Thickness determination of the tidemark of human articular cartilage using high-resolution micro-XRF imaging of zinc and lead.

Objective: The objective of the study was to specify the thickness of Zn and Pb accumulation within the tidemark (TM), a narrow structure between the non-calcified and the calcified articular cartilage. It is considered an active or resting calcification front. This banded structure of the cartilage-bone interface is known to undergo changes in osteoarthritis. Therefore, gaining knowledge about this structure is of interest. Methods: Femoral head samples were collected from patients suffering from various bone diseases, 6 samples have been investigated. Thin bone slices (3 â€‹µm thick) were measured with high resolution synchrotron micro-X-ray fluorescence (SR micro-XRF) analysis using a beam with dimensions of 500 â€‹× â€‹800 â€‹nm2. The tidemark region was found in all analyzed samples. The Savitzky-Golay filter was used to smooth the measured imaging data and Kaplan-Meier estimation to gain reliable tidemarks medians for Pb and Zn. To our knowledge this was the first time that these methods have been applied to gain information on histological structures obtained by elemental imaging. Results: The thickness of the Zn and Pb layer ranged from about 3 to 11 â€‹µm for Zn and 4-14.5 â€‹µm for Pb. Our Zn ratios (TM/matrix) were found to be 1.5-3-fold ratio between Zn tidemark values and in mineralized matrix and are similar in all samples. Conclusions: The determined thickness of the layer is much smaller than found in previous measurements with the beam having 20 â€‹× â€‹14 â€‹µm2 size. The Zn ratios agree with our previous findings.

A monochromatic confocal micro-x-ray fluorescence (µXRF) spectrometer for the lab.

Confocal micro-x-ray fluorescence (µXRF) is a powerful tool to analyze the spatial distribution of major, minor, and trace elements in three dimensions. Typical (confocal) µXRF measurements in the lab use polychromatic excitation, complicating quantification and fundamental parameter-based corrections and furthermore deteriorating peak-to-background ratios due to scattered bremsstrahlung. The goal for the new setup was to remedy these problems, without sacrificing spatial resolution, and keep it flexible for different excitation energies and transportation to other sources. The source assembly consists of a water-cooled fine-focus x-ray diffraction tube and a parallel beam-mirror, which produces a quasi-parallel, monochromatic beam. The presented results were obtained using a 2 kW molybdenum tube and a mirror for Mo-Kα. The confocal setup itself consists of two polycapillary half-lenses, one for the source side and the other for the detector side, where a 50 mm2 silicon drift detector is mounted. Both polycapillaries have a focus size of ∼15 µm for Mo-Kα. The second polycapillary can also be exchanged for a custom-designed collimator in order to perform non-confocal µXRF. Details of the technical setup and results from technical and biological samples are presented. Detection limits for selected elements from Ca to Pb in the confocal and non-confocal mode were established (e.g., 1 µg/g non-confocal and 20 µg/g confocal for As) using the NIST standard reference materials (SRMs) 621 and 1412. Furthermore, the results of the measurements of SRM 621 were evaluated using the fundamental parameter based quantification software ATI-QUANT. The results are compared with the certified values and generally are in good agreement.

Elemental imaging of trace elements in bone samples using micro and nano-X-ray fluorescence spectrometry.

Our group employs micro- and nano-X-ray fluorescence spectrometry (XRF) for the investigation of bone tissue. The manuscript presents the results of 3 various projects, in which spatial distribution of trace elements in bone samples was studied. The first study investigated the distribution of the elemental constituents of Mg-based implants at various stages of the degradation process in surrounding bone tissue with a focus on magnesium (Mg) and yttrium (Y). The analysis was performed in laboratory of Atominstitut using a special micro-XRF spectrometer for light element detection. The second study is devoted to the spatial distribution of Zn in high-grade sclerosing osteosarcoma mapped by confocal synchrotron radiation induced micro-XRF. Tumor zinc levels were compared with adjacent normal tissue. For discrimination between healthy and diseased bone quantitative backscattered electron imaging was used. The third experiment demonstrates the performance of synchrotron radiation induced nano-XRF with beam size of about 500 nm for bone tissue investigation. Special emphasis is set to advantages of micro- and nano-XRF in bone analysis as well as overcoming possible limitations.

