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X-rays can penetrate deeply into biological cells and thus allow for examination of their internal structures with high spatial resolution. In this study, X-ray phase-contrast imaging and tomography is combined with an X-ray-compatible optical stretcher and microfluidic sample delivery. Using this setup, individual cells can be kept in suspension while they are examined with the X-ray beam at a synchrotron. From the recorded holograms, 2D phase shift images that are proportional to the projected local electron density of the investigated cell can be calculated. From the tomographic reconstruction of multiple such projections the 3D electron density can be obtained. The cells can thus be studied in a hydrated or even living state, thus avoiding artifacts from freezing, drying or embedding, and can in principle also be subjected to different sample environments or mechanical strains. This combination of techniques is applied to living as well as fixed and stained NIH3T3 mouse fibroblasts and the effect of the beam energy on the phase shifts is investigated. Furthermore, a 3D algebraic reconstruction scheme and a dedicated mathematical description is used to follow the motion of the trapped cells in the optical stretcher for multiple rotations.

Background and Aim: Full‐thickness biopsies of the intestinal wall may be used to study and assess damage to the neurons of the enteric nervous system (ENS), that is, enteric neuropathy. The ENS is difficult to examine due to its localization deep in the intestinal wall and its organization with several connections in diverging directions. Histological sections used in clinical practice only visualize the sample in a two‐dimensional way. X‐ray phase‐contrast micro‐computed tomography (PC‐μCT) has shown potential to assess the cross‐sectional thickness and volume of the ENS in three dimensions (3D). The aim of this study was to explore the potential of PC‐μCT to evaluate its use to determine the size of the ENS. Methods: Full‐thickness biopsies of ileum obtained during surgery from five controls and six patients clinically diagnosed with enteric neuropathy and dysmotility were included. Punch biopsies of 1 mm in diameter and 1 cm in length, from an area containing myenteric plexus, were extracted from paraffin blocks, and scanned with synchrotron‐based PC‐μCT without any staining. Results: The microscopic volumetric structure of the neural tissue (consisting of both ganglia and fascicles) could be determined in all samples. The ratio of neural tissue volume/total tissue volume was higher in controls than in patients with enteric neuropathy (P = 0.013). The patient with the longest disease duration had the lowest ratio. Conclusion: The assessment of neural tissue can be performed in an objective, standardized way, to ensure reproducibility and comparison under physiological and pathological conditions. Further evaluation is needed to examine the role of this method in the diagnosis of enteric neuropathy. [ABSTRACT FROM AUTHOR]

X‐rays can penetrate deeply into biological cells and thus allow for examination of their internal structures with high spatial resolution. In this study, X‐ray phase‐contrast imaging and tomography is combined with an X‐ray‐compatible optical stretcher and microfluidic sample delivery. Using this setup, individual cells can be kept in suspension while they are examined with the X‐ray beam at a synchrotron. From the recorded holograms, 2D phase shift images that are proportional to the projected local electron density of the investigated cell can be calculated. From the tomographic reconstruction of multiple such projections the 3D electron density can be obtained. The cells can thus be studied in a hydrated or even living state, thus avoiding artifacts from freezing, drying or embedding, and can in principle also be subjected to different sample environments or mechanical strains. This combination of techniques is applied to living as well as fixed and stained NIH3T3 mouse fibroblasts and the effect of the beam energy on the phase shifts is investigated. Furthermore, a 3D algebraic reconstruction scheme and a dedicated mathematical description is used to follow the motion of the trapped cells in the optical stretcher for multiple rotations. [ABSTRACT FROM AUTHOR]

