Myotragus balearicus Bate, 1909 is an endemic Late Pleistocene to Holocene goat-like caprine that has been extensively studied for its morphological peculiarities related to a long isolation on the Balearic Islands. It is a classic example of the ‘Island Syndrome’ producing dwarf taxa with regards to their mainland relatives. Its sense organs (i.e., organs for sight and olfaction) have been investigated and generally testify to a strong impact of evolution in isolation. To the contrary, the bony labyrinth of its inner ear, where the sensory organs for hearing and balance are located, has never been published so far. We here describe the bony labyrinth of M. balearicus on a sample of four different specimens from two different Mallorcan caves and compare it to extant relatives inside the clade Caprinae. We show that the size of the bony labyrinth perfectly fits in the general scaling relationship between bony labyrinth size and body size within Bovidae, which is indicative of negative allometry. While our sample is small, morphological variation still seems limited to a slight undulation and shape difference of the lateral semicircular canal, a pattern already known in other ruminant species. Under the limitations of sample size, the effect of reduced resources, ecological interactions or environmental conditions on the island is not evidenced here. We further identify characters of the bony labyrinth that can be related to phylogenetic history linking all caprines together, and even specific to the clade gathering Myotragus and Oreamnos.

We describe an almost complete fossil cranium of a shrew, identified as Asoriculus gibberodon (Petényi, 1864) from the early Pliocene of Jradzor site, Armenia. The sedimentary unit, which yielded the specimen, is an 11-m-thick package composed of white thinly-parallel-laminated diatomite laying at the base of the Jradzor section. It was dated at 4.29 ± 0.09 Ma based on the magnetostratigraphy and 40Ar/39Ar radioisotopic dating of a tephra layer located at the top of the diatomite package. The skull from Jradzor shows several synapomorphies that allow its assignment to the Soricinae subfamily and Neomyini tribe. Among Neomyini, as far as the cranium anatomy is known, the specimen from Jradzor is most similar to that of Soriculus and Episoriculus. Both petrosal bones are preserved and are studied thanks to a 3D modelling of their morphology based on a CT-scan. Compared with other eulipotyphlans, the bony labyrinth of A. gibberodon from Jradzor shows a morphology typical of soricids. Its anatomy also indicates a high-frequency auditory capability similar to that of modern shrews but cannot confirm an echolocation system neither does it shows any feature that can be related to a specific locomotory adaption or ecological characteristic. The discovery of this cranium inside diatomites, corresponding to a distal lacustrine environment, raises the question of the possible semi-aquatic adaptation of this species (this adaptation being known for other extant species of the family). However, Soriculus and Episoriculus, the two genera closest to Asoriculus based on cranial anatomy are not semi-aquatic and are clearly distinguished from semi-aquatic Neomys shrews. The inner ear morphology is more similar to that of terrestrial shrews despite the general similarities among soricids and suggests an echolocation-based orientation using high frequencies to navigate through low vegetation, which is often essential in high metabolic rate organisms to reduce energy expenditure. We therefore propose a terrestrial locomotion for A. gibberodon, consistent with its previously proposed paleoecological model, depicting it was a terrestrial species inhabiting wet or humid environments in close proximity to permanent bodies of water.

Synchrotron radiation-based X-ray microtomography is uniquely suited for post-mortem 3D visualization of organs such as the mouse brain. Tomographic imaging of the entire mouse brain with isotropic cellular resolution requires an extended field-of-view and produces datasets of multiple terabytes in size. These data must be reconstructed, analyzed, and made accessible to domain experts who may have limited image processing knowledge. Extended-field X-ray microtomography is presented with 0.65 μ m $0.65 \,\umu \mathrm{m}$ voxel size covering an entire mouse brain. The 4495 projections from 8 × 8 offset acquisitions are stitched to reconstruct a volume of 150003 voxels. The microtomography volume was non-rigidly registered to the Allen Mouse Brain Common Coordinate Framework v3 based on a combination of image intensity and landmark pairs. The data were block-wise transformed and stored in a public repository with a hierarchical format for navigation and overlay with anatomical annotations in online viewers such as Neuroglancer or siibra-explorer. This study demonstrates X-ray imaging and data processing for a full mouse brain, augmenting current atlases by improving resolution in the third dimension by an order of magnitude. The 3.3-teravoxel dataset is publicly available and easily accessible for domain experts via browser-based viewers.