Dual energy-band excitation from a low power Rh anode X-ray tube for the simultaneous determination of low Z and high Z elements (Na-U) using total-reflection X-ray fluorescence analysis (TXRF).

This article presents results from an experimental setup for a dual energy-band vacuum spectrometer for total-reflection X-ray fluorescence analysis allowing simultaneous efficient excitation of low, medium, and high Z elements. The spectrometer is equipped with an air-cooled 35 W low power Rh X-ray tube and a 17 mm2 silicon drift detector with a thin 8 µm beryllium window. A Pd/B4C multilayer monochromator is used at the same time as a Bragg reflector for Rh-Kα radiation and as a high-energy cut-off reflector above 5 keV, where the characteristic Rh-L radiation is totally reflected and present in the spectrum of the exciting radiation. This leaves one broad low energy band below 5 keV and one high energy band around the energy of Rh-Kα. As Rh-L radiation would be absorbed on its path through air, a new beam entrance system was designed in order to guide the Rh-L photons into the vacuum chamber for efficient excitation of low Z elements. With this setup, elements down to sodium (Z = 11, E = 1.04 keV) could be detected. First results are presented, and spectra obtained in air as well as in vacuum are compared and discussed. Detection limits in the range of 1000 µg/kg for Na and around 140 µg/kg for Mg were achieved using the NIST SRM 1640 (trace elements in water).

Characterization of a submicro-X-ray fluorescence setup on the B16 beamline at Diamond Light Source.

An X-ray fluorescence setup has been tested on the B16 beamline at the Diamond Light Source synchrotron with two different excitation energies (12.7 and 17 keV). This setup allows the scanning of thin samples (thicknesses up to several micrometers) with a sub-micrometer resolution (beam size of 500 nm × 600 nm determined with a 50 µm Au wire). Sensitivities and detection limits reaching values of 249 counts s-1 fg-1 and 4 ag in 1000 s, respectively (for As Kα excited with 17 keV), are presented in order to demonstrate the capabilities of this setup. Sample measurements of a human bone and a single cell performed at B16 are presented in order to illustrate the suitability of the setup in biological applications.

Synchrotron radiation micro X-ray fluorescence spectroscopy of thin structures in bone samples: comparison of confocal and color X-ray camera setups.

In the quest for finding the ideal synchrotron-radiation-induced imaging method for the investigation of trace element distributions in human bone samples, experiments were performed using both a scanning confocal synchrotron radiation micro X-ray fluorescence (SR-µXRF) (FLUO beamline at ANKA) setup and a full-field color X-ray camera (BAMline at BESSY-II) setup. As zinc is a trace element of special interest in bone, the setups were optimized for its detection. The setups were compared with respect to count rate, required measurement time and spatial resolution. It was demonstrated that the ideal method depends on the element of interest. Although for Ca (a major constituent of the bone with a low energy of 3.69 keV for its Kα XRF line) the color X-ray camera provided a higher resolution in the plane, for Zn (a trace element in bone) only the confocal SR-µXRF setup was able to sufficiently image the distribution.

A setup for synchrotron-radiation-induced total reflection X-ray fluorescence and X-ray absorption near-edge structure recently commissioned at BESSY II BAMline.

An automatic sample changer chamber for total reflection X-ray fluorescence (TXRF) and X-ray absorption near-edge structure (XANES) analysis in TXRF geometry was successfully set up at the BAMline at BESSY II. TXRF and TXRF-XANES are valuable tools for elemental determination and speciation, especially where sample amounts are limited (<1 mg) and concentrations are low (ng ml(-1) to µg ml(-1)). TXRF requires a well defined geometry regarding the reflecting surface of a sample carrier and the synchrotron beam. The newly installed chamber allows for reliable sample positioning, remote sample changing and evacuation of the fluorescence beam path. The chamber was successfully used showing accurate determination of elemental amounts in the certified reference material NIST water 1640. Low limits of detection of less than 100 fg absolute (10 pg ml(-1)) for Ni were found. TXRF-XANES on different Re species was applied. An unknown species of Re was found to be Re in the +7 oxidation state.

