Combination Of Raman Microscopy Research Articles

A Spectroscopy-Based Multi-Analytical Approach for Studies in Conservation: Decorations in the Alexander Palace (Tsarskoye Selo)

We studied the painted decorations found during recent restoration work in the Alexander Palace in Tsarskoye Selo. Optical/laser spectroscopic methods were applied to obtain a characterization of the materials, pigments, and binders in use and, possibly, their degradation. We analyzed samples of the original Art Nouveau style decoration that was detached in 2019 during conservation work at the State Office of Emperor Nicholas II. A combination of Raman microscopy, infrared spectroscopy, and elemental analysis (obtained from the optical emission following laser plasma formation) allowed us to obtain detailed information on the materials used. The precious pigments of the artist’s green-blue palette and the binder used (drying oil) were identified. A mixture of blue (Prussian blue and ultramarine blue), white (lead white and barium white), and yellow (chrome yellow and zinc yellow) pigments determined the different blue hues used. The use of bronze paint in the dark blue area, which was identified as a brass powder applied with a drying oil as a binder, was also demonstrated.

Surface Molecular Imprinting with Bacteria: Visualizing Re-Binding and Selectivity

Introduction Surface molecular imprinting [1,2] is one of the preferred techniques when it comes to generating biomimetic recognition for sensing bioanalytes. Limiting oneself to the surface of the respective molecularly imprinted polymer (MIP) usually is no problem, because biospecies are usually large enough that few binding events lead to measurable sensor responses, e.g. for mass-sensitive bacteria detection [3]. However, the detection limits of quartz crystal microbalances (QCM) do usually not meet the needs for detecting contamination of real-life samples when it comes to pathogen detection. The (geometrical) limitation of surface MIP in that regard makes it necessary to think about suitable systems for pre-concentration prior to the sensor measurement. Herein, we report Raman Microscopy studies on bacteria surface MIP aiming at visualizing different areas of the polymer and distinguishing between different species bound to the surface. Furthermore, we assess the binding properties of MIP resulting from Pickering emulsion polymerization [4] as a possible approach to selectively pre-concentrate bacteria. Experimental For molecular imprinting, we chose a non-pathogenic strain of Escherichia coli (E. coli) as a model species. We prepared surface MIP of two different morphologies based on polystyrenes cross-linked with divinyl benzene utilizing azo-bisisobutyronitrile (AIBN) as a radical initiator. For MIP thin films, we prepared respective mixtures of monomer and crosslinker, pre-polymerized at 70°C, and then spin-coated onto the respective devices. In parallel, we prepared stamps comprising E. coli on their surfaces. Then, we pressed the stamp into the oligomer thin films on the respective surface and cured the system at 80°C over night. We took all spectra on a confocal Raman Microscopy system (WiTec alpha300RAS) at an excitation wavelength of l=532nm. Chemometric data evaluation relied on the software package Solo&Mia by Eigenvector. For Pickering emulsion, we prepared mixtures of styrene and divinylbenzene and used bacteria to emulsify them in either distilled water, or a suitable buffer system (e.g. 10mM PBS) and polymerized the bacteria stabilized droplets at 37°C. Then, we removed E. coli cells from the polymer surface via solvent extractions in acetic acid + SDS, water and methanol. Results and Discussion In a first step it was essential to clarify if one can use the Raman spectra of different bacteria species and polymer, to differentiate between those, respectively. Polymer and bacteria spectra obviously differ from each other enough to distinguish them by the naked eye. However, this is not the case for the Raman emission spectra of different bacteria species, because they all contain nucleic acids, proteins, and lipids. Therefore it is necessary to rely on chemometric strategies – modified partial least squares discriminant analysis (PLS-DA) in the concrete case – to achieve such goal. This is indeed possible: Fig. 1A shows the outcome for four different bacteria species, namely Escherichia coli, Lactococcus lactis, Bacillus cereus and Staphylococcus epidermidis that clearly demonstrates separation of the species according to their spectral properties. Knowing the specific spectral ranges also allows for generating false-color images showing which areas of a bacteria MIP surfaces reveal pure polymer and which parts contain bacteria of different species. It is even possible to go one step further: Comparing AFM and false color so-called RamanTV images (see Fig. 1B) for an example of the latter) demonstrates that the Raman microscope is even useful for distinguishing polymer surface and imprinted cavities. For such micrometer-sized analytes, the combination of Raman microscopy and AFM hence allows for generating maps showing all possible types of surfaces, i.e. polymer matrix as well as both unoccupied cavities and different bacteria species present at the polymer surface. This opens the perspective of directly visualizing occupancy and selectivity of the respective MIP.AFM turned out useful to visualize such imprints not only on flat surfaces, but also on curved ones: Figure 1C) and 1D) reveal an imprinted site on the surface of a miroparticle resulting from emulsion polymerization, Fig. 1E) an SEM image showing occupied and empty cavities. One can generate such cavities in different polymer matrices, for instance when replacing divinyl benzene as a cross-linker by trimethyolylpropane trimethacrylate (TRIM), or ethylene glycol dimethacrylate (EGDMA). In both cases, the respective imprinted surface reveal cavities that fit the size and shape of the template bacteria. Scanning electron microscopy also revealed that bacteria re-occupy those cavities if one exposes the particles to to bacteria suspensions. This leads to two conclusions: first, one can tune polymer properties without destroying the emulsion structure necessary for Pickering emulsion. This is of interest for bringing the artificial system closer to physiological conditions. Second, those particles are inherently suitable for selectively pre-concentrating those species from larger samples. Figure 1

