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Despite the increased brilliance of the new generation synchrotron sources, there is still a challenge with high‐resolution scanning of very thick and absorbing samples, such as a whole mouse brain stained with heavy elements, and, extending further, brains of primates. Samples are typically cut into smaller parts, to ensure a sufficient X‐ray transmission, and scanned separately. Compared with the standard tomography setup where the sample would be cut into many pillars, the laminographic geometry operates with slab‐shaped sections significantly reducing the number of sample parts to be prepared, the cutting damage and data stitching problems. In this work, a laminography pipeline for imaging large samples (>1 cm) at micrometre resolution is presented. The implementation includes a low‐cost instrument setup installed at the 2‐BM micro‐CT beamline of the Advanced Photon Source. Additionally, sample mounting, scanning techniques, data stitching procedures, a fast reconstruction algorithm with low computational complexity, and accelerated reconstruction on multi‐GPU systems for processing large‐scale datasets are presented. The applicability of the whole laminography pipeline was demonstrated by imaging four sequential slabs throughout an entire mouse brain sample stained with osmium, in total generating approximately 12 TB of raw data for reconstruction. [ABSTRACT FROM AUTHOR]

Recent trends in hard X‐ray micro‐computed tomography (microCT) aim at increasing both spatial and temporal resolutions. These challenges require intense photon beams. Filtered synchrotron radiation beams, also referred to as 'pink beams', which are emitted by wigglers or bending magnets, meet this need, owing to their broad energy range. In this work, the new microCT station installed at the biomedical beamline ID17 of the European Synchrotron is described and an overview of the preliminary results obtained for different biomedical‐imaging applications is given. This new instrument expands the capabilities of the beamline towards sub‐micrometre voxel size scale and simultaneous multi‐resolution imaging. The current setup allows the acquisition of tomographic datasets more than one order of magnitude faster than with a monochromatic beam configuration. [ABSTRACT FROM AUTHOR]

The detection system is a key part of any imaging station. Here the performance of the novel sCMOS-based detection system installed at the ID17 biomedical beamline of the European Synchrotron Radiation Facility and dedicated to high-resolution computed-tomography imaging is analysed. The system consists of an X-ray-visible-light converter, a visible-light optics and a PCO.Edge5.5 sCMOS detector. Measurements of the optical characteristics, the linearity of the system, the detection lag, the modulation transfer function, the normalized power spectrum, the detective quantum efficiency and the photon transfer curve are presented and discussed. The study was carried out at two different X-ray energies (35 and 50 keV) using both 2× and 1× optical magnification systems. The final pixel size resulted in 3.1 and 6.2 µm, respectively. The measured characteristic parameters of the PCO.Edge5.5 are in good agreement with the manufacturer specifications. Fast imaging can be achieved using this detection system, but at the price of unavoidable losses in terms of image quality. The way in which the X-ray beam inhomogeneity limited some of the performances of the system is also discussed. [ABSTRACT FROM AUTHOR]

Medical imaging and radiation therapy are widely used synchrotron-based techniques which have one thing in common: a significant dose delivery to typically biological samples. Among the ways to provide the experimenters with image guidance techniques indicating optimization strategies, Monte Carlo simulation has become the gold standard for accurately predicting radiation dose levels under specific irradiation conditions. A highly important hampering factor of this method is, however, its slow statistical convergence. A track length estimator (TLE) module has been coded and implemented for the first time in the open-source Monte Carlo code GATE/Geant4. Results obtained with the module and the procedures used to validate them are presented. A database of energy-absorption coefficients was also generated, which is used by the TLE calculations and is now also included in GATE/Geant4. The validation was carried out by comparing the TLE-simulated doses with experimental data in a synchrotron radiation computed tomography experiment. The TLE technique shows good agreement versus both experimental measurements and the results of a classical Monte Carlo simulation. Compared with the latter, it is possible to reach a pre-defined statistical uncertainty in about two to three orders of magnitude less time for complex geometries without loss of accuracy. [ABSTRACT FROM AUTHOR]

