The actin cytoskeleton plays multiple roles in structural colour formation in butterfly wing scales.

Vivid structural colours in butterflies are caused by photonic nanostructures scattering light. Structural colours evolved for numerous biological signalling functions and have important technological applications. Optically, such structures are well understood, however insight into their development in vivo remains scarce. We show that actin is intimately involved in structural colour formation in butterfly wing scales. Using comparisons between iridescent (structurally coloured) and non-iridescent scales in adult and developing H. sara, we show that iridescent scales have more densely packed actin bundles leading to an increased density of reflective ridges. Super-resolution microscopy across three distantly related butterfly species reveals that actin is repeatedly re-arranged during scale development and crucially when the optical nanostructures are forming. Furthermore, actin perturbation experiments at these later developmental stages resulted in near total loss of structural colour in H. sara. Overall, this shows that actin plays a vital and direct templating role during structural colour formation in butterfly scales, providing ridge patterning mechanisms that are likely universal across lepidoptera.

Non-destructive 3D exploration of silicate glass corrosion: a combined multiscale approach from the macro to the nanoscale.

In this research, a comprehensive multi-technique analysis, including synchrotron-based X-ray micro-computed tomography, is used to visualize the microstructure of alteration in a very particular Roman glass fragment, in which millennia of corrosion history have not significantly impacted the integrity of the fragment itself. This exceptionally rare occurrence has allowed for the maximization of meaningful data acquisition, by examining the alteration structures from the macro to the nanoscale. This study elucidates the intricate mechanisms underlying glass corrosion when in contact with soil, providing quantitative data and phase correlations in the alteration structures. These findings validate and refine existing predictive corrosion models.

Characterization of transient and progressive pulmonary fibrosis by spatially correlated phase contrast microCT, classical histopathology and atomic force microscopy.

Pulmonary fibrosis (PF) is a severe and progressive condition in which the lung becomes scarred over time resulting in pulmonary function impairment. Classical histopathology remains an important tool for micro-structural tissue assessment in the diagnosis of PF. A novel workflow based on spatial correlated propagation-based phase-contrast micro computed tomography (PBI-microCT), atomic force microscopy (AFM) and histopathology was developed and applied to two different preclinical mouse models of PF - the commonly used and well characterized Bleomycin-induced PF and a novel mouse model for progressive PF caused by conditional Nedd4-2 KO. The aim was to integrate structural and mechanical features from hallmarks of fibrotic lung tissue remodeling. PBI-microCT was used to assess structural alteration in whole fixed and paraffin embedded lungs, allowing for identification of fibrotic foci within the 3D context of the entire organ and facilitating targeted microtome sectioning of planes of interest for subsequent histopathology. Subsequently, these sections of interest were subjected to AFM to assess changes in the local tissue stiffness of previously identified structures of interest. 3D whole organ analysis showed clear morphological differences in 3D tissue porosity between transient and progressive PF and control lungs. By integrating the results obtained from targeted AFM analysis, it was possible to discriminate between the Bleomycin model and the novel conditional Nedd4-2 KO model using agglomerative cluster analysis. As our workflow for 3D spatial correlation of PBI, targeted histopathology and subsequent AFM is tailored around the standard procedure of formalin-fixed paraffin-embedded (FFPE) tissue specimens, it may be a powerful tool for the comprehensive tissue assessment beyond the scope of PF and preclinical research.

Deep learning to overcome Zernike phase-contrast nanoCT artifacts for automated micro-nano porosity segmentation in bone.

