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Elemental concentrations in micro-PIXE (Particle Induced X-ray Emission) maps of elements in biological tissue slices have been determined using auxiliary information on the sample matrix composition from EBS (Elastic Backscattering Spectroscopy) and STIM (Scanning Transmission Ion Microscopy). The thin sample approximation may be used for evaluating micro-PIXE data in cases, where X-ray absorption in the sample can be neglected and the mass of elements in a selected area can be estimated. The resulting sensitivity amounts to an impressive 10−12 g of the selected elements. Two cases are presented as examples. In the first, we determined the total mass of gold nanoparticles internalized by human monocyte-derived dendritic cells (MDDC). In the second, an inventory of the mass of elements in the micro-particulate material adsorbed at the wall of the lorica of the microzooplankton species Tintinnopsis radix has been created.

Recently we completed a construction of a cryostat at Jožef Stefan Institute (JSI) nuclear microprobe enabling us to analyze various types of biological samples in frozen hydrated state using micro-PIXE/STIM/RBS. Sample load-lock system was added to our existing setup to enable us to quickly insert a sample holder with frozen hydrated tissue samples onto a cold goniometer head cooled with liquid nitrogen inside the measuring chamber. Cryotome-cut slices of frozen hydrated plant samples were mounted between two thin silicon nitride foils and then attached to the sample holder. Sufficient thermal contact between silicon nitride foils and sample holder must be achieved, as well as between the sample holder and the cold goniometer head inside the measuring chamber to prevent melting of the samples. Matrix composition of frozen hydrated tissue is consisted mostly of ice. Thinning of the sample as well as water evaporation during high vacuum and proton beam exposure was inspected by the measurements with RBS and STIM method simultaneously with micro-PIXE. For first measuring attempts a standard micro-PIXE configuration for tissue mapping was used with proton beam cross section of 1.2 × 1.2 μm 2 and a beam current of 100 pA. The temperature of the cold goniometer head was kept below 130 K throughout the entire proton beam exposure. First measurements of thin plant tissue samples in frozen hydrated state show minute sample degradation during the 10 h period of micro-PIXE measurements.

Diverse physiological, biochemical and morphological adaptations enable plants to survive in extreme saline environments where osmotic and ionic stresses limit growth and development. Halophytes are salt-tolerant plants that can withstand extraordinarily high levels of Na and Cl in their leaves. The tissue and cellular distribution patterns of salt ions can be linked to the underlying mechanisms of salt tolerance. Application of fast, reliable, multi-elemental and quantitative techniques such as micro-proton-induced X-ray emission (micro-PIXE) will significantly contribute to and accelerate studies of plant salt tolerance, especially as micro-PIXE also provides spatially resolved quantitative data for light elements, such as Na and Mg. The spatial concentration distributions of Na, Mg, Cl, K, P and S in leaves of four halophytes (Bassia indica, Atriplex prostrata, Spartina maritima and Limonium angustifolium) were determined using micro-PIXE, to study the salt-tolerance strategies of the selected halophytes. Different distribution patterns of the studied elements were seen in the leaves; however, in all four of these plant species, Na was excluded from photosynthetically active chlorophyl tissues. With the exception of L. angustifolium, Cl, P and S contents (representing chloride, phosphate and sulphate ionic forms, respectively) did not ensure charge balance in the leaves, which suggests other anionic compounds, such as nitrate and organic anions, have crucial roles in maintaining electroneutrality in these halophytes. By increasing soil salinisation worldwide, the possibility to reliably complement spatial distributions of Na, Mg, Cl, K, P and S with plant structural morphology will contribute significantly to our understanding of plant tolerance mechanisms at the tissue and cell levels. In addition, these kinds of studies are of particular value for designing crop plants with high salt tolerance and for the development of phytoremediation technologies.

We demonstrate the stereo-PIXE method by measurement of surface topography of the relief details on 1€ coin. Two X-ray elemental maps were simultaneously recorded by two X-ray detectors positioned at the left and the right side of the proton microbeam. The asymmetry of the yields in the pixels of the two X-ray maps occurs due to different photon attenuation on the exit travel path of the characteristic X-rays from the point of emission through the sample into the X-ray detectors. In order to calibrate the inclination angle with respect to the X-ray asymmetry, a flat inclined surface model was at first applied for the sample in which the matrix composition and the depth elemental concentration profile is known. After that, the yield asymmetry in each image pixel was transferred into corresponding local inclination angle using calculated dependence of the asymmetry on the surface inclination. Finally, the quantitative topography profile was revealed by integrating the local inclination angle over the lateral displacement of the probing beam.

