Céline Daher
email: cel.daher@gmail.com

Ludovic Bellot-Gurlet
email: ludovic.bellot-gurlet@upmc.fr

Non-destructive characterization of archaeological resins: seeking alteration criteria through vibrational signatures

Keywords: FT-Raman, ATR-Infrared, archaeological resins, copal, ageing, spectral decomposition

Natural resins are prone, as all natural organic products, to ageing and degradation.

Characterizing this alteration provides a better knowledge about their chemistry, and when they are involved in cultural heritage artefacts, it helps to define relevant conservation protocols. In this study, a set of archaeological African copals that present specific surface degradations and colors were analyzed by FT-Raman and ATR-IR spectroscopies. A methodology based on the modifications of the vibrational features between the bulk and the resins surface was define. A relation between the evolution of the molecular structure of the copal samples and some vibrational bands could be established; and possible ageing reactions occurring at the surface of the samples could be proposed. This specific surface aspect is actually related to an alteration, and the surface states and the colors variability are linked to different alteration degrees. This could refer to whether different conservation contexts (presence of air, water, etc), possible preparation or different origins: for example the collection of fresh or partly fossilized resins.

A. Introduction

Natural resinous substances have been used, across time, for different purposes, and in different forms: in their raw form, such as frankincense or myrrh as incense [START_REF] Langenheim | Plant Resins: Chemistry, Evolution, Ecology, and Ethnobotany[END_REF] , or amber and copal for jewellery [START_REF] De Faria | [END_REF]3 ; or different tree exudates for healing purposes or religious ceremonials 4,5 . They also have been used after some treatments (heating, mixtures): as adhesives 6,7 or waterproofing materials for ceramics 8 or ship building [START_REF] Langenheim | Plant Resins: Chemistry, Evolution, Ecology, and Ethnobotany[END_REF]9 and finally as binders for paint 10 or part of a varnish composition 11,12 .

Besides, as all natural substances, resinous materials are highly exposed to ageing and degradation, which constitute an issue when it comes to Cultural Heritage artefacts. It is thus important to characterize the alteration and degradation processes in order to have, on one hand, a better understanding of the past and present chemistry of these substances; and to be able, on the other hand, to make viable and accurate diagnoses about their conservation state.

The identification of such substances is usually done by separative techniques often coupled to mass spectrometry 13,[START_REF]Organic Mass Spectrometry in Art and Archaeology[END_REF] which nevertheless require a destructive sampling and a reasoned choice of analytical and/or pre-treatment conditions. However, working on cultural heritage artefacts often requests non destructive techniques that perform rapid analyses with slight experimental constraints. Vibrational spectroscopies are increasingly employed for identification purposes by setting up reference databases, [START_REF] Vandenabeele | [END_REF]16 or to give spectroscopic clues to differentiate between organic substances 17,18,19,20,21,22 with some specific studies on archaeological resins 23,24 . Indeed, when ancient organic materials are involved, the question of ageing and degradation is raised. The molecular modifications cause changes in the vibrational signatures, and it becomes difficult to define spectral databases of substances that evolve over time 25 .

Previous studies using Raman spectroscopy 4,26,27 tried to find ageing criteria for natural resins mostly based on two bands intensity ratio (I 1650 /I 1450 ) that evolves from a fresh resin to an aged one. However they have based their work on a corpus of resins from different chemical families, origins, size and aspects. This large variety of parameters complicates the study; their respective influence is difficult to demonstrate.

This study focuses on artefacts with the same chemistry and origin, found in a same excavation site from the 13 th century: copals from Africa and/or Madagascar found in Sharma (Yemen) 28 . Besides, the altered and unaltered analyzed materials are part of a same sample, since their surface seems degraded, and the core of the resins has a fresh transparent aspect.

Furthermore, among all the copals found in this excavation site, three groups could be formed, based on their colors and aspects. This difference in the appearance might be related to different surface alteration states, and was the motivation for this work. We tried then to get chemical information about the alteration processes with samples matching different stages of the copal ageing.

In this work, FT-Raman and IR-microATR analyses were performed on a set of copals, whether on the internal or external samplings. We applied the I 1650 /I 1450 ratio as an ageing criterion but the obtained trend was opposite to the one obtained by Winkler et al. 26 . In order to understand this evolution and to get more precise degradation criteria, but also to propose some chemical interpretations to the spectral changes, spectral decomposition calculations were performed on different regions of the Raman and IR spectra. Finally, some hypotheses on the chemical evolution of the copals molecular structures are presented, and a progression of the ageing with the colors and surface aspects of the resins is highlighted with a proposition of new indexes.

B. Analyzed material

B.1. Samples

Ten samples of archeological copal from a medieval site in Sharma (Yemen) 28,[START_REF] Rougeulle | Proceedings of the Seminar for Arabian Studies[END_REF] have been analyzed. They have been previously identified as eastern African and/or Madagascar copals by their molecular markers thanks to GC/MS and py-GC/MS [START_REF] Regert | [END_REF] . This ensures, for the archaeological studied samples, a same biological origin and hence identical chemical characteristics. It is thus possible to attempt reliable statements about their degradation processes. These artefacts are raw fragments of copal which have a same hard and transparent bulk but different surface states and colors (Fig. 1): from yellow fragments with a thin dry surface, to orange fragments with a flaked-off surface and finally red fragments with a thick and very friable surface. Moreover, all the samples are wholly preserved, even sometimes with traces of sediments.

For these archaeological copals, a specific analyzing procedure was applied: small flakes from the internal and external parts of each artefact were sampled and separately analyzed to take into account this heterogeneity. Since the implemented analyses are nondestructive, each flake could be analyzed by FT-Raman then IR-microATR. For each fragments several points were analyzed and the very similar spectra were averaged in order to improve the signal-to-noise ratio.

B.2. Chemical composition

African and Madagascar copals are diterpenic resins that exude from trees of the Leguminosae family, and have as major components copalic and cis-ozic acids (Fig. 2) based on a labdane type skeleton [START_REF] Mills | The Organic Chemistry of Museum Objects[END_REF] . Copals are known to be highly polymerized resins, and most of the time, a pyrolysis step is needed for the separative methods analyses [START_REF] Osete-Cortina | [END_REF]33 .

C. Instrumentation

C.1. Raman spectroscopy

To overcome the high fluorescence of such organic samples which prevent the observation of Raman scattering, we favor a near infrared excitation at 1064 nm from an Nd-YAG laser diode. The FT-Raman spectrometer is a Bruker RFS 100/S based on a Michelsontype interferometer and equipped with a liquid nitrogen-cooled Ge detector.

The samples were analyzed using a microscope interface with a 40x IR objective, allowing a spot size of 30 µm approximately. In order to improve the collected Raman signal intensity, the samples were placed on a gold mirror. Laser power at the sample was adjusted at 250 mW (nominal power of 500 mW) and no damage or heating of the materials was observed. Spectra were recorded between 3500 and 50 cm -1 with a 4 cm -1 resolution and a low signal-to-noise ratio was achieved by accumulating 1500 scans.

C.2. Infrared spectroscopy

Non destructive micro-infrared spectroscopy analysis is carried out in micro Attenuated Total Reflectance mode (IR-microATR). Measurements by contact were performed with a 100 µm diameter Germanium crystal tip (mono-reflection) fixed on the 20x objective of an IRscope II microscope which also allows the visualization of the analyzed area. The microscope is linked to a Bruker Equinox 55 spectrometer equipped with a liquid nitrogen-cooled MCT detector. Spectra between 4000 and 600 cm -1 with a 4 cm -1 resolution were recorded with 200 scans accumulated.

D. Ageing characterization: methodology, results and discussion

D.1. The "vibrational features/molecular structures" relation

Spectra from internal and external fragments were first compared to underline their main differences and the effect of degradation on their molecular structure. Figure 3 presents the FT-Raman and IR-ATR spectra representative of an externally red archaeological copal.

On the FT-Raman spectrum (Fig. 3 a)) of the external part of the copal grain, in addition to a light fluorescence background due to the intense color of the surface, the bands are less intense and broader than the internal part one. The most important differences are located in the 1650-1500 cm -1 region assigned to the stretching vibrations of the CC double bonds.

Another region shows some decrease in the intensity, the CH stretching vibration massif, around 2900 cm -1 . For the IR spectra (Fig 3 b)), some bands decrease, for example the band at 887 cm -1 assigned to the bending vibration of the CH bonds in vinyl groups, others increase, especially a band around 1580 cm -1 , assigned to the C=C stretching vibration of aromatic groups. The alteration has also an impact on the CH stretching massif of the IR spectra, specifically the band at 3075 cm -1 assigned to CH bonds in vinyl groups.

The various observations made on the FT-Raman and IR spectra global profiles of a red copal underline the chemical transformations that occurred within the copal surface. These modifications can be considered as alteration, and can also be related to the color of the resin: more colored is the sample, more altered it will be. Moreover, it is correlated to the sample surface aspect, from dry in appearance to brittle, highlighting a mechanical de-cohesion derived from a chemical transformation. It is thus interesting to have an improved description and a better understanding of these spectral evolutions, and try to find specific alteration criteria that can be easily obtained.

