Diagnostic Raman spectroscopy for the forensic detection of biomaterials and the preservation of cultural heritage

This paper reviews the contributions of analytical Raman spectroscopy to the non-destructive characterisation of biological materials of relevance to forensic science investigations, including the sourcing of resins and the identification of the biodegradation of art and archaeological artefacts. The advantages of Raman spectroscopy for non-destructive analysis are well-appreciated; however, the ability to record molecular information about organic and inorganic species present in a heterogeneous specimen at the same time, the insensitivity of the Raman scattering process to water and hydroxyl groups, which removes the necessity for sample desiccation, and the ease of illumination for samples of very small and very large sizes and unusual shapes are also apparent. Several examples are used to illustrate the application of Raman spectroscopic techniques to the characterisation of forensic biomaterials and for the preservation of cultural heritage through case studies in the following areas: wall-paintings and rock art, human and animal tissues and skeletal remains, fabrics, resins and ivories.     

Raman spectroscopy in astrobiology

Raman spectroscopy is proposed as a valuable analytical technique for planetary exploration because it is sensitive to organic and inorganic compounds and able to unambiguously identify key spectral markers in a mixture of biological and geological components; furthermore, sample manipulation is not required and any size of sample can be studied without chemical or mechanical pretreatment. NASA and ESA are considering the adoption of miniaturised Raman spectrometers for inclusion in suites of analytical instrumentation to be placed on robotic landers on Mars in the near future to search for extinct or extant life signals. In this paper we review the advantages and limitations of Raman spectroscopy for the analysis of complex specimens with relevance to the detection of bio- and geomarkers in extremophilic organisms which are considered to be terrestrial analogues of possible extraterrestial life that could have developed on planetary surfaces.     

The ExoMars Raman spectrometer and the identification of biogeological spectroscopic signatures using a flight-like prototype

The molecular specificity of Raman spectroscopy provides a powerful tool for the analytical interrogation of mineralogical and many biological specimens. The Raman Laser Spectrometer (RLS) is a compact Raman spectrometer under development for deployment on the Martian surface as part of the forthcoming ESA ExoMars mission. This will be the first Raman instrument deployed in space. The scientific interpretation of the data emerging from such an instrument not only addresses the geological and mineral composition of the specimens but also enables an assessment to be made of organic biomaterials that may be preserved in the planetary geological record. The latter evidence centres on the residual and distinctive chemistry relating to the biological adaptation of the geological matrix that has occurred as a result of extremophilic organisms colonizing suitable geological niches for their survival in environmentally stressed habitats on Mars. These biogeological modifications have been studied terrestrially for Mars analogue sites and consist of both a geological component and residual key organic biomarkers, the recognition of which would be a prime factor in life detection surveys of a planetary surface and subsurface. In this paper, the protocols required for the Raman spectral discrimination of key biogeological features that may be detectable on the Martian planetary surface or subsurface are developed using the UK breadboard (UKBB) instrument. This instrument has been constructed to be functionally equivalent to the RLS flight instrument design in order to evaluate the feasible science return of the instrument which will finally be delivered to Mars. Initial Raman measurements using the UKBB are presented and compared with the performance of a commercial laboratory Raman microscope. The initial measurements reported here demonstrate this flight-like prototype achieves straightforward detection of biological signatures contained in geological matrices with Raman band signal to noise ratios high enough to determine sample composition by inspection and without the need for deconvolution or further processing.     

Surface-enhanced Raman scattering substrates fabricated using electroless plating on polymer-templated nanostructures

Surface-enhanced Raman scattering (SERS) has great potential as an analytical technique based on the unique molecular signatures presented even by structurally similar analyte species and the minimal interference of scattering from water when sampling in aqueous environments. Unfortunately, analytical SERS applications have been restricted on the basis of limitations in substrate design. Herein, we present a simple SERS substrate that exploits electroless deposition onto a nanoparticle-seeded polymer scaffold that can be fabricated quickly and without specialized equipment. The polymer-templated nanostructures have stable enhancement factors that are comparable to the traditional silver film over nanospheres (AgFON) substrate, broad localized surface plasmon resonance spectra that allow various Raman excitation wavelengths to be utilized, and tolerance for both aqueous and organic environments, even after 5 day exposure. These polymer-templated nanostructures have an advantage over the AgFON substrate based on the ease of fabrication; specifically, the ability to generate fresh SERS substrates outside the laboratory environment will facilitate the application of SERS to new analytical spectroscopy applications.     