The combination of Raman microscopy and electron microscopy – Practical considerations of the influence of vacuum on Raman microscopy

Due to the specific vacuum requirements for scanning electron microscopy (SEM), the Raman microscope has to operate in vacuum in a correlative Raman-SEM, which is a type of microscope combination that has recently increased in popularity. This works considers the implications of conducting Raman microscopy under vacuum, as opposed to operating in ambient air, the standard working regime of this technique. We show that the performance of the optics of the Raman microscope are identical in both conditions, but laser beam-sample interactions, such as fluorescent bleaching and beam damage, might be different due to the lack of oxygen in vacuum. The bleaching of the fluorescent background appears to be mostly unaffected by the lack of oxygen, except when very low laser powers are used. Regarding laser-beam damage, organic samples are more sensitive in vacuum than in air, whereas no definite verdict is possible for inorganic samples. These findings have practical implications for the application of correlative Raman-SEM, as low laser powers, or in extreme cases cryo-methods, need to be used for organic samples that appear only moderately beam sensitive under usual ambient air.

Surface Molecular Imprinting with Bacteria: Visualizing Re-Binding and Selectivity

Introduction Surface molecular imprinting [1,2] is one of the preferred techniques when it comes to generating biomimetic recognition for sensing bioanalytes. Limiting oneself to the surface of the respective molecularly imprinted polymer (MIP) usually is no problem, because biospecies are usually large enough that few binding events lead to measurable sensor responses, e.g. for mass-sensitive bacteria detection [3]. However, the detection limits of quartz crystal microbalances (QCM) do usually not meet the needs for detecting contamination of real-life samples when it comes to pathogen detection. The (geometrical) limitation of surface MIP in that regard makes it necessary to think about suitable systems for pre-concentration prior to the sensor measurement. Herein, we report Raman Microscopy studies on bacteria surface MIP aiming at visualizing different areas of the polymer and distinguishing between different species bound to the surface. Furthermore, we assess the binding properties of MIP resulting from Pickering emulsion polymerization [4] as a possible approach to selectively pre-concentrate bacteria. Experimental For molecular imprinting, we chose a non-pathogenic strain of Escherichia coli (E. coli) as a model species. We prepared surface MIP of two different morphologies based on polystyrenes cross-linked with divinyl benzene utilizing azo-bisisobutyronitrile (AIBN) as a radical initiator. For MIP thin films, we prepared respective mixtures of monomer and crosslinker, pre-polymerized at 70°C, and then spin-coated onto the respective devices. In parallel, we prepared stamps comprising E. coli on their surfaces. Then, we pressed the stamp into the oligomer thin films on the respective surface and cured the system at 80°C over night. We took all spectra on a confocal Raman Microscopy system (WiTec alpha300RAS) at an excitation wavelength of l=532nm. Chemometric data evaluation relied on the software package Solo&Mia by Eigenvector. For Pickering emulsion, we prepared mixtures of styrene and divinylbenzene and used bacteria to emulsify them in either distilled water, or a suitable buffer system (e.g. 10mM PBS) and polymerized the bacteria stabilized droplets at 37°C. Then, we removed E. coli cells from the polymer surface via solvent extractions in acetic acid + SDS, water and methanol. Results and Discussion In a first step it was essential to clarify if one can use the Raman spectra of different bacteria species and polymer, to differentiate between those, respectively. Polymer and bacteria spectra obviously differ from each other enough to distinguish them by the naked eye. However, this is not the case for the Raman emission spectra of different bacteria species, because they all contain nucleic acids, proteins, and lipids. Therefore it is necessary to rely on chemometric strategies – modified partial least squares discriminant analysis (PLS-DA) in the concrete case – to achieve such goal. This is indeed possible: Fig. 1A shows the outcome for four different bacteria species, namely Escherichia coli, Lactococcus lactis, Bacillus cereus and Staphylococcus epidermidis that clearly demonstrates separation of the species according to their spectral properties. Knowing the specific spectral ranges also allows for generating false-color images showing which areas of a bacteria MIP surfaces reveal pure polymer and which parts contain bacteria of different species. It is even possible to go one step further: Comparing AFM and false color so-called RamanTV images (see Fig. 1B) for an example of the latter) demonstrates that the Raman microscope is even useful for distinguishing polymer surface and imprinted cavities. For such micrometer-sized analytes, the combination of Raman microscopy and AFM hence allows for generating maps showing all possible types of surfaces, i.e. polymer matrix as well as both unoccupied cavities and different bacteria species present at the polymer surface. This opens the perspective of directly visualizing occupancy and selectivity of the respective MIP.AFM turned out useful to visualize such imprints not only on flat surfaces, but also on curved ones: Figure 1C) and 1D) reveal an imprinted site on the surface of a miroparticle resulting from emulsion polymerization, Fig. 1E) an SEM image showing occupied and empty cavities. One can generate such cavities in different polymer matrices, for instance when replacing divinyl benzene as a cross-linker by trimethyolylpropane trimethacrylate (TRIM), or ethylene glycol dimethacrylate (EGDMA). In both cases, the respective imprinted surface reveal cavities that fit the size and shape of the template bacteria. Scanning electron microscopy also revealed that bacteria re-occupy those cavities if one exposes the particles to to bacteria suspensions. This leads to two conclusions: first, one can tune polymer properties without destroying the emulsion structure necessary for Pickering emulsion. This is of interest for bringing the artificial system closer to physiological conditions. Second, those particles are inherently suitable for selectively pre-concentrating those species from larger samples.

Single‐cell Raman microscopy of microengineered cell scaffolds

AbstractStudying cells in a three‐dimensional (3D) environment has great potential in understanding cell behaviours such as morphology, proliferation, differentiation, and migration. Microengineered 3D cell scaffolds with precise defined geometries have offered a new approach to study cell behaviour and its interactions with scaffolds. The use of Raman spectroscopy to characterise biomolecules is a rapidly expanding area and has been implemented in numerous fields including pharmacology, microbiology, toxicology, and single‐cell studies. However, one area where it remains unexploited despite the vast potential of the technique is in the investigation of 3D cell scaffolds. A combination of Raman microscopy and chemometric approaches have employed to investigate the structure and biochemistry of nanofabricated scaffolds and a cell–scaffold complex. The 3D Raman mapping combined with the use of nanofabricated 3D scaffolds offers a unique opportunity to assess the influence of scaffold architecture on cell body and cell nuclei morphology and biochemistry. For the first time, we have cultured a human epithelial colorectal adenocarcinoma cell line on OrmoComp scaffolds and determined the structure and biochemistry of nanofabricated scaffolds and a cell–scaffold complex with the use of Raman microscopy combined with appropriate data analysis protocols. The results demonstrate the potential of 3D Raman mapping for identifying biochemical and physical variation within single cells as they grow and adhere to 3D scaffolds.

Raman Microspectroscopic Evidence for the Metabolism of a Tyrosine Kinase Inhibitor, Neratinib, in Cancer Cells.

Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label‐free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC‐MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics.

Nanoparticle-nanoparticle vs. nanoparticle-substrate hot spot contributions to the SERS signal: studying Raman labelled monomers, dimers and trimers.

