A SEM (DSM 962, Zeiss, Oberkochen, Germany) was employed to acquire qBEI images using 20 keV electrons leading to an information depth of about 1.5 μm [35]. Images at different magnifications 12-fold for overviews and 200-fold (pixel resolution of about 1 × 1 μm2) were obtained to select and define the region of interest (ROI) in bone for SR-μ-XRF analysis similar to a study done previously [32]. Especially areas (bone Alectinib supplier packets, osteons) containing mineralized bone matrix with different degrees of mineralization have been selected. The properties of synchrotron radiation (SR) including

high photon flux, natural collimation, polarization and the possibility to select the energy of the primary photons enabled sensitivities up to the femtogram range and a high spatial resolution in the micrometer range. In previous studies, the combination of a confocal geometry and SR allowed the analysis of trace elements in bone and articular cartilage at the micrometer range with high-sensitivity and high spatial distribution [11], [36] and [37]. Further details on confocal SR-μ-XRF can be found elsewhere [38], [39], [40], [41] and [42]. The present measurements have been carried out at the FLUO beamline of the ANKA

synchrotron facility at the Karlsruhe Institute of Technology Campus North [40] and [41] applying the same confocal setup as already described previously [32]. The actual excitation energy was 17 keV and the beam size was 17 μm × 12 μm (horizontal × vertical) Everolimus datasheet with a depth resolution of 19 μm at 9.71 keV (Au-Lα). Area scans in the sample surface were performed in the range of 500 μm × 500 μm up to 500 μm × 650 μm with a step size of 15 μm horizontal and 10 μm vertical. Acquisition times longer than 12 s per pixel were found not to show

any improvements in the signal to noise ratio of the obtained elemental maps. Especially, the low levels of Pb content required this relatively long acquisition time. The acquired spectra, an example of which is shown in Fig. 1, were processed according to the protocol described in [32]. The information about bone tissue structure and mineral content as obtained by qBEI was combined and correlated with the X-ray intensities of the corresponding Gefitinib elemental maps. The 2D data evaluation software ImageJ (v1.44, National Institutes of Health, USA) [43] and custom made routines were applied to pre-process the obtained data prior to statistical evaluation with GraphPad Prism (v4.0c, GraphPad Software, Inc., USA). First the qBEI images of high spatial resolution (1 μm per pixel) have been aligned with the corresponding SR μ-XRF maps. Secondly, the ROIs representing mineralized bone matrix and cement lines were indicated in the qBEI images. ROIs of mineralized bone matrix were marked within single structural units (osteon, bone packet) taking care that at least a distance of a few microns (5 to 10 μm) to cracks, cement lines, osteocyte lacunae, haversian canals or trabecular surface was kept.