Advanced Synchrotron Characterization of ...

Advanced Synchrotron Characterization of Paleontological Specimens Pierre Gueriau1,2, Sylvain Bernard3, and Loïc Bertrand1,2 1811-5209/16/0012-0045$2.50

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DOI: 10.2113/gselements.12.1.45

or the complex refractive index, from radiographic images that have been collected using hard X-rays. Furthermore, 2-D (and occasionally 3-D) images can be collected over a vast energy range from the hard X-ray to the infrared. Two-dimensional images are formed either by specimens being fast scanned in front of a macro- to micro- (typically a few μm or below) photon beam or by collecting images of the specimen– beam interaction on an area KEYWORDS : synchrotron, fossilization, biosignatures, biomolecules, trace detector. In general, X-ray absorpelements, paleoenvironments, taxonomy, conservation tion spectroscopy (XAS), X-ray fluorescence (XRF) and photoluminescence (PL) techniques provide element and species information, INTRODUCTION derived from the excitation of electrons in atomic core Fossils have been carefully documented since the time of levels, from molecular orbitals, and from the band structhe Classical Greek philosophers, in particular by Aristotle ture (in the case of metals and semiconductors). Data from and his disciples in the 4th century BC. Fossils consist of vibrational spectral imaging and X-ray scattering or X-ray highly heterogeneous materials. Such heterogeneity results diffraction (XRD) provides information on the structure, from their original complexity and the short-to-longbonding, supramolecular organization, and texture of term changes they experienced from their death, their materials. Here, we review the benefits and limits of these deposition, their entire postdeposition history to their new imaging approaches and see what information can (or discovery, storage, and display (i.e. taphonomy, geological cannot) be gleaned from the complementary investigation history, and conservation). Ever since the early microof fossil morphology, biogeochemical processes, paleoenviscopic observations of petrified wood by Robert Hooke ronmental conditions, and conservation strategies. (1635–1703) in 1665, cutting-edge imaging approaches have been used to investigate the fi ne-scale character of FINE ANATOMICAL DESCRIPTION fossils. The past ten years have witnessed a great increase Paleontology, as a discipline, was established in the in imaging studies of specimens. Synchrotron radiation (SR) 3-D X-ray microcomputed tomography (μCT) (together early 19th century following the work of George Cuvier (1769–1832) on comparative anatomy. Descriptions of termed SR-μCT) can image a fossils’ internal morphology in unprecedented detail and so help constrain phyloge- fossil anatomy are still the main tool for deciphering the netic relationships (Tafforeau et al. 2006). phylogenetic evolutionary history (phylogeny) of extinct organisms. relationships (Tafforeau et al. 2006). In parallel, a suite of Historically, paleontologists have used a variety of photographic techniques to document and enhance the contrast spectro-imaging methods have been developed for fi neof fossil morphologies, but many fossil forms remain diffiscale chemical and structural characterization. cult or impossible to describe using conventional imaging These spectro-imaging approaches rely on interactions methods. Recent developments in synchrotron-based between light and matter. In addition to absorbing it, atoms techniques now allow paleontologists to study specimens scatter an incident X-ray beam (B OX 1). Microcomputed in a way that provides a detailed view of a fossil’s anatomy tomography relies on reconstructing electron density, with no, or only limited, sample preparation.

haracterizing fossils and quantifying paleoenvironmental proxies at a detailed scale is a significant challenge. Three-dimensional tomographic reconstructions are becoming increasingly common, and new imaging approaches, such as synchrotron-based fast X-ray scanning and full-field multispectral imaging, now provide the means to (1) describe fossil morphology at a very fi ne scale, (2) decipher long-term alteration processes, and (3) better identify conservation requirements. These developments have opened new research avenues for the study of complex heterogeneous materials at the intersection between paleontology, biology, mineralogy, geochemistry, conservation science, materials science, and analytical instrumentation.