Bone material contains a hierarchical network of micro- and nano-cavities and channels, known as the lacuna-canalicular network (LCN), that is thought to play an important role in mechanobiology and turnover. The LCN comprises micrometer-sized lacunae, voids that house osteocytes, and submicrometer-sized canaliculi that connect bone cells. Characterization of this network in three dimensions is crucial for many bone studies. To quantify X-ray Zernike phase-contrast nanotomography data, deep learning is used to isolate and assess porosity in artifact-laden tomographies of zebrafish bones. A technical solution is proposed to overcome the halo and shade-off domains in order to reliably obtain the distribution and morphology of the LCN in the tomographic data. Convolutional neural network (CNN) models are utilized with increasing numbers of images, repeatedly validated by `error loss' and `accuracy' metrics. U-Net and Sensor3D CNN models were trained on data obtained from two different synchrotron Zernike phase-contrast transmission X-ray microscopes, the ANATOMIX beamline at SOLEIL (Paris, France) and the P05 beamline at PETRA III (Hamburg, Germany). The Sensor3D CNN model with a smaller batch size of 32 and a training data size of 70 images showed the best performance (accuracy 0.983 and error loss 0.032). The analysis procedures, validated by comparison with human-identified ground-truth images, correctly identified the voids within the bone matrix. This proposed approach may have further application to classify structures in volumetric images that contain non-linear artifacts that degrade image quality and hinder feature identification.

Osteoporosis and Covid-19: Detected similarities in bone lacunar-level alterations via combined AI and advanced synchrotron testing.

While advanced imaging strategies have improved the diagnosis of bone-related pathologies, early signs of bone alterations remain difficult to detect. The Covid-19 pandemic has brought attention to the need for a better understanding of bone micro-scale toughening and weakening phenomena. This study used an artificial intelligence-based tool to automatically investigate and validate four clinical hypotheses by examining osteocyte lacunae on a large scale with synchrotron image-guided failure assessment. The findings indicate that trabecular bone features exhibit intrinsic variability related to external loading, micro-scale bone characteristics affect fracture initiation and propagation, osteoporosis signs can be detected at the micro-scale through changes in osteocyte lacunar features, and Covid-19 worsens micro-scale porosities in a statistically significant manner similar to the osteoporotic condition. Incorporating these findings with existing clinical and diagnostic tools could prevent micro-scale damages from progressing into critical fractures.

Hard X-ray full-field nanoimaging using a direct photon-counting detector.

Full-field X-ray nanoimaging is a widely used tool in a broad range of scientific areas. In particular, for low-absorbing biological or medical samples, phase contrast methods have to be considered. Three well established phase contrast methods at the nanoscale are transmission X-ray microscopy with Zernike phase contrast, near-field holography and near-field ptychography. The high spatial resolution, however, often comes with the drawback of a lower signal-to-noise ratio and significantly longer scan times, compared with microimaging. In order to tackle these challenges a single-photon-counting detector has been implemented at the nanoimaging endstation of the beamline P05 at PETRA III (DESY, Hamburg) operated by Helmholtz-Zentrum Hereon. Thanks to the long sample-to-detector distance available, spatial resolutions of below 100 nm were reached in all three presented nanoimaging techniques. This work shows that a single-photon-counting detector in combination with a long sample-to-detector distance allows one to increase the time resolution for in situ nanoimaging, while keeping a high signal-to-noise level.

Novel setup for rapid phase contrast CT imaging of heavy and bulky specimens.

This work introduces a novel setup for computed tomography of heavy and bulky specimens at the SYRMEP beamline of the Italian synchrotron Elettra. All the key features of the setup are described and the first application to off-center computed tomography scanning of a human chest phantom (approximately 45 kg) as well as the first results for vertical helical acquisitions are discussed.

Mechanical characterization of miniaturized 3D-printed hydroxyapatite parts obtained through vat photopolymerization: an experimental study.

Hydroxyapatite is one of the materials of choice for tissue engineering bone scaffolds manufacturing. Vat photopolymerization (VPP) is a promising Additive Manufacturing (AM) technology capable of producing scaffolds with high resolution micro-architecture and complex shapes. However, mechanical reliability of ceramic scaffolds can be achieved if a high fidelity printing process is obtained and if knowledge of the intrinsic mechanical properties of the constituent material is available. As the hydroxyapatite (HAP) obtained from VPP is subjected to a sintering process, the mechanical properties of the material should be assessed with specific reference to the process parameters (e.g. sintering temperature) and to the specific characteristic size of the microscopic features in the scaffolds. In order to tackle this challenge the HAP solid matrix of the scaffold was mimicked in the form of miniaturized samples suitable for ad hoc mechanical characterization, which is an unprecedented approach. To this purpose small scale HAP samples, having a simple geometry and size similar to that of the scaffolds, were produced through VPP. The samples were subjected to geometric characterization and to mechanical laboratory tests. Confocal laser scanning and Computed micro-Tomography (micro-CT) were used for geometric characterization; while, micro-bending and nanoindentation were used for mechanical testing. Micro-CT analyses have shown a highly dense material with negligible intrinsic micro-porosity. The imaging process allowed quantifying the variation of geometry with respect to the nominal size showing high accuracy of the printing process and identifying printing defects on one specific sample type, depending on the printing direction. The mechanical tests have shown that the VPP produces HAP with an elastic modulus as high as approximately 100GPa and flexural strength of approximately 100MPa. The results of this study have shown that vat photopolymerization is a promising technology capable of producing high quality HAP with reliable geometric fidelity.

