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.

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.

Uncoupling of sexual reproduction from homologous recombination in homozygous Oenothera species.

Salient features of the first meiotic division are independent segregation of chromosomes and homologous recombination (HR). In non-sexually reproducing, homozygous species studied to date HR is absent. In this study, we constructed the first linkage maps of homozygous, bivalent-forming Oenothera species and provide evidence that HR was exclusively confined to the chromosome ends of all linkage groups in our population. Co-segregation of complementary DNA-based markers with the major group of AFLP markers indicates that HR has only a minor role in generating genetic diversity of this taxon despite its efficient adaptation capability. Uneven chromosome condensation during meiosis in Oenothera may account for restriction of HR. The use of plants with ancient chromosomal arm arrangement demonstrates that limitation of HR occurred before and independent from species hybridizations and reciprocal translocations of chromosome arms-a phenomenon, which is widespread in the genus. We propose that consecutive loss of HR favored the evolution of reciprocal translocations, beneficial superlinkage groups and ultimately permanent translocation heterozygosity.

Organ culture of human main and accessory lacrimal glands and their secretory behaviour.

The purpose of this study was to investigate the in vitro viability and secretory behaviour of human main and accessory lacrimal glands using an organ culture technique. We evaluated the influence of the second messengers cAMP and cGMP on secretion. Fragments less than 1 mm3 of main and accessory lacrimal glands as well as conjunctiva were cultured for 2-72 hr at 37 degrees C in an atmosphere consisting of 50% O2, 45% N2 and 5% CO2, using a specially devised culture medium (+/- cAMP or cGMP). The conjunctival tissue served as negative control. Supernatants were assayed for secretory-component-bound IgA, lactoferrin and lysozyme using ELISA. Cultured tissue pieces were embedded in paraffin, serially sectioned, stained and their volumes calculated using an image-analysis system. This enabled us to differentiate between secretory, connective and fatty tissue. Secreted exudate was correlated to the volume of secretory tissue. Viability of cultured organ pieces was determined by electron microscopic examination. Suitable organ culture conditions for human lacrimal glands were successfully established. Electron microscopic examinations proved that the structural characteristics of the organ and the polarity of the individual cells were well preserved up to 22 days of culture. Culture supernatants were assayed for secretory-component-bound IgA, lactoferrin, and lysozyme and showed that the amount of protein secreted increased with time. Upon addition of cAMP (1 x 10(-3) M) and cGMP (4 x 10(-3) M), secretion was elevated in both main and accessory lacrimal glands. An organ culture system for lacrimal glands was developed that maintains their structural and cellular characteristics as well as their secretory function for up to 22 days. We believe that this system mimics the in vitro state of the organ better than monolayer cultures and thus proves to be a valuable tool when examining lacrimal function in vitro. The fact that both cAMP and cGMP enhance secretion may help to shed some light on the cellular pathways human main and accessory lacrimal glands use for signal transduction.

Anterior chamber angle biometry: a comparison of Scheimpflug photography and ultrasound biomicroscopy.

Scheimpflug photography and ultrasound biomicroscopy allow measurement of anterior chamber angle width. To analyze the value of both diagnostic tools, measurements of the same subjects were correlated. Anterior chamber angle width in both eyes of 20 volunteers without any ophthalmological case history was measured in eight meridians. Scheimpflug photographs were recorded on Kodak T-max 400 black-and-white film using a Topcon SL-45 camera. Measurement of chamber angle was done with the aid of a self-programmed PC software. Ultrasound biomicroscopic measurements were obtained using a Humphrey UBM 840 system with a 50-MHz transducer. The chamber angle was measured as described by Pavlin et al. The data of both methods were correlated and analysed for statistically significant differences using a paired t test (level of significance p < or = 0.05). Both methods showed similar changes in chamber angle width and were able to document a variation in angle width with regard to location. The correlation averaged at 0.64. In 4 meridians significant differences were found between the two methods. Scheimpflug photography, however, is unable to visualize directly the angle itself. Both methods are limited by the fact that the angle is defined by curvilinear structures, i.e. posterior corneal surface and anterior iris surface. Scheimpflug photography is able to document changes in angle configuration. Ultrasound biomicroscopy, however, is a more precise method, because it allows direct documentation of all structures involved in chamber angle configuration.

Morphological investigations on the small intestinal mucosa of mouse after mild hyperthermic treatment.

Local hyperthermia, when applied as a therapeutic agent against radio-resistant tumours, can result in serious side effects in adjacent non-tumorous tissue, one especially thermo-sensitive organ being the small intestine. An experimental morphological study was therefore undertaken with mice to investigate whether "mild hyperthermia" (41 degrees C for 15, 30 and 60 min) causes alterations at the mucosa of the small intestine, and if so what are these effects and their likely pathomechanisms. Descriptive light and electron microscopical studies and morphometric evaluations are reported on the jejunal mucosa. The observed changes were most extensive after 60 min of 41 degrees C hyperthermia; but even 30 and 15 min hyperthermic treatment was followed by severe degenerative changes. The epithelium of the basal crypts and the stromal cells seemed to remain morphologically unaffected under these conditions, though the stroma can react with a temporary contraction. No "prime event" in the hyperthermia provoked tissue changes can be deducted from the combined light, electron microscopical, and morphometric studies. Comparing, however, the extent of the effects after mild hyperthermia of 60, 30 and 15 min, respectively, four phases of intestinal alterations due to mild hyperthermia and a likely pathomechanism of this treatment can be defined. The clinical implications of the findings are discussed.