When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever according to Hooke’s law. Depending on the situation, forces that are measured in AFM include mechanical contact force, van der Waals forces, capillary forces, chemical bonding, electrostatic forces, magnetic forces (see magnetic force microscope, MFM), Casimir forces, solvation forces, etc. Along with force, additional quantities may simultaneously be measured through the use of specialized types of probes. Atomic force microscope topographical scan of a glass surface. The micro and nano-scale features of the glass can be observed, portraying the roughness of the material. The image space is (x,y,z) = (20 µm × 20 µm × 420 nm).

The AFM can be operated in a number of modes, depending on the application. In general, possible imaging modes are divided into static (also called contact) modes and a variety of dynamic (non-contact or “tapping”) modes where the cantilever is vibrated or oscillated at a given frequency.^[7]

Imaging modes

AFM operation is usually described as one of three modes, according to the nature of the tip motion: contact mode, also called static mode (as opposed to the other two modes, which are called dynamic modes); tapping mode, also called intermittent contact, AC mode, or vibrating mode, or, after the detection mechanism, amplitude modulation AFM; non-contact mode, or, again after the detection mechanism, frequency modulation AFM.

Despite the nomenclature, repulsive contact can occur or be avoided both in amplitude modulation AFM and frequency modulation AFM, depending on the settings.

Contact mode

In contact mode, the tip is “dragged” across the surface of the sample and the contours of the surface are measured either using the deflection of the cantilever directly or, more commonly, using the feedback signal required to keep the cantilever at a constant position. Because the measurement of a static signal is prone to noise and drift, low stiffness cantilevers (i.e. cantilevers with a low spring constant, k) are used to achieve a large enough deflection signal while keeping the interaction force low. Close to the surface of the sample, attractive forces can be quite strong, causing the tip to “snap-in” to the surface. Thus, contact mode AFM is almost always done at a depth where the overall force is repulsive, that is, in firm “contact” with the solid surface.

Tapping mode

Single polymer chains (0.4 nm thick) recorded in a tapping mode under aqueous media with different pH.

In ambient conditions, most samples develop a liquid meniscus layer. Because of this, keeping the probe tip close enough to the sample for short-range forces to become detectable while preventing the tip from sticking to the surface presents a major problem for contact mode in ambient conditions. Dynamic contact mode (also called intermittent contact, AC mode or tapping mode) was developed to bypass this problem. Nowadays, tapping mode is the most frequently used AFM mode when operating in ambient conditions or in liquids.

In tapping mode, the cantilever is driven to oscillate up and down at or near its resonance frequency. This oscillation is commonly achieved with a small piezo element in the cantilever holder, but other possibilities include an AC magnetic field (with magnetic cantilevers), piezoelectric cantilevers, or periodic heating with a modulated laser beam. The amplitude of this oscillation usually varies from several nm to 200 nm. In tapping mode, the frequency and amplitude of the driving signal are kept constant, leading to a constant amplitude of the cantilever oscillation as long as there is no drift or interaction with the surface. The interaction of forces acting on the cantilever when the tip comes close to the surface, Van der Waals forces, dipole-dipole interactions, electrostatic forces, etc. cause the amplitude of the cantilever’s oscillation to change (usually decrease) as the tip gets closer to the sample. This amplitude is used as the parameter that goes into the electronic servo that controls the height of the cantilever above the sample. The servo adjusts the height to maintain a set cantilever oscillation amplitude as the cantilever is scanned over the sample. A tapping AFM image is therefore produced by imaging the force of the intermittent contacts of the tip with the sample surface.

Although the peak forces applied during the contacting part of the oscillation can be much higher than typically used in contact mode, tapping mode generally lessens the damage done to the surface and the tip compared to the amount done in contact mode. This can be explained by the short duration of the applied force, and because the lateral forces between tip and sample are significantly lower in tapping mode over contact mode. Tapping mode imaging is gentle enough even for the visualization of supported lipid bilayers or adsorbed single polymer molecules (for instance, 0.4 nm thick chains of synthetic polyelectrolytes) under liquid medium. With proper scanning parameters, the conformation of single molecules can remain unchanged for hours, and even single molecular motors can be imaged while moving.

When operating in tapping mode, the phase of the cantilever’s oscillation with respect to the driving signal can be recorded as well. This signal channel contains information about the energy dissipated by the cantilever in each oscillation cycle. Samples that contain regions of varying stiffness or with different adhesion properties can give a contrast in this channel that is not visible in the topographic image. Extracting the sample’s material properties in a quantitative manner from phase images, however, is often not feasible.

Non-contact mode

In non-contact atomic force microscopy mode, the tip of the cantilever does not contact the sample surface. The cantilever is instead oscillated at either its resonant frequency(frequency modulation) or just above (amplitude modulation) where the amplitude of oscillation is typically a few nanometers (<10 nm) down to a few picometers. The van der Waals forces, which are strongest from 1 nm to 10 nm above the surface, or any other long-range force that extends above the surface acts to decrease the resonance frequency of the cantilever. This decrease in resonant frequency combined with the feedback loop system maintains a constant oscillation amplitude or frequency by adjusting the average tip-to-sample distance. Measuring the tip-to-sample distance at each (x,y) data point allows the scanning software to construct a topographic image of the sample surface.

Non-contact mode AFM does not suffer from tip or sample degradation effects that are sometimes observed after taking numerous scans with contact AFM. This makes non-contact AFM preferable to contact AFM for measuring soft samples, e.g. biological samples and organic thin film. In the case of rigid samples, contact and non-contact images may look the same. However, if a few monolayers of adsorbed fluid are lying on the surface of a rigid sample, the images may look quite different. An AFM operating in contact mode will penetrate the liquid layer to image the underlying surface, whereas in non-contact mode an AFM will oscillate above the adsorbed fluid layer to image both the liquid and surface.

Schemes for dynamic mode operation include frequency modulation where a phase-locked loop is used to track the cantilever’s resonance frequency and the more common amplitude modulation with a servo loop in place to keep the cantilever excitation to a defined amplitude. In frequency modulation, changes in the oscillation frequency provide information about tip-sample interactions. Frequency can be measured with very high sensitivity and thus the frequency modulation mode allows for the use of very stiff cantilevers. Stiff cantilevers provide stability very close to the surface and, as a result, this technique was the first AFM technique to provide true atomic resolution in ultra-high vacuumconditions.

In amplitude modulation, changes in the oscillation amplitude or phase provide the feedback signal for imaging. In amplitude modulation, changes in the phase of oscillation can be used to discriminate between different types of materials on the surface. Amplitude modulation can be operated either in the non-contact or in the intermittent contact regime. In dynamic contact mode, the cantilever is oscillated such that the separation distance between the cantilever tip and the sample surface is modulated.

Amplitude modulation has also been used in the non-contact regime to image with atomic resolution by using very stiff cantilevers and small amplitudes in an ultra-high vacuum environment.

Topographic image

Image formation is a plotting method that produces a color mapping through changing the x–y position of the tip while scanning and recording the measured variable, i.e. the intensity of control signal, to each x–y coordinate. The color mapping shows the measured value corresponding to each coordinate. The image expresses the intensity of a value as a hue. Usually, the correspondence between the intensity of a value and a hue is shown as a color scale in the explanatory notes accompanying the image.

What is the topographic image of atomic force microscope?

Operation mode of image forming of the AFM are generally classified into two groups from the viewpoint whether it uses z-Feedback loop (not shown) to maintain the tip-sample distance to keep signal intensity exported by the detector. The first one (using z-Feedback loop), said to be “constant XX mode” (XX is something which kept by z-Feedback loop).

Topographic image formation mode is based on abovementioned “constant XX mode”, z-Feedback loop controls the relative distance between the probe and the sample through outputting control signals to keep constant one of frequency, vibration and phase which typically corresponds to the motion of cantilever (for instance, voltage is applied to the Z-piezoelectric element and it moves the sample up and down towards the Z direction.

Details will be explained in the case that especially “constant df mode”(FM-AFM) among AFM as an instance in next section.

Topographic image of FM-AFM

When the distance between the probe and the sample is brought to the range where atomic force may be detected, while a cantilever is excited in its natural eigen frequency (f₀), a phenomenon occurs that the resonance frequency (f) of the cantilever shifts from its original resonance frequency (natural eigen frequency). In other words, in the range where atomic force may be detected, the frequency shift (df=f-f₀) will be observed. So, when the distance between the probe and the sample is in the non-contact region, the frequency shift increases in negative direction as the distance between the probe and the sample gets smaller.

When the sample has concavity and convexity, the distance between the tip-apex and the sample varies in accordance with the concavity and convexity accompanied with a scan of the sample along x–y direction (without height regulation in z-direction). As a result, the frequency shift arises. The image in which the values of the frequency obtained by a raster scan along the x–y direction of the sample surface are plotted against the x–y coordination of each measurement point is called a constant-height image.

On the other hand, the df may be kept constant by moving the probe upward and downward (See (3) of FIG.5) in z-direction using a negative feedback (by using z-feedback loop) while the raster scan of the sample surface along the x–y direction. The image in which the amounts of the negative feedback (the moving distance of the probe upward and downward in z-direction) are plotted against the x–y coordination of each measurement point is a topographic image. In other words, the topographic image is a trace of the tip of the probe regulated so that the df is constant and it may also be considered to be a plot of a constant-height surface of the df.

Therefore, the topographic image of the AFM is not the exact surface morphology itself, but actually the image influenced by the bond-order between the probe and the sample, however, the topographic image of the AFM is considered to reflect the geographical shape of the surface more than the topographic image of a scanning tunnel microscope.

Force spectroscopy

Another major application of AFM (besides imaging) is force spectroscopy, the direct measurement of tip-sample interaction forces as a function of the gap between the tip and sample (the result of this measurement is called a force-distance curve). For this method, the AFM tip is extended towards and retracted from the surface as the deflection of the cantilever is monitored as a function of piezoelectric displacement. These measurements have been used to measure nanoscale contacts, atomic bonding, Van der Waals forces, and Casimir forces, dissolution forces in liquids and single molecule stretching and rupture forces. Furthermore, AFM was used to measure, in an aqueous environment, the dispersion force due to polymer adsorbed on the substrate. Forces of the order of a few piconewtons can now be routinely measured with a vertical distance resolution of better than 0.1 nanometers. Force spectroscopy can be performed with either static or dynamic modes. In dynamic modes, information about the cantilever vibration is monitored in addition to the static deflection.

Problems with the technique include no direct measurement of the tip-sample separation and the common need for low-stiffness cantilevers, which tend to ‘snap’ to the surface. These problems are not insurmountable. An AFM that directly measures the tip-sample separation has been developed. The snap-in can be reduced by measuring in liquids or by using stiffer cantilevers, but in the latter case a more sensitive deflection sensor is needed. By applying a small dither to the tip, the stiffness (force gradient) of the bond can be measured as well.

Biological applications and other

Force spectroscopy is used in biophysics to measure the mechanical properties of living material (such as tissue or cells) or detect structures of different stiffness buried into the bulk of the sample using the stiffness tomography. Another application was to measure the interaction forces between from one hand a material stuck on the tip of the cantilever, and from another hand the surface of particles either free or occupied by the same material. From the adhesion force distribution curve, a mean value of the forces has been derived. It allowed to make a cartography of the surface of the particles, covered or not by the material. AFM have been also used for mechanically unfolding proteins. In such experiments, the analyzes of the mean unfolding forces with the appropriate model leads to the obtainment of the information about the unfolding rate and free energy profile parameters of the protein.

Identification of individual surface atoms

The AFM can be used to image and manipulate atoms and structures on a variety of surfaces. The atom at the apex of the tip “senses” individual atoms on the underlying surface when it forms incipient chemical bonds with each atom. Because these chemical interactions subtly alter the tip’s vibration frequency, they can be detected and mapped. This principle was used to distinguish between atoms of silicon, tin and lead on an alloy surface, by comparing these ‘atomic fingerprints’ to values obtained from large-scale density functional theory (DFT) simulations.

The trick is to first measure these forces precisely for each type of atom expected in the sample, and then to compare with forces given by DFT simulations. The team found that the tip interacted most strongly with silicon atoms, and interacted 24% and 41% less strongly with tin and lead atoms, respectively. Thus, each different type of atom can be identified in the matrix as the tip is moved across the surface.

Probe

An AFM probe has a sharp tip on the free-swinging end of a cantilever that is protruding from a holder. The dimensions of the cantilever are in the scale of micrometers. The radius of the tip is usually on the scale of a few nanometers to a few tens of nanometers. (Specialized probes exist with much larger end radii, for example probes for indentation of soft materials.) The cantilever holder, also called holder chip – often 1.6 mm by 3.4 mm in size – allows the operator to hold the AFM cantilever/probe assembly with tweezers and fit it into the corresponding holder clips on the scanning head of the atomic force microscope.

This device is most commonly called an “AFM probe”, but other names include “AFM tip” and “cantilever” (employing the name of a single part as the name of the whole device). An AFM probe is a particular type of SPM (scanning probe microscopy) probe.

AFM probes are manufactured with MEMS technology. Most AFM probes used are made from silicon (Si), but borosilicate glass and silicon nitride are also in use. AFM probes are considered consumables as they are often replaced when the tip apex becomes dull or contaminated or when the cantilever is broken. They can cost from a couple of tens of dollars up to hundreds of dollars per cantilever for the most specialized cantilever/probe combinations.

Just the tip is brought very close to the surface of the object under investigation, the cantilever is deflected by the interaction between the tip and the surface, which is what the AFM is designed to measure. A spatial map of the interaction can be made by measuring the deflection at many points on a 2D surface.

Several types of interaction can be detected. Depending on the interaction under investigation, the surface of the tip of the AFM probe needs to be modified with a coating. Among the coatings used are gold – for covalent bonding of biological molecules and the detection of their interaction with a surface, diamond for increased wear resistance and magnetic coatings for detecting the magnetic properties of the investigated surface. Another solution exists to achieve high resolution magnetic imaging : having the probe equip with a microSQUID. The AFM tips is fabricated using silicon micro machining and the precise positioning of the microSQUID loop is done by electron beam lithography.

The surface of the cantilevers can also be modified. These coatings are mostly applied in order to increase the reflectance of the cantilever and to improve the deflection signal.

Forces vs tip geometry

The forces between the tip and the sample strongly depend on the geometry of the tip. Various studies were exploited in the past years to write the forces as a function of the tip parameters.

Among the different forces between the tip and the sample, the water meniscus forces are highly interesting, both in air and liquid environment. Other forces must be considered, like the Coulomb force, van der Waals forces, double layer interactions, solvation forces, hydration and hydrophobic forces.

Water meniscus

Water meniscus forces are highly interesting for AFM measurements in air. Due to the ambient humidity, a thin layer of water is formed between the tip and the sample during air measurements. The resulting capillary force gives rise to a strong attractive force that pulls the tip onto the surface. In fact, the adhesion force measured between tip and sample in ambient air of finite humidity is usually dominated by capillary forces. As a consequence, it is difficult to pull the tip away from the surface. For soft samples including many polymers and in particular biological materials, the strong adhesive capillary force gives rise to sample degradation and destruction upon imaging in contact mode. Historically, these problems were an important motivation for the development of dynamic imaging in air (e.g. ‘tapping mode’). During tapping mode imaging in air, capillary bridges still form. Yet, for suitable imaging conditions, the capillary bridges are formed and broken in every oscillation cycle of the cantilever normal to the surface, as can be inferred from an analysis of cantilever amplitude and phase vs. distance curves. As a consequence, destructive shear forces are largely reduced and soft samples can be investigated.

In order to quantify the equilibrium capillary force, it is necessary to start from the Laplace equation for pressure:Model for AFM water meniscus

{\displaystyle P=\gamma _{L}({\frac {1}{r}}_{1}+{\frac {1}{r}}_{0})\simeq {\frac {\gamma _{L}}{r_{eff}}}}

where γ_L is the surface energy and r₀ and r₁ are defined in the figure.

The pressure is applied on an area of

{\displaystyle A\simeq 2\pi R\simeq [r_{eff}(1+\cos \theta )+h]}

where d, θ, and h are defined in the figure.

The force which pulles together the two surfaces is

{\displaystyle F=2\pi R\gamma _{L}(1+\cos \theta +{\frac {h}{r_{eff}}})}

The same formula could also be calculated as a function of relative humidity.

Gao calculated formulas for different tip geometries. As an example, the forse decreases by 20% for a conical tip with respect to a spherical tip.

When these forces are calculated, a difference must be made between the wet on dry situation and the wet on wet situation.

For a spherical tip, the force is:

{\displaystyle f_{m}=-2\pi R\gamma _{L}(\cos \theta +\cos \phi )(1-{\frac {dh}{dD}})} for dry on wet

{\displaystyle f_{m}=-2\pi R\gamma _{L}{\frac {dr_{0}}{dD}}}for wet on wet

where θ is the contact angle of the dry sphere and φ is the immersed angle, as shown in the figure Also R,h and D are illustrated in the same figure.