X-ray imaging techniques employing diffractive and refractive lenses face the challenge of chromatic aberration if X-ray beams with a broad photon energy range are used. Recent advances combining a compound refractive lens and a Fresnel zone plate have enabled the development of achromatic lenses for X-rays, which exhibit a constant focal length over a wider range of photon energies. However, in this first demonstration, the potential of the achromatic X-ray lens was limited by the challenging task of aligning the two individual separate components. In this investigation, we designed, fabricated, and characterized monolithic X-ray achromatic lenses by integrating a Fresnel zone plate and a compound refractive lens onto a single substrate. This innovative approach inherently achieves precise alignment during fabrication, greatly simplifying and stabilizing the alignment for the X-ray imaging setups. Benefiting from an increased numerical aperture, the reported monolithic lens demonstrated state-of-the-art achromatic focusing down to approximately 200 nm for photon energies ranging from 6.6 keV to 7.7 keV. With these advancements, we present the first successful application of an achromatic lens in scanning and full-field transmission X-ray microscopy, as well as fluorescence spectroscopy, highlighting its potential for broad adoption across diverse X-ray imaging applications.

The acquisition of large tomography volumes, exceeding the typical detector field-of-view, requires advanced acquisition techniques. Current approaches are the tiling of local reconstructed volumes or the tiling in projection space, also known as mosaic tomography. Reconstruction tiling has the advantage that standard reconstruction software can be used and acquisition can be interrupted and resumed relatively easily. The disadvantage is that there is the need for volume registration and transformation. Projection tiling is faster and more dose efficient, however a custom reconstruction pipeline is required, registration in projection space is challenging due to lower contrast, and there is a high sensitivity to mechanical instabilities. In this work we propose a third, hybrid approach, to profit from the advantages of projection tiling, but limit the risks. The volume to be imaged is covered by overlapping cylinders, each corresponding to the reconstructed volume of one mosaic tomogram. The number of rings per cylinder and the total number of cylinders can be tuned to the specimen at hand. We demonstrate this approach for a 2 cm-wide section of a human brain stem, imaged at the ANATOMIX beamline of Synchrotron SOLEIL, France with 0.65 mu m voxel size, resulting in reconstructed slices 29,650 voxels wide. For mosaic reconstruction we used our team's existing pipeline. For stitching of volumes, image registration was performed in the overlap regions. As pairwise displacements between cylinders are not independent, we modified the registration approach to force a consistent solution. The results of the hybrid acquisition in seven tiles with four rings were compared to a pure projection tiling approach with eight rings and to local regions representing reconstruction tiling. In conclusion, we propose an extended field of view acquisition scheme building on the speed and dose efficiency of mosaic acquisition, but relaxing the requirements for mechanical and beam stability.

Imaging anatomical features of the human brain at cellular resolution currently relies on series of physical sections with related slicing artefacts. So far, microtomography has been employed to image an entire human brain at a voxel size of 20 iim and selected regions using 6 p.m. This study aims to demonstrate the feasibility of imaging the entire human brain with cellular resolution without the need for physical sectioning using hard X-ray computed tomography. 1.2 mm high sections of two human brains, one embedded in ethanol, the other in paraffin, were imaged using microtomography at the P07 beamline at DESY, Hamburg, Germany with a monochromatic beam at 67 keV. The extended field of view necessary to cover the ca. 10cm wide specimens at 2.54 p.m voxel size was realized by projection tiling with eight to ten rings. The resulting reconstructed slices measured 39,000 x 39,000 voxels. This synchrotron radiation-based study shows the feasibility of employing X-ray tomography to image the entire human brain with isotropic voxels of 2.54 p.m resolution. Next, we need to tackle the vertical stitching of several 10,000 slices of 6 GB each, posing the challenge of processing the big data of an entire PB-sized human brain and making it accessible to the research community.

