Plant-based red colouration of shell beads 15,000 years ago in Kebara Cave, Mount Carmel (Israel).

Decorating the living space, objects, body and clothes with colour is a widespread human practice. While the habitual use of red mineral pigments (such as iron-oxide, e.g., ochre) by anatomically modern humans started in Africa about 140,000 years ago, the earliest documentation of the use of organic plant or animal-based red pigments is known from only 6,000 years ago. Here, we report the oldest reliable evidence of organic red pigment use 15,000 years ago by the first sedentary hunter-gatherers in the Levant. SEM-EDS and Raman Spectroscopy analyses of 10 red-stained shell beads enabled us to detect and describe the use of a colourant made of Rubiaceae plants roots (Rubia spp., Asperula spp., Gallium spp.) to colour personal adornments from the Early Natufian of Kebara cave, Mount Carmel, Israel. This adds a previously unknown behavioural aspect of Natufian societies, namely a well-established tradition of non-dietary plant processing at the beginning of the sedentary lifestyle. Through a combined multidisciplinary approach, our study broadens the perspectives on the ornamental practices and the chaînes opératoires of pigmenting materials during a crucial period in human history.

Chemical characterization of inks in skin reactions to tattoo.

Skin reactions are well described complications of tattooing, usually provoked by red inks. Chemical characterizations of these inks are usually based on limited subjects and techniques. This study aimed to determine the organic and inorganic composition of inks using X-ray fluorescence spectroscopy (XRF), X-ray absorption spectroscopy (XANES) and Raman spectroscopy, in a cohort of patients with cutaneous hypersensitivity reactions to tattoo. A retrospective multicenter study was performed, including 15 patients diagnosed with skin reactions to tattoos. Almost half of these patients developed skin reactions on black inks. XRF identified known allergenic metals - titanium, chromium, manganese, nickel and copper - in almost all cases. XANES spectroscopy distinguished zinc and iron present in ink from these elements in endogenous biomolecules. Raman spectroscopy showed the presence of both reported (azo pigments, quinacridone) and unreported (carbon black, phtalocyanine) putative organic sensitizer compounds, and also defined the phase in which Ti was engaged. To the best of the authors' knowledge, this paper reports the largest cohort of skin hypersensitivity reactions analyzed by multiple complementary techniques. With almost half the patients presenting skin reaction on black tattoo, the study suggests that black modern inks should also be considered to provoke skin reactions, probably because of the common association of carbon black with potential allergenic metals within these inks. Analysis of more skin reactions to tattoos is needed to identify the relevant chemical compounds and help render tattoo ink composition safer.

Raman and ATR-FTIR analyses of medieval wall paintings from al-Qarawiyyin in Fez (Morocco).

A multi-analytical study has been undertaken to characterise wall paintings from the al-Qarawiyyin university in Fez, dating back to the 9th century and still in activity, with the aim of identifying the pigments used and going back to the painting techniques adopted by the Moroccan craftsmen of the time. The investigation crossed Raman and ATR-FTIR structural analyses along with XRF elemental ones, as well as colorimetric measurements. The colours and shades of six representative fragments collected during a recent excavation have been examined. Hence, red ochre (mainly hematite) was used to obtain brown-red colour, calcite for white, bistre for carbon-based black, while blue shades were achieved using azurite. Mixtures of pigments were created to widen the colour palette: cinnabar, minium and hematite were identified in orange hues while lapis lazuli and azurite were observed in grey-blue areas. The exploration of mortar layers revealed gypsum, gypsum/calcite and calcite type plasters. Furthermore, the observation of the morphological aspect of the interface between the plaster and the painting layer seems indicating that the lime-secco-painting skill had been the adopted painting technique.

Spectral characterisation of a traditional Mesoamerican dye: relationship between in situ identification on the 16th century Codex Borbonicus manuscript and composition of Justicia spicigera plant extract.