Understanding the uptake, accumulation and distribution of toxic elements in plants is crucial to the design of effective phytoremediation strategies, especially in the case of complex multi-element pollution. Using micro-proton induced X-ray emission, the spatial distribution of Na, Mg, Al, Si, P, S, Cl, K, Ca, Mn, Fe, Zn, As, Br, Rb, Sr, Cd and Pb have been quantitatively resolved in roots and rhizomes of an obligate wetland plant species, Typha latifolia, treated with a mixture of 100 μM each of As, Cd and Pb, together. The highest concentrations of As, Cd and Pb were found in the roots of the T. latifolia, with tissue-specific distributions. The As was detected in the root rhizodermis, and in the rhizome the majority of the As was within the vascular tissues, which indicates the high mobility of As within T. latifolia. The Cd was detected in the root exodermis, and in the vascular bundle and epidermis of the rhizome. The highest Pb concentrations were detected in the root rhizodermis and exodermis, and in the epidermis of the rhizome. These data represent an essential step in the resolution of fundamental questions in plant ionomics.

The present study was motivated by the paucity of reports on cellular internalization of ingested titanium dioxide (TiO2) nanoparticles (nano-TiO2). The model invertebrate (Porcellio scaber, Isopoda, Crustacea) was exposed to food dosed with nano-TiO2 containing 100, 1,000, 3,000, or 5,000 µg nano-TiO2 per gram of food. After 14 d of exposure, the amount of Ti in the entire body was analyzed by inductively coupled plasma–mass spectrometry, and elemental analyses of tissue cross sections were performed by particle induced X-ray emission. In addition, a series of toxicological markers including feeding parameters, weight change, and survival, as well as cytotoxic effects such as digestive gland cell membrane stability, were monitored. Internalization of ingested nano-TiO2 by the isopod's digestive gland epithelial cells was shown to depend on cell membrane integrity. Cell membranes were found to be destabilized by TiO2 particles, and at higher extracellular concentrations of nano-TiO2, the nanoparticles were internalized. Environ. Toxicol. Chem. 2012; 31: 1083–1090. © 2012 SETAC

Micro-proton induced X-ray emission (micro-PIXE) method was applied to study the micro-localization of silver (Ag) in digestive glands of a terrestrial arthropod (Porcellio scaber) after feeding on silver nanoparticles (nano-Ag) dosed food. The aim of our work was to assess whether feeding on nano-Ag results in the assimilation of silver (Ag) in digestive gland cells. To study micro-localization and elemental distribution of Ag, the animals were fed on food dosed with nanoparticles for 14 days under controlled laboratory conditions. At the end of the feeding exposure, the animals were dissected and digestive glands prepared for micro-PIXE analyses and TEM investigation. The results obtained by micro-PIXE documented high amounts of Ag inside S-cells of the digestive gland epithelium; however, TEM investigation did not show particle aggregates inside digestive gland cells. Also no adverse effect on feeding behavior was recorded what is a measure of toxic effects. We explain the presence of Ag inside the cells as a result of the assimilation of dissoluted Ag ions from ingested nano-Ag particles. Assimilation of excessive amounts of ingested metal ions in S-cells is a well known metal detoxification mechanism in isopods. We discuss the advantages of using micro-PIXE for the micro-localization of elements in biological tissue in studies of interactions between nanoparticles and biological systems.

A new confocal PIXE set-up at the Jožef Stefan Institute in Ljubljana was recently designed and built. It consists of a silicon-drift detector, a specially designed polycapillary lens and a snout-alignment interface for precise positioning. It allows detector movement in all directions and therefore precise alignment during the creation of the probing volume and the possibility of simultaneous use of other complementary techniques, including standard μ-PIXE measurements with another X-ray detector. A description of the new set-up is given, as well as a short presentation of the method itself. Two custom-designed types of X-ray lenses were tailored and manufactured for this application, a standard semi-lens and a polycapillary conic collimator; both were characterized and compared within the scope of development of the confocal PIXE system. First results of depth profiling with the beam scanning mode are shown.

