Synchrotron Radiation-based X-ray Microtomography Research Articles

Synchrotron radiation-based microcomputed tomography for three-dimensional growth analysis of Aspergillus niger pellets.

Filamentous fungi produce a wide range of relevant biotechnological compounds. The close relationship between fungal morphology and productivity has led to a variety of analytical methods to quantify their macromorphology. Nevertheless, only a µ-computed tomography (µ-CT) based method allows a detailed analysis of the 3D micromorphology of fungal pellets. However, the low sample throughput of a laboratory µ-CT limits the tracking of the micromorphological evolution of a statistically representative number of submerged cultivated fungal pellets over time. To meet this challenge, we applied synchrotron radiation-based X-ray microtomography at the Deutsches Elektronen-Synchrotron[German Electron Synchrotron Research Center], resulting in 19,940 3D analyzed individual fungal pellets that were obtained from 26 sampling points during a 48 hAspergillus niger submerged batch cultivation. For each of the pellets, we were able to determine micromorphological properties such as number and density of spores, tips, branching points, and hyphae. The computed data allowed us to monitor the growth of submerged cultivated fungal pellets in highly resolved 3D for the first time. The generated morphological database from synchrotron measurements can be used to understand, describe, and model the growth of filamentous fungal cultivations.

High-energy synchrotron-radiation-based X-ray micro-tomography enables non-destructive and micro-scale palaeohistological assessment of macro-scale fossil dinosaur bones.

Palaeohistological analysis has numerous applications in understanding the palaeobiology of extinct dinosaurs. Recent developments of synchrotron-radiation-based X-ray micro-tomography (SXMT) have allowed the non-destructive assessment of palaeohistological features in fossil skeletons. Yet, the application of the technique has been limited to specimens on the millimetre to micrometre scale because its high-resolution capacity has been obtained at the expense of a small field of view and low X-ray energy. Here, SXMT analyses of dinosaur bones with widths measuring ∼3 cm under a voxel size of ∼4 µm at beamline BL28B2 at SPring-8 (Hyogo, Japan) are reported, and the advantages of virtual-palaeohistological analyses with large field of view and high X-ray energy are explored. The analyses provide virtual thin-sections visualizing palaeohistological features comparable with those obtained by traditional palaeohistology. Namely, vascular canals, secondary osteons and lines of arrested growth are visible in the tomography images, while osteocyte lacunae are unobservable due to their micrometre-scale diameter. Virtual palaeohistology at BL28B2 is advantageous in being non-destructive, allowing multiple sampling within and across skeletal elements to exhaustively test the skeletal maturity of an animal. Continued SXMT experiments at SPring-8 should facilitate the development of SXMT experimental procedures and aid in understanding the paleobiology of extinct dinosaurs.

Micromorphology, mineralogy, and geochemistry of ferromanganese nodules in tropical soils and their impacts on heavy metal sequestration

Ferromanganese nodules (FMNs) are important products of soil-forming processes that can reflect the historical pedogenic environment and affect the elemental cycle in the soil. Despite extensive investigations on the morphology, microstructure, and components of FMNs, non-destructive observation of the 3D microstructure and elemental distribution feature of FMNs remains difficult, owing to the limitations of conventional measurement techniques and instrumental accuracy. In this study, the micromorphological, mineralogical and geochemical characteristics of FMNs in basalt-derived soils in tropical China were examined using different techniques, including scanning electron microscopy, X-ray diffraction, chemical extraction, and synchrotron radiation-based X-ray microtomography and microfluorescence mapping. Mn and Fe in FMNs were found to be inversely distributed in concentric rings, with Mn mainly enriched in the interior and Fe mainly distributed at the edge of the nodule. A line scan of the cross-section of FMNs revealed symmetrical distributions of the peak Fe and Mn contents and Fe:Mn ratios, indicating alternating dry/wet conditions and oxidation/reduction reactions during the formation of FMNs. FMNs were significantly enriched in P and various heavy metals, including Ba, Co, Cu, and Ni. The enrichment factors of P, Ba, Co, Cu, and Ni were in the range of 1.20–4.68, 8.34–27.12, 12.92–24.74, 2.51–3.55, and 2.75–3.58, respectively, reflecting the potential of FMNs to influence the soil nutrient cycle and heavy metal sequestration. Factor analysis further verified the adsorption of heavy metals by FMNs and indicated that the formation of FMNs was accompanied by an increase in Mn-bearing mineral and a decrease in phyllosilicate. Our study demonstrates that FMNs provide important information for understanding changes in pedogenic environment and heavy metal behavior in tropical soils.