Based on local contrasts in the absorption of X-rays in heterogeneous materials, noninvasive μCT provides unprecedented access to internal or histological structures of fragile materials, including entire matrix-encased specimens, such as insects embedded in amber. Synchroton radiation μCT phase contrast capabilities allow for fi ner discrimination of anatomical features than would be possible for laboratory or medical scanners (Tafforeau et al. 2006). Phase contrast brings improved edge detection

1 IPANEMA, CNRS, MCC, USR 3461 Université Paris-Saclay, 91192 Gif-sur-Yvette, France E-mails: [email protected], [email protected] 2 Synchrotron SOLEIL, 91192 Gif-sur-Yvette, France 3 IMPMC, CNRS UMR 7590, Sorbonne Universités

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nanotomography, illustrated by Matzke-Karasz et al. (2014) reporting on preserved organelles in 17 Ma ostracod sperm with 25-nm voxel size (a voxel being a 3-D pixel).

SYNCHROTRON ANALYSIS

BOX 1

fluorescence

characterisation of thin samples

characterisation of thick samples

fluorescence

Noninvasive mapping of major- and trace-elements using synchrotron radiation XRF (SR-XRF) enables anatomical details of fl at fossils to be defi ned because X-rays can penetrate materials to a depth of a few fractions of a millimeter (~250 and 300 μm for strontium and yttrium, respectively). Recently, SR-XRF mapping provided evidence that Archaeopteryx feathers are not just impressions but are actually remnant body fossil structures (Bergmann et al. 2010). The technique also allowed observation of the entire skull, vertebrae, and rib insertions of a newly discovered ~95 Ma ray-fi nned fish from Morocco (Gueriau et al. 2014; FIG. 2). While major- to minor-element maps using SEM–energy dispersive X-ray spectroscopy (EDX) requires between one to three hours to resolve millimeterscale details at a submicron spatial resolution, maps based on major- to trace-elements from SR-XRF (based on the measurement of integrated intensities in preselected spectral regions) can be collected over centimeter-scale regions at 100 μm resolution with a dwell time of 3 ms per pixel, and so take about 30 seconds per square centimeter. A critical improvement in SEM-EDX is fast raster-scanning (or ‘fly-scan’): by this means one can record the full-range of XRF spectra with a count time of milliseconds per pixel, while synchronously collecting complementary XRD and XAS data.

transmission elastic scattering inelastic scattering

incoming photons

diffraction

incoming photons

diffraction

excitation imaging raster-scanning

full-field imaging

In a synchrotron, light beams are generated by the deflection of high-energy particles travelling at close to the speed of light. Signals result from the interaction between the photon beam and the sample and are collected by detectors. The light source at most modern sources is white, meaning that its energy spectrum is continuous from the terahertz region to hard X-rays. Most synchrotron endstations work in the X-ray domain. The intensity of the absorbed, fluoresced or scattered beam will tell of sample composition, chemical properties, and structure. Scans of the incoming beam energy can be performed in many configurations to collect excitation spectra in speciation experiments (XAS, PL). In the X-ray range, focusing was once thought impossible because the optical index of most materials is close to unity. A century after the discovery of X-rays, focused spectroscopy now commonly uses several kinds of optics. Specimen information can now be collected at high spatial resolutions on (sub) micron spots. The high brightness of the synchrotron beam allows for both fast raster-scanning micro spectrometry and full-field imaging.

MINERAL ASSEMBLAGES Particular taphonomic and burial processes will lead to distinct patterns of preservation. These can include isolated bones in sands, highly flattened articulated specimens in shales, void cavities in calcareous nodules, or insects trapped in amber. Fossils mostly consist of biomineralized remains (the mineralized tissues produced by living organisms during their life), although altered biomolecules can sometimes resist long-term diagenesis (B OX 2). These minerals are either altered forms of original biominerals (e.g. bioapatite) or result from the replacement of original organic parts during diagenesis. The fidelity of such replication depends on the reactivity of the replacing minerals and on whether replication occurred via permineralization (mineral precipitation from early infi ltration and permeation of tissues by ion-charged water) or via authigenic mineralization (biologically induced mineral precipitation during decay; Briggs 2003). By providing innovative opportunities to assess the spatial distribution and properties of mineral phases, synchrotron-imaging techniques offer crucial information on processes that may have affected paleontological specimens after death.