In machine workshops and laboratories, specific materials and chemicals are processed and toxic aerosols may be released into air. For protection of the health of employees working in that environment, it is therefore important to gather knowledge about aerosol composition related to specific working activity and to monitor the aerosol concentration levels. An extensive study of this kind was performed in the machine workshop of Jožef Stefan Institute using a combination of time-, particle size- and location-resolved sampling. The majority of samples were analyzed by particle induced X-ray emission (PIXE) and energy dispersive X-ray spectrometry (EDX) techniques and the main results have been described in a separate paper. Here, we concentrate on presenting the results of individual particle analysis of an aerosol emission that is related to a short, unexpected episode related to the restoration of old furniture. Although the activity was identified by a strong correlation of elemental concentrations in a series of samples taken with a 2-h time resolution, the single microparticle analysis of the cascade impactor samples taken over the longer time period offered an additional insight into the corresponding aerosol composition. The analysis of the aerosol size-selected samples revealed strong pair correlations among concentration levels of Ba, Zn, S, Pb and Cl in a number of individual microparticles. The result is explained by the emission of Ba- and Pb-rich aerosol particles in course of removal of the old white paint layers. The former particles appear to originate from white pigment called lithopone, which is a mixture of BaSO4 and ZnS, and the latter group seems to be related to Pattinson's white lead PbCl·OH. Although the individual particle analysis by itself is in principle capable of assigning the emission source, in this case, it was used as an excellent tool to enhance the quality of source characterization. Copyright © 2011 John Wiley & Sons, Ltd.

A detailed localisation of elements within leaf tissues of hydroponically grown Cd/Zn hyperaccumulator Thlaspi praecox (Brassicaceae) was determined by micro-PIXE at Jozef Stefan Institute (Ljubljana, Slove- nia) in order to study accumulation patterns of Cd and other elements in the case of a single metal (Cd) pollution. Plants were treated with increasing concentrations of Cd in the solution (0 (control), 1, 10 and 100 lM). As expected, concentration of Cd in the leaves gradually increased with Cd concentration in the solution. In order to reveal the main Cd storage compartment space within the leaves a relative element distribution (pool) was calculated based on concentrations of elements in specific leaf tissues and their weight portions. Where present at detectable levels, Cd accumulated in the epidermal tissues (at 10 lM), but the contribution of epidermal pool decreased with increasing Cd concentration in solu- tion (at 100 lM). The opposite was observed for the mesophyll pool. In addition, in Cd treated plants, a significant decrease in mesophyll Fe pool and an increase in the epidermal Fe pool were observed. Sim- ilar effect was seen for Mn pool at 100 lM Cd treatment accompanied by increasing Zn epidermal pool with increasing Cd in nutrient solution. Altogether these results indicate repartitioning of essential mesophyll cation pools (e.g., Fe, Mn and possibly Zn) when increasing Cd contents, that are instead more intensively stored in the epidermal cells. These results confirmed micro-PIXE as effective and powerful technique providing essential information on metal localisation, repartitioning and major ele- mental stores in plants on the tissue levels that were not accessible using classical analytical tech- niques and thus complementing our current understanding of plant metal tolerance mechanisms as a whole.

We recorded a series of spectral maps of a hematite particle by combining a scanning proton microbeam excitation with detection of X-rays by a Si(Li) spectrometer equipped with polycapillary lens. The particle was driven through the sensitive microvolume to allow for a reconstruction of concentration in three dimensions. Beside the description of the experiment, important aspects of data analysis are emphasized to show the potential of the confocal micro-PIXE (Proton Induced X-ray Emission) imaging method: at present, this is limited by the focusing properties of the polycapillary lens.

A detailed report about the confocal experiment and the corresponding data analysis is presented to determine local atomic concentrations in a thick sample with a micrometer resolution. X-ray emission from a quartz substrate, loaded by aerosol particles, was induced by a scanning proton microbeam and observed by an Si(Li) spectrometer whose field of view was narrowed by a polycapillary lens. A series of X-ray images were recorded at different positions of the sample along the microbeam axis when the particles were driven through the sensitive microvolume. The concentrations reconstructed in three dimensions were used to extract penetration profiles of the strongest X-ray emitters (Fe, Ca, S) in an aerosol sample together with the surface profile of the matrix (Si). The results show exponentially dumped depth profiles with characteristic length depending on particle size. Copyright © 2009 John Wiley & Sons, Ltd.