D.2. Seeking degradation criteria

Previous studies showed the possibility of characterizing the maturation of such resins using Raman spectroscopy, by a band intensity ratio: the stretching vibration of the C=C bonds band around 1650 cm -1 with the deformation vibration of the C-H bonds band around 1450 cm -1 26,27 . We have already seen that the latter does not show an important intensity variation with degradation, but the former is highly influenced by the alteration process and significantly decreases. However this ratio has different meanings according to the different cited studies. Winkler et al. 26 underline that the resin is rather "fresh" or unaltered for higher intensity ratio (Iν C=C /Iδ C-H ) values, and that this ratio decreases with the sample age. Brody et al. 27 present the same ratio variation, but do not properly link it with the samples age. They evoke the influence of environmental factors in the resins evolution, as well as the influence of the different chemical compositions from a diterpenic resin to another (botanical origin).

The comparison between the internal and external parts of the studied copals shows an inverse trend in this ratio (see Table 1 and Fig. 4). First, for the internal parts of the ten samples, the ratio is located between 0.89 and 1.15 with a mean value at 1.05 for a standard deviation of about 9%. According to Winkler et al. 26 , all our copals have about the same age.

According to Brody et al. 27 , this very similar index value underlines comparable chemical states, which is consistent with the attested origin of our eastern Africa and/or Madagascar copals. However, both papers 26,27 do not specify the analyzed part of the samples (surface, bulk, or possible heterogeneities), but since they are interested by representative samples, we assume that only the "bulk" of their samples was considered. In our study, only one kind of resinous material was selected: same chemistry [START_REF] Regert | [END_REF] and thus same geological origin. Furthermore, our aim is to compare the internal and external altered parts in order to follow this degradation process. The expected results are therefore not related to the age, the maturation or fossilization processes of the resin.

By comparing the index values for the external parts of the three groups of copals, it can be noticed first that for the yellow samples, the ratios for the external samplings are consistently lower than for internal ones. However, the ratio for the external samplings increases significantly from yellow to red samples (from 1 to 1.76), according to the established degradation progression. A deeper look at the Raman bands profile of the copals external parts (Fig. 4) in the 1800-1400 cm -1 region, highlights a stable shape the 1500-1400 cm -1 area, while significant differences can be observed in the 1700-1500 cm -1 region. For the yellow copals, the C=C stretching band is less intense (Fig. 4 a) and b)), and seems slightly larger than the internal part spectra. For the orange copals (Fig. 4 c)), the 1650 cm -1 massif increases; and finally get very intense and broad for the red copals (Fig. 4 d)) with a maximum shifted to 1580 cm -1 . We can thus underline a clear global increase of the intensity of the I 1650 /I 1450 ratio related to the surface degradation state of these copals. This ratio index is here considered as an alteration marker of the samples surface. Besides, the evolution of the index all along the degradation process is not strictly linear, since a slight decrease is observed between the external and internal parts of the less altered samples (yellow copal).

However, even if this index is easily calculated and appears to be related with the degradation state, more detailed information about the chemical processes that occur should be extracted thanks to a deeper analysis of the bands profile.

D.3. Spectral decomposition, finding a general pattern

D.3.1. Fingerprint spectral region

A specific methodology for the spectral decomposition process was adopted, with the aim of monitoring the bands evolution, and trying to define the bands, and thus the specific bonds, that are most affected by the ageing. It was first applied on the fingerprint region (1800-1400 cm -1 ) since it seems to undergo several modifications, especially on the FT-Raman spectra. The spectra of all the internal and surface samples were truncated between 1925 and 1395 cm -1 , then a linear baseline was subtracted (set points: 1925, around 1515 and 1395 cm -1 ) and finally the cut spectra were normalized on the 1450 cm -1 massif intensity, since it appears unchanged with the resin ageing (Opus software, Bruker).

The decomposition procedure (Asrel2 homemade software, LADIR) was defined according to several parameters: the number of bands and their position (P), the area (A), the full width at half maximum (W) and the profile (K) of the band (which equals 0 if Gaussian, 1 if Lorentzian). The number and initial positions of the bands were determined by calculating the second derivative of the massif for each spectrum. Each minimum on the derivative indicates the position of a band. The same number of bands (11 bands) was defined for the internal and surface samples, 7 bands in the C=C vibration massif, and 4 bands in the C-H deformation massif. Concerning the adjustment procedure, it is based on a least squares method which adjusts the band parameters to fit the experimental spectrum.

Figure 5 illustrates the results of this procedure. For all the 10 internal samples, the decomposition result was the same, thus only one is presented (Fig. 5 a)). In general, for external samples same number of bands was obtained for all the fittings, but their position and profile differ from a sample group to another. Figure 6 shows a graph representing the evolution of the bands position between the surface sample fitting results and the internal sample ones, for the 3 groups of copals. For external samples (Fig. 5 b) to d)), it can be easily noticed that the positions of the 4 bands related to the C-H deformation massif are not really changing (bands 1 to 4), in contrast to the bands 5 to 11 that undergo important shifts especially for the orange and red copals. Using this spectral region for the study of the resins ageing is therefore currently compromised by the difficulty of finding an objective generic fitting pattern for the Raman spectra of the internal and external parts. It is thus difficult to distinctly underline and follow the bands that evolve with the resin alteration, and even harder to link this evolution to specific vibrators in this spectral region.

D.3.2. CH massif region

It was therefore necessary to focus on another spectral area that show an evolution of its vibrational features with the resin alteration, in order to point out these bands and then correlate these changes to chemical modifications at the resins surface. As it was said before, the CH stretching vibration massif (centered at 2900 cm -1 ) present several variations, whether on the IR or FT-Raman spectra. This region has been less studied, but can be very informative for substance discrimination 34,35 , or to assess if its features evolutions are characteristics of some organic binders alteration 36 .

The previous spectral decomposition methodology was applied to this region: the spectra have been truncated, baseline subtracted, normalized, and finally fitted with 7 bands as observed on the second derivative for both Raman and IR spectra and all samples (see example on Fig. 7). It can be noticed that the minima observed on the second derivatives are in the same positions for the internal and external fragments spectra. These 7 bands (numbered from 12 to 18 to avoid any confusion with the 1800-1400 cm -1 region bands) are assigned to C-H bonds in different chemical groups or environments: band 12 is assigned to ν(CH) s in CH 2 groups; bands 13 and 14 to ν(CH) s in CH 3 or CH groups; band 15 to ν(CH) as in CH 2 groups; band 16 to ν(CH) as in CH 3 groups; band 17 to ν(CH) in aromatic groups; and finally band 18 to ν(CH) in vinyl groups.

Comparing internal and external samplings signatures, the bands that generally vary the most are bands 12 and 18, at respectively 2849 and 3081 cm -1 for Raman and IR data (Fig. 7 c), colored bands). These two bands decrease significantly: indeed the 2849 cm -1 band that appears thin and relatively intense on the internal fragment spectrum, is more like a shoulder on the external sample spectrum; and the 3081 cm -1 band seems to decrease and gets broad, and tends to disappear. In order to quantify and characterize the spectral modifications, the fitting parameters (A, W and K) of the internal and external samples have been taken into account. Since the bands positions (P) are the same for the internal and external samples spectra, it was not taken into account for these calculations. Figure 8 represents the calculated differences for the areas (A) and the width at half maximum (W). Globally, the parameters do not show the same progress for all the bands. For Raman and IR data, the A and W parameters of bands 14 and 15 present an about 10% variation between the surface and the core of the resins: these two bands are in the middle of the CH stretching massif, and are not strongly affected by the resin ageing. Bands 12 and 18 are the most affected by the surface alteration: the areas are highly decreasing (up to 80% for band 18 on the IR spectra), and the widths at half maximum are increasing (up to 100% for band 18). These remarks are directly related to previous qualitative observation on the spectra fittings: if a band decrease and gets broader, its intensity necessarily decreases. Finally, A and W for bands 13 and 17 are slightly increasing: it is probably due to the fitting procedure during which the bands fulfill the massif ends to optimize the spectral adjustment. Finally, figure 8 shows that the extreme values for the relative differences are for the red copals, which are the most altered resins of the corpus.

The last parameter, K the band profile, tends to decrease for all the bands and all the samples, evolving towards a Gaussian profile.

D.4. Ageing and degradation: chemical interpretation

Band 18 is the most changing band and is assigned to CH vibrations in exocyclic CC double bonds which are highly present in the copal major components molecular structure (see Fig. 2). The important decrease of this band could be explained by an oxidation of the C=C bonds, and a cyclization reaction. These phenomena can occur more or less moderate, depending on the conservation environments: a resin exposed to the air is more prone to oxidation then a buried resin in a dry and close climate. Such reactions can explain the broadening of the 1630 cm -1 band on the Raman spectra and, on the IR spectra, the presence of a band around 1580 cm -1 , and also the decrease of the 887 cm -1 band, assigned to the bending vibration of olefinic CH. The two former observations can point out the formation of conjugated C=C aromatic bonds, which also explain the shift of the 1650 cm -1 band to lower wavenumbers; and the coloration of the samples surface as well.

Band 12 is assigned to the symmetric vibration of CH bonds in aliphatic CH 2 groups, and shows a relatively significant variation which can be related to cyclization reactions leading to the loss of CH 2 aliphatic chains; or, if the surface is highly altered, to chain breaking, explaining the brittleness of the surface for some samples.

Finally, the decrease of the K parameter, describing the evolution to a Gaussian profile of most of the bands, illustrates the emergence of disorder within the altered material. Indeed, a vibrator has normally a Lorentzian shape, but when a kind of disorder appears (for example random chain breakings) the vibrators are no longer identical because of the new different structural environments. Their characteristics are then randomly dispersed around a mean value, introducing a Gaussian feature in the band profile.

