Raman Spectroscopy and Microscopy

Raman Spectroscopy and Microscopy$ R Krishna, TJ Unsworth, and R Edge, The University of Manchester, Manchester, England, UK r 2016 Elsevier Inc. All rights reserved.

Introduction In 1928, physicists C. V. Raman and K. S. Krishnan first observed the classical phenomenon of inelastic scattering of monochromatic light. A shift in energy/wavelength of lights scattered by molecules from the incident light, commonly known as the ‘Raman effect’ corresponds to Raman scattering (Raman and Krishnan, 1928). The shift in wavelength depends upon structure and vibrational properties of the molecules responsible for scattering and is the basis of vibrational spectroscopic techniques. The wavelength shift is also known as ‘Raman shift’ and is also reported in relative wavenumbers (cm
1). The magnitude of the shift in energy/wavelength is independent of the excitation frequency and describes it as an intrinsic property of a molecule (Berciaud et al., 2009). Raman spectroscopy employs changes in energies of scattered monochromatic radiations to acquire the information regarding molecular vibrations for elucidating structure, composition, co-ordination, and bonding of sample molecules, thus providing qualitative and quantitative analyses of sample materials (Colthup et al., 1990). The most important vibrational spectroscopy includes Raman and infrared (IR) spectroscopy. Both techniques are complementary, since transitions allowed in Raman may be forbidden in IR or vice-versa and have a broad range of applications to provide characteristic fundamental vibrations that are used for elucidating structural and chemical components of a compound. Usually, both techniques are required to measure the two modes of energy including vibrational and vibrational-rotational. Some vibrational modes are both Raman and IR active and arise from different selection rules. In general, Raman spectroscopy is best at symmetric vibrations of nonpolar groups and is the topic of this article while IR spectroscopy is best at asymmetric vibrations of polar groups. On the other hand, an IR-active mode is one in which a particular vibration causes a change in the dipole moment of the molecule, while only those vibrations which change the molecular polarizability lead to Raman scattering. Therefore the activity of a certain vibrational mode depends highly on its symmetry and the symmetry of the molecule, and is the basic requirement for obtaining Raman spectra. Functional groups such as
C¼ C
,
CC
,
CN,
C
NO2,
C
S
,
S
S
,
C
X (X ¼ F, Cl, Br, or I) exhibit strong polarizability changes and therefore, evidence strong Raman signals (Dijkstra et al., 2005). Thus, with the revolutionary development in instrumentation, Raman spectroscope is now a powerful analytical tool applied in characterization and evaluation of vast range of materials for variety of scientific field applications. As a valuable technique for quantitative and qualitative measurements, it can include, but is not limited to, investigation of structural components, mechanical stresses, phase identification and composition, crystallographic orientation, chemical doping in 2D materials, amorphization/crystallization processes in covalent materials, organic/inorganic thin films for electronic devices, etc. In this technique, Raman spectral lines are obtained by illuminating a sample with a powerful laser source of infrared or visible monochromatic light source. A hybrid technique between Raman spectroscopy and microscopy is known as Raman microscopy and this technique is used to records Raman maps, using point mapping method, to represent spatial distribution of different chemical components/information within the sample. In Raman microscopy, a microscope objective is used to focus the incident light on the sample, exposing a small region in the sample. This allows investigation of the minor yet important changes in the microstructure with regards to composition, orientation, crystallinity, inclusions, and stress. The addition of a slit or pinhole aperture before the dispersive element increases the confocality of the system, by decreasing stray light. This article describes the techniques and various application related to Raman spectroscopy and microscopy in a wide range of material system and different field of science to better utilize the potential capability of this instrument.

Basic Principles and Instrumentation The elastic and inelastic scatterings are two important scattering processes that always happen when a monochromatic light impinges on a molecule. The elastic scattering occurs with no change in photon energy; whereas the inelastic scattering occurs with change in photon energy. Thus, categorically three types of scattering phenomena can be presented (shown in Figure 1; Colthup et al., 1990). First, if the light scattered has same wavelength and frequency, as the incident photon, the process is elastic in nature since the energy loss is negligible and such phenomenon is known as Rayleigh scattering. A small fraction of photons scatter inelastically due to a shift in frequency and hence energy, from the frequency of incident photons by the amount of vibrational energy that is lost or gained by the molecule, known as Raman scattering. Second, if the molecule gains vibrational energy from the ☆

Change History: August 2015. R. Krishna, T.J. Unsworth, and R. Edge updated and submitted the article.

Reference Module in Materials Science and Materials Engineering

doi:10.1016/B978-0-12-803581-8.03091-5

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Figure 1 (a) Scattering mechanism (b) spectrum shows Raman (stokes and anti-stokes) and Rayleigh scattering components (c) energy transitions for Rayleigh and Raman scattering.