A novel vacuum spectrometer for total reflection x-ray fluorescence analysis with two exchangeable low power x-ray sources for the analysis of low, medium, and high Z elements in sequence.

The extension of the detectable elemental range with Total Reflection X-ray Fluorescence (TXRF) analysis is a challenging task. In this paper, it is demonstrated how a TXRF spectrometer is modified to analyze elements from carbon to uranium. Based on the existing design of a vacuum TXRF spectrometer with a 12 specimen sample changer, the following components were renewed: the silicon drift detector with 20 mm(2) active area and having a special ultra-thin polymer window allowing the detection of elements from carbon upwards. Two exchangeable X-ray sources guarantee the efficient excitation of both low and high Z elements. These X-ray sources were two light-weighted easily mountable 35 W air-cooled low-power tubes with Cr and Rh anodes, respectively. The air cooled tubes and the Peltier-cooled detector allowed to construct a transportable tabletop spectrometer with compact dimensions, as neither liquid nitrogen cooling for the detector nor a water cooling circuit and a bulky high voltage generator for the X-ray tubes are required. Due to the excellent background conditions as a result of the TXRF geometry, detection limits of 150 ng for C, 12 ng for F, and 3.3 ng for Na have been obtained using Cr excitation in vacuum. For Rh excitation, the detection limits of 90 pg could be achieved for Sr. Taking 10 to 20 µl of sample volume, extrapolated detection limits in the ng/g (ppb) range are resulting in terms of concentration.

Combined evaluation of grazing incidence X-ray fluorescence and X-ray reflectivity data for improved profiling of ultra-shallow depth distributions.

The continuous downscaling of the process size for semiconductor devices pushes the junction depths and consequentially the implantation depths to the top few nanometers of the Si substrate. This motivates the need for sensitive methods capable of analyzing dopant distribution, total dose and possible impurities. X-ray techniques utilizing the external reflection of X-rays are very surface sensitive, hence providing a non-destructive tool for process analysis and control. X-ray reflectometry (XRR) is an established technique for the characterization of single- and multi-layered thin film structures with layer thicknesses in the nanometer range. XRR spectra are acquired by varying the incident angle in the grazing incidence regime while measuring the specular reflected X-ray beam. The shape of the resulting angle-dependent curve is correlated to changes of the electron density in the sample, but does not provide direct information on the presence or distribution of chemical elements in the sample. Grazing Incidence XRF (GIXRF) measures the X-ray fluorescence induced by an X-ray beam incident under grazing angles. The resulting angle dependent intensity curves are correlated to the depth distribution and mass density of the elements in the sample. GIXRF provides information on contaminations, total implanted dose and to some extent on the depth of the dopant distribution, but is ambiguous with regard to the exact distribution function. Both techniques use similar measurement procedures and data evaluation strategies, i.e. optimization of a sample model by fitting measured and calculated angle curves. Moreover, the applied sample models can be derived from the same physical properties, like atomic scattering/form factors and elemental concentrations; a simultaneous analysis is therefore a straightforward approach. This combined analysis in turn reduces the uncertainties of the individual techniques, allowing a determination of dose and depth profile of the implanted elements with drastically increased confidence level. Silicon wafers implanted with Arsenic at different implantation energies were measured by XRR and GIXRF using a combined, simultaneous measurement and data evaluation procedure. The data were processed using a self-developed software package (JGIXA), designed for simultaneous fitting of GIXRF and XRR data. The results were compared with depth profiles obtained by Secondary Ion Mass Spectrometry (SIMS).

Combination of grazing incidence x-ray fluorescence with x-ray reflectivity in one table-top spectrometer for improved characterization of thin layer and implants on/in silicon wafers.

As Grazing Incidence X-ray Fluorescence (GIXRF) analysis does not provide unambiguous results for the characterization of nanometre layers as well as nanometre depth profiles of implants in silicon wafers by its own, the approach of providing additional information using the signal from X-ray Reflectivity (XRR) was tested. As GIXRF already uses an X-ray beam impinging under grazing incidence and the variation of the angle of incidence, a GIXRF spectrometer was adapted with an XRR unit to obtain data from the angle dependent fluorescence radiation as well as data from the reflected beam. A θ-2θ goniometer was simulated by combining a translation and tilt movement of a Silicon Drift detector, which allows detecting the reflected beam over 5 orders of magnitude. HfO2 layers as well as As implants in Silicon wafers in the nanometre range were characterized using this new setup. A just recently published combined evaluation approach was used for data evaluation.

