Scattered high-energy synchrotron radiation at the KARA visible-light diagnostic beamline.

To characterize an electron beam, visible synchrotron light is often used and dedicated beamlines at synchrotron sources are becoming a more common feature as instruments and methods for the diagnostics are, along with the accelerators, further developed. At KARA (Karlsruhe Research Accelerator), such a beamline exists and is based on a typical infrared/visible-light configuration. From experience at such beamlines no significant radiation was expected (dose rates larger than 0.5 µSv h-1). This was found not to be the case and a higher dose was measured which fortunately could be shielded to an acceptable level with 0.3 mm of aluminium foil or 2.0 mm of Pyrex glass. The presence of this radiation led to further investigation by both experiment and calculation. A custom setup using a silicon drift detector for energy-dispersive spectroscopy (Ketek GmbH) and attenuation experiments showed the radiation to be predominantly copper K-shell fluorescence and is confirmed by calculation. The measurement of secondary radiation from scattering of synchrotron and other radiation, and its calculation, is important for radiation protection, and, although a lot of experience exists and methods for radiation protection are well established, changes in machine, beamlines and experiments mean a constant appraisal is needed.

Altered Elemental Distribution in Male Rat Brain Tissue as a Predictor of Glioblastoma Multiforme Growth-Studies Using SR-XRF Microscopy.

Glioblastoma multiforme (GBM) is a particularly malignant primary brain tumor. Despite enormous advances in the surgical treatment of cancer, radio- and chemotherapy, the average survival of patients suffering from this cancer does not usually exceed several months. For obvious ethical reasons, the search and testing of the new drugs and therapies of GBM cannot be carried out on humans, and for this purpose, animal models of the disease are most often used. However, to assess the efficacy and safety of the therapy basing on these models, a deep knowledge of the pathological changes associated with tumor development in the animal brain is necessary. Therefore, as part of our study, the synchrotron radiation-based X-ray fluorescence microscopy was applied for multi-elemental micro-imaging of the rat brain in which glioblastoma develops. Elemental changes occurring in animals after the implantation of two human glioma cell lines as well as the cells taken directly from a patient suffering from GBM were compared. Both the extent and intensity of elemental changes strongly correlated with the regions of glioma growth. The obtained results showed that the observation of elemental anomalies accompanying tumor development within an animal's brain might facilitate our understanding of the pathogenesis and progress of GBM and also determine potential biomarkers of its extension. The tumors appearing in a rat's brain were characterized by an increased accumulation of Fe and Se, whilst the tissue directly surrounding the tumor presented a higher accumulation of Cu. Furthermore, the results of the study allow us to consider Se as a potential elemental marker of GBM progression.

In vivo and in situ synchrotron radiation-based µ-XRF reveals elemental distributions during the early attachment phase of barnacle larvae and juvenile barnacles.

Barnacles are able to establish stable surface contacts and adhere underwater. While the composition of adult barnacle cement has been intensively studied, far less is known about the composition of the cement of the settlement-stage cypris larva. The main challenge in studying the adhesives used by these larvae is the small quantity of material available for analysis, being on the order of nanograms. In this work, we applied, for the first time, synchrotron radiation-based µ-X-ray fluorescence analysis (SR-µ-XRF) for in vivo and in situ analysis of young barnacles and barnacle cyprids. To obtain biologically relevant information relating to the body tissues, adhesives, and shell of the organisms, an in situ sample environment was developed to allow direct microprobe investigation of hydrated specimens without pretreatment of the samples. In 8-day-old juvenile barnacles (Balanus improvisus), the junctions between the six plates forming the shell wall showed elevated concentrations of calcium, potassium, bromine, strontium, and manganese. Confocal measurements allowed elemental characterization of the adhesive interface of recently attached cyprids (Balanus amphitrite), and substantiated the accumulation of bromine both at the point of initial attachment as well as within the cyprid carapace. In situ measurements of the cyprid cement established the presence of bromine, chlorine, iodine, sulfur, copper, iron, zinc, selenium, and nickel for both species. The previously unrecognized presence of bromine, iron, and selenium in the cyprid permanent adhesive will hopefully inspire further biochemical investigations of the function of these substances.

