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Carbon speciation in organic fossils using 2D to 3D x-ray Raman multispectral imaging

Carbon speciation in organic fossils using 2D to 3D x-ray Raman multispectral imaging SCIENCE ADVANCES RESEARCH ARTICLE APPLIED SCIENCES AND ENGINEERING Copyright © 2019 The Authors, some rights reserved; Carbon speciation in organic fossils using exclusive licensee American Association 2D to 3D x-ray Raman multispectral imaging for the Advancement of Science. No claim to 1,2 1,3 4 5 4 Rafaella Georgiou , Pierre Gueriau , Christoph J. Sahle , Sylvain Bernard , Alessandro Mirone , original U.S. Government 6 7 2,8 1,2 Romain Garrouste , Uwe Bergmann *, Jean-Pascal Rueff *, Loïc Bertrand * Works. Distributed under a Creative The in situ two-dimensional (2D) and 3D imaging of the chemical speciation of organic fossils is an unsolved problem Commons Attribution in paleontology and cultural heritage. Here, we use x-ray Raman scattering (XRS)–based imaging at the carbon NonCommercial K-edge to form 2D and 3D images of the carbon chemistry in two exceptionally preserved specimens, a fossil plant License 4.0 (CC BY-NC). dating back from the Carboniferous and an ancient insect entrapped in 53-million-year-old amber. The 2D XRS imaging of the plant fossil reveals a homogeneous chemical composition with micrometric “pockets” of preservation, likely inherited from its geological history. The 3D XRS imaging of the insect cuticle displays an exceptionally well preserved remaining chemical signature typical of polysaccharides such as chitin around a largely hollowed-out inclusion. Our results open up new perspectives for in situ chemical speciation imaging of fossilized organic materials, with the potential to enhance our understanding of organic specimens and their paleobiology. INTRODUCTION forward the search for traces of ancient biomolecules in the fossil The chemistry of ancient organic materials carries information of record (10). their original nature. Because this information is difficult to decode For instance, fourier transform infrared (FTIR) mapping revealed and limited by degradation, the depiction of chemical signatures the preservation of amide and thiol groups of the -keratin mole- preserved in the fossil record constitutes one of the essential challenges cule in ca. 50-million-year-old reptile skin from Utah, USA (11). for paleontologists. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) data In some rare cases, organic structures can be preserved in rocks. provided identification of hemoglobin-derived porphyrin molecules Emblematic cases of organic preservation include mammoths en- in a ca. 46-million-year-old blood-engorged mosquito from Montana, tombed in the permafrost (1,  2), insects trapped in amber (3–6), USA (12). In conjunction with immunohistochemical staining and colored dinosaur feathers (7), and charcolified or lignitic fossil plants FTIR imaging, ToF-SIMS identified endogenous proteinaceous and from the Carboniferous used as the main source for coal. Although lipid constituents, keratinocytes, and branched melanophores, which fascinating, the search for ancient biomolecules imposes stringent give evidence for homeothermy and crypsis in a ca. 180-million- interpretational and analytical challenges as (i) taphonomic and diage- year-old ichthyosaur from Germany (13). One of the most promising netic processes may strongly affect original chemistry, (ii) contaminants experimental approaches is scanning transmission x-ray microscopy are likely present at the surface of the samples, and (iii) carbon-based (STXM), a synchrotron-based soft x-ray technique that can probe compounds can be preserved as traces. speciation of light elements in micrometric samples at a spatial reso- Most fossil biogenic organic compounds have been detected in lution of a few tens of nanometers (14). Carbon K-edge spectra ob- their native form using invasive analysis such as gas chromatography/ tained on carbonaceous systems consist of spectral features that can mass spectrometry [GC/MS; e.g., (8, 9)] or amplified by polymerase differentiate organic compounds (15). Applied to paleontology, STXM chain reaction [e.g., (1,  2,  4)]. However, these measurements are identified partially degraded sporopollenin molecules within a performed on extracts and therefore only represent averaged informa- ca. 230-million-year-old lycophyte megaspore from France (16) tion over the sampling volume and do not yield the spatial complexity and partially preserved chitin-protein complexes within the cuticles of the chemistry of these specimens for which imaging is a requisite. of a ca. 310-million-year-old scorpion from Illinois, USA, and of a The development of new analytical tools and/or technical improve- ca. 420-million-year-old eurypterid from Canada (17). This technique ments toward higher sensitivity or resolution have recently pushed even allowed documenting the chemical nature of ancient (several billion years old) organic microfossils (18). However, these techniques present some limitations, the main one being the lack of bulk sensitivity. STXM-based x-ray absorption IPANEMA, CNRS, ministère de la culture, Université de Versailles Saint-Quentin- en- Yvelines, Université Paris-Saclay, BP 48 St. Aubin, 91192 Gif-sur-Yvette, France. near-edge structure (XANES) spectroscopy only allows probing thin Synchrotron SOLEIL, l’Orme des Merisiers, BP 48 St. Aubin, 91192 Gif-sur-Yvette, samples (i.e., samples transparent to x-rays at the transition energy France. Institute of Earth Sciences, University of Lausanne, Géopolis, CH-1015 4 of the element of interest). FTIR, Raman, and ToF-SIMS imaging Lausanne, Switzerland. ESRF–The European Synchrotron, 71, avenue des Martyrs, CS also only provide surface sensitivity. Thus, any contamination of the 40220, 38043 Grenoble, France. Muséum National d’Histoire Naturelle, Sorbonne Université, UMR CNRS 7590, IRD, Institut de Minéralogie, de Physique des Matériaux et surface by exogenous organic matter, and sample roughness as well, de Cosmochimie (IMPMC), 75005 Paris, France. Institut de Systématique Evolution can compromise data acquisition and interpretation. This “black Biodiversité (ISYEB), UMR 7205 MNHN/CNRS/Sorbonne Univ./EPHE/Univ. Antilles, and white” situation where organic compounds absorb either too Muséum National d’Histoire Naturelle, 57 rue Cuvier, CP 50, F-75005 Paris, France. Stanford PULSE Institute, SLAC National Accelerator Laboratory, Menlo Park, CA, USA. much or too little (hard x-rays) to allow meaningful imaging, de- Sorbonne Université, CNRS, Laboratoire de Chimie Physique–Matière et Rayonnement, pending on the nature of the probe, still poses numerous challenges LCPMR, F-75005 Paris, France. to the depiction of the three-dimensional (3D) chemical speciation *Corresponding author. Email: loic.bertrand@synchrotron-soleil.fr (L.B.); jean-pascal. rueff@synchrotron-soleil.fr (J.-P.R.); bergmann@slac.stanford.edu (U.B.) of primarily organic systems. A method providing spatially resolved Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 1 of 9 | SCIENCE ADVANCES RESEARCH ARTICLE information of organic carbon speciation in 3D and over large areas Noyelles-lez-Lens, France (Fig.  1,  A  to  C). The fossil fragment, appears critically required to overcome these limitations. easily recognizable by its characteristic diamond-shaped pattern, is Here, we report the unprecedented use of a hard x-ray probe for ca. 6 cm long and 2.5 cm wide and lies on a black shale, yellowish in the element-specific chemical bulk imaging of ancient materials. places, which also includes other plant fragments. Most of the Taking advantage of the capability of nonresonant x-ray Raman scatter- Lepidodendron trunk has the same appearance and color as the ing (XRS) for direct tomography with chemical bond contrast (19), shale, but it also contains a thicker, vitreous black to very shiny ma- we develop 2D and 3D XRS spectral imaging for cultural heritage terial (extremely similar to vitrinite) distributed along the edges of and geosciences. The large penetrative power of hard x-rays enables most diamond-shaped leaf scars. A few beige patches are irregularly the measurement to be done in a noninvasive way, with no particular distributed over the fossil. preparation nor specific experimental conditions, in air, and provides XRI produces carbon maps with a micrometric spatial resolution information that is not compromised by surface contamination by (down to a few micrometers as defined by the beam size) over large ensuring that the dominant signal contribution is from the bulk of the pluricentimetric objects. In contrast to carbon mapping using scan- probed material (20). XRS 2D and 3D imaging are demonstrated against ning electron microscopy with energy-dispersive x-ray spectroscopy, a fragment of Lepidodendron trunk from the Upper Carboniferous 2D XRI provides bulk mapping such that the carbon signal is not (or [ca. 305 million years (Ma) old] of Pas-de-Calais (France) and an minimally) hampered by contamination or surface roughness. Col- Eocene ant (ca. 53 Ma old) entrapped in amber from Oise (France), lecting a map before and after the K-edge allows reconstructing an respectively. The present results reveal local “pockets” of preservation edge jump map, a map of carbon concentration within the sample. in the chemical composition of the plant fossil, likely inherited