Purpose : Modern neuroimaging lacks the tools necessary for whole-brain, anatomically dense neuronal damage screening. An ideal approach would include unbiased histopathologic identification of aging and neurodegenerative disease. Methods: We report the postmortem application of multiscale X-ray phase-contrast computed tomography (X-PCI-CT) for the label-free and dissection-free organ-level to intracellular-level 3D visualization of distinct single neurons and glia. In deep neuronal populations in the brain of aged wild-type and of 3xTgAD mice (a triply-transgenic model of Alzheimer's disease), we quantified intracellular hyperdensity, a manifestation of aging or neurodegeneration. Results: In 3xTgAD mice, the observed hyperdensity was identified as amyloid-β and hyper-phosphorylated tau protein deposits with calcium and iron involvement, by correlating the X-PCI-CT data to immunohistochemistry, X-ray fluorescence microscopy, high-field MRI, and TEM. As a proof-of-concept, X-PCI-CT was used to analyze hippocampal and cortical brain regions of 3xTgAD mice treated with LY379268, selective agonist of group II metabotropic glutamate receptors (mGlu2/3 receptors). Chronic pharmacologic activation of mGlu2/3 receptors significantly reduced the hyperdensity particle load in the ventral cortical regions of 3xTgAD mice, suggesting a neuroprotective effect with locoregional efficacy. Conclusions: This multiscale micro-to-nano 3D imaging method based on X-PCI-CT enabled identification and quantification of cellular and sub-cellular aging and neurodegeneration in deep neuronal and glial cell populations in a transgenic model of Alzheimer's disease. This approach quantified the localized and intracellular neuroprotective effects of pharmacological activation of mGlu2/3 receptors. [ABSTRACT FROM AUTHOR]

Despite the diversity in fish auditory structures, it remains elusive how otolith morphology and swim bladder-inner ear (= otophysic) connections affect otolith motion and inner ear stimulation. A recent study visualized sound-induced otolith motion; but tank acoustics revealed a complex mixture of sound pressure and particle motion. To separate sound pressure and sound-induced particle motion, we constructed a transparent standing wave tube-like tank equipped with an inertial shaker at each end while using X-ray phase contrast imaging. Driving the shakers in phase resulted in maximised sound pressure at the tank centre, whereas particle motion was maximised when shakers were driven out of phase (180°). We studied the effects of two types of otophysic connections—i.e. the Weberian apparatus (Carassius auratus) and anterior swim bladder extensions contacting the inner ears (Etroplus canarensis)—on otolith motion when fish were subjected to a 200 Hz stimulus. Saccular otolith motion was more pronounced when the swim bladder walls oscillated under the maximised sound pressure condition. The otolith motion patterns mainly matched the orientation patterns of ciliary bundles on the sensory epithelia. Our setup enabled the characterization of the interplay between the auditory structures and provided first experimental evidence of how different types of otophysic connections affect otolith motion. [ABSTRACT FROM AUTHOR]

Following spinal cord injury (SCI) the degree of functional (motor, autonomous, or sensory) loss correlates with the severity of nervous tissue damage. An imaging technique able to capture non-invasively and simultaneously the complex mechanisms of neuronal loss, vascular damage, and peri-lesional tissue reorganization is currently lacking in experimental SCI studies. Synchrotron X-ray phase-contrast tomography (SXPCT) has emerged as a non-destructive three-dimensional (3D) neuroimaging technique with high contrast and spatial resolution. In this framework, we developed a multi-modal approach combining SXPCT, histology and correlative methods to study neurovascular architecture in normal and spinal level C4-contused mouse spinal cords (C57BL/6J mice, age 2–3 months). The evolution of SCI lesion was imaged at the cell resolution level during the acute (30 min) and subacute (7 day) phases. Spared motor neurons (MNs) were segmented and quantified in different volumes localized at and away from the epicenter. SXPCT was able to capture neuronal loss and blood–brain barrier breakdown following SCI. Three-dimensional quantification based on SXPCT acquisitions showed no additional MN loss between 30 min and 7 days post-SCI. In addition, the analysis of hemorrhagic (at 30 min) and lesion (at 7 days) volumes revealed a high similarity in size, suggesting no extension of tissue degeneration between early and later time-points. Moreover, glial scar borders were unevenly distributed, with rostral edges being the most extended. In conclusion, SXPCT capability to image at high resolution cellular changes in 3D enables the understanding of the relationship between hemorrhagic events and nervous structure damage in SCI. [ABSTRACT FROM AUTHOR]