The influence of the ketogenic diet on the elemental and biochemical compositions of the hippocampal formation.

A growing body of evidence demonstrates that dietary therapies, mainly the ketogenic diet, may be highly effective in the reduction of epileptic seizures. All of them share the common characteristic of restricting carbohydrate intake to shift the predominant caloric source of the diet to fat. Catabolism of fats results in the production of ketone bodies which become alternate energy substrates to glucose. Although many mechanisms by which ketone bodies yield its anticonvulsant effect are proposed, the relationships between the brain metabolism of the ketone bodies and their neuroprotective and antiepileptogenic action still remain to be discerned. In the study, X-ray fluorescence microscopy and FTIR microspectroscopy were used to follow ketogenic diet-induced changes in the elemental and biochemical compositions of rat hippocampal formation tissue. The use of synchrotron sources of X-rays and infrared allowed us to examine changes in the accumulation and distribution of selected elements (P, S, K, Ca, Fe, Cu, Zn, and Se) and biomolecules (proteins, lipids, ketone bodies, etc.) with the micrometer spatial resolution. The comparison of rats fed with the ketogenic diet and rats fed with the standard laboratory diet showed changes in the hippocampal accumulation of P, K, Ca, and Zn. The relations obtained for Ca (increased level in CA3, DG, and its internal area) and Zn (decreased areal density in CA3 and DG) were analogous to those that we previously observed for rats in the acute phase of pilocarpine-induced seizures. Biochemical analysis of tissues taken from ketogenic diet-fed rats demonstrated increased intensity of absorption band occurring at 1740 cm(-1), which was probably the result of elevated accumulation of ketone bodies. Moreover, higher absolute and relative (3012 cm(-1)/2924 cm(-1), 3012 cm(-1)/lipid massif, and 3012 cm(-1)/amide I) intensity of the 3012-cm(-1) band resulting from increased unsaturated fatty acids content was found after the treatment with the high-fat diet. This article is part of a Special Issue entitled "Status Epilepticus".

Discovering vanished paints and naturally formed gold nanoparticles on 2800 years old phoenician ivories using SR-FF-microXRF with the color X-ray camera.

Phoenician ivory objects (8(th) century B.C., Syria) from the collections of the Badisches Landesmuseum, Karlsruhe, Germany, have been studied with full field X-ray fluorescence microimaging, using synchrotron radiation (SR-FF-microXRF). The innovative Color X-ray Camera (CXC), a full-field detection device (SLcam), was used at the X-ray fluorescence beamline of the ANKA synchrotron facility (ANKA-FLUO, KIT, Karlsruhe, Germany) to noninvasively study trace metal distributions at the surface of the archeological ivory objects. The outstanding strength of the imaging technique with the CXC is the capability to record the full XRF spectrum with a spatial resolution of 48 µm on a zone of a size of 11.9 × 12.3 mm(2) (264 × 264 pixels). For each analyzed region, 69696 spectra were simultaneously recorded. The principal elements detected are P, Ca, and Sr, coming from the ivory material itself; Cu, characteristic of pigments; Fe and Pb, representing sediments or pigments; Mn, revealing deposited soil minerals; Ti, indicating restoration processes or correlated with Fe sediment traces; and Au, linked to a former gilding. This provides essential information for the assessment of the original appearance of the ivory carvings. The determined elemental maps specific of possible pigments are superimposed on one another to visualize their respective distributions and reconstruct the original polychromy and gilding. Reliable hypotheses for the reconstruction of the original polychromy of the carved ivories are postulated on this basis.

The applicability of Fourier transform infrared microspectroscopy for correction against matrix effects in X-ray fluorescence microimaging of tissues.