incident photons, the scattering is called Stokes Raman scattering and the energy of scattered photons will be lower than the incident photons. Third, if the molecule loses vibrational energy to the incident photons, the scattering is called anti-Stokes Raman scattering and the energy of scattered photons will be higher than the incident photons. Stokes Raman scattering has a higher probability of occurrence than anti-stokes Raman scattering and is therefore most often used in molecular vibrational studies (Colthup et al., 1990). Raman scattering is a fundamental component of Raman spectroscopic techniques and is used to obtain useful information about the structure and properties of molecules from their vibrational frequencies. As is well known, Raman scattering is a twophoton event and the process involved is the change in polarizability of a molecule with respect to its vibrational motion. The interaction of photons from the incident laser and the polarizability in the molecules creates induced dipole moments and the scattered light from such molecules provides unique information for frequency and vibration about the molecule or molecular composite in a matter. This makes Raman spectroscopy a powerful experimental tool for analytical study to diagnose the vibrational states of molecules in an ensemble. A Raman spectroscope consists of four important components: a laser excitation source, a fiber-optic Raman probe, an imaging spectrograph, and a charge-coupled device (CCD) detector. A sample is illuminated with a laser beam in the visible (Vis) or near infrared (NIR) range. The scattered light is received by a lens and analyzed through a spectrophotometer to obtain a Raman spectrum from a sample. Raman signal is very weak and there are different ways implemented to the technique for enhancing the Raman collection such as advances in optics, spectrophotometer, etc. On the other hand, development of laser technology, sensitive detectors in conjunction with optical fibers, robust spectrometers and automatic fluorescence removal enhanced the overall capabilities of spectrometer system. Different types of lasers are available commercially for use in Raman spectrometer as an excitation source like argon ion, krypton ion, He:Ne, Nd:YAG, and diode lasers operating at different excitation wavelengths. Further, this technique has unique advantages of simplicity in its usage, and fast and non-destructive way of analytical analyses. Sample inhomogeneity can be tackled by taking a series of measurements at different points on the surface to ensure representative data and a choice of excitation wavelengths can be used when samples tend to fluoresce or heat. In order to minimize fluorescence, the use of long wavelength excitation preferably in the near infrared region is desirable. Raman spectroscopy also offers adequate spectral resolution in the range of 3–9 cm
1 and provides excellent spatial resolution in the order of 0.5–1 mm. The spectral differences between two point measurements are directly correlated to the resolving power of the two points, which influences the spatial resolution (Lee and Whitely, 2007). A superior spatial resolution allows exploration of micrometer scale variations of chemical information within the specimen. The use of shorter (visible range) wavelength lasers is also an advantage, illuminating the sample with higher intensity light, producing a larger, and more powerful Raman signal in a small amount of time.

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Advanced Raman Techniques Confocal Raman Microscopy Confocal Raman microscopy integrates a Raman spectrometer and a standard optical microscope, in which an excitation laser from the probe-head is made and focused on the sample through the microscope objective (Figure 2). This instrument allows us to visualize a sample under a probe at a range of magnifications and carry out Raman analysis at microscopic spots. The important attribute of a confocal Raman microscope is a spatial filter (a pinhole aperture), which provides the instrumental ability to analyze controlled sampling volumes of micrometer size, individual particles, or few micrometer thick layers. The spatial resolution of the instrument is dependent upon the laser source, wavelength, and the type of microscope objective selected for analyses. This technique is an extremely powerful analytical method that possesses a number of applications in a variety of scientific fields by providing three dimensional spatial measurements for the analysis of samples, in the XY (lateral) and Z (depth) axes. A confocal Raman microscope can be applied to a variety of scientific areas such as mineralogy and petrography, carbon nanotubes and graphene technology, biological applications for cells, chromosomes and tissues studies, pharmaceutical development, polymeric and ceramic materials for high resolution imaging, rapid identification of samples, molecular compositions, stress determination, etc. As a non-invasive method it can be used to study the molecular level fingerprint of the biochemical composition and structure of cells with excellent spatial resolution (Bonnier and Byrne, 2012; Bonnier et al., 2010; Chen et al., 2014). In addition to this, a long-working-distance confocal Raman spectroscopy system may also offer a novel technique for the non-contact spatially resolved biochemical characterization of various tissue layers of the anterior segment of the eye (Jongsma et al., 1997). The applicability of this instrument in high-resolution imaging of calcium phosphate bone implant coatings has been demonstrated to get spectral information from the tissues and coating materials.

Surface-Enhanced Raman Spectroscopy (SERS) SERS is a sensitive Raman spectroscopic technique that provides enhanced Raman signal from Raman active molecules that have been adsorbed on certain metallic surfaces. This suggests that concentrations as low as pico- and femto-molar of Raman active analytes could be detected (Garrell, 1989; Kneipp et al., 1999). The importance of SERS is that it is surface selective and highly sensitive and has a broad wavenumber range, which was not available in the earlier Raman instruments. In SERS one can observe a large increase of the Raman cross-section of an analyte, which is approximately up to 15 orders of magnitude greater compared to the basic Raman scattering technique. The surface enhancement features make it capable of being utilized in a wide variety of interfacial systems such as electrochemical, catalytic, organic, modeled and actual biological systems, in-situ and ambient analyses, and adsorbate–surface interactions due to its capability for the detection of very low concentrations of analytes involving structural and molecular identification (Weaver and Norrod, 1998; Kneipp et al., 1999; Dougan et al., 2011; Kong et al., 2011). This technique achieves high specificity and sensitivity of surface signal due to surface enhancement mechanisms occurring at the surface. There are two primary mechanisms described in the literature: an electromagnetic (EM) and a chemical enhancement (Jeanmaire and Van Duyne, 1977; Das and Agrawal, 2011). The EM effect is more dominant and occurs due to the amplification of

Figure 2 Confocal Raman microscope (Das and Agrawal, 2011).

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Figure 3 SE micrograph of an alumina-modified silver film-over-nanosphere (AgFON) substrate. The substrate was obtained by the vapor deposition of a 200 nm layer of silver on the monolayer of surfactant-free white carboxyl-functionalized polystyrene latex nanospheres with diameters of 590 nm. On this, a layer of alumina with the average thickness of 2 Å was deposited (Zhang et al., 2006).