Wafer-Scale Fabrication of Hierarchically Porous Silicon and Silica by Active Nanoparticle-Assisted Chemical Etching and Pseudomorphic Thermal Oxidation.

Many biological materials exhibit a multiscale porosity with small, mostly nanoscale pores as well as large, macroscopic capillaries to simultaneously achieve optimized mass transport capabilities and lightweight structures with large inner surfaces. Realizing such a hierarchical porosity in artificial materials necessitates often sophisticated and expensive top-down processing that limits scalability. Here, an approach that combines self-organized porosity based on metal-assisted chemical etching (MACE) with photolithographically induced macroporosity for the synthesis of single-crystalline silicon with a bimodal pore-size distribution is presented, i.e., hexagonally arranged cylindrical macropores with 1 µm diameter separated by walls that are traversed by pores 60 nm across. The MACE process is mainly guided by a metal-catalyzed reduction-oxidation reaction, where silver nanoparticles (AgNPs) serve as the catalyst. In this process, the AgNPs act as self-propelled particles that are constantly removing silicon along their trajectories. High-resolution X-ray imaging and electron tomography reveal a resulting large open porosity and inner surface for potential applications in high-performance energy storage, harvesting and conversion or for on-chip sensorics and actuorics. Finally, the hierarchically porous silicon membranes can be transformed structure-conserving by thermal oxidation into hierarchically porous amorphous silica, a material that could be of particular interest for opto-fluidic and (bio-)photonic applications due to its multiscale artificial vascularization.

No Child Should Be Left Behind by COVID-19: A Report about the COVID-19 Pandemic Experience in Children and Adolescents with Acute or Chronic Disease Treated at a Pediatric Referral Hospital in Italy.

BACKGROUND: The pandemic of coronavirus disease 2019 (COVID-19) was undoubtedly a stressful experience for everyone. General opinion believed that children with acute or chronic illness could experience additional burden, but this is not confirmed. The aim of this study is to understand how children and adolescents already suffering from acute or chronic illness (e.g., cancer, cystic fibrosis, neuropsychiatric disorders) feel about the COVID-19 pandemic, and if the experience is significantly different between these children and children without illness. METHODS: Children and adolescents affected by acute or chronic illness (named the "fragile group") treated at the Regina Margherita Children Hospital in Italy, were enrolled in the study by filling a questionnaire about their pandemic experiences. Also, a group of children and adolescents without acute or chronic illness (named the "low-risk group") recruited in the emergency department of the hospital, participated in the study in order to compare experiences. RESULTS: The study group was composed of 166 children and adolescents (Median age = 12 yrs; 78% fragile group, 22% low-risk group). Participants experienced a general state of fear of the virus and of a potential infection for both themselves and their families, while feelings and thoughts that interfere with daily functioning were less frequent. The fragile group seems to be more resilient towards the pandemic situation than low-risk group and some differences on the basis of the type of illness were found in the fragile group. CONCLUSIONS: Dedicated psychosocial intervention must be proposed in order to support fragile children and adolescents' well-being during the pandemic, also on the basis of their clinical and mental history.

Morphological and Chemical Investigation of Ovarian Structures in a Bovine Model by Contrast-Enhanced X-ray Imaging and Microscopy.

