Obliteration of portal venules contributes to portal hypertension in biliary cirrhosis.

The effects of the obliteration of portal venules (OPV) in cirrhotic portal hypertension are poorly understood. To investigate its contribution to portal hypertension in biliary cirrhosis and its underlying mechanism, we evaluated OPV using two-dimensional (2D) histopathology in liver explants from patients with biliary atresia (BA, n = 63), primary biliary cholangitis (PBC, n = 18), and hepatitis B-related cirrhosis (Hep-B-cirrhosis, n = 35). Then, three-dimensional (3D) OPV was measured by X-ray phase-contrast CT in two parallel models in rats following bile duct ligation (BDL) or carbon tetrachloride (CCl4) administration, representing biliary cirrhosis and post-necrotic cirrhosis, respectively. The portal pressure was also measured in the two models. Finally, the effects of proliferative bile ducts on OPV were investigated. We found that OPV was significantly more frequent in patients with biliary cirrhosis, including BA (78.57 ± 16.45%) and PBC (60.00 ± 17.15%), than that in Hep-B-cirrhotic patients (29.43 ± 14.94%, p < 0.001). OPV occurred earlier, evidenced by the paired liver biopsy at a Kasai procedure (KP), and was irreversible even after a successful KP in the patients with BA. OPV was also significantly more frequent in the BDL models than in the CCl4 models, as shown by 2D and 3D quantitative analysis. Portal pressure was significantly higher in the BDL model than that in the CCl4 model. With the proliferation of bile ducts, portal venules were compressed and irreversibly occluded, contributing to the earlier and higher portal pressure in biliary cirrhosis. OPV, as a pre-sinusoidal component, plays a key role in the pathogenesis of portal hypertension in biliary cirrhosis. The proliferated bile ducts and ductules gradually take up the 'territory' originally attributed to portal venules and compress the portal venules, which may lead to OPV in biliary cirrhosis. © 2024 The Pathological Society of Great Britain and Ireland.

X-ray phase-contrast tomography of cells manipulated with an optical stretcher.

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.

BEATS: BEAmline for synchrotron X-ray microTomography at SESAME.

The ID10 beamline of the SESAME (Synchrotron-light for Experimental Science and Applications in the Middle East) synchrotron light source in Jordan was inaugurated in June 2023 and is now open to scientific users. The beamline, which was designed and installed within the European Horizon 2020 project BEAmline for Tomography at SESAME (BEATS), provides full-field X-ray radiography and microtomography imaging with monochromatic or polychromatic X-rays up to photon energies of 100 keV. The photon source generated by a 2.9 T wavelength shifter with variable gap, and a double-multilayer monochromator system allow versatile application for experiments requiring either an X-ray beam with high intensity and flux, and/or a partially spatial coherent beam for phase-contrast applications. Sample manipulation and X-ray detection systems are designed to allow scanning samples with different size, weight and material, providing image voxel sizes from 13 µm down to 0.33 µm. A state-of-the-art computing infrastructure for data collection, three-dimensional (3D) image reconstruction and data analysis allows the visualization and exploration of results online within a few seconds from the completion of a scan. Insights from 3D X-ray imaging are key to the investigation of specimens from archaeology and cultural heritage, biology and health sciences, materials science and engineering, earth, environmental sciences and more. Microtomography scans and preliminary results obtained at the beamline demonstrate that the new beamline ID10-BEATS expands significantly the range of scientific applications that can be targeted at SESAME.

Hard X-ray omnidirectional differential phase and dark-field imaging.

Ever since the discovery of X-rays, tremendous efforts have been made to develop new imaging techniques for unlocking the hidden secrets of our world and enriching our understanding of it. X-ray differential phase contrast imaging, which measures the gradient of a sample's phase shift, can reveal more detail in a weakly absorbing sample than conventional absorption contrast. However, normally only the gradient's component in two mutually orthogonal directions is measurable. In this article, omnidirectional differential phase images, which record the gradient of phase shifts in all directions of the imaging plane, are efficiently generated by scanning an easily obtainable, randomly structured modulator along a spiral path. The retrieved amplitude and main orientation images for differential phase yield more information than the existing imaging methods. Importantly, the omnidirectional dark-field images can be simultaneously extracted to study strongly ordered scattering structures. The proposed method can open up new possibilities for studying a wide range of complicated samples composed of both heavy, strongly scattering atoms and light, weakly scattering atoms.

Multiscale photonic imaging of the native and implanted cochlea.

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.

Three-dimensional virtual histology of the human hippocampus based on phase-contrast computed tomography.

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.

Feasibility and safety of synchrotron-based X-ray phase contrast imaging as a technique complementary to histopathology analysis.