from The map obtained appears unexpectedly contrasted, considering the its geological history, while they acquaint the exceptional preservation above description of the sample: Despite its similar appearance and of the insect cuticle by showing chemical signatures of polysaccharides color, the fossil is enriched in carbon, and no contrast is observed be- such as chitin. tween the vitreous material, the beige patches, and the rest of the fossil (Fig. 1, C and D). The carbon distribution can be used to pinpoint interesting areas for spectroscopy. We collected five full XRS-based RESULTS AND DISCUSSION carbon K-edge XANES spectra (Fig. 1E), one from the shale and four We collected XRS carbon K-edge intensities using mapping (or raster from the plant, targeting at the different materials observed (at the scanning) by sequentially moving objects across the photon beam at edge and inside of the diamond-shaped leaf scars) and at the different a given incident energy while measuring the scattered intensity. carbon amounts revealed by the map. The black shale matrix does not Several of these maps are acquired at different energy losses through contain notable amounts of organic carbon. Unexpectedly again, all the carbon K-edge to produce a hyperspectral data cube. four spectra from the fossil appear very similar after background- Illuminating a sample with an incident energy E and setting a corrected normalization. The spectra reveal two main absorption fixed analyzer energy E , the energy loss E = E − E can create elec- features at 285.4 and 293.0 eV attributed to 1s- * and 1s- * electronic f f tronic excitations. If E is tuned to a transition involving a bound transitions in aromatic and olefinic C═C carbons, respectively (23). In electron, then the resulting spectroscopy is called XRS spectroscopy. contrast to the usual low-energy carbon K-edge XANES spectroscopy, Beside a q-dependent background that is dominated by Compton bulk probing of the expected random-oriented polycrystalline material scattering and collective valence electron excitations such as plasmons allows comparing the intensity of these spectroscopic features. at high and low momentum transfers (q), respectively, the XRS With a (1s-*)/(1s-*) ratio [that is, 1s-*/(1s-* + arctangent), spectrum generally contains nondispersive features that are generated where the arctangent function models the edge jump in the carbon when a fraction of incident photon energy is transferred to the sample K-edge XANES spectra] of 0.45, these spectra reveal that the graphitic inner shell electrons, promoting them into unoccupied states (21). macromolecular organic carbon composing the Lepidendron trunk XRS therefore enables the measurement of the near-edge excitation is not highly ordered but rather similar to bituminous coals (24). spectrum in the energy loss domain. It combines the chemical sen- In contrast to lignite, the spectra of the Lepidodendron trunk do not sitivity of x-ray absorption spectroscopy (XAS) for the study of the exhibit any clear absorption feature at 288.7 eV attributed to carboxylic speciation of light elements such as carbon with the benefit of high functional groups (1s-* transition). photon energy (range, 6 to 13 keV), discarding the substantial ex- There is only a limited number of characteristic resonances at the perimental constraints of XAS at the low energy of the carbon K-edge carbon K-edge (20,  25), and the spectra at this edge from the (range, 280 to 350 eV). XRS has demonstrated a great potential to Lepidodendron trunk are well explained by a combination of two probe carbon speciation in homogeneous liquid and solid carbon– Gaussians centered at the 1s- * and 1s- * energies and of an arctangent- based samples that are poor in heavier elements (absorption of x-ray shaped contribution to account for transitions to the continuum from the latter represents the main limitation of this technique). (Fig. 2A). To further test the homogeneity of the carbon speciation XRS has been shown to be a promising means to identify the chemical over a very large sample area, we collected maps at different charac- speciation of light elements in a range of systems from oil cuts to teristic energies (270, 280, 285, 288, 293, and 350 eV). The spectral artists’ pigments (20–22). The proof of concept of imaging has been decomposition of the reduced XRS-based XANES spectra obtained established on a model object (19), yet XRS imaging (XRI) of real-life for each pixel using the same Gaussians and arctangent, as well as a materials has never been studied. linear fit of the Compton background, fits very well to the experi- mental data (Fig. 2C). The decomposition gives a (1s- *)/(1s- *) ratio Microscale 2D imaging of carbon on centimetric of 0.58, well comparable to that obtained for the full XRS-based Carboniferous plants XANES spectrum collected at the same location (0.45). We used XRI to study a fragment of Lepidodendron trunk collected The Gaussian distributions highlight two classes of pixels, those on an Upper Carboniferous (ca. 305  Ma old) coal slag heap in belonging to the fossil (high contribution) and those from the shale Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 2 of 9 | SCIENCE ADVANCES RESEARCH ARTICLE Fig. 1. Carbon XRS mapping and spectroscopy of a fragment of Lepidodendron trunk from the Upper Carboniferous (ca. 305 Ma ago) of Noyelles-lez-Lens, France. (A) Optical photograph of the studied object. (B) Schematic view of the experimental XRS setup. SDD, silicon drift detector. (C) Close-up on the studied area. The dashed line represents the boundaries identified in (D). (D) Carbon map from the dotted box area in (A) (scan area, 40 mm by 20 mm; 20,000 pixels; scan step, 200 m by 200 m; beam size, 15 m by 15 m). The box corresponds to the area analyzed in Fig. 2. a.u., arbitrary units. (E) Normalized background-corrected carbon K-edge XRS spectra from the locations indicated by asterisks in (D) (sum of four spectra; 500 ms per energy step; beam size, 15 m by 15 m), and pure graphite (denoted as “G”) for energy calibration and reference; spectra were vertically shifted for an increased readability. Scale bars, 1 cm. (Photo credit: Rafaella Georgiou, CNRS IPANEMA) matrix (low contribution, reflecting the lower carbon content of the high ratios indicate the local presence of much more thermally shale; Fig. 1E, spectrum 1). The distribution of the (1s-*)/(1s-*) altered compounds. ratio within the fossil appears quite homogeneous, with a mean ra- These results demonstrate the strong potential of 2D XRI to tio of 0.56 (Fig. 2D). This chemical map does not reveal any contrast evaluate carbon speciation in heterogeneous fossils. Observation of matching the optical morphology of the sample, as the vitreous micrometric “pockets” of preservation appears particularly promising material surrounding the diamond-shaped leaf scars remains spectro- for further molecular identification. scopically indistinguishable from the rest of the fossil. A few pixels yield significantly different ratios: Less than 5 and Revisiting the 3D preservation of insect in amber 2% of the pixels record ratios of <0.35 and >1, respectively, suggesting In dense samples such as rocks, x-rays (6 to 10 keV) penetrate a few a complex preservation history with local chemical heterogeneity. tens to hundreds of micrometers. In contrast, they will penetrate Pixels with lower ratios may indicate micrometric “pockets” of from millimeters to centimeters in organic matter. X-rays scattered preservation, where the plant material has not been turned into off the object at different depths along the incident beam direction coal but only as lignite, or even show the presence of less degraded will focus on different areas of the pixelated detector due to the plant compounds, such as cellulose or lignin, whereas pixels with point-to-point focusing properties of the bent analyzer crystals Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 3 of 9 | SCIENCE ADVANCES RESEARCH ARTICLE Fig. 2. 2D XRS carbon K-edge speciation mapping of a fragment of Lepidodendron trunk from the Upper Carboniferous (ca. 305 Ma ago) of Noyelles-lez-Lens, France. (A) Spectral decomposition in two Gaussians and an arctangent of edge features of the normalized background-corrected XRS carbon K-edge XANES spectrum from the location indicated as point “2” in Fig. 1D. (B) Carbon intensity maps collected at 270, 280, 285, 288, 293, and 350 eV from the solid box area in Fig. 1D (scan step, 300 m by 300 m; 6000 pixels; beam size, 15 m by 15 m). Note how accurately the intensities in the fossils match the full spectra collected and how the intensities decrease following the Compton scattering background in the shale. (C) Spectral decomposition of the reduced spectrum collected at the exact same location as spectrum point “2” in (A) and Fig. 1D. (D) Distribution of the (1s-*)/(1s-* + arctangent) ratio within the Lepidodendron trunk (calculated from the spectral decomposition of the reduced spectrum at each pixel). The white crosses indicate the location of the full spectra shown in Fig. 1E. (E) Histogram and kernel density of the ratio, allowing to pinpoint a few pixels with a speciation different from the full spectra collected. (F) Mean (reduced) spectra from the different classes of ratio identified by their respective colored boxes in (E). (Fig. 3A). The collection of data along the beam direction thereby tagmata are visible (head, mesosoma, and metasoma including the provides a 1D image where the contrast is governed by the inelastic petiole without showing segment details—the mandibles, maxillary scattering signal after proper integration, similar to confocal imaging. palps, and remnants of three legs), confirming that the hyperspectral The collection of successive sections by raster scanning the object dataset contains enough information to contrast different parts of the then makes it possible to construct a 3D tomographic volume at a fossils. A mean spectrum from 150 pixels in the bulk amber (Fig. 4D) resolution defined laterally by the dimensions of the projected exhibits a feature at 285.4 eV attributed to 1s- * transitions of aromatic- beam and by the detector projected pixel size along the beam [i.e., olefinic carbons (a broad feature in the energy range of 287.3 to direct tomography (19, 26)]. 