Alteration indexes can also be calculated or drawn based on the CH massif decomposition parameters. Figure 9 shows two plots of different fitting parameters of the external samplings spectra, involving the full width at half maximum of various bands. As said before, bands 12 and 18 are the two bands that evolve the most within the CH massif, and band 15 is the central and most intense one, and is the less influenced by the alteration.

Nevertheless, it evolves sufficiently to inform about the degradation state. We can notice, on these two plots, that the samples are sorted along the diagonal according to their colors, with an internal sample at the very beginning of the graphs, as a reference point. These parameters (or ratios) can thus be considered as new degradation indexes involving the CH massif features. It is thereby possible to monitor the copals alteration by considering the external parts of the samples. With this new approach, it appears that sampling is not necessary to characterize a sample degradation state; the analyses can be performed directly on the samples surface in a totally non destructive way.

E. Conclusion

The study of a collection of ancient copal artefacts from the same family tree and preserved in the same archaeological site allowed the observation of molecular scale alterations thanks to non destructive FT-Raman and IR-microATR spectroscopies. These artefacts, first classified by their apparent color (from yellow to red) and surface aspect (from dry to pulverulent, respectively), were sampled at their surface and in their bulk, enabling a direct comparison, and a characterization of their surface degradation. The restriction of the study to this samples set has prevented the introduction of additional parameters (variability of the chemical nature and uncertain origins) that can complicate the interpretations.

General observations of the vibrational signatures have underlined more important modifications of the molecular structure for the red copals than for the orange and yellow ones. The degradation state was first evaluated by FT-Raman using a previously proposed I 1650 /I 1450 ratio 26,27 . However, in our study, this ratio has a reverse evolution; lower values are obtained for less altered samples, and the index increases with the degradation state. This questions the chemical processes documented by this index. Is it related to long term fossilization processes affecting the whole sample, or some alterations starting from the surface? To meet some further detailed studies are required in order to specify the role of each parameter (chemistry, environment, time, etc) and their possible interactions.

Then, a relation between the copals molecular structures and the evolution of some vibrational features were investigated focusing of the spectral regions presenting the larger variations. Spectral decomposition was then implemented to identify and monitor the evolution of the bands assigned to specific structural features. A too important bands shift in the fingerprint region (1800-1400 cm -1 ) has hampered the built up of a unique decomposition model according to the resins degradation; and therefore the precise identification of the concerned bands and chemical bonds. However, the treatment of the CH stretching region (3100-2800 cm -1 ) has underlined bands variations that follow the resins degradation state.

Hypotheses on the chemical evolution of copal with ageing were then proposed, as well as other indexes involving the CH massif.

Moreover, knowing that these copals found in Yemen are from eastern Africa and/or Madagascar, they are a testimony of the ancient commercial routes during the 13 th century, and their aspects and their conservation states might then be related to specific collection and exploitation modalities. Are the resins freshly collected or gathered from partly fossilized deposits, or have they undergone some possible treatments? The conservation state could underline here if one or several geographical or technical origins are involved for these copals. Further studies with direct dating of the artefacts should bring further information.

Non destructive analytical approaches, with Raman and/or IR spectroscopies, could contribute to the study of resins degradation mechanisms, and propose ageing characterization and monitoring for conservation purposes. As resins are involved in various cultural heritage objects (in raw form, or in preparations as varnishes) such diagnostic tools could be easily implemented by respecting the conservation constraints.

Fig. 1 :

 1 Fig. 1: Representative samples of the studied copals illustrating the external aspect variability (Samples refs: MR1211, MS5673, MR1234).

Fig. 4 :

 4 Fig. 4: Representative FT-Raman spectra of 3 copals samples, one from each group: a) yellow (MR1211) -internal sampling, b) yellow (MR1211) -external sampling, c) orange (MS5673) -external sampling, and d) red (MR1234) -external sampling. The spectra have all been normalized on the 1450 cm -1 band intensity in order to monitor the 1650 cm -1 band evolution.

Fig. 6 :

 6 Fig. 6: Difference between the external and internal sample bands positions (∆ν) of the 11 decompostion bands of the 1900-1400 cm -1 region. These are mean values for each group.

Fig. 8 :Fig. 9 :

 89 Fig. 8: Evolution of some bands parameters (the area A, and the width at half maximum W) between internal and external samplings, mean values for the 3 groups of copals. These normalized relative differences were calculated by the following equation (for each parameter i): (i int. -i ext. ) x 100 / i int .

Table 1 :

 1 Intensity ratio of the 1650 cm -1 / 1450 cm -1 massifs (Iν (C=C) / Iδ (C-H) ) calculated for internal and external parts of 10 copals samples with different external aspects. The intensities were measured at the most intense point of each whole massif, and not at a specific wavenumber.

			Iν (C=C) / Iδ (C-H)
	Sample		Internal	External
		MR1210	0.94	0.83
	Yellow copals	MR1212	0.89	0.85
	Dry surface	MR1211	1.09	0.94
		MR1213	1.13	1.07
		MS5673	1.11	1.21
	Orange copals	MS5676	1.07	1.25
		MR1225	1.10	1.29
	Red copals	MR1226	1.15	1.31
	Pulverulent surface	MR1214	0.91	1.33
		MR1234	1.07	1.76

Acknowledgements

The authors would like to thank Axelle Rougeulle (UMR8167 Orient et Méditerranée, Textes -Archéologie -Histoire) for providing archaeological artefacts and allowing their sampling.

We thank also Martine Regert (CEPAM UMR7264 Cultures et Environnements. Préhistoire, Antiquité, Moyen-Âge) who gave us the opportunity to work on these samples. This work was partly supported by the ANR project EXSUDARCH "EXSUDats et goudrons végétaux en ARCHéologie, chimie, fabrication et utilisations".

Graphical TOC

New insights about copal degradation mechanisms are introduced and relevant analytical strategies for resinous substances through non destructive approaches by FT-Raman and ATR-IR are presented.


Céline Daher
email: cel.daher@gmail.com

Léa Drieu

Ludovic Bellot-Gurlet

Aline Percot

Céline Paris

Anne-Solenn Le Hô

Anne- Solenn Le Hô

Combined approach of FT-Raman, SERS and IR micro-ATR spectroscopies to enlighten ancient technologies of painted and varnished works of art

Keywords: FT-Raman, SERS, IR-microATR, lake pigments, varnish

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

Introduction

Cultural heritage paintings and painted objects are usually heterogeneous and complex. They consist of a support, on which preparatory grounds, paint layers, decorative settings and varnishes have been applied. So even if layers vary in their number, properties and function, these objects are built up by the successive applications, which thickness can be in the micrometer order. The complexity increases with the wide variety of used constituent materials: pigments, dyes, lakes, additives, adhesives, binders, medium, varnishes… Furthermore these materials have been transformed, mixed, subject to interactions, altered through time, either under manufacturing processes, ageing or restoring treatments. Concerning the 18 th century Decorative Arts objects, only few studies were done to document the emergence and development of painting and varnishing techniques in Paris or Europe [START_REF] Webb | Lacquer: Technology and Conservation[END_REF][START_REF] Hô | ICOM-CC 16th Triennial Conference[END_REF] . At this time, Paris was a bustling center for technological innovations to produce varnished objects, mainly thanks to four brothers "the Martins" and their descendants [START_REF] Watin | L'art de faire ou d'employer le vernis, ou l'art du vernisseur[END_REF] . Their productions were initiated to satisfy an increasing taste for Asian lacquers and their decors [START_REF] Ballardie | Painted wood: history and conservation[END_REF] that were highly imitated using black, red and golden oriental models. The Martin family gave its name to an enigmatic varnish that Watin, in his treatise (1772) "L'art de faire et d'employer les vernis (The art of making and using varnishes)" [START_REF] Watin | L'art de faire ou d'employer le vernis, ou l'art du vernisseur[END_REF] considers as a white luxurious and glossy varnish made of copal. Then, among the 18 th century, the painters and varnishers developed a totally new French style with a large palette of colors and ornaments using European raw materials and techniques. [START_REF] Koller | Japanische und europaische Lackarbeiten/Japanese and European Lacquerware: Adoption, Adaptation, Conservation[END_REF] The main existing studies are based on historical and stylistic approaches [START_REF] Czarnocka | [END_REF][START_REF] Koller | Lacke des Barock und Rokoko/Baroque and Rococo Lacquers[END_REF] , thus, the characterization of materials involved in this kind of painted works of art can provide a better knowledge of artworks through the understanding of these technological processes, and their evolution through time. However, the main constraints when working on cultural heritage artifacts are the availability and representativity of the probed matter. When sampling is possible, it is limited to very small amounts (sample can be as small as 200 µm in size and 20 micrometers thick).

Vibrational spectroscopies are widely used to characterize mineral pigments or organic dyes in the cultural heritage field. Indeed, they present the advantage of being fast without any sample preparation steps, non-destructive -the collected sample can be re-used for further analyses -and finally, the possibility of in-situ analyses with miniaturized portable devices [START_REF] Rosi | [END_REF]9] . Raman and infrared (IR) spectroscopies can also be coupled to a microscope allowing the reduction of the probed volume to a micrometric resolution in order to perceive heterogeneities, layering, and thus provide chemical information of a large class of amorphous or crystallized materials present in a same sample such as a pictorial layer.

In this study, some pieces of a large corpus of painted and varnished objects from 18 th century French Decorative Arts collections have been selected to be characterized using a combined approach of Raman, SERS and IR techniques to evaluate the ability of vibrational spectroscopies to identify the components of these complex mixture samples. Among the studied materials, we mainly focused on the most challenging analytical characterization, i.e.: layers containing organic materials, pictorial layers with red dye particles and binders, and pure organic layers as varnishes.