Characterisation of vegetal colourants in manuscripts is still a challenging task. Their identification using non-invasive techniques - since sampling is rarely possible - is valid only if there is evidence that the recorded signals are generated by compounds specific to the plant species. Otherwise, more extensive chemical characterisations are required to relate the non-invasive technique signals to the chemical composition of the dye extract and thus avoid misidentification. The case study of a traditional Mesoamerican dye found in the Codex Borbonicus is reported herein. The non-invasive identification of the colourant plant source and chemical characterisation of the coloured molecules were carried out through a multi-analytical technique approach. In situ Raman and UV-Vis emission fluorescence signals suggested the use of Justicia spicigera leaves to produce the brown paint layers of the manuscript. Analysis of the plant extract by liquid chromatography revealed that two compounds are mostly responsible for the colour, and these compounds were successfully isolated. Both contribute to the Raman spectra while only one of them is mainly responsible for the fluorescence recorded on Codex Borbonicus.

On-site contactless surface analysis of modern paintings from Galleria Nazionale (Rome) by reflectance FTIR and Raman spectroscopies.

Seven artworks representing the diversity of paints used around the 1960s and created by German and Italian painters (J. Albers, A. Bonalumi, L. Boille, T. Scialoja and M. Schifano) were studied on-site at the Galleria Nazionale d'Arte Moderna (Rome) with mobile instruments. We present a methodology based on Specular Reflectance Infrared Spectroscopy (SR-FTIR) adapted to unvarnished paintings. Complementary measurements have been performed by Raman spectroscopy. Characteristic bands regarding as-recorded infrared reflectance spectra and Kramers-Kronig Transformation-converted absorbance spectra are identified according to literature and reference spectra recorded on representative commercially available paints. To distinguish the different binders by SR-FTIR, we propose spectroscopic markers as the comparison of the intensity of carbonyl band around 1730-1735 cm-1 with bands at ~1160 (for acrylic), ~1230 (for PVAc), and 1270 cm-1 (for alkyds). On the other hand, oil/resin binders are characterized by intense and thin νCH2, νCH3 IR absorption bands around 2920-2850 cm-1, combined with an intense 1260 cm-1 band and a characteristic concave cradle shape (between ca. 1750 and 1260 cm-1). The results obtained establish the relevance of the implemented mobile non-invasive infrared spectroscopy analytical approach by successfully identifying acrylic, vinylic, oil media and enamel paints, with or without opacifiers, which is supplemented by Raman analyses for pigment identification.

Early anthropogenic use of hematite on Aurignacian ivory personal ornaments from Hohle Fels and Vogelherd caves, Germany.

The Aurignacian (ca. 43-35 ka) of southwestern Germany is well known for yielding some of the oldest artifacts related to symbolic behaviors, including examples of figurative art, musical instruments, and personal ornaments. Another aspect of these behaviors is the presence of numerous pieces of iron oxide (ocher); however, these are comparatively understudied, likely owing to the lack of painted artifacts from this region and time period. Several Aurignacian-aged carved ivory personal ornaments from the sites of Hohle Fels and Vogelherd contain traces of what appear to be red ocher residues. We analyzed these beads using a combination of macroanalytical and microanalytical methods, including scanning electron microscopy equipped with energy dispersive spectroscopy and Raman spectroscopy. We found that the residue is composed of the iron oxide mineral hematite (Fe2O3). Further analyses on associated archaeological sediments by X-ray diffraction revealed the absence of hematite and other iron oxide mineral phases, suggesting that the hematite residues were intentionally applied to the ivory personal ornaments by human agents. These findings have important implications as they represent evidence for the direct application of ocher on portable symbolic objects by early Homo sapiens in Europe. Furthermore, our results reveal shared behavioral practices from two key Aurignacian sites maintained over several millennia and illuminate aspects of pigment use and symbolic practices during a pivotal time in the cultural evolution of humans.

Correction: Divergence in the evolution of Paleolithic symbolic and technological systems: The shining bull and engraved tablets of Rocher de l'Impératrice.