The elemental composition of PM10 aerosols in the vicinity of the open-air bulk terminal in the Port of Koper, Slovenia, was determined. Using a combination of short but intense and longer but relatively simple set-up of sampling points and sampling periods, we studied elemental concentrations ranging from S to Br and Pb due to the presence of the terminal and related activities. We observed a decrease in concentrations with respect to the distance from the source and a long-term correlation of different elemental concentrations detected during the non-stop 2-year sampling period. Two types of filters, Nuclepore and quartz fibre, were used for sampling as being most appropriate for different aims of the project. The filters were irradiated with 2 MeV protons at the PIXE beamline of the Microanalytical Centre at Jozef Stefan Institute (JSI). The elemental concentrations were obtained by analysing the recorded x-ray spectra with the GUPIX code. We found a strong correlation between iron ore transfer and Fe concentration in PM10.

A better understanding of the mechanisms that govern copper (Cu) uptake, distribution and tolerance in Brassica carinata plants in the presence of chelators is needed before significant progress in chelate-assisted Cu phytoextraction can be made. The aims of this study were therefore to characterise (S,S)-N,N'-ethylenediamine disuccinic acid (EDDS)-assisted Cu uptake, and to compare the spatial distribution patterns of Cu in the roots and leaves of B. carinata plants. The plants were treated with 30 μM or 150 μM CuSO(4) or CuEDDS in hydroponic solution. Quantitative Cu distribution maps and concentration profiles across root and leaf cross-sections of the desorbed plants were obtained by micro-proton induced X-ray emission. In roots, the 30 μM treatments with both CuSO(4) and CuEDDS resulted in higher Cu concentrations in epidermal/cortical regions. At 150 μM CuSO(4), Cu was mainly accumulated in root vascular bundles, whereas with 150 μM CuEDDS, Cu was detected in endodermis and the adjacent inner cortical cell layer. Under all treatments, except with a H(+)-ATP-ase inhibitor, the Cu in leaves was localised mainly in vascular tissues. The incubation of plants with 150 μM CuEDDS enhanced metal translocation to shoots, in comparison to the corresponding CuSO(4) treatment. Inhibition of H(+)-ATPase activity resulted in reduced Cu accumulation in 30 μM CuEDDS-treated roots and 150 μM CuEDDS-treated leaves, and induced changes in Cu distribution in the leaves. This indicates that active mechanisms are involved in retaining Cu in the leaf vascular tissues, which prevent its transport to photosynthetically active tissues. The physiological significance of EDDS-assisted Cu uptake is discussed.

Bulk element concentrations of whole grain and element spatial distributions at the tissue level were investigated in wheat ( Triticum aestivum ) grain grown in Zn-enriched soil. Inductively coupled plasma mass spectrometry and inductively coupled plasma optical emission spectrometry were used for bulk analysis, whereas micro-proton-induced X-ray emission was used to resolve the two-dimensional localization of the elements. Soil Zn application did not significantly affect the grain yield, but did significantly increase the grain Ca, Fe and Zn concentrations, and decrease the grain Na, P and Mo concentrations; bulk Mg, S, K, Mn, Cu, Cd and Pb concentrations remained unchanged. These changes observed in bulk element concentrations are the reflection of tissue-specific variations within the grain, revealing that Zn application to soil can lead to considerable alterations in the element distributions within the grain, which might ultimately influence the quality of the milling fractions. Spatially resolved investigations into the partitioning of the element concentrations identified the tissues with the highest element concentrations, which is of utmost importance for accurate prediction of element losses during the grain milling and polishing processes.

Typha latifolia is a plant species widely used for phytoremediation. Accumulation, localization and distribution of Pb and mineral nutrients were investigated in roots, rhizomes and leaves of Typha latifolia grown at 0, 50, 100 and 250 mu M Pb concentrations in a pot experiment under controlled conditions. Bulk elemental concentrations were determined by X-ray fluorescence (XRF) spectroscopy whereas micro-proton-induced X-ray emission (micro-PIXE) was used for element localization in root and rhizome tissues. Gradual increase in bulk Pb concentrations was observed in Typha latifolia roots and rhizomes treated with increasing Pb concentrations, however in rhizomes Pb concentrations were an order of magnitude lower than in roots. In leaves Pb concentrations were around the limit of detection for XRF (similar to 20 mu g g(-1)). An increase in concentration of K and Ca in roots, rhizomes and leaves, of iron and zinc in roots and leaves, and of Mn in rhizomes was observed either at 50 and/or 100 mu M Pb treatments, whereas for K and Ca in roots, rhizomes and leaves, Fe and Zn in roots and leaves and Mn in rhizomes, or at 250 mu M Pb treatment the increase was seen for concentrations of Fe and Zn in rhizomes and Cu in roots. Mn concentrations decreased with Pb treatments in roots and leaves. Element localization using micro-PIXE analysis demonstrated Pb accumulation in epidermal and cortical tissues of treated roots and rhizomes, while in endodermis and vascular tissues Pb was not detected. A displacement of Ca from epidermal to cortical tissues was observed in Pb treated roots and rhizomes, pointing to cell wall immobilization of Pb as one of the tolerance mechanisms in Typha latifolia. High level of colocalization of Pb with P (r = 0.60), S (r = 0.37) and Zn (r = 0.70) was observed in Pb treated roots, while in rhizomes colocalization with the mentioned elements was still positive, but not that prominent. These results indicate that Pb may form complexes with phosphorus and sulfur compounds in roots and rhizomes, which may also represent attraction sites for binding Zn. Because of its large root and rhizome surface area acting as main sites for Pb adsorption, Typha latifolia may represent potentially efficient plant species for phytoremediation of Pb contaminated soils and waters.