Abstract Background and Aim Full‐thickness biopsies of the intestinal wall may be used to study and assess damage to the neurons of the enteric nervous system (ENS), that is, enteric neuropathy. The ENS is difficult to examine due to its localization deep in the intestinal wall and its organization with several connections in diverging directions. Histological sections used in clinical practice only visualize the sample in a two‐dimensional way. X‐ray phase‐contrast micro‐computed tomography (PC‐μCT) has shown potential to assess the cross‐sectional thickness and volume of the ENS in three dimensions (3D). The aim of this study was to explore the potential of PC‐μCT to evaluate its use to determine the size of the ENS. Methods Full‐thickness biopsies of ileum obtained during surgery from five controls and six patients clinically diagnosed with enteric neuropathy and dysmotility were included. Punch biopsies of 1 mm in diameter and 1 cm in length, from an area containing myenteric plexus, were extracted from paraffin blocks, and scanned with synchrotron‐based PC‐μCT without any staining. Results The microscopic volumetric structure of the neural tissue (consisting of both ganglia and fascicles) could be determined in all samples. The ratio of neural tissue volume/total tissue volume was higher in controls than in patients with enteric neuropathy (P = 0.013). The patient with the longest disease duration had the lowest ratio. Conclusion The assessment of neural tissue can be performed in an objective, standardized way, to ensure reproducibility and comparison under physiological and pathological conditions. Further evaluation is needed to examine the role of this method in the diagnosis of enteric neuropathy.

[Display omitted] Amyloid- β (A β) plaques from Alzheimer's Disease (AD) can be visualized ex vivo in label-free brain samples using synchrotron X-ray phase-contrast tomography (XPCT). However, for XPCT to be useful as a screening method for amyloid pathology, it is essential to understand which factors drive the detection of A β plaques. The current study was designed to test the hypothesis that A β -related contrast in XPCT could be caused by A β fibrils and/or by metals trapped in the plaques. Fibrillar and elemental compositions of A β plaques were probed in brain samples from different types of AD patients and AD models to establish a relationship between XPCT contrast and A β plaque characteristics. XPCT, micro-Fourier-Transform Infrared spectroscopy and micro-X-Ray Fluorescence spectroscopy were conducted on human samples (one genetic and one sporadic case) and on four transgenic rodent strains (mouse: APPPS1, ArcA β , J20; rat: TgF344). A β plaques from the genetic AD patient were visible using XPCT, and had higher β -sheet content and higher metal levels than those from the sporadic AD patient, which remained undetected by XPCT. A β plaques in J20 mice and TgF344 rats appeared hyperdense on XPCT images, while they were hypodense with a hyperdense core in the case of APPPS1 and ArcA β mice. In all four transgenic strains, β -sheet content was similar, while metal levels were highly variable: J20 (zinc and iron) and TgF344 (copper) strains showed greater metal accumulation than APPPS1 and ArcA β mice. Hence, a hyperdense contrast formation of A β plaques in XPCT images was associated with biometal entrapment within plaques. The role of metals in Alzheimer's disease (AD) has been a subject of continuous interest. It was already known that amyloid- β plaques (A β), the earliest hallmark of AD, tend to trap endogenous biometals like zinc, iron and copper. Here we show that this metal accumulation is the main reason why A β plaques are detected with a new technique called X-ray phase contrast tomography (XPCT). XPCT enables to map the distribution of A β plaques in the whole excised brain without labeling. In this work we describe a unique collection of four transgenic models of AD, together with a human sporadic and a rare genetic case of AD, thus exploring the full spectrum of amyloid contrast in XPCT. [ABSTRACT FROM AUTHOR]

The proximal caudal vertebrae and notochord in thick-toed geckos (TG) (Chondrodactylus turneri, Gray, 1864) were investigated after a 30-day space flight onboard the biosatellite Bion-M1. This region has not been explored in previous studies. Our research focused on finding sites most affected by demineralization caused by microgravity (G0). We used X-ray phase-contrast tomography to study TG samples without invasive prior preparation to clarify our previous findings on the resistance of TG's bones to demineralization in G0. The results of the present study confirmed that geckos are capable of preserving bone mass after flight, as neither cortical nor trabecular bone volume fraction showed statistically significant changes after flight. On the other hand, we observed a clear decrease in the mineralization of the notochordal septum and a substantial rise in intercentrum volume following the flight. To monitor TG's mineral metabolism in G0, we propose to measure the volume of mineralized tissue in the notochordal septum. This technique holds promise as a sensitive approach to track the demineralization process in G0, given that the volume of calcification within the septum is limited, making it easy to detect even slight changes in mineral content. [ABSTRACT FROM AUTHOR]

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.