from the differential refraction of the planar X-ray wave front at internal material boundaries: one can discriminate between similarly absorbing materials far better than before (Bertrand et al. 2012). For instance, μCT has provided insights into the embryology of the earliest animals that lived on Earth more than 500 million years ago (Donoghue et al. 2006); and it has revealed details on a complete gill skeleton of a 325 Ma shark-like fossil, challenging the view that sharks are primitive jawed vertebrates (Pradel et al. 2014; FIG. 1). Micro-CT has also allowed scientists do nondestructive, multiscale, 3-D virtual paleohistology on hominoid teeth (Tafforeau and Smith 2008). Paleohistology has also benefitted from the development of

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(B) Reconstructed slice following the transverse plan figured by the dashed line in A. (C) 3-D restoration of the braincase and associated visceral skeleton in right lateral view. M ODIFIED FROM PRADEL ET AL. (2014)

Propagation phase-contrast synchrotron radiation X-ray microcomputed tomography reveals the complete gill skeleton of a 325 Ma shark-like fossil Ozarcus mapesae. (A) Optical photograph of the Ozarcus mapesae fossil.

FIGURE 1

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Visualization of the anatomical details of a newly discovered fish from the Cretaceous of Morocco using XRF mapping. (A) Optical photograph of the anterior part of the fish (skull is on the right). (B) False color overlay showing the concentrations of iron (blue) and the rare earth elements

neodymium (red) and yttrium (green), which allow the entire skull, together with vertebrae (white arrows) and rib insertions (yellow arrows), to be observed. (C) Mean XRF spectra and main elemental contributions from the red box area in B: this spectrum is characteristic of fossil bone (100 pixels). M ODIFIED FROM G UERIAU ET AL. (2014)

Diagenetic mineral phases may not precisely replicate original biological structures, which can compromise the identification of fossils. An illustration of this is the reassessment of the Ediacaran Doushantuo fossils by Cunnigham et al. (2012). Among these fossils are the most ancient bilateral organisms on Earth, i.e. organisms without radial symmetry. By combining SR-μCT and electron probe microanalysis (EPMA), the authors reinterpreted these structures as artifacts of deposition caused by diagenetic minerals precipitating in the voids of the decayed tissues. Such a critical reappraisal illustrates the need for precise identification of the mineral assemblage in paleontological specimens.

ization process (B OX 2). Spatially resolved synchrotron techniques now allow documenting the biogeochemical nature of fossils.

FIGURE 2

Lebon et al. (2011) used SR-FTIR microspectroscopy to map the relative contents of, and the molecular structure of, the different constituents of fossil bones over millimeter-scale areas. Characteristic vibrational frequency maps allowed these authors to correlate the distribution of collagen biomarkers with the degree of preservation of histological structures in fossil bones from distinct ages and contexts. X-ray absorption spectroscopy (XAS) experiments at the carbon K-edge (i.e. at the energy necessary to excite electrons from the K shell of carbon atoms) can characterize the chemical markers of ancient biomolecules (FIG. 4). Cody et al. (2011) found evidence, via STXM experiments, for the presence of organic molecules that they identified as (partially) preserved chitin–protein complexes within ~310 Ma and ~420 Ma arthropod cuticles. Based on a similar approach, Ehrlich et al. (2013) demonstrated that chitin was preserved within ~500 Ma Burgess Shale sponges. Such molecular preservation is remarkable considering that this shale had been buried to depths of ~10 km. Even more remarkable is the persistence of partially degraded sporopollenin compounds within ~230 Ma lycophyte megaspores that had experienced intense metamorphism at burial depths of about 40 km (Bernard et al. 2007).

