Morphology of Palaeospondylus shows affinity to tetrapod ancestors.

Palaeospondylus gunni, from the Middle Devonian period, is one of the most enigmatic fossil vertebrates, and its phylogenetic position has remained unclear since its discovery in Scotland in 1890 (ref. 1). The fossil's strange set of morphological features has made comparisons with known vertebrate morphotype diversity difficult. Here we use synchrotron radiation X-ray micro-computed tomography to show that Palaeospondylus was a sarcopterygian, and most probably a stem-tetrapod. The skeleton of Palaeospondylus consisted solely of endoskeletal elements in which hypertrophied chondrocyte cell lacunae, osteoids and a small fraction of perichondral bones developed. Despite the complete lack of teeth and dermal bones, the neurocranium of Palaeospondylus resembles those of stem-tetrapod Eusthenopteron2 and Panderichthys3, and phylogenetic analyses place Palaeospondylus in between them. Because the unique features of Palaeospondylus, such as the cartilaginous skeleton and the absence of paired appendages, are present in the larva of crown tetrapods, our study highlights an unanticipated heterochronic evolution at the root of tetrapods.

Optical development in the murine eye lens of accelerated senescence-prone SAMP8 and senescence-resistant SAMR1 strains.

The eye lens is responsible for focusing objects at various distances onto the retina and its refractive power is determined by its surface curvature as well as its internal gradient refractive index (GRIN). The lens continues to grow with age resulting in changes to the shape and to the GRIN profile. The present study aims to investigate how the ageing process may influence lens optical development. Murine lenses of accelerated senescence-prone strain (SAMP8) aged from 4 to 50 weeks; senescence-resistant strain (SAMR1) aged from 5 to 52 weeks as well as AKR strain (served as control) aged from 6 to 70 weeks were measured using the X-ray interferometer at the SPring-8 synchrotron Japan within three consecutive years from 2020 to 2022. Three dimensional distributions of the lens GRIN were reconstructed using the measured data and the lens shapes were determined using image segmentation in MatLab. Variations in the parameters describing the lens shape and the GRIN profile with age were compared amongst three mouse strains. With advancing age, both the lens anterior and posterior surface flattens and the lens sagittal thickness increase in all three mouse strains (Anterior radius of curvature increase at 0.008 mm/week, 0.007 mm/week and 0.002 mm/week while posterior radius of curvature increase at 0.002 mm/week, 0.007 mm/week and 0.003 mm/week respectively in AKR, SAMP8 and SAMR1 lenses). Compared with the AKR strain, the SAMP8 samples demonstrate a higher rate of increase in the posterior curvature radius (0.007 mm/week) and the thickness (0.015 mm/week), whilst the SAMR1 samples show slower increases in the anterior curvature radius (0.002 mm/week) and its thickness (0.013 mm/week). There are similar age-related trends in GRIN shape in the radial direction (in all three types of murine lenses nr2 and nr6 increase with age while nr4 decrease with age consistently) but not in the axial direction amongst three mouse strains (nz1 of AKR lens decrease while of SAMP8 and SAMR1 increase with age; nz2 of all three models increase with age; nz3 of AKR lens increase while of SAMP8 and SAMR1 decrease with age). The ageing process can influence the speed of lens shape change and affect the GRIN profile mainly in the axial direction, contributing to an accelerated decline rate of the optical power in the senescence-prone strain (3.5 D/week compared to 2.3 D/week in the AKR control model) but a retardatory decrease in the senescence-resistant strain (2.1 D/week compared to the 2.3D/week in the AKR control model).

Volume X-Ray Micro-Computed Tomography Analysis of the Early Cephalized Central Nervous System in a Marine Flatworm, Stylochoplana pusilla.

Platyhelminthes are a phylum of simple bilaterian invertebrates with prototypic body systems. Compared with non-bilaterians such as cnidarians, the bilaterians are likely to exhibit integrated free-moving behaviors, which require a concentrated nervous system "brain" rather than the distributed nervous system of radiatans. Marine flatworms have an early cephalized 'central' nervous system compared not only with non-bilaterians but also with parasitic flatworms or freshwater planarians. In this study, we used the marine flatworm Stylochoplana pusilla as an excellent model organism in Platyhelminthes because of the early cephalized central nervous system. Here, we investigated the three-dimensional structures of the flatworm central nervous system by the use of X-ray micro-computed tomography (micro-CT) in a synchrotron radiation facility. We found that the obtained tomographic images were sufficient to discriminate some characteristic structures of the nervous system, including nerve cords around the cephalic ganglion, mushroom body-like structures, and putative optic nerves forming an optic commissure-like structure. Through the micro-CT imaging, we could obtain undistorted serial section images, permitting us to visualize precise spatial relationships of neuronal subpopulations and nerve tracts. 3-D micro-CT is very effective in the volume analysis of the nervous system at the cellular level; the methodology is straightforward and could be applied to many other non-model organisms.