Sub- and supercritical hydrothermal route for the synthesis of xonotlite nanofibers for application to green concrete materials

Despite a wide range of applications, naturally occurring minerals suffer from some limitations for industrial use. Consequently, many research efforts have been conducted to develop a fast, optimized, and sustainable methodology to produce synthetic minerals. In the case of calcium silicate hydrates (CSH), the hydrothermal flow approach allows to mimic the environment at high temperature and pressure of the natural geological processes for the synthesis of xonotlite under sub- and supercritical conditions in only few seconds. The ultra-fast, flexible, and effective production of xonotlite particles reported in this work expands its use towards industrial requirements, especially for applications to cement-based materials. In this context, CSH nanominerals can be used to impart new functionality or to accelerate the hydration process of cement for developing green cement materials. This study sheds light on the acceleration effect of crystalline xonotlite seed, measured using isothermal calorimetry and synchrotron radiation based X-ray microtomography, as a means of lowering the cement content without compromising the performance of the paste.

Virtual histology of an entire mouse brain from formalin fixation to paraffin embedding. Part 1: Data acquisition, anatomical feature segmentation, tracking global volume and density changes

BackgroundMicrometer-resolution neuroimaging with gold-standard conventional histology requires tissue fixation and embedding. The exchange of solvents for the creation of sectionable paraffin blocks modifies tissue density and generates non-uniform brain shrinkage. New methodWe employed synchrotron radiation-based X-ray microtomography for slicing- and label-free virtual histology of the mouse brain at different stages of the standard preparation protocol from formalin fixation via ascending ethanol solutions and xylene to paraffin embedding. Segmentation of anatomical regions allowed us to quantify non-uniform tissue shrinkage. Global and local changes in X-ray absorption gave insight into contrast enhancement for virtual histology. ResultsThe volume of the entire mouse brain was 60%, 56%, and 40% of that in formalin for, respectively, 100% ethanol, xylene, and paraffin. The volume changes of anatomical regions such as the hippocampus, anterior commissure, and ventricles differ from the global volume change. X-ray absorption of the full brain decreased, while local absorption differences increased, resulting in enhanced contrast for virtual histology. These trends were also observed with laboratory microtomography measurements. Comparison with existing methodsMicrotomography provided sub-10 μm spatial resolution with sufficient density resolution to resolve anatomical structures at each step of the embedding protocol. The spatial resolution of conventional computed tomography and magnetic resonance microscopy is an order of magnitude lower and both do not match the contrast of microtomography over the entire embedding protocol. Unlike feature-to-feature or total volume measurements, our approach allows for calculation of volume change based on segmentation. ConclusionWe present isotropic micrometer-resolution imaging to quantify morphology and composition changes in a mouse brain during the standard histological preparation. The proposed method can be employed to identify the most appropriate embedding medium for anatomical feature visualization, to reveal the basis for the dramatic X-ray contrast enhancement observed in numerous embedded tissues, and to quantify morphological changes during tissue fixation and embedding.

