X-ray Microfluorescence Research Articles

RNAi-Mediated Silencing of ITPK Gene Reduces Phytic Acid Content, Alters Transcripts of Phytic Acid Biosynthetic Genes, and Modulates Mineral Distribution in Rice Seeds

Phytic acid is the principal storage form of phosphorus in plant seeds and an essential signalling molecule in several regulatory processes of plant development. However, it is known as an anti-nutrient compound owing to its potent chelating property. Thus, reducing the phytic acid content in crops is desirable. Studies involving regulation of MIPS and IPK1 genes to generate low phytate rice have been reported earlier. However, the functional significance of OsITPK and the effect of its down-regulation on phytic acid content and the associated pleiotropic effects on rice have not yet been investigated. In this study, tissue specific RNA interference (RNAi)-mediated down-regulation of a major ITPK homolog (OsITP5/6K-1) resulted in 46.2% decrease in phytic acid content of T2 transgenic seeds with a subsequent 3-fold enhancement in the inorganic phosphorus content. Silencing of OsITP5/6K-1 altered the transcript levels of essential phytic acid pathway genes, without significantly affecting the transcript levels of other OsITPK homologs. Furthermore, the mapping of elements through X-ray microfluorescence analysis revealed significant changes in the spatial distribution pattern and translocation of elements in low phytate seeds. Additionally, low phytate polished seeds exhibited 1.3-fold and 1.6-fold enhancement in iron and zinc content in the grain endosperm, respectively. Silencing of OsITP5/6K-1 also altered the amino acid and myo-inositol content of the transgenic seeds. Our results successfully established that RNAi-mediated silencing of OsITP5/6K-1 gene significantly reduced the phytate levels in seeds without hampering the germination potential of seeds and plant growth. The present study provided an insight into the mechanism of phytic acid biosynthesis pathway.

Strength Development and Elemental Distribution of Dolomite/Fly Ash Geopolymer Composite under Elevated Temperature.

A geopolymer has been reckoned as a rising technology with huge potential for application across the globe. Dolomite refers to a material that can be used raw in producing geopolymers. Nevertheless, dolomite has slow strength development due to its low reactivity as a geopolymer. In this study, dolomite/fly ash (DFA) geopolymer composites were produced with dolomite, fly ash, sodium hydroxide, and liquid sodium silicate. A compression test was carried out on DFA geopolymers to determine the strength of the composite, while a synchrotron Micro-Xray Fluorescence (Micro-XRF) test was performed to assess the elemental distribution in the geopolymer composite. The temperature applied in this study generated promising properties of DFA geopolymers, especially in strength, which displayed increments up to 74.48 MPa as the optimum value. Heat seemed to enhance the strength development of DFA geopolymer composites. The elemental distribution analysis revealed exceptional outcomes for the composites, particularly exposure up to 400 °C, which signified the homogeneity of the DFA composites. Temperatures exceeding 400 °C accelerated the strength development, thus increasing the strength of the DFA composites. This appears to be unique because the strength of ordinary Portland Cement (OPC) and other geopolymers composed of other raw materials is typically either maintained or decreases due to increased heat.

Cerium anomalies in riverbanks: Highlight into the role of ferric deposits

In wetlands, stream riverbanks represent a large redox reactive front. At their surface, ferric deposits promote their capacity to trap nutrients and metals. Given that rare earth elements (REE) are now considered as emerging pollutants, it seems that the riverbank interface is a strategic area between wetlands and streams in terms of controlling the environmental dissemination of REE. Therefore, the evolutions of the REE distribution and cerium (Ce) anomaly (Ce/Ce*, i.e. depleted or enriched Ce concentration compared to the other REE) were studied at various locations on a riverbank. The positive Ce anomaly is related to a high Fe content, a low organic carbon/iron ratio ((OC)/Fe) and newly formed Fe oxyhydroxides independently of their interactions with organic matter. Micro-X ray fluorescence (μ-XRF) mapping confirms Ce accumulation with ferric deposits. The Ce speciation exhibits a mix of Ce(III) and Ce(IV) in the ferric deposits, almost 20% of Ce occurred as Ce(IV) due to oxidation by newly formed Fe oxyhydroxides, while the subsurface horizons only display Ce(III). These results provide evidence that the Ce anomaly variation observed in stream water between low and high flow periods is partly due to the erosion of ferric deposits exhibiting a positive Ce anomaly. Therefore, the Ce anomaly can be considered as a fingerprint of the release of Fe colloids in the rivers and streams connected to the wetland.