X-ray fluorescence (XRF) and Fourier transform infrared (FTIR) microscopy techniques are now considered popular for rapid and label-free complementary spectrochemical analysis of chemical elements and molecular systems in biological specimens. The morphological heterogeneity but also the inhomogeneities associated with the thickness/density of biological samples demonstrate challenges for the quantitative XRF microimaging. Therefore, in the present work, we proposed for the first time the application of the total absorbance under the FTIR spectra as a mass surface correction procedure for two-dimensional (2D) XRF microimaging of tissues. We also evaluated the equivalence of the developed correction method based on total absorbance of FTIR spectra with the proposed approaches based on incoherent scattering of primary X-rays as well as on the membrane Si-Kα transmission signal, on the example of selected rat organ tissues. Thin cryo-sections taken from various organs of Wistar rats were deposited on silicon nitride membranes (Si3N4). The FTIR microscopy studies were performed to collect infrared absorption spectra, used then for the determination of total absorbance values in the selected areas of tissue samples. In turn, hard X-ray imaging based on synchrotron radiation allowed the determination of characteristic radiation intensities of the elements detectable from the tissue, as well as the characteristic radiation of the membrane Si and incoherently scattered X-ray photons (Compton scattering). The latter served as correction factors for the surface mass of the sample alongside the FTIR total absorbance. The qualitative and quantitative analyses showed a high agreement between the results of elemental surface mass correction using total absorbance under FTIR spectra of tissues with those obtained using surface mass correction factors determined directly from XRF spectra. Therefore, the proposed procedure is a good alternative in cases where the surface mass effect of the sample cannot be eliminated based on the information provided directly by the XRF spectrum, as in the case of using polymer films as sample support. We have also proposed a procedure for synchronizing SRXRF and FTIR images, not limited to visual inspection of imaging/mapping data, but also enabling quantitative analysis. We found that the total absorbance determined from FTIR spectra can be successfully used as a correction factor for eliminating the surface mass effect in XRF microimaging of thin freeze-fried tissues and therefore to obtain the surface mass-independent elemental quantities. The proposed approach for 2D-FTIR-XRF analysis can also be a powerful and versatile tool for fostering a correlation and co-localization analysis to search for common distribution patterns between molecular arrangements and chemical elements.

A desktop X-ray monochromator for synchrotron radiation based on refraction in mosaic prism lenses.

Focusing planar refractive mosaic lenses based on triangular prism microstructures have been used as an alternative approach for wide-bandpass monochromatization of high-energy X-rays. The strong energy dependence of the refractive index of the lens material leads to an analogous energy dependence of the focal length of the lens. The refractive mosaic lens, in comparison with the refractive lens of continuous parabolic profile, is characterized by a higher aperture because of reduced passive material. In combination with a well defined pinhole aperture in the focal plane, the transmittance of photons of an appropriate energy can be relatively high and photons of deviating energy can be efficiently suppressed. The photon energy can be tuned by translating the pinhole along the optical axis, and the bandwidth changed by selecting appropriate pinhole aperture and beam stop. This method of monochromatization was realised at the ANKA FLUO beamline using a mosaic lens together with a 20 µm pinhole and beam stop. An energy resolution of 2.0% at 16 keV has been achieved.

In vitro and in ovo impact of the ionic dissolution products of boron-doped bioactive silicate glasses on cell viability, osteogenesis and angiogenesis.

Due to the pivotal role of angiogenesis in bone regeneration, the angiogenic properties of biomaterials are of high importance since they directly correlate with the biomaterials' osteogenic potential via 'angiogenic-osteogenic coupling' mechanisms. The impact of bioactive glasses (BGs) on vascularization can be tailored by incorporation of biologically active ions such as boron (B). Based on the ICIE16-BG composition (in mol%: 49.5 SiO2, 36.3 CaO, 6.6 Na2O, 1.1 P2O5, 6.6 K2O), three B-doped BGs have been developed (compositions in mol%: 46.5/45.5/41.5 SiO2, 36.3 CaO, 6.6 Na2O, 1.1 P2O5, 6.6 K2O, 3/4/8 B2O3). The influence of B-doping on the viability, cellular osteogenic differentiation and expression of osteogenic and angiogenic marker genes of bone marrow-derived mesenchymal stromal cells (BMSCs) was analyzed by cultivating BMSCs in presence of the BGs' ionic dissolution products (IDPs). Furthermore, the influence of the IDPs on angiogenesis was evaluated in ovo using a chorioallantoic membrane (CAM) assay. The influence of B-doped BGs on BMSC viability was dose-dependent, with higher B concentrations showing limited negative effects. B-doping led to a slight stimulation of osteogenesis and angiogenesis in vitro. In contrast to that, B-doping significantly enhanced vascularization in ovo, especially in higher concentrations. Differences between the results of the in vitro and in ovo part of this study might be explained via the different importance of vascularization in both settings. The implementation of new experimental models that cover the 'angiogenic-osteogenic coupling' mechanisms is highly relevant, for instance via extending the application of the CAM assay from solely angiogenic to angiogenic and osteogenic purposes.

