Micro-particle Induced X-ray Emission Research Articles

64Cu2+ Complexes of Tripodal Amine Ligands' In Vivo Tumor and Liver Uptakes and Intracellular Cu Distribution in the Extrahepatic Bile Duct Carcinoma Cell Line TFK-1: A Basic Comparative Study.

Copper (Cu) is a critical element for cancer cell proliferation and considerably accumulates in the nucleus. 64Cu2+ is an anticancer radiopharmaceutical that targets the copper requirement of cancer cells. However, intravenously injected 64Cu2+ ions primarily accumulate in the liver. Ligand complexation of 64Cu2+ may be a promising method for increasing tumor delivery by reducing liver uptake. In this study, we used three tripodal amine ligands [tris(2-aminoethyl)amine (Tren), diethylenetriamine (Dien), and tris(2-pyridylmethyl)amine (TPMA)] to enclose 64Cu2+ ions and compared their in vivo tumor and liver uptakes using a tumor-bearing xenograft mouse model of the extrahepatic bile duct carcinoma cell line TFK-1. We examined intracellular Cu distribution using microparticle-induced X-ray emission (micro-PIXE) analysis of these compounds. 64Cu2+-Tren and 64Cu2+-Dien showed higher tumor uptake than 64Cu2+-TPMA and 64Cu2+ ions in TFK-1 tumors. Among the three 64Cu2+ complexes and 64Cu2+ ions, liver uptake was inversely correlated with tumor uptake. Micro-PIXE analysis showed that in vitro cellular uptake was similar to in vivo tumor uptake, and nuclear delivery was the highest for 64Cu2+-Tren. Conclusively, an inverse correlation between tumor and liver uptake was observed using three 64Cu2+ complexes of tripodal amine ligands and 64Cu2+ ions. These results provide useful information for the future development of anticancer 64Cu radiopharmaceuticals.

Contribution of Mine-Derived Airborne Particulate Matter to Ca, Fe, Mn and S Content and Distribution in the Lichen Punctelia hypoleucites Transplanted to Bajo de la Alumbrera Mine, Catamarca (Argentina).

The aim of this work was to relate the contribution of mine-derived airborne particulate matter to Ca, Fe, Mn and S content and distribution in Punctelia hypoleucites transplanted to Bajo de la Alumbrera, an important open-pit mine in Catamarca, Argentina. Lichen samples were transplanted to four monitoring sites: two sites inside the mine perimeter and two sites outside the mine. After three months, elemental distribution in samples was analyzed by microparticle-induced X-ray emission (microPIXE), and elemental concentration was determined by specific techniques: Ca and Fe by instrumental neutron activation analysis, Mn by inductively coupled plasma atomic emission spectrometry and S by a turbidimetric method. A differential distribution of S and Ca in thalli transplanted in-mine sites was detected compared to that of samples transplanted outside-mine sites. An overlap of Fe and S in the upper cortex of the apothecium section was observed, leading to infer a mineral association of both elements. Similar association was observed for Ca and S. In addition to these results, the significantly higher concentration detected for S and Mn in in-mine site samples suggests a contribution of Fe, S, Ca and Mn of mining origin to the content and distribution of these elements in P. hypoleucites. MicroPIXE complemented with Mössbauer spectroscopy analysis determined the presence of pyrite particles together with other iron-bearing minerals displaying different degrees of oxidation. These results point to a mining origin of the airborne particulate matter trapped by the lichen thalli transplanted to Bajo de la Alumbrera. These findings indicate that P. Hypoleucites acts as an excellent air quality biomonitor in the Bajo de la Alumbrera mine area.

Radiation and Emission Phenomena in Combination

Radiation and Emission Phenomena in Combination

Improvement in the elution performance of an N,N,N’,N-tetraoctyl diglycolamide impregnated extraction chromatography adsorbent using neodymium via micro-particle-induced X-ray emission analysis

An adsorbent used for the recovery of trivalent minor actinides (MA(III); Am and Cm) from high level liquid waste generated from reprocessing of spent nuclear fuel was subjected to micro-particle induced X-ray emission (micro-PIXE) analysis to improve its elution performance. The experimental adsorbent comprised SiO2 particles, a polymer coating, and an N,N,N′,N′-tetraoctyl diglycolamide (TODGA). The particles to be analyzed were subjected to Nd adsorption and an elution operation, but Nd in the adsorbent was found to be uniformly distributed. This might have been caused by individual differences in the amount of impregnated TODGA. The remaining Nd species were not localized to a specific part of the adsorbent after the adsorption operation. Some Nd elements were retained in the adsorbent after elution, probably because of the poor diffusion of the mobile phase inside the adsorbent. An adsorbent having a different microstructure from the first was then evaluated, and rapid elution was observed on new adsorbent along micro-PIXE analysis.