For a conical tip, the formula becomes:

{\displaystyle f_{m}=-2\pi R\gamma _{L}{\frac {\tan \delta }{\cos \delta }}(\cos \theta +\sin \delta )(hD)(1-{\frac {dh}{dD}})} for dry on wet

{\displaystyle f_{m}=-2\pi R\gamma _{L}({\frac {1}{\cos \delta }}+\sin \delta )(r_{0})({\frac {dr_{0}}{dD}})} for wet on wet

where δ is the half cone angle and r₀ and h are parameters of the meniscus profile.

AFM cantilever-deflection measurement

Beam-deflection measurement

AFM beam-deflection detection

The most common method for cantilever-deflection measurements is the beam-deflection method. In this method, laser light from a solid-state diode is reflected off the back of the cantilever and collected by a position-sensitive detector (PSD) consisting of two closely spaced photodiodes, whose output signal is collected by a differential amplifier. Angular displacement of the cantilever results in one photodiode collecting more light than the other photodiode, producing an output signal (the difference between the photodiode signals normalized by their sum), which is proportional to the deflection of the cantilever. The sensitivity of the beam-deflection method is very high, a noise floor on the order of 10 fm Hz^{−​1⁄₂} can be obtained routinely in a well-designed system. Although this method is sometimes called the ‘optical lever’ method, the signal is not amplified if the beam path is made longer. A longer beam path increases the motion of the reflected spot on the photodiodes, but also widens the spot by the same amount due to diffraction, so that the same amount of optical power is moved from one photodiode to the other. The ‘optical leverage’ (output signal of the detector divided by deflection of the cantilever) is inversely proportional to the numerical aperture of the beam focusing optics, as long as the focused laser spot is small enough to fall completely on the cantilever. It is also inversely proportional to the length of the cantilever.

The relative popularity of the beam-deflection method can be explained by its high sensitivity and simple operation, and by the fact that cantilevers do not require electrical contacts or other special treatments, and can therefore be fabricated relatively cheaply with sharp integrated tips.

Other deflection-measurement methods

Many other methods for beam-deflection measurements exist.

• Piezoelectric detection – Cantilevers made from quartz (such as the qPlus configuration), or other piezoelectric materials can directly detect deflection as an electrical signal. Cantilever oscillations down to 10pm have been detected with this method.
• Laser Doppler vibrometry – A laser Doppler vibrometer can be used to produce very accurate deflection measurements for an oscillating cantilever (thus is only used in non-contact mode). This method is expensive and is only used by relatively few groups.
• Scanning tunneling microscope (STM) — The first atomic microscope used an STM complete with its own feedback mechanism to measure deflection. This method is very difficult to implement, and is slow to react to deflection changes compared to modern methods.
• Optical interferometry – Optical interferometry can be used to measure cantilever deflection. Due to the nanometre scale deflections measured in AFM, the interferometer is running in the sub-fringe regime, thus, any drift in laser power or wavelength has strong effects on the measurement. For these reasons optical interferometer measurements must be done with great care (for example using index matching fluids between optical fibre junctions), with very stable lasers. For these reasons optical interferometry is rarely used.
• Capacitive detection – Metal coated cantilevers can form a capacitor with another contact located behind the cantilever. Deflection changes the distance between the contacts and can be measured as a change in capacitance.
• Piezoresistive detection – Cantilevers can be fabricated with piezoresistive elements that act as a strain gauge. Using a Wheatstone bridge, strain in the AFM cantilever due to deflection can be measured. This is not commonly used in vacuum applications, as the piezoresistive detection dissipates energy from the system affecting Q of the resonance.

Piezoelectric scanners

AFM scanners are made from piezoelectric material, which expands and contracts proportionally to an applied voltage. Whether they elongate or contract depends upon the polarity of the voltage applied. Traditionally the tip or sample is mounted on a ‘tripod’ of three piezo crystals, with each responsible for scanning in the x,y and z directions. In 1986, the same year as the AFM was invented, a new piezoelectric scanner, the tube scanner, was developed for use in STM. Later tube scanners were incorporated into AFMs. The tube scanner can move the sample in the x, y, and z directions using a single tube piezo with a single interior contact and four external contacts. An advantage of the tube scanner compared to the original tripod design, is better vibrational isolation, resulting from the higher resonant frequency of the single element construction, in combination with a low resonant frequency isolation stage. A disadvantage is that the x–y motion can cause unwanted z motion resulting in distortion. Another popular design for AFM scanners is the flexurestage, which uses separate piezos for each axis, and couples them through a flexure mechanism.

Scanners are characterized by their sensitivity, which is the ratio of piezo movement to piezo voltage, i.e., by how much the piezo material extends or contracts per applied volt. Because of differences in material or size, the sensitivity varies from scanner to scanner. Sensitivity varies non-linearly with respect to scan size. Piezo scanners exhibit more sensitivity at the end than at the beginning of a scan. This causes the forward and reverse scans to behave differently and display hysteresis between the two scan directions.This can be corrected by applying a non-linear voltage to the piezo electrodes to cause linear scanner movement and calibrating the scanner accordingly. One disadvantage of this approach is that it requires re-calibration because the precise non-linear voltage needed to correct non-linear movement will change as the piezo ages (see below). This problem can be circumvented by adding a linear sensor to the sample stage or piezo stage to detect the true movement of the piezo. Deviations from ideal movement can be detected by the sensor and corrections applied to the piezo drive signal to correct for non-linear piezo movement. This design is known as a ‘closed loop’ AFM. Non-sensored piezo AFMs are referred to as ‘open loop’ AFMs.

The sensitivity of piezoelectric materials decreases exponentially with time. This causes most of the change in sensitivity to occur in the initial stages of the scanner’s life. Piezoelectric scanners are run for approximately 48 hours before they are shipped from the factory so that they are past the point where they may have large changes in sensitivity. As the scanner ages, the sensitivity will change less with time and the scanner would seldom require recalibration, though various manufacturer manuals recommend monthly to semi-monthly calibration of open loop AFMs.

Advantages and disadvantages

The first atomic force microscope

Advantages

AFM has several advantages over the scanning electron microscope (SEM). Unlike the electron microscope, which provides a two-dimensional projection or a two-dimensional image of a sample, the AFM provides a three-dimensional surface profile. In addition, samples viewed by AFM do not require any special treatments (such as metal/carbon coatings) that would irreversibly change or damage the sample, and does not typically suffer from charging artifacts in the final image. While an electron microscope needs an expensive vacuum environment for proper operation, most AFM modes can work perfectly well in ambient air or even a liquid environment. This makes it possible to study biological macromolecules and even living organisms. In principle, AFM can provide higher resolution than SEM. It has been shown to give true atomic resolution in ultra-high vacuum (UHV) and, more recently, in liquid environments. High resolution AFM is comparable in resolution to scanning tunneling microscopy and transmission electron microscopy. AFM can also be combined with a variety of optical microscopy and spectroscopy techniques such as fluorescent microscopy of infrared spectroscopy, giving rise to scanning near-field optical microscopy, nano-FTIR and further expanding its applicability. Combined AFM-optical instruments have been applied primarily in the biological sciences but have recently attracted strong interest in photovoltaics and energy-storage research, polymer sciences, nanotechnology and even medical research.

Disadvantages

A disadvantage of AFM compared with the scanning electron microscope (SEM) is the single scan image size. In one pass, the SEM can image an area on the order of square millimeters with a depth of field on the order of millimeters, whereas the AFM can only image a maximum scanning area of about 150×150 micrometers and a maximum height on the order of 10–20 micrometers. One method of improving the scanned area size for AFM is by using parallel probes in a fashion similar to that of millipede data storage.

The scanning speed of an AFM is also a limitation. Traditionally, an AFM cannot scan images as fast as an SEM, requiring several minutes for a typical scan, while an SEM is capable of scanning at near real-time, although at relatively low quality. The relatively slow rate of scanning during AFM imaging often leads to thermal drift in the imagemaking the AFM less suited for measuring accurate distances between topographical features on the image. However, several fast-acting designs were suggested to increase microscope scanning productivity including what is being termed videoAFM (reasonable quality images are being obtained with videoAFM at video rate: faster than the average SEM). To eliminate image distortions induced by thermal drift, several methods have been introduced.Showing an AFM artifact arising from a tip with a high radius of curvature with respect to the feature that is to be visualizedAFM artifact, steep sample topography

AFM images can also be affected by nonlinearity, hysteresis, and creep of the piezoelectric material and cross-talk between the x, y, zaxes that may require software enhancement and filtering. Such filtering could “flatten” out real topographical features. However, newer AFMs utilize real-time correction software (for example, feature-oriented scanning) or closed-loop scanners, which practically eliminate these problems. Some AFMs also use separated orthogonal scanners (as opposed to a single tube), which also serve to eliminate part of the cross-talk problems.

As with any other imaging technique, there is the possibility of image artifacts, which could be induced by an unsuitable tip, a poor operating environment, or even by the sample itself, as depicted on the right. These image artifacts are unavoidable; however, their occurrence and effect on results can be reduced through various methods. Artifacts resulting from a too-coarse tip can be caused for example by inappropriate handling or de facto collisions with the sample by either scanning too fast or having an unreasonably rough surface, causing actual wearing of the tip.

Due to the nature of AFM probes, they cannot normally measure steep walls or overhangs. Specially made cantilevers and AFMs can be used to modulate the probe sideways as well as up and down (as with dynamic contact and non-contact modes) to measure sidewalls, at the cost of more expensive cantilevers, lower lateral resolution and additional artifacts.

Other applications in various fields of study

AFM image of part of a Golgi apparatus isolated from HeLa cells

The latest efforts in integrating nanotechnology and biological research have been successful and show much promise for the future. Since nanoparticles are a potential vehicle of drug delivery, the biological responses of cells to these nanoparticles are continuously being explored to optimize their efficacy and how their design could be improved. Pyrgiotakis et al. were able to study the interaction between CeO₂ and Fe₂O₃ engineered nanoparticles and cells by attaching the engineered nanoparticles to the AFM tip. Studies have taken advantage of AFM to obtain further information on the behavior of live cells in biological media. Real-time atomic force spectroscopy (or nanoscopy) and dynamic atomic force spectroscopy have been used to study live cells and membrane proteins and their dynamic behavior at high resolution, on the nanoscale. Imaging and obtaining information on the topography and the properties of the cells has also given insight into chemical processes and mechanisms that occur through cell-cell interaction and interactions with other signaling molecules (ex. ligands). Evans and Calderwood used single cell force microscopy to study cell adhesion forces, bond kinetics/dynamic bond strength and its role in chemical processes such as cell signaling. Scheuring, Lévy, and Rigaud reviewed studies in which AFM to explore the crystal structure of membrane proteins of photosynthetic bacteria. Alsteen et al. have used AFM-based nanoscopy to perform a real-time analysis of the interaction between live mycobacteria and antimycobacterial drugs (specifically isoniazid, ethionamide, ethambutol, and streptomycine),^[58]which serves as an example of the more in-depth analysis of pathogen-drug interactions that can be done through AFM.

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The scanning electron microscope (SEM) uses a focused beam of high-energy electrons to generate a variety of signals at the surface of solid specimens. The signals that derive from electron-sample interactions reveal information about the sample including external morphology (texture), chemical composition, and crystalline structure and orientation of materials making up the sample.

In most applications, data are collected over a selected area of the surface of the sample, and a 2-dimensional image is generated that displays spatial variations in these properties. Areas ranging from approximately 1 cm to 5 microns in width can be imaged in a scanning mode using conventional SEM techniques (magnification ranging from 20X to approximately 30,000X, spatial resolution of 50 to 100 nm). The SEM is also capable of performing analyses of selected point locations on the sample; this approach is especially useful in qualitatively or semi-quantitatively determining chemical compositions (using EDS), crystalline structure, and crystal orientations (using EBSD). The design and function of the SEM is very similar to the EPMA and considerable overlap in capabilities exists between the two instruments.

Fundamental Principles of Scanning Electron Microscopy (SEM)

Accelerated electrons in an SEM carry significant amounts of kinetic energy, and this energy is dissipated as a variety of signals produced by electron-sample interactions when the incident electrons are decelerated in the solid sample. These signals include secondary electrons (that produce SEM images), backscattered electrons (BSE), diffracted backscattered electrons (EBSD that are used to determine crystal structures and orientations of minerals), photons (characteristic X-rays that are used for elemental analysis and continuum X-rays), visible light (cathodoluminescence–CL), and heat. Secondary electrons and backscattered electrons are commonly used for imaging samples: secondary electrons are most valuable for showing morphology and topography on samples and backscattered electrons are most valuable for illustrating contrasts in composition in multiphase samples (i.e. for rapid phase discrimination). X-ray generation is produced by inelastic collisions of the incident electrons with electrons in discrete ortitals (shells) of atoms in the sample. As the excited electrons return to lower energy states, they yield X-rays that are of a fixed wavelength (that is related to the difference in energy levels of electrons in different shells for a given element). Thus, characteristic X-rays are produced for each element in a mineral that is “excited” by the electron beam. SEM analysis is considered to be “non-destructive”; that is, x-rays generated by electron interactions do not lead to volume loss of the sample, so it is possible to analyze the same materials repeatedly.

Scanning Electron Microscopy (SEM) Instrumentation – How Does It Work?

Essential components of all SEMs include the following:

• Electron Source (“Gun”)
• Electron Lenses
• Sample Stage
• Detectors for all signals of interest
• Display / Data output devices
• Infrastructure Requirements:
  • Power Supply
  • Vacuum System
  • Cooling system
  • Vibration-free floor
  • Room free of ambient magnetic and electric fields

SEMs always have at least one detector (usually a secondary electron detector), and most have additional detectors. The specific capabilities of a particular instrument are critically dependent on which detectors it accommodates.

Applications

The SEM is routinely used to generate high-resolution images of shapes of objects (SEI) and to show spatial variations in chemical compositions: 1) acquiring elemental maps or spot chemical analyses using EDS, 2)discrimination of phases based on mean atomic number (commonly related to relative density) using BSE, and 3) compositional maps based on differences in trace element “activitors” (typically transition metal and Rare Earth elements) using CL. The SEM is also widely used to identify phases based on qualitative chemical analysis and/or crystalline structure. Precise measurement of very small features and objects down to 50 nm in size is also accomplished using the SEM. Backescattered electron images (BSE) can be used for rapid discrimination of phases in multiphase samples. SEMs equipped with diffracted backscattered electron detectors (EBSD) can be used to examine microfabric and crystallographic orientation in many materials.

Strengths and Limitations of Scanning Electron Microscopy (SEM)?

Strengths

There is arguably no other instrument with the breadth of applications in the study of solid materials that compares with the SEM. The SEM is critical in all fields that require characterization of solid materials. While this contribution is most concerned with geological applications, it is important to note that these applications are a very small subset of the scientific and industrial applications that exist for this instrumentation. Most SEM’s are comparatively easy to operate, with user-friendly “intuitive” interfaces. Many applications require minimal sample preparation. For many applications, data acquisition is rapid (less than 5 minutes/image for SEI, BSE, spot EDS analyses.) Modern SEMs generate data in digital formats, which are highly portable.

Limitations

Samples must be solid and they must fit into the microscope chamber. Maximum size in horizontal dimensions is usually on the order of 10 cm, vertical dimensions are generally much more limited and rarely exceed 40 mm. For most instruments samples must be stable in a vacuum on the order of 10^-5 – 10^-6 torr. Samples likely to outgas at low pressures (rocks saturated with hydrocarbons, “wet” samples such as coal, organic materials or swelling clays, and samples likely to decrepitate at low pressure) are unsuitable for examination in conventional SEM’s. However, “low vacuum” and “environmental” SEMs also exist, and many of these types of samples can be successfully examined in these specialized instruments. EDS detectors on SEM’s cannot detect very light elements (H, He, and Li), and many instruments cannot detect elements with atomic numbers less than 11 (Na). Most SEMs use a solid state x-ray detector (EDS), and while these detectors are very fast and easy to utilize, they have relatively poor energy resolution and sensitivity to elements present in low abundances when compared to wavelength dispersive x-ray detectors (WDS) on most electron probe microanalyzers (EPMA). An electrically conductive coating must be applied to electrically insulating samples for study in conventional SEM’s, unless the instrument is capable of operation in a low vacuum mode.

User’s Guide – Sample Collection and Preparation

Sample preparation can be minimal or elaborate for SEM analysis, depending on the nature of the samples and the data required. Minimal preparation includes acquisition of a sample that will fit into the SEM chamber and some accommodation to prevent charge build-up on electrically insulating samples. Most electrically insulating samples are coated with a thin layer of conducting material, commonly carbon, gold, or some other metal or alloy. The choice of material for conductive coatings depends on the data to be acquired: carbon is most desirable if elemental analysis is a priority, while metal coatings are most effective for high resolution electron imaging applications. Alternatively, an electrically insulating sample can be examined without a conductive coating in an instrument capable of “low vacuum” operation.

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It is the shift in wavelength of the inelastically scattered radiation that provides the chemical and structural information. Raman shifted photons can be of either higher or lower energy, depending upon the vibrational state of the molecule under study. A simplified energy diagram that illustrates these concepts is shown below.