Increased strontium uptake in trabecular bone of ovariectomized calcium-deficient rats treated with strontium ranelate or strontium chloride.

Based on clinical trials showing the efficacy to reduce vertebral and non-vertebral fractures, strontium ranelate (SrR) has been approved in several countries for the treatment of postmenopausal osteoporosis. Hence, it is of special clinical interest to elucidate how the Sr uptake is influenced by dietary Ca deficiency as well as by the formula of Sr administration, SrR versus strontium chloride (SrCl(2)). Three-month-old ovariectomized rats were treated for 90 days with doses of 25 mg kg(-1) d(-1) and 150 mg kg(-1) d(-1) of SrR or SrCl(2) at low (0.1% Ca) or normal (1.19% Ca) Ca diet. Vertebral bone tissue was analysed by confocal synchrotron-radiation-induced micro X-ray fluorescence and by backscattered electron imaging. Principal component analysis and k-means clustering of the acquired elemental maps of Ca and Sr revealed that the newly formed bone exhibited the highest Sr fractions and that low Ca diet increased the Sr uptake by a factor of three to four. Furthermore, Sr uptake in bone of the SrCl(2)-treated animals was generally lower compared with SrR. The study clearly shows that inadequate nutritional calcium intake significantly increases uptake of Sr in serum as well as in trabecular bone matrix. This indicates that nutritional calcium intake as well as serum Ca levels are important regulators of any Sr treatment.

Study of mural paintings using in situ XRF, confocal synchrotron-µ-XRF, µ-XRD, optical microscopy, and SEM-EDS--the case of the frescoes from Misericordia Church of Odemira.

In this work, we present the results of an analytical method developed for detailed pigment identification, stratigraphy, and degradation of the paint layers of mural paintings applied in the study of the 17th century frescoes from the Misericordia Church of Odemira (Southwest Portugal). In situ X-ray fluorescence spectrometry analyses were performed on three panels of the mural paintings and complemented by colorimetric measurements. The different color areas were also sampled as microfragments (approx. 1 mm2) that were studied as taken or mounted in epoxy resin to expose the different paint layers. The microfragments of paint layers and their cross sections were characterized by optical microscopy and scanning electron microscopy coupled with energy dispersive X-ray spectrometry. Furthermore, elemental analysis was obtained with spatially resolved confocal synchrotron radiation µ-X-ray fluorescence spectrometry performed at ANKA synchrotron FLUO beamline. Occasionally, phase analysis by µ-X-ray diffraction was also performed. Results from the different techniques allowed pigment identification and, in some cases, the evaluation of color changes due to degradation processes and, considering the Southern Portugal geology, the identification of their possible provenance. The pigments used were essentially yellow, brown and red ochres, smalt blue, copper green, and black earths, probably from local sources.

Human Platelet Lysate Can Replace Fetal Calf Serum as a Protein Source to Promote Expansion and Osteogenic Differentiation of Human Bone-Marrow-Derived Mesenchymal Stromal Cells.