Tissue-specific calcium and magnesium allocation to explain differences in bulk concentration in leaves of one-year-old seedlings of two olive (Olea europaea L.) cultivars

Olive tree (Olea europaea L.) leaves have recently been recognised as a valuable source in cosmetic and pharmaceutical industry as well as in preparation of health-supporting beverages. Little is known about the element composition of olive leaves and almost nothing about tissue-specific allocation of elements. Element composition and tissue-specific distribution were determined in leaves of two olive cultivars, Leccino and Istarska bjelica using micro-particle induced X-ray emission (micro-PIXE). In leaves of the Istarska bjelica cultivar larger bulk concentrations of potassium, sodium, molybdenum and boron, but smaller concentrations of calcium and magnesium were found than in leaves of the Leccino cultivar. Tissue-specific investigation revealed that larger concentration of calcium in epidermis and in leaf blade tissues (secondary veins, palisade and spongy mesophyll) contributed to the larger leaf bulk calcium concentration in the Leccino cultivar. For magnesium, all leaf tissues, except the bundle sheath cells and consequently the main vascular bundle, contributed to the larger bulk concentration in the Leccino cultivar. Potassium was not predominant in any of the leaf tissues examined, while sodium and molybdenum were below the limit of detection, and boron not detectable by micro-PIXE. The results indicate that sinks for calcium and magnesium are stronger in specific leaf tissues of the Leccino than of the Istarska bjelica cultivar. The new understanding of tissue-specific allocation of elements in leaves of olive will serve as a basis for detailed studies into the effects of foliar and/or soil fertilisers in olive.

An Ancient Egyptian Multilayered Polychrome Wooden Sculpture Belonging to the Museo Egizio of Torino: Characterization of Painting Materials and Design of Cleaning Processes by Means of Highly Retentive Hydrogels

This contribution focuses on the conservation of an Egyptian wooden sculpture (Inventory Number Cat. 745) belonging to the Museo Egizio of Torino in northwest Italy. A preliminary and interdisciplinary study of constituent painting materials and their layering is here provided. It was conducted by means of a multi-technique approach starting from non-invasive multispectral analysis on the whole object, and subsequently, on selected micro-samples. In particular, visible fluorescence induced by ultraviolet radiation (UVF), infrared reflectography (IRR) and visible--induced infrared luminescence were used on the whole object. The micro-samples were analysed by means of an optical microscope with visible and UV light sources, a scanning electron microscope (SEM) with an energy-dispersive X-ray spectrometer (EDX), Fourier transform infrared (FT-IR) spectrometer, pyrolysis-gas chromatography/mass spectrometer (Py-GC/MS) and micro-particle induced X-ray emission (PIXE). The characterization of the painting materials allowed the detection of Egyptian blue and Egyptian green, and also confirmed the pertinence of the top brown layer to the original materials, which is a key point to design a suitable surface treatment. In fact, due to the water sensitiveness of the original materials, only few options were available to perform cleaning operations on this artwork. To setup the cleaning procedure, we performed several preliminary tests on mockups using dry cleaning materials, commonly used to treat reactive surfaces, and innovative highly water retentive hydrogels, which can potentially limit the mechanical action on the original surface while proving excellent cleaning results. Overall, this study has proved fundamental to increase our knowledge on ancient Egyptian artistic techniques and contribute to hypothesize the possible provenance of the artefact. It also demonstrated that polyvinyl alcohol-based retentive gels allow for the safe and efficient cleaning of extremely water sensitive painted surfaces, as those typical of ancient Egyptian artefacts.

