Daniel Szczerba

Davin Tan

Jean-Louis Do

Hatem M Titi

Siham Mouhtadi

Denis Chaumont

María Del

Carmen Marco De Lucas

Nicolas Geoffroy

Michel Meyer

Yoann Rousselin

Marıá Del

Yoann Rous- Selin

Jessica M Hudspeth

Valeŕie Schwanen

Petra Spoerk-Erdely

Ann-Christin Dippel

Oleh Ivashko

Olof Gutowski

Philipp Glaevecke

Vasilli Bazhenov

Mihails Arhangelskis

Ivan Ha- Lasz

Tomislav Frisčǐć
email: tomislav.friscic@mcgill.ca

Simon A J Kimber
email: simon.kimber@u-bourgogne.fr

Real-Time Observation of "Soft" Magic-Size Clusters during Hydrolysis of the Model Metallodrug Bismuth Disalicylate

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Such unconventional nucleation and growth mechanisms imply metastable species trapped in local energetic minima [START_REF] Gebauer | Pre-nucleation clusters as solute precursors in crystallisation[END_REF][START_REF] Lee | Nonclassical nucleation and growth of inorganic nanoparticles[END_REF] . When intermediates can be crystallised, they are often found to be 'magic size' clusters, i.e. containing closed geometric shells of atoms or valence electrons [START_REF] Gary | Twostep nucleation and growth of InP quantum dots via magic-sized cluster intermediates[END_REF][START_REF] Luan | Four Types of CdTe Magic-Size Clusters from One Prenucleation Stage Sample at Room Temperature[END_REF] . However, the relevance of such 'frozen' solid-state structures to solution-phase reactivity is unclear. In fact, magic size gold nanoparticles have been shown to exist in multiple polymorphs [START_REF] Jensen | Polymorphism in magic-sized Au 144 (SR) 60 clusters[END_REF] , and CdS clusters undergo isomerisation driven by surface coordination [START_REF] Williamson | Chemically reversible isomerization of inorganic clusters[END_REF] . Such 'softness' might explain why clusters can be simultaneously stable enough to crystallise, but also sufuiciently labile to participate in further growth. This question is important for metallodrugs, and has much wider implications for e.g. bio-mineralisation [START_REF] Gebauer | Stable prenucleation calcium carbonate clusters[END_REF] , geological settings [START_REF] Furrer | The origin of aluminum ulocs in polluted streams[END_REF] and nuclear chemistry [START_REF] Soderholm | The structure of the plutonium oxide nanocluster [Pu38O56Cl54 (H2O) 8] 14[END_REF] .

Here, we use X-ray pair distribution function (PDF) analysis to directly measure the solution-phase structure of intermediate clusters during the hydrolysis of bismuth disalicylate (S1). This has been previously shown to form discrete bismuth oxide clusters capped by salicylate ions [START_REF] Andre | Mechanosynthesis of the metallodrug bismuth subsalicylate from Bi2O3 and structure of bismuth salicylate without auxiliary organic ligands[END_REF] .

Measurements were performed in anhydrous (Sigma, ⩽ 0.03 % H2O) DMF, (handled under Ar), and 'bench' conditions where DMF was handled in air. For the former, dynamic light scattering (DLS) detected only large aggregates (> 200 nm, Fig. 1a). However, after atmospheric exposure (24 hrs), 1.5 nm particles with a narrow size distribution were seen. Adding 10 % water by weight shifts the peak to 2.3 nm. This implies the presence of two magic size cluster species with tunable abundancies. Immediate growth of the small species was observed using bench DMF, even when sealed under Ar. The larger species again forms when more water is added. However, the size distributions were broader. The uirst step in the condensation hence occurs rapidly, and is perhaps catalysed with very small amounts of water. Growth of the larger species is either slower, or only favourable over a certain water concentration.

All solutions were a vivid pink, showing broad peaks in UV-Vis spectra (Fig. 1b). For dry solvents/early times, this is asymmetric, with a long tail extending up to > 900 nm, implying very small, or a mixture of species [START_REF] Beecher | Atomic structures and gram scale synthesis of three tetrahedral quantum dots[END_REF] . At low bismuth concentration, a clear two-peak structure at 450 and 540 nm resolves, which is insensitive to water concentration (S2). This is also seen after 14 months of ageing mixtures of Bi2O3 and HSal in DMF (S3), and may reulect an intrinsic instability of Bi2O3 to solvolysis. As the [Bi] and [H2O] increase, the 450 nm feature sharpens, and the signal above ca. 600 nm is quenched. Consistent with the DLS results, this indicates a well-deuined, larger species (which is photoluminescent, S4). Time-dependent experiments performed at 450 nm conuirm the link between solvent water content and kinetics. After an exponential decay caused by sedimentation, a rapid increase in intensity is found over 6-12 hrs for a sealed sample in bench DMF (Fig. 1c). In contrast, sealed samples in dry DMF show no reaction over periods of over 30 hrs.

To determine the structures of the species present, we used solution phase X-ray scattering. Brieuly, data were measured with high energy X-rays (90-100 keV), an experimentally determined solvent background was subtracted, then Fourier transformation to real space was performed. This yields the PDF, which is a histogram of interatomic distances in the solute. We developed a modelling technique using point-group symmetry constraints and Debye function calculations (SI.) Our initial experiments (S5) were performed in uive different solvents. The resulting PDFs fall in two groups, consistent with a small Bi-core (ca. 0.7 nm) and a large Bi-core (ca. 1.2 nm). Dry DMF pro-duced the former, whereas bench DMF showed the larger cluster. We uingerprinted the species present, by calculating the PDFs of a wide range of Bi-oxo clusters from the CSD database (S6). The position and intensity of the peaks for the small species were matched by a reported 10 solidstate [Bi9O7(HSal)13] cluster (Fig. 2a). This structure is triclinic, with 27 fractional Bi coordinates. However, the calculated peak widths (Γ) were substantially larger than in the data (Fig. 2b). The calculated Γ contains three contributions, thermal displacements (σ 2 ), broadening from the uinite momentum transfer (Qmax=14 A€ -1 ) and the dispersion of atomic distances in the model. Even when σ 2 was reduced to zero, we were unable to uit the peak widths, showing that the solid state model is measurably more distorted than the solvated cluster. Examining the {Bi9O7} core, we noticed that the Bi positions make up a partially stellated octahedron, with nearly three-fold symmetry along one axis. Displacements away from these positions originate from lone pair distortions. We converted the model to cartesian coordinates, and imposed three-fold symmetry (Fig. 2c). This model retains the oxide positions and salicylate ligands, and the Bi positions can be reuined with only two variables. This converged with a much better uit of the Bi-Bi peaks in the PDF, as the higher symmetry model reduces the peak widths (Fig. 2b). Complete ligand coverage fails to account for several sharp features at low-r, characteristic of organic bonding. We therefore added a contribution from free salicylate groups, consistent with a condensation reaction of smaller Bi species yielding free ligand and the [Bi9O7(HSal)13] cluster. Our time-resolved measurements (Fig. 3a,b) directly show that the {Bi9O7} cluster is the uirst product of hydrolysis. In a cyclohexanone slurry, sedimentation was slow enough to detect co-existence of polymer and [Bi9O7(HSal)13]. No evidence of smaller species, e.g. monomers or dimers was seen. However, in dry DMF open to air, we detected the initial formation of [Bi9O7(HSal)13] before the slow (over 17 hrs) emergence of peaks at distances of up to 10 A€ (Fig. 3b). This directly shows conversion into a larger species. Characteristic peaks in the PDF at 10 and 11.5 A€ , were found to match the known [START_REF] Andre | Mechanosynthesis of the metallodrug bismuth subsalicylate from Bi2O3 and structure of bismuth salicylate without auxiliary organic ligands[END_REF] Linear combination ruled out smaller species within our detection limit, giving the time evolution of the {Bi38O44} phase fraction in Fig. 3d. The calculated peak widths were again too broad when using the reported 11 solid-state structure for [Bi38O44(Hsal)26(H2O)4(DMF)18]. This has a >30000 A€ [START_REF] Sun | The biological and medicinal chemistry of bismuth[END_REF] unit cell with orthorhombic symmetry. The clusters have only two-fold symmetry, with 57 fractional coordinates for the Bi sites alone. We hence took inspiration from the observation that the {Bi38O44} core is a distorted Keplerate [START_REF] Chandrasekhar | Bi38 Oxocarboxylate Cages are Keplerates-Synthesis and Structural Characterization of Two Bi38 Oxocarboxylate Cages[END_REF] , made up of shells of a nested octahedron, cube and truncated octahedron. Assuming that only the amplitude of the distortion is reduced in the solution phase, we parameterised it using the shifts shown in Fig. 3c.

This generates a model with a single parameter which describes the amplitude of the symmetry breaking Bi displacements [START_REF] Perversi | Co-emergence of magnetic order and structural uluctuations in magnetite[END_REF] . While none of our samples showed purely the {Bi38O44} clusters after ageing times of up to 24 hours, two phase uits of the above model with [Bi9O7(HSal)13], gave excellent uits with a small number (7) of parameters (Fig. 3e). The reuined shifts of the Bi cations in the {Bi38O44} species are much smaller than that found in the solid state model (ca. 0.15 vs 0.3 A€ ), which seems ubiquitous for this class of clusters.