Trabecular architecture during the healing process of a tibial diaphysis defect

The adaptation of trabecular bone microstructure to mechanical loads has been intensively investigated. However, loading-unrelated aspects of trabecular architecture remain unclear. We used synchrotron radiation-based X-ray microtomography to study the 3D microarchitecture of newly formed trabecular tissue in a defect produced in the cortical region of the rat tibia diaphysis, in the absence (7, 14, and 21 days) or the presence (21 days) of carbonated hydroxyapatite/alginate (cHA) microspheres. This work provides the first evidence that the woven bone trabecular network, formed during the healing process, displays a well-organized 3D microarchitecture consisting of nodes with 3 (3-N), 4 (4-N) and 5 (5-N) connecting trabeculae, with a mean relative abundance of (3-N)/(4-N)/(5-N) = 66/24/7, for the analyzed periods. The measured inter-trabecular angles (ITA) distribution presented a Gaussian profile, with mean value at 115° for 3-N nodes, and 105° for 4-N nodes, close to the angles of idealized 3D regular structures (120° and 109.5°, respectively). Changes in the dispersion of ITA distribution suggested that a highly symmetric trabecular fabric organized under tensegrity principles is formed early during the bone healing process. Post-implantation, cHA disaggregated into multiple fragments (~20–400 μm), stimulating osteoconduction and bone growth toward the interior of the medullary cavity. The presence of biomaterials in bone defects affected the trabecular dimensions; however, it did not interfere with the formation of geometrical motifs with topological parameters similar to those found in the sham-defects. Statement of SignificanceThe trabecular bone microstructure enables the tissue to meet the necessary mechanical and functional demands. However, the process of trabecular microarchitecture formation during healing, in the absence or presence of a bone graft, is not yet well understood. This work demonstrated that, from the beginning of its formation in cortical bone defects, the woven-bone trabecular network is spatially organized according to the principle of tensegrity. This microarchitecture is comprised of highly symmetric geometric motifs and is an intrinsic characteristic of trabecular growth, regardless of hierarchical scale or mechanical stimulation. The addition of a biodegradable nanostructured calcium phosphate graft did not disrupt trabecular microarchitecture; however, graft biodegradation should be controlled to optimize the reproduction of intrinsic trabecular motifs throughout the defect.

Jawbone remodeling: a conceptual study based on Synchrotron High-resolution Tomography

One of the most important aspects of bone remodeling is the constant turnover mainly driven by the mechanical loading stimulus. The remodeling process produces changes not only in the bone microarchitecture but also in the density distribution of the mineralized matrix - i.e. in calcium concentrations- and in the osteocyte lacunar network. Synchrotron radiation-based X-ray microtomography (microCT) has proven to be an efficient technique, capable to achieve the analysis of 3D bone architecture and of local mineralization at different hierarchical length scales, including the imaging of the lacuno-canalicular network. In the present study, we used microCT within a conceptual study of jawbone remodeling, demonstratively focusing the investigation in two critical contexts, namely in the peri-dental and the peri-implant tissues. The microCT analysis showed that a relevant inhomogeneity was clearly present in both peri-dental and peri-implant biopsies, not only in terms of microarchitecture and mineralization degree, but also considering the lacunar network, i.e. size and numerical density of the osteocyte lacunae. The correlated histological results obtained on the same samples confirmed these observations, also adding information related to non-mineralized tissues. Despite its demonstrative nature, it was concluded that the proposed method was powerful in studying jawbone remodeling because it revealed a direct correlation of its rate with the lacunar density, as achieved by the analysis of the osteocyte lacunar network, and an inverse correlation with the local bone mineral density, as revealed with the Roschger approach.

Tooth replacement in early sarcopterygians.