X-ray phase contrast imaging (X-PCI) is a powerful technique for high-resolution, three-dimensional imaging of soft tissue samples in a non-destructive manner. In this technical report, we assess the quality of standard histopathological techniques performed on formalin-fixed, paraffin-embedded (FFPE) human tissue samples that have been irradiated with different doses of X-rays in the context of an X-PCI experiment. The data from this study demonstrate that routine histochemical and immunohistochemical staining quality as well as DNA and RNA analyses are not affected by previous X-PCI on human FFPE samples. From these data we conclude it is feasible and acceptable to perform X-PCI on FFPE human biopsies.

Comparative study of calcification in human choroid plexus, pineal gland, and habenula.

Choroid plexus, pineal gland, and habenula tend to accumulate physiologic calcifications (concrements) over a lifetime. However, until now the composition and causes of the intracranial calcifications remain unclear. The detailed analysis of concrements has been done by us using X-ray diffraction analysis (XRD), X-ray diffraction topography (XRDT), micro-CT, X-ray phase-contrast tomography (XPCT), as well as histology and immunohistochemistry (IHC). By combining physical (XRD) and biochemical (IHC) methods, we identified inorganic (hydroxyapatite) and organic (vimentin) components of the concrements. Via XPCT, XRDT, histological, and IHC methods, we assessed the structure of concrements within their appropriate tissue environment in both two and three dimensions. The study found that hydroxyapatite was a major component of all calcified depositions. It should be noted, however, that the concrements displayed distinctive characteristics corresponding to each specific structure of the brain. As a result, our study provides a basis for assessing the pathological and physiological changes that occur in brain structure containing calcifications.

A Novel Three-Dimensional Approach Towards Evaluating Endomyocardial Biopsies for Follow-Up After Heart Transplantation: X-Ray Phase Contrast Imaging and Its Agreement With Classical Histopathology.

Endomyocardial biopsies are the gold standard for surveillance of graft rejection following heart transplantation, and are assessed by classical histopathology using a limited number of previously stained slices from several biopsies. Synchrotron propagation-based X-ray phase contrast imaging is a non-destructive method to image biological samples without tissue preparation, enabling virtual 2D and 3D histopathology. We aimed to show the feasibility of this method to assess acute cellular rejection and its agreement to classical histopathology. Right ventricular biopsies were sampled from 23 heart transplantation recipients (20 males, mean age 54±14 years) as part of standard follow-up. The clinical diagnosis of potential rejection was made using classical histopathology. One additional study sample was harvested and imaged by X-ray phase contrast imaging, producing 3D datasets with 0.65 µm pixel size, and up to 4,320 images per sample. An experienced pathologist graded both histopathological and X-ray phase contrast images in a blinded fashion. The agreement between methods was assessed by weighted kappa, showing substantial agreement (kappa up to 0.80, p < 0.01) between X-ray phase contrast imaging and classical histopathology. X-ray phase contrast imaging does not require tissue processing, allows thorough analysis of a full myocardial sample and allows identification of acute cellular rejection.

Multimodal Image Fusion for X-ray Grating Interferometry.

X-ray grating interferometry (XGI) can provide multiple image modalities. It does so by utilizing three different contrast mechanisms-attenuation, refraction (differential phase-shift), and scattering (dark-field)-in a single dataset. Combining all three imaging modalities could create new opportunities for the characterization of material structure features that conventional attenuation-based methods are unable probe. In this study, we proposed an image fusion scheme based on the non-subsampled contourlet transform and spiking cortical model (NSCT-SCM) to combine the tri-contrast images retrieved from XGI. It incorporated three main steps: (i) image denoising based on Wiener filtering, (ii) the NSCT-SCM tri-contrast fusion algorithm, and (iii) image enhancement using contrast-limited adaptive histogram equalization, adaptive sharpening, and gamma correction. The tri-contrast images of the frog toes were used to validate the proposed approach. Moreover, the proposed method was compared with three other image fusion methods by several figures of merit. The experimental evaluation results highlighted the efficiency and robustness of the proposed scheme, with less noise, higher contrast, more information, and better details.

A novel fusion method for X-ray phase contrast imaging based on fast adaptive bidimensional empirical mode decomposition.