Comparison of two confocal micro-XRF spectrometers with different design aspects.

Two different confocal micro X-ray fluorescence spectrometers have been developed and installed at Osaka City University and the Vienna University of Technology Atominstitut. The Osaka City University system is a high resolution spectrometer operating in air. The Vienna University of Technology Atominstitut spectrometer has a lower spatial resolution but is optimized for light element detection and operates under vacuum condition. The performance of both spectrometers was compared. In order to characterize the spatial resolution, a set of nine specially prepared single element thin film reference samples (500 nm in thickness, Al, Ti, Cr, Fe Ni, Cu, Zr, Mo, and Au) was used. Lower limits of detection were determined using the National Institute of Standards and Technology standard reference material glass standard 1412. A paint layer sample (cultural heritage application) and paint on automotive steel samples were analyzed with both instruments. The depth profile information was acquired by scanning the sample perpendicular to the surface. © 2013 The Authors. X-Ray Spectrometry published by John Wiley & Sons, Ltd.

Spatial distribution of the trace elements zinc, strontium and lead in human bone tissue.

Trace elements are chemical elements in minute quantities, which are known to accumulate in the bone. Cortical and trabecular bones consist of bone structural units (BSUs) such as osteons and bone packets of different mineral content and are separated by cement lines. Previous studies investigating trace elements in bone lacked resolution and therefore very little is known about the local concentration of zinc (Zn), strontium (Sr) and lead (Pb) in BSUs of human bone. We used synchrotron radiation induced micro X-ray fluorescence analysis (SR µ-XRF) in combination with quantitative backscattered electron imaging (qBEI) to determine the distribution and accumulation of Zn, Sr, and Pb in human bone tissue. Fourteen human bone samples (10 femoral necks and 4 femoral heads) from individuals with osteoporotic femoral neck fractures as well as from healthy individuals were analyzed. Fluorescence intensity maps were matched with BE images and correlated with calcium (Ca) content. We found that Zn and Pb had significantly increased levels in the cement lines of all samples compared to the surrounding mineralized bone matrix. Pb and Sr levels were found to be correlated with the degree of mineralization. Interestingly, Zn intensities had no correlation with Ca levels. We have shown for the first time that there is a differential accumulation of the trace elements Zn, Pb and Sr in BSUs of human bone indicating different mechanisms of accumulation.

Differential accumulation of lead and zinc in double-tidemarks of articular cartilage.

INTRODUCTION: Long-term exposure to increased lead (Pb) concentrations is associated with several chronic diseases. The divalent cation zinc (Zn) is essential for numerous enzymes. In a recent study we found remarkably elevated concentrations of Pb and Zn in the tidemark (TM), which is the mineralization front of human articular cartilage. OBJECTIVE: Duplication or multiplication of TMs occurs with advancing age or degeneration. We hypothesized that trace elements accumulate in TMs as a function of time. Thus, in cases of double TMs, the deep (older) TM should contain higher Pb and Zn concentrations than the superficial (younger) TM. DESIGN: Undecalcified tissue from articular cartilage and subchondral bone of femoral heads and patellae was examined by synchrotron radiation induced confocal micro X-ray fluorescence analysis and by quantitative backscattered electron imaging to determine the local distribution of Ca, Zn, and Pb in this tissue. RESULTS: The evaluation of X-ray fluorescence intensities in double TMs revealed in average a 2.6-fold higher Pb level in the deep TM compared to the superficial TM while Zn concentrations were similar. Pb and Zn contents were significantly enhanced in the deep TM (Pb: 35-fold, Zn: five-fold) and in the superficial TM (Pb: 12-fold, Zn: five-fold) compared to the bone level. CONCLUSION: For the first time a differential accumulation of Pb and Zn is documented in regions with double TMs revealing various timescales for the accumulation of these elements. Increased amounts of Pb are present in the TMs (up to the 62-fold of the bone level) featuring a potential source of internal Pb release if the TM region is destroyed.