Stokes radiation occurs at lower energy (longer wavelength) than the Rayleigh radiation, and anti-Stokes radiation has greater energy. The energy increase or decrease is related to the vibrational energy levels in the ground electronic state of the molecule, and as such, the observed Raman shift of the Stokes and anti-Stokes features are a direct measure of the vibrational energies of the molecule. A schematic Raman spectrum may appear as shown below.

The energy of the scattered radiation is less than the incident radiation for the Stokes line and the energy of the scattered radiation is more than the incident radiation for the anti-Stokes line. The energy increase or decrease from the excitation is related to the vibrational energy spacing in the ground electronic state of the molecule and therefore the wavenumber of the Stokes and anti-Stokes lines are a direct measure of the vibrational energies of the molecule.

In the example spectrum, notice that the Stokes and anti-Stokes lines are equally displaced from the Rayleigh line. This occurs because in either case one vibrational quantum of energy is gained or lost. Also, note that the anti-Stokes line is much less intense than the Stokes line. This occurs because only molecules that are vibrationally excited prior to irradiation can give rise to the anti-Stokes line. Hence, in Raman spectroscopy, only the more intense Stokes line is normally measured – Raman scattering is a relatively weak process. The number of photons Raman scattered is quite small. However, there are several processes which can be used to enhance the sensitivity of a Raman measurement.

Simplified energy diagram

If the wavelength of the exciting laser coincides with an electronic absorption of a molecule, the intensity of Raman-active vibrations associated with the absorbing chromophore are enhanced by a factor of 102 to 104. This resonance enhancement or resonance Raman effect can be extremely useful, not just in significantly lowering the detection limits, but also in introducing electronic selectivety. Thus the resonance Raman technique is used for providing both structural and electronic insight into species of interest.

Metalloporphyrins, carotenoids and several other classes of biologically important molecules have strongly allowed electronic transitions in the visible, making them ideal candidates for resonance Raman spectroscopy. Resonance selectivity has a further practical use, in that spectrum of the chromophoric moiety is resonance enhanced and that of the surrounding environment is not. For biological chromophores, this means that absorbing active centres can be specifically probed by visible excitation wavelengths, and not the surrounding protein matrix (which would require UV lasers to bring into resonance).

Resonance Raman spectroscopy is also an important probe of the chemistry of metal centred complexes, fullerenes, polydiacetylenes and other “exotic” molecules which strongly absorb in the visible. Although many more molecules absorb in the ultraviolet, the high cost of lasers and optics for this spectral region have limited ultraviolet (UV) resonance Raman spectroscopy to a small number of specialist groups.

Schematic Raman spectrum

Vibrations which are resonantly enhanced fall into two or three general mechanistic classes. The most common case is Franck-Condon enhancement, in which a component of the normal coordinate of the vibration occurs in a direction in which the molecule expands during an electronic excitation. The more the molecule expands along this axis when it absorbs light, the larger the enhancement factor. The easily visualized ring breathing (in-plane expansion) modes of porphyrins fall into this class. Vibrations which couple two electronic excited states are also resonantly enhanced, through a mechanism called vibronic enhancement. In both cases, enhancement factors roughly follow the intensities of the absorption spectrum. The fuller theory of resonance enhancement is beyond the scope of this section.

Resonance enhancement does not begin at a sharply defined wavelength. In fact, enhancement of 5x to 10x is observed if the exciting laser is within even a few 100 wavenumbers below the electronic transition of a molecule. This “pre-resonance” enhancement can be experimentally useful.

The Raman scattering from a compound (or ion) adsorbed on or even within a few Angstroms of a structured metal surface can be 103 to 106x greater than in solution. This surface-enhanced Raman scattering is strongest on silver, but is observable on gold and copper as well. At practical excitation wavelengths, enhancement on other metals is unimportant.

SERS arises from two mechanisms:

1. The first is an enhanced electromagnetic field produced at the surface of the metal. When the wavelength of the incident light is close to the plasma wavelength of the metal, conduction electrons in the metal surface are excited into an extended surface electronic excited state called a surface plasmon resonance. Molecules adsorbed or in close proximity to the surface experience an exceptionally large electromagnetic field. Vibrational modes normal to the surface are most strongly enhanced.
2. The second mode of enhancement is by the formation of a charge-transfer complex between the surface and analyte molecule. The electronic transitions of many charge transfer complexes are in the visible, so that resonance enhancement occurs. Molecules with lone pair electrons or pi clouds show the strongest SERS. The effect was first discovered with pyridine.

Other aromatic nitrogen or oxygen containing compounds, such as aromatic amines or phenols, are strongly SERS active. The effect can also be seen with other electron-rich functionalities such as carboxylic acids. The intensity of the surface plasmon resonance is dependent on many factors including the wavelength of the incident light and the morphology of the metal surface. The wavelength should match the plasma wavelength of the metal. This is about 382 nm for a 5μm silver particle, but can be as high as 600nm for larger ellipsoidal silver particles. The plasma wavelength is to the red of 650nm for copper and gold, the other two metals which show SERS at wavelengths in the 350-1000 nm region. The best morphology for surface plasmon resonance excitation is a small (<100nm) particle or an atomically rough surface. SERS is commonly employed to study monolayers of materials adsorbed on metals, including electrodes.

Other popular surfaces include colloids, metal films on dielectric substrates and, recently, arrays of metal particles bound to metal or dielectric colloids through short linkages. Although SERS allows easy observation of Raman spectra from solution concentrations in the micromolar (10x-6) range,non-reproducability of quantitative measurements has in the past marred its utility for analytical purposes. However, standardization in production of SERS active media is steadily improving its potential in this area also.

UVRRS is a powerful tool in the molecular analysis of complex biological systems. Most biological systems absorb UV radiation and hence have the ability to offer resonance with UV Raman excitation. This results in the highly selective resonance Raman effect enabling enhancement of important biological targets such as protein or DNA. For example, excitation around 200nm enhances the Raman peaks from vibrations of amide groups; excitation around 220nm enhances peaks from certain aromatic residues. The Raman scatter from water is weak, allowing for analysis of very weak aqueous systems.

Fiber optic UVRRS configuration

Due to the selective nature of UVRRS, a tunable laser is typically required as the excitation source. Since truly tunable continuous-wave lasers are not yet available, a Nd:YAG-pumped dye laser with frequency-doubled output is one suitable UVRRS system. Depending on the dyes used, this laser setup can give almost any required UV wavelength. Intensified CCDs (ICCDs) with UV photocathodes, back-illuminated CCDs or CCDs with UV enhancing (BASF lumogen)coatings can be used as detectors for UVRRS. These detectors are used on account of their high detection efficiency and multichannel capabilities. The primary obstacle to the merging of the worlds of UVRRS and fiber-optic spectroscopy is solarization, the process by which UV radiation causes opacity of fiber-optics (even quite pure silica fibers). This opacity impairs transmission, rendering standard fiber-optics useless for UVRRS.

Species of Interest

Pulsed lasers are typically utilized in the study of short-lived species. A laser pulse can be supplied to a molecular system with enough energy to redistribute the electrons in a molecule causing the formation of an excited state as illustrated on the right. The Raman spectrum of this excited state molecule can be studied either using the same laser pulse or a different pulse from a second laser (single color and two-color pulsed Raman). Excited states of interest can have lifetimes, from picoseconds to milliseconds, but the majority can be studied using gating in the order of 5ns. As the majority of excited states are generated using UV and visible lasers, photocathodes with high UV and visible Quantum Efficiencies (QEs) are typically suitable.

Schematic of pump-probe (two color) Raman

The simplest pulsed laser experiments are so-called single-color experiments where high irradiance laser pulses are used both to initiate the photoreaction, and then to Raman probe the transient species created within the pulse width. By opening the intensifier tube as shown on the right, only the Raman spectrum of the excited state will be recorded. This pulse/ICCD gate combination will be repeated and accumulated hundreds to thousands of times in order to achieve a good overall signal-to-noise ratio with high dynamic range.

In Time Resolved Resonance Raman (TR3) spectroscopy, pairs of laser pulses of different wavelength are used to photolyse (optically “pump”) and then to Raman probe the transient species of interest. The spectral window of the spectrograph/detector is chosen so that it corresponds to the frequency range of the Raman scattering from the probe laser.

Pulsed two color Raman layout with delays under the control of a delay generator

In Time Resolved Resonance Raman (TR3) spectroscopy, pairs of laser pulses of different wavelength are used to photolyse (optically “pump”) and then to Raman probe the transient species of interest. The spectral window of the spectrograph/detector is chosen so that it corresponds to the frequency range of the Raman scattering from the probe laser.

The time evolution of the transient signal is monitored by recording a series of spectra at different delays after the photolysis event, i.e. at a series of time delays between the excitation and probe pulses. The ICCD camera or either of the lasers can supply the trigger. A delay generator is used to control the delays.

In Raman microscopy, a research grade optical microscope is coupled to the excitation laser and the spectrometer, thus producing a platform capable of obtaining both conventional images and in addition generating Raman Spectra from sample areas approaching the diffraction limit (~1 micron). Imaging and spectroscopy can be combined to generate “Raman cubes”, 3- dimensional data sets, yielding spectral information at every pixel of the 2D image.

A motorized xyz microscope stage can be used to automatically record spectral files, which will constitute the basis of Raman images, Raman maps or a set of Raman spectra recorded from preselected points. Specific software routines will allow the quick and easy reconstruction of these maps. The possibility of generating two-dimensional and three-dimensional images of a sample, using various special features, is an evident advantage over either traditional spectroscopy or microscopy.

Time delay sequences

The first ever Raman “instrument” was constructed in 1928. This instrument used monochromatized sunlight as a light source and a human eye as a detector. Raman instrumentation was developed (based around arc lamps and photographic plates) and soon became very popular up until the 1950s. Since these early days, Raman instrumentation has evolved markedly. Modern instrumentation typically consists of a laser, Rayleigh filter, a few lenses, a spectrograph and a detector (typically a CCD or ICCD).

Typical Continuous Wave (CW) Raman layout

One of the major advantages of dispersive Raman is that it offers the possibility to select the optimal laser excitation wavelength to permit the recording of the best Raman information. For example, wavelengths can be selected to offer the best resonance with the sample under investigation.

One might also need to tune wavelength to avoid fluorescence and thermal emission backgrounds. Nowadays, it is possible to use laser lines from UV, (down to 200nm) up to the infrared, (1.06μm Nd:YAG laser line), from microWatts up to several Watts.

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Driven by applications in chemical sensing, biological imaging and material characterisation, Raman spectroscopies are attracting growing interest from a variety of scientific disciplines. The Raman effect originates from the inelastic scattering of light, and it can directly probe vibration/rotational-vibration states in molecules and materials.

Despite numerous advantages over infrared spectroscopy, spontaneous Raman scattering is very weak, and consequently, a variety of enhanced Raman spectroscopic techniques have emerged. These techniques include stimulated Raman scattering and coherent anti-Stokes Raman scattering, as well as surface- and tip-enhanced Raman scattering spectroscopies. The present review provides the reader with an understanding of the fundamental physics that govern the Raman effect and its advantages, limitations and applications. The review also highlights the key experimental considerations for implementing the main experimental Raman spectroscopic techniques. The relevant data analysis methods and some of the most recent advances related to the Raman effect are finally presented. This review constitutes a practical introduction to the science of Raman spectroscopy; it also highlights recent and promising directions of future research developments.

Introduction

Raman Spectroscopy

There are numerous forms of light-matter interaction: fluorescence and phosphorescence are examples of absorption and subsequent emission of light by matter. Elastic scattering of light, such as Rayleigh scattering by atoms, molecules or phonons, and Mie/Tyndall scattering by dust particles are examples where the wavelength of the light is unchanged. Inelastic scattering such as Brillouin scattering by acoustic waves in crystals, Compton scattering by charged particles and Raman scattering by molecules or phonons are examples where the wavelength of the light does change [1]. Raman scattering of light by molecules was first predicted using classical quantum theory by Smekal in 1923 [2] and experimentally observed by Raman and Krishnan in 1928 [3, 4].

There are now more than 25 different types of known Raman spectroscopy techniques, such as spontaneous Raman, hyper-Raman scattering, Fourier transform Raman scattering [5], Raman-induced Kerr effect spectroscopy [6] and stimulated/coherent Raman scattering [1]. This review considers spontaneous and stimulated Raman scattering, coherent anti-Stokes Raman scattering (CARS), surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS).

Fifty years after its first observation, Raman spectroscopy started to become a prominent analysis technique among other optical metrology techniques, such as those involving absorption of infrared light; particularly when water and other useful polar solvents were present, because these media typically strongly absorb light in the infrared region. For example, in 1974, Fleischmann et al. [7] used Raman spectroscopy to distinguish two types of adsorbed pyridine (a basic cyclic heterodyne compound molecule) on the surface of a silver electrode to mitigate absorption effects. This experiment was incidentally the first serendipitous observation of SERS.

Raman spectroscopy is now an eminent technique for the characterisation of 2D materials (e.g. graphene [8,9,10] and transition metal dichalcogenides [11,12,13]) and phonon modes in crystals [14,15,16]. Properties such as number of monolayers [9, 12, 17, 18], inter-layer breathing and shear modes [19], in-plane anisotropy [20], doping [21,22,23], disorder [10, 24,25,26], thermal conductivity [11], strain [27] and phonon modes [14, 16, 28] can be extracted using Raman spectroscopy.

The biological and medical fields of research are greatly impacted by the development of Raman spectroscopy as it is a label-free (does not require fluorescent marker molecules [29, 30]) chemically selective hyperspectral imaging technique [31]. For instance, studying the transdermal delivery of drugs into skin often ordains ex vivo and invasive analysis techniques. Ex vivo transdermal delivery studies are unfavourable because skin regeneration stops, the immune response ceases, and metabolic activity is usually lost. Hence, the performance of transdermal drug delivery ex vivo is not an accurate reflection of the in vivo situation [32]. However, non-invasive in vivo measurements can be performed using Raman spectroscopy to gain detailed information about the molecular composition and concentration gradients in the skin [33]. In many biological processes, living microorganisms such as bacteria act as biocatalysts. Raman spectroscopy can probe inhomogeneity in the properties and physiological status of individual cells in biocatalytic processes [34]. Raman spectroscopy has also been used to identify and differentiate benign and malignant breast cancer lesions by probing their unique chemical compositions [35].

For biological samples, approximately 90% of the peaks are found in the ‘fingerprint’ spectral region, covering (Δν∼Δν∼ ~ 500 cm⁻¹ to ~ 1800 cm⁻¹; Δν∼Δν∼ is the wavenumber shift defined in the “Analysis methods” section), with the remaining found in the higher energy CH/OH stretching vibrational modes covering (Δν∼Δν∼ ~ 2700 cm⁻¹ to ~ 3300 cm⁻¹) [36].

Coherent Raman Spectroscopy

Coherent light-scattering events involving multiple incident photons simultaneously interacting with the scattering material was not observed until laser sources became available in the 1960s, despite predictions being made as early as the 1930s [37, 38]. The first laser-based Raman scattering experiment was demonstrated in 1961 [39]. Stimulated Raman scattering (SRS) and CARS have become prominent four-wave mixing techniques and are of interest in this review.

SRS is a coherent process providing much stronger signals relative to spontaneous Raman spectroscopy as well as the ability to time-resolve the vibrational motions. SRS is relevant to numerous areas of research such as plasma physics [40], atomic interferometry [41], supercontinuum generation [42], imaging of biomolecules in food products [43], imaging chemistry inside living cells [44], bulk media and nanoscale specimens [45]. The exchange of photon orbital angular momentum by SRS in plasma is gaining interest, particularly in the context of inertial fusion research [40, 46,47,48]. Supercontinuum generation is a complex nonlinear phenomenon that is characterized by the dramatic spectral broadening of intense light pulses passing through a nonlinear material [49]. Knight et al. [42] demonstrated flat ultrabroadband octave-spanning white-light supercontinuum generation by SRS and parametric four-wave mixing with 60-ps pump pulses of sub-kilowatt peak power in a photonic crystal fibre. Kasevich and Chu [41] demonstrated a matter-wave interferometer with laser-cooled sodium atoms using the mechanical effects of stimulated Raman transitions. SRS has even been used to observe time-resolved vibrational spectra of the primary isomerisation of retinal in the visual pigment rhodopsin [50].

Since its resurgence in 1999, CARS has become a prominent vibrational mode imaging tool in biological medicine [51, 52]. As anti-Stokes photons are blue shifted from the pump and Stokes frequencies, they are more easily detected in the presence of single-photon fluorescence [53]. CARS microscopy has been successfully applied to live-cell imaging [51, 54], skeletal stem cells [55], tracing toxic nanomaterials in biological tissues [56], volumetric imaging of human somatic cell division [57], flow cytometry [58, 59], detection of brain tumours [60] and tracking organelle transport in living cells [61]. Zirak et al. [62] has developed a CARS endoscope for in vivo imaging and demonstrated the instrument with murine adipose tissue and human nervus suralis samples. Evans et al. [63] have combined CARS with video rate microscopy to chemically image tissue in vivo. Potma and Xie [64] have directly visualised lipid phase segregation in single lipid bilayers with CARS. CARS can even be used as a high temporal and spatial resolution thermography technique and has found applications in electronic and opto-electronic device characterisation [65] and even turbomachinery [66].