We used a combination of Raman microscopy, AFM and TEM to quantify the influence of dimerization on the surface enhanced Raman spectroscopy (SERS) signal for gold and silver nanoparticles (NPs) modified with Raman reporters and situated on gold, silver, and aluminum films and a silicon wafer. The overall increases in the mean SERS enhancement factor (EF) upon dimerization (up by 43% on average) and trimerisation (up by 96% on average) of AuNPs and AgNPs on the studied metal films are within a factor of two, which is moderate when compared to most theoretical models. However, the maximum ratio of EFs for some dimers to the mean EF of monomers can be as high as 5.5 for AgNPs on a gold substrate. In contrast, for dimerization and trimerization of gold and silver NPs on silicon, the mean EF increases by 1-2 orders of magnitude relative to the mean EF of single NPs. Therefore, hot spots in the interparticle gap between gold nanoparticles rather than hot spots between Au nanoparticles and the substrate dominate SERS enhancement for dimers and trimers on a silicon substrate. However, Raman labeled noble metal nanoparticles on plasmonic metal films generate on average SERS enhancement of the same order of magnitude for both types of hot spot zones (e.g. NP/NP and NP/metal film).

Characterization of fine mode atmospheric aerosols by Raman microscopy and diffuse reflectance FTIR.

A combination of Raman microscopy and diffuse reflectance Fourier transform infrared spectroscopy (FTIR) has been used for the characterization of fine mode (<1 μm) tropospheric aerosols. Peak fitting was used to identify five overlapping bands in the Raman spectra. These bands have been identified as due to combustion generated carbon soot as well as large molecular organic carbon species. The fwhm of the D band at 1400 cm(-1) as well as the ratio of intensities of the D3 band at 1550 cm(-1) to the G band at 1580 cm(-1) can serve as a measure of the aerosol organic carbon content. Raman microscopy combined with spectral mapping capabilities was used to investigate the composition of the fine mode aerosols at the particle level, allowing for the direct determination of aerosol mixing state. Results showed that the fine aerosols were predominately internally mixed particles composed of carbon soot coated with molecular organic carbon species. Characterization of the aerosols by diffuse reflectance FTIR showed that the major organic carbon species were polycarboxylates and polysaccharide-like species typical of humic-like substances (HULIS).

Detailed spectroscopic analysis of complex multi‐component materials using a combination of Raman mapping with BTEM

AbstractThe combined application of Raman microscopy and self‐modeling curve resolution techniques can address a wide range of material characterization problems. In particular, the combination of Raman microscopy and the Band‐Target Entropy Minimization (BTEM) algorithm has been applied to various organic, inorganic, pharmaceutical and bio‐material related problems. In the present contribution, the principles behind this type of analysis are reviewed, followed by a number of case‐by‐case studies. For each of these examples, a Raman microscopic mapping measurement (consisting of 100 s up to 1000 s of spectra) is performed, followed by BTEM analysis which provides the underlying pure component spectra of the constituents present in the system without the use of any a priori information. In most cases, outstanding signal‐to‐noise ratios for components at the 0.1‐1.0 % level can be obtained, and sometimes trace constituents can also be detected. Subsequently, the identity of the components can be determined by comparison to spectral libraries. Finally, the reconstructed pure component spectra can be further used to obtain the spatial distribution of the constituents present in the sample. Copyright © 2012 John Wiley &amp; Sons, Ltd.

Analysis of the Residuals in Grave Goods From the Vaccaea Era at the Necropolis of “Las Ruedas” in Pintia

ABSTRACT The archaeological site of Pintia (Padilla de Duero/Peñafiel, Valladolid), considered one of the first cities on the Iberian Peninsula, has yielded very interesting archaeological records regarding the different functional areas that involve the oppidum. Pottery, metal, and bone/skeletal remains are very well preserved, being optimal for analysis and study of the ancient food contained, such as milk-based products, animal fats, beer, and wine. The combination of Raman Microscopy (RM) with other analytical techniques such as Light Microscopy (LM) and Environmental Scanning Electron Microscopy equipped with Energy Dispersive X-ray Spectroscopy (ESEM-EDX) becomes a powerful tool to obtain information on the possible content in the ceramic receptacles from historical-archaeological heritage. It can lead to an interpretation of their specific use or functionality. In this work, a unique sample preparation was used for all techniques, which lead to a very simple procedure and documentation. The degradation and aging of natural fibers and the presence of organic matter with low crystallinity and calcium oxalate–related residuals can be studied by these combined techniques. The archaeological residuals can be separated from modern contamination coming from agriculture processes, surrounding land minerals, or fresh vegetal matter.