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 PGalteration.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. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is one of the deadliest and most aggressive cancers, remarkably resilient to current therapeutic treatments. Here, we report preliminary in vivo studies of GBM treatments based on photo-nanotherapeutics to activate synergistic killing mechanisms. Core-shell nanoparticles have been weaponized by combining photophysical properties of a new generation PDT agent (Ir(III) complex) with the thermoplasmonic effects of resonant gold nanospheres. In order to investigate the damages induced in GBM treated with these photoactivable nanosystems, we employed X-ray phase-contrast tomography (XPCT). This high-resolution three-dimensional imaging technique highlighted a vast devascularization process by micro-vessels disruption, which is indicative of tumor elimination without relapse.

With growing molecular evidence for correlations between spatial arrangement of blood vasculature and fundamental immunological functions, carried out in distinct compartments of the subdivided lymph node, there is an urgent need for three-dimensional models that can link these aspects. We reconstructed such models at a 1.84 µm resolution by the means of X-ray phase-contrast imaging with a 2D Talbot array in a short time without any staining. In addition reconstructions are verified in immunohistochemistry staining as well as in ultrastructural analyses. While conventional illustrations of mammalian lymph nodes depict the hilus as a definite point of blood and lymphatic vessel entry and exit, our method revealed that multiple branches enter and emerge from an area that extends up to one third of the organ’s surface. This could be a prerequisite for the drastic and location-dependent remodeling of vascularization, which is necessary for lymph node expansion during inflammation. Contrary to corrosion cast studies we identified B-cell follicles exhibiting a two times denser capillary network than the deep cortical units of the T-cell zone. In addition to our observation of high endothelial venules spatially surrounding the follicles, this suggests a direct connection between morphology and B-cell homing. Our findings will deepen the understanding of functional lymph node composition and lymphocyte migration on a fundamental basis. [ABSTRACT FROM AUTHOR]

With growing molecular evidence for correlations between spatial arrangement of blood vasculature and fundamental immunological functions, carried out in distinct compartments of the subdivided lymph node, there is an urgent need for three-dimensional models that can link these aspects. We reconstructed such models at a 1.84 µm resolution by the means of X-ray phase-contrast imaging with a 2D Talbot array in a short time without any staining. In addition reconstructions are verified in immunohistochemistry staining as well as in ultrastructural analyses. While conventional illustrations of mammalian lymph nodes depict the hilus as a definite point of blood and lymphatic vessel entry and exit, our method revealed that multiple branches enter and emerge from an area that extends up to one third of the organ’s surface. This could be a prerequisite for the drastic and location-dependent remodeling of vascularization, which is necessary for lymph node expansion during inflammation. Contrary to corrosion cast studies we identified B-cell follicles exhibiting a two times denser capillary network than the deep cortical units of the T-cell zone. In addition to our observation of high endothelial venules spatially surrounding the follicles, this suggests a direct connection between morphology and B-cell homing. Our findings will deepen the understanding of functional lymph node composition and lymphocyte migration on a fundamental basis.

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. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is one of the deadliest and most aggressive cancers, remarkably resilient to current therapeutic treatments. Here, we report preliminary in vivo studies of GBM treatments based on photo-nanotherapeutics to activate synergistic killing mechanisms. Core-shell nanoparticles have been weaponized by combining photophysical properties of a new generation PDT agent (Ir(III) complex) with the thermoplasmonic effects of resonant gold nanospheres. In order to investigate the damages induced in GBM treated with these photoactivable nanosystems, we employed X-ray phase-contrast tomography (XPCT). This high-resolution three-dimensional imaging technique highlighted a vast devascularization process by micro-vessels disruption, which is indicative of tumor elimination without relapse. [ABSTRACT FROM AUTHOR]