EM fields near a metallic surface, whereas the chemical effect-contributing enhancement is only one to two orders of magnitude. The EM enhancement is dependent on the metallic surface roughness, while chemical enhancement involves change to the adsorbate electronic states due to chemisorption of the analyte (Weaver and Norrod, 1998). In order to improve the stability and reproducibility of SERS substrates and to increase the enhancement factors, there are a great variety of materials that can be used as SERS substrates. Metallic surfaces like gold, silver, and copper or alkali metals can provide the strongest enhancement in the visible range of excitation wavelength. These systems can provide enhancement in the Raman signal of the order of 104–106 folds which enables rapid and accurate detection of biological and chemical specimens (Zeisel et al., 1998). Elements like palladium and platinum also show enhancement in Raman scattering of about 102–103 in the near ultraviolet range of excitation wavelength (Zeisel et al., 1998). Metal substrates for SERS measurements must be nanostructured. Moreover, to achieve the highest enhanced SERS measurement, more shape-controlled and shape reproducible metallic nano-resonators on solid substrates have been introduced, such as metal-coated alumina nanoparticles. Figure 3 shows a scanning electron (SE) micrograph of an alumina-modified silver film-over-nanosphere (AgFON) substrate (Zhang et al., 2006). SERS measurements can also be conducted for analytes that illustrate absence of any SERS signals. Quantification of alkali metal ions can be based on the measurement of the SERS spectra of immobilized crown ethers that form complexes with the metal ions present in the ambient solution (Heyns et al., 1994). SERS can also be used for the identification of various biological agents such as fungi, virus, bacteria, bacterial spores, etc. Accurate and rapid detection of bioagents is critical in order to facilitate timely and appropriate actions in the occurrence of biological invasion. SERS can be used for the detection of bacillus anthracis spores, a dangerous pathogen for the disease anthrax. From a practical point of view, both the limit of detection and the time of the analyses of bacillus spores with the SERS measurements are acceptable (Zhang et al., 2006). Thus, SERS can be considered a label-free microorganism fingerprinting technique. Fluorescence spectroscopy is also used for impurity detection, but does not provide structural information. Therefore, SERS would be helpful to researchers not only in detection, but also identification of the molecules and their structural changes (Yin et al., 2012; Lin et al., 2012). Moreover, the efficiency of SERS has been recently increased to a detection limit of the order of 10
18 mol dm
3, using SERSsensors for some analytes. It has been observed that the recent usage of SERS has dramatically increased for the analysis of biological, medical, industrial, and environmental samples (Kudelski, 2008).

Coherent Anti-Stokes Raman Scattering (CARS) Microscopy CARS microscopy allows vibrational contrast imaging of chemical, biological, and biomedical systems with high speed, high sensitivity, and three-dimensional spatial and temporal resolution. The contrast mechanism is based on molecular vibrations intrinsic to a probing sample. Raman imaging requires a high average laser power and long integration times of 100 ms to 1 s per pixel because of the small cross section of Raman scattering. As a result, it necessitates spending several hours to acquire a Raman

Raman Spectroscopy and Microscopy

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image of living cells and molecular bonds in various biological systems. Such a long exposure time limits the application of Raman microscopy to the study of dynamic living cells and tissue in biological systems (Cheng and Xie, 2004). This difficulty in Raman microscopy can be overcome by vibrational microscopy based on the CARS technique. For molecules that cannot tolerate fluorophore probing, CARS microscopy permits vibrational contrast imaging of specific molecules in unstained samples (Zumbusch et al., 1999). Since the first systematic study conducted by Maker and Terhune at Ford Motor Co., CARS microscopy has become the most extensively used nonlinear Raman technique and a valuable tool for imaging molecular vibrations of live tissues, cell membranes and chromosomes (Maker and Terhune, 1965; Nafie, 2011; Cheng et al., 2002; Nan et al., 2003; Wang et al., 2005). Contrary to the incoherent spontaneous Raman process, CARS is a multi-photon optical process (four-wave mixing) in which the coherent signal, the result of coherent superposition of the signal waves generated in focus, can be used as an imaging tool for chemical mapping of cells and tissues with high sensitivity and speed. It is a key technique in vibrational contrast imaging for intrinsic biomolecules and deep tissues without chemical labeling in bioscience applications (Cheng et al., 2002). CARS microscopy avoids toxicity, photo bleaching of fluorophores, and artefacts associated with staining. The sensitivity of CARS microscopy is an order of magnitude greater than Raman microscopy. Therefore, it permits fast vibrational imaging at a moderate average excitation power, up to B10 mW, that is tolerable for biological samples. CARS microscopy has a 3D sectioning capability, making it useful for imaging cellular structures and thick tissues (Xie et al., 2006). Examples include imaging of C-H or H-O-H stretching vibration present in neuronal myelin under physiological conditions (Wang et al., 2005), vibrational imaging of phospholipid (Potma and Xie, 2003), imaging and study of chemical specificity in chemically amplified polymer films (Potma et al., 2004), selective probing of neutral lipid droplets in unstained live fibroblast cells and imaging of lipids in live cells due to the high density of C-H bonds (Nan et al., 2003) and measuring temperature and species concentration in reacting flows (Roy et al., 2010). These applications demonstrate that CARS microscopy provides exciting possibilities for chemical imaging of delicate biological and chemical samples and processes with chemical selectivity, bulksensitivity and non-invasiveness.