BACKGROUNDS: X-ray phase contrast imaging (XPCI) can separate the attenuation, refraction, and scattering signals of the object. The application of image fusion enables the concentration of distinctive information into a single image. Some methods have been applied in XPCI field, but wavelet-based decomposition approaches often result in loss of original data. OBJECTIVE: To explore the application value of a novel image fusion method for XPCI system and computed tomography (CT) system. METHODS: The means of fast adaptive bidimensional empirical mode decomposition (FABEMD) is considered for image decomposition to avoid unnecessary information loss. A parameter δ is proposed to guide the fusion of bidimensional intrinsic mode functions which contain high-frequency information, using a pulse coupled neural network with morphological gradients (MGPCNN). The residual images are fused by the energy attribute fusion strategy. Image preprocessing and enhancement are performed on the result to ensure its quality. The effectiveness of other image fusion methods has been compared, such as discrete wavelet transforms and anisotropic diffusion fusion. RESULTS: The δ-guided FABEMD-MGPCNN method achieved either the first or second position in objective evaluation metrics with biological samples, as compared to other image fusion methods. Moreover, comparisons are made with other fusion methods used for XPCI. Finally, the proposed method applied in CT show expected results to retain the feature information. CONCLUSIONS: The proposed δ-guided FABEMD-MGPCNN method shows potential feasibility and superiority over traditional and recent image fusion methods for X-ray differential phase contrast imaging and computed tomography systems.

Direct Conversion X-ray Detector with Micron-Scale Pixel Pitch for Edge-Illumination and Propagation-Based X-ray Phase-Contrast Imaging.

In this work, we investigate the potential of employing a direct conversion integration mode X-ray detector with micron-scale pixels in two different X-ray phase-contrast imaging (XPCi) configurations, propagation-based (PB) and edge illumination (EI). Both PB-XPCi and EI-XPCi implementations are evaluated through a wave optics model-numerically simulated in MATLAB-and are compared based on their contrast, edge-enhancement, visibility, and dose efficiency characteristics. The EI-XPCi configuration, in general, demonstrates higher performance compared to PB-XPCi, considering a setup with the same X-ray source and detector. However, absorption masks quality (thickness of X-ray absorption material) and environmental vibration effect are two potential challenges for EI-XPCi employing a detector with micron-scale pixels. Simulation results confirm that the behavior of an EI-XPCi system employing a high-resolution detector is susceptible to its absorption masks thickness and misalignment. This work demonstrates the potential and feasibility of employing a high-resolution direct conversion detector for phase-contrast imaging applications where higher dose efficiency, higher contrast images, and a more compact imaging system are of interest.

Generalized reverse projection method for grating-based phase tomography.

The reverse projection protocol results in fast phase-contrast imaging thanks to its compatibility with conventional computed-tomography scanning. Many researchers have proposed variants. However, all these reverse projection methods in grating-based phase-contrast imaging are built on the hypothesis of the synchronous phase of reference shifting curves in the whole field of view. The hypothesis imposes uniformity and alignment requirements on the gratings, thus the field of view is generally limited. In this paper, a generalized reverse projection method is presented analytically for the case of non-uniform reference in grating-based phase tomography. The method is demonstrated by theoretical derivation, numerical simulations and synchrotron radiation experiments. The influence of imaging position to sensitivity, and the phase-wrapping phenomenon are also discussed. The proposed method combines the advantages of the high efficiency of the reverse projection method and the universal applicability of the phase-stepping method. The authors believe that the method would be used widely in fast and dose-constrained imaging.

Multiscale X-ray phase contrast imaging of human cartilage for investigating osteoarthritis formation.

BACKGROUND: The evolution of cartilage degeneration is still not fully understood, partly due to its thinness, low radio-opacity and therefore lack of adequately resolving imaging techniques. X-ray phase-contrast imaging (X-PCI) offers increased sensitivity with respect to standard radiography and CT allowing an enhanced visibility of adjoining, low density structures with an almost histological image resolution. This study examined the feasibility of X-PCI for high-resolution (sub-) micrometer analysis of different stages in tissue degeneration of human cartilage samples and compare it to histology and transmission electron microscopy. METHODS: Ten 10%-formalin preserved healthy and moderately degenerated osteochondral samples, post-mortem extracted from human knee joints, were examined using four different X-PCI tomographic set-ups using synchrotron radiation the European Synchrotron Radiation Facility (France) and the Swiss Light Source (Switzerland). Volumetric datasets were acquired with voxel sizes between 0.7 × 0.7 × 0.7 and 0.1 × 0.1 × 0.1 µm3. Data were reconstructed by a filtered back-projection algorithm, post-processed by ImageJ, the WEKA machine learning pixel classification tool and VGStudio max. For correlation, osteochondral samples were processed for histology and transmission electron microscopy. RESULTS: X-PCI provides a three-dimensional visualization of healthy and moderately degenerated cartilage samples down to a (sub-)cellular level with good correlation to histologic and transmission electron microscopy images. X-PCI is able to resolve the three layers and the architectural organization of cartilage including changes in chondrocyte cell morphology, chondrocyte subgroup distribution and (re-)organization as well as its subtle matrix structures. CONCLUSIONS: X-PCI captures comprehensive cartilage tissue transformation in its environment and might serve as a tissue-preserving, staining-free and volumetric virtual histology tool for examining and chronicling cartilage behavior in basic research/laboratory experiments of cartilage disease evolution.

