3D renderings of ultrastructural bone features.

<p>[<b>a</b>] Rendering of osteocyte lacunae and canaliculi in the whole imaged volume overlayed over the bottom slice shown in grayscale. Colors correspond to connected components and grayscale to mass density. Note the difference in structure in the interstitia and osteon: the connected cells are all in the osteonal tissue, the others in the interstitial. The canaliculi are considerably reduced in the interstitia. [<b>b</b>] Zoom on the highlighted lacuna in A showing the interaction between the canaliculi [pink] and the cement line [green], and branching of the canaliculi.</p

Retrieved information in the reconstructed images.

<p>[<b>a</b>] Transverse, [<b>b</b>] frontal and [<b>c</b>] sagittal slices through the images reconstructed from phase data. Grayscale is proportional to local density. Osteocyte lacunae [Lc] and canaliculi [Ca] can clearly be seen. The heterogeneous organization of the matrix by mineralized collagen fibers can also be distinguished [box]. In this sample, a continuous change in collagen orientation can be seen between adjacent lamellae. The cement line [Cm], separating osteonal [On] and interstitial [It] tissue, can clearly be distinguished as more mineralized than the surrounding matrix. Tissue close to osteocyte lacunae is also hypermineralized. [<b>d</b>] Zoom on the boxed area in C. Matrix orientation is clearly visible and canaliculi are seen as black dots. [<b>e</b>] Mass density histograms in the three tissue types. [<b>f</b>] Samples were extracted from the mid diaphysis of a human femur. [<b>g</b>] The blue cylinder shows the imaged region inside the sample. [<b>h</b>] Schematic of a transverse section showing the organization of lamellar bone in osteons, interstitial tissue and cement lines. Blue circle shows the positioning of A. [<b>i</b>] Rendering of the electron density in the sample [blue] and porosity [yellow]. Structures such as osteocyte lacunae [Lc] and canaliculi [Ca], the cement line [Cm] and collagen fibers are revealed.</p

Experimental setup and image reconstruction.

<p>[<b>a</b>] Schematic of experimental setup. The X-ray beam [X] is monochromatized and focused into a focal spot [F] by X-ray reflective optics [KB]. The sample [S] is positioned on a translation-rotation stage downstream of the focus and imaged onto a stationary detector. Due to the resulting divergent beam, different spot-sample distances [D1] and different free space propagation distances [D2] imply different magnification factors on the detector. [<b>b</b>] Images were recorded at four focus-to-sample distances over a complete turn of the sample at 2999 projection angles. The images were used to reconstruct the phase shift at each angle [phase retrieval PR], which was used as input to a tomographic reconstruction algorithm to reconstruct the 3D local mass density.</p

Imaging of carbon nanotube contamination in lung tissue.

<p>(A) Slice from a low resolution electron density volume. (B) Low resolution 2D fluorescence image of the specimen showing iron, phosphorus and sulfur distributions. The circle indicates the ROI that was chosen for higher resolution imaging. The scanned area was about 0.2×0.3 mm² and the step size was 1.8 µm. (C) A schematic representation of the imaging geometry, showing the ROI inside the sample and the two slices that were chosen for further analysis. Phase contrast slice with 60 nm pixel size (D), Fe fluorescence slice with 500 nm pixel size (E) and correlative image of Fe and phase contrast (F) for slice #1 in the lung specimen. Numbers 1, 2, and 3 indicate positions of alveolar macrophages. Phase contrast (G), Fe fluorescence signal (H) and correlative image of Fe and phase contrast (I) for slice #2 in the lung specimen. Number 4 indicates the position of a type 1 pneumocyte, while number 5 indicates the position of an alveolar macrophage. Notice the factor of 10 difference in the relative fluorescence signal between parts (E, F) and (H, I).</p

Correlative Nanoscale 3D Imaging of Structure and Composition in Extended Objects

<div><p>Structure and composition at the nanoscale determine the behavior of biological systems and engineered materials. The drive to understand and control this behavior has placed strong demands on developing methods for high resolution imaging. In general, the improvement of three-dimensional (3D) resolution is accomplished by tightening constraints: reduced manageable specimen sizes, decreasing analyzable volumes, degrading contrasts, and increasing sample preparation efforts. Aiming to overcome these limitations, we present a non-destructive and multiple-contrast imaging technique, using principles of X-ray laminography, thus generalizing tomography towards laterally extended objects. We retain advantages that are usually restricted to 2D microscopic imaging, such as scanning of large areas and subsequent zooming-in towards a region of interest at the highest possible resolution. Our technique permits correlating the 3D structure and the elemental distribution yielding a high sensitivity to variations of the electron density via coherent imaging and to local trace element quantification through X-ray fluorescence. We demonstrate the method by imaging a lithographic nanostructure and an aluminum alloy. Analyzing a biological system, we visualize in lung tissue the subcellular response to toxic stress after exposure to nanotubes. We show that most of the nanotubes are trapped inside alveolar macrophages, while a small portion of the nanotubes has crossed the barrier to the cellular space of the alveolar wall. In general, our method is non-destructive and can be combined with different sample environmental or loading conditions. We therefore anticipate that correlative X-ray nano-laminography will enable a variety of <em>in situ</em> and <em>in operando</em> 3D studies.</p> </div

Correlative nanolaminography for materials research.

<p>A 2D slice of the structure and the elemental distribution in an aluminum foil, taken at 3 µm below the surface. (A) Structure obtained by full field phase laminography. The denser second-phase particles are dark. (B) Co-localization of Ni, Cu and Fe overlaid with the structure. (C) Co-localization of Ni, Cu and Fe shown in three dimensions.</p

Principle of correlative nanolaminography.

<p>(A) Schematic of the nanolaminography setup on ID22NI at the ESRF. The beam is focused by multilayer coated Kirkpatrick-Baez optics. The specimen is placed in the focus to raster scan the fluorescence spectra. The specimen is placed downstream of the focus to obtain a full-field magnified holographic image on the 2D detector. The tomographic rotation axis is inclined by the laminographic angle θ with respect to the beam direction. (B) 3D rendering of a lithographically fabricated Siemens star test pattern: the window reveals a single slice from the reconstructed 3D volume and the inset shows the center of the test pattern (scale bar 2 µm). (C) Profile plot along the circle shown in panel b. The inset shows a 135 nm half period achieved as lateral resolution in the 3D image.</p

Histograms of the lacunar volumes for the three different sites are shown.

<p>Histograms are normalized to the area under the total number of lacunae for each site. Bin size is set to 50 µm³. The transparent areas indicate the standard error for each site based on the individual samples.</p

Histograms of all jaw lacunae grouped in either BRONJ or healthy bone.

<p>The shaded areas correspond to the standard error based on the different samples. Histograms are normalized to the absolute amount of lacunae, bin size is 50 µm³. It should be noted that even though the histograms of the two groups look very similar, there are differences between the histograms of individual donors.</p

Investigated differences with respect to anatomical sites, assessed by analyses of variance (ANOVA), followed by post hoc multiple comparison Bonferroni tests are summarized.

<p>Significant differences between groups are indicated by a horizontal bar. If the significance level was reached, the p-Value and the F-Value are reported.</p