Palatoplasty in infants with cleft palate aims to reconstruct the intricate three-dimensional anatomy and restore the velopharyngeal function, which is essential for swallowing, speech, and ventilation of the middle ear through the opening of the Eustachian tube. The non-destructive analysis of the microarchitecture around the pterygoid hamulus using hard Xrays should enhance the existing knowledge from dissection and histological studies. Specifically, the micro -anatomical relationship between the palatine aponeurosis, the tendon of the tensor veli palatini muscle, and the pterygoid hamulus must be characterized to understand their structural relationship and functional implications. At the cellular level, the arrangement of fibers within muscle fascicles needs to be clarified. The right half of a historical plastinated infant cadaveric head was examined with two laboratory -based micro computed tomography (ILECT) systems: phoenixIxray nanotom m for imaging of the entire specimen with a pixel size of 55 lam; and Zeiss Xradia 610 Versa for local tomography with a pixel size of 3.4 lam. Using synchrotron radiation-based microtomography, additional measurements were performed with a pixel size of 3.24 lam. The resulting images were rigidly registered and analyzed. Automated threshold-based segmentation of bones and manual segmentation of muscles, tendons, and aponeurosis, were performed to visualize their topographic relationships in three dimensions. An unstained segment of a human gracilis muscle was examined using the Exciscope Polaris with a pixel size of 0.35 lam, and the fiber architecture was visually inspected. Laboratory -based X-ray CT systems are suitable for virtual-histology examination of soft tissues and visualization of subcellular structures therein. Synchrotron radiation-based CT with phase retrieval provided additional contrast within the plastinated soft tissues. The findings of this study support the hypothesis that the palatal muscles form a complex muscle sling around the pterygoid hamulus, underscoring the importance of preserving this bony protuberance during cleft palate repair.

Caries affects billions of individuals worldwide, thus pointing out the importance of advancements in restorative dentistry. Dental resin composites yield restorations with satisfying mechanical properties, therefore the focus of development has shifted to accelerated treatments and esthetic aspects. Challenges in matching tooth color arise due to limited options, application changes, and color variations over time. Single-shade composites with the 'chameleon effect' adapt their color to the surrounding enamel by closely matching the tooth's optical spectrum, enhancing color blending. Structural color, based on light interference, contributes to this effect. The study investigates the submicron filler particles' impact on optical properties and the chameleon effect. Four single-shade dental resin composite materials were investigated. Needle-like samples about 100 mu m in diameter were prepared and imaged in a scanning electron microscope. Light transmission through the materials for wavelengths between 200 and 900 nm was measured using a spectrophotometer. Threedimensional nanotomography data were obtained through transmission X-ray microscopy at the ANATOMIX beamline, Synchrotron SOLEIL, France in both absorption and Zernike phase contrast mode with 23 nm voxel size. The real space information was complemented with small-angle X-ray scattering. These experiments revealed substantial differences in the microscopic structure of the materials. In the case of Omnichroma, the filler consists of almost identical spheres with a diameter of 260 nm while Filtek Universal exhibits polydisperse, irregularly shaped fillers. Additionally, Venus Pearl One's fillers have a polyhedral shape and a wide size distribution. Finally, the setups used did not reveal any clearly identified microstructure of the Chroma Fill composite. Although all investigated materials are known to exhibit the chameleon effect, their differences in micro- and nanostructure call into question previous hypotheses on the chameleon effect's origin from structural color. While we have now a reasonable understanding of filler morphology, size distribution and spatial arrangement, more information is needed on the exact chemical composition of filler and matrix and their interaction with electromagnetic waves, including possible nonlinear effects.

