The stomatopod eye is a fascinating biological system capable of detecting both colour and polarization of light, making it a highly complex, mixed-tissue sample. In the investigation of complex biological systems, three-dimensional methods spanning multiple length scales with the power to resolve soft tissues are required. In this study, propagation-based phase contrast X-ray computed tomography with stitching at a 4th generation synchrotron was used to image a full stomatopod eye with sub-micron voxel size to illustrate how this method accommodates these demands. The images are based on natural X-ray contrast and without any added labels or staining agents. Key features of the eye were identified and segmented. Utilizing these segmentations, photo filter volumes, chitin porosity volumes, and muscle fiber periodicities were measured, demonstrating the ability to perform quantitative as well as qualitative investigations. Neural compartments and associated cells were discernable, showing the power of 4th generation synchrotron phase contrast for the study of soft tissues. The illustrated properties along with its non-invasive nature proves phase contrast synchrotron X-ray computed tomography with stitching to be a powerful tool for the investigation of biological materials.

The objective of this animal study was to evaluate the osseointegration of loaded strontium-functionalized orthodontic miniscrews (Ti-Sr-O) and the effect of the Sr coating on the elemental composition of the bone as well as the coating's effects on bone mineral size and orientation. Strontium-containing coated (Ti-Sr-O) miniscrews and grade 4 titanium miniscrews (Ti) were inserted into either tibia of thirty male Wistar rats and loaded through a coil spring. After a two-, four- and six-week healing period, specimens were analyzed via histomorphometry to measure bone-to-implant contact (BIC%) and peri-implant bone formation (BF%) in defined regions of interest (ROI-1; ROI-2). Furthermore, samples were investigated at the P06 synchrotron beamline to simultaneous measure the atomic composition with X-ray fluorescence (XRF) and the crystallite size/orientation with X-ray diffraction (XRD). After two weeks Ti-Sr-O functionalized miniscrews showed significantly more bone formation in ROI-1 than in the control group with Ti miniscrews at the tension (p**=0.005) and compression side (p*=0.018). The investigation by 2D XRF and XRD Mapping with a 0.4 mu m beam resulted in multiple significant changes in comparison to the controls. The XRF showed a significant increase in the Sr/Ca level in the peri implant bone, and XRD showed significant changes in degree of orientation of the crystallographic c-axis, and for the apparent crystallite size perpendicular to the crystallographic c direction. These changes depend on the treatment and Sr/Ca level. The results suggest advantageous osseointegration of Sr-coated miniscrews under immediate loading. This resulted in changes in the microscopic organization of the bone biomineral.

Accurate 3D characterization of osteocyte lacunae is important when investigating the role of osteocytes under various physiological and pathological conditions but remains a challenge. With the continued development of laboratory X-ray micro-computed tomography, an increasing number of studies employ these techniques beyond traditional bone morphometry to quantify osteocyte lacunae. However, there is a lack of knowledge on the effect of measurement parameters on the image quality and resolution and in turn the osteocyte lacunar quantification. Herein, we have examined the interplay between scan parameters and the resultant lacunar quantification in terms of lacunar size, shape, and density by comparison with a synchrotron benchmark dataset. We summarize our conclusions in a guide for use of μ-CT for osteocyte lacunar quantification: (1) Identification of the measurement requirements to address the research questions. (2) Collection and preparation of suitable sample(s) that fulfills these requirements. (3) Experimental considerations including determination of the required voxel size, in turn dictating the maximum FOV and by extension the maximum size of the sample(s). The experimental parameters chosen should ensure optimal image contrast, sufficient signal to noise, angular sampling etc. Usually, it is advisable to measure as well as possible within the limits of time, budget, data storage and analysis capabilities. (4) Data analysis and reporting of the results, including visual examination of the data at multiple steps in the analysis, to ensure correct feature identification and suitable reporting approaches. (5) Cross study comparisons, which may be unsuitable if the experimental conditions and analysis strategies are not comparable.

Stomatopods are ferocious hunters that use weaponized appendages to strike down their pray. The clubs of species such as Odontodactylus scyllarus undergo tremendous forces, and in consequence they have intricate structures, consisting of hydroxyapatite, chitin, amorphous calcium phosphate and carbonate, and occasionally calcite. These materials are distributed differently across the four major zones of the dactyl club: the impact, periodic lateral and medial, and striated regions. While stomatopod clubs and their structure have been studied for a long time, studies have thus far been constrained to 2D mapping experiments with moderate resolution due to difficulties in preparing whole club thin sections, and absorption tomography that gives information on densities but not molecular length scales. To address this problem, and shed light on the structure of entire clubs, we herein used X-ray powder diffraction computed tomography (XRD-CT) using high energy X-rays at the P07 beamline of PETRA-III to allow penetrating the large samples whilst still obtaining high resolution information. This allowed mapping the 3D distribution of diffraction phases including the biomineral apatite and the semi-crystal chitin matrix. This showed that hydroxyapatite forms an envelope around the club, and that chitin forms 2D sheets in the periodic region of the club.