A number of additional synchrotron-based imaging techniques can be used to map inorganic crystalline phases. For instance, scanning transmission X-ray microscopy (STXM) allows mapping the oxidation state and speciation of a number of elements within mineral phases in complex mixed-valence systems, in the soft X-ray energy range covering the L-edges of K, Ca, Ti–Zn, Ga, Ge, As, Se, Rb and Sr (Bernard et al. 2009). Nowak et al. (2005) combined SR-XRF and XRD to document the spatial distribution of diagenetic minerals within petrified wood. By coupling SR-XRF imaging and X-ray absorption near-edge structure (XANES) spectroscopy on the ~500 Ma Burgess Shale arthropods, Pushie et al. (2014; FIG. 3) identified Cu-enriched phases, mostly in the form of chalcopyrite, which the authors postulate to be the remnants of bloodcarrying Cu-hemocyanin, a protein with a role similar to the hemoglobin that is produced by some arthropods and mollusks today.

X-ray photoelectron emission microspectroscopy (X-PEEM) is a technique comparable to STXM (the electron current emitted from a sample surface being related to X-ray absorbance; FIG. 4). By performing X-PEEM experiments

Laboratory taphonomic experiments combined with synchrotron analyses are increasingly being used to investigate the role played by mineral phases in maintaining the structural and chemical integrity of fossils under diagenetic conditions. For instance, using STXM and micro-Fourier transform infrared spectroscopy (μ-FTIR), Li et al. (2014) found evidence that the calcium phosphates that encrust bacterial cells help in the morphological and chemical preservation of these cells during fossilization. Similarly, Picard et al. (2015) suggested that the presence of Fe-rich phases prevents the degradation of organomineral biostructures, even under pressure and temperature conditions typical of low-grade metamorphism.

BOX 2

Unambiguously identifying biogenic remnants in sediments or rocks requires multiple lines of evidence based on a coherent pattern of presumptive biosignatures, i.e. specific mineral, molecular, structural and/or isotopic proxies. The identification of biosignatures strongly depends on the level of deterioration of the investigated fossils, the quality of the preparation of such heterogeneous samples, and the selectivity of the analytical methods used. Biosignatures are not immutable but rather prone to degradation. Biodegradation, decay, burial, increased pressure and temperature, and weathering may all affect the original mineral, chemical, and isotopic nature of the fossilized remains. Furthermore, biogenic patterns may be difficult to distinguish from abiogenic ones. The usefulness of biosignatures thus relies not only on the probability of life creating it but also on the improbability of nonbiological (abiotic) processes producing it. As paleontology tends to rely on imperfect signals, making interpretations subject to controversy, the use of moderating terms such as “suggestive,” “permissive,” “presumptive,” “persuasive,” and “compelling” are needed to qualify any evidence.

BIOGEOCHEMICAL SIGNATURES A significant complication for interpreting biogeochemical signatures within paleontological specimens is the systematic alteration of biomolecules during the fossil-

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distributions. (C). Cu K-edge XAS spectrum (dots) collected within the dark stain of the fossil compared to that of several cupriferous minerals (lines), indicating that Cu is mostly in the form of chalcopyrite. MODIFIED FROM PUSHIE ET AL. (2014)

XRF and Cu K-edge XAS evidences for mineral remnants of blood carrying Cu-hemocyanin in Burgess Shale arthropods (~500 Ma, British Columbia, Canada). (A) Optical photograph of a Marrella splendens specimen and (B) corresponding false color overlay of iron (red), sulfur (green) and copper (blue)

FIGURE 3

at the carbon K-edge, Boyce et al. (2010) investigated the chemical composition of a partially permineralized ~300 Ma Lycopsid tree. In addition to reporting the signature of ancient biomolecules with no homologue among living plants, they observed a prevalence of aromatic carbon molecules in all tissues investigated resulting from the degradation of original aliphatic compounds during burial-induced processes.