Fetal calf serum (FCS) is frequently used as a growth factor and protein source in bone-marrow-derived mesenchymal stromal cell (BMSC) culture media, although it is a xenogenic product presenting multiple disadvantages including but not limited to ethical concerns. A promising alternative for FCS is human platelet lysate (hPL), which is produced out of human platelet concentrates and happens to be a stable and reliable protein source. In this study, we investigated the influence of hPL in an expansion medium (ESM) and an osteogenic differentiation medium (ODM) on the proliferation and osteogenic differentiation capacity of human BMSC. Therefore, we assessed population doublings during cell expansion, performed alizarin red staining to evaluate the calcium content in the extracellular matrix and determined the activity of alkaline phosphatase (ALP) as osteogenic differentiation correlates. The proliferation rate of BMSC cultured in ESM supplemented with hPL exceeded the proliferation rate of BMSC cultured in the presence of FCS. Furthermore, the calcium content and ALP activity was significantly higher in samples incubated in hPL-supplemented ODM, especially in the early phases of differentiation. Our results show that hPL can replace FCS as a protein supplier in cell culture media and does not negatively affect the osteogenic differentiation capacity of BMSC.

Detection and imaging of gadolinium accumulation in human bone tissue by micro- and submicro-XRF.

Gadolinium-based contrast agents (GBCAs) are frequently used in patients undergoing magnetic resonance imaging. In GBCAs gadolinium (Gd) is present in a bound chelated form. Gadolinium is a rare-earth element, which is normally not present in human body. Though the blood elimination half-life of contrast agents is about 90 minutes, recent studies demonstrated that some tissues retain gadolinium, which might further pose a health threat due to toxic effects of free gadolinium. It is known that the bone tissue can serve as a gadolinium depot, but so far only bulk measurements were performed. Here we present a summary of experiments in which for the first time we mapped gadolinium in bone biopsy from a male patient with idiopathic osteoporosis (without indication of renal impairment), who received MRI 8 months prior to biopsy. In our studies performed by means of synchrotron radiation induced micro- and submicro-X-ray fluorescence spectroscopy (SR-XRF), gadolinium was detected in human cortical bone tissue. The distribution of gadolinium displays a specific accumulation pattern. Correlation of elemental maps obtained at ANKA synchrotron with qBEI images (quantitative backscattered electron imaging) allowed assignment of Gd structures to the histological bone structures. Follow-up beamtimes at ESRF and Diamond Light Source using submicro-SR-XRF allowed resolving thin Gd structures in cortical bone, as well as correlating them with calcium and zinc.

Increased zinc accumulation in mineralized osteosarcoma tissue measured by confocal synchrotron radiation micro X-ray fluorescence analysis.

Abnormal tissue levels of certain trace elements such as zinc (Zn) were reported in various types of cancer. Little is known about the role of Zn in osteosarcoma. Using confocal synchrotron radiation micro X-ray fluorescence analysis, we characterized the spatial distribution of Zn in high-grade sclerosing osteosarcoma of nine patients (four women/five men; seven knee/one humerus/one femur) following chemotherapy and wide surgical resection. Levels were compared with adjacent normal tissue. Quantitative backscattered electron imaging as well as histological examinations was also performed. On average, the ratio of medians of Zn count rates (normalized to calcium) in mineralized tumor tissue was about six times higher than in normal tissue. There was no difference in Zn levels between tumor fraction areas with a low fraction and a high fraction of mineralized tissue, which were clearly depicted using quantitative backscattered electron imaging. Moreover, we found no correlation between the Zn values and the type of tumor regression according to the Salzer-Kuntschik grading. The underlying mechanism of Zn accumulation remains unclear. Given the emerging data on the role of trace elements in other types of cancer, our novel results warrant further studies on the role of trace elements in bone cancer. Copyright © 2016 The Authors. X-Ray Spectrometry published by John Wiley & Sons Ltd.

Efficient and rapid labeling of transplanted cell populations with superparamagnetic iron oxide nanoparticles using cell surface chemical biotinylation for in vivo monitoring by MRI.