Orientational order is a salient feature of many soft matter systems. Detail in structural molecular organisation is a prevailing goal in the field of biology, biomedicine, material sciences and molecular physics [67]. Polarisation-resolved optical microscopy is becoming a powerful tool to address molecular orientational distributions into the focal volume of a microscope [68]. In coherent nonlinear optics, polarised second harmonic generation [69,70,71], polarised third harmonic generation [72] and polarised four-wave mixing [73, 74] have already been used to recover orientational information on endogeneous proteins and lipids in biological tissues. In addition to the orientational information, coherent Raman scattering (CRS) processes are sensitive to molecular bond vibrations, allowing chemical specificity without the need for fluorescence labelling/dyes [75,76,77]. CARS microscopy can be used to image chemical and orientational order of liquid crystalline (commonly used in display technology) samples [78]. Polarisation-resolved hyperspectral SRS microscopy has also been demonstrated as a label-free biomolecular imaging technique with teeth [79]. In addition, polarised-CARS has been used to study the molecular order of lipids in myelin at sub-diffraction scales in mice [80].

Enhanced Raman Spectroscopy

The sensitivity of Raman spectroscopy can be enhanced through various techniques such as resonance Raman spectroscopy [81], TERS [82, 83] or SERS [84]. SERS is particularly interesting since it allows an enhancement of several orders of magnitude of the Raman signal by modifying the surface upon which an analyte material is to be placed. The enhanced light-matter interaction in TERS and SERS is tuneable (to some extent) by modifying the surface nanostructure of metallic films on dielectric surfaces [85, 86]. The wavelength of charge density oscillations, known as plasmons, is dependent on these surface nanostructures and can enhance the light-matter interaction locally [87]. Plasmons are a prominent topic in physics and plasmonic devices such as filters [88], waveguides [88, 89], polarisers [90] and nanoscale light sources [91] have now been realised.

Fleischmann et al. [7] first observed SERS in 1974 when investigating pyridine on the rough surface of a silver electrode [92]. The sensitivity of SERS makes it well-suited to study electron transfer reactions, which lie at the heart of numerous fundamental processes: electro-catalysis, solar energy conversion, energy storage in batteries, and biological events such as photosynthesis [93]. SERS has also been identified as a valuable technique for the detection of explosives/chemical weapons [94], unmodified DNA [95], aerosol pollutants [96] and pathogens [97].

TERS is a technique that provides spectral information with a spatial resolution on the nanometre scale [98]. Since the first reports of TERS emerged in 2000 [99, 100], TERS has become a powerful technique for studying thin crystalline materials [101], carbon nanotubes[86, 102, 103], single strands of RNA/DNA [104, 105], redox reactions [106], mapping of individual molecules [83], semi-conductor nanostructures and microcavities [107].

In the following sections, the fundamental physics that underpins the spontaneous Raman effect, stimulated- and coherent Raman spectroscopy, SERS and TERS are detailed in the context of their applications. Experimental considerations are discussed, and examples of Raman spectroscopy instrumentation setups are presented. The conventions for presenting spectra are detailed and examples of analysis techniques are given for each of the applications of Raman spectroscopy. In the final sections, the recent advances that constitute the current frontiers of Raman spectroscopy are presented from various fields of research worldwide.

Fundamental Principles

When light interacts with matter, the oscillatory electro-magnetic (EM) field of the light perturbs the charge distribution in the matter which can lead to the exchange of energy and momentum leaving the matter in a modified state. Examples include electronic excitations and molecular vibrations or rotational-vibrations (ro-vibrations) in liquids and gases, electronic excitations and optical phonons in solids, and electron-plasma oscillations in plasmas [108].

Spontaneous Raman

When an incident photon interacts with a crystal lattice or molecule, it can be scattered either elastically or inelastically. Predominantly, light is elastically scattered (i.e. the energy of the scattered photon is equal to that of the incident photon). This type of scattering is often referred to as Rayleigh scattering. The inelastic scattering of light by matter (i.e. the energy of the scattered photon is not equal to that of the incident photon) is known as the Raman effect[1, 4, 6]. This inelastic process leaves the molecule in a modified (ro-)vibrational state. In the case of a crystal lattice, the energy transfer creates a quantum of vibration in the lattice known as a phonon (a quasi-particle). Raman scattering in crystals can also lead to paramagnetic ions, surface plasmons (which are discussed later in this review) and spin waves [15]. The shift in angular frequency of the scattered light can be described by the following equation:ωscat=ωp±ωosc,ωscat=ωp±ωosc,(1)

where subscripts osc denotes the lattice or molecule vibration, p denotes the incident photon (often referred to as the pump photon) and scat denotes the scattered light [1]. The binary operator (±) is determined by energy conservation. When the energy of the scattered photon is lower than that of the incident photon (i.e. red shifted), the process is referred to as Stokes Raman scattering. Conversely, when the energy of the scattered photon is higher than that of the incident photon (i.e. blue shifted), the process is referred to as anti-Stokes Raman scattering. The Raman process must also conserve momentum, which is expressed in wave vector form as:k⇀scat=k⇀p±q⇀,k⇀scat=k⇀p±q⇀,(2)

where k⇀scatk⇀scat, k⇀pk⇀p and q⇀q⇀ are the wave vectors of the scattered light, the incident light and the phonon or molecular (ro-)vibration, respectively.

In molecules and crystals, the charge distribution has an equilibrium state to which it tends. An externally applied field can modify or perturb the charge distribution but only in accordance with the molecule or crystal’s ability to form dipoles which may be anisotropic. This anisotropic property of molecules and crystals is called the polarisability and dielectric susceptibility, respectively. The classical approach theorises that the existence of the Raman effect is associated with the modulation of the polarisability (for molecular (ro-)vibrations) or dielectric susceptibility (for crystal lattice vibrations) due to the oscillatory nature of their interatomic displacement [6, 109]. For crystal lattice vibrations, consider the polarisation vector of the material, P⇀P⇀. If the suffixes j and k represent the vector components in the x, yand z directions, the j^th component of P⇀P⇀ (to first-order) is related to the oscillatory electric field vector E⇀E⇀ associated with the light by [110]:P(1)j=ε0χ(1)jkEk,Pj(1)=ε0χjk(1)Ek,(3)

where ε₀ is the permittivity of free space, χ_jk is the dielectric susceptibility of the material (a rank two tensor) and the convention of summation over repeated indices is implied [109]; the superscript (1) signifies that this is the first-order contribution to the polarisation [1]. The polarisability tensor is a function of the nuclear coordinates which, by extension, means that it will also depend on the (ro-)vibrational frequency. Assuming the modulation is small, the dependence can be expressed in a Taylor series with respect to the coordinates of vibration as follows:χ(1)jk(k⇀p,ωp)≈χ(1)jk(k⇀p,ωp)u⇀=0+ul⎛⎝⎜∂χ(1)jk(k⇀p,ωp)∂ul⎞⎠⎟u⇀=0+ulum⎛⎝⎜∂2χ(1)jk(k⇀p,ωp)∂ul∂um⎞⎠⎟u⇀=0+…,χjk(1)(k⇀p,ωp)≈χjk(1)(k⇀p,ωp)u⇀=0+ul(∂χjk(1)(k⇀p,ωp)∂ul)u⇀=0+ulum(∂2χjk(1)(k⇀p,ωp)∂ul∂um)u⇀=0+…,(4)

where u⇀u⇀ is the nuclear displacement vector, the indices j, k, l and m indicate different spatial coordinates with repeated indices in any of the terms implying the summation of the constituents of that index. If we write the electric field associated with the light as follows:E⇀(r⇀,t)=E⇀(k⇀p,ωp)cos(k⇀p∙r⇀−ωpt),E⇀(r⇀,t)=E⇀(k⇀p,ωp)cos⁡(k⇀p∙r⇀−ωpt),(5)

and the nuclear displacement as follows:u⇀(r⇀,t)=u⇀(q⇀,ωosc)cos(q⇀∙r⇀−ωosct),u⇀(r⇀,t)=u⇀(q⇀,ωosc)cos⁡(q⇀∙r⇀−ωosct),(6)

an explicit expression for time dependence of P(1)jPj(1) can be found by substitution of these two mathematical equations of the monochromatic light and displacement. The numerous resulting terms pertain to optical processes such as Rayleigh scattering, optical absorption and Raman scattering. The term which pertains to the first-order Raman scattering is derived from the second term on the right-hand side of Eq. 4 and yields:Pj(r⇀,t,u⇀)=12ε0⎛⎝⎜∂χ(1)jk(k⇀p,ωp)∂ul⎞⎠⎟u⇀=0ul(q⇀,ωosc)Ek(k⇀p,ωp)×{cos[(k⇀p+q⇀)∙r⇀−(ωp+ωosc)t]∙+cos[(k⇀p−q⇀)∙r⇀−(ωp−ωosc)t]}Pj(r⇀,t,u⇀)=12ε0(∂χjk(1)(k⇀p,ωp)∂ul)u⇀=0ul(q⇀,ωosc)Ek(k⇀p,ωp)×{cos⁡[(k⇀p+q⇀)∙r⇀−(ωp+ωosc)t]∙+cos⁡[(k⇀p−q⇀)∙r⇀−(ωp−ωosc)t]}(7)

This term contains sum (anti-Stokes) and difference (Stokes) frequencies and demonstrates conservation of momentum as per Eqs. 1 and 2. This formulation follows the classical description from refs. [1, 109].

The quantum mechanical description of the Raman process states that the (ro-)vibrational energy of the molecules/phonons are discrete quanta. Figure 1a shows an energy level diagram illustrating the Raman processes with Stokes emission at ω_S and anti-Stokes emission at ω_AS.

In Raman scattering, the intermediate states of the perturbation imposed by the incident pump photon (| r 〉 and | l 〉 in Fig. 1a, b) generally do not correspond to electronic states of the system and are said to be virtual energy states. These virtual intermediate states do not represent a well-defined energy state of the system. As the frequency of the pump photon approaches the energy of the electronic states, the strength of the Raman effect increases due to resonance effects and is termed pre-resonance Raman. If the intermediate state corresponds to a discrete electronic energy state, the interaction is described as resonance Raman scattering and the signal strength is expected to exceed that of virtual-intermediate-state Raman scattering by orders of magnitude. If the energy of the incident light is in the range of dissociative energy levels, the process is described as continuum resonance Raman scattering [1].

Raman scattering transitions between certain quantum states are forbidden. In materials with inversion symmetry (i.e. centrosymmetric crystal structure [113]), the initial and final states must have the same parity and are mutually exclusive with absorptive transitions (optically active transitions). In other words, transitions can be either Raman active or optically active. For linear molecules, the symmetric stretching modes of vibration or bending are Raman active and are optically inactive; those with anti-symmetric modes are Raman inactive and optically active (i.e. mutually exclusive) [1]. This rule is general and for nonlinear molecules, mutual exclusion is relaxed. In materials without inversion symmetry, (ro-)vibrational mode transition can be both Raman and optically active [1, 108].

The Stokes Raman signal for molecules is more intense than the anti-Stokes signal as the population of energy states is governed by thermal statistics [1, 108]. For bosonic systems, such as phonons in crystals, the probability of the scattering target occupying a given vibrational quantum energy state obeys Bose-Einstein statistics. Under nonresonant Raman scattering and thermal equilibrium, the ratio of the anti-Stokes and Stokes scattered intensity is given by [109]:IASIS=(ωp+ωoscωp−ωosc)4e(−ℏωosckT)IASIS=(ωp+ωoscωp−ωosc)4e(−ℏωosckT)(8)

where I_S and I_AS are the intensity of the Stokes and anti-Stokes light, respectively, ℏ is Planck’s constant divided by 2π, k is the Boltzmann constant and T is the temperature associated with the scattering species. This equation is sometimes used to measure the temperature via Raman spectroscopy [65]. This relation becomes inaccurate for resonance Raman scattering because the Stokes and anti-Stokes processes occur at different pump photon frequencies [109].

In the case of spontaneous Raman scattering, the Raman effect is very weak; typically, 1 in 10⁸of the incident radiation undergoes spontaneous Raman scattering [6]. The transition from the virtual excited state to the final state can occur at any point in time and to any possible final state based on probability. Hence, spontaneous Raman scattering is an incoherent process. The output signal power is proportional to the input power, scattered in random directions and is dependent on the orientation of the polarisation. For example, in a system of gaseous molecules, the molecular orientation relative to the incident light is random and hence their polarisation wave vector will also be random. Furthermore, as the excited state has a finite lifetime, there is an associated uncertainty in the transition energy which leads to natural line broadening of the wavelength as per the Heisenberg uncertainty principle (∆E∆t ≥ ℏ/2) [1]. The scattered light, in general, has polarisation properties that differ from that of the incident radiation. Furthermore, the intensity and polarisation are dependent on the direction from which the light is measured [1]. The scattered spectrum exhibits peaks at all Raman active modes; the relative strength of the spectral peaks are determined by the scattering cross-section of each Raman mode [108]. Photons can undergo successive Rayleigh scattering events before Raman scattering occurs as Raman scattering is far less probable than Rayleigh scattering.

Nonlinear Susceptibility

The polarisation described by Eq. 3 is in agreement with first-order (i.e. linear) optics and describes the single-photon scattering process (two-wave mixing process). In wave mixing processes with more than two waves, nonlinear optical polarisation must be considered due to the products of the mixed electric field components. Nonlinear optical polarisation can be described by [110] the following:Pj=ε0[χ(1)jkEk+χ(2)jklEkEl+χ(3)jklmEkElEm+…],Pj=ε0[χjk(1)Ek+χjkl(2)EkEl+χjklm(3)EkElEm+…],(9)

where χ⁽²⁾ is the second-order susceptibility (rank three tensor), χ⁽³⁾ is the third-order susceptibility (rank four tensor) and the sum over repeated subscript indices is again implied. Each of the terms in Eq. 9 can be written in shorthand by P⇀(1)P⇀(1), P⇀(2)P⇀(2), P⇀(3)P⇀(3), etc. The physical processes that occur because of the second-order polarisation, P⇀(2)P⇀(2), tend to be distinct from those arising from the third-order polarisation, P⇀(3)P⇀(3). This polarisation can have electric dipole, quadrupolar, octupolar, (etc.) contributions. Under the electric dipole approximation, the second-order polarisation can only occur in crystals that are noncentrosymmetric (lack inversion symmetry). Hence, χ⁽²⁾ vanishes for media such as fluids (e.g. liquid/gas) and amorphous solids (e.g. glass). Third-order nonlinear optical interactions (i.e. those described by a χ⁽³⁾ susceptibility) can occur for both centrosymmetric and noncentrosymmetric systems [109, 110]. Electric quadrupolar, octupolar, (etc.) χ⁽²⁾ contributions do not disappear under inversion symmetry.

Stimulated Raman Scattering

While spontaneous Raman scattering is an incoherent process, SRS is a coherent four-wave nonlinear optical mixing process. The modes of oscillation are in phase forming a coherent modulation of polarisation in the sample with susceptibility χ⁽³⁾(ω_S; ω_p + ω_S − ω_p) [110]. The scattered light is also coherent [45]. The SRS process is dependent on the spontaneous Raman cross-section, the spectral linewidth, the path length of the light-field-matter interaction, the input intensity and optical feedback (light generation) of Stokes frequency light [110].

When photons of frequency ω_p and ω_S simultaneously interact with a molecule or crystal lattice in the ground state, the system vibrates with an induced frequency: ω_osc = ω_p − ω_S. Unlike spontaneous Raman scattering, the de-excitation (relaxation) time to and energy of the final state are determined by the stimulation effect. The interaction results in the transfer of energy from the pump photon to the molecule/lattice, and the molecule/crystal scatters a new photon with frequency and phase matching that of the incident light of frequency ω_S. Figure 2a shows the process schematically.

It is common to employ an external radiation source tuned to the Stokes frequency in tandem with the pump laser beam to provoke this effect. This technique can lead to exponential gain in the Stokes signal, by transferring energy from the pump radiation, and rapid population of the final (ro-)vibrational state |f 〉 [115]. However, if the intensity of the incident light of frequency ω_p is sufficient, the generation of Stokes frequency photons within the material can self-promote SRS without the need for an external ω_S source. The intensity threshold of incident light in organic liquids, such as ethanol, for this kind of self-generated SRS typically requires an incident peak intensity of pump light > 10⁹ W/cm² for an optical path length of a few centimetres. However, the SRS threshold can be significantly reduced by extending the length of the pump and Stokes field interaction with an optical resonator, such as internal reflection in a droplet of liquid. The example shown in Fig. 2b is the SRS spectrum taken with droplets of ethanol directly compared to the spontaneous Raman spectrum of bulk ethanol. The droplets act to confine the light by internal reflection which feeds back the Stokes light as a self-SRS inducing optical resonator [114].