[This corrects the article DOI: 10.1371/journal.pone.0173037.].

Rediscovering Ducos du†Hauron's Color Photography through a Review of His Three-Color Printing Processes and Synchrotron Microanalysis of His Prints.

Louis Ducos du Huron (1837-1920) dedicated his entire life to the elaboration of physical-chemical processes for color photography. This study aimed at highlighting his unique contribution to three-color printing through 1) an in-depth review of the many protocols he published and 2) the synchrotron-based IR and X-ray microanalysis of fragments sampled in three artworks. Ducos du Hauron's method relied on the preparation and assembly of three monochromes (red, blue, yellow). This study brings to light complex multistep recipes based on photochemistry (carbon print), organic, and inorganic chemistry. The various ingredients involved (e.g., pigments, dichromate gelatin, collodion, resin) were identified and localized through their spectroscopic signature, confirming the relevance of synchrotron spectromicroscopy for the characterization of historical photographs. The impressive correlation between texts and chemical analyses calls for a wider application to the history of photography.

Nanoscale modifications in the early heating stages of bone are heterogeneous at the microstructural scale.

Nanoscale studies of bone provide key indicators to evidence subtle structural changes that may occur in the biomedical, forensic and archaeological contexts. One specific problem encountered in all those disciplines, for which the identification of nanostructural cues could prove useful, is to properly monitor the effect of heating on bone tissue. In particular, the mechanisms at work at the onset of heating are still relatively unclear. Using a multiscale approach combining Raman microspectroscopy, transmission electron microscopy (TEM), synchrotron quantitative scanning small-angle X-ray scattering imaging (qsSAXSI) and polarized light (PL) microscopy, we investigate the ultrastructure of cortical bovine bone heated at temperatures < 300°C, from the molecular to the macroscopic scale. We show that, despite limited changes in crystal structure, the mineral nanoparticles increase in thickness and become strongly disorganized upon heating. Furthermore, while the nanostructure in distinct anatomical quadrants appears to be statistically different, our results demonstrate this stems from the tissue histology, i.e. from the high degree of heterogeneity of the microstructure induced by the complex cellular processes involved in bone tissue formation. From this study, we conclude that the analysis of bone samples based on the structure and organization of the mineral nanocrystals requires performing measurements at the histological level, which is an advantageous feature of qsSAXSI. This is a critical aspect that extends to a much broader range of questions relating to nanoscale investigations of bone, which could also be extended to other classes of nanostructured heterogeneous materials.

Divergence in the evolution of Paleolithic symbolic and technological systems: The shining bull and engraved tablets of Rocher de l'Impératrice.

The development of the Azilian in Western Europe 14,000 years ago is considered a "revolution" in Upper Paleolithic Archaeology. One of the main elements of this rapid social restructuring is the abandonment of naturalistic figurative art on portable pieces or on cave walls in the Magdalenian in favor of abstract expression on small pebbles. Recent work shows that the transformation of human societies between the Magdalenian and the Azilian was more gradual. The discovery of a new Early Azilian site with decorated stones in France supports this hypothesis. While major changes in stone tool technology between the Magdalenian and Azilian clearly mark important adaptive changes, the discovery of 45 engraved schist tablets from archaeological layers at Le Rocher de l'Impératrice attests to iconographic continuity together with special valorization of aurochs as shown by a "shining" bull depiction. This evidence suggests that some cultural features such as iconography may lag far behind technological changes. We also argue that eventual change in symbolic expression, which includes the later disappearance of figurative art, provides new insight into the probable restructuring of the societies.

Raman and NMR kinetics study of the formation of amidoamines containing N-hydroxyethyl groups and investigations on their Cu(II) complexes in water.