Resonance Raman Spectroscopy (RSS) Resonance Raman scattering is the basis of Resonance Raman spectroscopy (RSS) and can be obtained when the incident radiation is at frequency near the frequency of an electronic transition of the molecule of interest. This provides enough energy to excite the electrons of a molecule to a higher electronic state and results in a resonance enhancement of a subset of Raman-active modes (Robert, 2009). This is known as the resonance effect and can be employed to selectively observe a molecule or directly probe the molecular events in a complex biological and chemical medium. This technique has been extensively exploited in the analyses of various biological chromophores such as enzymes, various parts of biomolecules, and protective pigments of photosynthetic organisms (Robert, 1996, 2009; Spiro, 1974). Resonance Raman spectroscopy has many features that make it an attractive analytical technique. By making use of this technique, it is possible to relate the electronic properties of molecules involved in photosynthetic processes, such as polyyne encapsulated inside single-wall carbon nanotubes, whether isolated in solvents, embedded in soluble or membrane proteins or in vivo, to their structure and/or physical properties of their environment or to determine subtle changes of their conformation associated with rudimentary processes (Robert, 2009; Malard et al., 2007). Discussions with the understanding of a few rather interesting examples of applications that have been reported in the literature (Morris and Wallan, 1979; Strommen and Nakamoto, 1977). This has explored the potential utility of RRS as a reliable technique to increase sensitivity and decrease measurement times in the analyses of microbial species, carotenoids in biological matrices or to monitor the physiological state of a cell, pigments and dye analyses in forensic investigations, the study of cytochromes, etc. There is also quantification on the enhancement factor associated by combining RRS and SERS, where excitation wavelength not only coincides with the plasmonic band, but also with the absorption wavelength of the substrates/analytes of interest (White, 2003; Smith et al., 2001). The advantages of advanced Raman techniques and their potentiality have been discussed from a practical viewpoint in this article. These techniques are extremely flexible and can be configured in many different ways. The continued improvements in modern optics including diode lasers, improved miniaturized detector capabilities and fiber optic coupling have led to a rapid increase in the use of Raman spectroscopy (Kong Chong et al., 1992). Although the Raman effect is weak, this weakness can be overcome to an extent with the use of advanced techniques where power density is high, and by the use of a microscope or fiber optic systems, in which laser excitation is transmitted along one fiber and the scattered radiations are transmitted to the detector along different fibers (Utzinger and Richards-Kortum, 2003). Thus, the advanced Raman spectroscopy techniques appear ready for analytical purposes and offer easy interpretation without the need for sample preparation.

Raman Applications Raman spectroscopy is gradually becoming a mature technique in analytical science. The technique is now applied to a broad range of scientific disciplines including biology and medicine, revealing the molecular signature of diseases, and individual

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bacteria and yielding chemical images of a sample. Some of these interesting applications of Raman spectroscopy in various fields of science have been discussed below.

Analytical Sciences Forensic science Forensic sciences are always keen to adopt fast, non-destructive, selective, and sensitive investigation methods. Raman spectroscopy has already proved to be a promising technique and has found a wide range of applications in forensic science. Man-made polymeric fibers (Zieba-Palus et al., 2008), modern inks (in documents) (Zieba-Palus and Kunicki, 2006; Claybourn and Ansell, 2000), multilayer paint samples (microchips, attrition on fabric), plastics, glass, bodily fluids, and explosives (Bonnier and Byrne, 2012; Bonnier et al., 2010; Boyd et al., 2011; Virkler and Lednev, 2008, 2010) are the most commonly encountered pieces of evidence during forensic investigations and have been evaluated using Raman spectroscopy and other related techniques. Other uses include the examination of drugs, lubricants, and detection of anthrax residues (Farquharson et al., 2004). Analyses of inks have been very difficult for the professional document examiner due difficulties in the comparison of black ballpoint inks and the chronological sequencing of crossed ink lines. The conventional approaches apply chemical methods, which normally yield poor chemical selectivity and hence a lack of specificity. Raman spectroscopy offers an excellent solution since it provides direct identification of chemical signatures and determination of chronological sequences of crossed ink lines on the questioned and fraudulent documents. This could be possible due to characteristics of ink such as luminescence in visible or in infrared light (Souder, 1955). Thus, the investigations are most often aimed at authentication of documents or at determination of the chronological sequence of two lines, which cross within a document. A good discrimination of chemically and compositionally different inks, and inks of different sources with the same chemical composition, has been obtained by Raman spectroscopy (Braz et al., 2013; Mazzella and Buzzini, 2005). The Raman spectroscopic technique is used for the examination and identification of various types of biological fluids, such as semen stains, vaginal fluid, sweat, saliva, and blood. This approach offers the confirmatory non-destructive, rapid, and on-scene identification of various body fluid evidence (Boyd et al., 2011; Virkler and Lednev, 2008, 2010). These fluids are different from one another as each body fluid has a unique composition and can be differentiated by visual comparison of their Raman signatures (Virkler and Lednev, 2008). Experimental design and methodology needs to be tailored to the task, as samples may be in trace amounts (Tripathi et al., 2011; Ali et al., 2009b), hazardous (Izake et al., 2013) and photosensitive (López-López et al., 2013). Thus, the important challenge in such cases of identification is substrate interference in Raman signals. This issue becomes more complicated if interference is from heterogeneous substrates. Nevertheless, this challenge has been overcome by subtracting the substrate contribution using a reference spectrum of the neat substrate (McLaughlin et al., 2013). Statistical analyses and identification of body fluid using an automatic subtraction algorithm in a MATLAB environment for treating Raman spectra has been developed for spectroscopic signatures of trace amounts of samples during forensic analyses (Beier and Berger, 2009). Advances in instrumentation for the detection of Raman scattering have established the technique for the analyses of explosive and hazardous environmental samples, with a good flexibility for measurement. Several analytical techniques have been established for the identification of explosives under various conditions, however many of these methods involve the partial or complete destruction of the samples (Shaw et al., 2005; Keller et al., 2006; Justes et al., 2007; Na et al., 2007). Raman spectroscopy allows the non-destructive analysis of samples, which is particularly important with regard to the speed of analysis, prevention of sample contamination and preservation of evidential materials (Ali et al., 2009a). A detailed vibrational analysis for the detection and examination of 2,4,6-trinitro-1-methylbenzene, propanetriol trinitrate, and 1,3,5-trinitro-1,3,5-triazacyclohexane (NG and RDX) have been published (Carver and Sinclair, 1983). Confocal Raman microscopy is used to detect trace amounts of explosives from contaminated surfaces as a result of manual handling, packaging, or transportation of explosive substances such as pentaerythritol tetranitrate (PETN), 2,4,6-trinitrotoluene (TNT), ammonium nitrate, and hexamethylenetetramine (HMTA) particles (Ali et al., 2009a). Raman spectroscopy in conjunction with several other spectroscopic and chromatographic techniques such as IR, FTR, nuclear magnetic resonance, SERS, mass and X-ray fluorescence spectrometry, and high pressure chromatography have been employed for screening and characterization of explosives and hazardous materials (Cheng et al., 1995; Hatab et al., 2010). Fibers are also one of the important pieces of evidence encountered during forensic investigation. Raman microscopy can be used as an analytical tool for measuring different aspects of fiber analyses in both dyed and non-dyed fibers such as orientation, variation of coated reactive dyes, and degradation (Lang et al., 1986; Keen et al., 1998). Raman measures vibrational states of nonpolar bonds of both dyes and fibers. Spectra from dyed cotton fibers can provide information on molecular signatures of the dyes without interference from cotton substrates (Thomas et al., 2005). These results suggest Raman spectroscopy is a powerful investigational tool for fibers (Lepot et al., 2008; Abbott et al., 2010). Raman spectroscopy is widely used as a complementary method with Fourier transform infrared spectroscopy (FT-IR) for the detection and analysis of drugs of abuse. Identification and quantification of drugs of abuse by Raman spectroscopy has been well documented in different types of forensic evidence (West and Went, 2011). The important advantage related to the Raman technique is that it allows the sample measurement through translucent plastic and glass containers avoiding the need to open sealed bags/bottles, therefore reducing any contamination to the sample (West and Went, 2011; Moreno et al., 2014). The