An improved understanding of an ovary's structures is highly desirable to support advances in folliculogenesis knowledge and reproductive medicine, with particular attention to fertility preservation options for prepubertal girls with malignant tumors. Although currently the golden standard for structural analysis is provided by combining histological sections, staining, and visible 2D microscopic inspection, synchrotron radiation phase-contrast microtomography is becoming a new challenge for three-dimensional studies at micrometric resolution. To this aim, the proper use of contrast agents can improve the visualization of internal structures in ovary tissues, which normally present a low radiopacity. In this study, we report a comparison of four staining protocols, based on iodine or tungsten containing agents, applied to bovine ovarian tissues fixed in Bouin's solution. The microtomography (microCT) analyses at two synchrotron facilities under different set-ups were performed at different energies in order to maximize the image contrast. While tungsten-based agents allow large structures to be well identified, Iodine ones better highlight smaller features, especially when acquired above the K-edge energy of the specific metal. Further scans performed at lower energy where the setup was optimized for overall quality and sensitivity from phase-contrast still provided highly resolved visualization of follicular and intrafollicular structures at different maturation stages, independent of the staining protocol. The analyses were complemented by X-ray Fluorescence mapping on 2D sections, showing that the tungsten-based agent has a higher penetration in this type of tissues.

Three-Dimensional Study of Polymer Composite Destruction in the Early Stages.

The investigation of destruction processes in composite materials is a current problem for their structural application and the improvement of their functional properties. This work aimed to visualize structural changes induced in layered carbon fiber reinforced plastics (CFRP) with the help of synchrotron X-ray microtomography. This article presents the details of destructive processes in the early stages of the deformation of reinforced polymers under uniaxial stretching, investigated at the micro level. Individual structural elements of the composite-filaments, parallel fiber bundles, the nonuniformity of the polymer binder distribution, and continuity defects-were observed under an external load. We have considered the influence of the material architecture and technological defects on fracture evolution in cross-ply and quasi-isotropic fiber-reinforced plastics. The results indicate the sequence of irreversible structural changes before the destruction of the material.

On stars and spikes: Resolving the skeletal morphology of planktonic Acantharia using synchrotron X-ray nanotomography and deep learning image segmentation.

Acantharia (Acantharea) are wide-spread marine protozoa, presenting one of the rare examples of strontium sulfate mineralization in the biosphere. Their endoskeletons consist of 20 spicules arranged according to a unique geometric pattern named Müller's principle. Given the diverse mineral architecture of the Acantharia class, we set out to examine the complex three-dimensional skeletal morphology at the nanometer scale using synchrotron X-ray nanotomography, followed by image segmentation based on deep learning methods. The present study focuses on how the spicules emanate from the robust central junction in the orders Symphyacanthida and Arthracanthida, the geometry of lateral spicule wings as well as pockets of interspicular space, which may be involved in cell compartmentalization. Through these morphometric studies, we observed subtle deviations from the previously described spatial arrangement of the spicules. According to our data, spicule shapes are adjusted in opposite spicules as to accommodate the overall spicule arrangement. In all types examined, previously unknown interspicular interstices were found in areas where radial spicules meet, which could have implications for the crystal growth mechanism and overall endoskeletal integrity. A deeper understanding of the spiculogenesis in Acantharia can provide biomimetic routes towards complex inorganic shapes. STATEMENT OF SIGNIFICANCE: Morphogenesis, the origin and control of shape, provides an avenue towards tailored inorganic materials. In this work, we explored the intricate skeletal organization of planktonic Acantharia, which are amongst the few strontium sulfate biomineralizing organisms in nature. By using nanoscale X-ray imaging and deep learning image segmentation, we found deviations from previously described geometric patterns and undiscovered skeletal features. The bio-inspired synthesis of inorganic materials with complex shape has important ramifications for solid-state chemistry and nanotechnology.

Micromorphology of pineal gland calcification in age-related neurodegenerative diseases.