Insight into the Effect of Mg(OH)2 Films vs. Noble Element Enrichment on the Global and Local Cathodic Activation of Corroding Mg

The anodically-enhanced cathodic activation of high-purity Mg was studied by global and local electrochemical measurements in unbuffered 0.6 M NaCl, 0.6 M NaCl saturated in Mg(OH)2, 0.1 M MgCl2, 0.1 M Na2SO4, and 0.1 M tris(hydroxymethyl)aminomethane hydrochloride (TRIS). The selection of diverse aqueous environments was deliberate in order to explore the effect of dissolution product upon subsequent cathodic activation. Cathodic activation was found to be strong in all of the chloride-containing environments where a porous hydroxide forms; weak in Na2SO4 where a denser, more compact hydroxide forms; and negligible in TRIS where no hydroxide forms. Elemental enrichment, mapped using microparticle-induced x-ray emission, revealed the enrichment of Fe only in Cl−-containing environments, suggesting that noble metal enrichment provides a stronger influence on cathodic activation than that of Mg(OH)2 films.

Contrasting allocation of magnesium, calcium and manganese in leaves of tea (Camellia sinensis (L.) Kuntze) plants may explain their different extraction efficiency into tea

During tea preparation mineral elements are extracted from the dried leaves of tea (Camellia sinensis (L.) Kuntze) plants into the solution. Micro-particle induced X-ray emission was employed to investigate the spatial distribution of magnesium (Mg), calcium (Ca) and manganese (Mn) in the young and old leaves of tea plants grown in the absence and presence of aluminium (Al) in the substrate. Results revealed that in tea leaves the largest concentrations of Mg occurred in the epidermis, of Ca in oxalate crystals and of Mn in epidermis and oxalate crystals; there was a leaf-age effect on tissue-specific concentrations of Mg, Ca and Mn with all tissues of old leaves containing larger concentrations of Mg, Ca and Mn than young leaves; supplementation of substrate with Al reduced concentrations of Mg, Ca and Mn in the old leaves, and a link between the distribution of Mg, Ca and Mn in the tea leaves with the extraction efficiencies of these elements into the tea was possible. We conclude that old leaves of tea plants cultivated under conditions of low Al availability will have the largest concentrations of Mg, Ca and Mn and may represent most acceptable ingredient for the preparation of tea.

Advanced Micro-chemical Investigation of Golden Threads from Romanian Byzantine Embroideries by Micro-Particle Induced X-ray Emission (micro-PIXE)

In the present study, golden threads from two, apparently identical, medieval epitrachelions considered masterpieces of the Romanian Byzantine embroidery art, were investigated by using Micro-Particle Induced X-ray Emission (micro-PIXE) and optical microscopy. PIXE measurements allowed to accurate identify the elemental composition, distribution of the trace elements, the layered structures (depth profiling) and thicknesses of the gold layer. Useful information for the characterization of the gilded silver threads due to elemental maps concerning the constituent elements spatial distribution in the sample was also obtained.

Complexation of Eu(III), Pb(II), and U(VI) with a Paramecium glycoprotein: Microbial transformation of heavy elements in the aquatic environment

This study investigated the interaction of inorganic aqueous Eu(III), Pb(II), and U(VI) with Paramecium sp., a representative single-celled protozoan that lives in freshwater. Living and prekilled Paramecium cells were tested. The prekilled cells were killed with a fixative. After 24 h exposure of the cells to inorganic aqueous solutions containing Eu(III) or U(VI), analyses by microparticle-induced X-ray emission with a focused beam (<1 μm) did not detect Eu and U in the living cells, whereas Eu and U were detected in the prekilled cells. Size exclusion chromatography coupled with on-line ultraviolet–visible detection and elemental detection by inductively coupled plasma mass spectrometry of the aqueous phases collected after the living cell experiments revealed that a fraction of the Eu, Pb, and U in the aqueous phase bound to a large (ca. 250 kDa) Paramecium biomolecule and formed a metal-organic complex. The characteristics of the biomolecule were consistent with those of the soluble glycoproteins covering the surfaces of Paramecium cells. These results show that Paramecium cells transform inorganic aqueous Eu, Pb, and U to organic complexes. This paper discusses the relation between this novel complexation and the sorption of these heavy elements on Paramecium cells.

XRF and micro-PIXE studies of inhomogeneity of ancient bronze and silver alloys

New results regarding alloy composition and microstructure for a series of ancient bronze and silver items by X-ray Fluorescence and micro-Particle Induced X-ray Emission spectrometry were obtained in the framework of an extensive numismatic project (Scythian-type arrowheads, arrowhead-shaped monetary signs and wheel coins produced by Histria, 7th–4th century of BCE, and Dacian Radulesti-Hunedoara-type silver tetradrachms, 2nd–1st century of BCE). In Histria, warfare arrowheads were used for trade with Barbarian neighbors at first, then mechanically modified, next melted and cast as dedicated monetary signs, being, in the end, replaced by wheel coins. Three different types of alloys have been identified, and Cu-Mn and Cu-Pb segregation shown. In a blank for Radulesti-Hunedoara-type coins, Ag-(Cu+Pb) segregation has been demonstrated, suggesting an imperfectly alloyed silver-leaded bronze.