Red organic dyes have already been successfully identified using Raman and infrared spectroscopies in many contexts: dyed fibers, [10][11][12] manuscripts inks, [13,14] ancient cosmetics, [15] frescoes, [16,17] paintings, [18][19][20][21] violin varnishes [22] or polychrome sculptures. [23] These techniques have also highly been employed to characterize natural organic media that can be used in painting layers or vanishes, [24,[START_REF] Derrick | Infrared Spectroscopy in Conservation Science[END_REF] to differentiate between these materials, [START_REF] Osticioli | [END_REF][27][28] or to study the ageing and degradation of such materials. [29][30][31][32] However, one major issue to Raman spectroscopy when organic products are analyzed is the important fluorescence emission that may hide the Raman features. [33] Sometimes, fluorescence can be hindered when using a near IR excitation wavelength at 1064 nm for dyes, [16,34] or other organic media [35][36][37] . Some studies show new mathematical developments in order to remove the fluorescence background and get the vibrational features; [11,38] or new instrumental developments allowing to improve the signal by Resonance Raman Spectroscopy (RRS) [39] . But the mostly used technique to get rid of this luminescence phenomenon is Surface Enhanced Raman Spectroscopy (SERS) [40] with sometimes extraction of the dye, [10,41] the application of colloids on small samplings [42,43] , after HF hydrolysis [44] or even directly on the work of art [12,45] . Several preparation protocols for SERS surfaces are being developed, [46][47][48][49] and new improvements allow now non-invasive in situ study of the works of art using specific gels in which metallic colloids are embedded [50,51] or using tip-enhanced SERS (TERS) and advanced functionalized SERS-active optical fibers. [52][53][54] IR spectroscopy can be used to get complementary information about the studied materials since it does not generate fluorescence emission. It can be used to characterize binders and minerals in multilayered samples such as painting layers or glazes [55,56] , or varnish layers using synchrotron radiation [57][58][59] . Mathematical developments can also be considered to determine mixtures' components such as first and second derivatives of the spectra [60] , spectral decomposition [61] , or Principal Component Analyses. [62][63][64] The aim of the present study was to develop a convenient analytical strategy, as non-destructive as possible, that allows the identification of a wide range of materials involved in painted works of art from organic substances to mineral materials. A multi-step analytical methodology, based on the use of complementary Raman, SERS and infrared microATR-spectroscopy and the optimization of the experimental settings used was elaborated; and mathematical treatments such as spectral decomposition were applied.

In this work, reference materials -alizarin red, cochineal lake, and home-made varnishes consisting of drying oil and colophony -and samples collected from four painted and varnished objects from French 18 th century collections were investigated: a commode, a weaving shuttle, a blue silver case and finally a glass holder. The obtained results report the performance of the complementary approach involving vibrational spectroscopies, and enlighten the painting and varnishing techniques during the 18 th century in France.

Experimental Reference samples

Dyes and lake pigments

Natural organic dyes can be extracted from plants or insects, and the resulting molecules are mainly, for red dyes, a mix of anthraquinone type skeleton. These molecules may be complexed by mineral substrates or inorganic salts called "mordents" as for example alum (KAl(SO 4 ) 2 .12H 2 O) or calcium carbonate (CaCO 3 ) leading thus to what is called lake pigments [20] .

For this study, we mainly focused on two different commercial references of lake pigments taken from the C2RMF materials library. First, red carmine lake (Mermeri, Milan), extracted from cochineal insects with major component carminic acid (fig. 1a), and second, madder lake (Lefranc) mainly composed of alizarin dye (fig. 1b).

Organic materials: model varnishes and binders

Model varnishes prepared on glass slides during the 1970's from commercial materials were used as reference materials. Since these organic substances are known to evolve during the curing and ageing of the varnish, it is preferable to compare the spectra obtained on the studied varnished museum objects with already dried and slightly oxidized varnishes. Three experimental films were thus considered, according to the most famous varnishes employed during the 18 th century for furniture or luxury objects in France: linseed oil, colophony and copal, referring to the "vernis Martin". Linseed oil is made of triglycerides with 68% of polyunsaturated fatty acids that play a major role in the varnish drying process. Linoleic acid (C18:2) and linolenic acid (C18:3) are the main compounds of linseed oil (fig. 1d) [START_REF] Mills | The Organic Chemistry of Museum Objects[END_REF] .

Colophony is a diterpenic resin that exudates from Pinus sp. trees. Its major component is abietic acid (fig. 1c) and is known to easily undergo oxidation reactions, leading to degradation products built on the same chemical skeleton [START_REF] Langenheim | Plant Resins: Chemistry, Evolution, Ecology, and Ethnobotany[END_REF] .

Copal is a diterpenic resin as well, that have various geographical and so botanical origins. Some are from Araucariaceae sp. trees (Manila copal) with sandaracopimaric and agathic acids as main components; and others from Fabaceae sp. trees (Madagascar or African copals) with copalic and osic acids as main compounds.

Concerning the different binders -or different chemical families of binders -that could have been used in these 18 th century paintings, different films were analyzed. They were made of linseed oil as oil binder, gum Arabic as polysaccharidic binder and rabbit skin glue as proteinaceous binder. The sample were prepared during the early 2000 from commercial materials (from Laverdure, Paris and Sennelier, Paris) dissolved in ethanol and then applied on glass blades and left for drying.

Samples collected from painted and varnished works of art

In order to get information on the whole multi-layered system, it was necessary to sample the different museum objects and to work on selected levels. Therefore, samples were collected from 35 18 th century painted and varnished objects issued from French Decorative Arts museum collections. Among all these samples, and according to the samples size and shape, 16 could be analyzed using vibrational spectroscopies and only a few were successfully characterized, the others leading to poor quality spectra. They belong to the following objects: a weaving shuttle (inv13377, musée des Arts Décoratifs, Paris; fig. 2 a)), a commode "Adelaide" (invV1965, musée national du château de Versailles, Versailles; fig. 2 b)) with a beige background and decorated with pink floral motifs, a blue silver case « Carnet de bal » (inv11854, musée des Arts Décoratifs, Paris; fig. 2 c)), and a golden glass holder (invPR.2013.2.515, musée des Arts Décoratifs, Paris, fig. 2 d)).

For the two former objects, the pink pictorial layers were investigated. Fig. 2 a') and b') present the stratigraphy of these two objects, where the pink layers appear to be a mixture of a white pigment with red particles of variable size. Some inclusions of blue grains can also be noticed in these layers.

On the weaving shuttle's stratigraphy (fig. 2 a')), a strong discoloration is observed on the surface: the pink particles seem absent from the upper part of the stratigraphy. Indeed, these particles are organic pigments which were probably discolored due to a strong lighting. On the commode's sampling (fig. 2 b')), we can observe two different pink layers: the lowest one seems to be the original ornament, and the second was probably applied during a later intervention.

For the blue case and the glass holder, merely organic layers were sampled. Flakes of about 300 µm in size were taken from the upper layer -the final varnish -of these two objects (fig. 2 c') and d')), but also from the lowest layer -the underlayer -that is directly in contact with the support (fig. 2 c'') and d'')). This thick underlayer was probably applied on their metallic support as a waterproofing treatment (to protect the support from any corrosion) or as an adhesion level for the following painting layers.

Raman instrumentation

Visible excitation wavelength: Raman and SERS analyses

Raman spectroscopy measurements were performed using a LabRam HR800 (Horiba-Jobin Yvon) instrument characterized by a focal length of 800 mm and edge filters. The analyses were done using the 458 nm excitation wavelength of a water-cooled Ar+ laser and a 600 lines/mm grating. The detector is a CCD camera with a Peltier effect cooling system. Spectral resolution was around 2 cm -1 , and calibration was checked with respect to the 520.5 cm -1 silicon band. The spectrometer is coupled to an Olympus microscope equipped with a 100x Olympus objective which allows a ~1µm diameter spot size. The laser power was adjusted depending on the analyzed sample (nearly 50 µW at the sample for most of them) and the counting time was between 30 sec to 1 min. Silver colloids were prepared following the procedure of Lee and Meisel [START_REF] Lee | [END_REF] by reduction of silver nitrate with sodium citrate adapted from Van Elslande et al [15] .

Near Infrared excitation wavelength: FT-Raman analyses

FT-Raman analyses were performed using a near infrared excitation at 1064 nm provided by an Nd-YAG laser diode available on a Bruker RFS 100/S spectrometer based on a Michelson-type interferometer, and equipped with a liquid nitrogen-cooled germanium detector. The commercial references were analyzed using a microscopic interface equipped with a 40x objective, allowing a spot size of 30 µm approximately. The sampled objects were analyzed using a 100x objective leading to an about 15µm spot size. The laser power was adjusted depending on the analyzed sample. Reference materials and the objects' samples were placed on a gold mirror to improve the collected Raman signal intensity. Spectra were recorded between 3500 and 50 cm -1 with a 4 cm -1 resolution, and with 1000 to 1500 scans to optimize the signal-to-noise ratio.

Infrared instrumentation

Micro-infrared spectroscopy analyses were performed in an Attenuated Total Reflectance mode (mono reflection-ATR) using a Bruker Equinox 55 spectrometer coupled to an IRscope II microscope equipped with a 20x ATR objective with a Germanium crystal. This crystal needs a contact with the analyzed object, and depending on the material hardness it can lead to some damage, but the sample can still be collected for further analyses. A liquid nitrogen-cooled MCT detector was used, and a background was collected before each sample's spectra. 200 scans were accumulated between 4000 and 600 cm -1 with a 4 cm -1 resolution.