High-energy X-ray micro-laminography to visualize microstructures in dense planar objects.

High-energy X-ray micro-laminography has been developed to observe inner- and near-surface structures in dense planar objects that are not suitable for observation by X-ray micro-tomography. A multilayer-monochromator-based high-intensity X-ray beam with energy of 110 keV was used for high-energy and high-resolution laminographic observations. As a demonstration of high-energy X-ray micro-laminography for observing dense planar objects, a compressed fossil cockroach on a planar matrix surface was analyzed with effective pixel sizes of 12.4 µm and 4.22 µm for wide field of view and high-resolution observations, respectively. In this analysis, the near-surface structure was clearly observed without undesired X-ray refraction-based artifacts from outside of the region of interest, a problem typical in tomographic observations. Another demonstration visualized fossil inclusions in a planar matrix. Micro-scale features of a gastropod shell and micro-fossil inclusions in the surrounding matrix were clearly visualized. When observing local structures in the dense planar object with X-ray micro-laminography, the penetrating path length in the surrounding matrix can be shortened. This is a significant advantage of X-ray micro-laminography where desired signals generated at the region of interest including optimal X-ray refraction effectively contribute to image formation without being disturbed by undesired interactions in the thick and dense surrounding matrix. Therefore, X-ray micro-laminography allows recognition of the local fine structures and slight difference in the image contrast of planar objects undetectable in a tomographic observation.

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.

Observation of water droplets in microporous layers for polymer electrolyte fuel cells by X-ray computed nano-tomography.

An X-ray computed nano-tomography (nano-CT) system has been established at the BL33XU beamline of SPring-8. The optical system consists of pseudo-Köhler illumination with a sector condenser zone plate, an apodization Fresnel zone plate as the objective lens, and a Zernike phase plate. The imaging detector is a fiber-coupling type X-ray camera. The performance of the X-ray nano-CT system was confirmed by imaging an X-ray test chart. The system was subsequently applied to the observation of a microporous layer for polymer electrolyte fuel cells and a simulated microporous layer including liquid water. The nano-CT system, which can perform a computed tomography measurement in less than 4 min, allowed visualization of a spherical water droplet produced in the microporous layer. In the present study, the shape of water droplets in a nanoscale porous structure is investigated.

Double-multilayer monochromators for high-energy and large-field X-ray imaging applications with intense pink beams at SPring-8 BL20B2.

In this study, double-multilayer monochromators that generate intense, high-energy, pink X-ray beams are designed, installed and evaluated at the SPring-8 medium-length (215 m) bending-magnet beamline BL20B2 for imaging applications. Two pairs of W/B4C multilayer mirrors are designed to utilize photon energies of 110 keV and 40 keV with bandwidths of 0.8% and 4.8%, respectively, which are more than 100 times larger when compared with the Si double-crystal monochromator (DCM) with a bandwidth of less than 0.01%. At an experimental hutch located 210 m away from the source, a large and uniform beam of size 14 mm (V) × 300 mm (H) [21 mm (V) × 300 mm (H)] was generated with a high flux density of 1.6 × 109 photons s-1 mm-2 (6.9 × 1010 photons s-1 mm-2) at 110 keV (40 keV), which marked a 300 (190) times increase in the photon flux when compared with a DCM with Si 511 (111) diffraction. The intense pink beams facilitate advanced X-ray imaging for large-sized objects such as fossils, rocks, organs and electronic devices with high speed and high spatial resolution.

In Situ Observation of the Movement of Magnetic Particles in Polyurethane Elastomer Densely Packed Magnetic Particles Using Synchrotron Radiation X-ray Computed Tomography.