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By offering the ability to detect in situ the molecular components within fossils, SR-based imaging techniques led to the conclusion that biogeochemical remnants of ancient biomolecules may be preserved in rocks even after they have experienced intense metamorphic conditions (e.g. Bernard and Papineau 2014). However, the study of biosignatures is not restricted to organics. For instance, based on SR-XRF mapping and XANES spectroscopy at the copper K-edge, Wogelius et al. (2011) proposed a coloration pattern for the ~125 Ma fossil bird Confuciusornis sanctus.

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(A) STXM compositional map of a pollen grain showing amide- and aliphatic-rich organics (yellow) and aromatic- and ketone-rich organics (blue). B–D. STXM absorption maps (optical density) of a pollen grain wall showing (in white) the spatial distribution of (B) ketone-rich organics, (C) aliphatic-rich organics and (D) amide-rich organics. IMAGES A–D FROM B ERNARD ET AL. (2009). E–F. X-PEEM maps of vascular tissues of fossilized plants showing in white the spatial distribution of (E) carboxyl-rich organics and (F) carbonates. IMAGES E–F from BOYCE ET AL. (2010).

FIGURE 4

PALEOENVIRONMENTAL INFORMATION Reconstructing ancient environments and climates requires a range of indirect proxies to be used, including the biological or physical imprints archived in rocks and fossils. For instance, isotopic compositions and trace elemental contents in ocean sediments may provide information on ocean salinity, temperature, and biological productivity; lake sediments, speleothems, and tree rings may record information related to atmospheric temperatures; ice cores are used to estimate paleoatmospheric gas levels. In addition, depending on the extent of diagenetic degradation, isotopes and trace elements in bioapatite from vertebrate hard tissues (bones, teeth, and scales) may provide significant information about paleodiet, paleoclimate, and paleoenvironment.

As(III) oxidation and As(V) reduction being caused by microbes living in permanently anoxic conditions. Using the same technique, Frisia et al. (2012) demonstrated that microbial activity may control/modify P concentration within speleothems, thereby highlighting the caution needed when interpreting climate proxies. Microscale mapping of strontium over unprecedented sample areas of fossil fi sh otoliths provided evidence for fluctuations in Sr concentration from the core to the periphery (Cook et al. 2015; FIG. 5). These fluctuations provide essential information on the fish’s living environment. Combining synchrotron micro XRF, XAS and XRD, Gueriau et al. (2015) mapped the distribution and speciation of rare earth elements (REEs), in particular cerium, in exceptionally preserved, Cretaceous-aged fossil fi shes and shrimps from Morocco. Cerium adopts the +III and +IV oxidation states (most other REEs are purely trivalent with similar reactivity and transport properties), and this led to a depletion/enrichment of Ce compared to other

Although isotopic information cannot be obtained from X-ray methods, high spatial resolution and high signalto-noise ratio SR-XRF spectroscopy allows semiquantitative mapping the distribution of trace elements at the micrometer scale (Gueriau et al. 2014). For instance, SR-XRF raster-scanning allowed detection of oxidized and reduced As species in 2.72 Ga fossil stromatolites from Western Australia (Sforna et al. 2014). This observation led the authors to propose that As cycling widely occurred on Earth before the oxygenation of the atmosphere, with E LEMENTS

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P (μmol•g-1) Ca (mmol•g-1) Sr (μmol•g-1) 1.0 14 7.1 12 30 80

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SPECIMEN CONSERVATION, SAMPLE PREPARATION, AND RADIATION DAMAGE

AA

Counts (a.u.) 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1

REEs. This ‘cerium anomaly’ is used as a key proxy to assess paleoredox conditions. Therefore, microscale synchrotron mapping and direct probing of the speciation of trace elements open new avenues to better describe and constrain, or even challenge, paleo-environmental proxies.