A method of sample surface topography reconstruction is described based on the asymmetry in the X-ray yields acquired by a pair of X-ray detectors positioned at opposite sides of the probing beam. The yield asymmetries in the two simultaneously acquired elemental maps are caused by differences in the X-ray absorption along the X-ray exit route in the sample with a topographically structured surface. The approach presented introduces an asymmetry factor of the X-ray intensity in each of the image pixels to obtain an image asymmetry matrix. The dependence of the asymmetry factor on the local target inclination in the detector plane is calculated for a flat sample surface model. The image asymmetry matrix is then converted into the local inclination angle matrix. As the last step, the surface topography is then reconstructed from the local inclination angle. The method is based on X-ray absorption phenomena and could be applied equivalently in X-ray elemental mapping methods using excitation beams with a well-defined direction in the sample. This includes micro-X-Ray Fluorescence analysis (micro-XRF) and micro-Proton Induced X-ray Emission (micro-PIXE). We demonstrated the method by topographic analysis of engraved metallic samples with stereo-PIXE and compared it with the results of stylus profilometry. The smallest value of the X-ray production depth and the characteristic X-ray attenuation length determines the lateral resolution of the proposed topography reconstruction method.

In a typical micro-PIXE set-up the insertion of an X-ray optic such as a polycapillary half lens, in front of the X-ray detector in combination with the focused charged particle micro-beam creates a confocal geometry and thus a probing-volume. By moving the sample across the probing-volume, spatially resolved characteristic X-ray intensities are detected. The aim of this work is to present a theoretical model that describes the micro-PIXE intensities in confocal geometry in the case of either layered-structured materials or materials composed of major–minor elements exhibiting concentration gradients within depth. The influence of various experimental parameters such as the beam energy, spatial resolution of the X-ray lens, micro-beam dimensions, irradiation and detection angles, as well as the structure (thickness, composition) of the individual layers that compose the analyzed sample, is investigated by means of simulations of the PIXE yield at various positions of the analyzed sample within the probing volume. The advantages and limitations of the micro-PIXE analysis in confocal geometry are presented through examples and discussed, in particular with respect to the differential PIXE analysis.

We have reconstructed three-dimensional (3D) element-specific distributions of aerosol microparticles captured in a thick quartz filter. The characteristic x rays were induced by a proton microprobe and detected by a Si(Li) spectrometer equipped with a polycapillary lens. Combining a fine microbeam scanning in the lateral plane with the sample movement parallel to the beam axis, the aerosol particles were driven across the sensitive microvolume and for Fe, Ca, S, and Si a sequence of two-dimensional lateral x-ray images was recorded. This is the first example of an x-ray element selective 3D imaging of a few micrometer sized objects with a proton beam.

A novel experimental technique, 3D micro-particle-induced X-ray emission (PIXE) is described in the present paper. 3D Micro-PIXE is realized by using an X-ray optic in front of the detector, thus creating a confocal arrangement together with the focused proton micro-beam. This confocal setup defines a probing volume from which information on elemental distribution is obtained. If a sample is moved through the probing volume, depth-resolved measurements become possible. The confocal setup was characterized with respect to its spatial and depth resolution. As an example of application for this new non-destructive analytical technique, an archaeological ceramic fragment was examined. A first approach to simulate the complex experimental results is performed. The potential of 3D micro-PIXE to provide advanced qualitative information on the elemental distribution in the sample is discussed.