Results and discussion

This part presents the obtained results on several objects and samples. Pictorial layers of the two pink objects were investigated using SERS for the red dyes particles, FT-IR and FT-Raman for binders and charges. Pure organic layers (protective underlayers and upper varnishes) found on metallic objects were analyzed using FT-Raman, and mathematical treatments of the signals obtained were performed in order to identify the precise composition of such organic layers.

Pink objects: characterization of the pictorial layers

Red lake dyes

On samples taken from the two pink objects (weaving shuttle and commode), we observed heterogeneous pictorial layers composed of a mixture of white and deep red spots (see fig. 2 a')) and b')). Each analysis on these samples using Raman or infrared spectroscopy was performed pointing at these red particles.

Generally, the complexation of the dye molecule on the inorganic substrate, implied by the making of the lake pigment, involves spectral modifications compared to the pure organic dye spectrum. Since only lake pigments are used in pictorial layers, and to ensure a proper comparison with the museum samples spectra we choose to analyze commercial lake pigments as references, instead of pure dyes.

The samples taken from the colored layers of the two pink objects have been first analyzed by Raman spectroscopy using a 458 nm excitation laser. However, the high fluorescence background observed prevents the observation of the Raman features of the lake pigment on each sample. This fluorescence background may be due to the organic dye itself or the organic binder used in the pictorial layer. One of these spectra is shown in fig. 3 a), in the case of the weaving shuttle red particles.

In order to access the most colored areas and avoid the binder contribution to the Raman spectra, the samples were cut using a scalpel. A silver colloids aliquot was deposited directly on the sample to produce a SERS effect. This procedure is easily implemented as it does not require any additional preparation treatments. Fig. 3 shows the SERS spectra obtained from samples of the two objects (fig. 3 b) andc)) and from the two suspected lake pigment commercial references (carmine and madder, fig. 3 d) and e)) analyzed in the same conditions.

The spectrum of the weaving shuttle sample has a close similarity to the spectrum of the carmine lake reference, displaying particularly a band at 1109 cm -1 attributed to bond vibrations (C-C and C-OH) of the glucose group of carminic acid (fig. 3 b) and d) plain dots, see also fig. 1 a)) [10,34] .

The spectrum obtained from a sample of the commode shows a profile similar to the madder lake reference, and in particular three bands characteristic of the C=C stretching in madder lake skeleton at 1291 cm -1 , 1326 cm -1 and 1355 cm -1 (fig. 3 c) and e), stars) [10,34] . Moreover, on this sample, both of the two pink layers (see fig. 2 b')) were analyzed, and exactly same spectra were obtained (not shown here).

By using silver colloids and a 458 nm Raman excitation wavelength, we were able to properly characterize by SERS the lake pigments (cochineal and madder) used in the two pink objects. On the commode sample, both original and restoration pink layers were done using same pigment, madder lake. However, SERS enhances the Raman signal of molecules adsorbed on the silver colloids, hiding thereby the other compounds signals. If we want to go beyond pigment identification and try to determine the other constituents, further techniques must be used: infrared and FT-Raman spectroscopies.

Binder and charges

Analyses on samples taken from the weaving shuttle were performed using ATR infrared spectroscopy. The corresponding spectrum presented in fig. 4 a) reveals a band at 1707 cm -1 assigned to the stretching vibration of the C=O bonds, and bands appearing at 1241 cm -1 , 1159 cm -1 and 1097 cm -1 assigned for the first one to the bending vibration of COH bonds [27] , and for the two other bands to the stretching vibrations of CO bonds. These bands suggest the presence of siccative oil used as a binder in this pictorial layer, confirmed by the comparison with the spectrum of a linseed oil reference film (fig. 4 d)). Indeed, the comparison with other kinds of binders, such as rabbit skin glue (proteins, fig. 4 e)) or gum Arabic (polysaccharides, fig. 4 f)) shows that the bands specific of these two materials such as the two amides bands between 1500 and 1600 cm -1 , and the NH stretching vibration at 3250 cm -1 for the glue, and the C-O stretching band at 1050 cm -1 for Arabic gum are totally absent from the sample spectrum.

On the same spectrum (fig. 4 a)), a band of small intensity appearing around 2087 cm -1 is attributed to the C≡N vibration of Prussian blue (M + Fe 3+ Fe 2+ (CN) 6 .nH 2 O, M=Na, K, NH 4 ) which might have been added to the pictorial layer to slightly modify the reddish color to a more purple color (see also fig. 2 a')). The shuttle IR spectrum reveals also a broad band at 1414 cm -1 , a band of weak intensity at 1045 cm -1 and a sharp band at 683 cm -1 . These bands are attributed to the CO 3 2-vibration of lead white (PbCO 3 ) 2 Pb(OH) 2 (white pigment) which is employed in mixture with carmine lake, as shown in the stratigraphic view of the sample (see fig. 2 a')).

Most of these results are confirmed by the analysis performed on this sample using FT-Raman spectroscopy with a 1064 nm excitation wavelength. The resulting Raman spectrum presented in fig. 5 has a high similarity to the spectrum of a linseed oil reference film, particularly concerning the C-H bending vibrations at 1305 cm -1 , 1445 cm -1 and stretching vibrations around 2900 cm -1 , and the stretching of the C=C bonds at 1602 cm -1 . It is also clearly different from the two other binders spectra presented in fig. 5 c) and d). However, since Raman and IR spectroscopies can not differentiate between siccative oils [28] and since linseed oil is the most commonly used, an assumption can be made that linseed oil was employed for this painting. [START_REF] Webb | Lacquer: Technology and Conservation[END_REF] The sample Raman spectrum also reveals a strong and sharp signal related to lead white at 1051 cm -1 , characteristic of the CO 3 2-group vibration.

Two infrared spectra were obtained from samples taken from the commode (see fig. 4 spectra b) and c)). One of the spectra (fig. 4 b)) shows the characteristics bands of siccative oil: the CH stretching band at 2900 cm -1 which profile is characteristic of an oil as well as the 1700 cm -1 C=C stretching band. More intense bands located at 1449 cm -1 and 875 cm -1 are assigned to the vibration of the CO 3 2-group in calcium carbonate (CaCO 3 ). The presence of lead white is also demonstrated in this case by the presence of its typical bands at 1414 cm -1 , 1045 cm -1 and 683 cm -1 on spectrum c) in fig. 4.

We were able to characterize, using IR and FT-Raman spectroscopies, the different pictorial layers of the pink objects. Siccative oil was used as binder, and lead white, Prussian blue and calcium carbonate have been employed to alter the red color, giving pink and lilac shades to the objects' pictorial layers.

Pure organic layers: underlayers and superficial varnishes

General remarks

Pure organic layers were found at different stratigraphic levels of the multilayered systems: a first thick protective or waterproofing layer for some objects, and a thinner upper varnish for all the studied objects. These two different layers were sampled from the blue silver case (see also fig. 2 c') and c")) and the glass holder (see also fig. 2 d') and d")) and analyzed using FT-Raman spectroscopy. show vibrational features, characteristics of natural organic materials [27] . On spectrum a) a heating bump between 3000 and 2500 cm -1 is observed; but the sample does not show any visible damage after the analysis.

The spectra were baseline corrected to improve the observation of vibrational features and are presented in fig. 6 a') to c'). These spectra were compared to the spectra of two reference media that were highly employed during the 18 th century in Europe for varnishes production: a linseed oil film (fig. 6 e)) a colophony resin film (fig. 6 f)) and a copal resin film (fig. 6 g)). The observed vibrational features are very similar to the reference media ones, in terms of bands position and profile. These bands are underlined in fig. 6: the CH stretching vibrations band around 2900 cm -1 , the C=C stretching vibration band at 1640 cm -1 , the CH bending vibration at 1440 cm -1 , the OH bending vibration band at 1300 cm -1 and finally the C-O stretching band at 1090 cm -1 . [27] However, this data comparison is insufficient to precisely characterize the nature of these two organic compounds and especially to identify which resin was employed for the mixture preparations.

It is thus necessary to go further in the data treatment and work on the bands morphology and profile.

Indeed a previous study [28] shows that using a spectral decomposition procedure of specific bands can give important and precise information. We chose to work on one specific region, the CH stretching band, for several reasons: it is the most intense band of the spectra, it is observed for all references and museum samples, it presents rather different profiles for the three organic reference compounds, and finally seems to be poorly influenced by the oxidation process that may occur in these materials.

Moreover, this band is being more and more used to identify and characterize organic compounds in the cultural heritage field. [START_REF] Osticioli | [END_REF]28,31,63] Data treatment: spectral decomposition

The spectra were thus cut between 3180 and 2500 cm -1 , and spectral decomposition software (PALME, MONARIS, D. Baron) was used to process the CH band. This homemade software considers the mixture spectra as a linear combination of the pure material ones, so the sum of the pure materials spectra must fit the experimental mixture spectrum. [68][69][START_REF] Salpin | Glass holder Underlayer Fit with linseed oil only 3100 3000 2900 2800 2700 2600 2500 Raman Intensity Wavenumber / cm -1 Glass holder Underlayer Fit with colophony only Experimental spectrum Calculated spectrum Baseline Linseed oil Colophony 3100 3000 2900 2800 2700 2600 2500 Raman Intensity Wavenumber / cm -1 Glass holder Underlayer Fit with linseed oil only[END_REF] Moreover, if one pure material spectrum does not contribute well to the mixture spectrum, it leads to an incorrect adjustment (see supplementary data S1), or appears negatively which means it has been rejected by the software (see supplementary data S2). This was applied to the spectra of the glass holder's upper varnish and underlayer, and the spectrum of the silver blue case's upper varnish as well. The fitting results are presented on fig. 7. It can be noticed on these three examples that the experimental varnishes spectra are well adjusted by linseed oil and colophony reference spectra, despite a noticeable noise for two of them. The shape of the CH band thus appears as quite conserved even with spectra from ancient micro samples that are not always well defined, especially in the fingerprint region. Adding this spectral decomposition step allows us to confirm the use of linseed oil and colophony in these 18 th century French Decorative Art painted and varnished objects. On Supplementary Data S2, an adjustment with a copal spectrum shows a negative fit, proving the absence of copal in this varnish. Finally, further than these FT-Raman data, some destructive GC/MS analyses were performed after the vibrational techniques on the same micro-samples. These results confirmed the identification of a mix of (linseed) oil and colophony and therefore the suitability of the proposed approach for further studies.