To examine in more detail the structural changes upon solvating or crystallising the two clusters, we directly compared the distribution of Bi-Bi distances. For the solvated {Bi9O7} and {Bi38O44} cores, a much increased multiplicity is found for the distances in each coordination shell (Fig. 4a,4b). However, there is little evidence for volume changes, since the average values remain similar. The main difference between the solution and solid state forms of these clusters appears to be a reduction in Bi 3+ lone pair displacements. We note that other measurements, more sensitive to oxide displacements, may be needed to rule out alternative hypotheses related to disorder of this sub-lattice. This result is not unexpected, since anisotropic packing interactions constrain molecular structures in the solid state [START_REF] Steed | Should solid-state molecular packing have to obey the rules of crystallographic symmetry[END_REF] . It is however, extremely difuicult to prove experimentally by any method other than solution phase X-ray scattering as developed here. We gained further insight into the electronic origins of distortions in [Bi9O7(HSal)13] by DFT structure relaxation, which used a continuum solvent model and simpliuied ligands (S7). This T = 0 K calculation retains the distorted Bi-O substructure, which is hence shown to be an intrinsic Jahn-Teller like electronic instability. We speculate that the observed higher-symmetry solvated structure is higher energy, and that electronic degeneracy is lifted upon crystallisation [START_REF] O‰ Pik | Studies of the Jahn-Teller effect. I. A survey of the static problem[END_REF] . The importance of such inter-cluster interactions is supported by our vibrational spectroscopy measurement. In the simple octahedral species [Bi6OH12] 6+ , the low-energy vibrational spectrum is almost unchanged on solvation [START_REF] Maroni | The Vibrational Spectrum of the Hydrolytic Hexamer of Bismuth (III) 1a[END_REF] . However, for [Bi38O44(Hsal)26(H2O)4(DMF)18], the metal-oxo modes are highly sensitive to the cluster environment. While clearly visible in single crystals, gentle grinding wipes out all but one Raman mode below 200 cm -1 (S7). In the solution phase, none are visible (S8). Together with the broad UV-Vis absorption bands, this shows that the solvated clusters undergo large and incoherent motions at room temperature [START_REF] Beecher | Atomic structures and gram scale synthesis of three tetrahedral quantum dots[END_REF][START_REF] Voznyy | Computational study of magic-size CdSe clusters with complementary passivation by carboxylic and amine ligands[END_REF] .

In summary, detailed solution phase speciation detects new and previously unexplored complexity in the solvolysis of bismuth salicylate. The sensitivity of the reaction to small perturbations supports the hypothesis of a ulat freeenergy surface in unconventional nucleation and growth [START_REF] Gebauer | Pre-nucleation clusters as solute precursors in crystallisation[END_REF][START_REF] Lee | Nonclassical nucleation and growth of inorganic nanoparticles[END_REF] . We directly probe the solute structure using Xray scattering and symmetry constrained minimal models. This shows that, while magic-size clusters are trivially similar to frozen solid state analogues, they are signiuicantly higher symmetry and higher energy. These clusters are hence structurally 'soft', explaining why they can be crystallised under appropriate conditions, while also remaining labile enough to grow into larger species. We speculate that the energetic barrier which renders such clusters metastable in solution, is comparable to the energy gain when the distorted solid state form is reached.

ASSOCIATED CONTENT

Supporting information is available free of charge, and consists of: Experimental section/methods Supporting uigures S1 to S9 and details on the calculation of the pair distribution functions from models.

Notes

The authors declare no competing uinancial interests.

FIGURE 2 .

 2 FIGURE 2. a, Solid-state model 10 for [Bi9O7(HSal)13], atoms are bismuth (purple), oxygen (red), carbon (grey) and hydrogen (white). b, The Bi positions have nearly three-fold point group symmetry. This symmetry was added for the solution phase uit. c, (top) The solid-state structure (blue) produces too broad Bi-Bi peaks (i.e. is too distorted), compared to the hydrolysis product (red) in cyclohexanone; (middle) The symmetric Bi positions produces the correct peak widths. (bottom) Adding free salicylic acid (∼160 per cluster) uits the low-r features including the intense C-C distance at ∼1.5 A€ .

FIGURE 1 .

 1 FIGURE 1. a, Samples in dry DMF (under Ar), show only large aggregates by DLS. Atmospheric exposure, or adding 10 % H2O by volume yields {Bi9O7} or {Bi38O44} species. Solvent exposed to air (bottom) broadens the size distributions. b, Formation of the larger species in panel a causes an increase in intensity and sharpening of the UV-VIS peak at 450 nm. c, No change in the intensity at 450 nm is seen for sealed samples in dry DMF for > 30 hrs.

  uinal hydrolysis product, [Bi38O44(Hsal)26-(H2O)4(DMF)18].

Figure 3 .

 3 Figure 3. a, Time-resolved X-ray PDF data for a slurry of coordination polymer in cyclohexanone (open to the atmosphere). This reaction produces [Bi9O7(HSal)13] only. b, X-ray PDF data for a slurry of polymer in dry DMF (open to the atmosphere). This reaction rapidly produces [Bi9O7(HSal)13], which then converts to [Bi38O44(Hsal)26(H2O)4(DMF)18]. c, Parameterization of the lone-pair distortions of {Bi38O44} in the solid state. The amplitude of this mode was reuined against the experimental data. d, Time dependence of the {Bi38O44} phase fractions in DMF. e, Final uit of the model described in the text to data from a sample produced in 10 % H2O in DMF. This sample was sealed, and has aged for ∼24 hours. A contribution from {Bi9O7} is included at the ∼30 % level.

FIGURE 4 .

 4 FIGURE 4. a, Bi-Bi distances determined from the {Bi9O7} solid state structure model 10 with those from our reuinements. The latter model not only uits our data better, but is higher symmetry. b, Comparison of the Bi-Bi distances determined from the {Bi38O44} solid state structure model 10 with those from our reuinements. The main structural change on solvation is a reduction in Bi 3+ lone-pair displacements.

ACKNOWLEDGMENT

We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron beam time on ID15B. We acknowledge DESY (Hamburg, Germany), a member of the Helmholtz Association HGF, for the provision of experimental facilities. Parts of this research were carried out at P21.1. We thank Sakura Pascarelli for her kind help in Hamburg, and the staff of ID15 at the ESRF for assistance with consumables. Agnieszka Poulain is acknowledged for support on the beam line. VS and PSE were funded by the ESRF-ILL International Summer Student Program. This work was supported by the EIPHI Graduate School (contract ANR-17-EURE-0002). Ce travail a eté soutenu par le programme "Investissements d' Avenir", ISITE-BFC project (contract ANR-15-IDEX-0003). We thank Agneś Birot for administrative support.

AUTHOR INFORMATION Corresponding Authors

Tomislav Friščić -Department of Chemistry, McGill University, Montreál, QC H3A 0B8, Canada. Email: tomislav.friscic@mcgill.ca orcid.org/0000-0002-3921-7915


Keywords: Complex craniosynostosis, Hydrocephalus, Chiari, Crouzon, Apert, Pfeiffer

Background: Patients with syndromic faciocraniosynostosis due to the mutation of the fibroblast growth factor receptor (FGFR) 2 gene present premature fusion of the coronal sutures and of the cranial base synchondrosis. Cerebrospinal fluid (CSF) circulation disorders and cerebellar tonsil prolapse are frequent findings in faciocraniosynostosis. Objective: We reviewed the medical literature on the pathophysiological mechanisms of CSF disorders such as hydrocephalus and of cerebellar tonsil prolapse in FGFR2-related faciocraniosynostosis. Discussion: Different pathophysiological theories have been proposed, but none elucidated all the symptoms present in Apert, Crouzon and Pfeiffer syndromes. The first theory that addressed CSF circulation disruption was the constrictive theory (cephalocranial disproportion): cerebellum and brain stem are constricted by the small volume of the posterior fossa. The second theory proposed venous hyperpressure due to jugular foramens stenosis. The most recent theory proposed a pressure differential between CSF in the posterior fossa and in the vertebral canal, due to foramen magnum stenosis.

INTRODUCTION

Craniostenosis refers to premature closure of the cranial suture. Incidence is estimated at between 1 per 2,100 and 1 per 2,500 births [START_REF] Lajeunie | FGFR2 mutations in Pfeiffer syndrome[END_REF]. While the vast majority of craniostenoses involve only one of the cranial sutures [START_REF] Azoury | Fibroblast Growth Factor Receptor 2 (FGFR2) Mutation Related Syndromic Craniosynostosis[END_REF], children with syndromic faciocraniostenosis, such as Apert, Crouzon-Pfeiffer, Muenke or Saethre Chotzen syndromes, usually have premature fusion of one or two coronal sutures but also of the sutures of the skull and synchondroses of the skull base [START_REF] Coll | Pattern of Closure of Skull Base Synchondroses in Crouzon Syndrome[END_REF][START_REF] Rijken | The formation of the foramen magnum and its role in developing ventriculomegaly and Chiari I malformation in children with craniosynostosis syndromes[END_REF]. Mutations in the gene coding for fibroblast growth factor receptor 2 (FGFR2) may be responsible for the various syndromes, according to the type of amino acid alterations, such as Crouzon, Apert or Pfeiffer syndromes but also

McGillivray family scaphocephalia, lacrimotorino-dento-digital, Antley-Bixler, cutis gyrata-acanthosis nigricans-craniosynostosis, or Jackson-Weiss syndromes [START_REF] Azoury | Fibroblast Growth Factor Receptor 2 (FGFR2) Mutation Related Syndromic Craniosynostosis[END_REF][START_REF] Collet | Crouzon syndrome and Bent bone dysplasia associated with mutations at the same Tyr-381 residue in FGFR2 gene[END_REF][START_REF] Apra | FGFR2 splice site mutations in Crouzon and Pfeiffer syndromes: two novel variants[END_REF]. The association between CSF disorder, cerebellar tonsil prolapse and syndromic craniosynostosis is frequent and well-documented, especially for the Crouzon, Apert and Pfeiffer syndromes. For instance, 30% to 70% of children with Crouzon or Pfeiffer syndrome [START_REF] Cinalli | Hydrocephalus and craniosynostosis[END_REF][START_REF] Proudman | Central nervous system imaging in Crouzon's syndrome[END_REF][START_REF] Noetzel | Hydrocephalus and mental retardation in craniosynostosis[END_REF][START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF] and 40 to 90% of children with Apert syndrome show ventriculomegaly [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF][START_REF] Hanieh | Apert's syndrome[END_REF][START_REF] Renier | Prognosis for mental function in Apert's syndrome[END_REF]. This increase in volume results in progressive ventriculomegaly, requiring neurosurgical treatment [START_REF] Collmann | Hydrocephalus in craniosynostosis: a review[END_REF]. Hydrocephalus, defined as an increase in the volume occupied by the CSF with an increase in intracranial pressure, is also common [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. 9% to 17% of patients with Crouzon syndrome, 28-64% of those with Pfeiffer syndrome and 4-7% of those with Apert syndrome have authentic hydrocephalus [START_REF] Cinalli | Hydrocephalus and craniosynostosis[END_REF][START_REF] Proudman | Central nervous system imaging in Crouzon's syndrome[END_REF][START_REF] Noetzel | Hydrocephalus and mental retardation in craniosynostosis[END_REF][START_REF] Hanieh | Apert's syndrome[END_REF][START_REF] Renier | Prognosis for mental function in Apert's syndrome[END_REF][START_REF] Moore | Hydrocephalus in Pfeiffer syndrome[END_REF][START_REF] Murovic | Hydrocephalus in Apert syndrome: a retrospective review[END_REF][START_REF] Collmann | Hydrocephalus in craniosynostosis[END_REF] . A previous article reported hydrocephalus in 29% of children with Crouzon syndrome and 33% with Pfeiffer syndrome [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]; all syndromes were FGFR-2 induced and genetically confirmed. No children with Apert syndrome had an active fluid disorder [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. Discrepancies in percentages for Pfeiffer syndrome in most series probably come from the difficulty of clinically differentiating between different forms of faciocraniostenosis, such as Crouzon, Jackson-Weiss and Pfeiffer syndrome, in the absence of genetic mutation analysis. This problem is absent in Apert syndrome thanks to its morphological characteristics (syndactyly); which explains the small percentage discrepancy between reports.