Nuclear microprobe performance in high-current proton beam mode for micro-PIXE

The performance of a nuclear microprobe is dominantly determined by the brightness of the injected ion beam. At Jožef Stefan Institute (JSI), negative hydrogen ion beams are created in a multicusp ion source and injected into a 2MV tandetron accelerator. The output characteristics of the multicusp ion source were tuned in order to obtain matching proton beam intensities for the ion accelerator and for the object slits as well. For the optimal focusing of the proton beam in a high-current mode (I>100pA) to the sub-micrometer dimensions, dedicated thin nanostructures with sharp edges have been manufactured. Set of nanostructures was micromachined by focused ion beam (FIB) at film reference material, produced by Institute for Reference Materials and Measurements (IRMM) and constituted of 57μg/cm2 of titanium on vitreous carbon substrate. The proton beam profiles were measured by beam scans across the nanostructures over long measuring times, indicating eventual slow drifts of the sample from a reference beam direction. Overall, proton beam dimensions of 600nm were obtained, demonstrating appropriate stability for micro-PIXE (micro-Particle Induced X-ray Emission) at sub-micrometer resolution for elemental analysis of biological tissue samples prepared in a freeze-dried state or in a frozen-hydrated state. The resulting performance required for micro-PIXE analysis in a high current mode with a 3MeV proton beam is presented.

Encapsulated protamine-hyaluronic acid particles for targeting carboplatin directed by radiation

Encapsulated protamine-hyaluronic acid particles containing carboplatin were prepared and their ability to release carboplatin was tested in vivo. Protamine–hyaluronic acid particles containing carboplatin were prepared by mixing protamine (1.6 mg) and hyaluronic acid (1.28 mg) into a 5 mg/mL carboplatin solution for 30 min at room temperature. A 1 mL solution of protamine–hyaluronic acid particles was poured into an ampule of COATSOME[Formula: see text] EL-010 (Nichiyu, Tokyo, Japan), shaken three times by hand, and allowed to incubate at room temperature for 15 min. Following that, 10 or 20 Gy of 100 kiloelectronvolt (KeV) soft X-ray was applied. The release of carboplatin was imaged using a microparticle-induced X-ray emission (PIXE) camera. The amount of carboplatin released was expressed as the amount of platinum released and measured via quantitative micro-PIXE analysis. The diameter of the generated encapsulated particles measured [Formula: see text] nm (mean ± standard error). The release of carboplatin from the encapsulated protamine–hyaluronic acid particles was observed under a micro-PIXE camera. The amount of carboplatin released was [Formula: see text] under 10 Gy of radiation, and [Formula: see text] under 20 Gy of radiation, which was a sufficient dose for cancer treatment. However, 10 or 20 Gy of radiation is much greater than the dose used for clinical cancer treatment (2 Gy). Further research to reduce the radiation dose to 2 Gy in order to release sufficient carboplatin for cancer treatment is required.

Elemental and immunohistochemical analysis of the lungs and hilar lymph node in a patient with asbestos exposure, a pilot study.

Studies have shown that inhaled mine dust, such as asbestos, can be translocated to various organs including the lymph nodes. Recently, we have established a protocol that enables us to identify inhaled elements using paraffin embedded lung specimens by in-air microparticle-induced X-ray emission (micro-PIXE). However, little research has examined the concentration of these inhaled fibers in various organs or the mechanisms of their translocation. In this study, we compared the concentration of inhaled fibers in the lung parenchyma to the concentration in the hilar lymph node as well as to determine the elemental spatial distribution of the inhaled fibers in a patient with occupational asbestos exposure. Lung tissues and hilar lymph node in a patient with asbestos exposure were used in this study. Elemental analysis was performed by in-air micro-PIXE. Immunohistochemical analysis was performed using anti CD163, smooth muscle actin, vimentin and β-catenin antibody. The analysis revealed that the amount of inhaled silicon was approximately 6 times higher in the lymph node than in the lungs. The spatial analysis showed that silicon, iron and aluminium were co-localized in the hilar lymph node. The immunohistochemical analysis showed localized agreement of the inhaled fibers with macrophages, smooth muscle actin, and vimentin in the hilar lymph node. This study showed that in-air micro-PIXE could be useful for analyzing the elemental distribution and quantification of inhaled fibers in the human body. Furthermore, immunohistochemistry in combination with in-air micro-PIXE analyses may help to determine the mechanism of mine dust distribution in vivo.