The effectiveness of a series of optically transparent aligners for orthodontic treatments depends on the anchoring of each tooth. In contrast with the roots, the crowns’ positions and orientations are measurable with intraoral scans, thus avoiding any X-ray dose. Exemplified by two patients, we demonstrate that three-dimensional crown movements could be determined with micrometer precision by registering weekly intraoral scans. The data show the movement and orientation changes in the individual crowns of the upper and lower jaws as a result of the forces generated by the series of aligners. During the first weeks, the canines and incisors were more affected than the premolars and molars. We detected overall tooth movement of up to about 1 mm during a nine-week active treatment. The data on these orthodontic treatments indicate the extent to which actual tooth movement lags behind the treatment plan, as represented by the aligner shapes. The proposed procedure can not only be used to quantify the clinical outcome of the therapy, but also to improve future planning of orthodontic treatments for each specific patient. This study should be treated with caution because only two cases were investigated, and the approach should be applied to a reasonably large cohort to reach strong conclusions regarding the efficiency and efficacy of this therapeutic approach.

Imaging natural history collections is becoming an important conservation tool that also serves research purposes. Herbaria are at the forefront of this new area, where automatic conveyor belts can scan thousands of sheets per day. The production of high quality images is used as a tool for inventory, monitoring, communication, data exchange between scientists and new taxonomic identifications. Microtomography of collection items with these aims is much more time-consuming and expensive. While it has been so far limited to rare and important specimens such as types or reference specimens (i.e., historically or scientifically important specimens; see in Ref. [1]), the data generated takes conservation to another level. This is because nit captures not only surface features, but also very fine texture and internal structures are digitally recorded, depicting the object in almost all its complexity and dimensions. Generating this kind of data helps researchers achieve their goals, provides firsth-and scientific data, limits further handling of sometimes fragile specimens and, can help reduce the ecological footprint of scientific travel. In this work, we illustrate the power of microtomography in conservation work by imaging fossil type specimens (i.e. remains of extinct organisms used to designate new species) which are witnesses of past life on our planet. They provide information on how today's biodiversity has evolved and are a good indicator for the past climates. In addition, they often fascinate a wide audience and are therefore good ambassadors for communicating scientific findings. Recording them with the help of X-ray microtomography should therefore be a general goal, which we illustrate here with examples.

Amber captures a snapshot of life and death from millions of years in the past. Here, the fate of three fossil Darwin wasps in Baltic amber is virtually dissected with the help of micro-CT scanning, to better understand the taphonomic processes that affected their preservation. The states of the fossils range from nearly perfect preservation, including remains of internal organs, to empty casts that were strongly affected by decomposition. We describe the three specimens as new taxa, Osparvis aurorae gen. et sp. nov., Grana harveydenti gen. et sp. nov. and Xorides? romeo sp. nov. Based on the taphonomic and morphological interpretations, we conclude that two specimens were trapped alive, and the third ended up in resin post-mortem. The morphology and classification of the specimens provide clues regarding their ecology, and we discuss their likely hosts and parasitation modes. Taken together, our three wasp fossils showcase how an integrative analysis of amber taphonomy, taxonomic association and morphology can shed light onto past biodiversity and offer valuable insights for interpreting their evolutionary history.

Noise-induced hearing loss can be caused by sudden or prolonged exposure to loud noise. Noise exposure is known to contribute to the degeneration of sensory cells, disrupting the conversion of mechanical sound waves into electrical impulses and further their transmission to the brain. To determine the pathophysiological condition of the inner ear cells in animal models, the measurement of the animals' hearing is essential. The follow-up examination of the cochlea is particularly important as it provides information on the cellular morphology changes. Our aim was therefore to investigate the hair cell survival in the inner ear of mice exposed to two high noise levels using synchrotron radiation-based microtomography. Its spatial resolution allows for the reconstruction of three-dimensional images of unstained cochlea at the cellular level. We segmented the basilar membrane via automatic cell segmentation and fast manual cell removal, and determined its length using a one-dimensional Isomap embedding. After extracting its middle region and image slices aligned with it, surviving inner and outer hair cell locations were semi-automatically determined and then manually corrected using the ImageJ plugin PointPicker. These results were compared with the confocal microscopy data. The data collected provides meaningful information about healthy and damaged hair cells in the adult cochlea.