BACKGROUND: The formation of concrements in human pineal gland (PG) is a physiological process and, according to many researchers, is associated with the involution of PG structures. The majority of scientific publications concern progressive calcification of PG, leaving out studies on the destruction of already formed calcified concrements. Our study fills the gap in knowledge about calcified zones destruction in PG in normal aging and neuropathological conditions, which has not been addressed until now. PURPOSE: Our objective is to gain insight into human PG tissue impairment in both normal aging and neurodegenerative conditions. X-ray phase-contrast tomography (XPCT) allowed us to study PG tissue degeneration at high spatial resolution and, for the first time, to examine the damaged PG concrements in detail. Our research finding could potentially enhance the understanding of the PG involvement in the process of aging as well as in Alzheimer's disease (AD) and vascular dementia (VD). METHODS: The research was carried out on human PG autopsy material in normal aging, VD, and AD conditions. Laboratory-based micro-computed tomography (micro-CT) was used to collect and evaluate samples of native, uncut, and unstained PG with different degrees of pineal calcification. The detailed high-resolution 3D images of the selected PGs were produced using synchrotron-based XPCT. Histology and immunohistochemistry of soft PG tissue confirmed XPCT results. RESULTS: We performed via micro-CT the evaluation of the morphometric parameters of PG such as total sample volume, calcified concrements volume, and percentage of concrements in the total volume of the sample. XPCT imaging revealed high-resolution details of age-related PG alteration. In particular, we noted signs of moderate degradation of concrements in some PGs from elderly donors. In addition, our analysis revealed noticeable degenerative change in both concrements and soft tissue of PGs with neuropathology. In particular, we observed a hollow core and separated layers as well as deep ragged cracks in PG concrements of AD and VD samples. In parenchyma of some samples, we detected wide pinealocyte-free fluid-filled areas adjacent to the calcified zones. CONCLUSION: The present work provides the basis for future scientific research focused on the dynamic nature of PG calcium deposits and PG soft tissue in normal aging and neurodegenerative diseases.

Deep Learning-Based Segmentation of Post-Mortem Human's Olfactory Bulb Structures in X-ray Phase-Contrast Tomography.

The human olfactory bulb (OB) has a laminar structure. The segregation of cell populations in the OB image poses a significant challenge because of indistinct boundaries of the layers. Standard 3D visualization tools usually have a low resolution and cannot provide the high accuracy required for morphometric analysis. X-ray phase contrast tomography (XPCT) offers sufficient resolution and contrast to identify single cells in large volumes of the brain. The numerous microanatomical structures detectable in XPCT image of the OB, however, greatly complicate the manual delineation of OB neuronal cell layers. To address the challenging problem of fully automated segmentation of XPCT images of human OB morphological layers, we propose a new pipeline for tomographic data processing. Convolutional neural networks (CNN) were used to segment XPCT image of native unstained human OB. Virtual segmentation of the whole OB and an accurate delineation of each layer in a healthy non-demented OB is mandatory as the first step for assessing OB morphological changes in smell impairment research. In this framework, we proposed an effective tool that could help to shed light on OB layer-specific degeneration in patients with olfactory disorder.

Machine learning denoising of high-resolution X-ray nanotomography data.

High-resolution X-ray nanotomography is a quantitative tool for investigating specimens from a wide range of research areas. However, the quality of the reconstructed tomogram is often obscured by noise and therefore not suitable for automatic segmentation. Filtering methods are often required for a detailed quantitative analysis. However, most filters induce blurring in the reconstructed tomograms. Here, machine learning (ML) techniques offer a powerful alternative to conventional filtering methods. In this article, we verify that a self-supervised denoising ML technique can be used in a very efficient way for eliminating noise from nanotomography data. The technique presented is applied to high-resolution nanotomography data and compared to conventional filters, such as a median filter and a nonlocal means filter, optimized for tomographic data sets. The ML approach proves to be a very powerful tool that outperforms conventional filters by eliminating noise without blurring relevant structural features, thus enabling efficient quantitative analysis in different scientific fields.

3D Spatial Distribution of Nanoparticles in Mice Brain Metastases by X-ray Phase-Contrast Tomography.

Characterizing nanoparticles (NPs) distribution in multiple and complex metastases is of fundamental relevance for the development of radiological protocols based on NPs administration. In the literature, there have been advances in monitoring NPs in tissues. However, the lack of 3D information is still an issue. X-ray phase-contrast tomography (XPCT) is a 3D label-free, non-invasive and multi-scale approach allowing imaging anatomical details with high spatial and contrast resolutions. Here an XPCT qualitative study on NPs distribution in a mouse brain model of melanoma metastases injected with gadolinium-based NPs for theranostics is presented. For the first time, XPCT images show the NPs uptake at micrometer resolution over the full brain. Our results revealed a heterogeneous distribution of the NPs inside the melanoma metastases, bridging the gap in spatial resolution between magnetic resonance imaging and histology. Our findings demonstrated that XPCT is a reliable technique for NPs detection and can be considered as an emerging method for the study of NPs distribution in organs.