Flower-Shaped Trapiche Ruby from Mong Hsu, Myanmar: A Revised Growth Mechanism

Two polished slices of flower-shaped trapiche ruby from Mong Hsu were analysed by X-ray computed tomography and X-ray micro-fluorescence. They are characterised by the presence of a core overgrown by two layers of ruby (with the outer one forming the ‘petal’ shape), and these three domains were previously attributed by Liu (2015) to a multi-stage growth mechanism. The present research indicates that these specimens show textural sector zoning associated with chemical sector zoning. In agreement with literature data, three different growth sectors are identified: a pinacoidal growth sector (corresponding to the core) and two sets of dipyramidal growth sectors with different inclinations with respect to the c-axis. The core, middle and outer layers are thus growth sectors of the same crystal, and no successive stages are needed to explain their formation. Therefore, the growth model proposed for other trapiche rubies from Myanmar also applies to the formation of these flower-shaped specimens. The distinctive shape of these rubies is due to weathering of the growth sectors and the sector boundaries.

Phosphorus homeostasis in Populus alba L. under excess phosphate conditions, assessed by 31P nuclear magnetic resonance spectroscopy and X-ray microfluorescence.

The phosphates (Pi) are nowadays recognized as pollutants. We studied the effect of Pi (0.625-12.500mM KH2PO4) in the culture medium on in vitro grown 2-month-old Populus alba trees. The levels of sugar phosphates and vacuolar and cytoplasmic Pi in cell compartments of roots and stems were determined using 31P NMR, while tissue-specific micro- and macroelements mapping on stem cross-sections were performed using synchrotron-based X-ray microfluorescence. Plants grown on 0.625mM Pi (MS/2 medium) showed a survival rate of 70%. With the increase in Pi concentrations up to 6.250mM, plant growth and survival increased, without changes in total P content per mass or in the levels of cytoplasmic and vacuolar phosphates, in both stems and roots, while the levels of Fe, Cu, Zn, Ca and Mn in stems increased. Further increase in Pi to 9.375 and 12.500mM in the medium resulted in inhibited growth comparable with plants grown on MS/2, with the increase in total P content per mass up to 50%, in both stems and roots, but with no changes in cytoplasmic and vacuolar phosphates; 12.500mM Pi affected even plant survival (70%) and thus might be considered as mildly toxic. 31P NMR results indicate that the high tolerance of P. alba to increased Pi could result from its ability to maintain an intracellular P homeostasis, despite P accumulation up to 50%, in both stems and roots, indicating P. alba as a promising wood species for dendroremediation.

Visible cellular distribution of cadmium and zinc in the hyperaccumulator Arabidopsis halleri ssp. gemmifera determined by 2-D X-ray fluorescence imaging using high-energy synchrotron radiation.

The striking sub-cellular distribution of cadmium (Cd) and zinc (Zn) in the Cd and Zn hyperaccumulator Arabidopsis halleri ssp. gemmifera was revealed by microbeam X-ray microfluorescence analysis (μ-XRF) using high-energy synchrotron radiation. Plants were grown in hydroponics with various Cd and Zn concentrations. The concentration of Cd in the aerial portions of the plants increased with increasing Zn exposure and the transportation efficiency of Cd from the root to the shoot was affected by both the Cd and Zn concentrations in the nutrient solution. The μ-XRF imaging clearly showed that Cd and Zn were preferentially accumulated in trichomes on the leaf, while the distribution of Cd in the leaf was changed by Zn treatment. It was observed that Cd treated with a higher Zn concentration (20 μM Cd + 100 μM Zn) was distributed in the mesophyll tissue at high concentrations. In addition, μ-XRF imaging clarified that the distribution of Zn inside the leaf was different from that of Cd at a cellular level. Zn was primarily distributed in the mesophyll tissue of the leaf blade. In contrast, Cd was localized in the vascular bundle of the main vein. That is, Zn was transported to mesophyll tissue from the vascular bundle more efficiently than Cd. As seen above, we were able to study the difference of the distribution of Cd and Zn, which are congeners and behave similarly, inside the plant body at the cellular level in detail by high-energy μ-XRF.