Raman spectroscopy of <Emphasis Type="Italic">natron</Emphasis>: shedding light on ancient Egyptian mummification

The mummification ritual in ancient Egypt involved the evisceration of the corpse and its desiccation using natron, a naturally occurring evaporitic mineral deposit from the Wadi Natrun, Egypt. The deposit typically contains sodium carbonate, sodium bicarbonate and impurities of chloride and sulfate as its major elemental components. It is believed that the function of the natron was to rapidly remove the water from the cadaver to prevent microbial attack associated with subsequent biological tissue degradation and putrefaction. Several specimens of natron that were recently collected from the Wadi Natrun contained coloured zones interspersed with the mineral matrix that are superficially reminiscent of extremophilic cyanobacterial colonisation found elsewhere in hot and cold deserts. Raman spectroscopy of these specimens using visible and near-infrared laser excitation has revealed not only the mineral composition of the natron, but also evidence for the presence of cyanobacterial colonies in several coloured zones observed in the mineral matrix. Key Raman biosignatures of carotenoids, scytonemin and chlorophyll have been identified. Figure The mummification ritual in ancient Egypt involved the evisceration of the corpse and its desiccation using natron, a naturally occurring evaporitic mineral deposit from the Wadi Natrun, Egypt. The deposit typically contains sodium carbonate, sodium bicarbonate and impurities of chloride and sulfate as its major elemental components. It is believed that the function of the natron was to rapidly remove the water from the cadaver to prevent microbial attack associated with subsequent biological tissue degradation and putrefaction. Several specimens of natron that were recently collected from the Wadi Natrun contained coloured zones interspersed with the mineral matrix that are superficially reminiscent of extremophilic cyanobacterial colonisation found elsewhere in hot and cold deserts. Raman spectroscopy of these specimens using visible and near-infrared laser excitation has revealed not only the mineral composition of the natron, but also evidence for the presence of cyanobacterial colonies in several coloured zones observed in the mineral matrix. Key Raman biosignatures of carotenoids, scytonemin and chlorophyll have been identified.     

Ancient biodeterioration: an FT–Raman spectroscopic study of mammoth and elephant ivory

Raman spectra of mammoth ivory specimens have been recorded using near-infrared excitation, and comparisons made with modern Asian and African elephant ivories. Whereas the most ancient mammoth ivory (60–65 ky) showed no evidence for an organic collagen component, more recent samples of mammoth ivory indicated that some preservation had occurred, although with biodeterioration of the protein structure exhibited by the amide I and III bands in the 1200–1700 cm⁻¹ region of the Raman spectrum. The consequent difficulties encountered when applying chemometrics methods to ancient ivory analysis (which are successful for modern specimens) are noted. In the most ancient mammoth ivory specimens, which are extensively fragmented, evidence of mineralization is seen, with the production of gypsum, calcite and limonite; Raman microscopic analysis of crystalline material inside the fissures of the mammoth ivory shows the presence of gypsum as well as cyanobacterial colonisation. The application of Raman spectroscopy to the nondestructive analysis of archaeological materials in order to gain information of relevance to their preservation or restoration is highlighted.     

Advances in surface-enhanced Raman spectroscopy for analysis of pharmaceuticals: A review

Recently, Raman spectroscopy become a popular and potential analytical technique for the analysis of pharmaceuticals as a result of its advancement. The innovation of laser technology, Fourier Transform-Raman spectrometers with charge coupled device (CCD) detectors, ease of sample preparation and handling, mitigation of sub-sampling problems using different geometric laser irradiance patterns and invention of different optical components of Raman spectrometers are contributors of the advancement of Raman spectroscopy. Transmission Raman Spectroscopy is a useful tool in pharmaceutical analysis to address the problems related with sub-sampling in conventional Raman back scattering. More importantly, the development of surface-enhanced Raman scattering (SERS) has been a prominent advancement for Raman spectroscopy to be applied for pharmaceuticals analysis as it avoids the inherent insensitivity and fluorescence problems. As the active pharmaceutical ingredients (APIs) contain aromatic or conjugated domains with strong Raman scattering activity, Raman spectroscopy is an attractive alternative conventional analytical method for pharmaceuticals. Coupling of Raman spectroscopy with separation techniques is also another advancement applied to reduce or avoid possible spectral interferences. Therefore, in this review, transmission Raman spectroscopy, SERS, and SERS coupled with various separation techniques for pharmaceutical analysis are presented.     