Innovative combination of spectroscopic techniques to reveal nanoparticle fate in a crop plant

Nanotechnology is the new industrial revolution of our century. Its development leads to an increasing use of nanoparticles and thus to their dissemination. Their fate in the environment is of great concern and especially their possible transfer in trophic chains might be an issue for food safety. However, so far our knowledge on this topic has been restricted by the lack of appropriate techniques to characterize their behavior in complex matrices. Here, we present in detail the use of cutting-edge beam-based techniques for nanoparticle in situ localization, quantification and speciation in a crop plant species (Lactuca sativa). Lettuce seedlings have been exposed to TiO2 and Ag nanoparticles and analyzed by inductively coupled plasma spectrometry, micro-particle induced X-ray emission coupled to Rutherford backscattering spectroscopy on nuclear microprobe, micro-X-ray fluorescence spectroscopy and X-ray absorption near edge structure spectroscopy. The benefits and drawbacks of each technique are discussed, and the types of information that can be drawn, for example on the translocation to edible parts, change of speciation within the plant, detoxification mechanisms, or impact on the plant ionome, are highlighted. Such type of coupled approach would be an asset for nanoparticle risk assessment.

An integrated approach for assessing the bioreceptivity of glazed tiles to phototrophic microorganisms

A laboratory-based methodology was designed to assess the bioreceptivity of glazed tiles. The experimental set-up consisted of multiple steps: manufacturing of pristine and artificially aged glazed tiles, enrichment of phototrophic microorganisms, inoculation of phototrophs on glazed tiles, incubation under optimal conditions and quantification of biomass. In addition, tile intrinsic properties were assessed to determine which material properties contributed to tile bioreceptivity. Biofilm growth and biomass were appraised by digital image analysis, colorimetry and chlorophyll a analysis. SEM, micro-Raman and micro-particle induced X-ray emission analyses were carried out to investigate the biodeteriorating potential of phototrophic microorganisms on the glazed tiles. This practical and multidisciplinary approach showed that the accelerated colonization conditions allowed different types of tile bioreceptivity to be distinguished and to be related to precise characteristics of the material. Aged tiles showed higher bioreceptivity than pristine tiles due to their higher capillarity and permeability. Moreover, biophysical deterioration caused by chasmoendolithic growth was observed on colonized tile surfaces.

The investigation of prostatic calcifications using μ-PIXE analysis and their dosimetric effect in low dose rate brachytherapy treatments using Geant4

Low dose rate brachytherapy is a widely used modality for the treatment of prostate cancer. Most clinical treatment planning systems currently in use approximate all tissue to water, neglecting the existence of inhomogeneities, such as calcifications. The presence of prostatic calcifications may perturb the dose due to the higher photoelectric effect cross section in comparison to water. This study quantitatively evaluates the effect of prostatic calcifications on the dosimetric outcome of brachytherapy treatments by means of Monte Carlo simulations and its potential clinical consequences.Four pathological calcification samples were characterised with micro-particle induced x-ray emission (μ-PIXE) to determine their heavy elemental composition. Calcium, phosphorus and zinc were found to be the predominant heavy elements in the calcification composition. Four clinical patient brachytherapy treatments were modelled using Geant4 based Monte Carlo simulations, in terms of the distribution of brachytherapy seeds and calcifications in the prostate. Dose reductions were observed to be up to 30% locally to the calcification boundary, calcification size dependent. Single large calcifications and closely placed calculi caused local dose reductions of between 30–60%. Individual calculi smaller than 0.5 mm in diameter showed minimal dosimetric impact, however, the effects of small or diffuse calcifications within the prostatic tissue could not be determined using the methods employed in the study. The simulation study showed a varying reduction on common dosimetric parameters. D90 showed a reduction of 2–5%, regardless of calcification surface area and volume. The parameters V100, V150 and V200 were also reduced by as much as 3% and on average by 1%. These reductions were also found to relate to the surface area and volume of calcifications, which may have a significant dosimetric impact on brachytherapy treatment, however, such impacts depend strongly on specific factors in the patient’s individual treatment. These factors include the number, size, composition and spatial distribution of calcifications in the prostate as well as the distribution of brachytherapy seeds.