Despite new classifications for Chiari malformation [START_REF] Haddad | The newer classifications of the chiari malformations with clarifications: An anatomical review[END_REF], the present study focused on Chiari type-I malformation, which we prefer to call ectopia of the cerebellar tonsils (ECT). It is defined as >5 mm protrusion of the cerebellar tonsils under the basionopisthion plane, and is found in 70% of Crouzon, 50% of Pfeiffer and 1.9% of Apert syndromes [START_REF] Friede | Chronic tonsillar herniation: an attempt at classifying chronic herniations at the foramen magnum[END_REF][START_REF] Cinalli | Chiari malformation in craniosynostosis[END_REF].

METHOD

Three literature searches were conducted on PubMed. The first used the following keywords: fibroblast growth factor type 2 receptor, craniosynostoses, hydrocephalus.

The second used: fibroblast growth factor type 2 receptor, craniosynostoses, Chiari. The third used: fibroblast growth factor type 2 receptor, posterior fossa. Respectively 12, 9 and 7 articles were retrieved, and were all analyzed. The complete reference lists of all 28 articles were exhaustively analyzed.

PATHOPHYSIOLOGY OF FLUID DISORDER

Several theories have been developed in recent years to explain fluid disorders in syndromic craniosynostosis.

1.Constrictive theory

According to constrictive theory, a small posterior fossa results in a mechanical increase in resistance to the flow of cerebrospinal fluid (CSF). This postulate was based on the finding, in children with craniosynostosis and active CSF disorder, of an association between compressed subarachnoid spaces in the posterior fossa and a small "Chiari like" 4 th ventricle anomaly [START_REF] Cinalli | Hydrocephalus and craniosynostosis[END_REF][START_REF] Collmann | Hydrocephalus in craniosynostosis: a review[END_REF][START_REF] Cinalli | Chronic tonsillar herniation in Crouzon's and Apert's syndromes: the role of premature synostosis of the lambdoid suture[END_REF][START_REF] Cinalli | Occipital remodeling and suboccipital decompression in severe craniosynostosis associated with tonsillar herniation[END_REF][START_REF] Thompson | Aetiology of herniation of the hindbrain in craniosynostosis. An investigation incorporating intracranial pressure monitoring and magnetic resonance imaging[END_REF][START_REF] Francis | Chronic tonsillar herniation and Crouzon's syndrome[END_REF]. This posterior fossa constriction was first referred to in 1976 by Hoffman and Tucker as "cephalocranial disproportion" [START_REF] Hoffman | Cephalocranial disproportion. A complication of the treatment of hydrocephalus in children[END_REF]. Hydrocephalus would in that case be the consequence of progressive blocking of the subarachnoid spaces secondary to insufficient posterior fossa development compared to cerebellar growth [START_REF] Cinalli | Hydrocephalus and craniosynostosis[END_REF][START_REF] Venes | Arnold-Chiari malformation in an infant with Kleeblattschadel: an acquired malformation?[END_REF]. It was hypothesized that, in reduced 4 th ventricle volume, these posterior fossa modifications impacted Sylvius aqueduct permeability [START_REF] Proudman | Central nervous system imaging in Crouzon's syndrome[END_REF][START_REF] Noetzel | Hydrocephalus and mental retardation in craniosynostosis[END_REF][START_REF] Collmann | Hydrocephalus in craniosynostosis: a review[END_REF]; but MRI studies generally reported an open aqueduct for patients with Crouzon, Apert or Pfeiffer syndrome [START_REF] Cinalli | Hydrocephalus and craniosynostosis[END_REF][START_REF] Collmann | Hydrocephalus in craniosynostosis: a review[END_REF][START_REF] Cinalli | Chronic tonsillar herniation in Crouzon's and Apert's syndromes: the role of premature synostosis of the lambdoid suture[END_REF][START_REF] Thompson | Aetiology of herniation of the hindbrain in craniosynostosis. An investigation incorporating intracranial pressure monitoring and magnetic resonance imaging[END_REF]. Reduced posterior fossa volume was demonstrated on isotopic cisternography [START_REF] Scarfò | Contribution to the study of craniostenosis: disturbance of the cerebrospinal fluid flow in oxycephaly[END_REF] and several volumetric studies [START_REF] Sgouros | Skull base growth in craniosynostosis[END_REF][START_REF] Wang | Beare-Stevenson cutis gyrata syndrome with Chiari malformation[END_REF]. Sgouros et al. showed that the posterior fossa was smaller in the first 2 years of life in both girls and boys with craniosynostosis: the posterior fossa had a smaller anterior posterior diameter but greater width [START_REF] Sgouros | Skull base growth in craniosynostosis[END_REF]. In 2015, we compared the volumes of the posterior fossa and the cerebellum (figure 1) between children suffering from FGFR2-induced craniosynostosis ( 14Crouzon, 6 Pfeiffer and 11 Apert syndromes) and a population of healthy subjects, using millimetric computed tomography (CT) acquisitions [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. Posterior fossa volume (figure 1) was measured by contouring the space between the cerebellar tent, occipital bone, clivus and temporal bone [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. The anterolateral boundary was the ridge of the petrous part of the temporal bone and the anterior boundary was its connection with the posterior petroclinoid ligament [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. No difference in either posterior fossa or cerebellum volume emerged between the healthy population and the syndromic patients [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF].

Venous theory

Venous theory was developed by Sainte Rose et al. in 1984 [START_REF] Sainte-Rose | Intracranial venous sinus hypertension: cause or consequence of hydrocephalus in infants?[END_REF]. The alleged pathophysiology consists in venous sinus hyperpressure, following observation of jugular foramen stenosis in hydrocephalic children with Crouzon syndrome [START_REF] Cinalli | Hydrocephalus and craniosynostosis[END_REF][START_REF] Sainte-Rose | Intracranial venous sinus hypertension: cause or consequence of hydrocephalus in infants?[END_REF]. The jugular foramen has a large lateral portion, the sigmoidal part, which receives drainage from the sigmoidal sinus, and a smaller medial part, the petrous part, which receives drainage from the lower petrous sinus [START_REF] Rhoton | Jugular foramen[END_REF]. This venous hyperpressure results in defective CSF resorption in venous sinuses [START_REF] Cinalli | Hydrocephalus and craniosynostosis[END_REF][START_REF] Sainte-Rose | Intracranial venous sinus hypertension: cause or consequence of hydrocephalus in infants?[END_REF]. Angiographic studies demonstrated the presence of a highly developed collateral venous network in a majority of children suffering from Crouzon syndrome and in fewer children with Apert syndrome [START_REF] Cinalli | Hydrocephalus and craniosynostosis[END_REF]. A previous study compared jugular foramina diameter between FGFR2-mutant children (Crouzon, Apert and Pfeiffer syndromes) and a healthy population, using millimetric CT slices (figure 2) [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. Only children with Apert syndrome showed truly reduced jugular foramina [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF], corroborating the finding that children with Apert syndrome had tetraventriculomegaly with a wide open aqueduct while none showed active hydrocephalus [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. These findings are similar to the physiopathology of ventriculomegaly in children suffering from achondroplasia, another FGFR-related pathology with reduced jugular foramen, showing large ventricles with widening of the subarachnoid spaces secondary to increased venous pressure but without any active CSF disorder [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF][START_REF] Sainte-Rose | Intracranial venous sinus hypertension: cause or consequence of hydrocephalus in infants?[END_REF][START_REF] Reina | Craniovertebral junction anomalies in achondroplastic children[END_REF][START_REF] King | Neurosurgical implications of achondroplasia[END_REF]. The theory of venous hyperpressure, related to stenosis of the jugular foramen, may explain ventriculomegaly in Apert syndrome, but cannot explain hydrocephalus in Crouzon or Pfeiffer syndromes, where ventricular dilation is associated with reduced subarachnoid spaces [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. All these differences reflect the fact that jugular foramen stenosis cannot completely explain the pathophysiology of hydrocephalus in children with Crouzon or Pfeiffer syndrome.