In situ observation of the migration and structure formation of magnetic particles in polyurethane elastomers was carried out by X-ray computed tomography using synchrotron radiation. The mean diameter of the magnetic particles was 7.0 µm, and the volume fraction was ϕ= 0.24 at its maximum. The exposure time was 100 ms/frame, and the pixel size was 0.458 µm/pixel. The orientation angle and the volume fraction of the maximum aggregate were analyzed using commercial software for image analysis. The orientation angle for magnetic elastomers with ϕ = 0.24 was approximately 55° at 0 mT and decreased remarkably with the magnetic field. At magnetic fields above 150 mT, the orientation angle gradually decreased with the field and showed a constant value of 38° at 300 mT, suggesting that magnetic particles move and form a chain-like structure although the chains do not align perfectly in the direction of the magnetic field. On the other hand, the volume fraction of the maximum aggregate was constant at magnetic fields below 100 mT, and it significantly increased with the field, indicating that magnetic particles were connected to each other and developed into a macroscopic structure with anisotropy. Dynamic viscoelastic measurements revealed that the storage modulus of the magnetic elastomers cannot be simply scaled by the orientation angle. It was also found that the volume fraction of the maximum aggregate is a good parameter for explaining the huge increase in the storage modulus. The dynamic movement of magnetic particles when a magnetic field of 300 mT was switched on and off was also successfully observed. When the field was switched on, magnetic particles connected instantly and their aggregates were rapidly elongated in the direction of the magnetic field. When the field was switched off, some of the connections between aggregates were broken; however, most of the aggregates did not return to the original position even 5 min after being switched off.

Optical development in the zebrafish eye lens.

The optics of the eye is the key to a functioning visual system. The exact nature of the correlation between ocular optics and eye development is not known because of the paucity of knowledge about the growth of a key optical element, the eye lens. The sophisticated optics of the lens and its gradient of refractive index provide the superior optical quality that the eye needs and which, it is thought, has a major influence on the development of proper visual function. The nature of a gradient refractive index lens, however, renders accurate measurements of its development difficult to make and has been the reason why the influence of lens growth on visual function remains largely unknown. Novel imaging techniques have made it possible to investigate growth of the eye lens in the zebrafish. This study shows measurements using X-ray Talbot interferometry of three-dimensional gradient index profiles in eye lenses of zebrafish from late larval to adult stages. The zebrafish lens shows evidence of a gradient of refractive index from the earliest stages measured and its growth suggests an apparent coincidence between periods of rapid increase in refractive index in the lens nucleus and increased expression of a particular crystallin protein group.

In vivo monitoring of bone microstructure by propagation-based phase-contrast computed tomography using monochromatic synchrotron light.

Hard X-ray phase-contrast imaging is sensitive to density variation in objects and shows a dose advantage for in vivo observation over absorption-contrast imaging. We examined the capability of propagation-based phase-contrast tomography (PB-PCT) with single-distance phase retrieval for tracking of bone structure and mineral changes using monochromatic synchrotron light. Female mice underwent ovariectomy and drill-hole surgery in the right tibial diaphysis and were divided into two groups: OVX and OVX-E (n = 6 each); the latter group was treated with intraperitoneal administration of 14,15-epoxyeicosatrienoic acid (14,15-EET) for promoting bone repair. Age-matched mice subjected to sham ovariectomy and drill-hole surgery (Sham) were also prepared (n = 6). In vivo CT scans of the drilled defect were acquired 3, 7, and 11 days after surgery, and tomographic images were matched by three-dimensional registration between successive time points for monitoring the process of defect filling. In addition, using absorption-contrast CT as the reference method, the validity of PB-PCT was evaluated in one mouse by comparing images of tibial metaphyseal bone between the two methods in terms of bone geometry as well as the measure of mineralization. Although phase retrieval is strictly valid only for single-material objects, PB-PCT, with its lower radiation dose, could provide a depiction of bone structure similar to that from absorption-contrast CT. There was a significant correlation of linear absorption coefficients between the two methods, indicating the possibility of a rough estimate of the measure of mineralization by PB-PCT. Indeed, delayed bone regeneration (OVX vs. Sham) and the efficacy of 14,15-EET for improving osteoporotic bone repair (OVX-E vs. OVX) could be detected in both bone volume and mineralization by PB-PCT. Thus, in combination with single-distance phase retrieval, PB-PCT would have great potential for providing a valuable tool to track changes in bone structure and mineralization, and for evaluating the effects of therapeutic interventions as well.

Development of an X-ray imaging detector for high-energy X-ray microtomography.

A dedicated X-ray imaging detector for 200 keV high-energy X-ray microtomography was developed. The novelty of the detector is a large-format camera lens employed for a wide field of view. Several scintillators were evaluated in terms of the degree of efficiency of detection for high-energy X-ray photons and the modulation transfer function. For tomographic measurement, a high-definition CMOS camera was incorporated in the detector to achieve a high spatial resolution while keeping the field of view wide. Rocks with fossil inclusions were imaged to demonstrate the applicability of the detector to high-energy X-ray microtomography.