Geochemical compositional mapping of Lower Jurassic trace fossils: Palaeoenvironmental significance and methodological implications

Analyses of X-ray microfluorescence (XRF) elemental maps of the ichnofossil assemblages from the upper Pliensbachian-lower Toarcian marl-limestone rhythmite of the South Iberian Palaeomargin (Betic Cordillera, SE Spain) show that the compositions of burrow infills are geochemically and mineralogically different with respect to the surrounding sediment. XRF elemental maps and back-scattered electron images show pyrite framboids within the burrows and enrichment in Fe and S relative to the host sediment. This is congruent with the position of burrows beneath the redox boundary at the time of their active or passive sedimentary infilling. Organic matter that accumulated at the seafloor was not preserved due to oxidation and consumption by benthic organisms. However, burrows located beneath the redox boundary served as traps for organic matter preservation. The composition of burrow fills reflects the original signal of the redox conditions of pore-waters below the sediment-water interface, which was not preserved in the surrounding sediment. The XRF elemental maps also show that some trace fossils from limestone and marly-limestone intervals contain high concentrations of Si, Al, K, Zn, Cr, and Ti with respect to the surrounding sediment. This may reflect enrichment of clay minerals in the burrow fills derived from overlying marly layers. This work proposes the use of elemental imaging of trace fossils prior bulk-rock geochemical analyses in order to evaluate the potential for biogenic heterogeneity and elemental partitioning before sampling. Geochemical analysis of trace fossils represents an additional tool that may be of use in hydrocarbon exploration.

Microscale Analyses of Mineral Particles in Sugar Cane Bagasse and Straw Shed Light on How Debris Can Be Incorporated into Biomass

Sugar cane bagasse and straw are vast, inexpensive renewable resources that can be used as solid fuels as well as feedstocks for production of materials, chemicals, and advanced biofuels. However, industrial valorization of bagasse and straw can be hampered by the mineral impurities commonly present in the biomass. In this work, we use synchrotron X-ray computed microtomography (μCT) and scanning X-ray microfluorescence (μXRF) to observe and analyze mineral particles in sugar cane bagasse and straw. On the basis of the microscale analyses, we suggest possible mechanisms that contribute to the incorporation of extraneous inorganic matter into biomass, especially soil debris. Our analyses discriminate between (1) mineral particles having a characteristic range of size (circa 5–27 μm) found at surfaces as well as inside biomass particles, (2) the contribution of water that keeps larger mineral particles adhered to the biomass, and (3) soil aggregates that are adhered to the surfaces of straw, with no equival...

Analysis of two meteorite fragments (lunar and martian) using X-Ray microfluorescence and X-Ray computed microtomography techniques

Meteorites have been arousing the curiosity of mankind since antiquity. However, the interest in these objects goes far beyond mere curiosity in the study of such materials, which has great importance due essentially to the information they can provide. The importance of studying meteorites is associated about the earliest conditions and processes during the formation and earliest history of the solar system. So, in this study, the characterization of two meteorite fragments was performed using X-ray computed microtomography (micro-CT) and X-ray microfluorescence (micro-XRF). These techniques were used for their non-destructive characteristics and the ability to provide information about the structure and composition the meteorites. The micro-CT images showed encrusted structures within both samples. However, while in Lunar meteorites spheroidal structures very similar to small grains internally grouped in clusters were found, in the Martian meteorite a very peculiar structure was identified. Besides that, the micro-CT it was also possible to evaluate the different density materials that compose the samples. The micro-XRF results accounted for the presence of the elements Si, Ca, Ti, Cr, Mn, Fe, Ni and Sr in the Lunar sample, as well as of Si, S, K, Ca, Ti, Cr, Mn, Fe, Cu, Zn, Sr and Y in the Martian sample. The results obtained are effective for the characterization of meteorites, proving thus that it is possible to obtain important information about the chemical composition, as well as about the distribution and the internal structure of these materials, evaluating aspects such as density and porosity.

Characterization of scale deposition in oil pipelines through X-Ray Microfluorescence and X-Ray microtomography

The formation of scales consists in one of the most relevant problems in the oil prospecting field and occurs when incompatible types of water (injected sea water and formation water) are mixed in the reservoir, unavoidably undergoing chemical interaction followed by mineral precipitation. In this work, scale samples extracted from obstructed oil pipes were characterized through X-Ray Microfluorescence and X-Ray microtomography by analyzing their elemental and structural composition. Different types of scale were found according to their elemental distribution (mainly BaSO4 and CaCO3) and to the way that they were deposited inside the pipes. The results of both techniques provided data that can be used to optimize the prevention and removal methods of such materials from pipes and equipments used in oil facilities.