Evaluating the morphology of the degradation layer of pure magnesium via 3D imaging at resolutions below 40 nm.

Magnesium is attractive for the application as a temporary bone implant due to its inherent biodegradability, non-toxicity and suitable mechanical properties. The degradation process of magnesium in physiological environments is complex and is thought to be a diffusion-limited transport problem. We use a multi-scale imaging approach using micro computed tomography and transmission X-ray microscopy (TXM) at resolutions below 40 nm. Thus, we are able to evaluate the nanoporosity of the degradation layer and infer its impact on the degradation process of pure magnesium in two physiological solutions. Magnesium samples were degraded in simulated body fluid (SBF) or Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) for one to four weeks. TXM reveals the three-dimensional interconnected pore network within the degradation layer for both solutions. The pore network morphology and degradation layer composition are similar for all samples. By contrast, the degradation layer thickness in samples degraded in SBF was significantly higher and more inhomogeneous than in DMEM+10%FBS. Distinct features could be observed within the degradation layer of samples degraded in SBF, suggesting the formation of microgalvanic cells, which are not present in samples degraded in DMEM+10%FBS. The results suggest that the nanoporosity of the degradation layer and the resulting ion diffusion processes therein have a limited influence on the overall degradation process. This indicates that the influence of organic components on the dampening of the degradation rate by the suppression of microgalvanic degradation is much greater in the present study.

Discovery of Lebambromyia in Myanmar Cretaceous Amber: Phylogenetic and Biogeographic Implications (Insecta, Diptera, Phoroidea).

Lebambromyia sacculifera sp. nov. is described from Late Cretaceous amber from Myanmar, integrating traditional observation techniques and X-ray phase contrast microtomography. Lebambromyia sacculifera is the second species of Lebambromyia after L. acrai Grimaldi and Cumming, described from Lebanese amber (Early Cretaceous), and the first record of this taxon from Myanmar amber, considerably extending the temporal and geographic range of this genus. The new specimen bears a previously undetected set of phylogenetically relevant characters such as a postpedicel sacculus and a prominent clypeus, which are shared with Ironomyiidae and Eumuscomorpha. Our cladistic analyses confirmed that Lebambromyia represented a distinct monophyletic lineage related to Platypezidae and Ironomyiidae, though its affinities are strongly influenced by the interpretation and coding of the enigmatic set of features characterizing these fossil flies.

Collagen Fiber Orientation Is Coupled with Specific Nano-Compositional Patterns in Dark and Bright Osteons Modulating Their Biomechanical Properties.

Bone continuously adapts to its mechanical environment by structural reorganization to maintain mechanical strength. As the adaptive capabilities of bone are portrayed in its nano- and microstructure, the existence of dark and bright osteons with contrasting preferential collagen fiber orientation (longitudinal and oblique-angled, respectively) points at a required tissue heterogeneity that contributes to the excellent fracture resistance mechanisms in bone. Dark and bright osteons provide an exceptional opportunity to deepen our understanding of how nanoscale tissue properties influence and guide fracture mechanisms at larger length scales. To this end, a comprehensive structural, compositional, and mechanical assessment is performed using circularly polarized light microscopy, synchrotron nanocomputed tomography, focused ion beam/scanning electron microscopy, quantitative backscattered electron imaging, Fourier transform infrared spectroscopy, and nanoindentation testing. To predict how the mechanical behavior of osteons is affected by shifts in collagen fiber orientation, finite element models are generated. Fundamental disparities between both osteon types are observed: dark osteons are characterized by a higher degree of mineralization along with a higher ratio of inorganic to organic matrix components that lead to higher stiffness and the ability to resist plastic deformation under compression. On the contrary, bright osteons contain a higher fraction of collagen and provide enhanced ductility and energy dissipation due to lower stiffness and hardness.