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The extraction, transport, storage, study, and exhibition of paleontological specimens constitute dramatic environmental changes for those specimens. For example, drying, exposure to oxygen, and humidity will lead to mechanical degradation (flaking, cracking) and chemical alteration (oxidation, efflorescence, discoloration). 0 1 2 3 4 5 6 7 8 0 250 0 250 0 250 0 250 Paleontology benefits from research Energy (keV) X (μm) X (μm) X (μm) X (μm) done by the conservation sciences (e.g. alteration of paints in mural paintings, degradation FIGURE 5 A 4.1 ka fossil fish otolith from Huaca Prieta (Chicama of stones in historical buildings). For instance, Odin et al. Valley, Peru) was mapped using XRF. (A) Light microscopy image of the otolith. The white box outlines the section (2015) used sulfur K-edge XANES to follow the degradaanalyzed by XRF. (B) XRF spectrum of a single pixel (blue line) and tion of pyritic fossils from the Permian rocks of the Autun the average spectrum over the entire section shown in A (black Basin (France). In addition to the inorganic sulfide pyrite line). (C) LEFT: Detailed views of the otolith section taken in (FeS2 ), organic sulfides were found in shale fractions and reflected and transmitted light. RIGHT: SR-XRF maps of phosphorous, maceral layers. When exposed to air and moisture, shale- calcium, and strontium; Sr banding corresponds to subannual and fraction sulfides oxidize to sulfates, whereas maceral layer annual temporal variations in the individual’s local environment. SOURCE : COOK ET AL. (2015). sulfides remain unaltered. While storage in a cold anoxic environment will slow oxidation processes, it may also Investigations using ultraviolet (UV) and X-ray light induce degradation (as occurs in paint pigments). may, of course, lead to radiation-induced changes in a For decades, paleontological specimens have been handled specimen. Even in the absence of visual damage, irradiawithout gloves, dipped in ethanol to enhance contrast for tion may still lead to atomic and molecular changes, observation, or stored in paper, wood, or plastic vessels resulting in immediate or delayed alteration that can bias exposed to numerous sources of contamination. Similarly, future analyses. Organics are highly sensitive to X-rays: the application of consolidants and varnishes significantly amorphization of polysaccharides has been reported at impairs the study of fossils as unique scientific archives. moderate irradiation doses, and racemization of amino Truly noninvasive experiments, such as μCT of whole acids occurs on the timescale of minutes under high X-ray samples or XRD/XRF macroscanning of compressed fluxes (Moini 2014). This duration significantly exceeds fossils, only require the transfer of specimens onto a highcounting times in XRF and XRD, but it is comparable to precision rotating stage. Still, specific sample preparations that used for XAS or μCT. Contrasting results were reported are needed for high-resolution imaging techniques. Only regarding the decay of DNA amplification potential in flat specimens can be measured using XRF, XRD, PL, and archaeological bones upon X-ray irradiation. Formation X-PEEM because the X-rays are extracted from the fi rst tens of color centers during μCT experiments leads to the blackto hundreds of micrometers only. In contrast, when using ening of fossil tooth enamel. It can be reversed visually by STXM, samples have to be transparent to soft X-rays and bleaching under UV light, although occasional darkening sample preparation challenges are similar to those required may remain (Richards 2012). for transmission electron microscopy (TEM). Damage formation is a complex phenomenon that is Embedding samples in organic resins is a common practice influenced by many parameters such as dose, flux, sample to avoid their disintegration during precision sawing, composition and structure, atmosphere composition, polishing, and handling. However, this procedure leads etc. (Bertrand et al. 2015). Long exposure favors the to organic contamination, which limits the study of any appearance of radiation-induced side effects, while high inherent fossil organics. As alternatives, argon-beam sample absorbance (at low energy or close to absorption milling may be used to sputter the sample surface until edges, and for high electron density) may strongly alter nanoscale polishing is obtained and a focused ion beam the sample’s surface. Decreasing exposure duration (fast can facilitate the high-precision extraction of ultrathin shutters, flyscan), optimizing flux, characterizing material 3 sample foils (15 × 5 × 0.1 μm ). These techniques maintain behavior under irradiation, and ensuring a proper curation textural integrity even for fragile materials, and prevent of analytical data are essential measures to mitigate radiashrinkage and deformation of microscale to nanoscale tion effects on unique or valuable specimens. pores. Milling at relatively low ion currents at the fi nal stages of sample preparation efficiently minimizes artifacts due to implantation, amorphization, or the redeposition of sputtered material. F EBRUARY 2016

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Synchrotron imaging techniques, used in conjunction with the more classic paleontological techniques, therefore, provide a rich source of morphological and chemical clues to the life of the past, and are a means of gathering data on past environments and climates. It can reasonably be expected that the increasing use of synchrotron approaches in paleontology, together with continual technological and methodological improvements, will lead to important new discoveries in the coming years.