Foramen magnum stenosis theory

Foramen magnum (FM) area was found to be reduced in children with Crouzon syndrome [START_REF] Coll | Pattern of Closure of Skull Base Synchondroses in Crouzon Syndrome[END_REF][START_REF] Rijken | The formation of the foramen magnum and its role in developing ventriculomegaly and Chiari I malformation in children with craniosynostosis syndromes[END_REF][START_REF] Coll | The growth of the foramen magnum in Crouzon syndrome[END_REF][START_REF] Rijken | Foramen magnum size and involvement of its intraoccipital synchondroses in Crouzon syndrome[END_REF][START_REF] Milhorat | Mechanisms of cerebellar tonsil herniation in patients with Chiari malformations as guide to clinical management[END_REF][START_REF] Goodrich | Skull base growth in craniosynostosis[END_REF][START_REF] Assadsangabi | Morphology of the foramen magnum in syndromic and non-syndromic brachycephaly[END_REF]. A previous study confirmed this in populations with FGFR2induced Crouzon and Pfeiffer syndrome [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. The reduction in area was significantly associated with CSF disorder [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. The pathophysiology alleged to explain hydrocephalus in FM stenosis is a craniospinal pressure gradient: blockage of the subarachnoid spaces linked to the reduction in FM area increases pressure in the cranial compartment according to Bernouilly's theorem and the Ventury effect [START_REF] Levine | The pathogenesis of syringomyelia associated with lesions at the foramen magnum: a critical review of existing theories and proposal of a new hypothesis[END_REF]. If fluid flow is constant and diameter decreases, flow-rate increases and pressure decreases downstream of the stenosis while the opposite occurs upstream. As previously demonstrated [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF][START_REF] Coll | The growth of the foramen magnum in Crouzon syndrome[END_REF], and in agreement with the theory of Greitz [START_REF] Greitz | assessment of hydrocephalus: new theories and implications for therapy[END_REF][START_REF] Greitz | Unraveling the riddle of syringomyelia[END_REF] and Levine [START_REF] Levine | The pathogenesis of syringomyelia associated with lesions at the foramen magnum: a critical review of existing theories and proposal of a new hypothesis[END_REF], reduced CSF flow between the intracranial subarachnoidal and spinal subarachnoidal spaces plays a key role in the development of hydrocephalus in syndromic craniosynostosis. Interruption of CSF flow, by generating fluctuations independent of fluid pressure on each side of the FM, induces a dissociation of craniospinal pressure, resulting in a decrease in subarachnoid craniospinal chamber compliance [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF][START_REF] Levine | The pathogenesis of syringomyelia associated with lesions at the foramen magnum: a critical review of existing theories and proposal of a new hypothesis[END_REF][START_REF] Greitz | assessment of hydrocephalus: new theories and implications for therapy[END_REF][START_REF] Greitz | Unraveling the riddle of syringomyelia[END_REF]. This reduced craniospinal compliance with higher pressure gradient at the cranial stage leads, according to Greitz's hydrodynamic theory [START_REF] Greitz | assessment of hydrocephalus: new theories and implications for therapy[END_REF][START_REF] Greitz | Unraveling the riddle of syringomyelia[END_REF] and Levine's theory [START_REF] Levine | The pathogenesis of syringomyelia associated with lesions at the foramen magnum: a critical review of existing theories and proposal of a new hypothesis[END_REF], to abnormal pressure transmission to brain capillaries, with increases in ventricular pulse pressure and pulsatile CSF flow in the aqueduct, and finally global ventricular dilation [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF][START_REF] Greitz | assessment of hydrocephalus: new theories and implications for therapy[END_REF]. In 2013, Preus et al. developed the pulsatile vector theory, according to which arterial and venous blood flow are at the origin of three vector forces responsible for CSF pulsatility and circulation [START_REF] Preuss | Updated physiology and pathophysiology of CSF circulation--the pulsatile vector theory[END_REF]. By altering arterio-venous flow following a decrease in craniospinal compliance, as in the Greitz model [START_REF] Greitz | assessment of hydrocephalus: new theories and implications for therapy[END_REF], craniospinal pressure dissociation increases the "interstitial fluid shockwave", as in Preuss's model [START_REF] Preuss | Updated physiology and pathophysiology of CSF circulation--the pulsatile vector theory[END_REF], leading to an increase in ventricular CSF pulse wave and ultimately to ventricular dilation [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. All these mechanisms explain the presence of communicating hydrocephalus in children with Crouzon or Pfeiffer syndrome, who show a reduced FM. The absence of active hydrocephalus in Apert syndrome, where FM is not reduced, supports this theory, as does the absence of subarachnoid effusion in Crouzon and Pfeiffer FGFR2-mutants in previous reports [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF].

Clinical implications

The variable efficacy of endoscopic 3 rd ventriculostomy (ETV) in some children with faciocraniosynostosis [START_REF] Dirocco | The role of endoscopic third ventriculostomy in the treatment of hydrocephalus associated with faciocraniosynostosis[END_REF] is an additional argument in favor of craniospinal pressure dissociation [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. The stoma increases the low compliance as a result of craniospinal dissociation, by creating an alternative CSF pathway to the subarachnoid space [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF].

Pediatric craniofacial neurosurgeons usually perform craniospinal decompression surgeries, associated or not to posterior cranial vault expansion in children with FGFR2induced craniosynostosis [START_REF] Nowinski | Posterior cranial vault expansion in the treatment of craniosynostosis. Comparison of current techniques[END_REF]. It is usually easier to perform ETV than posterior cranial vault expansion, and future statistical analysis may assess the efficiency of these techniques in controlling hydrocephalus.

PATHOPHYSIOLOGY OF ECTOPIA OF THE CEREBELLAR TONSILS

The pathophysiological explanation of ECT given by Cinalli et al. [START_REF] Cinalli | Chiari malformation in craniosynostosis[END_REF][START_REF] Cinalli | Chronic tonsillar herniation in Crouzon's and Apert's syndromes: the role of premature synostosis of the lambdoid suture[END_REF] consists in premature closure of lambdoidal sutures in Crouzon and Pfeiffer syndromes compared to Apert syndrome, resulting in smaller posterior fossa volume. The conflict between neural growth and reduced bone growth, especially during the first 2 years of life when cerebellar growth is greater than that of other posterior fossa neural structures, is believed to be the origin of cerebellar tonsil ectopia [START_REF] Cinalli | Chiari malformation in craniosynostosis[END_REF][START_REF] Cinalli | Chronic tonsillar herniation in Crouzon's and Apert's syndromes: the role of premature synostosis of the lambdoid suture[END_REF]. In Crouzon syndrome, the lambdoid sutures are usually closed at a younger age than in the general population [START_REF] Coll | Pattern of Closure of Skull Base Synchondroses in Crouzon Syndrome[END_REF].

Chronologically, the first sutures to close in Crouzon syndrome are the posterior intraoccipital sutures, then the lambdoid sutures at the age of 10 months [START_REF] Coll | Pattern of Closure of Skull Base Synchondroses in Crouzon Syndrome[END_REF]. It is generally impossible to assess sutures ossification for patients with Apert or Pfeiffer syndrome, because most are operated on before 2 years of age [START_REF] Coll | Pattern of Closure of Skull Base Synchondroses in Crouzon Syndrome[END_REF][START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. Moreover, any craniofacial surgery can bias analysis of these children [START_REF] Coll | Pattern of Closure of Skull Base Synchondroses in Crouzon Syndrome[END_REF]. An FGFR2-induced craniosynostosis series of children under 2 years of age found no growth differential between neural and posterior fossa structures to explain ECT [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF] (figure3). While there was no statistically significant correlation between presence or absence of ECT and the anatomical measurements we performed, hydrocephalus and ECT were significantly associated. The literature data show that, for Crouzon's syndrome, all hydrocephalic patients have ECT and 53% of patients with ECT are hydrocephalic [START_REF] Cinalli | Chronic tonsillar herniation in Crouzon's and Apert's syndromes: the role of premature synostosis of the lambdoid suture[END_REF]. In an FGFR2-induced craniosynostosis series, 75% of Crouzon syndrome children with hydrocephalus had ECT whereas 60% of Crouzon syndrome children with ECT had hydrocephalus [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF].

The same series also showed that 83% of hydrocephalic children with Crouzon or Pfeiffer syndrome had ECT whereas only 62.5% of Crouzon or Pfeiffer children with ECT were hydrocephalic [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. Children with Apert syndrome did not have reduced FM, were not hydrocephalic and had no ECT [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. The hypothesis of aggravation of FM stenosis by ECT seems more coherent than a descent of the cerebellar tonsils secondary to hydrocephalus, although this has not been confirmed statistically.

CONCLUSION

Children with FGFR2-induced craniosynostosis can have hydrocephalus and ECT.

Hydrocephalus and tonsillar prolapse are statistically associated [START_REF] Rijken | The formation of the foramen magnum and its role in developing ventriculomegaly and Chiari I malformation in children with craniosynostosis syndromes[END_REF][START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF][START_REF] Coll | The growth of the foramen magnum in Crouzon syndrome[END_REF]. A correlation between small foramen magnum area and hydrocephalus has been demonstrated in children with genetically confirmed Crouzon or Pfeiffer syndrome [START_REF] Rijken | The formation of the foramen magnum and its role in developing ventriculomegaly and Chiari I malformation in children with craniosynostosis syndromes[END_REF][START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF][START_REF] Coll | The growth of the foramen magnum in Crouzon syndrome[END_REF]. Children with Apert syndrome have ventriculomegaly without decreased foramen magnum area [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. While the most likely mechanism for active fluid disorder in children with Crouzon or Pfeiffer syndrome appears to be craniospinal dissociation resulting from foramen magnum stenosis, ventriculomegaly in Apert syndrome appears to be secondary to vascular stenosis at the jugular foramen [START_REF] Coll | Skull Base Morphology in Fibroblast Growth Factor Receptor Type 2-Related Faciocraniosynostosis: A Descriptive Analysis[END_REF]. As the clinical phenotype varies greatly in FGFR2-related synostosis, the pathophysiological mechanisms underlying CSF disorders such as cerebellar tonsil prolapse may also differ according to the type of mutation.

Figure 1 .

 1 Figure 1. 3D reconstruction, axial, sagittal and coronal millimetric CT slices showing, in

Figure 2 .

 2 Figure 2. Multiplanar CT reconstruction perpendicular to the major axis of the left

Figure 3 .