We have studied the three-dimensional (3D) cytoarchitecture of the human hippocampus in neuropathologically healthy and Alzheimer’s disease (AD) individuals, based on phase-contrast X-ray computed tomography of postmortem human tissue punch biopsies. In view of recent findings suggesting a nuclear origin of AD, we target in particular the nuclear structure of the dentate gyrus (DG) granule cells. Tissue samples of 20 individuals were scanned and evaluated using a highly automated approach of measurement and analysis, combining multiscale recordings, optimized phase retrieval, segmentation by machine learning, representation of structural properties in a feature space, and classification based on the theory of optimal transport. Accordingly, we find that the prototypical transformation between a structure representing healthy granule cells and the pathological state involves a decrease in the volume of granule cell nuclei, as well as an increase in the electron density and its spatial heterogeneity. The latter can be explained by a higher ratio of heterochromatin to euchromatin. Similarly, many other structural properties can be derived from the data, reflecting both the natural polydispersity of the hippocampal cytoarchitecture between different individuals in the physiological context and the structural effects associated with AD pathology. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

The cochlea of our auditory system is an intricate structure deeply embedded in the temporal bone. Compared with other sensory organs such as the eye, the cochlea has remained poorly accessible for investigation, for example, by imaging. This limitation also concerns the further development of technology for restoring hearing in the case of cochlear dysfunction, which requires quantitative information on spatial dimensions and the sensorineural status of the cochlea. Here, we employed X-ray phase-contrast tomography and light-sheet fluorescence microscopy and their combination for multiscale and multimodal imaging of cochlear morphology in species that serve as established animal models for auditory research. We provide a systematic reference for morphological parameters relevant for cochlear implant development for rodent and nonhuman primate models. We simulate the spread of light from the emitters of the optical implants within the reconstructed nonhuman primate cochlea, which indicates a spatially narrow optogenetic excitation of spiral ganglion neurons. [ABSTRACT FROM AUTHOR]

Background: Enteric neuropathy is described in most patients with gastrointestinal dysmotility and may be found together with reduced intraepidermal nerve fiber density (IENFD). The aim of this pilot study was to assess whether three‐dimensional (3d) imaging of skin biopsies could be used to examine various tissue components in patients with gastrointestinal dysmotility. Material and methods: Four dysmotility patients of different etiology and two healthy volunteers were included. From each subject, two 3‐mm punch skin biopsies were stained with antibodies against protein gene product 9.5 or evaluated as a whole with two X‐ray phase‐contrast computed tomography (CT) setups, a laboratory µCT setup and a dedicated synchrotron radiation nanoCT end‐station. Results: Two patients had reduced IENFD, and two normal IENFD, compared with controls. µCT and X‐ray phase‐contrast holographic nanotomography scanned whole tissue specimens, with optional high‐resolution scans revealing delicate structures, without differentiation of various fibers and cells. Irregular architecture of dermal fibers was observed in the patient with Ehlers‐Danlos syndrome and the patient with idiopathic dysmotility showed an abundance of mesenchymal ground substance. Conclusions: 3d phase‐contrast tomographic imaging may be useful to illustrate traits of connective tissue dysfunction in various organs and to demonstrate whether disorganized dermal fibers could explain organ dysfunction. [ABSTRACT FROM AUTHOR]

Light microscopical analysis in two dimensions, combined with immunohistochemistry, is presently the gold standard to describe the enteric nervous system (ENS). Our aim was to assess the usefulness of three-dimensional (3D) imaging by X-ray phase-contrast tomography in evaluating the ENS of the human bowel. Myenteric ganglia were identified in full-thickness biopsies of the ileum and colon by hematoxylin & eosin staining. A1-mm biopsy punch was taken from the paraffin blocks and placed into a Kapton® tube for subsequent tomographic investigation. The samples were scanned, without further preparation, using phase-contrast tomography at two different scales: overview scans (performed with laboratory setups), which allowed localization of the nervous tissue (∼1µm effective voxel size); and high-resolution scans (performed with a synchrotron endstation), which imaged localized regions of 320x320x320 µm3 (176 nm effective voxel size). The contrast allowed us to follow the shape and the size changes of the ganglia, as well as to study their cellular components together with the cells and cellular projections of the periganglional space. Furthermore, it was possible to show the 3D network of the myenteric plexus and to quantify its volume within the samples. Phase-contrast X-ray tomography can be applied for volume analyses of the human ENS and to study tissue components in unstained paraffin-embedded tissue biopsies. This technique could potentially be used to study disease mechanisms, and to compare healthy and diseased tissues in clinical research. [ABSTRACT FROM AUTHOR]