Coherent Anti-Stokes Raman Scattering

CARS is a third-order nonlinear four-wave optical mixing process. Figure 1b shows the energy level diagram for the process. A pump beam and probe beam of frequency ω_p and ω_pr are mixed with a third beam of frequency ω_S (Stokes frequency) and incident on the sample. The frequency difference (ω_p − ω_S) needs to match the frequency associated with the Raman active (ro-) vibrational mode ω_osc = ω_p − ω_S [4, 53, 56, 116]. The frequency of the Stokes beam is usually adjusted/tuned to satisfy this criterion [117]. Next, a probe photon of frequency ω_prprovides a perturbation for the anti-Stokes scattering process to occur at frequency ω_AS = ω_p − ω_S + ω_pr [5]. A macroscopic third-order polarisation, P⁽³⁾, is induced due to the coherent superposition of the microscopic dipole oscillations [53]. Hence, CARS is governed by the third-order susceptibility of the form: χ⁽³⁾(ω_AS; ω_p − ω_S + ω_pr).

There are numerous treatments and approaches to formulating expressions for χ⁽³⁾. If one assumes that the excitation field is much weaker than the intramolecular forces, then a perturbative approach can be adopted [5, 110]. If this is not the case, non-perturbative treatments can be considered [118]. By considering the density matrix equation of the system and expressing the external field interaction as a perturbation in the Hamiltonian, the semi-classical nonlinear optics theory generates an expression for χ⁽³⁾ with 48 terms, each of which contribute to the third-order susceptibility [5]. A generalised expression for dominant terms in resonant CARS is given by the following [51, 119]:χ(3)=ARωosc−(ωp−ωS)−iΓR+χ(3)NR,χ(3)=ARωosc−(ωp−ωS)−iΓR+χNR(3),(10)

where Γ_R is the half width at half maximum for the Raman line [51]; A_R is a constant representing the Raman scattering cross-section. The first term is the contribution due to CARS vibrational resonance as in Fig. 1b (ω_osc = ω_p − ω_S). The second term is the nonresonant background signal and is independent of the Raman shift (ω_osc ≠ ω_p − ω_S). The nonresonant background occurs because not all quantum pathways of the scattering process involve a resonance with a (ro-)vibrational state. This nonresonant contribution interferes with the resonant part of the signal. The nonresonant background causes distinctive distortions of CARS spectra in comparison with spontaneous Raman spectra and has prevented CARS from becoming a widespread technique [120].

The incident light beams of differing frequency move in and out of phase with each other in both time and space. Hence, the CARS signal reaches its first maximum when the field-sample interaction length scale is less than the coherence length scale to yield constructive interference [121, 122]. For plane-wave pump and Stokes beams, the intensity of the anti-Stokes signal is as follows [53, 123]:IAS∝∣∣χ(3)∣∣2IpIprIS⎛⎝⎜⎜sin(Δk⇀∙z⇀2)∣∣Δk⇀∣∣2⎞⎠⎟⎟2,IAS∝|χ(3)|2IpIprIS(sin⁡(Δk⇀∙z⇀2)|Δk⇀|2)2,(11)

where z⇀z⇀ is the sample thickness (vector normal to the lattice cell surface), k⇀k⇀ is the wavevector of light, Δk⇀=k⇀p−k⇀S+k⇀pr−k⇀ASΔk⇀=k⇀p−k⇀S+k⇀pr−k⇀AS is the wavevector mismatch (the velocity difference between the four waves) and I_i is the intensity of the wave denoted by the subscript. Phase matching is achieved when Δk⇀=0Δk⇀=0 and the intensity of the anti-Stokes signal is maximised because the energy and momentum transfer processes correspond to allowed transitions. As the magnitude of χ⁽³⁾ is linearly proportional to the number oscillators involved in the process, the intensity of the anti-Stokes signal is quadratically proportional to the number/concentration of oscillators [53].

Researchers typically employ the pump beam to provide the second virtual excitation (i.e. the probe light shown in Fig. 1b; i.e ω_pr = ω_p and ω_AS = 2ω_p − ω_S) [119]. The intensity of the CARS signal is therefore quadratically proportional to the intensity of the pump beam (see Eq. 11). The CARS signal is monodirectional due to the phase-matching condition [120]. However, high numerical aperture (NA) lenses or microscope objectives (confocal light) are commonly employed to satisfy the phase-matching condition without the need for complex mechanical systems to achieve phase-matched beam alignment [5, 117].

Raman resonances typically have coherence times of ~ 1 ps. Hence, the pump and Stokes beams are typically pulsed in picosecond timescales to obtain coherent excitation [124] and to inhibit multiphoton effects [53]. The CARS process takes place in the immediate vicinity of the beam’s focal spot. The signal produced is typically 10⁶ times that of spontaneous Raman scattering. CARS microscopy offers non-invasive characterisation and imaging of (ro-)vibrational spectra with high sensitivity and spectral resolution as well as three dimensional sectioning capabilities [116].

Surface Plasmons and Polaritons

Surface plasmons can occur at the interface between a dielectric and conducting material, such as a metal or degenerate semi-conductor [88, 125]. They are the light-induced coherent oscillations of surface conduction electrons about their equilibrium position. The nanoscale volume of opposing charge that remains acts as a restoring force on the electrons. The result can be described with a damped simple harmonic oscillator model, in which the oscillations of the free-charge carriers have an associated resonance. Surface plasmons can be excited by EM radiation and plasmonics is the study of these light-matter interactions [126].

Plasmonic nanoparticles that are much smaller than the wavelength of the incident light can support non-propagating surface plasmons that oscillate with a frequency known as the local surface plasmon resonance (LSPR) [127, 128]. The wavelength of surface plasmons is much shorter than that of the associated propagating light for a given frequency [129]. The LSPR wavelength is dependent on the nanoparticle’s size, shape, material, external dielectric properties and inter-nanoparticle separation [85, 127, 128, 130,131,132,133].

Surface plasmons that propagate are referred to as surface plasmon polaritons (SPPs) [134,135,136]. They are essentially light waves that are trapped at the interface due to their interaction with the free electrons of the conducting material [88]. For a planar dielectric-conducting interface, polaritons propagate in 2-dimensional space along the surface interface for length scales of tens to hundreds of micrometres [126, 134,135,136]. They decay evanescently in the direction perpendicular to the surface interface with 1/e decay lengths of up to 200 nm [126, 137]. The field intensity in this evanescent decay region is amplified by orders of 10 to 100 relative to the incident radiation [136]. Hence, light-matter interactions with adsorbed molecules on the surface are also enhanced.

In the case of LSPR, the surface roughness or surface nanoparticles cause local concentrations of charge carriers which further amplify the evanescent EM field due to the lightning rod effect [138]. Even larger field-enhancements (up to 10⁶) can be observed in gap plasmons (in the gap between two neighbouring plasmonic nanoparticles; see Fig. 1c) [85, 111]. This enhanced near-field effect gives rise to the technique known as SERS and is discussed in the next section.

Surface-Enhanced Raman Scattering

Raman is generally a very weak process; it is estimated that approximately one in every 10⁸photons undergo Raman scattering spontaneously [6]. This inherent weakness poses a limitation on the intensity of the obtainable Raman signal. Various methods can be used to increase the Raman throughput of an experiment, such as increasing the incident laser power and using microscope objectives to tightly focus the laser beam into small areas. However, this can have negative consequences such as sample photobleaching [139]. Placing the analyte on a rough metal surface can provide orders of magnitude enhancement of the measured Raman signal, i.e. SERS.

Two mechanisms have been proposed to explain the increase in Raman signal provided by SERS. The first is via EM enhancements where local surface plasmons concentrate the local electric field near the surface of the metal in ‘hot spots’ located on the sharp edges of nanostructures or in regions of concentrated free-charge carriers due to the lighting rod effect [140]. Figure 1c, illustrates the SERS process. This process can increase Raman generation by a factor of 10⁸ to 10¹¹ [86, 141,142,143,144]. The second method is chemical enhancement via charge transfer between the metal surface and the analyte, which enhances Raman scattering by a factor of approximately 10² to 10³ [86, 145,146,147]. However, the charge transfer mechanism only applies to specific molecules, whereas the EM mechanism is applicable for all analytes [4, 92, 102, 148].

The ubiquity of EM enhancements has led to the development of numerous SERS substrates, which can be divided into two groups: metallic nanostructures fabricated on a solid substrate [85] and colloidal suspensions of plasmonic nanoparticles [96, 141]. The most common materials used to fabricate SERS substrates are gold and silver because of their good plasmonic response [149]. Gold also benefits from chemical stability as it is a noble metal. Other metals are also being investigated, such as aluminium for UV Raman spectroscopy [150, 151].

Tip-Enhanced Raman Scattering

The diffraction limit of light restricts the focus spot size in standard optical techniques (such as Raman spectroscopy) to be at least half of the wavelength of the light according to Abbe’s criterion [152,153,154]. Light from the sample is composed of both propagating and non-propagating radiation. The non-propagating evanescent waves remain in the vicinity of their sources and do not participate in image formation in the far field. Instead, they extend laterally on the sample among the plasmon-active sites. Hence the spatial resolution is restricted by the size of the focal spot of the light. Even with a focal spot size of a half-wavelength (~ 250 nm for visible light), any objects that are much smaller than the half-wavelength would appear as a defuse shape.

TERS is a relatively new optical nanoimaging technique that combined Raman spectroscopy with scattering (or apertureless) scanning near-field optical microscopy. TERS offers spatial resolution far beyond the diffraction limit of the probing light. In the context of the a priori description, this is achieved by forcing the near-field evanescent light into the far-field image formation [86]. At the present date, the spatial resolution of TERS is typically reported to be 10–30 nm and is largely assumed to scale with the size of the tip’s apex [103, 155,156,157]. Incremental improvements to this resolution have been reported [104, 158]. Enhancement factors for TERS are significantly weaker than SERS due to the relative size of the probed signal volume. The enhancement factor (relative to spontaneous Raman scattering) is typically reported to be 10³ to 10⁶. As with SERS, two field enhancement mechanisms are thought to contribute to the Raman signal: EM and chemical enhancement [86].

TERS is implemented by positioning a plasmon-active (plasmonic) nanotip approximately 50 nm above the sample’s region of interest. The Raman probe light is focused onto the tip-surface cavity to induce LSPR within the tip’ apex and (in some circumstances) the sample surface. The surface plasmons may then enhance evanescent or near-field light with the incident probe light and/or the Raman scattered light. Hence, the LSPRs both confine and enhance the light field in the vicinity of the tip’s apex. The enhanced local EM field is most concentrated at the tip apex due to the lightning rod effect. This evanescent light at the tip apex can then excite or stimulate Raman, two-photon or second harmonic scattering from a nanoscale volume of the sample under the tip. A Raman image of the sample surface can be obtained by raster scanning the sample under the nanometric tip.

Experimental Considerations

Instrumentation

The nonresonant Raman effect is a very weak process. Hence, monochromatic, narrow-beam and high-intensity lasers are preferable to produce quality Raman spectra. The exploitation of microelectronics, such as stepper motor drives, photon counters, digital data acquisition and computational processing systems can further enhance the quality of spectra. As spontaneous Raman spectroscopy is naturally an incoherent process, continuous-wave laser sources are commonly used because pulsed lasers require higher peak powers for sufficient signal-to-noise ratio, which can photobleach/damage samples.

The choice of wavelength of the laser source depends on the required application. Lower visible wavelengths and UV cause strong photoluminescence in organic materials, which can mask the Raman peaks. Therefore, a longer visible or near-IR wavelength (500—830 nm) laser source is often suited for studying organic materials, because of the reduced photoluminescence. However, the Raman signal intensity is inversely proportional to the wavelength of the pump light. Hence, longer wavelengths of light require longer acquisition times [1, 6].

Raman spectroscopy is most often performed using laser sources at λ = 785 nm. This wavelength source is often selected as it balances the competing factors between Raman signal intensity, fluorescence, detector sensitivity and cost, and cost-effective/compact high-quality laser sources. However, visible lasers in the blue and green (e.g. λ = 532 nm) are becoming more common in Raman spectroscopy [159].

Raman scattering is measured in terms of the wavelength shift from the source wavelength. Ideally the illumination source for Raman measurements should be purely monochromatic, in other words, a single wavelength. However, all laser sources possess a spectrum of wavelengths known as a linewidth. The linewidth of a laser is usually measured in Hertz and is typically > 1 MHz for solid-state lasers used in Raman applications. A narrow linewidth is preferable for Raman spectroscopy because the measured shift in the Raman scattering process is limited by the laser’s linewidth.

Laser sources for Raman spectroscopy need to be stable in wavelength and power over extended periods of time and from use to use. Raman spectra are usually collected over long integration times and for many acquisitions. If the wavelength of the source drifts during a measurement, then the Raman peaks will drift as well, because Raman is measured as a shift relative to the pump light. Wavelength drift is also problematic from measurement to measurement as it causes peaks to shift, in turn making comparisons between measurements difficult. The output power stability of the source is important for similar reasons. If the laser power drifts from measurement to measurement, then quantitative comparisons cannot be made easily.

Spectral purity is another key criterion for Raman laser sources. The spectral purity of laser sources often requires side-mode suppression better than 60 dB. In many cases, side-mode suppression is sufficient if > 60 dB spectral purity is reached at ~ 1–2 nm from the laser wavelength peak. However longer wavelength (near-IR) Raman spectroscopy requires side-mode suppression ratios within a few hundreds of pm from the main peak. These criteria are discussed in the context of common Raman laser sources in the following paragraphs [159].

Most modern Raman systems use solid-state laser sources rather than gas lasers because of their spectral quality and stability. There are three main categories of continuous-wave solid-state laser sources used in Raman spectroscopy: Diode-pumped single-longitudinal mode (SLM) lasers; single-mode diode lasers (distributed feedback (DFB) or distributed Bragg reflection (DBR)); and volume Bragg-grating (VBG) frequency-stabilised diode lasers. These laser sources have varying optical characteristics.

Diode-pumped SLM lasers are readily available in compact form from the UV to the near-IR. Power levels of several Watts are achievable at 1064 nm in the near-IR. In the visible range, numerous lines in the blue-green-red region (457 to 660 nm) are available with output powers of ~ 100 mW. In the UV spectral range, power outputs of 10–50 mW at 355 nm are available. Hermite-Gaussian laser beam modes are described by their transverse electro-magnetic mode (TEM): TEM_m,n, where m and n represent the Hermite-Gaussian mode index [46]. Diode-pumped SLM lasers provide excellent TEM₀₀ mode beams, precise wavelengths with low drift, and a single-frequency linewidth > 1 MHz. The spectral purity of diode-pumped SLM lasers is typically > 60 dB in terms of their side-mode suppression ratio. Weak emissions that neighbour the laser’s main peak several nanometres in spectral shift can occur in diode-pumped SLM lasers. However, these neighbouring lines can be mitigated with dielectric band-pass filters. The wavelength of diode-pumped SLM lasers is typically stable to within 4 pm over a temperature change of 30 °C.

Single-mode diode lasers are compact and cost-effective pump illumination sources with single-frequency linewidth (> 1 MHz), single-TEM beam quality and output powers of up to ~ 100 mW. Wavelengths of λ = 785, 830, 980 and 1064 nm are most common in Raman spectroscopy. The side-mode suppression ratio is typically limited by sideband emission to ~ 50 dB at ~ 100 pm from the main peak.

VBG frequency-stabilised diode lasers use a narrow-linewidth VBG element with a diode-laser emitter to achieve narrow-line emission. These lasers are often used for applications requiring narrow-line emission at wavelengths that are not available for DFB or DBR laser sources. Frequency-locking multi-TEM diode lasers can be used to increase the output power of the narrow-linewidth emission. The stability of the output wavelength and linewidth requires careful thermomechanical control and high-precision alignment inside VBG frequency-stabilised diode lasers. Linewidths can range from single-frequency emission to ~ 10s of pm, depending on the wavelength and the output power. The side-mode suppression ratio is limited to ~ 50 dB, ~ 250 pm from the main peak emission. However, this can be improved using filters.

In confocal Raman imaging applications, it is necessary to use diffraction-limited TEM₀₀beams for optimum spatial resolution. However, this is relaxed for probe-based quantitative Raman analysis. In addition, confocal Raman setups require laser beam isolation as samples may generate optical feedback that is well aligned to the incident pump light. This counter-propagating feedback can induce power and noise instability and can even damage the laser source. Optical isolators are often integrated into the laser system itself because careful alignment must be achieved in the output after the isolator [4, 6, 159].

The spectrometer is a core component of any set-up used for measuring Raman spectra. The spectrometer should match the wavelength(s) of the laser source(s) used. The spectral range and resolution required will depend on the application. For example, the spectral range is determined by the position of the Raman peaks of interest (i.e. at large Δν∼Δν∼ ~ 3000 cm⁻¹or low Δν∼Δν∼ ~ 1 cm⁻¹). If the application requires closely spaced Raman peaks to be resolved, then spectral resolution is key. The spectral resolution of a spectrometer is largely determined by the slit width at the spectrometer entrance, the focal length of the spectrometer, the dispersion, the size of the grating (or prism) and the size and sensitivity/quality of the detector. There is a trade-off between the overall spectral range and resolution when considering the design of the experiment for a given application. In the case of weak Raman signals, optimising the signal-to-noise ratio is a priority.