289.0 eV) and a feature at 292.4 eV related to C─C 1s-* contribu- We performed 3D XRI of a block of Eocene amber containing a tions (23). Ambers are formed during the polymerization of morphologically well preserved ant worker, without wings [Oise, non-volatile terpenoids, which are the major components of the France (ca. 53  Ma old); Fig.  3B]. Experimental conditions were resins produced as a protective metabolism by many angiosperms selected to yield a voxel size of 50 × 50 × 50 m , each voxel being and gymnosperms while the volatile terpenoids escape to the atmo- associated to its own XRS-based XANES spectrum. sphere. The Eocene Oise amber, an Ic-type resin typical of angiosperms The image of the total signal intensity leads to direct observation derived from Fabaceae sp. (27), is characterized by the presence of of most of the morphology of the fossil (Figs. 3C and 4A). The three aromatic-olefinic carbons, attributed to terpenoids, biomarkers of Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 4 of 9 | SCIENCE ADVANCES RESEARCH ARTICLE Fig. 3. XRS 3D carbon K-edge speciation mapping of an Eocene (ca. 53 Ma ago) ant entrapped in amber from Oise, France. (A) Schematic view of the experimental XRS setup. (B) Optical photograph of the specimen. (C) Isosurface of the raw, energy-integrated, intensity data. (D) 3D rendering of the ant cuticle (brown) and internal void (transparent gray) classes of voxels based on the total signal intensity; image with interpolation [voxel size, 50 m ; 23.409 voxels (amber voxels not shown); beam size, 10 m by 20 m). Oblique, dorsal, lateral right, and ventral views of the 3D rendering after smoothing (averaged voxel-distance interpolation). (E) Clustering of the ant cuticle voxels [brown voxels shown in (D)] based on the A parameter allows chemically distinguishing two classes of voxels: one in dorsal right position (negative chit A values in blue) and the other in ventral left position (positive A values in red), here shown in oblique, dorsal, lateral right, and ventral 3D views (after smoothing). chit chit (Photo credit: Rafaella Georgiou, CNRS IPANEMA) where  E is the energy transfer with respect to the K-edge of the the botanical origin of the resins (28). The broad feature likely corre- element under study. When the model is complete (all reference sponds to the superimposition of the 1s- * electronic transitions of compounds identified), the A parameters reflect the quantity of aliphatic carbons (287.3 to 288.0 eV) (25), formed during polymer- ref,norm ization (28), and of the 1s- * transitions of carbonyl groups. each species n of normalized XRS spectrum I ( E) in the voxel Contrasts observed in the specimen indicate the presence of dif- probed. In the restricted E range used, the tail of the background ferent chemical compounds. Spectra from the perimeter of the in the XRS spectrum is expected to be quasi-affine in energy and is insect show features markedly different from the bulk amber. The modeled as C (X) + C (X)E. 1 2 XRS spectrum I(E, X) at a given voxel of coordinates X = (x, y, z) The decomposition of the XRS data was performed as described is a linear combination of the spectra of the different compounds in Material and Methods, using as initial guess the averaged spectrum present and a background signal from valence electron background of amber measured in the bulk amber (Fig. 4D). As shown on the and Compton scattering map of the quadratic error of the fit, the spectral data differ consider- ably from the amber for a 1-pixel-wide line at the location of the ref,norm insect’s former cuticle (Fig. 4B). Spectra from these pixels show I(E, X ) = ∑ A (X ) I (E ) + C (X ) + C (X ) E (1) n n 1 2 notably different characteristics compared to those of the amber n=1 Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 5 of 9 | SCIENCE ADVANCES RESEARCH ARTICLE AB x (beam direction) x (beam direction) C D 285.4 286.6 288.3 289.2 292.4 ExV ExV ExD Amber ExD Chitin standard 285 290 295 300 x (beam direction) Energy transfer (eV ) Fig. 4. XRS virtual cross section and spectra of an Eocene ant entrapped in amber from Oise (France, ca. 53 Ma old). (A) Total intensity virtual cross-sectional image (pixel size, 50 m; beam size, 10 m by 20 m) of the ant entrapped in the Eocene amber (image with interpolation). (B) Spatial distribution of the quadratic error showing the diverse chemical regions of the sample when performing a fit based on the amber reference (image with interpolation). (C) Spatial distribution of A when performing chit a fit based on the reference compounds amber and chitin (image with interpolation). (D) From top to bottom, distinctive normalized mean XRS spectral profiles corresponding to the ventral (A > 0.007, ExV) and the dorsal (A < −0.007, ExD) areas of the exoskeleton, as indicated with the arrows in (C), Oise amber collected from the bulk chit chit specimen (amber), and chitin standard used as a reference material (chitin standard). Scale bars, 500 m. matrix. In particular, some show a more intense feature at 288.3 eV The clustering of the data based on A allows discussing the chit attributed to 1s-* transitions from amide groups, while others are chemistry of the arthropod’s cuticle. The spectrum ExV is charac- characterized by the absence of spectral feature at 285.4 eV, which terized by the presence of 1s-* transitions due to the presence of points to the absence of aromatic-olefin carbons. aromatic and/or olefinic carbons (Fig. 4D). Both spectra ExV and Arthropods’ cuticle is a hierarchically structured material com- those from the chitin reference are dominated by a broad complex posed of an external thin epicuticle layer, rich in lipids and proteins, feature centered at 288.3 eV, which has been attributed to the presence and the exocuticle and endocuticle composed mainly of chitin- of carbonyl associated with the amidyl group of the glycosyl ring protein complexes. Chitin [(C H O N) ], a linear polymer of (17). The complex feature centered at 289.2 eV of the XRS spectrum 8 13 5 n -1,4–linked N-acetyl glucosamine, unlike most carbohydrate com- of the chitin standard is attributed to the 1s-3p/ * transition of pounds, is not water soluble and known to be quite decay resistant O-alkyl (C─OH) moieties (15). The feature at 286.6 eV, present in (17, 29, 30). We therefore compared the latter spectra to a reference the chitin standard, is assigned to the presence of vinyl ketone moieties, chitin sample measured with XRS spectroscopy under identical a possible result of radiation-induced changes (31). The feature at conditions. Since both show similar features, we added a chitin 285.4 eV, assigned to the presence of 1s- * transition of olefinic reference spectrum to our fitting model. The parameters A and moieties, may be indicative of the specimen chemistry and/or result amb A predicted from linear least-squares fitting reflect the possible from the elimination of hydroxyl groups from polysaccharides during chit presence of the compounds in each voxel. Positive values of A irradiation as reported by Cody et al. (31). chit (A > 0.007) are observed in the ventral exoskeleton (Fig.  4C, In contrast, 1s- * transitions are totally absent from the arthropod’s chit arrow ExV). In contrast, significantly different values (A < −0.007) cuticle close to the specimen dorsal right surface (Fig. 4, C and D, chit are observed for pixels denoted by arrow ExD, which leads to a spectrum ExD). The less intense feature of this spectrum, centered second spectrum when segmented (Fig. 4C). We speculate that at 288.3 eV, is attributed to the overlapping contribution of 1s- * the negative A values in the least-squares regression indicate transitions in amide groups (15) and the possible presence of 1s-* chit the presence of an additional chemical compound not included transitions of aliphatic groups in the energy region of 287.3 to 288.0 eV in the model. (25). The presence of aliphatic carbons is consistent with the seminal Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 6 of 9 y (scanning direction) y (scanning direction) y (scanning direction) Normalized intensity (a.u.) | SCIENCE ADVANCES RESEARCH ARTICLE work of Stankiewicz et al. (32), which identifies through invasive in 3D with reasonable resolution opens entirely new avenues for means aliphatic geopolymers in amber inclusions’ exoskeleton, the chemical characterization of organisms preserved in amber. attributed to burial diagenetic transformation of the original chemistry of the organism. Chitin and lignocellulose