Teeth were an important innovation in vertebrate evolution but basic aspects of early dental evolution remain poorly understood. Teeth differ from other odontode organs, like scales, in their organized, sequential pattern of replacement. However, tooth replacement patterns also vary between the major groups of jawed vertebrates. Although tooth replacement in stem-osteichthyans and extant species has been intensively studied it has been difficult to resolve scenarios for the evolution of osteichthyan tooth replacement because of a dearth of evidence from living and fossil sarcopterygian fishes. Here we provide new anatomical data informing patterns of tooth replacement in the Devonian sarcopterygian fishes Onychodus, Eusthenopteron and Tiktaalik and the living coelacanth Latimeria based on microfocus- and synchrotron radiation-based X-ray microtomography. Early sarcopterygians generated replacement teeth on the jaw surface in a pattern similar to stem-osteichthyans, with damaged teeth resorbed and replacement teeth developed on the surface of the bone. However, resorption grades and development of replacement teeth vary spatially and temporally within the jaw. Particularly in Onychodus, where teeth were also shed through anterior rotation and resorption of bone at the base of the parasymphyseal tooth whorl, with new teeth added posteriorly. As tooth whorls are also present in more stem-osteichthyans, and statodont tooth whorls are present among acanthodians (putative stem-chondrichthyans), rotational replacement of the anterior dentition may be a stem-osteichthyan character. Our results suggest a more complex evolutionary history of tooth replacement.

Osteo-regeneration personalized for children by rapid maxillary expansion: an imaging study based on synchrotron radiation microtomography

BackgroundPersonalized maxillary expansion procedure has been proposed to correct maxillary transversal deficiency; different protocols of stem cell activation have been suggested and rapid maxillary expansion (RME) is the most commonly used among clinicians. The present study aimed to quantify in three-dimensions (3D) the osteo-regeneration of the midpalatal suture in children submitted to RME.MethodsThree patients (mean age 8.3 ± 0.9 years) were enrolled in the study to preform biopsy of midpalatal suture. Two patients (subjects 1 and 2) were subjected to RME before biopsy. The third patient did not need maxillary expansion treatment and was enrolled as control (subject 3). Midpalatal suture samples were harvested 7 days after RME in subject 1, and 30 days after RME in subject 2. The samples were harvested with the clinical aim to remove bone for the supernumerary tooth extraction. When possible, maxillary suture and bone margins were both included in the sample. All the biopsies were evaluated by complementary imaging techniques, namely Synchrotron Radiation-based X-ray microtomography (microCT) and comparative light and electron microscopy.ResultsIn agreement with microscopy, it was detected by microCT a relevant amount of newly formed bone both 7 days and 30 days after RME, with bone growth and a progressive mineralization, even if still immature respect to the control, also 30 days after RME. Interestingly, the microCT showed that the new bone was strongly connected and cross-linked, without a preferential orientation perpendicular to the suture’s long axis (previously hypothesized by histology), but with well-organized and rather isotropic 3D trabeculae.ConclusionsThe microCT imaging revealed, for the first time to the authors’ knowledge, the 3D bone regeneration in children submitted to RME.

Earliest Onychophoran in Amber Reveals Gondwanan Migration Patterns

The anomalous occurrence of supposedly Gondwanan taxa in Laurasian-derived regions remains an intriguing chapter of paleobiogeographical history. Representatives of Peripatidae, a major subgroup ofvelvet worms (Onychophora), show a disjointed distribution in the neotropics, tropical Africa, and Southeast Asia, the latter being the only landmass previously associated with Laurasia [1, 2]. The arrival of these animals in Southeast Asia is explained bytwoalternative, albeit not mutually exclusive, hypotheses: an early migration via Europe before continental drift (Eurogondwana hypothesis) or transportation via insular India during the Cretaceous and Paleogene ("out-of-India" hypothesis) [3-6]. The latter hypothesis is based on a single extant species of Peripatidae,Typhloperipatus williamsoni, in India. †Cretoperipatus burmiticus from Myanmar is the oldest fossil onychophoran found in amber [7], dating to sometime between the two proposed scenarios, and hence crucial for clarifying how Gondwanan lineages of these low-vagility animals reached Southeast Asia (see also Supplemental Information). Based on the anatomical reconstruction of †C.burmiticus using synchrotron radiation-based X-ray microtomography (SRμCT) and comparisons with extant taxa, we resolved this fossil species within Onychophora, particularly within Peripatidae, with T.williamsoni as its closest extant relative. This suggests that an early Eurogondwanan migration of peripatids was the most likely event, as Burmese amber is too old to becompatible with the out-of-India hypothesis. Moreover, peripatids probably colonized India only recently from Myanmar, refuting the putative Gondwanan relict status of Indian onychophorans. Finally, preservation artifacts identified in the novel amber material might have a major impact on studies of onychophoran stem and/or crown groups.