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application of surface-enhanced Raman spectroscopy (SERS) to detect trace amounts of drugs of abuse in saliva and the use of Raman microscopy to detect drug particles trapped between fibers, fingerprints, on human nails, or banknotes are a few examples of the potentiality of this technique (Inscore et al., 2011; Ali et al., 2008a,b; Day et al., 2004; Frederick et al., 2004). Likewise, quantitative determination of caffeine in commercial energy drink samples has been achieved by vibrational spectroscopic techniques. FT-IR provides a rapid and alternative method to the most frequently used techniques such as liquid, ion, and gas chromatography and electrophoresis (Armenta et al., 2005). Vibrational spectroscopy (IR, NIR, and Raman) has also been proved as a powerful tool in the analysis of adulterated fuel samples like gasoline, diesel, alcohol fuel, and kerosene. These spectroscopic methods of analysis are fast, present accuracy, and precision in data and can be used in remote quality control (Oliveira et al., 2007; Fodor et al., 1996; Flecher et al., 1996). The design of multivariate analyses using calibration models such as principal component regression (PCR), partial least square (PLS) regression, and artificial neutral network (ANN) has been applied with vibrational spectroscopy for examining adulteration with precise control and accuracy within the spectral regions of importance (Oliveira et al., 2007).

Environmental materials Hazardous and heavy metal contamination has become a significant issue in recent years due to developments in industrial, domestic, and agricultural activities. In some environments, materials are plagued by particulate contamination, which deteriorates the quality of the product. Heavy and hazardous materials in trace amounts can find a way to reach aquifers due to human activities. The effect of toxicity becomes more severe if contaminated species are highly soluble in environmental materials and their concentrations are below the detection limit. The properties of serious toxicity and accumulation in alimentary systems have raised the importance of their detection. Even though they are present at very low concentrations in environmental materials, heavy metal ions can be fatal once they reach dangerous levels in the human body. Therefore monitoring of heavy metal ions in food, soil, and water is a requisite for human safety. Trace amounts of hazardous agents or contaminants can be detected using optical detection methods in combination with Raman and fluorescence spectroscopy and Raman digital imaging by their vibrational Raman fingerprints (Kalasinsky et al., 2007). SERS can be applied to arsenate and arsenite sensing in aqueous solutions with a detection limit of 1 ppb using the Lanmuir– Blodgett assemblies of polyhedral Ag nanocrystal substrates (Mulvihill et al., 2008). Even Al3 þ , Sb2 þ , As2 þ , Cd3 þ , Pb2 þ , and Cr3 þ ions in very little amounts can be detected using the surface enhancement technique in SERS. Even uranium can be detected in aqueous solution by using (aminomethyl) phosphonic acid (APA)-modified gold nanoparticles. The uranyl band was observed at a wavenumber of 830 cm
1, which is proportional to uranium concentration in aqueous media up to a detection limit of 8  10–7 M. This allows the detection of uranium in a highly contaminated groundwater containing dissolved salts (Ruan et al., 2007). Quantitative determination of TNT in-field and real-time analyses of soil and ground-water is required for when contamination is beyond the human levels of tolerance; prolonged exposure of humans to TNT can result in anemia and liver abnormalities (van Dillewijn et al., 2008). It has been demonstrated that SERS is a rapid and selective approach for the detection of trace amounts of TNT in aqueous media when utilizing 6-aminohexanethiol (AHT) as the new TNT recognition molecule to confirm the trace contaminants in environmental samples (Jamil et al., 2015).

Archeological materials Raman spectroscopy provides a non-destructive method for characterizing the structure and chemical composition and determining the authenticity and provenance of objects of archeological and historical importance. Non-destructive analyses of pigments in rock art and frescoes, textiles, mummified tissues, hair, skeletal fragments, resins, corroded items, ancient skin tissue samples, etc., have been extensively studied by Raman spectroscopy. Clay mineral constituents and firing temperature estimation of archeological pottery shreds were analyzed using FT-IR and micro Raman spectroscopy (Kiruba and Ganesan, 2015). Regarding the mummification processes, a varying degree of degradation in skin under soil environments has been observed in Raman spectra. A comparison based on Raman spectra between deceased and healthy skin makes it possible to identify skin preservation techniques, whether the technique is chemically assisted or natural. This information is of great importance from an archeological conservation point and in terms of gaining knowledge of past societies (Edwards et al., 2002).