Chemical signatures of fossilized resins and recent plant exudates

Amber is one of the few gemstones based on an organic structure. Found over most of the world, it is the fossil form of sticky plant exudates called resins. Investigation of amber by modern analytical techniques provides structural information and insight into the identity of the ancient plants that produced the source resin. Mass spectrometric analysis of materials separated by gas chromatography has identified specific compounds that are the basis of a reliable classification of the different types of amber. NMR spectroscopy of bulk, solid amber provides a complementary classification. NMR spectroscopy also can be used to characterize modern resins as well as other types of plant exudates such as gums, gum resins, and kinos, which strongly resemble resins in appearance but have very different molecular constitutions.     

Combined FT–Raman spectroscopic and mass spectrometric study of ancient Egyptian sarcophagal fragments

The application of combined Raman spectroscopic and GC–MS analytical techniques for the characterisation of organic varnish residues from Egyptian Dynastic funerary sarcophagal and cartonnage fragments from the Graeco-Roman period, ca. 2200 BP, is described. The nondestructive use of Raman spectroscopy was initially employed to derive information about the specific location of organic material on the specimens, which were then targeted in specific areas using minimal sampling for GC–MS analysis. In the case of the sarcophagal fragment, a degraded yellow-brown surface treatment was identified as a Pistacia spp. resin; this provides additional evidence for the use of this resin, which has previously been identified in Canaanite transport amphorae, varnishes and “incense” bowls in an Egyptian Late Bronze Age archaeological context. The cartonnage fragment also contained an organic coating for which the Raman spectrum indicated a degradation that was too severe to facilitate identification, but the GC–MS data revealed that it was composed of a complex mixture of fatty acid residues. The combined use of GC–MS and Raman spectroscopy for the characterisation of organic materials in an archaeological context is advocated for minimisation of sampling and restriction to specifically identified targets for museum archival specimens.     

Raman identification of yellow synthetic organic pigments in modern and contemporary paintings: reference spectra and case studies

The characterization of the binding media and pigments in modern and contemporary paintings is important for designing safe conservation treatments, as well as for determining suitable environmental conditions for display, storage and transport. Raman spectroscopy is a suitable technique for the in situ non-destructive identification of synthetic organic pigments in the presence of the complex binding media characteristic of synthetic resin paints or colour lithographic inks. The precise identification of a pigment by comparing its spectrum to that of a reference is necessary when conservation treatments with aqueous solutions or organic solvents are being considered for a work of art, since solubility properties can sometimes vary within the same pigment group. The Raman spectra of 21 yellow synthetic organic pigments, belonging to the monoazo, monoazo lakes, diarylide, disazo condensation, benzimidazolone, bisacetoacetarylide, azo-methine metal complex, isoindolinone and isoindoline groups are presented. Since modern artists frequently mixed paint developed for other applications, in addition to colorants developed as artists' paints, other synthetic organic pigments were included in the spectral database. Two monoazo pigments, Pigment Yellow 1 and Pigment Yellow 3, a benzimidazolone, Pigment Yellow 154 and a phthalocynanine, Pigment Green 7, were identified in sample cross-sections from four modern and contemporary paintings in the collection of The Museum of Modern Art in Ljubljana, Slovenia.     

Analyses of the surfaces of concrete by Raman and FT‐IR spectroscopies: comparative study of hardened samples after demoulding and after organic post‐treatment