Amyloid-β (Aβ) plaques from Alzheimer's Disease (AD) can be visualized ex vivo in label-free brain samples using synchrotron X-ray phase-contrast tomography (XPCT). However, for XPCT to be useful as a screening method for amyloid pathology, it is essential to understand which factors drive the detection of Aβ plaques. The current study was designed to test the hypothesis that Aβ-related contrast in XPCT could be caused by Aβ fibrils and/or by metals trapped in the plaques. Fibrillar and elemental compositions of Aβ plaques were probed in brain samples from different types of AD patients and AD models to establish a relationship between XPCT contrast and Aβ plaque characteristics. XPCT, micro-Fourier-Transform Infrared spectroscopy and micro-X-Ray Fluorescence spectroscopy were conducted on human samples (one genetic and one sporadic case) and on four transgenic rodent strains (mouse: APPPS1, ArcAβ, J20; rat: TgF344). Aβ plaques from the genetic AD patient were visible using XPCT, and had higher β-sheet content and higher metal levels than those from the sporadic AD patient, which remained undetected by XPCT. Aβ plaques in J20 mice and TgF344 rats appeared hyperdense on XPCT images, while they were hypodense with a hyperdense core in the case of APPPS1 and ArcAβ mice. In all four transgenic strains, β-sheet content was similar, while metal levels were highly variable: J20 (zinc and iron) and TgF344 (copper) strains showed greater metal accumulation than APPPS1 and ArcAβ mice. Hence, a hyperdense contrast formation of Aβ plaques in XPCT images was associated with biometal entrapment within plaques. STATEMENT OF SIGNIFICANCE: The role of metals in Alzheimer's disease (AD) has been a subject of continuous interest. It was already known that amyloid-β plaques (Aβ), the earliest hallmark of AD, tend to trap endogenous biometals like zinc, iron and copper. Here we show that this metal accumulation is the main reason why Aβ plaques are detected with a new technique called X-ray phase contrast tomography (XPCT). XPCT enables to map the distribution of Aβ plaques in the whole excised brain without labeling. In this work we describe a unique collection of four transgenic models of AD, together with a human sporadic and a rare genetic case of AD, thus exploring the full spectrum of amyloid contrast in XPCT., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

To quantitatively evaluate brain tissue and its corresponding function, knowledge of the 3D cellular distribution is essential. The gold standard to obtain this information is histology, a destructive and labor-intensive technique where the specimen is sliced and examined under a light microscope, providing 3D information at nonisotropic resolution. To overcome the limitations of conventional histology, we use phase-contrast X-ray tomography with optimized optics, reconstruction, and image analysis, both at a dedicated synchrotron radiation endstation, which we have equipped with X-ray waveguide optics for coherence and wavefront filtering, and at a compact laboratory source. As a proof-of-concept demonstration we probe the 3D cytoarchitecture in millimeter-sized punches of unstained human cerebellum embedded in paraffin and show that isotropic subcellular resolution can be reached at both setups throughout the specimen. To enable a quantitative analysis of the reconstructed data, we demonstrate automatic cell segmentation and localization of over 1 million neurons within the cerebellar cortex. This allows for the analysis of the spatial organization and correlation of cells in all dimensions by borrowing concepts from condensed-matter physics, indicating a strong short-range order and local clustering of the cells in the granular layer. By quantification of 3D neuronal "packing," we can hence shed light on how the human cerebellum accommodates 80% of the total neurons in the brain in only 10% of its volume. In addition, we show that the distribution of neighboring neurons in the granular layer is anisotropic with respect to the Purkinje cell dendrites. [ABSTRACT FROM AUTHOR]