Life Science Raman spectroscopy has been an extremely promising technique in biological research as it provides spectra, which are information rich and give detailed insight about the elemental/compositional variations in tissues and cells without the need of stains or labeling. It also provides analytical solutions for skin care, drug-cell interactions, bacteriology, dentistry, cancer diagnosis, etc., (Krafft et al., 2003). FT Raman and Raman spectroscopy are used in the characterization and examination of the outermost layer of human skin, quantization of biochemical constituents, disease diagnosis, histochemical analysis of biological tissues and cancer studies in soft tissues, colon, bladder, breast, and brain (Manoharan et al., 1996). Raman micro-spectroscopy can provide biochemical information regarding a molecular specificity within a diffraction limit resolution of 1 mm in vivo conditions without the need of fixations, markers, or stains (Krafft et al., 2003). The biochemical

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information in Raman spectra displays the fingerprint for any toxic agent interaction(s), cell degradation, and differentiation of tissues and live cells. The Raman spectrum of a cell can also provide a monogram of biochemical changes related to any drug effects (Notingher, 2007). The spectral features in Raman spectra of macromolecules lie in the region between 200 and 3500 cm
1 and arise from the molecular vibrations of either the polypeptide backbone or side chains (Wallach et al., 1970). Advances in technology have increased the competence of Raman microscopy in bio-medical and bio-chemical analyses. It could also become a useful tool in the detection of certain DNA target sequences of interest, without any replication and modification of the parent DNA material. Moreover, multiple gene targets can be identified in a single SERS measurement that produces a string Raman signal upon laser excitation (Wabuyele and Vo-Dinh, 2005). Raman spectroscopy is a technique for rapid quality screening, as it provides structural information about changes in proteins, water, and lipids of muscle food, which occur during deterioration. The technique may be preferable in the study of food systems containing molecules in solution, in solid phases and in turbid or opaque materials because inherently high protein concentrations usually yield Raman spectra with good signal-to-noise ratios (Li-Chan, 1996). It is used to evaluate quantitative measurements of fat content in ground muscle tissues of salmon, cod, sea bass, and Pacific halibut (Marquardt and Wold, 2004). In addition to its use in the analysis of food constituents, Raman spectroscopy could be potentially applied to many other areas of food science, including studies of trace components, nucleic acids or their constituents, whole cells and tissues, microorganisms, and even the packing in which foods are contained (Li-Chan, 1996; Herrero, 2008). Direct observation of products in sugar fermentation for ethanol production, analysis of polyolefin materials often used in packaging, such as high-density polyethylene, polypropylene, or polyvinylchloride, studies of the behavior of polymer fibers and composites under stress and strain have been demonstrated (Gerrard, 1994). Advances in Raman techniques coupled with recently developed sampling techniques have enabled probing of tiny samples of micrometer thickness. This allows the identification of bio-membrane monolayer systems at interfaces by providing information on the molecular structure of the membrane (Dluhy et al., 1995). Thus, Raman technique can provide useful information about the molecular and supramolecular structure of living tissues, including hard tissues like bone (Schrader et al., 1997). Diagnostic applications of Raman spectroscopy have been reviewed in detail (Tu and Chang, 2012; Haka et al., 2009). This technique has been validated for the discrimination of normal and malignant tissues in cervical cancers (Crow et al., 2005). It was observed that non-contaminated cervix tissues were characterized by sharp, broad amide I, broader amide III, and sharp peaks at 853 and 938 cm
1 wavenumbers, which are structural proteins such as collagen. While the malignant tissue spectra with respect to normal tissue was relatively weaker and sharper amide I, the minor red shift in –CH2, and sharper amide III, indicated the presence of Deoxyribonucleic acid (DNA), lipids, and non-collagenous proteins (Krishna et al., 2006). These results and discussions show spectroscopic techniques are rapidly becoming invaluable tools for the life sciences, allowing biologists a better understanding of living processes and providing a new dimension of information, based upon biochemical and histochemical processes and compositions.

Materials Science Raman spectroscopy and microscopy provide the opportunity to characterize the macroscopic properties of a material by exploring its microscopic structure. Being a non-destructive characterization procedure, the Raman technique is readily applicable to any material system including very complex systems in analytical science. This section explores the potential applications of Raman technique in the field of material science.

Carbonaceous materials Carbonaceous materials have a wide range of physical and chemical properties derived from the spatial arrangements of carbon atoms in a hexagon ring and their chemical covalent and van der Waals bonds. Diamonds, nanodiamonds, single-walled carbon nanotubes, double-walled carbon nanotubes carbon nanowires, and carbon nanofibres are all important carbonaceous materials and are characterized by an impressive mechanical strength. One-dimensional carbon nanotubes, grapheme, and graphite are characterized by good electrical and thermal conductivity, while diamond and nanodiamond are electrical and thermal insulators. This wide range of physical properties explains the complex and abundant applications of carbonaceous materials. Raman scattering is one of the prime spectroscopic techniques for the characterization of carbonaceous materials, which includes study related to their vibrational, electronic, and optical properties. Several Raman spectral bands have been reported in carbonaceous materials (Krishna et al., 2015). These spectra can provide various information regarding structure and properties. The prime first order Raman peaks in graphite are optical phonons at 1582 cm
1 corresponding to the E2g mode, called the graphite band (G-band) and at 1349 cm
1 corresponding to the A1g mode, called the disorder band (D-band) (Tuinstra and Koenig, 1970). A strong Raman peak in diamond related structures can be observed at 1332 cm
1, which originates from sp3 carbon bonds and represents defect structures in the presence of sp3 bonds (Ferrari and Robertson, 2004). Thus, it is possible to study the defective structure of similar kinds of materials. Typically, in carbonaceous materials the degree of disorder is measured by the ratio of the integrated areas under D and G bands. Raman lines are also sensitive to temperature, pressure, and stress or strain, and