could be identified using pyrolysis-GC/MS in subfossil (2 to 20 thousand years old) insect and CONCLUSIONS AND IMPLICATIONS plant specimens entrapped in resins from Kenya, yet no trace of these Over the past two decades, XRS spectroscopy has been successfully macromolecules could be identified in Dominican amber inclusions applied for chemical speciation of light element systems and conditions (>25 Ma); instead, aliphatic polymers and sulfur-containing moieties not suited for a soft x-ray probe. Our results shown here extend this were identified (32). Aliphatic signatures were also identified in fossil capability to the hyperspectral 2D and 3D imaging of organic-rich arthropods preserved in sediments [e.g., (30)]. In all the previous paleontological specimens and geological objects. The possibility to examples mentioned, pyrolysis-GC/MS was used at a semiquantitative work on these samples without any specific preparation and under level; selective sampling does not ensure that the sampling area is ambient conditions is the critical advantage of this hard x-ray probe. representative of the whole specimen. Here, the identification of two While other imaging methods, most notably x-ray phase-contrast distinct chemical fingerprints in the exoskeleton (dorsal right versus tomography, provide better spatial resolution and are less restricted ventral left distributions; Fig. 3E) points to the importance of discrim- in terms of sample size and composition, the XRS method shown inating the spatial distribution of organic compounds in 3D, at a here uniquely complements such information by providing un- global scale, to provide complete information about the specimens’ precedented insights into 3D carbon speciation. Given the small cross biochemistry, physiology, molecular evolution or function, or chemical section and resulting weak signal strength, XRS-based speciation interactions between the organism and the depositional setting. characterization and imaging pose some restrictions for sample size Inclusions in amber occur in several flow sequences and often form and composition. For 2D imaging, the main restriction is the pres- multiple inclusions (i.e., syninclusions). Each flow leads to a preservation ence of heavy elements in the sample, which reduce the scattering mode that depends on several unknown factors including time. volume. As current XRS setups operate in the range of 6 to 10 keV, Coty et al. (33) highlighted the need for a 3D approach of taphonomy to 3D imaging is furthermore restricted to sample sizes and compositions constrain paleobiological interpretations. Their computed tomography that permit the penetration at these x-ray energies. Further work scan of multiple inclusions in the same sample revealed different levels will aim at minimizing the source of potential radiation-induced of preservation depending on the resin layer and on the type of organism. damage (37). Several directions can be pursued as follows: using Although amber and copal (fossil tree resin) preserve three- cryogenic and anaerobic conditions for some samples, increasing dimensionally and, in superb detail, numerous organisms (insects, x-ray energy to reduce the photoelectric cross section while increasing feathers, plants, etc.) as a result of entrapment that rapidly dehydrates the XRS intensity and penetration length, and defocusing the beam the inclusions and protects them from water and microbial decay in the vertical direction using the so-called 2D sectioning mode (19). (3), many localities only preserve cuticle or hollow molds (such as In addition to ancient fossils, the development of this approach to the ant we studied herein) or no fossils at all, and the processes that the study of organic-rich heterogeneous materials shows promise control this range in preservation (nature of the resin producer and for numerous fields of research, including geosciences, life sciences, of the inclusions, environmental conditions, maturation, and re- materials sciences, and cultural heritage. working) remain essentially unknown (34, 35). The clustering of the data based on the total intensity allows the discrimination of the inclusion from the surrounding organic sub- MATERIALS AND METHODS strate. Further decomposition of the cuticle voxels (Fig. 3D, brown Paleontological samples voxels) based on the A parameter allows the identification of chitin Two paleontological samples were examined as follows: (i) a fragment chit traces distributed in the cuticle (Fig. 3E, red voxels). This wealth of of Lepidodendron trunk collected by one of us (P.G.) on an Upper information allows us to report the first ever fully noninvasive 3D Carboniferous (ca. 305 Ma ago) coal slag heap in Noyelles-lez-Lens, speciation of carbonaceous compounds inside a paleontological France, on 15 April 2012; (ii) an insect (inv. no. PA-3822, Hymenoptera: specimen (Fig. 3). Cuticle voxels with A positive and negative Formicidae) entrapped in amber, an Ic-type resin typical of angio- chit values are represented in red and blue colors, respectively. The lack sperms and derived from a Fabaceae. The amber deposit of Oise, of carbon signal in the internal part of the abdomen and the head found in 1996 at the Quesnoy locality in the Oise River area of Paris (Fig. 3, D and E, transparent gray voxels) points to the poor preserva- Basin in France, has been dated back from the earliest Eocene tion state of the internal soft tissue structures, while the voxels with (approximately 53 million years ago) (38). The specimen belongs to chitin affinities (positive A values; Fig. 3E, red voxels) confirm the paleontological collections of the Muséum National d’Histoire chit the preservation of the cuticular anatomy in the ventral surface of Naturelle (Paris). For comparison, we collected data on purified the insect. Until now, chemical characterization has mainly focused powder of chitin from shrimp shells (C9752, CAS: 1398-61-4, on the amber, and very few studies have investigated the inclusions Sigma-Aldrich, St. Louis, USA). because of the following challenges (36): Spectral features from the inclusions are predicted to be very close in wave number to those of X-ray Raman-based spectral imaging the resin, and because compounds of the resin have largely penetrated All experiments were performed at the GALAXIES and ID20 beamlines the inclusions (32), sampling and/or spectroscopy techniques only at the SOLEIL and the ESRF (European Synchrotron Radiation Facili- provide average information, which involves destructive sample ty) synchrotrons, respectively. The spectrometer at the GALAXIES preparation, restricting their use for rare specimens. The fact that beamline was equipped with four spherically bent Si(444) analyzer we were able to detect polysaccharide traces and distinguish the pre- crystals operated at a Bragg angle of 86° (39). The ESRF spectrometer served insect’s exoskeleton from its encasing amber and hollow parts is equipped to house 72 spherically bent Si(660) analyzer crystals, 36 Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 7 of 9 | SCIENCE ADVANCES RESEARCH ARTICLE to Paleogenomics: Large-scale sequencing of mammoth DNA. Science 311, of which were used in the forward scattering geometry (median 392–394 (2006). scattering angle, 30°) and 36 in the backscattering geometry (median 3. G. O. Poinar Jr., R. Hess, Ultrastructure of 40-million-year-old insect tissue. Science 215, scattering angle, 122°) (40), resulting in average momentum transfers 1241–1242 (1982). −1 −1 of 2.6 ± 0.6 Å and 8.8 ± 0.6 Å , respectively. 4. R. DeSalle, J. Gatesy, W. Wheeler, D. 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Science 276, 1541–1543 (1997). and statistical accuracy in x-ray Raman scattering based direct tomography. 30. N. S. Gupta, O. E. Tetlie, D. E. G. Briggs, R. D. Pancost, The fossilization of Eurypterids: A result J. Synchrotron. Radiat. 24, 476–481 (2017). of molecular transformation. Palaios 22, 439–447 (2007). 31. G. D. Cody, J. Brandes, C. Jacobsen, S. Wirick, Soft x-ray induced chemical modification Acknowledgments: Q. Guériau performed the 3D rendering of the fossil ant. R.Ge. and L.B. of polysaccharides in vascular plant cell walls. J. Electron Spectrosc. Relat. Phenom. 170, thank S. Cohen (IPANEMA) for discussion on the processing of the spectral data and L. Beck 57–64 (2009). (LSCE/LMC14). P.G. thanks the Société Amicale des Géologues Amateurs for the 32. B. A. Stankiewicz, H. N. Poinar, D. E. G. Briggs, R. P. Evershed, G. O. 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Peñalver, Taphonomy of insects in carbonates the Région Île-de-France/DIM Matériaux anciens et patrimoniaux and the European and amber. Palaeogeogr. Palaeoclimatol. Palaeoecol. 203, 19–64 (2004). Commission programs IPERION CH (GA. 654028) and E-RIHS PP (GA. 739503). U.B. and L.B. 35. V. E. McCoy, C. Soriano, S. E. Gabbott, A review of preservational variation of fossil acknowledge support from the France–Stanford Center for Interdisciplinary Studies inclusions in amber of different chemical groups. Earth Environ. Sci. Trans. R. Soc. Edinb. Program and the LabEx PATRIMA (Agence Nationale de la Recherche, 10-LABX-0094)/ 107, 203–211 (2018). Fondation des Sciences du Patrimoine. Author contributions: L.B. and U.B. conceived the 36. H. G. Edwards, D. W. Farwell, S. E. J. Villar, Raman microspectroscopic studies of amber original idea. L.B. and J.