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.

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.

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.

[The monitoring of laboratory indices of immune status of healthy children residing in conditions of intensive immunotropic zone of industrial town.]

The complex evaluation was implemented concerning to quantitative and functional indices of immune-competent cells of peripheral blood, cytokine and immunoglobulin profile of blood serum with detection of confidence ranges of immunologic parameters of healthy children aged 3-11 years (n=98) residing in intensive immunotropic zone with the highest ecological risk. The multi-parametric flow cytofluorimeter COULTER EPICS XL (Beckman Coulter Inc.) with selection of of panels of monoclonal antibodies with multi-color combination of fluorichromуes was applied to analyze leukocytes, sub-population composition of lymphocites and processes of activation of cells of peripheral blood. The levels of cytokines were detected using immunoenzyme technique applying reagents by «R&D Diagnostics Inc.¼ (USA). The reagents' kit of «Vektor-Best¼ (Novosibirsk) was applied to detect spontaneous and mitogen-induced production of cytokines by cells of total blood The immuneturbidimetry technique was applied to detect content of immunoglobulins in blood serum. The total and specific IgE was detected using solid-phase immune enzyme analysis with reagents' kits by «Alkor Bio Company¼ (St. Petersburg). The statistical data processing was implemented using software Statistica 10 with data verification 95%-99%. In junior school children as compared with children aged 3-6 years, statistically reliable greater number of leukocytes were detected with preservation of high ratio of neutrophils, prevalence of helper sub-population of T-lymphocytes, expressed activation of B-lymphocytes, higher levels of serum IgA, IgG, common and specific IgE (latent sensibilization by domestic allergens), prevalence of intensity of production of interleukins IL-13 over IL-4 and significantly higher provision with natural killers.

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.