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studies on graphite under the effect of these parameters have been carried out (Krishna et al., 2015; Peña-Álvarez et al., 2014). Tuinstra and Koening (Tuinstra and Koenig, 1970) have proposed an empirical relationship between the intensity ratio of D and G bands and crystallite size La in carbon based materials as: IIDG ¼ CðlÞ La ; Cðl ¼ 514:5 nmÞ ¼ 44. This correlation has been used to qualitatively control carbon structural transformations in amorphous carbon, Diamond-Like Carbon (DLC) and films (Tomasella et al., 2003). Figure 4 illustrates the first-order stokes Raman spectrum of highly ordered pyrolytic graphite (HOPG) exhibiting a single Gband and a diamond sample which shows a D-band. Some additional bands can also be noticed at 2700 and 3250 cm
1 in a second-order Raman process in the HOPG sample. Figure 4 also shows a bright field transmission electron micrograph of the pristine HOPG sample. This information is needed to characterize the graphite sample in quantifying residual stresses and their nature, identifying constituents, crystallite size of different constituents and structural crystallinity, and studying the disorder in graphite based systems (Krishna et al., 2015; Pimenta et al., 2007; Cancado et al., 2008; Figure 5).

Figure 4 The first and second order Raman spectra in HOPG and a diamond sample. The TEM micrograph of the HOPG sample represents an absence of defects resulting in a total disappearance of a D-peak in the Raman spectrum.

Figure 5 Dispersive Raman spectra of graphite grade Gilsocarbon (shown in inset). The spectra are from the constituents of the graphite filler – binder regions. The spectra show first order fundamental bands (D, G, and D0 ) and their overtones (Krishna et al., 2015).

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Micro-Raman spectroscopy has been used to study the spatial variation of internal stresses in mono- and polycrystalline synthetic diamond. It can be directly used to get information on the state of stress distribution around the indentation in the diamond (Fish et al., 1999).

Semiconductors Raman scattering techniques can be applied for the structural characterization and investigation of properties of semiconductors. The shape, size, and position of the first order phonon bands in Raman spectra have been used for the study of residual stress, lattice disordering, crystallization, and homogeneity in semiconductors (Nesheva, 2005). Nanocrystalline silicon semiconductors and microcrystallite semiconductors have been widely studied through use of Raman spectroscopy. Moreover, Raman has the potential of being developed as a tool for probing the location of dopants within nanocrystals, like P-doped Si nanocrystals (Islam and Kumar, 2001; Khoo and Chelikowsky, 2014). In general, a stress of 1 GPa introduces a Raman line shift of about 2 cm
1 in the polycrystalline silicon wafers, which appear large enough to cause cracking in the film. In one of recent study, authors reported that Raman enhancement is dependent on the diameter, the excitation wavelength, and the incident polarization, which is expanding opportunities for engineered photonic and sensing properties of nanowires (Cao et al., 2007).

Nanotechnology Raman spectroscopy has been successfully applied to probing and characterization of nanoscale materials like nanosensors, nanotubes, nanowires, and nanoparticles. Based on their physical, chemical, mechanical, electrical, optical, catalytic, and magnetic properties, including their high surface area per mass that emerges at the nanoscale, several classes of materials can be studied. The use of nanomaterials in existing Raman based analytical techniques has resulted in a high rate of signal enhancement (Wang et al., 2010). SERS is one of these techniques based on the enhancement of the Raman signal of molecules using nanomaterials with unique properties. As discussed earlier, Raman signals are enhanced by a factor of up to 1014 using this technique. Thus, SERS provides in situ detection of molecules with high level of sensitivity and detailed information about the molecular composition and chemical structure of the molecules (Temiz et al., 2013). Nanoparticle-based environmental sensors are attractive options in spectroscopy and have the potential to detect toxins, heavy metal ions, and organic pollutants in water, air, and soil. They not only improve the detection and sensing of pollutants, but are set to emerge as new remediation technologies (Wang et al., 2010). Raman spectroscopy has already been proven as a characterization tool used in better understanding and controlling of nanoscale-related properties in nanomaterials such as nanoceramics, nanocomposites, glassy materials, and relaxor ferroelectrics (Gouadec and Colomban, 2007). Raman spectra produce similar information from single crystal nanomaterials to facilitate direct identification of the phases, and are used to identify and characterize phase transitions in nanoscale materials. Barborini et al. showed using the Raman technique that the structure of gas phase-deposited TiO2 clusters turned from rutile to anatase as particle diameter decreased to less than 5 nm (Barborini et al., 2002).

Chemical Science Polymers There have been many studies conducted using Raman and FT Raman spectroscopy for the characterization of polymeric materials in terms of structural information, composition, interfacial environments, and dynamics. A variety of spectroscopic techniques are used to study the bulk properties of polymeric materials such as homogeneity and composition, but when more detailed information is needed about local variations in compositions or properties, Raman microscopy is used. Specific peak parameters (position, width, shape, and relative integral amplitude) can provide sensitive information about the physical and chemical modification of the surface, dopants, and the effect on the dispersion of material within polymers. Raman imaging can be used to study polymer changes in the crystalline phase through a sample. Syndiotactic polystyrene is an industrial polymer that can exist in several well-defined crystalline phases as well as in an amorphous form. The determination of crystallinity in a polymer is important because most physical and mechanical properties are strongly affected by the degree of crystallinity. The crystallinity distribution in the syndiotactic polystyrene sample was derived by factor analysis obtained from band area integration using high definition hyperspectral Raman imaging (Zhang et al., 1998). Raman spectroscopy also allows the orientation of polymers to be studied. The amount of band shift owing to an applied load depends on the degree of orientation; the more oriented the sample, the more shift is seen when a load is applied (Rodriguez-Cabello et al., 1995). The quantitative details regarding the experimental design for orientation mapping and crystallinity in uniaxially drawn polymer using polarized confocal Raman microscopy have been well established (Everall, 1998; Lapersonne et al., 1992).