Pure component Raman spectral reconstruction from glazed and unglazed Yuan, Ming, and Qing shards: a combined Raman microscopy and BTEM study

AbstractRaman mapping measurements were performed on the glazed and unglazed surfaces of shards excavated from Yuan, Ming, and Qing dynasty strata. A number of areas on each surface were chosen. Circa 21 × 21 pixels were measured for each area using both 514 and 785‐nm laser as the Raman excitation. Data were collected from 100–3600 cm−1. Many sets of spectra exhibited very intense fluorescence. In spite of the intense fluorescence, the resulting sets of spectra were collated and analyzed together using the band‐target entropy minimization (BTEM) algorithm. Pure component spectral estimates of many of the major components were achieved, without the use of any a priori information such as spectral libraries. These include α‐silica quartz, carbon, anatase, cobalt oxides, hematite, glassy silicate, and lanthanide complexes. In addition, two further unidentified pure component spectra A and B were recovered as well as an interference pattern due to the microscopic texture of the shards (associated with small particle/thin layer domains). The carbon was primarily present in elemental form, i.e. mixture of amorphous and graphitic (unordered and ordered domains); however there is an evidence of some partial oxidation, i.e. formation of carboxylates. The interference patterns and the lanthanide complexes were only observed when using the longer wavelength red laser. The cobalt oxides and the anatase were only observed when using the green laser. In summary, the combination of Raman microscopy and BTEM has allowed the enumeration of many of the underlying spectral patterns present and hence unambiguous identification of the major individual components present in the archaeological samples. This approach would appear applicable to other classes of archaeological materials as well. Limitations and extensions of the present approach are discussed. Copyright © 2010 John Wiley &amp; Sons, Ltd.

Towards a nondestructive chemical characterization of biofilm matrix by Raman microscopy

In this study, the applicability of Raman microscopy (RM) for nondestructive chemical analysis of biofilm matrix, including microbial constituents and extracellular polymeric substances (EPS), has been assessed. The examination of a wide range of reference samples such as biofilm-specific polysaccharides, proteins, microorganisms, and encapsulated bacteria revealed characteristic frequency regions and specific marker bands for different biofilm constituents. Based on received data, the assignment of Raman bands in spectra of multispecies biofilms was performed. The study of different multispecies biofilms showed that RM can correlate various structural appearances within the biofilm to variations in their chemical composition and provide chemical information about a complex biofilm matrix. The results of RM analysis of biofilms are in good agreement with data obtained by confocal laser scanning microscopy (CLSM). Thus, RM is a promising tool for a label-free chemical characterization of different biofilm constituents. Moreover, the combination of RM with CLSM analysis for the study of biofilms grown under different environmental conditions can provide new insights into the complex structure/function correlations in biofilms.

A Raman spectroscopic study of thermally treated glushinskite—the natural magnesium oxalate dihydrate

Raman spectroscopy has been used to study the thermal transformations of natural magnesium oxalate dihydrate known in mineralogy as glushinskite. The data obtained by Raman spectroscopy was supplemented with that of infrared emission spectroscopy. The vibrational spectroscopic data was complimented with high resolution thermogravimetric analysis combined with evolved gas mass spectrometry. TG–MS identified two mass loss steps at 146 and 397 °C. In the first mass loss step water is evolved only, in the second step carbon dioxide is evolved. The combination of Raman microscopy and a thermal stage clearly identifies the changes in the molecular structure with thermal treatment. Glushinskite is the dihydrate phase in the temperature range up to the pre-dehydration temperature of 146 °C. Above 397 °C, magnesium oxide is formed. Infrared emission spectroscopy shows that this mineral decomposes at around 400 °C. Changes in the position and intensity of the CO and CC stretching vibrations in the Raman spectra indicate the temperature range at which these phase changes occur.