Conclusions

Our combined approach allows exploring, in greater depth and with details, painted and varnished works of art. Using Raman, SERS and IR spectroscopies we could get new and original information about the techniques (see supplementary data S3) used to produce several objects taken from a large corpus of the 18 th century French Decorative Arts collections. Furthermore, the obtained data are highly informative on the material composition in different layers.

We were thus able to characterize -for two objects -the whole composition of some painting layers: very small (10 µm in size) particles of red dyes as madder and cochineal lakes using SERS (458 nm excitation wavelength and silver colloids) without any extraction or pretreatment; lead white or calcium carbonate as major mineral pigments, and Prussian blue added to slightly modify the pink color to a more lilac or purple color using IR-ATR mode; and finally the binder as a siccative oil using IR-ATR and FT-Raman.

Some objects presented a thick organic underlayer directly in contact with the support, that was identify, using a specific data treatment procedure on the FT-Raman spectra, as a mixture of siccative oil and pine resin, as well as the thin upper varnishes.

When several experimental settings are available such as different Raman excitation wavelengths, different sets of objectives, silver colloids for SERS analyses, it is possible to choose the right setup, according to the analyzed sample or material, in order to have the most complete characterization of the multi-layered studied objects. Moreover, these techniques can provide nondestructive analyses, a condition that is highly recommended for the study of Cultural Heritage artifacts. Finally, for some very complex samples such as mixtures, having the correct set of experimental devices is not sufficient, and further data treatments using mathematical approaches are required to get the most detailed information contained in the vibrational spectra, as here the identification of a colophony/siccative oil varnish.

Fig. 6

 6 Fig. 6 presents the obtained raw spectra for these two objects: a) and b) are respectively the upper varnish and the underlayer of the glass holder, and c) and d) are respectively the upper varnish and the underlayer of the silver blue case. First, we can notice that spectrum d) in fig. 6 does not show any Raman bands, all being hidden by a very intense fluorescence background. The a), b) and c) spectra

Fig. 1 :Fig. 2 :Fig. 3 :Fig. 4 :Fig. 5 : 1 (Fig. 6 :

 1234516 Fig. 1 : Main compounds of the reference materials. a) carminic acid (carmine lake), b) alizarin (madder lake), c) abietic acid (colophony), and d) linolenic acid (linseed oil).

Fig. 7 :

 7 Fig. 7 : Spectral decomposition of FT-Raman CH band for three organic layers (mixtures) using linseed oil and colophony films as pure reference materials. Misfitting examples are presented as supplementary data.

Acknowledgements

The authors would like to thanks the "Sciences du Patrimoine" foundation and "Labex Patrima" for financially supporting this work through the project ViVa: "Les Vies de Vernis

Artistiques: procédés techniques et vieillissement d'un corpus d'objets vernis des Arts Décoratifs du

XVIIIe siècle". Museum conservators Mrs. Anne Forray-Carlier (musée des Arts Décoratifs, Paris) and Mr. Pierre-Xavier Hans (musée national du château de Versailles, France) are thanked for providing the studied objects and allowing some sampling. Finally, Ms. Juliette Langlois and Mr.

Yannick Vandenberghe and Ms. Fériel Soualmia are thanked for their help for the GC/MS analyses, for the SEM-EDS study of the samples' mineral parts and for the colloids preparation respectively.

Support

French Decorative Arts objects from 18 th century museum collections 585 were studied. These complex materials were analyzed using FT-IR, 586

FT-Raman and SERS, and particularly organic materials such as lake 587 pigments, binders and varnishes were identified thanks to the right 588 choice of analytical parameters and, for some complex mixtures 589 specific data treatments. 590 591


Oussama Haddad

Julien Leboucher

Jocelyne Troccaz

Eric Stindel

Initialized Iterative Closest Point for bone recognition in ultrasound volumes

Ultrasound (US) probes have been used as guiding tools for Computer Assisted Orthopedic Surgeries (CAOS) [1]. Because of the US data uncertainty, the process of recognition -the localization of regions of interest in the image-requires a registration to a more precise, but invasive, imaging modality such as Computed Tomography (CT). A millimetric precision and a real-time processing are intraoperative requirements. Iterative Closest Point (ICP) [2] is a simple and non symmetric rigid registration algorithm that is sensitive to the initial position of the point sets. The aim of this study is to show the contribution of initializing ICP in rigid US-CT registration and to illustrate it on data of a proximal femur. First, an iterative initialization of the model (CT) to the partial view (US) is performed using ICP with annealed filtering. The first obtained local minimum is

then used to initialize a refinement step that maps the partial view to the model. One femur phantom was imaged both in a water bath using a calibrated 3D ultrasound probe and by CT. For each of the ten US acquisitions (five in the Anterior neck A, and five in the Posterior neck P), the CT scan is brought by means of fiducials pair-point matching. The initialization step improves ICP successful registrations from (A:25%, P:21%) to (A:76%, to P:52%) and the registration takes about 3s in average whilst ICP takes about 1s.

Introduction

Skin markers [START_REF] Renkawitz | Leg Length and Offset Measures with a Pinless Femoral Reference Array during THA[END_REF], time of flight cameras [START_REF] Veron-Tocquet | A study of accuracy for a single Time of Flight camera capturing knee flexion movement[END_REF] and ultrasound probes [START_REF] Schers | 3D/3D Ultrasound registration for panoramic volume reconstruction[END_REF] have been proposed as guiding tools in non invasive CAOS. US based guidance for CAOS generally relies on a prior knowledge on the bone shape that is elaborated in a preoperative step of the surgery. In the Operating Room (OR), a fast and accurate transfer of a preoperative plan helps the surgeon in locating regions of interests in the bone.

ICP is a simple and fast rigid registration method. It is based on the iterative search of point to point correspondences in sets of 3D points. It is used to map a partial view (source) to a model (destination). Non suitable initial state and incorrect correspondences are major sources of ICP failure. Points in the model that have no homologues are ignored in the iterative registration process. The full model information can be used in the initialization. Obtaining a suitable initial state for ICP can be done by matching anatomical landmarks [START_REF] Betke | Automatic 3D Registration of Lung Surfaces in Computed Tomography Scans[END_REF]. Landmarks can be extracted manually, which hinders the "real-time" constraint. Curvilinear features (such as the curvature) of the meshes [START_REF] Krsek | Differential invariants as the base of triangulated surface registration[END_REF], challenging to extract from US data, have also been proposed. Matching principal axes of the meshes was also used [START_REF] Fakhfakh | Automatic registration of pre-and intraoperative data for long bones in Minimally Invasive Surgery[END_REF][START_REF] Haddad | Robust rigid registration for non invasive Computer Assisted Orthopedic Surgery[END_REF], but non overlapping regions in CT scans and US noise change the estimate of the principal axes. Genetic algorithms are a possible way to initialize ICP [START_REF] Lomonosov | Pre-registration of arbitrarily oriented 3D surfaces using a genetic algorithm[END_REF] but complexity is a critical issue. Recently [START_REF] Linha | Global iterative closet point using nested annealing for initialization[END_REF], nested annealing was used to initialize ICP. High convergence rates and running times were reported. It was also reported that the proposed method experiences troubles aligning symmetric shapes, since local minima would resemble to the global one. Generally, the annealing process can be used as a tool to help iterative algorithms traverse local minima. In previous work [START_REF] Fakhfakh | Automatic registration of pre-and intraoperative data for long bones in Minimally Invasive Surgery[END_REF], we showed that it helps ICP in the non-overlapping regions detection.

Registration performances are also affected by ultrasound noise. Preprocessing steps were proposed in order to better reconstruct US data: Poisson [START_REF] Fakhfakh | Automatic registration of pre-and intraoperative data for long bones in Minimally Invasive Surgery[END_REF], anisotropic diffusion [START_REF] Fieten | An ICP variant with anisotropic weighting to accommodate measurement errors in A-Mode ultrasound-based registration[END_REF] and weighting the reconstruction using the cosine of the incidence angle [START_REF] Ye | 3-D Freehand Echocardiography for Automatic Left Ventricle Reconstruction and Analysis Based on Multiple Acoustic Windows[END_REF].

In this paper, an initialized ICP approach is tested for US-CT registration of proximal femur. An iterative initialization maps the model to the partial view using ICP. The minimized cost function is made convex by the use of US point's confidence in an annealing process. The first obtained local minimum is then used to initialize a refinement step that maps the partial view to the model. Data are collected using a CT scanner and a localized 3D ultrasound probe imaging a femur phantom fixed in a water bath. Registration results are evaluated using a ground truth given by fiducial pair-point matching.