Study of composition and structure of demineralized bone using X-ray techniques

The aim of this study is to compare the composition and structure of cortical bones with different stages of in-vitro demineralization from the micro (10–500 μm) to the nano-scale level (below 1 μm) using X-ray techniques, such as Small Angle X-ray Scattering (SAXS), X-ray Microfluorescence (μ-XRF) and Synchrotron Microtomography (SR-μCT). In total, 8 fresh cortical bone samples of different ages from 2 diaphyseal femora were selected. They were submitted to a 4 days demineralization experiment with 2 concentrations of EDTA acid (0.1 mol L−1 pH 10, and 0.5 mol L−1 pH 7.4, at 25 °C, without agitation). EDTA is a slow demineralization agent and permits better tissue preservation of bone specimens. μ-XRF elemental mapping showed that EDTA 0.1 mol L−1 demineralized more homogeneously the samples than EDTA 0.5 mol.L−1 (pH 7.4). SR-μCT data have demonstrated the changes that occurred in the 3D internal microstructure of cortical bone during the demineralization and besides the results concluded that both EDTA concentrations had similar effect on the intracortical porosity. From SAXS analysis EDTA 0.1 mol L−1 had a better performance in the demineralization of the samples without damaging the nanostructures.

The speciation and distribution characteristics of Cu in Phragmites australis (Cav.) Trin ex. Steudel.

Heavy metal allocation and the mechanism(s) of metal sequestration in different clonal organs, micro-domains and subcellular structures has not been systematically studied for rhizomatous perennial plants. It is thus pertinent to investigate knowledge of the speciation and distribution characteristics of Cu in Phragmites australis to elucidating the mobility of metals in wetland plants after their uptake via root systems so as to facilitate development of strategies to enhance Cu tolerance. This study investigated the distributions of Cu in P.australis root, stem and leaf using ICP-MS, synchrotron-based X-ray micro-fluorescence and X-ray absorption spectroscopy, then evaluated the effects of Cu on cellular structure and ultrastructure via transmission electron microscopy. The results indicate a clear preferential localisation of Cu in the roots as compared with the shoots (stems and leaves). The intensity of Cu in the vascular bundles was higher than that in the surrounding epidermis and the endodermis and parenchyma outside the medullary cavity. The dominant chemical form of Cu in P.australis was similar to Cu citrate. The results suggest that although Cu can be easily transported into the vascular tissues in roots and stems via Cu citrate, most of the metal absorbed by plants is retained in the roots because if its high binding to the cell wall, thus preventing metal translocation to aerial parts of the plants. Therefore, P.australis showed a high capacity toaccumulate Cu in roots, being therefore a suitable species for phytostabilisation interventions.

In vitro and in vivo evaluations of nanocrystalline Zn-doped carbonated hydroxyapatite/alginate microspheres: zinc and calcium bioavailability and bone regeneration.

Background: Zinc-doped hydroxyapatite has been proposed as a graft biomaterial for bone regeneration. However, the effect of zinc on osteoconductivity is still controversial, since the release and resorption of calcium, phosphorus, and zinc in graft-implanted defects have rarely been studied.Methods: Microspheres containing alginate and either non-doped carbonated hydroxyapatite (cHA) or nanocrystalline 3.2 wt% zinc-doped cHA (Zn-cHA) were implanted in critical-sized calvarial defects in Wistar rats for 1, 3, and 6 months. Histological and histomorphometric analyses were performed to evaluate the volume density of newly formed bone, residual biomaterial, and connective tissue formation. Biomaterial degradation was characterized by transmission electron microscopy (TEM) and synchrotron radiation-based X-ray microfluorescence (SR-µXRF), which enabled the elemental mapping of calcium, phosphorus, and zinc on the microsphere-implanted defects at 6 months post-implantation.Results: The bone repair was limited to regions close to the preexistent bone, whereas connective tissue occupied the major part of the defect. Moreover, no significant difference in the amount of new bone formed was found between the two microsphere groups. TEM analysis revealed the degradation of the outer microsphere surface with detachment of the nanoparticle aggregates. According to SR-µXRF, both types of microspheres released high amounts of calcium, phosphorus, and zinc, distributed throughout the defective region. The cHA microsphere surface strongly adsorbed the zinc from organic constituents of the biological fluid, and phosphorus was resorbed more quickly than calcium. In the Zn-cHA group, zinc and calcium had similar release profiles, indicating a stoichiometric dissolution of these elements and non-preferential zinc resorption.Conclusions: The nanometric size of cHA and Zn-cHA was a decisive factor in accelerating the in vivo availability of calcium and zinc. The high calcium and zinc accumulation in the defect, which was not cleared by the biological medium, played a critical role in inhibiting osteoconduction and thus impairing bone repair.