Concrete surfaces were studied by two spectroscopic techniques, FT‐IR (in ATR mode) and Raman, to establish a nondestructive method to analyze the distribution of hydrated and organic phases. The surface composition of ordinary clinker, polished concrete, concrete after demoulding, and coated concrete as used in building construction was studied. The clinker's mineral phases and the polished concrete were first analyzed by Raman spectroscopy to determine a spectrum database of the specific phases located on the surface of the concrete. Then, both spectroscopic techniques were used to analyze, directly, the surface of hardened concrete after demoulding. No impact of roughness or porosity was highlighted using Raman spectroscopy; many cementitious, or hydrated phases (alite, belite, tricalcium aluminate, ferrite, portlandite and ettringite) were clearly identified. FT‐IR in ATR mode only identified some hydrated phases: portlandite and CaO? SiO₂? H₂O (C? S? H), but organic residues from the demoulding oil were characterized by this technique. Furthermore, the convenience of using these techniques together was tested by analyzing the composition of concrete surfaces coated by different organic post‐treatments. FT‐IR spectroscopy was useful to identify the main organic groups at the concrete surface, whereas Raman spectroscopy was especially able to characterize the mineral/hydrated phases under a thick post‐treatment layer (constituted of polyester varnish). Due to their own specificities, these complementary techniques should be used together to easily identify all the mineral phases and organic residues/coatings on concrete surfaces. Copyright © 2010 John Wiley & Sons, Ltd.     

Micro-Raman analysis of coloured lithographs

Raman micro-spectroscopy was chosen for analysis and identification of the pigments present in four nineteenth-century hand-coloured lithographs, as this technique has several advantages over others for this purpose. The possibility of performing completely non-destructive analysis without any sampling is probably one of its most favourable qualities for art analysis. Raman spectroscopy can also be used to determine some pigments that cannot be detected using FTIR, such as vermilion, carbon blacks, cadmium pigments, etc. Among others, Prussian blue, ultramarine blue, carbon black, chrome yellow, yellow ochre, red lead, red iron oxide, burnt Sienna, indigo blue, chrome orange, phthalocyanine green, and some other organic pigments, were determined in the specimens. The results obtained have led to doubts about the age of the lithographs.     

FT-NIR spectroscopy for non-invasive identification of natural polymers and resins in easel paintings

In the present study, the analytical strengths and limitations of near-infrared (NIR) spectroscopy to non-invasively characterize organic components in painting materials have been investigated. In spite of the increased amount of information available today from advanced modern analytical instrumentations dedicated to cultural heritage, the non-invasive identification of materials belonging to the wide class of organic compounds historically used in paintings is still a challenging task. Near-infrared spectroscopy offers several attractive features that make this technique particularly suitable to this purpose. In fact, it is non-invasive, allows for non-contact measurements in reflectance mode, gives molecular information on complex macromolecules, and can be performed on-site by means of portable devices. First-derivative transformation of reflectance spectroscopic data has been applied to provide a simple and fast way to deduce more information from NIR spectra. This approach has allowed spectral features to be identified that can be useful to distinguish different compounds belonging to the classes of lipids, proteins, and resins. To this purpose, at first, a spectral database of pure standard has been collected. Our analytical approach was then successfully validated on pictorial models reproducing the typical stratigraphy of an easel painting. As final step, the study of a real painting has been attempted and a drying oil, animal glue, and a terpenic natural resin, as well as an earth pigment were clearly identified, as cross-validated by GC-MS analysis.     

Anatase—a pigment in ancient artwork or a modern usurper?

Fragments of wall-paintings from Roman villas in Easton Maudit, which date from ca 150 AD have been studied by Raman spectroscopy. An intact ancient Roman paint pot discovered in the remains of a villa in Castor, Cambridgeshire, still containing a mixture of white and red pigment was also analysed and the pigments identified as haematite and anatase. The discovery of anatase in the intact artist’s paint pot, particularly, and also on fragments of broken paint pots from the Easton Maudit villa site, is a unique contribution to current knowledge of ancient European pigment history, because the presence of this mineral has not hitherto been recognised fully in an ancient artist’s palette. The relative spectral response of anatase and haematite in the Raman data is compared with that of anatase and other red pigments such as minium, cinnabar, and litharge.     

Optical fibre SERS sensors

Surface-enhanced Raman scattering (SERS) has established itself as an important analytical technique. However, efforts to transfer the technology from the laboratory to the production line, clinic or field have been frustrated by the lack of robust affordable substrates and the complexity of interfacing between sample and spectrometer. Prompted by the success of optical fibre systems for implementing normal Raman scattering spectroscopy in remote locations and biomedical applications, attention has now shifted to the development of SERS-active optical fibres. Other workers have attempted to develop SERS probes with extended interaction lengths and both far-field and near-field SERS imaging techniques for high-resolution chemical mapping of surfaces. This review discusses the development of these technologies and presents the current state of the art. Although recent developments show great promise, some outstanding challenges and opportunities remain to be addressed.     