Spectral filtering plays a vital role in the acquisition of Raman spectra. Firstly, the incident laser light must be spectrally pure, which is accomplished with a narrow-linewidth laser source as discussed previously. However, if the laser light is delivered to the sample by an optical fibre, then it is inevitable that Raman generation will occur in the fibre. Therefore, it is important to use a narrow band-pass filter to reject any Raman signal generated in delivering the laser to the sample. Narrow band-pass filters can provide transmission > 90 % at the laser wavelength while suppressing light to an optical density of OD > 5 at wavelengths differing by just 1% from the laser wavelength.

Importantly, light collected for detection requires filtering to block the laser wavelength. If the laser light is not filtered out, it can go on to generate Raman in the detection arm of the set-up and drown out the desired Raman signal when it reaches the spectrometer. The type of filter required depends on whether Stokes, anti-Stokes or both are to be measured. To only detect anti-Stokes Raman, a short-pass filter should be used as anti-Stokes Raman light has a higher energy and hence shorter wavelength than the laser source. To only detect Stokes Raman, a long-pass filter should be used as the Stokes Raman light has a lower energy and hence longer wavelength than the laser source. Long pass edge filters with edge-transition widths of < 3 nm and edge steepness < 40 cm⁻¹ are available. To detect both Stokes and anti-Stokes Raman light, a notch filter centred on the laser wavelength should be used as it allows both shorter and longer wavelengths to be detected. Notch filters with OD > 6 at the laser line wavelength are available. Multi-notch filters are also available and can block multiple laser lines simultaneously. Holographic notch filters significantly outperform dielectric notch filters, providing excellent attenuation of the Rayleigh line while passing light as near as 50 cm^–1from the Rayleigh line. Acousto-optic modulators can also be used in conjunction with an excitation laser to select emissions with a desired wavelength (as a filter) [160] or as a time-gated illumination system in tapping mode atomic force microscopy (AFM)-based TERS [161].

The quantum efficiency of standard room-temperature silicon-based CCD devices for Raman signal detection degenerates above λ = 800 nm. For longer wavelengths, indium gallium arsenide array devices can be used, but these are less sensitive with higher noise levels and cost.

The visible to near-infrared wavelength range (λ = 500–830 m) is particularly suitable for inorganic materials (e.g. graphene, carbon nanotubes (CNTs) and fullerenes) and SERS. UV lasers are attractive for organic materials (e.g. pathogens, proteins, DNA, and RNA). For materials with strong fluorescence that require near-IR illumination, it is common to use a 1064-nm wavelength.

Spontaneous and Coherent Raman Scattering Setups

Spontaneous Raman spectroscopy is most commonly used for modes with forbidden single-photon absorption or emission experiments [108]. SRS is sometimes used for wavelength shifting of coherent light, light amplification, pulse compression, phase conjugation and beam combining [108]. Unlike spontaneous Raman scattering, SRS is highly directional and offers enhanced signal strength and the ability to time-resolve the evolution and dephasing of coherent (ro-)vibrational motion [45].

Figure 3a shows a typical Raman setup based on a confocal geometry used by Wiedemeier et al. [162]. Confocal setups of this type are commonly used and employ an infinity-corrected objective lens (large numerical aperture (NA) lens) to focus the pump light. Wiedemeier et al. [162] used a diode-pumped solid-state laser as a monochromatic light source centred at 532 nm. Confocal mode is achieved by the use of a pinhole module in front of the spectrometer to spatially filter the light. The pinhole only passes light that originates from the focal plane to the detector. For detection of the Raman signal, a holographic-imaging spectrometer with an attached CCD camera is used. A holographic transmission grating with high light throughput served as a dispersive element, which enables large spectral ranges in a comparatively short time period to be acquired. Raster scanning of the sample in a confocal setup needs to be precise. Hence, a piezo actuated nano-positioner is used for positioning of the specimen.

Spontaneous anti-Stokes scattering is weaker than Stokes Raman scattering due to the relatively low probability of thermal excitation. Hence, anti-Stokes Raman spectroscopy is typically used with stimulated or coherent spectroscopy. CARS spectroscopy offers a 10⁵increase in conversion efficiency, spectral and spatial discrimination against fluorescence and, most importantly, does not require a monochromator. Due to the required coherence of the process, high-peak power pulsed tuneable laser sources are employed. These peaks are readily available using picosecond or femtosecond light lasers, the choice of which is determined by the spectral resolution required and the timescale of interest [139].

Avoiding direct electronic excitations in the sample is an important consideration as photochemical damage (due to photobleaching) can occur in samples. Djaker et al. [139], for example, use near-infrared laser sources to mitigate photobleaching in their samples of polystyrene beads.

Figure 3b shows a typical CARS setup that measures both forward scattered light (F-CARS) and back- or epi-scattered light (E-CARS) [116, 139, 163]. The system has two synchronised picosecond pulse trains. The pump and Stokes beams are generated by two picosecond Ti:Sapphire lasers operating at 80 MHz and are tuneable from 700 to 1000 nm to cover the entire spectrum of molecular (ro-)vibrations in biological systems (up to Δν∼Δν∼ ~ 3000 cm⁻¹). The ps pulse duration is adjustable by a Gires-Tournois interferometer. The Ti:Sapphire lasers are pumped by a frequency-doubled CW Nd:Vanadate laser that provides monochromatic light at 532 nm. The two pulse trains were polarised with a pulse duration of 3 ps, corresponding to a spectral width of 1.76 cm⁻¹. The pump and Stokes beams are synchronously pulse picked through two Bragg cells to reduce the repetition rate of the pulse trains to several hundred kilohertz, thus avoiding photodamage of the sample while still maintaining high-peak power for CARS generation. The pump and Stokes beams are temporally synchronised by a SynchroLock system, which electronically adjusts the time delay between the two pulse trains. A small part of the output of the lasers are launched in optical fibres coupled to photodiodes and connected to a SynchroLock controller, which measures the lasers frequency or phase difference between the master and the slave; the timing jitter was reported to be ~ 250 fs. The spectral resolution was estimated to be 2.5 cm⁻¹, which is high enough to resolve Raman spectral features of biological samples. The use of a broadband Stokes wave enables the acquisition of a full CARS spectrum in only one measurement, with this configuration being known as multiplex or broadband CARS [164,165,166,167].

The two pulse trains are spatially filtered, collinearly combined and expanded through beam expanders. They are then sent into an inverted microscope and focused onto the sample by a water-immersion objective lens with a large NA. The E-CARS signal is collected by the same objective lens while the F-CARS signal is collected by a condenser lens with a lower NA. The E-CARS and F-CARS signals are filtered through a set of band-pass filters and detected by two avalanche photodiodes with a 200 μm × 200 μm active area. The CARS images are collected by raster scanning the sample, using an XYZ piezo flexure stage.

Several methods have been developed to suppress the nonresonant background associated with CARS. E-CARS is relatively insensitive to the nonresonant background of sample solvents [168]. Polarisation-sensitive CARS can differentiate the resonant and nonresonant signals by their polarisation [169]. However, these two techniques reduce the anti-Stokes signal strength [120]. Time-resolved CARS [170], temporal or spectral interferometry CARS [52, 171] and frequency-modulated CARS [172] can also suppress the nonresonant background. However, the setup in terms of both optics and electronics is challenging [120].

SERS Specific Considerations

A variety of nanostructures, such as bowtie antennas [173], nano-rings [174], nanovoids [175], nanoparticle aggregates [87, 176, 177], nanoflower [178], nanorod arrays [97] and nanowells [179] can be used for SERS. Each nanostructure can have a number of plasmonic resonances, and matching the excitation laser to these wavelengths can greatly enhance the SERS intensity [141, 180, 181]. Matching the plasmonic resonance to the pump laser can be done either by tuning the laser wavelength or by tuning the LSPR of the nanostructures [85, 182,183,184].

The difficulty faced in producing SERS substrates is consistency in fabrication and repeatability in measurements due to the inhomogeneity and randomness of SERS active hot spots [85, 185, 186]. For SERS substrates produced by top-down methods, such as electron beam lithography [187], the main challenge is scaling the fabrication. Conventional top-down methods limit the active area of the SERS substrate and are not conducive to large-area manufacturing. Bottom-up fabrication methods have their own set of problems. Even though bottom-up approaches allow wafer scale fabrication, consistency across the wafer is usually lacking [188]. This inconsistency hinders the repeatability of measurements, which is problematic for quantitative analysis. Colloidal SERS schemes suffer from complications introduced by stabilising agents at the surface of the nanoparticles, which help to keep nanoparticles in suspension. These stabilising agents can either impede or augment the measured Raman signal [189]. The chemical synthesis for nanoparticle colloids also requires precise optimisation. The poor reproducibility of nanoparticle colloidal synthesis hampers batch-to-batch consistency.

Often, only very few sites exhibit the highest SERS enhancement and the variability in size and shape can alter the plasmonic properties from the desired LSPR [85]. Figure 3c, shows a setup which combines SERS with dark-field spectroscopy. The dark-field spectrometer analyses the light scattered from the nanostructures (illuminated by the white-light source) to select nanostructures with the desired plasmonic properties.

TERS Specific Considerations

Scanning probe microscopy (SPM) techniques, such as atomic force microscopy (AFM), scanning tunnelling microscopy (STM) or shear force microscopy (SFM), are usually the tools of choice for TERS [86]. TERS has the ability to simultaneously measure topography by the conventional SPM mode of the system and obtain corresponding spectral information from a sample with nanometric spatial resolution and high sensitivity [86]. Certain SPM techniques ordain probe modifications for the plasmonically induced nanoscale evanescent light to activate/enhance the Raman signal. The tips can either be made of a metal or coated with a thin layer of metal to modify them for TERS. When the apex of a metallic or a metal-coated nanotip is illuminated with focused light at the LSPR wavelength, local surface plasmons around the tip apex are excited, and evanescent light is produced at the tip apex. This evanescent light can generate Raman scattering from a sample placed right under the tip apex. The process of Raman scattering takes place in the near-field and the spectral signal is scattered and converted back to the far-field by the tip apex, which is then collected by the usual optics and spectrometer in the far-field. Figure 3d shows such a TERS setup with a modified AFM. The setup consists of largely similar equipment shown in Fig. 3a (discussed in an earlier section). An inverted microscope illuminates the sample from underneath and the tip is placed at the top surface of the sample. The Raman back-scattered signal is then directed to the spectrometer. An evanescent mask blocks the central part of the laser beam inhibiting the low NA component of the incident light, so that only the high-NA component of the incident light reaches the sample so that total internal reflection occurs. This limits the transmitted light that falls onto the tip and, hence, only the evanescent light participates in the Raman scattering signal. Suppressing the participation of transmitted far-field light reduces the unfavourable background signal.

Polarisation-dependent TERS can be performed with light polarisation parallel to the tip apex in addition to the in-plane linear and radial polarisations. Polarisation dependent TERS is enabled by the large incidence angle from the high-NA objective lens and the use of devices that modify the polarisation state of the light such as a λ/2 waveplate [190]. The Raman scattered light is then collected in the low NA region through an apertured mask, which inhibits any residual laser light. As the tip apex approaches the sample within the focal spot, evanescent light is created at the tip’s apex [86]. Since the intensity distribution within laser focus is not uniform, it is very important to lock the relative position of laser focus to the tip [191, 192].

The strength and resolution of TERS depends on the ability of the tip to enhance and confine the light field at the tip’s apex, respectively. In STM systems, the tips are made of solid metal and the substrates need to be conductive in order to control the tunnelling current [193]. The STM tip resembles a long and smooth nanocone, with an apex diameter of ~ 20 nm. The length of the tip (~ tens of micrometres) makes them plasmonically unfavourable for visible light enhancement. However, the tunnelling gap between the tip and the sample can be tuned to the desired LSPR wavelength, creating a strong hotspot within the gap [143, 194, 195]. Some of the more advanced STM systems allow high-vacuum and low-temperature measurements [196]. As the substrate in STM needs to be conductive (often opaque in the visible wavelength range), the setup shown in Fig. 3d would not be suitable. Hence, a side illumination and side collection configuration is more common with STM-based TERS. To prevent the objective from mechanically interfering with the STM tip, a lens with a long working distance is required. It is therefore not trivial to tightly focus the incident light on the tip apex. A parabolic mirror can be used to mitigate mechanical interference and tightly focus the incident light to the tip apex as well as to collect the Raman signal [196, 197].

The spatial resolution in TERS is comparable to the size of the metallic nanostructure at the tip apex [86]. The gain in spatial resolution comes at a cost to overall signal enhancement (relative to SERS) due to the reduction of the Raman active volume.

In AFM systems, the tips are usually semiconductor cantilevers, with an apex diameter of ~ 5 nm. Figure 4 shows five examples of AFM-based TERS tips that have been demonstrated in the literature. The semiconductor tips are usually coated with metal either by thermal evaporation under high-vacuum [202] or electroless metal plating (mirror reaction) [203] techniques. Figure 4a shows an example of a smooth AFM TERS tip. As the substrate does not need to be conductive, AFM-based TERS can be performed in either bottom-up transmissive illumination (as in Fig. 3d) or in side/top reflective illumination configurations; the transmissive configuration in Fig. 3d is more common.

The surface of AFM tips becomes nanostructured during the coating process resembling aggregated nanoparticles on the semiconducting tip (Fig. 4b) [198]. These nanostructures are suitable for the resonant excitation of LSPR and SPPs. The smooth tip shown in Fig. 4a has been fabricated by subsequently depositing a thin granular layer of additional metal. Other researchers have tested AFM tips with a metallic nanoparticle attached to the tip apex (Fig. 4c) [198], or a segregation in the tips coating to form an antenna (Fig. 4d) by focused ion beam lithography [115]. Tips can also be created by electrochemical deposition [204].

For transparent dielectric substrates, a thin metal film (thin enough to be transparent) can be coated onto the substrate to further enhance the field in the tip-sample gap [205]. It is also possible to perform TERS in liquids with AFM-based systems, which is favourable for biological specimens which require liquid environments to function [206]. Performing TERS in liquid with STM systems is much more difficult [86, 106]. SFM-based TERS is also an attractive technique and maintains many of the properties of AFM-based TERS with the exception of the tip material which resembles similar TERS properties of STM-based TERS [86, 156, 207, 208].

Some TERS setups have demonstrated vastly improved signal-to-noise ratio in TERS by SPP nanofocusing [201, 209, 210]. This technique focuses the laser onto a plasmon-coupling nanostructure (in the form of a grating) on the upper area of the tip, usually at a distance of ~ 10 μm from the tip apex. Figure 4e shows a typical nanofocused SPP-based TERS setup (i), the process of SPP nanofocusing by coupling the incident light to a focused ion beam-fabricated grating (ii), and an example SEM image of a SPP-nanofocusing tip (iii). The excited plasmons then propagate toward the tip apex through the process of adiabatic compression and create a confined EM field at the tip apex [209].

Tuning the Plasmon Resonance

The size, shape, composition of the nanostructures and inter-nanostructure spacing all affect the wavelength of the surface plasmon resonance [85, 86]. Metals are most often used as the conducting medium for surface plasmons; however, semiconductors also possess plasmonic characteristics [125]. Gold shows strong enhancement factors in the red spectral region [111, 177, 211, 212], silver in the blue-green spectral region [132, 213] and aluminium in the UV and deep UV spectral regions [150, 151, 175]. The blue-green spectral region is the most commonly used Raman spectroscopy range. However, silver is prone to oxidation which degrades the plasmonic characteristics within a few hours of exposure to atmosphere. For this reason, silver is often mixed with other metals, such as titanium [214].

The range of plasmon resonance can be tuned by the thickness and choice of coating metal, e.g. tungsten, gold, silver or aluminium. In TERS, the grain size of the metal coating corrugations (Fig. 4b) is roughly comparable to the wavelength of the LSPR/SSP. Unlike STM tips, it is possible to control the LSPR/SPP wavelength by adjusting the size of the nanoparticles. The surface plasmon resonance wavelength is also dependent on the refractive index of the dielectric material. In AFM-based TERS, for example, the silicon cantilever tip can be heated to ~ 1000 °C in the presence of water vapour to oxidise the silicon into silicon dioxide [215]. As SiO₂ has a lower refractive index than Si, the surface plasmon resonance is blue shifted [86].

The size and shape of the metal-coated AFM tip apex can also be modified to tune the LSPR [199, 200]. Fabricating a single metallic nanoparticle attached to the tip’s apex (Fig. 4c) or segregated antenna-shaped tip (Fig. 4d) has been demonstrated as a means to finely tune the surface plasmon resonance in AFM-based TERS [199, 200, 208, 216]. However, the most commonly used tips for AFM-based TERS are the tips that have disconnected metal nanoparticles evaporated on a semiconductor cantilever in the standard coating process (Fig. 4b) described a priori [198].