 3 Figure 3. Posterior fossa volume and cerebellar volume plotted by age. Crouzon and


Carole Biron
email: carole.biron@u-bordeaux-montaigne.fr

Aurélie Mounier

Josefina Pérez Arantegui

Gwénaëlle Le Bourdon

Laurent Servant

Rémy Chapoulie

Clodoaldo Roldán

David Almazán

Nerea Díez-De-Pinos

Floréal Daniel

Colours of the " images of the floating world ". Non-invasive analyses of Japanese ukiyo-e woodblock prints (18th and 19th centuries) and new contributions to the insight of oriental materials

Keywords: Pigments, dyes, non-invasive methods, characterisation, Japanese woodblock prints

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2

The first Japanese woodblock prints date back to the 7 th -8 th century thanks to the importation from China of the woodblock engraved technique. From the beginning of the Edo era (1615 -1868), a new style of woodblock prints, the ukiyo-e, appears following the social and economic changes of this period of peace and prosperity, which sees the growth of an urban culture.

Ukiyo-e literally means the « picture of the floating world ». The term ukiyo, referring to the ephemeral side of life, was described by the 17 th century Japanese writer Asai Ryoi. The ukiyo-e prints adopt a new iconography depicting scenes of everyday life and of the pleasures of life, such as beautiful women and courtesans, kabuki1 actors or landscapes.

Following these new trends and evolutions, the techniques and materials used by artists were also changing. The first coloured prints appeared in the early 18 th century, first with few colours (red, green, yellow). In the second half of the 18 th century, the nishiki-e technique allows the use of a large palette of colours and the production of brocade picture [START_REF] Delay | L'estampe japonaise[END_REF]. This technique involved several woodblocks used for printing, one for each colour, thanks to the presence of the kentō, guides engraved in the woodblock to maintain the paper sheets [START_REF] Yoshida | Japanese Print-Making. A handbook of traditional & modern techniques[END_REF]. The traditional colour palette was mainly composed of natural organic dyes (safflower, madder, indigo, gamboge, etc.) and some inorganic pigments (orpiment, red lead, ochres, etc.). From the 19 th century, with Japanese economic opening, various chemical pigments imported from the West enriched the available palette, such as Prussian blue [START_REF] Winter | East Asian Paintings -Materials, Structures and Deterioration Mechanisms[END_REF].

Few studies have been conducted on the material aspect of these objects. Information on the kind of materials, either paper or colouring material, are, however, of interest for different reasons. Analyses conducted on these artworks mainly aimed to improve our knowledge on the history of art and techniques of the ukiyo-e by identifying the matters used to produce them. Thus, information such as technical, aesthetic or economic choices (for example natural or synthetic pigments, local or imported pigments) can be gathered and it may shed light on the artistic trends at that time and on politic and social history of Japan. Previous studies were focused on the identification of the pigments used in the ukiyo-e prints and paintings. However, they mainly considered some specific aspects or materials, such as colour (blue [START_REF] Leona | Fiber Optics Reflectance Spectroscopy: A Unique Tool for the Investigation of Japanese Paintings[END_REF][START_REF] Leona | The identification of Indigo and Prussian Blue on Japanese Edo-Period Paintings[END_REF][START_REF] Sasaki | Dayflower Blue: Its appearance and lightfastness in Traditional Japanese Prints[END_REF][START_REF] Shimoyama | Non-Destructive Identification of Blue Colorants in Ukiyo-e Prints by Visible-Near Infrared Reflection Spectrum Obtained with a Portable Spectrophotometer Using Fiber Optics[END_REF][START_REF] Kida | Deterioration of Ferric Ferrocyanide pigment in Ukiyo-e printed in the Late Edo Period[END_REF], yellow [START_REF] Luo | Synthetic arsenic sulfides in Japanese prints of the Meiji period[END_REF][START_REF] Zaleski | Natural and synthetic arsenic sulfide pigments in Japanese woodblock prints of the late Edo period[END_REF], for example), particular types of materials using specific analytical methods (organic colourants with spectrofluorimetry [START_REF] Shimoyama | Non-destructive determination of colorants used for traditional Japanese ukiyo-e woodblock prints by the three-dimensional fluorescence spectrum using fibre optics[END_REF][START_REF] Derrick | Characterization of Yellow and Red Natural Organic Colorants on Japanese Woodblock Prints by EEM Fluorescence Spectroscopy[END_REF]; synthetic organic dyes with Raman spectroscopy [START_REF] Cesaratto | A complete Raman study of common acid red dyes: application to the identification of artistic materials in polychrome prints[END_REF][START_REF] Cesaratto | A timeline for the introduction of synthetic dyestuffs in Japan during the late Edo and Meiji periods[END_REF][START_REF] Cesaratto | The cultural meanings of color: Raman spectroscopic studies of red, pink and purple dyes in late Edo and early Meiji period prints[END_REF]), and sometimes considering limited time (early prints 1650 -1800 [START_REF] Derrick | Characterization of Yellow and Red Natural Organic Colorants on Japanese Woodblock Prints by EEM Fluorescence Spectroscopy[END_REF] or from the second half of the 19 th century [START_REF] Cesaratto | A timeline for the introduction of synthetic dyestuffs in Japan during the late Edo and Meiji periods[END_REF]).

The main part of this work consisted in the global identification of the colouring matters employed to create the Japanese woodblock prints in order to follow the evolution of the use of pigments and dyes throughout the late 18 th century to the second half of the 19 th century. Non-invasive techniques, such as X-Ray fluorescence spectroscopy, visible reflectance spectroscopy, infrared spectroscopy or spectrofluorimetry have proven to be suitable for the identification of the colourants used on prints on paper and specifically ukiyo-e [START_REF] Biron | Probing some organic ukiyo-e Japanese pigments and mixtures using non-invasive and mobile infrared spectroscopies[END_REF][START_REF] Biron | Characterisation of organic colourants in ukiyo-e prints by Fourier transform near infrared fibre optics reflectance spectroscopy[END_REF][START_REF] Mounier | Red and blue colours on 18th-19th century Japanese woodblock prints: In situ analyses by spectrofluorimetry and complementary non-invasive spectroscopic methods[END_REF][START_REF] Pérez-Arantegui | Colours and pigments in late ukiyo-e art works: A preliminary non-invasive study of Japanese woodblock prints to interpret hyperspectral images using in-situ point-by-point diffuse reflectance spectroscopy[END_REF]. Thus, analyses have been conducted on a corpus of ten ukiyo-e prints belonging to the Museum of Zaragoza, Spain.

Material and methods

Prints of the Asian art collection (Museum of Zaragoza, Spain)

The "Federico Torralba" collection, kept in the Museum of Zaragoza (Spain), is a collection of Asian arts gathering more than a thousand of Asian artworks, such as bronze sculptures, Chinese paintings, pieces of lacquerware and a hundred of Japanese woodblock prints. Among them, there are prints from the 18 th and the 19 th centuries signed by some of the greatest Japanese artists such as Harunobu, Utamaro or Koryûsai (figure 1). Among the selected woodblock prints to be studied (see supplementary material), as well as some artworks already described in previous works [START_REF] Mcglinchey | Handheld XRF for the examination of paintings: proper use and limitations[END_REF][START_REF] Roldán | Characterization of Sorolla's gouache pigments by means of spectroscopic techniques[END_REF], one of the most ancient woodblock prints in the collection by Suzuki Harunobu 鈴木春信 (1725-1770), dedicated to poetess Ise and dated 1767-1768 (n.º49571), was included (figure 1.left) [START_REF] Almazán | El grabado japonés ukiyo-e del periodo Edo (1615-1868) en la Colección de Arte Oriental de Federico Torralba del Museo de Zaragoza : el género bijin-ga[END_REF]. In order to span the period from the second half of the 18 th century to the end of the 19 th c., also three other woodblocks prints were studied: one by Teisai Senchō 貞斎 泉晁 (1812-?), Sugatano Sugata Ebi-ya, dated in the 1830s (n.º49909); another by Utagawa Kuniyoshi 歌川国芳 (1797-1861), Shimazoroi onna Benkei, in c.1844 (n.º49872); and the third one by Tsukioka Yoshitoshi 月岡芳年 (1839-1892), Tsuki no haykushi, published in 1885-1891 (n.º49861) [START_REF] Almazán | El grabado japonés ukiyo-e de era Meiji (1868-1912) en la Colección de Arte Oriental de Federico Torralba del Museo de Zaragoza[END_REF]. To complete the corpus, a triptych print, designed by Kunisada/Toyokuni III during the year 1855 (collection of the IRAMAT-CRP2A laboratory), has been added to the study (figure 1. right).

Non-invasive analyses employed

X-Ray Fluorescence spectroscopy (XRF)

Non-destructive analysis of the paintings was made in situ at the building of the Museum of Zaragoza. The Energy Dispersive X-Ray Fluorescence (XRF) analyses were performed by means of a portable spectrometer equipped with a Peltier cooled semiconductor SDD (25 mm 2 detection area, 500 µm thickness, 12.5 µm Be window) with an energy resolution of 135 eV (FWHM at 5.9 keV). The spectrometer used included a low power X-ray transmission tube (silver anode) operating with an excitation potential of 30 kV and an acquisition time of 200 s. The X-ray beam impinges orthogonally on the sample surface and it is collimated by an aluminium pinhole, resulting an analysed area of about 15 mm 2 at the sample surface. The X-ray tube and detector were fitted on a mechanical device with excitation-detection geometry of 45º and a sample-detector distance of 1.5 cm. The focus of two laser beams controlled the sample position and the point of analysis. All XRF measurements were performed in ambient air and only qualitative identification of the elemental composition of the samples was accomplished. The fluorescence X-ray spectrum of every given measurement point was normalized to the total counts of the spectrum in order to facilitate the qualitative comparison with other spectra minimizing geometrical effects and fluctuations of the tube intensity. The XRF spectra process was performed by the PyMCA software [START_REF] Solé | A multiplatform code for the analysis of energydispersive X-ray fluorescence spectra[END_REF].