Characterization of fresh and aged natural ingredients used in historical ointments by molecular spectroscopic techniques: IR,Raman and fluorescence

Natural organic materials used to prepare pharmaceutical mixtures including ointments and balsams have been characterized by a combined non-destructive spectroscopic analytical approach. Three classes of materials which include vegetable oils (olive, almond and palm tree), gums (Arabic and Tragacanth) and beeswax are considered in this study according to their widespread use reported in ancient recipes. Micro-FTIR, micro-Raman and fluorescence spectroscopies have been applied to fresh and mildly thermally aged samples. Vibrational characterization of these organic compounds is reported together with tabulated frequencies, highlighting all spectral features and changes in spectra which occur following artificial aging. Synchronous fluorescence spectroscopy has been shown to be particularly useful for the assessment of changes in oils after aging; spectral difference between Tragacanth and Arabic gum could be due to variations in origin and processing of raw materials. Analysis of these materials using non-destructive spectroscopic techniques provided important analytical information which could be used to guide further study.     

Spectral analysis of pharmaceutical formulations prepared according to ancient recipes in comparison with old museum remains

A study of the composition of the remains of ancient ointments from museums was undertaken to enable understanding of the preparation techniques. Comparison of ancient recipes from different historical periods and spectroscopic characteristics of inorganic and/or organic remains recovered in museum vessels enabled preparation of ancient pharmaceutical–cosmetic formulations. Farmacopea Augustana by Occo was one the most important books studied for the 14 formulations prepared in the laboratory. Three formulations are discussed in detail and raw materials and new preparations were proposed for ozone ageing. The most important micro Raman results are discussed. The spectra of the raw materials lipids, beeswax, and resins are discussed; beeswax and pig suet (axŭngia) Raman spectra were found to be similar, but different from those of the aged oils. SERS was applied to ancient ointments and galbanum and the Raman spectra are reported and discussed for the first time.     

Raman spectroscopy of stercorite H(NH4)Na(PO4)·4H2O--a cave mineral from Petrogale Cave, Madura, Eucla, Western Australia

Raman spectroscopy complimented with infrared spectroscopy has been used to characterise the mineral stercorite H(NH4)Na(PO4)·4H2O. The mineral stercorite originated from the Petrogale Cave, Madura, Eucla, Western Australia. This cave is one of many caves in the Nullarbor Plain in the South of Western Australia. These caves have been in existence for eons of time and have been dated at more than 550 million years old. The mineral is formed by the reaction of bat guano chemicals on calcite substrates. A single Raman band at 920 cm(-1) defines the presence of phosphate in the mineral. Antisymmetric stretching bands are observed in the infrared spectrum at 1052, 1097, 1135 and 1173 cm(-1). Raman spectroscopy shows the mineral is based upon the phosphate anion and not the hydrogen phosphate anion. Raman and infrared bands are found and assigned to PO4(3-), H2O, OH and NH stretching vibrations. The detection of stercorite by Raman spectroscopy shows that the mineral can be readily determined; as such the application of a portable Raman spectrometer in a 'cave' situation enables the detection of minerals, some of which may remain to be identified.     

Raman spectroscopic analysis of dragon's blood resins-basis for distinguishing between Dracaena(Convallariaceae), Daemonorops(Palmae) and Croton(Euphorbiaceae)

"Dragon[prime or minute]s blood" is the name applied to the deep-red coloured resin obtained from various plants. The original source in Roman times, used by many cultures and esteemed for its depth of colour and mystical association, was the dragon tree Dracaena cinnabari(Convallariaceae), found only on the Indian Ocean island of Socotra, (Yemen). Additional sources emerged later, including another species of Dracaena, D. draco, from the Canary Islands and Madeira, and species in the genera Daemonorops(Palmae) from South East Asia and Croton(Euphorbiaceae) from tropical parts of both the New and Old Worlds. In this study, examples of dragon's blood resins from the Economic Botany Collections at the Royal Botanic Gardens, Kew, dating from 1851 to 1993, have been analysed non-destructively using Raman spectroscopy. The Raman spectra of well-documented, provenanced specimens have been used to establish the source of specimens of questionable or unknown origin. It has also been possible from the Raman spectra to indicate whether processing of the resins has been undertaken in the preparation of the specimens before their deposition at Kew.