Inline X-ray phase tomography has emerged as one of the most suitable imaging techniques for the three-dimensional examination of soft tissue at the microscopic level. Historically, this method was constrained to synchrotron radiation due to its specific requirements, such as beam coherence. However, recent advancements in detector technology (optical magnification) and X-ray sources (e.g., smaller source sizes and liquid metal sources) have enabled the transfer of this technology to laboratory settings. In this study, we investigated selected parts of an ethanol-fixated mosquito-specifically the head, abdomen, and proboscis-at the sub-cellular level using the Xradia 610 Versa (Carl Zeiss X-ray Microscopy, Inc., Dublin, California, United States) with voxel sizes as small as 180 nm. A single lens of the compound eye was segmented from the data set of the head, and the focal length was calculated to be 22 mu m. These results demonstrate the capability of laboratory-based X-ray phase tomography for high-resolution imaging of soft tissues, facilitating detailed structural analyses previously achievable only with synchrotron radiation.

ABSTRACT Background The roles of hypoxia and hypoxia inducible factor (HIF) during chronic kidney disease (CKD) are much debated. Interventional studies with HIF-α activation in rodents have yielded contradictory results. The HIF pathway is regulated by prolyl and asparaginyl hydroxylases. While prolyl hydroxylase inhibition is a well-known method to stabilize HIF-α, little is known about the effect asparaginyl hydroxylase factor inhibiting HIF (FIH). Methods We used a model of progressive proteinuric CKD and a model of obstructive nephropathy with unilateral fibrosis. In these models we assessed hypoxia with pimonidazole and vascularization with three-dimensional micro-computed tomography imaging. We analysed a database of 217 CKD biopsies from stage 1 to 5 and we randomly collected 15 CKD biopsies of various severity degrees to assess FIH expression. Finally, we modulated FIH activity in vitro and in vivo using a pharmacologic approach to assess its relevance in CKD. Results In our model of proteinuric CKD, we show that early CKD stages are not characterized by hypoxia or HIF activation. At late CKD stages, some areas of hypoxia are observed, but these are not colocalizing with fibrosis. In mice and in humans, we observed a downregulation of the HIF pathway, together with an increased FIH expression in CKD, according to its severity. Modulating FIH in vitro affects cellular metabolism, as described previously. In vivo, pharmacologic FIH inhibition increases the glomerular filtration rate of control and CKD animals and is associated with decreased development of fibrosis. Conclusions The causative role of hypoxia and HIF activation in CKD progression is questioned. A pharmacological approach of FIH downregulation seems promising in proteinuric kidney disease.

The performance of machine learning algorithms, when used for segmenting 3D biomedical images, does not reach the level expected based on results achieved with 2D photos. This may be explained by the comparative lack of high-volume, high-quality training datasets, which require state-of-the-art imaging facilities, domain experts for annotation and large computational and personal resources. The HR-Kidney dataset presented in this work bridges this gap by providing 1.7 TB of artefact-corrected synchrotron radiation-based X-ray phase-contrast microtomography images of whole mouse kidneys and validated segmentations of 33 729 glomeruli, which corresponds to a one to two orders of magnitude increase over currently available biomedical datasets. The image sets also contain the underlying raw data, threshold- and morphology-based semi-automatic segmentations of renal vasculature and uriniferous tubules, as well as true 3D manual annotations. We therewith provide a broad basis for the scientific community to build upon and expand in the fields of image processing, data augmentation and machine learning, in particular unsupervised and semi-supervised learning investigations, as well as transfer learning and generative adversarial networks.

Nephrotoxicity is an important drug safety aspect to be assessed during drug discovery and development. To study renal toxicity, in vitro cell-based assays are often used. Unfortunately, translating the results of such cell assays to vertebrates including human remains challenging. Therefore, we aim to evaluate whether zebrafish larvae (ZFL) could serve as a vertebrate screening model to detect gentamicin-induced changes of kidney glomeruli and proximal tubules. To validate the model, we compared the results of ZFL with those obtained from kidney biopsies of gentamicin-treated mice. We used transgenic zebrafish lines expressing enhanced green fluorescent proteins in the glomerulus to visualize glomerular damage. Synchrotron radiation-based computed tomography (SRμCT) is a label-free approach providing three-dimensional representations of renal structures with micrometre resolution. Clinically used gentamicin concentrations induce nephrotoxicity and affect glomerular and proximal tubular morphology. Findings were confirmed in mice and ZFL. There was a strong correlation between fluorescent signals in ZFL, SRμCT- derived descriptors of glomerular and proximal tubular morphology and the histological analysis of mouse kidney biopsies. A combination of SRμCT and confocal microscopy provides unprecedented insights into anatomical structures of the zebrafish kidney. Based on our findings, we suggest to use ZFL as a predictive vertebrate screening model to study drug-induced nephrotoxicity and to bridge the gap between cell culture-based test systems and experiments in mammals.