Confocal micro-x-ray fluorescence spectrometer for light element analysis.

An existing micro-x-ray fluorescence (micro-XRF) spectrometer designed for light element analysis (6 ≤ Z ≤ 14) has been extended to confocal geometry: a second polycapillary x-ray optic has been introduced in front of the energy dispersive x-ray detector. New piezo positioners for optimum alignment of both optics have been installed inside the vacuum chamber. The spectrometer offers now the possibility of true 3D elemental analysis in the micrometer regime. Depth resolution varies between 100 µm at 1 keV fluorescence energy (Na-Kα) and 30 µm for 17.5 keV (Mo). To further extend analytical capabilities a second x-ray tube with a Rh anode has been acquired to supplement to existing Mo anode tube. Lower limits of detection have been determined to be in the ppm region for confocal geometry. The spectrometer has been characterized and tested using different samples. Furthermore, results have been compared with SR micro-XRF to show the capabilities and limitations of this spectrometer.

Investigation of element distribution and homogeneity of TXRF samples using SR-micro-XRF to validate the use of an internal standard and improve external standard quantification.

Total reflection X-ray fluorescence analysis (TXRF) offers a nondestructive qualitative and quantitative analysis of trace elements. Due to its outstanding properties TXRF is widely used in the semiconductor industry for the analysis of silicon wafer surfaces and in the chemical analysis of liquid samples. Two problems occur in quantification: the large statistical uncertainty in wafer surface analysis and the validity of using an internal standard in chemical analysis. In general TXRF is known to allow for linear calibration. For small sample amounts (low nanogram (ng) region) the thin film approximation is valid neglecting absorption effects of the exciting and the detected radiation. For higher total amounts of samples deviations from the linear relation between fluorescence intensity and sample amount can be observed. This could be caused by the sample itself because inhomogeneities and different sample shapes can lead to differences of the emitted fluorescence intensities and high statistical errors. The aim of the study was to investigate the elemental distribution inside a sample. Single and multi-element samples were investigated with Synchrotron-radiation-induced micro X-ray Fluorescence Analysis (SR-µ-XRF) and with an optical microscope. It could be proven that the microscope images are all based on the investigated elements. This allows the determination of the sample shape and potential inhomogeneities using only light microscope images. For the multi-element samples, it was furthermore shown that the elemental distribution inside the samples is homogeneous. This justifies internal standard quantification.

Analytical possibilities of total reflection X-ray spectrometry (TXRF) for trace selenium determination in soils.

Selenium content of soils is an important issue due to the narrow range between the nutritious requirement and toxic effects upon Se exposure. However, its determination is challenging due to low concentrations within complex matrices that hamper the analysis in most spectroscopic techniques. In this study, we explored the possibilities of several analytical approaches combined with total reflection X-ray (TXRF) spectrometry for soil Se determinations. The direct analysis of a solid suspension using 20 mg of fine ground material (<50 µm) has a relatively high Se limit of detection (LOD) of 1 mg/kg (worldwide Se average in soils = 0.4 mg/kg) and is therefore only suitable for seleniferous soils. Several fast and simple analytical strategies were developed to decrease matrix effects and improve the LOD for Se determination in soil digests. On one hand, the application of a liquid-liquid extraction procedure using ethyl ether and the introduction of a Cr absorbent in the instrument configuration were carried out to avoid the associated problems on TXRF analysis of soil extracts due to the high Fe concentrations (∼700 mg/L). On the other hand, a dispersive liquid-liquid microextraction procedure (DLLME) before the TXRF analysis of the soil digest was also developed. The effects of various experimental parameters such as sample volume, effect of major elements present in the soil matrix (Fe), and Se concentration in the sample were investigated. The LOD using this analytical methodology (0.05 mg/kg of Se) was comparable to or lower than those obtained in previous works using other popular spectrometric techniques such as GFAAS, ICPMS, and AFS. The calculated Se concentration for JSAC-0411 ([Se] = 1.32 ± 0.27 mg/kg) using the combination of DLLME and TXRF ([Se] = 1.40 ± 0.23 mg/kg) was in agreement with the certified value.