Because the X-rays applied to the area of interest can penetrate through the layered structure of the analysed artworks (paint layers and paper backing), the discrimination between the characteristic elements of the pigment and those of the paper backing was made by spectra comparison. XRF spectra of paper backings showed a similar elemental composition constituted by Ca, Fe, S and K. The relative intensity of these elements varied for each print. The XRF spectra of a painted surface were directly compared to those derived from the paper backing, and the detected elements were also correlated with the named 'key elements' in a list of plausible pigments [START_REF] Mcglinchey | Handheld XRF for the examination of paintings: proper use and limitations[END_REF][START_REF] Roldán | Characterization of Sorolla's gouache pigments by means of spectroscopic techniques[END_REF][START_REF] Janssens | Non-Invasive and Non-Destructive Examination of Artistic Pigments, Paints, and Paintings by Means of X-Ray Methods[END_REF]. Argon and silver were identified in all XRF spectra, but these elements were artefacts linked to the air (Ar) and the Xray tube (Ag) and not to the sample composition. Several works make reference to the XRF pigment identification and are available by consulting scientific and academic publications (see bibliographic sections of the references [START_REF] Mcglinchey | Handheld XRF for the examination of paintings: proper use and limitations[END_REF][START_REF] Roldán | Characterization of Sorolla's gouache pigments by means of spectroscopic techniques[END_REF][START_REF] Janssens | Non-Invasive and Non-Destructive Examination of Artistic Pigments, Paints, and Paintings by Means of X-Ray Methods[END_REF] or online databases [START_REF] Larsen | Free XRF Spectroscopy Database of Pigments Checker[END_REF].

For the analyses of the print of Kunisada (BDX), we used an energy dispersive X-ray fluorescence spectrometer Seiko 6000vx equipped with a rhodium source (50 kV/mA) and a SDD Vortex detector. A helium flush is used in order to detect light elements (> Na). The SDD type detector (50 mm2 ; resolution 155 eV) is cooled by Peltier effect. The specimen holder is motorized and the repositioning accuracy is about 5 µm. This allows qualitative and quantitative analyses (with standards) and qualitative mapping. Only qualitative analyses at 15 kV without filter for light elements (≥ Na) and 50 kV with a lead filter for the heavy elements (≤ U) have been done on the woodblock print. Two collimators were used (3x3 mm and 1.2x1.2 mm) depending on the size of the analysed area. As for the in-situ data, the spectra recorded on the coloured areas were compared with the spectra of the underlying paper and a list of 'key elements' was established.

Hyperspectral imaging (HSI)

Hyperspectral remote sensors collect image data simultaneously in hundreds of narrow, adjacent spectral bands giving a continuous spectrum for each image cell. In this research, the hyperspectral CCD camera (HS-XX-V10E), developed by SPECIM, has a 1600 × 840 pixels resolution, a 2.8 nm spectral resolution and a wavelength range from 400 to 1000 nm. The painting was illuminated by two halogen lamps oriented at 45° from the sample. Spectral IDAQ software provided data acquisition, storage and wavelength calibration. The treatment of the data cube was performed with ENVI 5.2+IDL software. This technique generates data (reflectance spectra) which can be treated to obtain images (RGB, IRFC, grey colours…) [START_REF] Mounier | Hyperspectral imaging, spectrofluorimetry, FORS and XRF for the non-invasive study of medieval miniatures materials[END_REF]. By a procedure named "classification," the software compares the reference spectra of our database with those obtained from the painting [28-30] 2 . Several algorithms allow to get the spatial distribution (supervised, unsupervised, endmembers collection. . .) of the pigments. One of the most common spectral mapping methods is the Spectral Angle Mapper (SAM). This method considers each spectrum as a vector in an n-dimensional scatter plot (or n-D space). The mathematical treatment computes an angle between the reference and the observed spectrum. Smaller angles represent closer matches to the reference spectrum, and endmember spectra used by SAM can come from our spectral library. SAM is a similarity measure, not an identifier. A supervised classification of the HSI data spectra was done using the SAM algorithm (with an angle of 0.4, a good compromise for the mapping of most of the pigments) in order to obtain the spatial distribution of the pigments. An example is given in complementary material. The database must be comprehensive to provide good mapping. Studies have shown that reflectance spectra vary with mixtures or mixing proportions [START_REF] Biron | A blue can conceal another! Non-invasive multi-spectroscopic analyses of mixtures of indigo and Prussian blue[END_REF], which sometimes makes mapping difficult. Thus, other complementary methods (EDXRF, FORS-NIR, ...) are necessary to refine the results.

Diffuse Reflectance Spectroscopy (point-by-point)

Reflectance spectra and colour were measured in several points of the prints. Reflectance UV-Vis spectra were recorded using a Minolta CM-2600d model portable spectrophotometer, equipped with a 52-mm barium sulphate integrating sphere, dual-beam geometry, di:8°, de:8°, a 360-740 nm wavelength range with 10 nm measurement intervals, and irradiating a 3-mm diameter area. The standard illuminant was D65, using a CIE 1964 10° standard observer. A plate of barium sulphate was used as white reference to calibrate reflectance spectra. From the dispersion of the reflectance measurements, colour coordinates (L*, a*, b*) of the CIELab 1976 colour space were calculated.

Portable Reflectance FTIR

In situ mid-FTIR reflectance analyses were carried out using a portable instrument ALPHA FTIR Spectrometer (Bruker) equipped with a DTGS detector and using an external reflection module. The spectra were obtained by averaging 200 scans, at a resolution of 4 cm -1 in the 7500-400 cm -1 range and expressed as function of pseudo-absorbance (log(1/R), R being the reflectance measured by calculating the ratio of the intensity of the beam reflected by the sample to the intensity of the beam reflected by a reference gold mirror). The colour charts were positioned vertically on an easel in front of the reflection module of the spectrometer.

Fibre Optic Reflectance Spectroscopy in Near Infrared (FT-NIR FORS)

A portable FT-NIR Rocket spectrometer (ARCoptix), equipped with an InGaAs photodiode detector and an HL2000 halogen lamp (Ocean Optic, 20 Watts) was used. Measurements were collected through an optical fibre bundle constituted of seven optical fibres (400 µm diameter -6 illumination fibres around one collecting fibre) resulting in a beam diameter of approximately 3 mm. The probe was positioned perpendicularly upon the surface at a distance between 3 and 5 mm. Spectra were measured in the 11000 -4000 cm -1 range (900 -2500 nm) by averaging 200 scans and at a resolution of 8 cm -1 . NIR reflectance (R) spectra were obtained using a white Spectralon® standard as reference. They were expressed as pseudo-absorbance via log(1/R) conversion.

Spectrofluorimetry

The fluorescence emission spectra were acquired using the LEDµSF. It is composed of a Thorlabs spectrometer (CCS200/M) with a spectral range from 200 to 1000 nm and an optical fibre (Ø 400 μm) [START_REF] Mounier | LEDμSF: A new portable device for fragile artworks analyses. Application on medieval pigments[END_REF]. The UV excitation sources are composed of two low power LEDs at 285 nm (300 μW) and 375 nm (5 mW). They are used with associated filters to remove the signal due to the reflection of the LED and the second order effects (320 nm for the first LED and 455 nm for the second one, respectively). The investigated area on the sample was about 1-2 mm in diameter for a working distance of 4 cm. The analysis time was set between 10 μs to 50 s. For each measure, a background was recorded and subtracted to the sample spectrum. This background constituted the environmental noise during measurement. The subtraction allowed to increase the signal-to-noise ratio. Then a spectral smoothing was performed. The LEDμSF is powered and controlled by USB connection to a portable computer and by means of a graphic interface. The instrument stands on a tripod equipped with a micrometric device and two red lasers in order to adjust the working distance and the positioning without any contact.

Results

The analytical results for each print are summarised in table 1 (the localisation of the analysed zones is proposed in supplementary material). Three colours are predominant in Japanese woodblock prints: red, blue and yellow. One or more colourants can be used to get the primary colours or different hues (green, violet, etc.).

Blue colours

Three different blues have been identified in the studied prints. Indigo, whose Japanese name is ai, was used in Japan since, at least, the ninth-century [START_REF] Winter | East Asian Paintings -Materials, Structures and Deterioration Mechanisms[END_REF][START_REF] Yamasaki | Pigments used on Japanese Paintings from the Protohistoric Period through the 17th century[END_REF]. It was obtained from the indigenous indigo plant Polygonum tinctorium, from dyed fabrics as described by Hokusai [START_REF] Perrin | Le Traité illustré du coloris de Hokusai[END_REF] or as already-made sticks, imported from China [START_REF] Takamatsu | On Japanese pigments[END_REF].

Indigo was identified thanks to its specific visible feature, a maximum of absorbance at around 660 nm, and infrared bands, a doublet at about 1480 and 1460 cm -1 (figure 2.b and 2.d). On each print where indigo was identified, it involved also at least another colourant (table 1) as Prussian blue for blue areas (n°49148, figure 2. b), orpiment to obtain green hues (n°49571 and 49868, figure 2.d)) or with vermilion (n°49225). Fluorimetry allowed also the identification of indigo (emission band at about 730 nm) sometimes mixed with Prussian blue, while the latter is not detected by UV fluorescence because of the iron presence [START_REF] Mounier | Red and blue colours on 18th-19th century Japanese woodblock prints: In situ analyses by spectrofluorimetry and complementary non-invasive spectroscopic methods[END_REF][START_REF] Biron | A blue can conceal another! Non-invasive multi-spectroscopic analyses of mixtures of indigo and Prussian blue[END_REF]. Unfortunately, portable XRF is not a suitable technique to identify organic pigments, because working in air it does not detect light elements with Z lower than 13 (Al).