CONCLUDING REMARKS The various synchrotron methods discussed here are well adapted to the sort of multiscale heterogeneous materials that must be studied as is. Capturing information at various successive length scales (100 nm, 1 μm, 10 μm, 100 μm) is crucial for deciphering an animal’s taxonomic status and for correlating high-resolution morphologic (evolutionary) information with paleoenvironmental and taphonomic markers. A potential consequence of this new and exciting field is that it will become harder to get synchrotron beam time. There are currently ~60 synchrotron light sources operating worldwide, sixteen of which entered operation in the past 15 years. Compact X-ray sources are expected to provide increased capacities in the future. Novel synchrotron sources are today being considered or developed to increase X-ray tunability for chemical speciation analysis, to increase the flux rate for micro- or submicroscale imaging, and to more efficiently couple the different types of analytical equipment.

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ACKNOWLEDGMENTS The IPANEMA platform benefitted from a CPER grant (MESR, Région Île-de-France). Support from the Research Infrastructures activity IPERION CH (EU Horizon2020, GA n° 654028), the ERC project PaleoNanoLife (PI: F. Robert), the PATRIMA LabEx and within the MNHN/IPANEMA agreement is acknowledged. We warmly thank Dr Alan Pradel (Muséum national d’Histoire naturelle, Paris), Dr Jake Pushie, Prof. Brian Pratt (University of Saskatchewan, Canada), and Phil Cook (IPANEMA) for providing original fi les for FIGS 1, 3, and 5, respectively.

Cunningham JA and 7 coauthors (2012) Distinguishing geology from biology in the Ediacaran Doushantuo biota relaxes constraints on the timing of the origin of bilaterians. Proceedings of the Royal Society B 279: 2369-2376 Donoghue PC and 9 coauthors (2006) Synchrotron X-ray tomographic microscopy of fossil embryos. Nature 442: 680-683 Ehrlich H and 19 coauthors (2013) Discovery of 505-million-year old chitin in the basal demosponge Vauxia gracilenta. Scientific Reports 3, doi: 10.1038/ srep03497 Frisia S and 5 coauthors (2012) A re-evaluation of the palaeoclimatic significance of phosphorus variability in speleothems revealed by high-resolution synchrotron micro XRF mapping. Climate of the Past 8: 2039-2051 Gueriau P and 8 coauthors (2014) Trace elemental imaging of rare earth elements discriminates tissues at microscale in flat fossils. Plos One 9: e86946 Gueriau P, Mocuta C, Bertrand L (2015) Cerium anomaly at microscale in fossils. Analytical Chemistry 87: 8827-8836. Lebon M and 7 coauthors (2011) Imaging fossil bone alterations at the microscale by SR-FTIR microspectroscopy. Journal of Analytical Atomic Spectrometry 26: 922-929 Li J and 6 coauthors (2014) Impact of biomineralization on the preservation of microorganisms during fossilization: An experimental perspective. Earth and Planetary Science Letters 400: 113-122 Matzke-Karasz R and 8 coauthors (2014) Subcellular preservation in giant ostracod sperm from an early Miocene cave deposit in Australia. Proceedings of the Royal Society B 281: doi: 10.1098/ rspb.2014.0394. Moini M, Rollman CM, Bertrand L (2014) Assessing the impact of synchrotron X-ray irradiation on proteinaceous specimens at macro and molecular levels. Analytical Chemistry 86: 9417-9422

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