Microbeam X-ray fluorescence mapping of Cu and Fe in human prostatic carcinoma cell lines using synchrotron radiation

Cancer is a worldwide public health problem and prostate cancer continues to be one of the most common fatal cancers in men. Copper plays an important role in the aetiology and growth of tumours however, whether intratumoral copper is actually elevated in prostate cancer patients has not been established. Iron, an important trace element, plays a vital function in oxygen metabolism, oxygen uptake, and electron transport in mitochondria, energy metabolism, muscle function, and hematopoiesis. The X-ray microfluorescence technique (μXRF) is a rapid and non-destructive method of elemental analysis that provides useful elemental information about samples without causing damage or requiring extra sample preparations. This study investigated the behavior of cells in spheroids of human prostate cells, tumour cell line (DU145) and normal cell line (RWPE-1), after supplementation with zinc chloride by 24 hours using synchrotron X-ray microfluorescence (μSRXRF). The measurements were performed with a standard geometry of 45º of incidence, excited by a white beam using a pixel of 25 μm and a time of 300 ms/pixel at the XRF beamline at the Synchrotron Light.

Distributions of cadmium, zinc, and polyphenols in Gamblea innovans

Gamblea innovans is a Cd- and Zn-accumulating deciduous tree widely distributed in the secondary forests of Japan. We aimed to understand the characteristics of Cd and Zn accumulation in G. innovans in order to effectively utilize the species for phytoremediation. To accomplish that, we studied the relationship between secondary metabolite concentrations and the accumulation and distributions of Cd and Zn in G. innovans leaves and basal stems using micro-X ray fluorescence (µ-XRF). Our results showed a negative correlation between Zn leaf concentrations and polyphenol/2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. This finding might be related to stress or the manifestation of a mechanism for tolerance to Cd and Zn accumulation. In addition, we observed that Cd accumulated primarily in the apoplastic region of surface tissues such as bark and the epidermis of leaves, whereas Zn accumulated in both apoplastic and symplastic regions. Thus, it might be possible that G. innovans can distinguish between Cd and Zn and control their translocation.

Thiosulphate-induced phytoextraction of mercury in Brassica juncea: Spectroscopic investigations to define a mechanism for Hg uptake

Thiosulphate is extensively used to enhance mercury (Hg) phytoextraction due to its efficient in prompting plant Hg uptake. However, the mechanism by which thiosulphate promotes Hg uptake is poorly understood. We determined the concentrations of Hg and potassium (K), and their spatial distribution, in the tissues of Brassica juncea grown in Hg-contaminated soils treated by thiosulphate and compared this to a non-treated soil (control). The spatial distribution of Hg and K was characterized using micro-X ray fluorescence spectroscopy. The subcellular localization and speciation of Hg in the root of plant treated by thiosulphate were elucidated using Transmission electron microscope coupled energy-dispersive X-ray (TEM-EDX) spectroscopy. Thiosulphate increased significantly the Hg concentration in the roots (mainly in the epidermis and xylem) and shoots (mainly in the vascular bundles), while Hg was accumulated in the root (mainly in the epidermis) of the control plant. Thiosulphate promoted the movement of Hg from the epidermis to the xylem of roots, with subsequent loading into the stem via vascular bundles. Thiosulphate decreased the K concentration in plant tissues, relative to the control plant, and we propose this is due to leakage of electrolyte from roots via increased plasma membrane permeability as a consequence of physiological damage caused by the added thiosulphate. Mercury was distributed mainly at the extracellular space in the roots and was shown by TEM-EDX to be predominately amorphous nano-clusters of HgS. We conclude that thiosulphate-promoted Hg accumulation in the plant may happen through increased plasma membrane permeability, a changed pathway of Hg movement within plants, and extracellular co-transportation of Hg-S complexes in the roots. Our results may underpin the ongoing development of phytomanagement as an environmental strategy for Hg contaminated soils around the world.