Analysis Methods

A Note on Units

By convention, Raman spectra are considered in terms of the wavenumber ν∼ν∼ in units of cm⁻¹. The conversion from angular frequency is as follows:ν∼=ω2πc0,ν∼=ω2πc0,(12)

where c₀ is the speed of light in vacuum and ω is the angular frequency. Raman spectra are usually plotted in terms of the wavenumber shift from the incident excitation radiation. This shift is defined as follows:Δν∼=ν∼p−ν∼scat,Δν∼=ν∼p−ν∼scat,(13)

where ν∼pν∼p is the wavenumber of the pump beam with angular frequency ω_p and ν∼scatν∼scat is the wavenumber of the scattered light accordingly. For Stokes Raman scattering, ν∼scat=ν∼p−ν∼oscν∼scat=ν∼p−ν∼osc (where ν∼oscν∼osc is the molecule or lattice vibration wavenumber) and Δν∼Δν∼ is positive. By contrast, for anti-Stokes Raman scattering, ν∼scat=ν∼p+ν∼oscν∼scat=ν∼p+ν∼osc and Δν∼Δν∼ is negative [1].

Raman spectra are (by standard) presented with the wavenumber shift linearly increasing from right to left on the horizontal axis. The vertical axis ordinate is linear and proportional to intensity. However, researchers also present Raman spectra with wavenumber shift denoted simply as wavenumber and/or increasing from left to right instead of right to left [1].

Spontaneous Raman Spectra

Figure 5a shows the Rayleigh and the Raman spectrum of carbon tetrachloride (liquid) excited by an argon ion laser, ν∼1ν∼1 ~ 20,487 cm⁻¹ (487.99 nm). This spectrum is presented according to recommendations of the International Union of Pure and Applied Chemistry. It contains a strong band at ν∼1ν∼1 ~ 20,487 cm⁻¹ due to the Rayleigh scattering of the incident laser radiation and a number of weaker bands with wavenumbers, ν∼1±ν∼oscν∼1±ν∼osc: ν∼oscν∼osc = 218, 314, 459, 762 and 790 cm⁻¹. The Stokes Raman lines are shown on the left-hand side of the plot (Fig. 5a); the anti-Stokes Raman lines are shown on the right. The ν∼oscν∼osc values relate to the fundamental vibrations of the carbon tetrachloride molecule [1]. In the original work by Raman and Krishnan [220], the same spectrum was measured using mercury arc radiation (ν∼1ν∼1 = 22,938 cm⁻¹, 435.83 nm). In this seminal work, the anti-Stokes bands at ν∼1+762ν∼1+762 and ν∼1+790ν∼1+790 cm⁻¹ were not observed. Hence, after the invention of the laser, Rayleigh and Raman scattering experiments are preferably performed using monochromatically intense lasers.

Layered Two-Dimensional Systems

Raman spectroscopy can be used to determine the layer thickness in two-dimensional materials with atomic level precision, using either the inter-layer or intra-layer vibrational modes [19]. Lee et al. [12] demonstrated the technique with two intra-layer Raman modes of molybdenum disulphide (MoS₂). Figure 5b shows representative Raman spectra for single- and few-layer MoS₂ samples. Among the four Raman-active modes of bulk 2H phase MoS₂crystal (shown in Fig. 5b v), Lee et al. [12] only observed the E12gE2g1 and A_1g modes near Δν∼Δν∼ = 400 cm⁻¹. The authors surmised that the other modes were not observed either because of the selection rules for the scattering geometry (E_1g) [217] or because of the limited rejection of the Rayleigh scattering radiation (E22gE2g2) [13]. The authors [12] report that single-layer MoS₂exhibits a strong in-plane vibrational mode at Δν∼Δν∼ ~ 384 cm⁻¹, corresponding to the E12gE2g1mode of the bulk 2H-MoS₂ crystal. For all film thickness, the Raman spectra in Fig. 5b i show strong in-plane E12gE2g1 and out-of-plane A_1gvibration signals. As the sample thickness increases (Fig. 5b i and ii), the E12gE2g1 mode red shifts and the A_1g mode blue shifts. For films of four of more layers, the E12gE2g1 and A_1g modes converge on the bulk values. Spatial maps of a MoS₂ film sample for the E12gE2g1 mode is shown in Fig. 5b iii; that of the A_1g mode is shown in Fig. 5b iv. These maps demonstrate that the frequency of the two modes only slightly vary in regions of the sample with a given layer thickness. Hence, Raman spectra can provide a convenient and reliable means of determining the layer thickness in two-dimensional crystalline materials with atomic level precision.

Enhanced Raman Scattering Through SERS

Ault et al. [221] were the first to use SERS to enhance the Raman scattering signal of previously undetectable secondary organic aerosol particles on Ag nanoparticle-coated quartz substrates. Fu et al. [96] demonstrated enhancement factors of 6 for the Raman spectra of ammonium sulphate (AS) at the Raman active mode ν∼sν∼s(SO₄²⁻) at 970 cm⁻¹ with Klarite. Figure 5c shows a microscope image of a large AS particle on the surface of Klarite (Fig. 5c i), the corresponding Raman mapping image is in Fig. 5c ii. Figure 5c iii shows another sample of AS particle but on a silicon wafer. The corresponding Raman mapping image is shown in Fig. 5c iv. Aside from the three larger AS particles, small (sub-micron) AS particles are apparent in Fig. 5c iii. However, in the absence of SERS, these smaller particles are undetectable. On the other hand, the SERS Raman mapping image (Fig. 5c ii) shows a vastly enhanced signal intensity, as is evident from the scale bars, to the point where a number of small spots yield a signal at the ν∼sν∼s(SO₄²⁻) Raman mode. Such spots most likely correspond to small AS particles that are observable in Fig. 5c ii but are not apparent in Fig. 5c i.

Insights into Cellular Structure with CARS

CARS microscopy is relevant to the chemical [64, 222,223,224,225], materials [78, 226, 227], biological and medical fields [29, 36, 61, 63, 167, 228] and can provide unparalleled insights into cellular structures [53]. Spontaneous Raman and infrared micro/spectroscopy can provide adequate chemical specificity and sensitivity to delineate a variety of neoplasms [229,230,231,232,233,234,235,236,237] but require long integration times and have a coarse spatial resolution, which may limit accurate tumour-boundary identification and early-stage tumour detection. However, coherent Raman imaging techniques have demonstrated high-speed, high-spatial-resolution imaging, but with contrast limited to single or few Raman peaks [36, 53, 167, 232]. Figure 5d presents images of orthotopic xenograft brain tumours from within a murine brain [167]. Figure 5d i shows a broadband CARS image with nuclei in blue (Δν∼Δν∼ = 730 cm⁻¹), lipid content in red (Δν∼Δν∼ = 2850 cm⁻¹) and red blood cells in green (Δν∼Δν∼ = 1548 cm⁻¹ + 1565 cm⁻¹: C-C stretch from haemoglobin [238]). The large tumour mass and a projection of neoplastic cells within healthy tissue are clearly shown (Fig. 5d i). Figure 5ii shows several small regions of main tumour mass migrating into the healthy brain matter. Figure 5 iii shows the boundary between normal brain tissue, white matter and tumour masses, which contrasts lipids in red (Δν∼Δν∼ = 2850 cm⁻¹); CH₃ stretch-CH₂ stretch (Δν∼Δν∼ = 2944 − 2850 cm⁻¹), a general contrast; and nuclei in blue (Δν∼Δν∼ = 785 cm⁻¹). The image shows the fibrous texture of the white matter and strands of myelination around cancer cell clusters. Figure 5d iv presents a set of single-pixel spectra from an intra-tumoural nucleus, the white matter and normal brain, respectively. The spectra indicate that lipids are most concentrated in the white matter and least in the tumour regions.

Raman Thermography

Advances in electronic and opto-electronic semiconductor devices, such as high electron mobility transistors (HEMTs), have led to thermal management challenges [65]. Conventional thermal characterisation approaches such as infrared thermography are often no longer applicable for the accurate characterisation of high-power density devices due to limited spatial resolution which can result in the underestimation of the device peak temperature [239]. Batten et al. [218] have demonstrated temperature profiling in AlGaN/GaN HEMTs using Raman thermography by exploiting the E₂ and A₁ (LO) phonon modes. Both the E₂ and A₁ (LO) modes shift to lower frequency when operating the device. Figure 5e shows a comparison of the temperature rise in a AlGaN/GaN HEMT on a SiC substrate from Raman thermography and thermal simulations. The device was operated at a source-drain voltage of 40 V and a power density of 25 W/mm and had a thermal resistance of 8 °C/(W/mm).

Measuring Strain on the Nanoscale Using TERS

TERS microscopy is an effective means of imaging nanostructures beyond the spatial resolution of the so-called light diffraction limit [152,153,154, 219]. Nanostructures such as DNA molecules [240], carbon nanotubes (CNTs) [241, 242], silicon devices [101, 243], dye molecules [244] and single molecules [83] can be imaged using TERS. The technique can even be used to measure the local molecular strain in nanostructured materials. For example, AFM can be used to manipulate CNTs with nanoscale precision to develop a local strain [245,246,247,248]. Figure 5f (left) illustrates the process of CNT manipulation using contact-mode AFM. Although local strain in CNTs has previously been studied using AFM and transmission electron microscopy [245], TERS microscopy is the only optical technique that can provide images of such local structural distribution of nanomaterials. When a straight CNT is deformed by manipulation, a local breakdown in symmetry is induced. This causes the selections rules of Raman scattering to become relaxed, allowing forbidden Raman modes to become visible in the vicinity of the local curvature [219]. The position of the characteristic G-mode Raman scattering line in graphene can be used to deduce local strain using TERS [249]. Figure 5f (right) shows a TERS image of a deformed CNT which has been constructed from the peak positions of the G⁺-mode [219]. The image has a spatial resolution better than 20 nm which is about 25 times finer than the diffraction limit of the excitation wavelength of light (488 nm). The colour variation (as indicated by the scale bar) corresponds to the local peak position of the G⁺-mode and represents the variation of strain along the CNT.

Recent Results

Stimulated Raman Scattering Microscopy

Unlike CARS, SRS microscopy does not contain a nonresonant background signal that degrades image contrast. However, SRS can be affected by cross-phase modulation (where light at one wavelength modulates the refractive index in the medium affecting another wavelength of light), transient-absorption (which is characteristic of femtosecond light pulses) and photo-thermal effects which can modify the vibrational energy levels and reduce hyperspectral image contrast [250,251,252]. SRS is quantified by the amount of energy transfer from the pump light to the Stokes light when the difference frequency between the pump and Stokes light matches a specific vibrational frequency, ω_osc. In addition, the resulting signal from SRS is strongly sensitive to the incident polarisations when the orientation of the probed vibrating species is ordered. This polarisation dependence can be exploited to probe the orientational order of vibrational modes in samples. However, currently developed techniques are not able to perform large-field fast time scale dynamics instantaneously due to the requirement of point-wise scanning over the sample space. Conventional polarisation-resolved techniques take minutes because each point of the scanning area must be polarisation tuned sequentially [74, 80, 253, 254].

Multi-lamellar myelin plays a crucial role for efficient transmission of nerve impulses as an electrical insulator [255]. The lipids and proteins in myelin self-assemble into a highly ordered and stable structure to form a tightly packed membrane [256]. In neurological disorders, this compact structure is highly perturbed leading to dysfunctions of the central nervous system [257, 258]. As these biological processes are highly dynamic, researchers seek to observe the dynamics of molecular order with sufficient resolution and frame rate. Hofer et al. [259] have recently demonstrated fast-polar-SRS by exploiting high-speed amplitude- and polarisation modulation with an acousto-optic modulator (AOM) and electro-optical polarisation modulation, respectively, to read out the molecular order and orientation at a fast rate. They therefore obtain both amplitude and phase information. The authors report the ability to retrieve density maps of molecular bonds with the absolute value of molecular order. The linear polarisation direction of the pump beam is rapidly rotated while the Stokes polarisation is circularly polarised to avoid polarisation dependence from the Stokes beam. The polarisation is further modified by a quarter-wave plate. The polarisation modulation leads to an α-dependant response of the signal intensity given by the following:I(α)∝a0+S2cos2(α−φ2)I(α)∝a0+S2cos⁡2(α−φ2)(14)

where α is the rotating pump polarisation direction in the sample plane, a₀ is the total measured intensity, and S₂and φ₂ are the amplitude and phase of the second-order induced modulation [259].

Figure 6a.i shows a comparison of conventional polarisation SRS with that from Hofer’s fast-polarisation SRS on a multi-lamellar lipid vesicle (MLV). The fast-polarisation SRS image in the bottom of Fig. 6a i was obtained in 1 s which is two orders of magnitude faster than the conventional-SRS image (top) using the same incident powers, number of pixels and dwell time per pixel. Figure 6a ii shows sub-second frame-rate imaging of a MLV using double EOM-AOM modulation SRS at two instances in time. The measurement technique was remarked to have little effect on the lipid order properties during the measurement. Hofer et al.[259] were able to observe second-timescale dynamics in thin lipid membranes down to the cell plasma membrane using fast-polarisation-resolved SRS as shown in Fig. 6a iii.

Flow cytometry (FC) is one of the most important technologies for high-throughput single-cell analysis. FC is a technique used to measure physical/chemical characteristics of a population of cells or particles suspended in a fluid [59, 261]. The fluid suspension flows through the instrument detectors for fluorescent labelling which is the primary approach for cellular analysis in FC. Figure 6b i shows an optical FC setup [260]. However, for small molecules, the fluorescent tags can perturb the biological function of the species. In addition, non-specific binding of fluorescent labels as well as cellular autofluorescence can also reduce the clarity of the result. SRS flow cytometry (SRS-FC) non-invasively detects chemical cell content but conventional techniques suffer slow acquisition rates.

Zhang et al. [260] have recently demonstrated label-free high-throughput single-particle SRS-FC with a 32-channel multiplexing technique. Their technique measured single-particle chemistry at a rate of 5 μs per SRS spectrum, approaching that of standard fluorescence-based FC. The SRS-FC technique was based on broadband laser excitation and a multiplex spectral detection system. The systems allowed the acquisition of 200,000 spectra per second, more than 11,000 particles per second. The subpopulations of species, such as mixed polymer beads and 3T3-L1 cells, could be separated and distinguished through compositional principle component analysis (CPCA) of the SRS signals. The principle components were designated according to their Raman spectra. An agglomerative clustering procedure was performed on the resulting CPCA spectral matrix. This procedure assumed the number of cluster groups (κ) was known to separate the clusters of principle components in the CPCA analysis. Figure 6b ii shows the CPCA of the SRS spectra for a mixture of three types of beads: poly-methyl-methacrylate (PMMA), polystyrene (PS) and polycaprolactone (PCL), all with a 10-μm mean diameter, mixed at a 2:1:1 ratio of PMMA:PS:PCL and a final concentration of 2% solids in the fluid. The flow speed was ≈ 0.16 ms⁻¹, the SRS-FC data was acquired in 6 s. The CPCA plot (Fig. 6b ii) shows three distinct clusters of principle components. The agglomerative clustering procedure (κ = 3) allowed the quantification of ~ 7100 PMMA bead (red), ~ 3400 PS beads (blue) and ~ 3600 PCL beads (green) as shown in Fig. 6b ii. Their measurement demonstrated the ratio of ≈ 2:1:1 (PMMA:PS:PCL) at a throughput rate of ~ 2350 particles per second and that their multiplex SRS-FC system, paired with the CPCA analysis, could distinguish different chemical components with small spectral differences. Zhang et al. [260] were able to detect beads as small as 1 μm and were even able to detect single Staphylococcus aureus bacteria flowing through the laser focus highlighting the potential to characterise subcellular organelles with SRS-FC.

Zhang et al. [260] also demonstrated the discrimination of 3T3-L1 cells at different stages of cell differentiation according to their difference in lipid amount using SRS-FC. After insulin-induced differentiation, 3T3-L1 cells acquire an adipocyte-like phenotype with a significantly increased amount of triglycerides which aggregate to form large lipid droplets. This aggregation of triglycerides causes the intensity of the methylene symmetric vibration at Δν∼Δν∼= 2850 cm⁻¹ from fatty-acid acyl chains to become stronger compared to that of non-differentiated cells which provides the means for CPCA analysis. Figure 6b iii shows the CPCA scatter plot of the cell mixture measured by Zhang et al. [260] which were separated using the agglomerative clustering approach. The insert SRS images (Fig. 6b iii, right) show a non-differentiated 3T3-L1 cell and a differentiated cell with the formation of large lipid droplets.

Twisted Laguerre-Gaussian lasers, with orbital angular momentum (OAM) and characterised by doughnut-shaped intensity profiles, are of great interest to a number of growing research fields such as ultra-cold atoms [262,263,264], microscopy and imaging [265, 266], atomic and nanoparticle manipulation [267, 268], ultra-fast optical communication [269, 270], quantum computing [271], astrophysics [272] and plasma accelerators [47]. Spiral phase plates or computer-generated holograms are usually used to generate visible light with OAM [273,274,275,276]. Spiral phase plates can produce light with predefined OAM modes. By using plasma as an optical medium to generate and amplify laser pulses with OAM and relativistic intensities, well above the damage threshold of optical devices, could provide for high-energy-density science and applications. Plasmas also allow for greater flexibility in the level of OAM in the output laser beam than conventional optics. Vieira et al. [46] have shown that SRS in nonlinear optical media with Kerr nonlinearity (e.g. plasmas, optical fibres and nonlinear optical crystals) can be used to generate and amplify OAM light. The authors show that it is possible to generate and amplify light with OAM when no net OAM is initially present. Figure 6a i illustrates the process in which the pump EM fields can have different OAM components in both transverse directions x and y (blue and orange in Fig. 6a i). l_0x is the pump electric field component of OAM in the x direction. Likewise, l_0y is the pump electric field component of OAM in the y direction. The initial seed electric field component has an OAM component l_1x. After interacting with the plasma, the pump is depleted, and a new electric field component appears in the seed with OAM l_1y = l_1x + l_0x − l_0y.