The most common form of epilepsy among adults is mesial temporal lobe epilepsy (mTLE), with seizures often originating in the hippocampus due to abnormal electrical activity. The gold standard for the histopathological analysis of mTLE is histology, which is a two-dimensional technique. To fill this gap, we propose complementary three-dimensional (3D) X-ray histology. Herein, we used synchrotron radiation-based phase-contrast microtomography with 1.6 μm-wide voxels for the post mortem visualization of tissue microstructure in an intrahippocampal-kainate mouse model for mTLE. We demonstrated that the 3D X-ray histology of unstained, unsectioned, paraffin-embedded brain hemispheres can identify hippocampal sclerosis through the loss of pyramidal neurons in the first and third regions of the Cornu ammonis as well as granule cell dispersion within the dentate gyrus. Morphology and density changes during epileptogenesis were quantified by segmentations from a deep convolutional neural network. Compared to control mice, the total dentate gyrus volume doubled and the granular layer volume quadrupled 21 days after injecting kainate. Subsequent sectioning of the same mouse brains allowed for benchmarking 3D X-ray histology against well-established histochemical and immunofluorescence stainings. Thus, 3D X-ray histology is a complementary neuroimaging tool to unlock the third dimension for the cellular-resolution histopathological analysis of mTLE.

The correct interpretation of fossils and their reliable taxonomic placements are fundamental for understanding the evolutionary history of biodiversity. Amber inclusions often preserve more morphological information than compression fossils, but are often partially hidden or distorted, which can impede taxonomic identification. Here, we studied four new fossil species of Darwin wasps from Baltic and Dominican amber, using micro computed tomography (micro-CT) scans and 3D reconstructions to accurately interpret and increase the availability of morphological information. We then infer their taxonomic placement in a Bayesian phylogenetic analysis by combining morphological and molecular data of extant and fossil Darwin wasps and evaluate the impact and usefulness of the additional information from micro-CT scanning. The results show that although we gained significant morphological information from micro-CT scanning, especially concerning measurements and hidden dorsal and ventral structures, this did not impact subfamily-level placement for any of the four fossils. However, micro-CT scanning improved the precision of fossil placements at the genus level, which might be key in future dating and diversification analyses. Finally, we describe the four new fossil species as Rhyssa gulliveri sp. nov. in Rhyssinae , Triclistus levii sp. nov. in Metopiinae, Firkantus freddykruegeri gen. et. sp. nov. in Pimplinae and Magnocula sarcophaga gen. et sp. nov. in Phygadeuontinae. The first two species are the first known representatives of the subfamilies Rhyssinae and Metopiinae in amber.

Joint tissues consist of trabecular and cortical bone as well as calcified and hyaline cartilage, which presents a challenge for hard X-ray-based visualization on the sub-cellular level due to the wide range of local X-ray absorption values. The density of the calcified tissues requires rather high photon energy, which often leads to insufficient contrast within the cartilage and impedes the visualization of individual biological cells. Decalcification of the tissues reduces the total and local X-ray absorption values and allows for selecting a lower photon energy. Further contrast enhancement can be achieved by ethanol fixation and paraffin tissue embedding. In this study, we (i) searched for an appropriate visualization method to investigate lesions generated by a laser osteotome and (ii) visualized a decalcified porcine joint after ethanol fixation and subsequent paraffin embedding using laboratory- and synchrotron radiation-based microtomography. The experiments at the ANATOMIX beamline of Synchrotron SOLEIL were performed in off-axis scan mode with a pixel size of 1.3 mu m. Individual cells in all layers of the joint could be made visible and the effect of ethanol fixation and paraffin embedding demonstrated.