Methods for dynamic synchrotron X-ray respiratory imaging in live animals.

Small-animal physiology studies are typically complicated, but the level of complexity is greatly increased when performing live-animal X-ray imaging studies at synchrotron and compact light sources. This group has extensive experience in these types of studies at the SPring-8 and Australian synchrotrons, as well as the Munich Compact Light Source. These experimental settings produce unique challenges. Experiments are always performed in an isolated radiation enclosure not specifically designed for live-animal imaging. This requires equipment adapted to physiological monitoring and test-substance delivery, as well as shuttering to reduce the radiation dose. Experiment designs must also take into account the fixed location, size and orientation of the X-ray beam. This article describes the techniques developed to overcome the challenges involved in respiratory X-ray imaging of live animals at synchrotrons, now enabling increasingly sophisticated imaging protocols.

Photon-counting, energy-resolving and super-resolution phase contrast X-ray imaging using an integrating detector.

This work demonstrates the use of a scientific-CMOS (sCMOS) energy-integrating detector as a photon-counting detector, thereby eliminating dark current and read-out noise issues, that simultaneously provides both energy resolution and sub-pixel spatial resolution for X-ray imaging. These capabilities are obtained by analyzing visible light photon clouds that result when X-ray photons produce fluorescence from a scintillator in front of the visible light sensor. Using low-fluence monochromatic X-ray projections to avoid overlapping photon clouds, the centroid of individual X-ray photon interactions was identified. This enabled a tripling of the spatial resolution of the detector to 6.71 ± 0.04 µm. By calculating the total charge deposited by this interaction, an energy resolution of 61.2 ± 0.1% at 17 keV was obtained. When combined with propagation-based phase contrast imaging and phase retrieval, a signal-to-noise ratio of up to 15 ± 3 was achieved for an X-ray fluence of less than 3 photons/mm2.

Cell compaction is not required for the development of gradient refractive index profiles in the embryonic chick lens.

The development of the eye requires the co-ordinated integration of optical and neural elements to create a system with requisite optics for the given animal. The eye lens has a lamellar structure with gradually varying protein concentrations that increase towards the centre, creating a gradient refractive index or GRIN. This provides enhanced image quality compared to a homogeneous refractive index lens. The development of the GRIN during ocular embryogenesis has not been investigated previously. This study presents measurements using synchrotron X-ray Talbot interferometry and scanning electron microscopy of chick eyes from embryonic day 10: midway through embryonic development to E18: a few days before hatching. The lens GRIN profile is evident from the youngest age measured and increases in magnitude of refractive index at all points as the lens grows. The profile is parabolic along the optic axis and has two distinct regions in the equatorial plane. We postulate that these may be fundamental for the independent central and peripheral processes that contribute to the optimisation of image quality and the development of an eye that is emmetropic. The spatial distributions of the distinct GRIN profile regions match with previous measurements on different fibre cell groups in chick lenses of similar developmental stages. Results suggest that tissue compaction may not be necessary for development of the GRIN in the chick eye lens.

Origin of ecdysis: fossil evidence from 535-million-year-old scalidophoran worms.

With millions of extant species, ecdysozoans (Scalidophora, Nematoida and Panarthropoda) constitute a major portion of present-day biodiversity. All ecdysozoans secrete an exoskeletal cuticle which must be moulted periodically and replaced by a larger one. Although moulting (ecdysis) has been recognized in early Palaeozoic panarthropods such as trilobites and basal groups such as anomalocaridids and lobopodians, the fossil record lacks clear evidence of ecdysis in early scalidophorans, largely because of difficulties in recognizing true exuviae. Here, we describe two types of exuviae in microscopic scalidophoran worms from the lowermost Cambrian Kuanchuanpu Formation ( ca 535 Ma) of China and reconstruct their moulting process. These basal scalidophorans moulted in a manner similar to that of extant priapulid worms, extricating themselves smoothly from their old tubular cuticle or turning their exuviae inside out like the finger of a glove. This is the oldest record of moulting in ecdysozoans. We also discuss the origin of ecdysis in the light of recent molecular analyses and the significance of moulting in the early evolution of animals.

Development of an X-ray imaging detector to resolve 200 nm line-and-space patterns by using transparent ceramics layers bonded by solid-state diffusion.