The authors [46] use an analytical theory for arbitrary transverse laser field envelope profiles and particle-in-cell simulations for plasma. Stimulated Raman backscattering in plasma is a three-wave mode coupling process in which a pump pulse decays into an electrostatic (Langmuir) plasma wave as well as a counter-propagating seed laser. The plasma can be viewed as a high-intensity mode converter. The presence of OAM in the pump and/or seed results in additional matching conditions that ensure the conservation of angular momentum of the pump when the pump decays into a scattered electro-magnetic wave and a Langmuir wave [40].

Particular superpositions of Hermite-Gaussian modes TEM modes are mathematically equivalent to Laguerre-Gaussian modes [277]. Vieira et al. [46] therefore explore the use of Stimulated Raman backscattering to generate and amplify light with OAM using TEM laser beams with no initial net OAM. Each Hermite-Gaussian beam in the simulation is described by TEM_m,n, where m and n represent the Hermite-Gaussian mode index. Figure 6c ii and iii show the 3D simulation results from the setup shown in Fig. 6c i. The simulations show that SRS results in a new OAM mode with l₁ = 1 linearly polarised at 45°. The field topology of the seed normalised vector potential changes from plane isosurfaces in Fig. 6c ii, to helical isosurfaces in (Fig. 6c iii). Hence, light with OAM has been generated from light with no net OAM. The authors [46] note that their results could be extended to other nonlinear optical media with Kerr nonlinearity. In the case of plasma, the interaction between the seed light and the pump light occurs via an electron Langmuir wave. This interaction ensures that the frequency, wavenumber and OAM matching conditions are conserved.

Coherent Anti-Stokes Raman Scattering Microscopy

CARS results from an induced anti-Stokes scattering of radiation, ω_AS, which is enhanced when ω_p − ω_S = ω_OSC. One of the main challenges with CARS microscopy is the nonresonant background. The existence of the nonresonant background can either distort or even saturate the resonant signal of Raman peaks, which reduces the image contrast. Qin et al. [278] have recently demonstrated multi-colour background-free coherent anti-Stokes Raman scattering microscopy using an all-fibre, low-cost, multi-wavelength time lens source. A time lens, in analogy to a spatial lens, is simply a quadratic optical phase modulator in time, which can be approximated by a portion of a sinusoidal phase modulator [279,280,281]. Three different wavelength picosecond pulse trains were provided by the time lens source, at 1064.3 nm (stable), 1052–1055 nm (tuneable) and 1040–1050 nm (tuneable). The time lens was used to apply temporal quadratic phase modulation to a continuous-wave laser to broaden its spectral bandwidth [279, 282,283,284,285,286,287,288]. In this instance, the time lens was applied with fibre-integrated electro-optic radio-frequency phase modulators. The phase modulation and pulse synchronisation were derived from a mode-locked Ti:Sapphire laser that provided synchronised multi-colour picosecond pulses with dispersion compensation. Electronic tuning of the pulse delay was used to achieve temporal overlap between the pump and Stokes laser pulse trains, which is a convenient substitution for mechanical optical delay paths. Two of the three wavelengths of light from the time lens source were used for two-colour on-resonance imaging and the third wavelength for off-resonance (nonresonant background subtraction) imaging. Pixel-to-pixel wavelength switching was achieved, which provided simultaneous two-colour CARS imaging with real-time nonresonant background subtraction. Qin et al. [278] demonstrated the technique with an excised fresh tissue sample from a mouse ear and imaged molecular stretching vibrations at 2845 cm⁻¹ (CH₂) and 2940 cm⁻¹ (CH₃) and non-resonance background at Δν∼Δν∼ = 2770 cm⁻¹. Figure 7a i–iii shows the process applied to the Raman peak of CH₃ stretching vibration from the mouse ear tissue sample.

The nonresonant background signal in CARS can also be suppressed by applying an external static electric field to the sample known as electro-CARS. Capitaine et al. [120] demonstrate this electro-optical technique on n-alkanes in solution with broadband multiplex coherent anti-Stokes Raman scattering spectroscopy. The nonresonant background is suppressed due to the orientation response of the molecules to the electric field. The molecular orientation is related to the induced electric dipole moment. The enhancement of the CARS signal-to-noise ratio was achieved in the case of the CH₂ and CH₃ symmetric/asymmetric stretching vibrational modes.

Conventional CARS provides information about the chemical nature but not about the molecular organisation or symmetry in the system. The Cartesian components of the nonlinear susceptibility tensor χ⁽³⁾ represent the vibrational symmetry properties of the material [290, 291]. These tensor elements can be extracted with polarisation-resolved coherent Raman scattering schemes [72, 74, 169, 259, 292,293,294,295]. However, these schemes often involve the acquisition of multiple images from different polarisation angles requiring long acquisition times due to limits imposed by polarisation tuning [259] and time-consuming post-processing [67].

Cleff et al. [289] have recently demonstrated a label-free microscopy technique that uses circularly polarised light to probe the symmetry as well as the chemical fingerprint of the probed sample in a single acquisition. This symmetry-resolved CARS (SR-CARS) depends on both the presence of (ro-)vibrational modes as well as their local organisation. By switching between combinations of left- and right-handed circular polarisation states for the involved fields, the individual symmetry contributions of the sample can be imaged. This technique offers a straightforward means to access the local organisation of (ro-)vibrational bonds with improved image contrasts (with 1 to 2 orders of magnitude) for anisotropic samples, as well as improved chemical selectivity without post-processing and independently of sample orientation in the transverse plane. In addition, SR-CARS provides higher chemical selectivity with the contrast in symmetry characteristics, which are not accessible with conventional spontaneous Raman or SRS microscopy.

Multi-lamellar lipid vesicles (MLVs) are made of a tight packing of lipid layers forming a ring of highly ordered matter with twofold symmetry and a lipid orientation distribution close to a Gaussian angular shape [74].

Figure 7b i shows a conventional CARS image of an aqueous MLV at Δν∼Δν∼ = 1133 cm⁻¹ (C-C stretching vibration) which illustrates the expected poor contrast due to the nonresonant background [289]. Figure 7b ii and iii show the zeroth and second-order mF¯¯¯¯mF¯-value image of the same MLV as in Fig. 7b ii. The mF¯¯¯¯mF¯-value is the summation of the light circular polarisation handedness quantum numbers of the incident light beams:mF¯¯¯¯=mp−ms+mpr−masmF¯=mp−ms+mpr−mas(15)

When light with field tensor F¯¯¯¯F¯ probes matter with nonlinear susceptibility tensor χ⁽³⁾, in a CARS process, the light probes only the parts of the matter with identical rotational invariant symmetries (i.e. identical mF¯¯¯¯mF¯). Hence, by engineering the field tensor of the light, specific sample symmetries can be directly read out, creating a symmetry-based image contrast mechanism [289]. The aqueous solution surrounding the MLV is only visible in the mF¯¯¯¯=0mF¯=0image (Fig. 7b ii) due to its purely isotropic nature. Background-free imaging of the MLV with superior contrast with respect to conventional CARS is shown in Fig. 7b iii at mF¯¯¯¯=2mF¯=2, which results from the symmetric microscopic organisation of the lipids in the MLV. Imaging at mF¯¯¯¯=4mF¯=4 (not shown) lacked sufficient signal strength to provide an image of the MLV due to the lack of anti-symmetry in the lipid organisation.

As with SRS, the CARS signal is sensitive to the polarisation of the incident light when the orientation of the scattering species is ordered. Polarisation-resolved CARS (PR-CARS) requires monitoring of the CARS signal response depending on the relative rotation of the incident light polarisations (pump and Stokes) to the sample, in species with ordered orientations. Provided that the molecular bonds are oriented, the detected intensity of the anti-Stokes signal is maximised when the incident polarisations lie along the averaged direction of the bonds. The ability to monitor lipid order without the need for fluorescent labels can provide information on lipid packing properties. As mentioned, PR-CARS schemes often involve long acquisition times due to limits imposed by polarisation tuning and time-consuming post-processing.

In addition to fast-polar-SRS, Hofer et al. [259] have recently demonstrated fast-polar-CARS imaging with combined electro-optic polarisation and acousto-optic amplitude modulations. Figure 7c shows fast-polarisation CARS with similar sensitivity to that of SRS (shown in Fig. 6a). Despite the requirement of lock-in amplification for the detection of low modulation over a large nonresonant background, the fast-polarisation technique demonstrated by Hofer et al. [259] can considerably improve the signal-to-noise ratio in CARS imaging. Despite the robustness of MLVs, occasional alteration of molecular order in MLVs could be observed at the time scale accessible in Hofer’s experiment (0.25–1 s per image). MLVs could detach from the sample surface, inducing motion (Fig. 7c) or shape change. The modifications observed in Fig. 7c were attributed to a local membrane disruption, followed by its spontaneous reformation. Hofer et al. [259] demonstrated the possibility of visualising local modification during MLV displacement that was not accessible using the minute-time-scale conventional polarisation Raman experiments.

There have also been a number of developments in CARS flow cytometry (CARS-FC) [58, 296, 297]. However, these techniques were shown to be much slower than fluorescence-based FC. Out-of-focus microparticles can randomly impede CARS-FC and the fluid often generates a strong nonresonant background limiting CARS-FC from achieving high-throughput single-cell analysis. Recently, however, O’Dwyer et al. have demonstrated that it is possible to significantly enhance the fraction of unambiguously and instantly recognised in-focus microparticles, in unconstrained flows by co-monitoring CARS-FC with linear scattering of light.

CARS is invariably performed with two synchronised picosecond laser sources owing to the coherence life time of Raman resonance. Ti:Sapphire oscillators [168] or optical parametric oscillators pumped by a picosecond frequency-doubled Nd:Vanadate laser [53] are the instruments of choice, which are generally very expensive and the synchronisation mechanisms can be challenging. In addition, the spectral drift in the pump wavelength can introduce errors in the calculation of ω_osc. Langbein et al. [124] have demonstrated CARS micro-spectroscopy using a single Ti:Sapphire laser oscillator and simple passive optical elements. Vibrational excitation, tuneable over a large spectral range with adjustable spectral resolution, was achieved by spectral selection with dichroic mirrors and linear chirping by glass elements.

Tip-Enhanced Dual Wavelength Coherent Anti-Stokes Raman Scattering Microscopy

TERS offers spatial resolution far beyond the diffraction limit of the probing light. The more conventional technique is to directly illuminate the tip-sample cavity [86]. This technique achieves the desired resolution (beyond the diffraction limit) by forcing the evanescent light into the far field image formation. However, the far field light presence in the tip-sample cavity generates an unfavourable background light source. It is possible to perform TERS by coupling the far-field excitation light to the tip a few tens of micrometres from the tip apex [298]. Femtosecond laser pulses can be coupled to the tip surface by shining the light on a grating fabricated on the tip surface. The SPPs then propagate to the tip apex and generate background-free localised optical excitation [210].

Toma et al. [298] previously demonstrated selective excitation of a single Raman mode and its CARS imaging of CNT using ultra-fast SPP pulse nanofocusing using an Au tapered tip. In a more recent publication, seminal work by Tomita et al. [155] demonstrated simultaneous nanofocusing of ultra-fast SPP pulses at 440 and 800 nm, which were coupled with a common diffraction grating structure. Figure 8a i, illustrates the scheme. The Al-tapered tip had an apex radius of ≈ 35 nm. Selective CARS microscopy that combined an 800 nm (ω) SPP pump pulse and a 440-nm (2ω) SPP probe pulse was achieved. Figure 8a ii illustrates the energy level process of ω– and 2ω-CARS. The pump pulse achieves selective vibrational excitation by spectral focusing [299,300,301,302]. Raman shift intensities with this 2ω-CARS scheme were reported to increase by as much as 4 compared with that of ω-CARS for monolayer graphene. The selectivity of vibration band excitation and background noise suppression were confirmed on the CARS intensity probed by a 2ω-SPP plasmon pulse for a monolayer graphene sample. Venezuela et al. [8] reported the Raman lines in graphene associated with both phonon-defect processes (such as the D line at Δν∼Δν∼ ~ 1350 cm⁻¹) and two-photon processes (such as the 2D line). The 2D-band intensity in graphene was reduced monotonously when the defect concentration was increased, contrary to the D-band. Tomita et al. [155] applied their multi-vibrational-mode 2ω-CARS imaging method to a multi-walled CNT (MWCNT) at the D, G and 2D bands. This dual-wavelength nanofocusing technique could open new nanoscale micro-spectroscopy and optical excitation schemes in SPM, such as sum frequency mixing, two-photon excitation (ω+2ω) and pump-probe schemes.

Figure 8b i shows a topography image of a MWCNT with a diameter of ~ 175 nm measured by the Al-tapered tip. Figure 8b ii shows a composite image of three 2ω-CARS images of the MWCNT using the 2ω-CARS spectrum from D- (red), G- (blue), and 2D- (green) bands. In ref. [155], the D- and 2D-band showed a negative correlation (in agreement with ref. [8]) except for the central part of the MWCNT. The 2D- and G-bands were intense near the central part of the MWCNT. Tomita et al. [155] indirectly estimated the spatial resolution of their technique to be less than 90 nm by taking the profile of the 2D-band signal across the axis of the MWCNT.

Conclusions

This review detailed the numerous applications of Raman spectroscopy and its advanced derivatives: stimulated Raman scattering, coherent anti-Stokes Raman scattering and surface- and tip-enhanced Raman spectroscopy. A description of the fundamental physics that underpins these techniques has been provided. Experimental considerations have been discussed with examples of typical instrumentation used. Examples of the analysis techniques employed to interpret the Raman spectroscopic data were presented and discussed.

The Raman effect now underpins prominent spectroscopic techniques in biology, medicine, crystallography and flow cytometry and has gained interest in plasma physics. It is employed as a non-invasive label-free chemically selective hyperspectral imaging technique with recent advances enabling the probing of molecular orientation and chemical composition. SRS and CARS are used to enrich signal detection at specified wavelengths associated with vibrational modes that are prescribed for spectral-selective imaging. Unlike SRS, CARS carries with it a nonresonant background contribution to the spectrum. This review detailed some of the efforts to suppress this unfavourable contribution.

Surface-enhanced Raman scattering is an ultrasensitive Raman technique that has enabled the detection of trace amounts of molecular species in samples that would otherwise be undetectable in spontaneous- or coherent Raman scattering techniques. The enhancement effect is largely associated with the plasmonic activity of the sample surface, which augments the light-matter interaction. This enhancement effect is optimised by tuning the plasmons associated wavelength with plasmonically active surface nanostructures.

Tip-enhanced Raman scattering spectroscopy is a relatively new technique that can capture hyperspectral images with spatial resolution beyond the diffraction limit of light. As light is fundamental to the Raman effect, the spatial resolution offered by TERS is so far unparalleled by other Raman scattering techniques. The surface plasmon wavelength can also be tuned for TERS techniques and recent advances have exploited surface plasmon polaritons to focus evanescent light at the tip apex with light coupled remotely from the tip apex. This technique has yielded enhanced the signal-to-noise ratio by removing the far-field light from the apex region. Recent advances have demonstrated this technique with dual-wavelength CARS.

Abbreviations

AFM:

Atomic force microscopyAOM:

Acousto-optic modulatorAS:

Ammonium sulphateCARS:

Coherent anti-Stokes Raman scatteringCARS-FC:

CARS flow cytometryCNT:

Carbon nanotubeCPCA:

Compositional principle component analysisCRS:

Coherent Raman scatteringDBR:

Distributed Bragg reflectionDFB:

Distributed feedbackE-CARS:

Epi-scattered CARSEM:

Electro-magneticFC:

Flow cytometryF-CARS:

Forward scattered CARSHEMTs:

High electron mobility transistorsLSPR:

Local surface plasmon resonanceMLV:

Multi-lamellar lipid vesicleMoS₂ :

Molybdenum disulphideMWCNT:

Multi-walled carbon nanotubeNA:

Numerical apertureOAM:

Orbital angular momentumPCL:

PolycaprolactonePMMA:

Poly-methyl-methacrylatePR-CARS:

Polarisation-resolved CARSPS:

PolystyreneSERS:

Surface-enhanced Raman scatteringSFM:

Shear force microscopySLM:

Single-longitudinal modeSPM:

Scanning probe microscopySPP:

Surface plasmon polaritonSR-CARS:

Symmetry-resolved CARSSRS:

Stimulated Raman scatteringSTM:

Scanning tunneling microscopyTEM:

Transverse electro-magnetic modeTERS:

Tip-enhanced Raman scatteringVBG:

Volume Bragg-grating