Co-localization of iron binding on silica with p62/sequestosome1 (SQSTM1) in lung granulomas of mice with acute silicosis.

The cellular mechanisms involved in the development of silicosis have not been fully elucidated. This study aimed to examine influence of silica-induced lung injury on autophagy. Suspensions of crystalline silica particles were administered transnasally to C57BL/6j mice. Immunohistochemical examination for Fas and p62 protein expression was performed using lung tissue specimens. Two-dimensional and quantitative analysis of silica deposits in the lungs were performed in situ using lung tissue sections by an in-air microparticle induced X-ray emission (in-air micro-PIXE) analysis system, which was based on irrradiation of specimens with a proton ion microbeam. Quantitative analysis showed a significant increase of iron levels on silica particles (assessed as the ratio of Fe relative to Si) on day 56 compared with day 7 (p<0.05). Fas and p62 were expressed by histiocytes in granulomas on day 7, and the expressions persisted for day 56. Fas- and p62-expressing histiocytes were co-localized in granulomas with silica particles that showed an increase of iron levels on silica particles in mouse lungs. Iron complexed with silica induces apoptosis, and may lead to dysregulations of autophagy in histiocytes of granulomas, and these mechanisms may contribute to granuloma development and progression in silicosis.

Se(IV) uptake by Äspö diorite: Micro-scale distribution

Risk assessment of deep geological repositories (DGR) for high level radioactive waste demands radionuclide (RN) sorption data, with minimised uncertainties, on repository barriers. Sorption data in crystalline rocks, are principally obtained on crushed material and reported as distribution coefficients, relative to the mass of solid (Kd in m3/kg). Main sources of uncertainty on available sorption data are due to differences in experimental approach (comparison of crushed and intact material), groundwater and rock composition, redox conditions or the high heterogeneity of crystalline rock.In this study, the effect of rock mineral heterogeneity on selenium(IV) surface distribution on diorite crystalline rock was analysed under oxic and anoxic conditions. The micro-Particle Induced X-ray Emission (μPIXE) ion beam technique was selected because it allows quantifying tracer distribution directly on intact rock samples, at mineral micro-scale.Diorite samples were extracted from the Äspö underground research laboratory (Sweden), handled and transported under anoxic conditions. Maintaining controlled redox conditions during the whole experiment is considered particularly relevant to preserve real repository conditions.Selenite distribution on Äspö diorite surface was heterogeneous, its retention being higher under anoxic conditions. By μPIXE analyses the main Se retentive regions were identified, equivalent in both oxic/anoxic conditions, and surface distribution coefficients (Ka) were determined on main diorite minerals. Sorption values ranged from near zero, on quartz or K-feldspars, to higher values on Fe-bearing minerals like biotite (Ka∼7×10−5m) under anoxic conditions. Experimental Ka values determined here are compared to reported distribution coefficients (Kd).

Prehistoric pigment characterisation of the Abri Pataud rock-shelter (Dordogne, France)

Iron oxide pigments found in archaeological context constitute an important source of information for the understanding of cultural and subsistence activities of ancient human cultures. In order to complete archaeological contextual information, many analytical methods have been applied to characterise pigments and to provide further information on this material (&lt;em&gt;e.g.&lt;/em&gt; supplies, selections, mechanical or physical transformations of raw material, use and application processes). Several studies have demonstrated that the elemental composition of iron oxide pigments can be used to discriminate between several geological provenances. In this study, non-destructive micro-particle induced Xray emission analysis was applied in order to distinguish different kinds of reddish pigments from the prehistoric site of Abri Pataud, more especially from the Layer 2 attributed to Final Gravettian period (22,000 BP). By using an external beam, this technique required no sampling, and enabled us to perform localised analyses directly on raw material, on ochre residues applied on artefacts or on fragments of the wall of this rock-shelter. The results obtained by this technique demonstrate that the pigments covering the decorated fragments of the rock-shelter wall, found during the excavation of the Layer 2, have elemental compositions similar to the composition of a raw pigment found in the same layer. These results suggest that the shelter was decorated during the Final Gravettian period and thus provide new insights for the understanding of the archaeological context of this occupation layer.