Identification by RAMAN Microscopy of magnesian vivianite formed from Fe2+, Mg, Mn2+and P043-’ in a Roman camp near fort Vechten, Utrecht, The Netherlands

AbstractThe presence of a magnesian vivianite (Fe2+)2.5(Mg,Mn,Ca)0.5(PO4)2·8H2O, has been identified in a soil sample from a Roman camp near Fort Vechten, The Netherlands, using a combination of Raman microscopy and scanning electron microscopy. An unsubstituted vivianite and baricite were characterised for comparative reasons. The split phosphate-stretching mode is recognised around 1115, 1062 and 1015 cm−1, while the corresponding bending modes are found around 591, 519, 471 and 422 cm−1. The substitution of Mg and Mn for Fe2+in the crystal structure causes a shift towards higher wavenumbers compared to pure vivianite. As shown by the baričite sample substitution causes a broadening of the bands. The observed broadening however is larger than can be explained by substitution alone. The low intensity of the water bands, especially in the OH-stretching region between 2700 and 3700 cm−1indicates that the magnesian vivianite is partially dehydrated, which explains the much larger broadening than the observed broadening caused by substitution of Mg and Mn in vivianite and baričite.

Thermal treatment of weddellite—a Raman and infrared emission spectroscopic study

Thermal transformations of natural calcium oxalate dihydrate known in mineralogy as weddellite have been undertaken using a combination of Raman microscopy and infrared emission spectroscopy. The vibrational spectroscopic data was complimented with high resolution thermogravimetric analysis combined with evolved gas mass spectrometry. TG–MS identified three mass loss steps at 114, 422 and 592 °C. In the first mass loss step water is evolved only, in the second and third steps carbon dioxide is evolved. The combination of Raman microscopy and a thermal stage clearly identifies the changes in the molecular structure with thermal treatment. Weddellite is the phase in the temperature range up to the pre-dehydration temperature of 97 °C. At this temperature, the phase formed is whewellite (calcium oxalate monohydrate) and above 114 °C the phase is the anhydrous calcium oxalate. Above 422 °C, calcium carbonate is formed. Infrared emission spectroscopy shows that this mineral decomposes at around 650 °C. Changes in the position and intensity of the CO and CC stretching vibrations in the Raman spectra indicate the temperature range at which these phase changes occur.

Taking A Closer Look At Art

Spectroscopy can help in art conservation efforts by providing a means to identify the pigments in a work of art. At last month's Leermakers Symposium, titled 'Where Chemistry Meets Art and Archaeology, at Wesleyan University, Middletown, Conn., Robin J. H. Clark, an inorganic chemist at University College London who claims a lifelong love of color, described the combination of Raman microscopy with laser-induced breakdown spectroscopy (LIBS) to analyze painted artworks. His group used these techniques to analyze a 19th-century Russian icon, a painting of Saint Nicholas on a wooden panel. They undertook the analysis to determine what pigments were used in the original painting and in subsequent restoration efforts, which Clark said have been extensive and aggressive. LIBS is an atomic emission spectroscopic technique in which nanosecond laser pulses ablate a small amount of material from the surface. It can be performed on the artwork itself without taking a sample, but because the laser pulses ...

Fourier transform Raman spectroscopic study of prehistoric rock paintings from the Big Bend region, Texas

Samples of prehistoric rock paintings from the Big Bend region of Texas were analysed using Raman microscopy. This was the first chemical/mineralogical study of ancient paints from the region, and thus allowed us to test the utility of Raman microscopy as the principal tool for analysing pictograph rock paints. The method is non-destructive and non-invasive, two extremely important criteria for the study of these artifacts. The Raman study was followed by scanning electron microscopic, Fourier transform IR spectroscopic, powder x-ray diffractometric, and gas chromatographic–mass spectrometric analyses to test the results obtained from the Raman analysis. It was concluded that the combination of Raman microscopy and scanning electron microscopy provided the most information and required the least amount of sample. Mineralogical, chemical and elemental information was obtained on the paint materials, including the pigments and binders. Furthermore, it was possible to identify and establish the relationship between the paints and the natural surrounding matrix, which included calcium sulphate (gypsum) and calcium oxalate monohydrate (whewellite). Copyright © 1999 John Wiley & Sons, Ltd.