Prussian blue, or bero-ai (Fe4[Fe(CN)6]3), was easily identified using mid-infrared spectroscopy thanks to the strong cyano stretching vibration ν(C≡N) at around 2080 -2100 cm -1 , combined with characteristic narrow near infrared bands, including the most intense about 4220 cm -1 (figure 2.a and 2.b) [START_REF] Biron | Probing some organic ukiyo-e Japanese pigments and mixtures using non-invasive and mobile infrared spectroscopies[END_REF]. It was mainly used alone for blue areas thanks to its ability to provide very different hues of blue, from lighter sky blue to a dark, almost black, blue [START_REF] Biron | A blue can conceal another! Non-invasive multi-spectroscopic analyses of mixtures of indigo and Prussian blue[END_REF]. Prussian blue was also employed in mixture to get mainly green shades, combined with orpiment (table 1, n°49872, 49861, 49148 and BDX), identified thanks to visible reflectance spectra (figure 2.d, inflection point at about 490 nm) and confirmed by the presence in the XRF spectra of the K-lines of iron at 6.40 and 7.06 keV. Due to the high tinting strength of the Prussian blue, often present in low concentrations, the X-ray fluorescence lines of Fe may be weak. We have compared the intensity of the XRF lines of Fe from the blue zones with the XRF lines of Fe in the paper backing areas. In the case of the blue zones identified as Prussian blue, the intensity of the XRF lines of Fe in the pigment is always higher than in the paper backing. Dayflower, also known as Japanese tsuyukusa (wild species) or aobana (hybridised species), gives a blue colourant, extracted with water from paper sheets soaking with the blue liquid (aigami), only used in Japan. Dayflower showed a characteristic visible spectrum with two apparent maxima of absorption at 590 and 640 nm (Figure 2.c). In the studied prints, dayflower was not identified alone but always in mixtures with a red colourant to get a purple hue.

Red colours

Several red colourants have been found in the prints. Red safflower is an organic red obtained from the petals of the specie carthamus tinctorius and it showed an apparent absorption band at 530 nm in the visible range [START_REF] Aceto | Characterisation of colourants on illuminated manuscripts by portable fibre optic UVvisible-NIR reflectance spectrophotometry[END_REF] as several bands in the mid-infrared range, principally at 1736 and 1603 cm -1 [START_REF] Biron | Probing some organic ukiyo-e Japanese pigments and mixtures using non-invasive and mobile infrared spectroscopies[END_REF].

Red safflower was also identified by fluorimetry with a characteristic emission band at 580-590 nm on several prints (spectra not shown, n°49571, 49572, 49868, 49861, 49872, 49909, BDX). These spectral features have allowed the identification of red safflower on six of the woodblock prints studied. The mid infrared bands ascribed to red safflower (2920, 2850, 1730 and 1630 cm -1 ) have also been highlighted using spectral subtraction of the paper signal, for example for the most ancient analysed print of the collection (print n°49571, figure 3.a). It was used alone to get pinkish red to red colours or in mixture with dayflower for the purple colours, as found in the prints n°49868 or BDX (figure 2.c). Three kinds of inorganic pigments were also identified thanks to visible reflectance spectroscopy and XRF analyses. Red lead (tan in Japanese, Pb3O4), vermilion (shu, HgS) and red ochre (bengara) were recognised using their specific visible reflectance features, which are inflection points at respectively 560 nm, 590-600 nm and 580 nm (figure 3.b) [START_REF] Aceto | Characterisation of colourants on illuminated manuscripts by portable fibre optic UVvisible-NIR reflectance spectrophotometry[END_REF], and confirmed by the main XRF spectra L-lines of Pb in red lead at 10.54 and 12.63 keV, by the main XRF spectra L-lines of Hg in vermillion at 9.98 and 11.86 keV, and the K-lines of Fe at 6.40 and 7.06 keV in red ochre.

Finally, cochineal and synthetic red were identified in the recent prints.

Cochineal was revealed in a Meiji reprint, following a design by Koryûsai (n°49148). The organic pigment was mixed with vermilion [START_REF] Mounier | Red and blue colours on 18th-19th century Japanese woodblock prints: In situ analyses by spectrofluorimetry and complementary non-invasive spectroscopic methods[END_REF]. The reflectance spectrum presented two absorption bands at 530 and 565 nm (Table 1), characteristic of this insect-based pigment. The ultraviolet (UV) excited fluorescence emission spectra were disturbed by the fluorescence emitted by the paper at about 500 nm and therefore the maximum of fluorescence emission of cochineal was shifted towards the short wavelengths (from 640 nm down to 610 nm), which limits the identification of the cochineal based only on the UV fluorescence emission spectra [START_REF] Mounier | Red and blue colours on 18th-19th century Japanese woodblock prints: In situ analyses by spectrofluorimetry and complementary non-invasive spectroscopic methods[END_REF].

Among the most recent woodblock prints, new synthetic red pigments were also identified. Naphthol red was detected in the dark red of the Yoshitoshi design (nº 49861, dated in 1886) by its visible reflectance spectrum, with its characteristic inflection point at 615 nm. However, the use of this red pigment was mainly confirmed because of its association to the presence of barium sulphate (probably barium white) [START_REF] Mounier | Red and blue colours on 18th-19th century Japanese woodblock prints: In situ analyses by spectrofluorimetry and complementary non-invasive spectroscopic methods[END_REF][START_REF] Pérez-Arantegui | Colours and pigments in late ukiyo-e art works: A preliminary non-invasive study of Japanese woodblock prints to interpret hyperspectral images using in-situ point-by-point diffuse reflectance spectroscopy[END_REF][START_REF] Almazán | El grabado japonés ukiyo-e del periodo Edo (1615-1868) en la Colección de Arte Oriental de Federico Torralba del Museo de Zaragoza : el género bijin-ga[END_REF], a common ingredient of lithographic inks used as an extender or as a substrate of the lake pigment, detected by the presence of the characteristics XRF L-lines of Ba, at 4.46, 4.84 and 5.16 keV, and the K-line of S, at 2.31 keV (figure 4).

Yellow colours

As mentioned above, yellow pigments and colourants were employed in the ukiyo-e woodblock prints. Orpiment (or arsenic sulphide) was mainly detected in mixtures with indigo and Prussian blue, using XRF, by the presence of the K-lines of As at 10.53 and 11.72 keV, and its inflection point around 490 nm on visible reflectance spectra (Figure 2.d). Arsenic sulphide was found alone only in the most recent woodblock prints (Yoshitoshi nº 49861, figure 5.a, and Shuntei nº 49770). In both cases visible reflectance spectra exhibited two inflection points at 490-500 nm and 550-560 nm, suspecting a mixture of As pigments and then the use of synthetic As sulphides [START_REF] Luo | Synthetic arsenic sulfides in Japanese prints of the Meiji period[END_REF][START_REF] Pérez-Arantegui | Colours and pigments in late ukiyo-e art works: A preliminary non-invasive study of Japanese woodblock prints to interpret hyperspectral images using in-situ point-by-point diffuse reflectance spectroscopy[END_REF].

Infrared analyses have revealed the presence of yellow ochre in one print (n°49148) with the identification of the specific bands of kaolin, known as an accessory mineral of ochre (bands observed at 475 cm -1 , 540 cm -1 and between 3600 and 3700 cm -1 ) (figure 5.b) [START_REF] Miliani | Reflection infrared spectroscopy for the noninvasive in situ study of artists' pigments[END_REF][START_REF] Zaffino | Exploiting external reflection FTIR spectroscopy for the in-situ identification of pigments and binders in illuminated manuscripts. Brochantite and posnjakite as a case study[END_REF].

Organic colourants were also part of the palette of the woodblock printer and yellow dyes are challenging to discriminate. Indeed, all of them present almost the same spectral features in the visible range, as absorptions occur mainly between 200 and 400 nm [START_REF] Aceto | Characterisation of colourants on illuminated manuscripts by portable fibre optic UVvisible-NIR reflectance spectrophotometry[END_REF]. However, this is a first step to characterise the nature of the yellow colourant as it is recognised as an organic one. In some cases, infrared can define the nature of the colourant used, when sufficient pigment was used to be detected and when the signal of the paper does not overlap entirely with the signal of the colourant [START_REF] Biron | Probing some organic ukiyo-e Japanese pigments and mixtures using non-invasive and mobile infrared spectroscopies[END_REF].

Thus, organic yellow colourants were detected in five woodblock prints of the studied corpus. Among them, infrared analyses allowed the particular identification of turmeric, bands at 1600 and 1520 cm -1 (n°49572, figure 5.c) and gamboge, bands at 1735 and 1598 cm -1 (n°49861, figure 5.d) [START_REF] Biron | Probing some organic ukiyo-e Japanese pigments and mixtures using non-invasive and mobile infrared spectroscopies[END_REF]. The UV fluorescence spectra on yellow zones also suggested the presence of gamboge on the print n° 49872 with a maximum at about 550 nm (table 1). Besides, visible reflectance spectra in yellow areas of this print (n° 49872) showed an inflection point at 510 nm, similar to that of gamboge, but undistinguishable from As sulphide spectrum; in fact, XRF analyses detected traces of As and S, proving that a mixture of organic and inorganic pigments has been employed. Another organic product such yellow safflower could have been used (maximum of fluorescence at 525-535 nm, and visible reflectance spectrum with the inflection point around 500 nm), but it was not possible to confirm this attribution for the moment (n°49909, for instance).

Discussion

The prints studied exhibit vivid and varied hues of colours covering almost all the rainbow colours, from deep violet to bright red. The colour-skilled craftsmen obtained these shades using pure colourants or mixtures of pigments. The non-invasive analyses demonstrated the use of a restricted palette, limited to around fifteen pigments and dyes. Only primary colours were used, except for the most recent woodblock prints and the possibility of getting pink and purple colours directly with synthetic dyes, like eosin, methyl violet or crystal violet (Shuntei, n°49770, see also [START_REF] Pérez-Arantegui | Colours and pigments in late ukiyo-e art works: A preliminary non-invasive study of Japanese woodblock prints to interpret hyperspectral images using in-situ point-by-point diffuse reflectance spectroscopy[END_REF]). No one of the green pigments as malachite or copper-based green pigments were employed. However, all the colours of the rainbow were obtained with these materials from purple to dark blue. Thus, the identification of the materials was less simple than it seemed, because of the presence of several mixtures and the possible combinations of various pigments. For instance, purple corresponded mainly to a mixture of dayflower and red safflower, whereas greens were obtained either with a mixture of orpiment with blue (indigo or Prussian blue) (n°49571, 49868, 49872, BDX, 49861, 49148) or by printing a layer of Prussian blue over a yellow ochre one (n°49148). Even for primary colours, mixtures were evidenced by the combined use of Prussian blue and indigo (n°49225 or 49148) or cochineal and vermilion (n°49148). Colourants were therefore used for their proper colours, and prepared and mixed in the printers' workshops for this purpose, following the artist or publisher directives.