Registration in ultrasound based CAOS

Non invasive means to track bones include: skin markers, Time of Flight or RGB-D cameras, ultrasound probes. This study focuses on 3D US. US to CT image registration aims at transferring pre-operative information and planning in the OR. The

Preoperative steps

Medical decisions prior to the surgery and a prior knowledge on the bone shape need to be available to the guidance system in the OR. Statistical Shape Models (SSM) or CT/MRI acquisitions are used for this purpose. The intraoperative imaging tool is a tracked 3D ultrasound probe. A probe to tracker calibration is then required [START_REF] Baumann | 3-D Ultrasound Probe Calibration for Computer-Guided Diagnosis and Therapy[END_REF].

Intraoperative guidance

The matching between intraoperative US and preoperative data helps localizing a target visible on the preoperative data in the OR. Acquiring and processing intraoperative data is constrained by accuracy (millimetric to be able to replace existing invasive fiducials [START_REF] Clavé | Comparison of the reliability of leg length and offset data generated by three hip replacement CAOS systems using EOS™ imaging[END_REF]) and speed (processing must not take longer than the acquisition). The process has to be repeatable within the surgery time frame if required.

Methods

ICP

Consider the problem of positioning a shape X in a model Y (represented by n and m points respectively). ICP iteratively provides an estimate of the optimal transformation T that maps X (source) to Y (destination). It minimizes a Surface Registration Error (SRE):

(1)

Where

is the nearest point in set Y to . Equation (1) yields to a lack of robustness to noise which is mainly ultrasound noise in this study. A commonly used strategy is to introduce a weighting parameter related to the quality of the match [START_REF] Fakhfakh | Automatic registration of pre-and intraoperative data for long bones in Minimally Invasive Surgery[END_REF][START_REF] Billings | Iterative Most Likely Oriented Point Registration[END_REF]. We use the cosine of the incidence angle ( [START_REF] Clavé | Comparison of the reliability of leg length and offset data generated by three hip replacement CAOS systems using EOS™ imaging[END_REF] between the line joining the US emitter to the surface point and the local surface normal at this point.

(2)

A visualization of the obtained reference positions is provided in Fig 4 . A color map shows the value of (3) at each US point. Important factors influence the registration accuracy: The surface areas selected for registration (which can be controlled by the FoV of the US emitter), the quality of the meshes (which can be improved by elaborating a more sophisticated segmentation, for example local phase features for US bones detection [START_REF] Hacihaliloglu | Bone Segmentation and Fracture Detection in Ultrasound Using 3D Local Phase Features[END_REF]), and the initial estimate of the correspondences.

Iterative initialization

In ICP, using the model as the source shape can provide more information to the registration process, where all the model points participate in the estimate of the optimal transform. In this case, the minimized SRE includes nonoverlapping regions, and convergence is not guaranteed. We propose to use the model as the source shape in an iterative initialization. The registration is guided by the confidences accorded to the US points. Initially, all correspondences are given the same weight 1. Iteratively, the filtering effect increases using deterministic annealing and following:

Where is the annealing rate, a compromise between accuracy and runtime. We fix similarly to [START_REF] Haddad | Robust rigid registration for non invasive Computer Assisted Orthopedic Surgery[END_REF]. Mapping CT points to US correspondences using ordered weights is done using Singular Value Decomposition [START_REF] Bellekens | A Survey of Rigid 3D Pointcloud Registration Algorithms[END_REF]. The first obtained local minimum (SRE starts increasing or stabilizing) is used to initialize the refinement step: The obtained optimal transformation that maps the CT to US, is inverted and applied on the US mesh (Initialized US). The latter is registered to the original CT using standard ICP. This way a comparison to classic ICP becomes undemanding.

Data

CT Data

A CT scan of a femur phantom (sawbones®) (Siemens Biograph, with an imaging resolution of 0.7mm) is used as the model in the study. Thirteen 1-mm metal fiducials were attached to the bone. Thresholding segmentation was performed and a 3D surface mesh was extracted from the segmented CT volume. The mesh was then filtered using Laplace smoothing [START_REF] Cignoni | MeshLab: an Open-Source Mesh Processing Tool P[END_REF]. We only keep the proximal part of the femur ( over the surface of the entire bone, 100 cm²

Ultrasound data

Ultrasound data are obtained using a tracked 3D transducer (Voluson 730 ultrasound machine, GE Medical Systems) imaging the femur phantom fixed in a water tank. The Field of View (FoV) is 90°, the depth is 7 cm and the resolution is 0.47 mm. Probe calibration [START_REF] Baumann | 3-D Ultrasound Probe Calibration for Computer-Guided Diagnosis and Therapy[END_REF] was performed using a membrane phantom in nylon. It enables to define a reference attached to the US volume in the reference frame of the tracking device. A point, virtually corresponding to the center of the arc of piezo elements is defined and named "US emitter". The proximal neck of the femur is the focus of this study. 10 US volumes were taken: 5 from the anterior and 5 from the posterior femur.

A region growing segmentation, using manual thresholds was first applied on US images. The obtained rough and thick US surface, were then smoothed using Poisson surface reconstruction [START_REF] Fakhfakh | Automatic registration of pre-and intraoperative data for long bones in Minimally Invasive Surgery[END_REF]. We chose to use a ray-casting of the volumes using the spatial location of the US emitter to extract the external surface: points in the volume that are closer to the US emitter. Ray-casting was performed using vtkCellLocator [START_REF] Schroeder | Visualizing with VTK: A Tutorial[END_REF]. Segmentation steps are summarized in

Reference positions

In order to evaluate the registration results, a ground truth is computed using fiducials attached to the sawbone. Their position is extracted from CT data. Fiducials are represented in the CT volumes as round shapes, and we estimate a fiducial location at the center of mass of its corresponding shape. Then they are digitized using an NDI Spectra Polaris system and transferred to the US reference frame using the US probe calibration information. Fiducials paired-points matching enables bringing the CT scan to the US image in a single reference system (Fig 3 and4). The mean fiducials matching error is equal to 1.29 mm. The calibration had an average error of (0.50 mm, 0.88°). We propose to perform a local systematic research of the minimum US-CT SRE [START_REF] Mozes | 3D A-Mode Ultrasound Calibration and Registration of the Tibia and Femur for Computer-Assisted Robotic Surgery[END_REF], in [+-2mm, +-2°]. The final mean (US-CT) SRE at GT is 0.66 mm.

Experiments and results

Initial relative positions of the CT and US are randomly generated from the ground truth positions. 500 random transformations following normal distribution are generated and applied on the US surfaces in the reference positions. The tested translation parameters are in [-20 mm, 20mm] and the rotational angles are in [-90°, 90°] (around the 3 principal axes of the US meshes, each transformation parameter is generated independently). Standard ICP and the proposed initialized ICP (I_ICP) are compared.

Target Registration Error (TRE) is defined as the SRE between the registered US mesh and the US mesh on the reference position and this is done knowing the correspondences. The SRE is given following (1) where the set X is the US mesh and the set Y is the CT model. Only "successful" registrations (defined as: TRE < 2 mm) are considered. Registration is tested on 10 US volumes; 5 are anterior (resp. posterior) and named Ai (resp. Pi). Quantitative measures of accuracy and runtime are shown in Table 1. With an average of 23 (resp. 15) iterations for A (resp. P), ICP success rates increase from 25% (resp. 21.1%) for A (resp. P) to more than 76% (resp. 52%) for A (resp.P). ICP accuracy, both with and without initialization, can be clinically accepted. On a 4th generation i5 processor, the registration takes about 3s, and the mean initialization runtime is nearly twice classical ICP runtime approach. Visualization of random initial states, ICP and I_ICP results are shown in Fig 5 . Symmetry descriptors for a 3D shape are provided in [START_REF] Kazhdan | Symmetry Descriptors and 3D Shape Matching[END_REF]. This study does not include such features, but it is clear that the iterative initialization profits from the closed form of the femur.

Conclusion

Once calibrated US data are registered to CT, the location of femoral structures in the OR and preoperative plan transfer become possible. Clinical applications of femur recognition in ultrasound volumes are: ultrasound guided femur-tumor ablation, total hip and knee arthroplasty. ICP is a fast and iterative rigid registration method that is sensitive to its initial state. An iterative initialization using combined ICP with annealed filtering is proposed and has been tested for proximal femur recognition in US volumes. Five US acquisitions, by femur side and focusing on the neck of a fixed femur phantom in a water bath, were used in this study along with a CT scan. We show that the use of the full model of the bones as the source helps initializing ICP. Posterior US acquisitions, where the trochanteric crest can hinder the US beam spread, had less successful registrations to CT than anterior acquisitions. Registration runtimes are equivalent to US volume acquisition and construction.

The tested range of capture was significant [±20mm, ±90°], and the obtained success rates make ICP a good candidate for transferring a preoperative plan in CAOS. Among feature based registrations, a sequential least squares estimation technique, the Unscented Kalman Filter (UKF) [START_REF] Hedjazi Moghari | A Novel Incremental Technique for Ultrasound to CT Bone Surface Registration Using Unscented Kalman Filtering[END_REF], overpasses ICP on a general case of US bones to CT registration. UKF showed quite worse results than ICP on scapula data [START_REF] Tyryshkin | Identification of anatomical landmarks for registration of CT and ultrasound images in computer-assisted shoulder arthroscopy[END_REF]. UKF is also computationally demanding compared to ICP. A Genetic algorithm for registering femur data was proposed [START_REF] Uddanwadikar | Fabrication of Accurate Bone Implant Geometry Using Puzzle Solving Technqiue[END_REF] and it was reported that the obtained results are not impressive as compared to ICP.