Purpose: Assessing the complex three-dimensional (3D) structure of the cochlea is crucial to understanding the fundamental aspects of signal transduction in the inner ear and is a prerequisite for the development of novel cochlear implants. X-ray phase-contrast computed tomography offers destruction-free 3D imaging with little sample preparation, thus preserving the delicate structure of the cochlea. The use of heavy metal stains enables higher contrast and resolution and facilitates segmentation of the cochlea., Approach: For μ-CT of small animal and human cochlea, we explore the heavy metal osmium tetroxide (OTO) as a radiocontrast agent and delineate laboratory μ - CT from synchrotron CT. We investigate how phase retrieval can be used to improve the image quality of the reconstructions, both for stained and unstained specimens., Results: Image contrast for soft tissue in an aqueous solution is insufficient under the in-house conditions, whereas the OTO stain increases contrast for lipid-rich tissue components, such as the myelin sheaths in nervous tissue, enabling contrast-based rendering of the different components of the auditory nervous system. The overall morphology of the cochlea with the three scalae and membranes is very well represented. Further, the image quality of the reconstructions improves significantly when a phase retrieval scheme is used, which is also suitable for non-ideal laboratory μ - CT settings. With highly brilliant synchrotron radiation (SR), we achieve high contrast for unstained whole cochleae at the cellular level., Conclusions: The OTO stain is suitable for 3D imaging of small animal and human cochlea with laboratory μ - CT , and relevant pathologies, such as a loss of sensory cells and neurons, can be visualized. With SR and optimized phase retrieval, the cellular level can be reached even for unstained samples in aqueous solution, as demonstrated by the high visibility of single hair cells and spiral ganglion neurons., (© 2023 The Authors.)

Purpose: X-ray phase-contrast tomography (XPCT) is a non-destructive, three-dimensional imaging modality that provides higher contrast in soft tissue than absorption-based CT and allows one to cover the cytoarchitecture from the centi- and millimeter scale down to the nanoscale. To further increase contrast and resolution of XPCT, for example, in view of addressing connectivity issues in the central nervous system (CNS), metal staining is indispensable. However, currently used protocols, for example, based on osmium and/or uranium are less suited for XPCT, due to an excessive β / δ -ratio. In this work, we explore the suitability of different staining agents for XPCT. Particularly, neodymium(III)-acetate (NdAc), which has recently been proposed as a non-toxic, non-radioactive easy to use alternative contrast agent for uranyl acetate (UAc) in electron microscopy, is investigated. Due to its vertical proximity to UAc in the periodic table, similar chemical but better suited optical properties for phase contrast can be expected., Approach: Differently stained whole eye samples of wild type mouse and tissues of the CNS are embedded into EPON epoxy resin and scanned using synchrotron as well as with laboratory radiation. Phase retrieval is performed on the projection images, followed by tomographic reconstruction, which enables a quantitative analysis based on the reconstructed electron densities. Segmentation techniques and rendering software is used to visualize structures of interest in the sample., Results: We show that staining neuronal samples with NdAc enhances contrast, in particular for laboratory scans, allowing high-resolution imaging of biological soft tissue in-house. For the example of murine retina, specifically rods and cones as well as the sclera and the Ganglion cell layer seem to be targeted by the stain. A comparison of electron density by the evaluation of histograms allowed to determine quantitative measures to describe the difference between the examined stains., Conclusion: The results suggest NdAc to be an effective stain for XPCT, with a preferential binding to anionic groups, such as phosphate and carboxyl groups at cell surfaces, targeting certain layers of the retina with a stronger selectivity compared to other staining agents. Due to the advantageous X-ray optical properties, the stain seems particularly well-suited for phase contrast, with a comparably small number density and an overall superior image quality at laboratory sources., (© 2023 The Authors.)