-P.R. coordinated the research. P.G. selected the carboniferous fossil resins with insect inclusions. Spectrochim. Acta A Mol. Biomol. Spectrosc. 68, 1089–1095 plant. R.Ga. selected the Eocene Oise amber. R.Ge., R.Ga., and P.G. provided the paleontological (2007). interpretation. J.-P.R. and C.J.S. developed the experimental setups at the GALAXIES and ID20 37. L. Bertrand, S. Schöeder, D. Anglos, M. B. H. Breese, K. Janssens, M. Moini, A. Simon, beamlines, respectively. A.M. wrote the software to integrate the data at the ESRF. R.Ge. Mitigation strategies for radiation damage in the analysis of ancient materials. developed the code to process the 3D XRI data with L.B. C.J.S., S.B., and U.B. contributed Trends Anal. Chem. 66, 128–145 (2015). toward the interpretation of the XRS spectra. R.Ge., P.G., and L.B. wrote the manuscript and 38. A. Nel, G. de Plöeg, J. Dejax, D. Dutheil, D. de Franceschi, E. Gheerbrant, M. Godinot, prepared the figures. R.Ge., P.G., C.J.S., S.B., U.B., J.-P.R., and L.B. took part in the experiments. All S. Hervet, J.-J. Menier, M. Augé, G. Bignot, C. Cavagnetto, S. Duffaud, J. Gaudant, S. Hua, authors read and approved the final manuscript. Competing interests: All authors declare A. Jpssang, F. L. de Broin, J.-P. Pozzi, J.-C. Rage, Un gisement sparnacien exceptionnel à that they have no competing interests. Data and materials availability: All data needed to plantes, arthropodes et vertébrés (Éocène basal, MP7) : Le Quesnoy (Oise, France). evaluate the conclusions in the paper have been made publicly available in open access Comptes Rendus de l’Académie des Sciences - Series IIA - Earth and Planetary Science 329, through the following DOIs: 10.5281/zenodo.3238621 and 10.5281/zenodo.3238615. 65–72 (1999). 39. J. M. Ablett, D. Prieur, D. Céolin, B. Lassalle-Kaiser, B. Lebert, M. Sauvage, T. Moreno, Submitted 23 February 2019 S. Bac, V. Balédent, A. Ovono, M. Morand, F. Gélebart, A. Shukla, J.-P. Rueff, The GALAXIES Accepted 25 July 2019 inelastic hard x-ray scattering end-station at Synchrotron SOLEIL. J. Synchrotron. Radiat. Published 30 August 2019 26, 263–271 (2019). 10.1126/sciadv.aaw5019 40. S. Huotari, C. J. Sahle, C. Henriquet, A. Al-Zein, K. Martel, L. Simonelli, R. Verbeni, H. Gonzalez, M.-C. Lagier, C. Ponchut, M. M. Sala, M. Krisch, A large-solid-angle x-ray Citation: R. Georgiou, P. Gueriau, C. J. Sahle, S. Bernard, A. Mirone, R. Garrouste, U. Bergmann, Raman scattering spectrometer at ID20 of the European Synchrotron Radiation Facility. J.-P. Rueff, L. Bertrand, Carbon speciation in organic fossils using 2D to 3D x-ray Raman J. Synchrotron Radiat. 24, 521–530 (2017). multispectral imaging. Sci. Adv. 5, eaaw5019 (2019). Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 9 of 9 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Science Advances Pubmed Central

Carbon speciation in organic fossils using 2D to 3D x-ray Raman multispectral imaging

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SCIENCE ADVANCES RESEARCH ARTICLE APPLIED SCIENCES AND ENGINEERING Copyright © 2019 The Authors, some rights reserved; Carbon speciation in organic fossils using exclusive licensee American Association 2D to 3D x-ray Raman multispectral imaging for the Advancement of Science. No claim to 1,2 1,3 4 5 4 Rafaella Georgiou , Pierre Gueriau , Christoph J. Sahle , Sylvain Bernard , Alessandro Mirone , original U.S. Government 6 7 2,8 1,2 Romain Garrouste , Uwe Bergmann *, Jean-Pascal Rueff *, Loïc Bertrand * Works. Distributed under a Creative The in situ two-dimensional (2D) and 3D imaging of the chemical speciation of organic fossils is an unsolved problem Commons Attribution in paleontology and cultural heritage. Here, we use x-ray Raman scattering (XRS)–based imaging at the carbon NonCommercial K-edge to form 2D and 3D images of the carbon chemistry in two exceptionally preserved specimens, a fossil plant License 4.0 (CC BY-NC). dating back from the Carboniferous and an ancient insect entrapped in 53-million-year-old amber. The 2D XRS imaging of the plant fossil reveals a homogeneous chemical composition with micrometric “pockets” of preservation, likely inherited from its geological history. The 3D XRS imaging of the insect cuticle displays an exceptionally well preserved remaining chemical signature typical of polysaccharides such as chitin around a largely hollowed-out inclusion. Our results open up new perspectives for in situ chemical speciation imaging of fossilized organic materials, with the potential to enhance our understanding of organic specimens and their paleobiology. INTRODUCTION forward the search for traces of ancient biomolecules in the fossil The chemistry of ancient organic materials carries information of record (10). their original nature. Because this information is difficult to decode For instance, fourier transform infrared (FTIR) mapping revealed and limited by degradation, the depiction of chemical signatures the preservation of amide and thiol groups of the -keratin mole- preserved in the fossil record constitutes one of the essential challenges cule in ca. 50-million-year-old reptile skin from Utah, USA (11). for paleontologists. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) data In some rare cases, organic structures can be preserved in rocks. provided identification of hemoglobin-derived porphyrin molecules Emblematic cases of organic preservation include mammoths en- in a ca. 46-million-year-old blood-engorged mosquito from Montana, tombed in the permafrost (1,  2), insects trapped in amber (3–6), USA (12). In conjunction with immunohistochemical staining and colored dinosaur feathers (7), and charcolified or lignitic fossil plants FTIR imaging, ToF-SIMS identified endogenous proteinaceous and from the Carboniferous used as the main source for coal. Although lipid constituents, keratinocytes, and branched melanophores, which fascinating, the search for ancient biomolecules imposes stringent give evidence for homeothermy and crypsis in a ca. 180-million- interpretational and analytical challenges as (i) taphonomic and diage- year-old ichthyosaur from Germany (13). One of the most promising netic processes may strongly affect original chemistry, (ii) contaminants experimental approaches is scanning transmission x-ray microscopy are likely present at the surface of the samples, and (iii) carbon-based (STXM), a synchrotron-based soft x-ray technique that can probe compounds can be preserved as traces. speciation of light elements in micrometric samples at a spatial reso- Most fossil biogenic organic compounds have been detected in lution of a few tens of nanometers (14). Carbon K-edge spectra ob- their native form using invasive analysis such as gas chromatography/ tained on carbonaceous systems consist of spectral features that can mass spectrometry [GC/MS; e.g., (8, 9)] or amplified by polymerase differentiate organic compounds (15). Applied to paleontology, STXM chain reaction [e.g., (1,  2,  4)]. However, these measurements are identified partially degraded sporopollenin molecules within a performed on extracts and therefore only represent averaged informa- ca. 230-million-year-old lycophyte megaspore from France (16) tion over the sampling volume and do not yield the spatial complexity and partially preserved chitin-protein complexes within the cuticles of the chemistry of these specimens for which imaging is a requisite. of a ca. 310-million-year-old scorpion from Illinois, USA, and of a The development of new analytical tools and/or technical improve- ca. 420-million-year-old eurypterid from Canada (17). This technique ments toward higher sensitivity or resolution have recently pushed even allowed documenting the chemical nature of ancient (several billion years old) organic microfossils (18). However, these techniques present some limitations, the main one being the lack of bulk sensitivity. STXM-based x-ray absorption IPANEMA, CNRS, ministère de la culture, Université de Versailles Saint-Quentin- en- Yvelines, Université Paris-Saclay, BP 48 St. Aubin, 91192 Gif-sur-Yvette, France. near-edge structure (XANES) spectroscopy only allows probing thin Synchrotron SOLEIL, l’Orme des Merisiers, BP 48 St. Aubin, 91192 Gif-sur-Yvette, samples (i.e., samples transparent to x-rays at the transition energy France. Institute of Earth Sciences, University of Lausanne, Géopolis, CH-1015 4 of the element of interest). FTIR, Raman, and ToF-SIMS imaging Lausanne, Switzerland. ESRF–The European Synchrotron, 71, avenue des Martyrs, CS also only provide surface sensitivity. Thus, any contamination of the 40220, 38043 Grenoble, France. Muséum National d’Histoire Naturelle, Sorbonne Université, UMR CNRS 7590, IRD, Institut de Minéralogie, de Physique des Matériaux et surface by exogenous organic matter, and sample roughness as well, de Cosmochimie (IMPMC), 75005 Paris, France. Institut de Systématique Evolution can compromise data acquisition and interpretation. This “black Biodiversité (ISYEB), UMR 7205 MNHN/CNRS/Sorbonne Univ./EPHE/Univ. Antilles, and white” situation where organic compounds absorb either too Muséum National d’Histoire Naturelle, 57 rue Cuvier, CP 50, F-75005 Paris, France. Stanford PULSE Institute, SLAC National Accelerator Laboratory, Menlo Park, CA, USA. much or too little (hard x-rays) to allow meaningful imaging, de- Sorbonne Université, CNRS, Laboratoire de Chimie Physique–Matière et Rayonnement, pending on the nature of the probe, still poses numerous challenges LCPMR, F-75005 Paris, France. to the depiction of the three-dimensional (3D) chemical speciation *Corresponding author. Email: loic.bertrand@synchrotron-soleil.fr (L.B.); jean-pascal. rueff@synchrotron-soleil.fr (J.