Regarding the results obtained on the analysed woodblock prints, an evolution of the amount of hues chosen and the palette of colourants used is noticeable. Indeed, latest prints showed more complex colour associations and a larger palette of colourants, composed of five or more colourants identified, without taking into account black inks and white pigments. On the contrary, woodblock prints dated between 1780s and 1840s were achieved using no more than four pigments. This long period encompassed notably eras of austerity, Kansei era (1789 -1801) and Tempō era (1830 -1843), which hit also artistic production, so of course occurred in ukiyo-e production. Moral and artistic edicts were issued in order to forbid the production of ukiyo-e, considered luxurious products, by mainly limiting the authorised colours and colourants [START_REF] Takahashi | The evolution of ukiyoé : the artistic, economic and social significance of Japanese wood-block prints[END_REF][START_REF] Tinios | Diversification and Further Popularization of the Full-colour Woodblock Print, c. 1804-1868[END_REF]. To a certain extent, this might explain the limited palette encountered in the woodblock prints dated from this period.

The evolution of the palette of the printer can be summarised in figure 6, involving results of this study [START_REF] Mounier | Red and blue colours on 18th-19th century Japanese woodblock prints: In situ analyses by spectrofluorimetry and complementary non-invasive spectroscopic methods[END_REF][START_REF] Pérez-Arantegui | Colours and pigments in late ukiyo-e art works: A preliminary non-invasive study of Japanese woodblock prints to interpret hyperspectral images using in-situ point-by-point diffuse reflectance spectroscopy[END_REF] as well as summarised results from the literature [START_REF] Winter | East Asian Paintings -Materials, Structures and Deterioration Mechanisms[END_REF][START_REF] Luo | Synthetic arsenic sulfides in Japanese prints of the Meiji period[END_REF][START_REF] Zaleski | Natural and synthetic arsenic sulfide pigments in Japanese woodblock prints of the late Edo period[END_REF][START_REF] Derrick | Characterization of Yellow and Red Natural Organic Colorants on Japanese Woodblock Prints by EEM Fluorescence Spectroscopy[END_REF][START_REF] Cesaratto | A timeline for the introduction of synthetic dyestuffs in Japan during the late Edo and Meiji periods[END_REF][START_REF] Keyes | Japanese woodblock prints. A catalogue of the Mary A. Ainsworth Collection[END_REF][START_REF] Fitzhugh | A database of pigments on Japanese ukiyo-e paintings in the Freer Gallery of Art[END_REF][START_REF] Connors | The identification and light sensitivity of Japanese woodblock print colorants: The impact on art history and preservation[END_REF]. To complete the previous comments, only primary colours, except modern synthetic pink and violet dyes, were used by the ukiyo-e printmakers. The colourants considered as 'traditional' and part of the palette from the beginning of the ukiyo-e printing, namely red lead, ochre, red safflower, orpiment, organic yellow, dayflower and indigo [START_REF] Takahashi | The evolution of ukiyoé : the artistic, economic and social significance of Japanese wood-block prints[END_REF][START_REF] Sasaki | Materials and Techniques[END_REF], were almost used until the end of the 19th century. The new imported pigments, as Prussian blue, organic red (cochineal and synthetic reds), did not replace the old ones, but enlarged the colour chart available. Even woodblock printers seemed to have conserved old practices for usual hues of colours, such as mixture of indigo and orpiment for green colours, until the second half of the 19 th century and the arrival of new synthetic pigments [START_REF] Cesaratto | A timeline for the introduction of synthetic dyestuffs in Japan during the late Edo and Meiji periods[END_REF], easy-to-use because of their high tinting power and their very-high water solubility, completely suitable for printing process. Changes occurred during this period, and especially from the 1870s. Some colourants turned out not to be used anymore, such as dayflower or red safflower. New synthetic red and violet dyes prevailed in the palette, but used in the same way as the old colourants. These facts revealed the changes in the Japanese society between traditions (red and purple were important colours, generally associated with high-society clothes since the Heian period) and the westernisation process (new materials were considered as new technology and, by extent, progress) [START_REF]The Impact of Synthetic Dyes on the Luxury Textiles of Meiji Japan[END_REF].

Conclusion

A holistic non-invasive analytical approach was proposed to study Japanese ukiyo-e woodblock print.

Results obtained with this different method turned out to be complementary. Most of the identifications of the colourants remained based on the visible reflectance spectroscopy, whose results were extended and confirmed by XRF, near-and mid-infrared and fluorescence analyses. By combining all these methods, all the inorganic compounds were identified, even in the case of mixtures. Regarding the organic colourants, which were largely used in Japanese ukiyo-e, the results were also conclusive especially for the blue and red areas. Yellow organic pigments were also detected, but determining their nature remains challenging. Infrared investigations allowed to improve the assignments for the yellow colourants.

Finally, a general chronology of the use of the colourants in the ukiyo-e woodblock prints between 1700 and 1900 is proposed. Traditional Japanese organic pigments (red safflower and yellow dyes, such as gamboge, and to some extent dayflower), as well as inorganic pigments (red lead, orpiment) were used almost until the end of the 19 th century, in other words, until the end of the production time of ukiyo-e woodblock prints. New colourants, such as Prussian blue, cochineal or synthetic red and violet dyes, enriched the palette. However, they did not entirely replace the old colourants and printmakers could use the available colours following their needs and the taste of the time.

Table 1 .

 1 Summary of the analytical results by HSI, point-by-point diffuse reflectance spectroscopy, FT-NIR FORS, FTIR, spectrofluorimetry and XRF spectroscopy in the coloured areas of the studied prints.

	Museum no.	Artist	Estimated date	Analysed Colour	L*; a*; b*		HSI VNIR (nm)	VIS DRS (nm)	FT-NIR FORS (cm -1 )	FTIR (cm -1 )	LEDµSF (nm)	XRF	Analyses interpretation
	49571	Harunobu	1767-1768	Bluish kimono	-		520, 560, 640 a						indigo
				Greenish-blue obi	54.4; -1.27;		660 a	600 r , 650 a				-	Indigo + orpiment
					14.67
				Greenish mountains	67.17; 0.55;	67.17;	660 a	630 r , 650 a				As, S	Indigo + orpiment
					17.51	0.55;
						17.51
				Grey mountains	55.7; 1.15;		660 a	650(?) a , 690 i					Indigo + black ink (?)
					13.03
				Greyish-green floor	57.29; 1.30;			650(?) a , 690 i				-	Indigo + black ink (?)
					15.81
				Brown kimono	41.1; 10.13;		~580 i	575 i	11000-8000 /		570	As, S,	Orpiment + Ochre
					15.84				6950			Fe
				Pinkish-red balcony	66.5; 11.76; 17.33		560 i	560 i	4330	2960, 2920, 2850, 1740 sh	570, 690	Pb	Red lead + red safflower
	49572	Koryûsai	1776-1781	Yellow courtesan's	54.42; 9.37;			No Max., no		1600 sh , 1150,			Turmeric
				obi	29.61			Min.		1105, 1075 & 1025 inv
				Orange left kamuro's	54.8; 26.3;		565 i *	565 i *			595*	Pb*	Red lead*
				kimono	33.06*
				Light-red courtesan's kimono sleeve	52.2; 13.84; 20.2*		530 a & 570 i *	515 a , 575 & 440 i *			595*		Safflower*
	49868	Eisen	c. 1825	Violet kimono (bottom)	48.06; 1.29; 4.31*		530, 594 / 645 a *	540 & 590 & 640 a *			685* 560, 610,		Dayflower* + red safflower
				Green obi	49.1; -9.15;		480 i , 660 a	480 i , 660 a *				As, S*	Indigo + As sulphide*
				background	9.88*
				Red fold kimono	53.97; 25.5; 23.4*		530 a , 580 i *	520 a , 455 & 585 i *			590*		Red safflower*
				Orange obi flowers	52.6; 23.05; 25.8*		530 a / 580 i *	520 a , 450 & 585 i *				As, S*	Red safflower + As sulphide*
	49909	Senchô	1830s	Light-blue kimono	44.83; -		490 r	500 r	11000-7500	~2090		Fe	Prussian blue
					8.79; -0.43
				Dark blue kimono	24.75; -		455 r	450 r	11000-7500,	~2090		Fe	Prussian blue
					3.50; -13.5				4220
				Light red kimono	43.45; 23.17; 19.6		530 a	520 a , 440 & 580 i			590	-	Red safflower
				Dark red kimono	44.89;		530 a	520 a , 440 &				-	Red safflower
					26.47; 20.8			580 i
				Yellow headdress	65.95; 4.78;			500 i			535	-	Organic yellow
					27.98
	49872	Kuniyoshi	c.1844	Yellow hand-mirror	65.66; 6.84;		520 i	510 i			550	(As)	Organic yellow / gamboge (?) / traces of
					35.91								arsenic sulphide
				Light-blue kimono	46.71; -		525 r , 690 a	525 r , 690 a		2095		Fe, Ca	Prussian blue + calcite (?)
					4.61; 3.06
				Blue cartouche	39.72; -9.19; -13.0		465 r , 690 a	460 r , 690 a	4220 sh	2100		Fe	Prussian blue

Kabuki is a Japanese traditional and popular theatre

The quality of the hyperspectral data interpretation (images and spectra) is based on that of the reference database. That of the CRP2A considers 200 organic and inorganic pigments used from medieval to contemporary times and prepared on parchment, Japanese paper… according to the ancient recipes. These "palettes" of reference pigments also allowed us to define the optimum analytical parameters (exposition time, working distance, power of beams, etc.) for the study of these types of fragile objects.

Acknowledgements

We are also grateful to A. Queffelec and to the laboratory PACEA (Université de Bordeaux-CNRS) for the access to portable mid-infrared spectrometer.

Fundings

This project (INDIGO, coord. F. Daniel) has received support from LaScArBx, a research programme supported by the National Research Agency (ANR-10-LabX-52). This study was also part of the AOBANA and MURASAKI projects (2018-2020, Nouvelle Aquitaine/Aragon Region fundings), coordinated by Floréal Daniel (IRAMAT-CRP2A) and Josefina Pérez Arantegui (Zaragoza University).