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.

Purpose: X-ray phase-contrast tomography (PCT) is a rapidly emerging imaging modality for reconstructing estimates of an object’s three-dimensional x-ray refractive index distribution. Unlike conventional x-ray computed tomography methods, the statistical properties of the reconstructed images in PCT remain unexplored. The purpose of this work is to quantitatively investigate noise propagation in PCT image reconstruction. Methods: The authors derived explicit expressions for the autocovariance of the reconstructed absorption and refractive index images to characterize noise texture and understand how the noise properties are influenced by the imaging geometry. Concepts from statistical detection theory were employed to understand how the imaging geometry-dependent statistical properties affect the signal detection performance in a signal-known-exactly/background-known-exactly task. Results: The analytical formulas for the phase and absorption autocovariance functions were implemented numerically and compared to the corresponding empirical values, and excellent agreement was found. They observed that the reconstructed refractive images are highly spatially correlated, while the absorption images are not. The numerical results confirm that the strength of the covariance is scaled by the detector spacing. Signal detection studies were conducted, employing a numerical observer. The detection performance was found to monotonically increase as the detector-plane spacing was increased. Conclusions: The authors have conducted the first quantitative investigation of noise propagation in PCT image reconstruction. The reconstructed refractive images were found to be highly spatially correlated, while absorption images were not. This is due to the presence of a Fourier space singularity in the reconstruction formula for the refraction images. The statistical analysis may facilitate the use of task-based image quality measures to further develop and optimize this emerging modality for specific applications. [ABSTRACT FROM AUTHOR]

Single-shot X-ray phase-contrast imaging can simplify acquisition processes and reduce the dose delivered to a patient, and also reduce the imaging time, which increases the usability of the system for computed tomography (CT) and dynamic planar imaging. Edge illumination (EI) is a promising method of X-ray phase-contrast imaging that is expected to be implemented in compact devices using conventional X-ray sources. EI-based single-shot X-ray phase-contrast tomography (XPCT) has attracted much attention in recent years. Although single-shot XPCT is possible, further studies are needed before this method is translated into real-world applications. In this paper, a simulation model was built to study EI-baesd single-shot XPCT, including the main hardware conditions (such as the source, mask, detector, objects, and geometric conditions) and some key factors (such as photon statistics, X-ray spectrum, pixel crosstalk, and X-ray absorption). The extraction of information and reconstruction in the absorption, phase, and differential phase signals were implemented, and the effects of pixel crosstalk, photon statistics, and mask thickness on the imaging results were studied. The results suggest that XPCT has significant potential for improving imaging quality and reducing the radiation dose compared to traditional absorption-based CT techniques. The effects of pixel crosstalk are negligible when σ c , the standard deviation of the Gaussian function, is much smaller than the detector pixel as a considerable top width remains in the pixel point spread function. Besides, a mask of considerable thickness is required to ensure the reconstruction accuracy of the quantitative information. [ABSTRACT FROM AUTHOR]

The understanding of structure-function relationships in normal and pathologic mammalian tissues is at the basis of a tissue engineering (TE) approach for the development of biological substitutes to restore or improve tissue function. In this framework, it is interesting to investigate engineered bone tissue, formed when porous ceramic constructs are loaded with bone marrow stromal cells (BMSC) and implanted in vivo. To monitor the relation between bone formation and vascularization, it is important to achieve a detailed imaging and a quantitative description of the complete three-dimensional vascular network in such constructs. Here, we used synchrotron X-ray phase-contrast micro-tomography to visualize and analyze the three-dimensional micro-vascular networks in bone-engineered constructs, in an ectopic bone formation mouse-model. We compared samples seeded and not seeded with BMSC, as well as samples differently stained or unstained. Thanks to the high quality of the images, we investigated the 3D distribution of both vessels and collagen matrix and we obtained quantitative information for all different samples. We propose our approach as a tool for quantitative studies of angiogenesis in TE and for any pre-clinical investigation where a quantitative analysis of the vascular network is required.