-P.R.); bergmann@slac.stanford.edu (U.B.) of primarily organic systems. A method providing spatially resolved Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 1 of 9 | SCIENCE ADVANCES RESEARCH ARTICLE information of organic carbon speciation in 3D and over large areas Noyelles-lez-Lens, France (Fig.  1,  A  to  C). The fossil fragment, appears critically required to overcome these limitations. easily recognizable by its characteristic diamond-shaped pattern, is Here, we report the unprecedented use of a hard x-ray probe for ca. 6 cm long and 2.5 cm wide and lies on a black shale, yellowish in the element-specific chemical bulk imaging of ancient materials. places, which also includes other plant fragments. Most of the Taking advantage of the capability of nonresonant x-ray Raman scatter- Lepidodendron trunk has the same appearance and color as the ing (XRS) for direct tomography with chemical bond contrast (19), shale, but it also contains a thicker, vitreous black to very shiny ma- we develop 2D and 3D XRS spectral imaging for cultural heritage terial (extremely similar to vitrinite) distributed along the edges of and geosciences. The large penetrative power of hard x-rays enables most diamond-shaped leaf scars. A few beige patches are irregularly the measurement to be done in a noninvasive way, with no particular distributed over the fossil. preparation nor specific experimental conditions, in air, and provides XRI produces carbon maps with a micrometric spatial resolution information that is not compromised by surface contamination by (down to a few micrometers as defined by the beam size) over large ensuring that the dominant signal contribution is from the bulk of the pluricentimetric objects. In contrast to carbon mapping using scan- probed material (20). XRS 2D and 3D imaging are demonstrated against ning electron microscopy with energy-dispersive x-ray spectroscopy, a fragment of Lepidodendron trunk from the Upper Carboniferous 2D XRI provides bulk mapping such that the carbon signal is not (or [ca. 305 million years (Ma) old] of Pas-de-Calais (France) and an minimally) hampered by contamination or surface roughness. Col- Eocene ant (ca. 53 Ma old) entrapped in amber from Oise (France), lecting a map before and after the K-edge allows reconstructing an respectively. The present results reveal local “pockets” of preservation edge jump map, a map of carbon concentration within the sample. in the chemical composition of the plant fossil, likely inherited from The map obtained appears unexpectedly contrasted, considering the its geological history, while they acquaint the exceptional preservation above description of the sample: Despite its similar appearance and of the insect cuticle by showing chemical signatures of polysaccharides color, the fossil is enriched in carbon, and no contrast is observed be- such as chitin. tween the vitreous material, the beige patches, and the rest of the fossil (Fig. 1, C and D). The carbon distribution can be used to pinpoint interesting areas for spectroscopy. We collected five full XRS-based RESULTS AND DISCUSSION carbon K-edge XANES spectra (Fig. 1E), one from the shale and four We collected XRS carbon K-edge intensities using mapping (or raster from the plant, targeting at the different materials observed (at the scanning) by sequentially moving objects across the photon beam at edge and inside of the diamond-shaped leaf scars) and at the different a given incident energy while measuring the scattered intensity. carbon amounts revealed by the map. The black shale matrix does not Several of these maps are acquired at different energy losses through contain notable amounts of organic carbon. Unexpectedly again, all the carbon K-edge to produce a hyperspectral data cube. four spectra from the fossil appear very similar after background- Illuminating a sample with an incident energy E and setting a corrected normalization. The spectra reveal two main absorption fixed analyzer energy E , the energy loss E = E − E can create elec- features at 285.4 and 293.0 eV attributed to 1s- * and 1s- * electronic f f tronic excitations. If E is tuned to a transition involving a bound transitions in aromatic and olefinic C═C carbons, respectively (23). In electron, then the resulting spectroscopy is called XRS spectroscopy. contrast to the usual low-energy carbon K-edge XANES spectroscopy, Beside a q-dependent background that is dominated by Compton bulk probing of the expected random-oriented polycrystalline material scattering and collective valence electron excitations such as plasmons allows comparing the intensity of these spectroscopic features. at high and low momentum transfers (q), respectively, the XRS With a (1s-*)/(1s-*) ratio [that is, 1s-*/(1s-* + arctangent), spectrum generally contains nondispersive features that are generated where the arctangent function models the edge jump in the carbon when a fraction of incident photon energy is transferred to the sample K-edge XANES spectra] of 0.45, these spectra reveal that the graphitic inner shell electrons, promoting them into unoccupied states (21). macromolecular organic carbon composing the Lepidendron trunk XRS therefore enables the measurement of the near-edge excitation is not highly ordered but rather similar to bituminous coals (24). spectrum in the energy loss domain. It combines the chemical sen- In contrast to lignite, the spectra of the Lepidodendron trunk do not sitivity of x-ray absorption spectroscopy (XAS) for the study of the exhibit any clear absorption feature at 288.7 eV attributed to carboxylic speciation of light elements such as carbon with the benefit of high functional groups (1s-* transition). photon energy (range, 6 to 13 keV), discarding the substantial ex- There is only a limited number of characteristic resonances at the perimental constraints of XAS at the low energy of the carbon K-edge carbon K-edge (20,  25), and the spectra at this edge from the (range, 280 to 350 eV). XRS has demonstrated a great potential to Lepidodendron trunk are well explained by a combination of two probe carbon speciation in homogeneous liquid and solid carbon– Gaussians centered at the 1s- * and 1s- * energies and of an arctangent- based samples that are poor in heavier elements (absorption of x-ray shaped contribution to account for transitions to the continuum from the latter represents the main limitation of this technique). (Fig. 2A). To further test the homogeneity of the carbon speciation XRS has been shown to be a promising means to identify the chemical over a very large sample area, we collected maps at different charac- speciation of light elements in a range of systems from oil cuts to teristic energies (270, 280, 285, 288, 293, and 350 eV). The spectral artists’ pigments (20–22). The proof of concept of imaging has been decomposition of the reduced XRS-based XANES spectra obtained established on a model object (19), yet XRS imaging (XRI) of real-life for each pixel using the same Gaussians and arctangent, as well as a materials has never been studied. linear fit of the Compton background, fits very well to the experi- mental data (Fig. 2C). The decomposition gives a (1s- *)/(1s- *) ratio Microscale 2D imaging of carbon on centimetric of 0.58, well comparable to that obtained for the full XRS-based Carboniferous plants XANES spectrum collected at the same location (0.45). We used XRI to study a fragment of Lepidodendron trunk collected The Gaussian distributions highlight two classes of pixels, those on an Upper Carboniferous (ca. 305  Ma old) coal slag heap in belonging to the fossil (high contribution) and those from the shale Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 2 of 9 | SCIENCE ADVANCES RESEARCH ARTICLE Fig. 1. Carbon XRS mapping and spectroscopy of a fragment of Lepidodendron trunk from the Upper Carboniferous (ca. 305 Ma ago) of Noyelles-lez-Lens, France. (A) Optical photograph of the studied object. (B) Schematic view of the experimental XRS setup. SDD, silicon drift detector. (C) Close-up on the studied area. The dashed line represents the boundaries identified in (D). (D) Carbon map from the dotted box area in (A) (scan area, 40 mm by 20 mm; 20,000 pixels; scan step, 200 m by 200 m; beam size, 15 m by 15 m). The box corresponds to the area analyzed in Fig. 2. a.u., arbitrary units. (E) Normalized background-corrected carbon K-edge XRS spectra from the locations indicated by asterisks in (D) (sum of four spectra; 500 ms per energy step; beam size, 15 m by 15 m), and pure graphite (denoted as “G”) for energy calibration and reference; spectra were vertically shifted for an increased readability. Scale bars, 1 cm. (Photo credit: Rafaella Georgiou, CNRS IPANEMA) matrix (low contribution, reflecting the lower carbon content of the high ratios indicate the local presence of much more thermally shale; Fig. 1E, spectrum 1). The distribution of the (1s-*)/(1s-*) altered compounds. ratio within the fossil appears quite homogeneous, with a mean ra- These results demonstrate the strong potential of 2D XRI to tio of 0.56 (Fig. 2D). This chemical map does not reveal any contrast evaluate carbon speciation in heterogeneous fossils. Observation of matching the optical morphology of the sample, as the vitreous micrometric “pockets” of preservation appears particularly promising material surrounding the diamond-shaped leaf scars remains spectro- for further molecular identification. scopically indistinguishable from the rest of the fossil. A few pixels yield significantly different ratios: Less than 5 and Revisiting the 3D preservation of insect in amber 2% of the pixels record ratios of <0.35 and >1, respectively, suggesting In dense samples such as rocks, x-rays (6 to 10 keV) penetrate a few a complex preservation history with local chemical heterogeneity. tens to hundreds of micrometers. In contrast, they will penetrate Pixels with lower ratios may indicate micrometric “pockets” of from millimeters to centimeters in organic matter. X-rays scattered preservation, where the plant material has not been turned into off the object at different depths along the incident beam direction coal but only as lignite, or even show the presence of less degraded will focus on different areas of the pixelated detector due to the plant compounds, such as cellulose or lignin, whereas pixels with point-to-point focusing properties of the bent analyzer crystals Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 3 of 9 | SCIENCE ADVANCES RESEARCH ARTICLE Fig. 2. 