A high-resolution lens-coupled X-ray imaging detector equipped with a thin-layer transparent ceramics scintillator has been developed. The scintillator consists of a 5 µm thick Ce-doped Lu3Al5O12 layer (LuAG:Ce) bonded onto the support substrate of the non-doped LuAG ceramics by using a solid-state diffusion technique. Secondary electron microscopy of the bonded interface indicated that the crystal grains were densely packed without any pores in the optical wavelength scale, indicating a quasi-uniform refractive index across the interface. This guarantees high transparency and minimum reflection, which are essential properties for X-ray imaging detectors. The LuAG:Ce scintillator was incorporated into an X-ray imaging detector coupled with an objective lens with a numerical aperture of 0.85 and an optical magnification of 100. The scintillation light was imaged onto a complementary metal-oxide-semiconductor image sensor. The effective pixel size on the scintillator plane was 65 nm. X-ray transmission images of 200 nm line-and-space patterns were successfully resolved. The high spatial resolution was demonstrated by X-ray transmission images of large integrated circuits with the wiring patterns clearly visualized.

Contributions of shape and stiffness to accommodative loss in the ageing human lens: a finite element model assessment.

Ageing changes to the various components of the accommodative system of the eye lens contribute to the loss of focusing power. The relative contributions of each ageing component, however, are not well defined. This study investigates the contribution of geometric parameters and material properties on accommodation, simulated using models based on human lenses aged 16, 35, and 48 years. Each model was tested using two different sets of material properties and a range of zonular fiber angles and was compared to results from in vivo measurements. The geometries and material parameters of older and younger lens models were interchanged to investigate the role of shape and material on accommodative capacity. Results indicate that geometry has the greater role in accommodation.

Direct observation of grain rotations during coarsening of a semisolid Al-Cu alloy.

Sintering is a key technology for processing ceramic and metallic powders into solid objects of complex geometry, particularly in the burgeoning field of energy storage materials. The modeling of sintering processes, however, has not kept pace with applications. Conventional models, which assume ideal arrangements of constituent powders while ignoring their underlying crystallinity, achieve at best a qualitative description of the rearrangement, densification, and coarsening of powder compacts during thermal processing. Treating a semisolid Al-Cu alloy as a model system for late-stage sintering-during which densification plays a subordinate role to coarsening-we have used 3D X-ray diffraction microscopy to track the changes in sample microstructure induced by annealing. The results establish the occurrence of significant particle rotations, driven in part by the dependence of boundary energy on crystallographic misorientation. Evidently, a comprehensive model for sintering must incorporate crystallographic parameters into the thermodynamic driving forces governing microstructural evolution.

Fresnel zone plate with apodized aperture for hard X-ray Gaussian beam optics.

Fresnel zone plates with apodized apertures [apodization FZPs (A-FZPs)] have been developed to realise Gaussian beam optics in the hard X-ray region. The designed zone depth of A-FZPs gradually decreases from the center to peripheral regions. Such a zone structure forms a Gaussian-like smooth-shouldered aperture function which optically behaves as an apodization filter and produces a Gaussian-like focusing spot profile. Optical properties of two types of A-FZP, i.e. a circular type and a one-dimensional type, have been evaluated by using a microbeam knife-edge scan test, and have been carefully compared with those of normal FZP optics. Advantages of using A-FZPs are introduced.

Selection and constraint underlie irreversibility of tooth loss in cypriniform fishes.

The apparent irreversibility of the loss of complex traits in evolution (Dollo's Law) has been explained either by constraints on generating the lost traits or the complexity of selection required for their return. Distinguishing between these explanations is challenging, however, and little is known about the specific nature of potential constraints. We investigated the mechanisms underlying the irreversibility of trait loss using reduction of dentition in cypriniform fishes, a lineage that includes the zebrafish (Danio rerio) as a model. Teeth were lost from the mouth and upper pharynx in this group at least 50 million y ago and retained only in the lower pharynx. We identified regional loss of expression of the Ectodysplasin (Eda) signaling ligand as a likely cause of dentition reduction. In addition, we found that overexpression of this gene in the zebrafish is sufficient to restore teeth to the upper pharynx but not to the mouth. Because both regions are competent to respond to Eda signaling with transcriptional output, the likely constraint on the reappearance of oral teeth is the alteration of multiple genetic pathways required for tooth development. The upper pharyngeal teeth are fully formed, but do not exhibit the ancestral relationship to other pharyngeal structures, suggesting that they would not be favored by selection. Our results illustrate an underlying commonality between constraint and selection as explanations for the irreversibility of trait loss; multiple genetic changes would be required to restore teeth themselves to the oral region and optimally functioning ones to the upper pharynx.