Pharmaceuticals Raman and mid-range IR (MIR) spectroscopy are versatile analytical tools in pharmaceutical environments due to their inherently high chemical specificity, ability to probe samples in aqueous media, and potential for high penetration depth into non-absorbing or weakly absorbing turbid samples. These techniques are used to characterize the bulk chemical constituents of intact samples in a

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rapid, non-invasive, and non-destructive way. Applications exist in characterization of drug formulations and elucidation of kinetic processes in drug delivery by noting shift in Raman bands and changes in band intensity. Fast quantitative analysis of pharmaceutical formulations, detection, and identification of counterfeit medicines is also possible (Eliasson and Matousek, 2007). Quantitative analysis and identification of active pharmaceutical ingredients (API) in drugs is an important part of pharmaceutical analysis. Application of Raman spectroscopy in studying batch sizes for quality control sampling in tablet manufacturing, and pharmaceutical drug discovery and development has been analyzed (Gala and Chauhan, 2015). The technique can reduce the time required to analyze active ingredients in the composition of tablets, while increasing the number of tablets testing cycle. Thus, it has enabled the estimation of the molecular activity of drugs and to establish a drug’s physicochemical properties such as its partition coefficient, compatibility studies during the drug formulation process, in vivo studies, and characterization of the structure of colloidal drug carrier systems (Torreggiani and Fini, 1999). Raman spectroscopy is employed as a real-time analytical technique to monitor the transformation process of APIs in an aqueous environment, solution-mediated polymorphic transformation of sulfathiazole, studies of API polymorphism, etc. Various substances that are not the part of drugs but have profound use in pharmaceutical industries like detergents, surfactants, cosmetics, etc., can also be analyzed with the vibrational spectroscopy technique without any adverse effect on spectra, and quantitative analysis can be conducted without background interference (Das and Agrawal, 2011).

Advantages Raman spectroscopy could be applied easily to the analysis of wide range of sample types and may be examined either in bulk or in macroscopic amounts over a range of temperature and physical states, for example, thin films, powders, fibers, embedded layers, gases, liquids, bulk surface. Some major advantages of Raman spectroscopy are its ability to record spectra with minimal sample preparation and minimum time required for spectra collection. It is also compatible for aqueous, biological, and chemical samples. In recent times, Raman spectroscopy has become an important analytical tool and is commonly used in almost all laboratories. Its application is growing rapidly and it is becoming more widely used in many fields of science with new developments in laser technology, detector capability, and spectrometer robustness.

Limitations The limitation in adopting Raman microscopy for routine analysis is the cost of the equipment. High levels of fluorescence are a major issue with Raman measurements as this will overlay the Raman scattering peaks. This can be avoided by using advanced instrumentation or shifting the excitation wavelength to near the IR region. There is always possibility for thermal damage of the sample if excitation power is too high.

Summary Recent developments of Raman spectroscopy have made Raman techniques some of the most versatile analytical tools for the analyses of a variety of materials. The non-invasive characteristics of Raman measurements are of significant practical importance, especially for biological samples and materials/objects of cultural heritage. Raman spectroscopy is being successfully employed in the analysis of a wide range of materials. (a) Raman techniques provide a molecular fingerprint of samples measured and provide non-destructive identification of materials in chemical sciences. Raman can be used in the application of raw material verification and identification, which is of primary importance for finished products in pharmaceutical, nutraceutical, and cosmetic industries. It has proven valuable in quantitating active substances in pharmaceutical formulations and finished products, crystallinity measurements, and concentration measurements of different polymorphic ingredients and chemical species. (b) Raman spectrometers are potentially useful in analytical sciences as they provide a handful of sources of information concerning the composition, structure, and stability of coordination compounds. They can provide valuable information quickly, thus reducing the time and cost needed for measurements. Raman can therefore be a potential solution for the rapid identification of multiple forensic sample types, ideal for law enforcement personnel. (c) Raman instruments provide non-destructive, non-contact identification of explosive and hazardous substances, allowing users to get real-time actionable identification of materials while reducing operational uncertainty and response time. (d) Raman spectroscopy is well recognized as an analytical tool for the discrimination of similar substances. Therefore, it can be used not only for the identification of adulterants but also for providing quantitative results. (e) Raman technology is used to predict crystallographic orientation during growth through the ratio of Raman peak intensities in different positions. (f) Raman spectroscopy has been also been used in the field of coating and thin film technology for structural characterization and for identifying surface properties of materials.

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(g) Raman spectroscopy is the perfect tool to determine synthetic and natural materials; it can be used in geology and gemmology studies as a non-destructive technique that requires no sample preparation to analyze samples in great detail. (h) Raman spectroscopy could be a promising tool in the development of both existing and next generation energy technologies. It can be used in battery manufacturing industries to test components for optimal electrochemical performance. (i) Raman spectroscopy is the most important analytical tool available for investigating the structures produced from carbon. It can be used to identify and analyze all forms of carbonaceous materials including graphite, diamond, diamond-like carbon (DLC), carbon nanotubes (CNT), fullerene, grapheme, etc. (j) Raman spectroscopy is a label-free analytical technique that can be used for the analysis of the chemical and structural information of cells, micro-organisms, tissues, etc.

Acknowledgments The materials contained in this article are taken from the publications listed below.

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