Three amidoamines containing the N-hydroxyethyl group (HOEt), namely (HOEt)2N(CH2)2C(O)NH2 (1), [(HOEt)2N(CH2)2C(O)NH]2CH2 (2) and HOEtN[(CH2)2C(O)NH2]2 (3) have been synthesized by reacting diethanolamine HOEt2NH with acrylamide and N,N'-methylenebisacrylamide (respectively 1 and 2) and ethanolamine HOEtNH2 with acrylamide (3). Four other compounds corresponding to 1 and 2, but derived from sec-amines Me2NH (4 and 5) and Et2NH (6 and 7) have been prepared for the sake of comparison of the spectroscopic features. All compounds have been obtained by the well-known aza-Michael addition between an N-nucleophile and an activated vinyl group. The reaction in water between diethanolamine and acrylamide leading to 1 has been monitored in situ by Raman and NMR spectroscopy, both techniques confirming second order kinetics and giving values for kinetic constants in excellent agreement. The coordination ability of 1 and 2 towards Cu2+ in water has been studied by the Job's plot method. Spectroscopic data indicate that ligand 1 prevalently forms a 4:1 Ligand/Metal complex with a (N,O3) coordination set on the equatorial plane of Cu2+, whereas ligand 2, containing two amide functionalities bridged by a methylene group, appears able to form a 1:1 Ligand/Metal chelate species, again with a (N,O3) donor set around copper.

Early Evidence for the Extensive Heat Treatment of Silcrete in the Howiesons Poort at Klipdrift Shelter (Layer PBD, 65 ka), South Africa.

Heating stone to enhance its flaking qualities is among the multiple innovative adaptations introduced by early modern human groups in southern Africa, in particular during the Middle Stone Age Still Bay and Howiesons Poort traditions. Comparatively little is known about the role and impact of this technology on early modern human behaviors and cultural expressions, due, in part, to the lack of comprehensive studies of archaeological assemblages documenting the heat treatment of stone. We address this issue through an analysis of the procedure used for heating and a technological analysis of a lithic assemblage recovered from one Howiesons Poort assemblage at Klipdrift Shelter (southern Cape, South Africa). The resulting data show extensive silcrete heat treatment, which adds a new dimension to our understanding of fire-related behaviors during the Howiesons Poort, highlighting the important role played by a heat treatment stage in the production of silcrete blades. These results are made possible by our new analytical procedure that relies on the analysis of all silcrete artifacts. It provides direct evidence of a controlled use of fire which took place during an early stage of core exploitation, thereby impacting on all subsequent stages of the lithic chaîne opératoire, which, to date, has no known equivalent in the Middle Stone Age or Middle Paleolithic record outside of southern Africa.

Raman Spectroscopy of cultural heritage Materials: Overview of Applications and New Frontiers in Instrumentation, Sampling Modalities, and Data Processing.

Rooted in the long tradition of Raman spectroscopy of cultural heritage materials, in this work we provide a personal perspective on recent applications and new frontiers in sampling modalities, data processing, and instrumentation.

A previously undescribed organic residue sheds light on heat treatment in the Middle Stone Age.

South Africa has in recent years gained increasing importance for our understanding of the evolution of 'modern human behaviour' during the Middle Stone Age (MSA). A key element in the suite of behaviours linked with modern humans is heat treatment of materials such as ochre for ritual purposes and stone prior to tool production. Until now, there has been no direct archaeological evidence for the exact procedure used in the heat treatment of silcrete. Through the analysis of heat-treated artefacts from the Howiesons Poort of Diepkloof Rock Shelter, we identified a hitherto unknown type of organic residue - a tempering-residue - that sheds light on the processes used for heat treatment in the MSA. This black film on the silcrete surface is an organic tar that contains microscopic fragments of charcoal and formed as a residue during the direct contact of the artefacts with hot embers of green wood. Our results suggest that heat treatment of silcrete was conducted directly using an open fire, similar to those likely used for cooking. These findings add to the discussion about the complexity of MSA behaviour and appear to contradict previous studies that had suggested that heat treatment of silcrete was a complex (i.e., requiring a large number of steps for its realization) and resource-consuming procedure.