The contractile sponge epitheliumsensu lato– body contraction of the demospongeTethya wilhelmais mediated by the pinacoderm

Sponges constitute one of the two metazoan phyla that are able to contract their bodies despite a complete lack of muscle cells. Two competing hypotheses on the mechanisms behind this have been postulated to date: (1) mesohyl-mediated contraction originating from fusiform smooth muscle-like actinocytes ('myocytes') and (2) epidermal contraction originating in pinacocytes. No direct support exists for either hypothesis. The question of agonist-antagonist interaction in sponge contraction seems to have been completely neglected so far. In the present study we addressed this by studying sponge contraction kinetics. We also tested both hypotheses by carrying out volumetric studies of 3D synchrotron radiation-based x-ray microtomography data obtained from contracted and expanded specimens of Tethya wilhelma. Our results support the pinacoderm contraction hypothesis. Should mesohyl contraction be present, it is likely to be part of the antagonist system. We conclude that epithelial contraction plays a major role in sponges. Contractile epithelia sensu lato may be regarded as part of the ground pattern of the Metazoa.

Development of high pressure apparatus for X-ray microtomography at SPring-8

A new high-pressure apparatus has been developed for studies of synchrotron radiation-based X-ray microtomography at SPring-8 of Japan. The high pressure tomography apparatus at SPring-8 is a compact hydraulic press with a 0.8 MN capacity and is equipped with an opposed anvil device. It has two wide windows for X-ray access with a 160-degree opening in the equatorial plane to the compression axis. Radiographs are acquired over 180 degree rotation for reconstruction of 3D image, in which some shadows occur, because the press frame blocks a 20-degree angular region. 3D tomography image computed from radiographs obtained using the high pressure tomography apparatus has a reasonably good quality enough to measure physical properties of materials.

Sponge budding is a spatiotemporal morphological patterning process: Insights from synchrotron radiation-based x-ray microtomography into the asexual reproduction of Tethya wilhelma

BackgroundPrimary agametic-asexual reproduction mechanisms such as budding and fission are present in all non-bilaterian and many bilaterian animal taxa and are likely to be metazoan ground pattern characters. Cnidarians display highly organized and regulated budding processes. In contrast, budding in poriferans was thought to be less specific and related to the general ability of this group to reorganize their tissues. Here we test the hypothesis of morphological pattern formation during sponge budding.ResultsWe investigated the budding process in Tethya wilhelma (Demospongiae) by applying 3D morphometrics to high resolution synchrotron radiation-based x-ray microtomography (SR-μCT) image data. We followed the morphogenesis of characteristic body structures and identified distinct morphological states which indeed reveal characteristic spatiotemporal morphological patterns in sponge bud development. We discovered the distribution of skeletal elements, canal system and sponge tissue to be based on a sequential series of distinct morphological states. Based on morphometric data we defined four typical bud stages. Once they have reached the final stage buds are released as fully functional juvenile sponges which are morphologically and functionally equivalent to adult specimens.ConclusionOur results demonstrate that budding in demosponges is considerably more highly organized and regulated than previously assumed. Morphological pattern formation in asexual reproduction with underlying genetic regulation seems to have evolved early in metazoans and was likely part of the developmental program of the last common ancestor of all Metazoa (LCAM).