3d phase-contrast nanotomography of unstained human skin biopsies may identify morphological differences in the dermis and epidermis between subjects.

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.

Three-dimensional virtual histology of human cerebellum by X-ray phase-contrast tomography.

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.

Development of X-ray phase CT with a hybrid configuration of Lau and Talbot-Lau interferometers.

X-ray phase computed tomography (CT) is used to observe the inside of light materials. In this paper, we report a new study to develop and test a laboratory assembled X-ray phase CT system that comprises an X-ray Lau interferometer, a rotating Mo anode X-ray tube, and a detector with high spatial resolution. The system has a high spatial resolution lower than 10 µm, which is evaluated by differentiating neighbouring carbon fibres in a polymer composite material. The density resolution is approximately 0.035 g/cm3, which enables to successfully distinguish the high-density polyethylene (HDPE, 0.93 g/cm3) from the ultra-low-density polyethylene (ULDPE, 0.88 g/cm3) in the sample. Moreover, the system can be switched to operate on another mode based on a Talbot-Lau interferometer that provides a wider field of view with a moderate spatial resolution (approximately 100 µm). By analyzing sample images of the biological, this study demonstrates the feasibility and advantages of using hybrid configuration of this X-ray phase CT system.

Investigation of the human pineal gland 3D organization by X-ray phase contrast tomography.

Pineal gland (PG) is a part of the human brain epithalamus that plays an important role in sleep, circadian rhythm, immunity, and reproduction. The calcium deposits and lesions in PG interfere with normal function of the organ and can be associated with different health disorders including serious neurological diseases. At the moment, the detailed mechanisms of PG calcifications and PG lesions formation as well as their involvement in pathological processes are not fully understood. The deep and comprehensive study of the structure of the uncut human PG with histological details, poses a stiff challenge to most imaging techniques, due to low spatial resolution, low visibility or to exceedingly aggressive sample preparation. Here, we investigate the whole uncut and unstained human post-mortem PGs by X-ray phase contrast tomography (XPCT). XPCT is an advanced 3D imaging technique, that permits to study of both soft and calcified tissue of a sample at different scales: from the whole organ to cell structure. In our research we simultaneously resolved 3D structure of parenchyma, vascular network and calcifications. Moreover, we distinguished structural details of intact and degenerated PG tissue. We discriminated calcifications with different structure, pinealocytes nuclei and the glial cells processes. All results were validated by histology. Our research clear demonstrated that XPCT is a potential tool for the high resolution 3D imaging of PG morphological features. This technique opens a new perspective to investigate PG dysfunction and understand the mechanisms of onset and progression of diseases involving the pineal gland.

X-ray phase contrast tomography for the investigation of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons. Pre-clinical studies drive the development of animal models that well mimic ALS disorder and enable both the dissection of disease processes and an early assessment of therapy efficacy. A comprehensive knowledge of neuronal and vascular lesions in the brain and spinal cord is an essential factor to understand the development of the disease. Spatial resolution and bidimensional imaging are important drawbacks limiting current neuroimaging tools, while neuropathology relies on protocols that may alter tissue chemistry and structure. In contrast, recent ex vivo studies in mice demonstrated that X-ray phase-contrast tomography enables study of the 3D distribution of both vasculature and neuronal networks, without sample sectioning or use of staining. Here we present our findings on ex vivo SOD1G93A ALS mice spinal cord at a micrometric scale. An unprecedented direct quantification of neuro-vascular alterations at different stages of the disease is shown.

The versatile X-ray beamline of the Munich Compact Light Source: design, instrumentation and applications.

Inverse Compton scattering provides means to generate low-divergence partially coherent quasi-monochromatic, i.e. synchrotron-like, X-ray radiation on a laboratory scale. This enables the transfer of synchrotron techniques into university or industrial environments. Here, the Munich Compact Light Source is presented, which is such a compact synchrotron radiation facility based on an inverse Compton X-ray source (ICS). The recent improvements of the ICS are reported first and then the various experimental techniques which are most suited to the ICS installed at the Technical University of Munich are reviewed. For the latter, a multipurpose X-ray application beamline with two end-stations was designed. The beamline's design and geometry are presented in detail including the different set-ups as well as the available detector options. Application examples of the classes of experiments that can be performed are summarized afterwards. Among them are dynamic in vivo respiratory imaging, propagation-based phase-contrast imaging, grating-based phase-contrast imaging, X-ray microtomography, K-edge subtraction imaging and X-ray spectroscopy. Finally, plans to upgrade the beamline in order to enhance its capabilities are discussed.