Synchrotron X-ray Microdiffraction and Fluorescence Imaging of Mineral and Rock Samples

In this report, we describe a detailed procedure for acquiring and processing x-ray microfluorescence (μXRF), and Laue and powder microdiffraction two-dimensional (2D) maps at beamline 12.3.2 of the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. Measurements can be performed on any sample that is less than 10 cm x 10 cm x 5 cm, with a flat exposed surface. The experimental geometry is calibrated using standard materials (elemental standards for XRF, and crystalline samples such as Si, quartz, or Al2O3 for diffraction). Samples are aligned to the focal point of the x-ray microbeam, and raster scans are performed, where each pixel of a map corresponds to one measurement, e.g., one XRF spectrum or one diffraction pattern. The data are then processed using the in-house developed software XMAS, which outputs text files, where each row corresponds to a pixel position. Representative data from moissanite and an olive snail shell are presented to demonstrate data quality, collection, and analysis strategies.

Synchrotron X-ray Microdiffraction and Fluorescence Imaging of Mineral and Rock Samples

In this report, we describe a detailed procedure for acquiring and processingx-ray microfluorescence (μXRF), and Laue and powder microdiffraction two-dimensional (2D) maps at beamline 12.3.2 of the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. Measurements can be performed on any sample that is less than 10 cm x 10 cm x 5 cm, with a flat exposed surface. The experimental geometry is calibrated using standard materials (elemental standards for XRF, and crystalline samples such as Si, quartz, or Al2O3 for diffraction). Samples are aligned to the focal point of the x-ray microbeam, and raster scans are performed, where each pixel of a map corresponds to one measurement, e.g., one XRF spectrum or one diffraction pattern. The data are then processed using the in-house developed software XMAS, which outputs text files, where each row corresponds to a pixel position. Representative data from moissanite and an olive snail shell are presented to demonstrate data quality, collection, and analysis strategies.

Distribution, redox state and (bio)geochemical implications of arsenic in present day microbialites of Laguna Brava, Salar de Atacama

Understanding how microorganisms adapted to the high arsenic concentration present on early Earth requires understanding of the processes involved in the arsenic biogeochemical cycle operating in living microbial mats. To this end, we investigated a living microbial mat from Laguna Brava (Salar de Atacama, Chile), a hypersaline lake with high arsenic concentration, using an array of conventional geochemical techniques, such as X-ray diffraction, SEM-EDX and Confocal Laser Scanning Microscopy (CLSM), combined with state-of-the-art high resolution scanning imaging techniques, including X-ray micro-fluorescence (μXRF) and X-ray Absorption Near Edge Structure (XANES) mapping. This experimental approach allowed us to unravel the relationship between the microbial mat activity, mineral occurrence, arsenic speciation, distribution of major and trace elements and their relationship with the mineralogy and the exopolymeric substances (EPS). We show that As was not linked to Ca or Si, and only moderately related to Fe, resulting from sorption onto an iron (oxy)hydroxide mineral. In addition, we were able to identify organic-rich globules containing significant As but no other trace metal(loid)s, and determine the co-existence of As(III) and As(V). These observations strongly support the occurrence of microbially-mediated arsenic cycling in these microbial mats.

Estudio de pátinas procedentes de hornos de fundición en la Real Fábrica de Artillería de Sevilla (s. XVII)

El objeto principal del presente trabajo es determinar la estructura de los estratos que componen el revestimiento del paramento de las zonas identificadas en la Fábrica de Artillería de Sevilla como “fundición vieja” y “fundición nueva” y realizar la caracterización química de los mismos con la finalidad concreta de identificar los posibles depósitos metálicos que pudieran haberse acumulado durante la actividad fabril de fundición llevada a cabo en el edificio desde finales del siglo XVI hasta el XX (Sobrino 2011). Hay que señalar que las posibles pátinas han sido sucesivamente ocultadas mediante el pintado de los paramentos, de ahí que la serie estratigráfica acumulada a lo largo del tiempo corresponda a una secuencia alterna (Barbero, 2013). Las muestras fueron evaluadas inicialmente mediante microscopía óptica para la determinación de los estratos existentes. Mediante microfluorescencia de rayos X y microscopía electrónica se identificaron las partículas que contenían metales y se determinó su composición química (Berthold, 2009). Este análisis confirmó la hipótesis de la presencia de elementos metálicos pesados, destacando especialmente la presencia de hierro, habiéndose detectado también en las distintas muestras en proporciones minoritarias, aunque relevantes (cobre, cinc y, en casos aislados, plomo y níquel). Todos ellos responden a su posible presencia en los materiales utilizados en la fundición.