Functional morphology of Tethya species (Porifera): 2. Three-dimensional morphometrics on spicules and skeleton superstructures of T. minuta

The biomechanics of body contraction in Porifera is almost unknown, although sponge contraction has been observed already in ancient times. Some members of the genus Tethya represent the most contractile poriferan species. All of them show a highly ordered skeleton layout. Based on three main spicule types, functional units are assembled, termed skeleton superstructures here. Using synchrotron radiation based x-ray microtomography and quantitative image analysis with specially developed particle and structure recognition algorithms allowed us to perform spatial allocation and 3D-morphometric characterizations of single spicules and skeleton superstructures in T. minuta. We found and analyzed three skeleton superstructures in the investigated specimen: (1) 85 megasclere bundles, (2) a megaster sphere, composed by 16,646 oxyasters and (3) a pinacoderm–tylaster layer composed by micrasters. All three skeleton superstructures represent composite materials of siliceous spicules and extracellular matrix. From structure recognition we developed an abstracted mathematical model of the bundles and the sphere. In addition, we analyzed the megaster network interrelation topology and found a baso-apical linear symmetry axis for the megaster density inside the sphere. Based on our results, we propose a hypothetical biomechanical contraction model for T. minuta and T. wilhelma, in which the skeleton superstructures restrain physical stress generated by contraction in the tissue. While skeletal structures within the genus Tethya have been explained using R. Buckminster Fullers principle of tensegrity by other authors, we prefer material science based biomechanical approaches, to understand skeletal superstructures by referring to their composite material properties.

Functional morphology of Tethya species (Porifera): 1. Quantitative 3D-analysis of Tethya wilhelma by synchrotron radiation based X-ray microtomography

Rhythmic body contraction is a phenomenon in the Porifera, which is only partly understood. As a foundation for the understanding of the functional morphology of the highly contractile Tethya wilhelma, we performed a qualitative and quantitative volumetric 3D-analysis of the morphology of a complete non-contracted specimen at resolutions of 5.2 and 6.9 μm, using synchrotron radiation based X-ray computed microtomography (SR-μCT). For the first time, we were able to visualize all three major body structures of a complete poriferan without dissection of the shock-frozen, fixed and contrasted specimen in a near-to-life confirmation: poriferan tissue, mineral skeleton and aquiferous system. Applying a ‘virtual cast’ technique allowed us to analyze the structural details of the complete canal structure. Our results imply an extensive re-circulation of water inside the poriferan due to well-developed by-pass-canals, connecting excurrent and incurrent system. Nevertheless, the oscule region is strictly separated from the incurrent system. Based on our data, we developed a hypothetical flow regime for T. wilhelma, which explains the necessity of by-pass canals to minimize pressure boosts in the canal system during contraction. Additionally, re-circulation optimizes nutrient uptake, within small-sized poriferans, like T. wilhelma. Quantitative analysis allowed us to measure volumes and surfaces, displaying remarkable organizational differences between choanosome and cortex, by means of distribution of morphological elements. The surface-to-volume ratio proved to be very high, underlining the importance of the poriferan pinacoderm. We support a pinacoderm-contraction hypothesis.

Distribution Characteristics of Constituent Particles in Thick Plate of 2024 AI-T351 / Verteilungscharakteristika von Einschlüssen in 2024 AI-T351 Grobblech

Constituent particles and precipitations in 2024 Al-T351 were analysed in order to investigate their spatial distribution characteristics using various methods that are optical microscopy (OM), scanning electron microscopy (SEM) and synchrotron-radiation based X-ray microtomography (XTM). The OM method provides quantitative or half-quantitative results of the size, distance and shape of the particles. The SEM method produces graded information on the particles and precipitations with wider magnification scales. XTM method provides a clear view of the spatial distribution of the coarse particles and particle clusters. It was found that the particle clusters form interfaces, which separate the material into disk-like matrix domains. The interfaces are expected to have a low mechanical strength, which promotes damage and fracture in the material.