2D XRS carbon K-edge speciation mapping of a fragment of Lepidodendron trunk from the Upper Carboniferous (ca. 305 Ma ago) of Noyelles-lez-Lens, France. (A) Spectral decomposition in two Gaussians and an arctangent of edge features of the normalized background-corrected XRS carbon K-edge XANES spectrum from the location indicated as point “2” in Fig. 1D. (B) Carbon intensity maps collected at 270, 280, 285, 288, 293, and 350 eV from the solid box area in Fig. 1D (scan step, 300 m by 300 m; 6000 pixels; beam size, 15 m by 15 m). Note how accurately the intensities in the fossils match the full spectra collected and how the intensities decrease following the Compton scattering background in the shale. (C) Spectral decomposition of the reduced spectrum collected at the exact same location as spectrum point “2” in (A) and Fig. 1D. (D) Distribution of the (1s-*)/(1s-* + arctangent) ratio within the Lepidodendron trunk (calculated from the spectral decomposition of the reduced spectrum at each pixel). The white crosses indicate the location of the full spectra shown in Fig. 1E. (E) Histogram and kernel density of the ratio, allowing to pinpoint a few pixels with a speciation different from the full spectra collected. (F) Mean (reduced) spectra from the different classes of ratio identified by their respective colored boxes in (E). (Fig. 3A). The collection of data along the beam direction thereby tagmata are visible (head, mesosoma, and metasoma including the provides a 1D image where the contrast is governed by the inelastic petiole without showing segment details—the mandibles, maxillary scattering signal after proper integration, similar to confocal imaging. palps, and remnants of three legs), confirming that the hyperspectral The collection of successive sections by raster scanning the object dataset contains enough information to contrast different parts of the then makes it possible to construct a 3D tomographic volume at a fossils. A mean spectrum from 150 pixels in the bulk amber (Fig. 4D) resolution defined laterally by the dimensions of the projected exhibits a feature at 285.4 eV attributed to 1s- * transitions of aromatic- beam and by the detector projected pixel size along the beam [i.e., olefinic carbons (a broad feature in the energy range of 287.3 to direct tomography (19, 26)]. 289.0 eV) and a feature at 292.4 eV related to C─C 1s-* contribu- We performed 3D XRI of a block of Eocene amber containing a tions (23). Ambers are formed during the polymerization of morphologically well preserved ant worker, without wings [Oise, non-volatile terpenoids, which are the major components of the France (ca. 53  Ma old); Fig.  3B]. Experimental conditions were resins produced as a protective metabolism by many angiosperms selected to yield a voxel size of 50 × 50 × 50 m , each voxel being and gymnosperms while the volatile terpenoids escape to the atmo- associated to its own XRS-based XANES spectrum. sphere. The Eocene Oise amber, an Ic-type resin typical of angiosperms The image of the total signal intensity leads to direct observation derived from Fabaceae sp. (27), is characterized by the presence of of most of the morphology of the fossil (Figs. 3C and 4A). The three aromatic-olefinic carbons, attributed to terpenoids, biomarkers of Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 4 of 9 | SCIENCE ADVANCES RESEARCH ARTICLE Fig. 3. XRS 3D carbon K-edge speciation mapping of an Eocene (ca. 53 Ma ago) ant entrapped in amber from Oise, France. (A) Schematic view of the experimental XRS setup. (B) Optical photograph of the specimen. (C) Isosurface of the raw, energy-integrated, intensity data. (D) 3D rendering of the ant cuticle (brown) and internal void (transparent gray) classes of voxels based on the total signal intensity; image with interpolation [voxel size, 50 m ; 23.409 voxels (amber voxels not shown); beam size, 10 m by 20 m). Oblique, dorsal, lateral right, and ventral views of the 3D rendering after smoothing (averaged voxel-distance interpolation). (E) Clustering of the ant cuticle voxels [brown voxels shown in (D)] based on the A parameter allows chemically distinguishing two classes of voxels: one in dorsal right position (negative chit A values in blue) and the other in ventral left position (positive A values in red), here shown in oblique, dorsal, lateral right, and ventral 3D views (after smoothing). chit chit (Photo credit: Rafaella Georgiou, CNRS IPANEMA) where  E is the energy transfer with respect to the K-edge of the the botanical origin of the resins (28). The broad feature likely corre- element under study. When the model is complete (all reference sponds to the superimposition of the 1s- * electronic transitions of compounds identified), the A parameters reflect the quantity of aliphatic carbons (287.3 to 288.0 eV) (25), formed during polymer- ref,norm ization (28), and of the 1s- * transitions of carbonyl groups. each species n of normalized XRS spectrum I ( E) in the voxel Contrasts observed in the specimen indicate the presence of dif- probed. In the restricted E range used, the tail of the background ferent chemical compounds. Spectra from the perimeter of the in the XRS spectrum is expected to be quasi-affine in energy and is insect show features markedly different from the bulk amber. The modeled as C (X) + C (X)E. 1 2 XRS spectrum I(E, X) at a given voxel of coordinates X = (x, y, z) The decomposition of the XRS data was performed as described is a linear combination of the spectra of the different compounds in Material and Methods, using as initial guess the averaged spectrum present and a background signal from valence electron background of amber measured in the bulk amber (Fig. 4D). As shown on the and Compton scattering map of the quadratic error of the fit, the spectral data differ consider- ably from the amber for a 1-pixel-wide line at the location of the ref,norm insect’s former cuticle (Fig. 4B). Spectra from these pixels show I(E, X ) = ∑ A (X ) I (E ) + C (X ) + C (X ) E (1) n n 1 2 notably different characteristics compared to those of the amber n=1 Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 5 of 9 | SCIENCE ADVANCES RESEARCH ARTICLE AB x (beam direction) x (beam direction) C D 285.4 286.6 288.3 289.2 292.4 ExV ExV ExD Amber ExD Chitin standard 285 290 295 300 x (beam direction) Energy transfer (eV ) Fig. 4. XRS virtual cross section and spectra of an Eocene ant entrapped in amber from Oise (France, ca. 53 Ma old). (A) Total intensity virtual cross-sectional image (pixel size, 50 m; beam size, 10 m by 20 m) of the ant entrapped in the Eocene amber (image with interpolation). (B) Spatial distribution of the quadratic error showing the diverse chemical regions of the sample when performing a fit based on the amber reference (image with interpolation). (C) Spatial distribution of A when performing chit a fit based on the reference compounds amber and chitin (image with interpolation). (D) From top to bottom, distinctive normalized mean XRS spectral profiles corresponding to the ventral (A > 0.007, ExV) and the dorsal (A < −0.007, ExD) areas of the exoskeleton, as indicated with the arrows in (C), Oise amber collected from the bulk chit chit specimen (amber), and chitin standard used as a reference material (chitin standard). Scale bars, 500 m. matrix. In particular, some show a more intense feature at 288.3 eV The clustering of the data based on A allows discussing the chit attributed to 1s-* transitions from amide groups, while others are chemistry of the arthropod’s cuticle. The spectrum ExV is charac- characterized by the absence of spectral feature at 285.4 eV, which terized by the presence of 1s-* transitions due to the presence of points to the absence of aromatic-olefin carbons. aromatic and/or olefinic carbons (Fig. 4D). Both spectra ExV and Arthropods’ cuticle is a hierarchically structured material com- those from the chitin reference are dominated by a broad complex posed of an external thin epicuticle layer, rich in lipids and proteins, feature centered at 288.3 eV, which has been attributed to the presence and the exocuticle and endocuticle composed mainly