More phantoms and US focus zones need to be tested. The effect of the registration error (TRE, SRE) on the intraoperative system feed-back uncertainty is a crucial information for the validation. This requirement depends on the surgery into focus: Bone-tumor localization uncertainty for navigated bone tumor ablation and offset (with the leg length) for navigated total hip arthroplasty.

  Fig 1 shows major steps in US-based CAOS.

Fig 1 :

 1 Fig 1: Pre-Intraoperative steps of an ultrasound based CAOS

  sum of the triangles surfaces, and 29.000 points).

.

Fig 2 :

 2 Fig 2: Segmentation steps: A) US image (Top left), B) Threshold US image (Top right), C) Poisson surface reconstruction (Bottom left), D) Ray-casting (Bottom right).

Fig 3 :

 3 Fig 3: Visualization of the CT surface superimposed on the US volume

Fig 4 :

 4 Fig 4: Visualization of the reference positions of the registrations and corresponding mean residuals

Fig 5 :

 5 Fig 5: Qualitative results

Table 1 :

 1 Quantitative results

		Percentage of successful	Mean run time	Mean TRE	Mean SRE	Mean number of
	US	registrations (Success criteria:	(seconds)	(mm)		(mm)			iterations
	acquisition	TRE < 2 mm)							(round to nearest)
		ICP	I-ICP	ICP	I-ICP	ICP	I-ICP	ICP	I-ICP	ICP	I-ICP
	A1	24.6	75.8	1.16	3.15	0.37	0.21	0.612	0.610	89	21 + 57
	A2	29.8	72.8	1.56	3.88	0.21	0.17	0.641	0.640	122	26 + 82
	A3	25	79.2	1.12	3.14	0.22	0.25	0.683	0.685	88	22 + 63
	A4	22.4	81.6	0.79	2.59	0.37	0.23	0.768	0.767	71	22 + 49
	A5	23.2	75.2	1.17	3.63	0.34	0.28	0.737	0.736	87	25 + 58
	Mean (A)	25	76.92	1.16	3.27	0.30	0.22	0.688	0.687	91	23 + 62 (85)
	P1	16.8	33.4	1.45	2.54	0.61	0.52	0.568	0.573	112	12 + 92
	P2	22.4	59.2	1.70	2.80	0.59	0.60	0.541	0.540	140	11 + 115
	P3	21.1	62.4	1.99	3.47	0.66	0.78	0.520	0.515	155	10 + 158
	P4	18.4	37	1.49	2.55	0.44	0.22	0.614	0.608	127	13 + 98
	P5	26.8	70.4	1.27	3.27	0.99	1.18	0.875	0.888	103	27 + 72
	Mean (P)	21.1	52.48	1.58	2.92	0.65	0.66	0.623	0.624	127	15 + 107 (122)

Acknowledgments: Thanks to Johan Sarrazin, TIMC-IMAG, for providing calibration material and assistance. This work was partly supported by the French ANR within the Investissements d'Avenir program (Labex CAMI) under reference ANR-11-LABX-0004. Computer Assisted Medical Intervention Tool Kit.


B Veron

N Hungr

J Troccaz

Making a clinical device from a laboratory prototype: from Prosper to Prosper OR

INTRODUCTION

Prostate brachytherapy consists in inserting radioactive seeds in the prostate in case of localized cancer in order to kill cancerous cells whilst sparing healthy tissue or organ at risks (e.g. urethra, rectal wall). These seeds are inserted into the prostate using needles introduced through a guiding grid connected to a transrectal ultrasound probe. To handle limitations or difficulties of the conventional procedure, many systems have been designed worldwide (see [START_REF] Podder | AAPM and GEC-ESTRO guidelines for image-guided robotic brachytherapy: Report of Task Group 192[END_REF] for a broad overview of systems existing in 2015). Developed systems provide different levels of assistance: from guiding the needle by positioning and orienting a guide out of the patient [START_REF] Fichtinger | Robotic assistance for ultrasound-guided prostate brachytherapy[END_REF], to automatic insertion of the needle [START_REF] Hungr | A 3-d ultrasound robotic prostate brachytherapy system with prostate motion tracking[END_REF] or even injection of radioactive seeds through the needles into the patient [START_REF] Yu | Robotic system for prostate brachytherapy[END_REF]. Some of the systems also include ultrasound probe positioning and motion. Despite a very large number of prototypes, very few systems entered the operation room (OR). As far as we know, regarding brachytherapy applications, only the B-Rob system [START_REF] Fichtinger | Robotic assistance for ultrasound-guided prostate brachytherapy[END_REF] developed by Johns Hopkins University and Queens University has been evaluated on patients. A feasibility study (NCT00381966 on ClinicalTrials.gov) was ended in 2012. The results of this study [START_REF] Song | Robotic needle guide for prostate brachytherapy: clinical testing of feasibility and performance[END_REF] confirm the interest of the robotic assistance in the needle placement. The EUCLIDEAN system [START_REF] Yu | Robotic system for prostate brachytherapy[END_REF], a fully automated device with needle insertion and seed injection, received FDA clearance for investigational studies. Reliability on phantoms was thoroughly studied [START_REF] Podder | Reliability of EUCLIDIAN: An autonomous robotic system for image-guided prostate brachytherapy[END_REF] but, to the best of our knowledge, no clinical studies have been published. The reasons for such limited number of clinical evaluations and industrial developments come from the many issues to be solved such as safety, sterilizability, electromagnetic compatibility. The more invasive and autonomous is the robot, the most constraining and demanding are the regulations. In this paper, we will describe how the Prosper robot [START_REF] Hungr | A 3-d ultrasound robotic prostate brachytherapy system with prostate motion tracking[END_REF] has been modified based on our laboratory and cadaver experiments to be turned into a version usable in the OR for a clinical evaluation.

MATERIALS AND METHODS

PROSPER laboratory version: this system (shown in Figure . 1) was designed as a proof of concept for automatic brachytherapy. It allowed us to develop methods to calibrate the robot with the 3D transrectal ultrasound (TRUS) probe and to automatically correct positioning errors due to prostate motion and deformation using 3DUS to 3DUS registration. It consist of two modules: one 5 degrees of freedom (DoFs) module for positioning and orienting the needle close to the patient skin and a 2 DoFs module for needle insertion in the prostate. It was studied on both phantoms and cadavers [START_REF] Hungr | A 3-d ultrasound robotic prostate brachytherapy system with prostate motion tracking[END_REF]. These studies showed very promising results allowing to position a needle with an error lower than 2 mm considering prostate motion up to 7 mm. Nevertheless, the system also showed some critical limitations. First, cadaver studies showed a lack of rigidity of the robot while inserting the needle. Moreover, from a clinical point of view, the system showed sterilization issues, and safety concerns because of the automatic needle insertion and the way the robot handled the needle (impossibility to quickly remove the needle).

PROSPER OR version: this new system (shown in Figure . 2) was designed to deal with the limitations previously mentioned. Off-the-shelf components (Faullhaber brushless motors couple with THK linear carriage for instance) were re-designed. A global quality approach and risk analysis were undertaken leading to this new system with extra capabilities.

RESULTS

First, the robot workspace was increased to allow treatment of prostate with a larger volume and extra passive DoFs were added to ease the robot positioning with respect to the patient. Moreover, the structure of the system was reinforced and a fixation system to the OR table was added. This allows to make the system more rigid and ensure an accurate needle insertion. To tackle with security and sterilization, protection hoods covering all the mechanical parts of the robot were designed. All hoods are made of sterilizable biocompatible plastic and the hoods being in the close environment of the needle and/or in contact with it are designed to be removable and autoclave compatible. Thus, the sterilization process is simple, fast and safe as it relies on the most standard sterilization procedure. Moreover, the needle holder was modified to enable a safe and fast removal of the needle at any time. The insertion procedure was reviewed to enable manual and/or automatic insertion. In both insertion modes, the robot acts as a needle guide positioning the needle in accordance with the trajectory chosen on the ultrasound images, and a programmable mechanical stop limits the insertion depth based on the target planned. If the manual insertion mode is selected, the needle insertion is let to the urologist.

Finally, for the system to adapt with a number of TRUS probes (brachytherapy side-fire or biopsy end-fire), a probe holder was designed. It allows to adjust the probe position and orientation in the sagittal plane. The system was delivered in 2015 and is currently under tests to validate its electromagnetic compatibility and its sterilization process.

CONCLUSION AND DISCUSSION

A very strict quality approach and risk analysis were led to bring the new PROSPER OR system to the patients. Specific tests were designed to ensure the quality and safety of the system on both mechanical and software point of view. The system is currently under strong quality tests, including registration with the TRUS probe, accuracy measurement on synthetic phantoms, evaluation of the sterilization process and electromagnetic compatibility.

To ensure the proper use of the PROSPER OR system, a new state-machine working with the CamiTK framework [START_REF] Fouard | CamiTK: a Modular Framework Integrating Visualization, Image Processing and Biomechanical Modeling[END_REF] has been designed. It allows, first to simply and rapidly implement complex medical protocols, second to ensure compliance with the established protocol in use.

Finally, a medical protocol is currently under discussion with urologists to bring the robot on patients and evaluate its accuracy and benefits to patients.

Figure. 1 .

 1 Figure. 1. Laboratory set-up of PROSPER (left: experiments on a deformable phantom; right: cadaver experiment).

Figure. 2 .

 2 Figure. 2.Set-up of the PROSPER OR systemin the OR.