Towards a non-invasive quantitative analysis of the organic components in museum objects varnishes by vibrational spectroscopies: methodological approach.

The compositions of ancient varnishes are mainly determined destructively by separation methods coupled to mass spectrometry. In this study, a methodology for non-invasive quantitative analyses of varnishes by vibrational spectroscopies is proposed. For that, experimental simplified varnishes of colophony and linseed oil were prepared according to 18th century traditional recipes with an increasing mass concentration ratio of colophony/linseed oil. FT-Raman and IR analyses using ATR and non-invasive reflectance modes were done on the "pure" materials and on the different mixtures. Then, a new approach involving spectral decomposition calculation was developed considering the mixture spectra as a linear combination of the pure materials ones, and giving a relative amount of each component. Specific spectral regions were treated and the obtained results show a good accuracy between the prepared and calculated amounts of the two compounds. We were thus able to detect and quantify from 10% to 50% of colophony in linseed oil using non-invasive techniques that can also be conducted in situ with portable instruments when it comes to museum varnished objects and artifacts.

Advanced discriminating criteria for natural organic substances of cultural heritage interest: spectral decomposition and multivariate analyses of FT-Raman and FT-IR signatures.

Natural organic substances are involved in many aspects of the cultural heritage field. Their presence in different forms (raw, heated, mixed), with various conservation states, constitutes a real challenge regarding their recognition and discrimination. Their characterization usually involves the use of separative techniques which imply destructive sampling and specific analytical preparations. Here we propose a non destructive approach using FT-Raman and infrared spectroscopies for the identification and differentiation of natural organic substances. Because of their related functional groups, they usually present similar vibrational signatures. Nevertheless the use of appropriate signal treatment and statistical analysis was successfully carried out to overcome this limitation, then proposing new objective discriminating methodology to identify these substances. Spectral decomposition calculations were performed on the CH stretching region of a large set of reference materials such as resins, oils, animal glues, and gums. Multivariate analyses (Principal Component Analyses) were then performed on the fitting parameters, and new discriminating criteria were established. A set of previously characterized archeological resins, with different surface aspects or alteration states, was analyzed using the same methodology. These testing samples validate the efficiency of our discriminating criteria established on the reference corpus. Moreover, we proved that some alteration or ageing of organic materials is not an issue to their recognition.

Alteration of Asian lacquer: in-depth insight using a physico-chemical multiscale approach.

Oriental lacquer has been used in Asian countries for thousands of years as a durable and aesthetic coating material for its adhesive, consolidating, protective and decorative properties. Although these objects are made from an unusual material in Occident, Western museum collections host many lacquerwares. Curators, restorers and scientists are daily confronted with questions of their conservation and their alteration. The characterization of their conservation state is usually assessed through visual observations. However deterioration often starts at the microscopic level and cannot be detected by a simple visual inspection. Often, ageing and deterioration of artworks are connected to physical, mechanical and chemical transformations. Thus new insight into alteration of lacquer involves the monitoring of macro-, microscopic and molecular modifications, and this can be assessed from physico-chemical measurements. Non-invasive (microtopography and Scanning Electron Microscopy - SEM) and micro-invasive (infrared micro-spectroscopy using a synchrotron source - SR-µFTIR) investigations were performed to study the degradation processes of lacquers and evaluate their level of alteration. In particular, spectral decomposition and fitting procedure were performed in the 1820-1520 cm(-1) region to follow the shift of the C=O and C=C band positions during lacquer ageing. The present work proves the potential of this physico-chemical approach in conservation studies of lacquers and in the quantification of the state of alteration. It evidences chemical phenomena of alteration such as oxidation and decomposition of a lacquer polymeric network. It also demonstrates for the first time the degradation front of artificially aged lacquer and the chemical imaging of a more than 2000 years old archaeological lacquer by using SR-µFTIR.