Computed tomography spans a versatile set of techniques that range several length scales and modalities. It is common to them all that the sample must be prepared in a way which allows for addressing the scientific question(s) posed as well as being suitable for the specific setup of the experiment. We present two lathe-based sample preparation workflows developed to prepare biomineralized samples (here bone) for two very different experiments in terms of setup, types of questions asked, and sample requirements. The first experiment, involving the measurement of high throughput (synchrotron) micro-tomography, required the preparation of many samples with homogeneity in size, shape, and bone site. This was achieved through a particular sequence of cutting and embedding steps finalized by lathe milling. The resulting samples were cylindrical in shape with diameters close to the field of view of the ensuing tomography experiment, which allowed maximizing the investigated sample volumes. The second experiment was a combined ptychography and X-ray fluorescence nano-tomography experiment, which required preparation of a few-micrometer-sized sample. Moreover, the scientific interest was in a specific, localized feature in bone. Thus, the sample had to be extracted from a precise location from within the whole bone. Again, the developed workflow comprised many steps, including both lathe milling and focused ion beam milling. Importantly, localized preparation was enabled by measuring in-house X-ray micro-computed tomography at crucial points in the workflow. The presented workflows provide examples of preparation pathways that can be standardized and strongly increase the throughput, quality, and success rate of tomography experiments.

Bone has a complex hierarchical structure with structural integration from nm to cm. The understanding of bone structure is developing rapidly due to improvements in available methodologies that allow unravelling structures across several length scales. These methods include advances in electron microscopy, in particular, focused ion beam scanning electron microscopy (FIB‐SEM), confocal laser scanning microscopy techniques, X‐ray imaging, X‐ray diffraction tomography (XRD‐CT), and tensor tomography (small angle X‐ray scattering tensor tomgraphy, SAXS‐TT and wide angle X‐ray scattering tensor tomgraphy, WAXS‐TT). Special emphasis is placed on the latter X‐ray techniques that are emerging into powerful tools. Through a review of selected recent results on the structure of the bone matrix as well as the lacuno‐canalicular network housing the osteocyte cells of bone, it is proposed that bone is more heterogeneous than typically described and that local variation in composition and crystallography may play a significant role in bone biology in health and disease.

Propagation-based phase contrast, for example in the form of edge enhancement contrast, is well established within X-ray imaging but is not widely used in neutron imaging. This technique can help increase the contrast of low-attenuation samples but may confuse quantitative absorption measurements. Therefore, it is important to understand the experimental parameters that cause and amplify or dampen this effect in order to optimize future experiments properly. Two simulation approaches have been investigated, a wave-based simulation and a particle-based simulation conducted in McStas [Willendrup & Lefmann (2020). J. Neutron Res. 22, 1–16], and they are compared with experimental data. The experiment was done on a sample of metal foils with weakly and strongly neutron absorbing layers, which were measured while varying the rotation angle and propagation distance from the sample. The experimental data show multiple signals: attenuation, phase contrast and reflection. The wave model reproduces the sample attenuation and the phase peaks but it does not reproduce the behavior of these peaks as a function of rotation angle. The McStas simulation agrees better with the experimental data, as it reproduces attenuation, phase peaks and reflection, as well as the change in these signals as a function of rotation angle and distance. This suggests that the McStas simulation approach, where the particle description of the neutron facilitates the incorporation of multiple effects, is the most convenient way of modeling edge enhancement in neutron imaging.

Guanine crystals are frequently encountered in nature in the beta-polymorph to create structural colors, to enhance the vision of creatures, and for camouflage. Unfortunately, it is challenging to control the crystallization of guanine in aqueous conditions in the laboratory because of its low solubility in water. Here, we crystallize guanine in an aqueous environment under confinement. We employ water-oil-water double emulsions stabilized with a metal-ligand functionalized surfactant as selectively permeable containers to crystallize guanine by dynamically adjusting the pH and guanine concentration. If formed under high osmotic pressures that result in high guanine concentrations within emulsion cores, guanine crystallizes into the anhydrous beta-polymorph with a spherical morphology. In contrast, if crystals form within emulsion cores containing low guanine concentrations, they attain the monohydrate form possessing a needle-like morphology. These findings demonstrate for the first time that the structure and morphology of guanine crystals formed in the laboratory under confinement in an aqueous environment can be tuned by the local guanine concentration and to some extent by the solution pH.

The use of a phase-retrieval technique for propagation-based phase-contrast neutron imaging with a polychromatic beam is demonstrated. This enables imaging of samples with low absorption contrast and/or improving the signal-to-noise ratio to facilitate e.g. time-resolved measurements. A metal sample, designed to be close to a phase pure object, and a bone sample with canals partially filled with D2O were used for demonstrating the technique. These samples were imaged with a polychromatic neutron beam followed by phase retrieval. For both samples the signal-to-noise ratios were significantly improved and, in the case of the bone sample, the phase retrieval allowed for separation of bone and D2O, which is important for example for in situ flow experiments. The use of deuteration contrast avoids the use of chemical contrast enhancement and makes neutron imaging an interesting complementary method to X-ray imaging of bone.