Analytical approaches for Hg determination in wastewater samples by means of total reflection X-ray fluorescence spectrometry.

At present, there is a considerable interest in Hg monitoring in wastewater samples due to its widespread occurrence and the high toxicity of most of its compounds. Hg determination in water samples by means of total reflection X-ray fluorescence spectrometry (TXRF) entails some difficulties due to the high vapor pressure and low boiling point of this element that produce evaporation and loss of Hg from the surface of the reflector during the drying process, commonly used for sample preparation in TXRF analysis. The main goal of the present research was to develop a fast and simple chemical strategy to avoid Hg volatilization during the analysis of wastewater samples by TXRF spectrometry. Three different analytical procedures were tested for this purpose: (i) increasing the viscosity of the wastewater sample by adding a non-ionic surfactant (Triton X-114), (ii) Hg immobilization on the quartz reflectors using the extractant tri-isobutylphosphine (Cyanex 471X) and (iii) formation of a stable and non-volatile Hg complex into the wastewater sample. The best analytical strategy was found to be the formation of a Hg complex with thiourea (pH=10) before the deposition of 10 microL of sample on the reflector for following TXRF analysis. Analytical figures of merit such as linearity, limits of detection, accuracy and precision were carefully evaluated. Finally, the developed methodology was applied for the determination of Hg in different types of wastewater samples (industrial effluents, municipal effluents from conventional systems and municipal effluents from constructed wetlands).

Lead accumulation in tidemark of articular cartilage.

OBJECTIVE: Determination of the spatial distribution of the toxic element lead (Pb) and other trace elements in normal articular cartilage and subchondral bone from adult humans with no history of work-related exposure to Pb. METHODS: Four macroscopically normal femoral heads and three patellas were harvested from randomly selected forensic autopsies. All subjects died of acute illnesses, had no history of work-related exposure to Pb and had no metabolic bone disease. The elemental distribution of lead (Pb) together with zinc (Zn), strontium (Sr) and calcium (Ca) in the chondral and subchondral region was detected using high resolution synchrotron radiation induced micro X-ray fluorescence (SR mu-XRF) analysis. SR mu-XRF line scans in conventional and SR mu-XRF area scans in confocal geometry were correlated to backscattered electron (BE) images visualizing the mineralized tissue. RESULTS: In all samples, we found a highly specific accumulation of Pb in the tidemark, the transition zone between calcified and non-calcified articular cartilage. Pb fluorescence intensities in the tidemark, which is thought to be a metabolically active mineralization front, were 13-fold higher when compared to subchondral bone. Pb intensities in the subchondral region were strongly correlated with Zn, but were distinctly different from Ca and Sr. CONCLUSIONS: The finding of the highly specific accumulation of lead in the tidemark of human articular cartilage is novel. However at this point, the exact mechanisms of the local Pb accumulation as well as its clinical implications are unknown.

Laser technology: source of coherent kiloelectronvolt X-rays.

Generating X-rays that have the properties of laser light has been a long-standing goal for experimental science. Here we describe the emission of highly collimated, spatially coherent X-rays, at a wavelength of about 1 nanometre and at photon energies extending to 1.3 kiloelectronvolts, from atoms that have been ionized by a 5-femtosecond laser pulse. This means that a laboratory source of laser-like, kiloelectronvolt X-rays, which will operate on timescales relevant to many chemical, biological and materials problems, is now within reach.

Femtosecond X-Ray fluorescence

Using few-cycle-driven coherent laser harmonics, K-shell vacancies have been created in light elements, such as boron (E(B) = 188 eV) and carbon (E(B) = 284 eV), on a time scale of a few femtoseconds for the first time. The capability of detecting x-ray fluorescence excited by few-femtosecond radiation with an accuracy of the order of 1 eV paves the way for probing the evolution of the microscopic environment of selected atoms in chemical and biochemical reactions on previously inaccessible time scales (<100 fs) by tracing the temporal evolution of the "chemical shift" of peaks associated with inner-shell electronic transitions in time-resolved x-ray fluorescence and photoelectron spectra.