Determination of the dynamics of specific cell populations in vivo is essential for the development of cell-based therapies. For cell tracking by magnetic resonance imaging (MRI), cells need to internalize, or be surface labeled with a MRI contrast agent, such as superparamagnetic iron oxide nanoparticles (SPIOs): SPIOs give rise to signal loss by gradient-echo and T(2)-weighted MRI techniques. In this study, cancer cells were chemically tagged with biotin and then magnetically labeled with anti-biotin SPIOs. No significant detrimental effects on cell viability or death were observed following cell biotinylation. SPIO-labeled cells exhibited signal loss compared to non-SPIO-labeled cells by MRI in vitro. Consistent with the in vitro MRI data, signal attenuation was observed in vivo from SPIO-labeled cells injected into the muscle of the hind legs, or implanted subcutaneously into the flanks of mice, correlating with iron detection by histochemical and X-ray fluorescence (XRF) methods. To further validate this approach, human mesenchymal stem cells (hMSCs) were also employed. Chemical biotinylation and SPIO labeling of hMSCs were confirmed by fluorescence microscopy and flow cytometry. The procedure did not affect proliferation and multipotentiality, or lead to increased cell death. The SPIO-labeled hMSCs were shown to exhibit MRI signal reduction in vitro and was detectable in an in vivo model. In this study, we demonstrate a rapid, robust, and generic methodology that may be a useful and practical adjuvant to existing methods of cell labeling for in vivo monitoring by MRI. Further, we have shown the first application of XRF to provide iron maps to validate MRI data in SPIO-labeled cell tracking studies.

Bone material quality in transiliac bone biopsies of postmenopausal osteoporotic women after 3 years of strontium ranelate treatment.

Strontium ranelate (SrR) is a relatively new treatment for osteoporosis. In this study we investigated its potential impact on human bone material quality in transiliac bone biopsies from postmenopausal osteoporotic women treated 3 years with calcium and vitamin D plus either 2 g SrR per day or placebo. Bone mineralization density distribution (BMDD), strontium (Sr) concentration, collagen cross-link ratio, and indentation modulus were analyzed by quantitative backscattered electron imaging, electron-induced X-ray fluorescence analysis, synchrotron radiation induced micro X-ray fluorescence elemental mapping, Fourier transform infrared imaging, and nanoindentation, respectively. The BMDD of SrR-treated patients was shifted to higher atomic numbers (Z(mean) +1.5%, p < .05 versus placebo). We observed Sr being preferentially incorporated in bone packets formed during SrR treatment up to 6% atom fraction [Sr/(Sr + Ca)] depending on the SrR serum levels of the individuals (correlation r = 0.84, p = .018). Collagen cross-link ratio was preserved in SR-treated bone. The indentation modulus was significantly decreased in younger versus older bone packets for both placebo- (-20.5%, p < .0001) and SrR-treated individuals (-24.3%, p < .001), whereas no differences were found between the treatment groups. In conclusion, our findings indicate that after SrR treatment, Sr is heterogeneously distributed in bone and preferentially present in bone packets formed during treatment. The effect of SrR on BMDD seems to be due mainly to the uptake of Sr and not to changes in bone calcium content. Taken together, these data provide evidence that the investigated bone quality determinants at tissue level were preserved in postmenopausal osteoporotic women after 3-year treatment with 2 g SrR per day plus calcium and vitamin D.

Microanalysis (micro-XRF, micro-XANES, and micro-XRD) of a tertiary sediment using microfocused synchrotron radiation.

Micro-focused synchrotron radiation techniques to investigate actinide elements in geological samples are becoming an increasingly used tool in nuclear waste disposal research. In this article, results using mu-focus techniques are presented from a bore core section of a U-rich tertiary sediment collected from Ruprechtov, Czech Republic, a natural analog to nuclear waste repository scenarios in deep geological formations. Different methods are applied to obtain various, complementary information. Elemental and element chemical state distributions are obtained from micro-XRF measurements, oxidation states of As determined from micro-XANES, and the crystalline structure of selected regions are studied by means of micro-XRD. We find that preparation of the thin section created an As oxidation state artifact; it apparently changed the As valence in some regions of the sample. Results support our previously proposed hypothesis of the mechanism for U-enrichment in the sediment. AsFeS coating on framboid Fe nodules in the sediment reduced mobile groundwater-dissolved U(VI) to less-soluble U(IV), thereby immobilizing the uranium in the sediment.