Liquid plays an important role in bone that has a complex 3D hierarchical pore structure. However, liquid (water) is difficult to discern from e.g. an organic matrix by X-ray imaging. Therefore, we use a correlative approach using both high resolution X-ray and neutron imaging. Human femoral bone with liquid adsorbed into some of the pores was imaged with both the Neutron Microscope at the ICON beamline, SINQ at PSI, and by lab-based μCT using 2.7 μm voxel size. Segmentation of the two datasets showed that, even though the liquid was clearly distinguishable in the neutron data and not in the X-ray data, it remained challenging to segment it from bone due to overlaps of peaks in the gray level histograms. In consequence, segmentations from X-ray and neutron data varied significantly. To address this issue, the segmented X-ray porosities was overlaid on the neutron data, making it possible to localize the liquid in the vascular porosities of the bone sample and use the neutron attenuation to identify it as H2O. The contrast in the neutron images was lowered slightly between the bone and the liquid compared to the bone and the air. This correlative study shows that the complementary use of X-rays and neutrons is very favorable, since H2O is very distinct in the neutron data, while D2O, H2O, and organic matter can barely be distinguished from air in the X-ray data.

This is an X-ray dataset from a table-top PHYWE instrument recorded at the CINEMAX Summer School on 3D Imaging, August 2021, Fuglsang Manor, Lolland, Denmark. The scanner was operated in offset mode, ie the centre-of-rotation was deliberately moved to the side to enable a larger field of view to image an object larger than the instrument's normal field of view, using a 360 degree scan. The object scanned is an apple collected in the Fuglsang Manor garden. The data contains, in the raw/ folder the raw projections and in the reconstruction/ folder the reconstructed slices computed by the instrument's own software. These reconstructed slices do not contain the full apple and also shows double illumination from the offset scan geometry. The file settings.cto contain all required geometrical information to set up the geometry for reconstruction using other software.

Biominerals typically have complex hierarchical structures traversing many length scales. This makes their structural characterization complicated, since it requires 3D techniques that can probe full specimens at down to nanometer-resolution, a combination that is difficult - if not impossible - to achieve simultaneously. One challenging example is bone, a mineralized tissue with a highly complex architecture that is replete with a network of cells. X-ray computed tomography techniques enable multiscale structural characterization through the combination of various equipment and emerge as promising tools for characterizing biominerals. Using bone as an example, we discuss how combining different X-ray imaging instruments allow characterizing bone structures from the nano- to the organ-scale. In particular, we compare and contrast human and rodent bone, emphasize the importance of the osteocyte lacuno-canalicular network in bone, and finally illustrate how combining synchrotron X-ray imaging with laboratory instrumentation for computed tomography is especially helpful for multiscale characterization of biominerals.

The biomineralization of bone remains a puzzle. During Haversian remodeling in the dense human cortical bone, osteoclasts excavate a tunnel that is then filled in by osteoblasts with layers of bone of varying fibril orientations, resulting in a lamellar motif. Such bone represents an excellent possibility to increase our understanding of bone as a material as well as bone biomineralization by studying spatio/temporal variations in the biomineral across an osteon. To this end, fluorescence computed tomography and diffraction scattering computed tomography with sub-micrometer resolution is applied to obtain position resolved fluorescence spectra and diffraction patterns in a 3D volume. The microstructural properties of the apatite biomineral are not homogeneous but depend critically on the time point at which it was laid down. This indicates that the nature of bone biomineral is highly dependent on the microenvironment during bone formation and remodeling.

Bone has a complex hierarchical structure, which is essential for its performance. Bone is typically replete with cells called osteocytes that are embedded in the mineralized bone matrix in osteocyte lacunae, which are interconnected by canaliculi only a few hundred nanometer wide to form a vast cellular network. Our understanding of the osteocyte lacuno-canalicular network has been limited because of difficulties to image the cellular network within the opaque bone matrix in 3D. Synchrotron X-ray computed tomography is ideally suited to study the lacuno-canalicular network in bone because it combines the high penetration power of X rays with sub-micron resolution while retaining a fast acquisition time and thus high throughput. We discuss how synchrotron radiation-based tomography techniques have given insights into the osteocyte network in bone both in the form of regular tomography and, for higher resolution studies, in the form of nanotomography such as holotomography. These studies have provided quantitative measures of osteocyte lacunar properties and their relation to location within bones and bone challenges such as immobilization or lactation. Nanotomography revealed new features of the canalicular network that we term canalicular junctions, which are likely to play an important but hitherto hidden role in fluid flow dynamics within the bone cellular network. The examples illustrate how tomography provides information on complex biological materials like bone and we foresee that these capabilities will continue to improve with future/upgraded synchrotron X-ray sources.