of chitin- of carbonyl associated with the amidyl group of the glycosyl ring protein complexes. Chitin [(C H O N) ], a linear polymer of (17). The complex feature centered at 289.2 eV of the XRS spectrum 8 13 5 n -1,4–linked N-acetyl glucosamine, unlike most carbohydrate com- of the chitin standard is attributed to the 1s-3p/ * transition of pounds, is not water soluble and known to be quite decay resistant O-alkyl (C─OH) moieties (15). The feature at 286.6 eV, present in (17, 29, 30). We therefore compared the latter spectra to a reference the chitin standard, is assigned to the presence of vinyl ketone moieties, chitin sample measured with XRS spectroscopy under identical a possible result of radiation-induced changes (31). The feature at conditions. Since both show similar features, we added a chitin 285.4 eV, assigned to the presence of 1s- * transition of olefinic reference spectrum to our fitting model. The parameters A and moieties, may be indicative of the specimen chemistry and/or result amb A predicted from linear least-squares fitting reflect the possible from the elimination of hydroxyl groups from polysaccharides during chit presence of the compounds in each voxel. Positive values of A irradiation as reported by Cody et al. (31). chit (A > 0.007) are observed in the ventral exoskeleton (Fig.  4C, In contrast, 1s- * transitions are totally absent from the arthropod’s chit arrow ExV). In contrast, significantly different values (A < −0.007) cuticle close to the specimen dorsal right surface (Fig. 4, C and D, chit are observed for pixels denoted by arrow ExD, which leads to a spectrum ExD). The less intense feature of this spectrum, centered second spectrum when segmented (Fig. 4C). We speculate that at 288.3 eV, is attributed to the overlapping contribution of 1s- * the negative A values in the least-squares regression indicate transitions in amide groups (15) and the possible presence of 1s-* chit the presence of an additional chemical compound not included transitions of aliphatic groups in the energy region of 287.3 to 288.0 eV in the model. (25). The presence of aliphatic carbons is consistent with the seminal Georgiou et al., Sci. Adv. 2019; 5 : eaaw5019 30 August 2019 6 of 9 y (scanning direction) y (scanning direction) y (scanning direction) Normalized intensity (a.u.) | SCIENCE ADVANCES RESEARCH ARTICLE work of Stankiewicz et al. (32), which identifies through invasive in 3D with reasonable resolution opens entirely new avenues for means aliphatic geopolymers in amber inclusions’ exoskeleton, the chemical characterization of organisms preserved in amber. attributed to burial diagenetic transformation of the original chemistry of the organism. Chitin and lignocellulose could be identified using pyrolysis-GC/MS in subfossil (2 to 20 thousand years old) insect and CONCLUSIONS AND IMPLICATIONS plant specimens entrapped in resins from Kenya, yet no trace of these Over the past two decades, XRS spectroscopy has been successfully macromolecules could be identified in Dominican amber inclusions applied for chemical speciation of light element systems and conditions (>25 Ma); instead, aliphatic polymers and sulfur-containing moieties not suited for a soft x-ray probe. Our results shown here extend this were identified (32). Aliphatic signatures were also identified in fossil capability to the hyperspectral 2D and 3D imaging of organic-rich arthropods preserved in sediments [e.g., (30)]. In all the previous paleontological specimens and geological objects. The possibility to examples mentioned, pyrolysis-GC/MS was used at a semiquantitative work on these samples without any specific preparation and under level; selective sampling does not ensure that the sampling area is ambient conditions is the critical advantage of this hard x-ray probe. representative of the whole specimen. Here, the identification of two While other imaging methods, most notably x-ray phase-contrast distinct chemical fingerprints in the exoskeleton (dorsal right versus tomography, provide better spatial resolution and are less restricted ventral left distributions; Fig. 3E) points to the importance of discrim- in terms of sample size and composition, the XRS method shown inating the spatial distribution of organic compounds in 3D, at a here uniquely complements such information by providing un- global scale, to provide complete information about the specimens’ precedented insights into 3D carbon speciation. Given the small cross biochemistry, physiology, molecular evolution or function, or chemical section and resulting weak signal strength, XRS-based speciation interactions between the organism and the depositional setting. characterization and imaging pose some restrictions for sample size Inclusions in amber occur in several flow sequences and often form and composition. For 2D imaging, the main restriction is the pres- multiple inclusions (i.e., syninclusions). Each flow leads to a preservation ence of heavy elements in the sample, which reduce the scattering mode that depends on several unknown factors including time. volume. As current XRS setups operate in the range of 6 to 10 keV, Coty et al. (33) highlighted the need for a 3D approach of taphonomy to 3D imaging is furthermore restricted to sample sizes and compositions constrain paleobiological interpretations. Their computed tomography that permit the penetration at these x-ray energies. Further work scan of multiple inclusions in the same sample revealed different levels will aim at minimizing the source of potential radiation-induced of preservation depending on the resin layer and on the type of organism. damage (37). Several directions can be pursued as follows: using Although amber and copal (fossil tree resin) preserve three- cryogenic and anaerobic conditions for some samples, increasing dimensionally and, in superb detail, numerous organisms (insects, x-ray energy to reduce the photoelectric cross section while increasing feathers, plants, etc.) as a result of entrapment that rapidly dehydrates the XRS intensity and penetration length, and defocusing the beam the inclusions and protects them from water and microbial decay in the vertical direction using the so-called 2D sectioning mode (19). (3), many localities only preserve cuticle or hollow molds (such as In addition to ancient fossils, the development of this approach to the ant we studied herein) or no fossils at all, and the processes that the study of organic-rich heterogeneous materials shows promise control this range in preservation (nature of the resin producer and for numerous fields of research, including geosciences, life sciences, of the inclusions, environmental conditions, maturation, and re- materials sciences, and cultural heritage. working) remain essentially unknown (34, 35). The clustering of the data based on the total intensity allows the discrimination of the inclusion from the surrounding organic sub- MATERIALS AND METHODS strate. Further decomposition of the cuticle voxels (Fig. 3D, brown Paleontological samples voxels) based on the A parameter allows the identification of chitin Two paleontological samples were examined as follows: (i) a fragment chit traces distributed in the cuticle (Fig. 3E, red voxels). This wealth of of Lepidodendron trunk collected by one of us (P.G.) on an Upper information allows us to report the first ever fully noninvasive 3D Carboniferous (ca. 305 Ma ago) coal slag heap in Noyelles-lez-Lens, speciation of carbonaceous compounds inside a paleontological France, on 15 April 2012; (ii) an insect (inv. no. PA-3822, Hymenoptera: specimen (Fig. 3). Cuticle voxels with A positive and negative Formicidae) entrapped in amber, an Ic-type resin typical of angio- chit values are represented in red and blue colors, respectively. The lack sperms and derived from a Fabaceae. The amber deposit of Oise, of carbon signal in the internal part of the abdomen and the head found in 1996 at the Quesnoy locality in the Oise River area of Paris (Fig. 3, D and E, transparent gray voxels) points to the poor preserva- Basin in France, has been dated back from the earliest Eocene tion state of the internal soft tissue structures, while the voxels with (approximately 53 million years ago) (38). The specimen belongs to chitin affinities (positive A values; Fig. 3E, red voxels) confirm the paleontological collections of the Muséum National d’Histoire chit the preservation of the cuticular anatomy in the ventral surface of Naturelle (Paris). For comparison, we collected data on purified the insect. Until now, chemical characterization has mainly focused powder of chitin from shrimp shells (C9752, CAS: 1398-61-4, on the amber, and very few studies have investigated the inclusions Sigma-Aldrich, St. Louis, USA). because of the following challenges (36): Spectral features from the inclusions are predicted to be very close in wave number to those of X-ray Raman-based spectral imaging the resin, and because compounds of the resin have largely penetrated All experiments were performed at the GALAXIES and ID20 beamlines the inclusions (32), sampling and/or spectroscopy techniques only at the SOLEIL and the ESRF (European Synchrotron Radiation Facili- provide average information, which involves destructive sample ty) synchrotrons, respectively. The spectrometer at the GALAXIES preparation, restricting their use for rare specimens. 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