Objectives: Neoadjuvant chemotherapy is the state-of-the-art treatment in advanced breast cancer. A correct visualization of the post-therapeutic tumor size is of high prognostic relevance. X-ray phase-contrast computed tomography (PC-CT) has been shown to provide improved soft-tissue contrast at a resolution formerly restricted to histopathology, at low doses. This study aimed at assessing ex-vivo the potential use of PC-CT for visualizing the effects of neoadjuvant chemotherapy on breast carcinoma. Materials and Methods: The analysis was performed on two ex-vivo formalin-fixed mastectomy samples containing an invasive carcinoma removed from two patients treated with neoadjuvant chemotherapy. Images were matched with corresponding histological slices. The visibility of typical post-therapeutic tissue changes was assessed and compared to results obtained with conventional clinical imaging modalities. Results: PC-CT depicted the different tissue types with an excellent correlation to histopathology. Post-therapeutic tissue changes were correctly visualized and the residual tumor mass could be detected. PC-CT outperformed clinical imaging modalities in the detection of chemotherapy-induced tissue alterations including post-therapeutic tumor size. Conclusions: PC-CT might become a unique diagnostic tool in the prediction of tumor response to neoadjuvant chemotherapy. PC-CT might be used to assist during histopathological diagnosis, offering a high-resolution and high-contrast virtual histological tool for the accurate delineation of tumor boundaries. [ABSTRACT FROM AUTHOR]

Simple Summary: This study aims at using a multi-technique approach to detect and analyze the effects of high dose rate spatially fractionated radiation therapies and to compare them to seamless broad beam irradiation targeting healthy and glioblastoma-bearing rat brains and delivering three different doses per each irradiation geometry. Brains were analyzed post mortem by multi-scale X-ray phase contrast imaging–computed tomography, histology, immunohistochemistry, X-ray fluorescence, and small- and wide-angle X-ray scattering to achieve detailed visualization, characterization and classification in 3D of the radio-induced effects on brain tissues. The original results bring new insights to the understanding of the response of cerebral tissue and tumors treated with high dose rate spatially fractioned radiotherapies and put the basis for channeling studies of in-vivo applications for monitoring RT effects. The purpose of this study is to use a multi-technique approach to detect the effects of spatially fractionated X-ray Microbeam (MRT) and Minibeam Radiation Therapy (MB) and to compare them to seamless Broad Beam (BB) irradiation. Healthy- and Glioblastoma (GBM)-bearing male Fischer rats were irradiated in-vivo on the right brain hemisphere with MRT, MB and BB delivering three different doses for each irradiation geometry. Brains were analyzed post mortem by multi-scale X-ray Phase Contrast Imaging–Computed Tomography (XPCI-CT), histology, immunohistochemistry, X-ray Fluorescence (XRF), Small- and Wide-Angle X-ray Scattering (SAXS/WAXS). XPCI-CT discriminates with high sensitivity the effects of MRT, MB and BB irradiations on both healthy and GBM-bearing brains producing a first-time 3D visualization and morphological analysis of the radio-induced lesions, MRT and MB induced tissue ablations, the presence of hyperdense deposits within specific areas of the brain and tumor evolution or regression with respect to the evaluation made few days post-irradiation with an in-vivo magnetic resonance imaging session. Histology, immunohistochemistry, SAXS/WAXS and XRF allowed identification and classification of these deposits as hydroxyapatite crystals with the coexistence of Ca, P and Fe mineralization, and the multi-technique approach enabled the realization, for the first time, of the map of the differential radiosensitivity of the different brain areas treated with MRT and MB. 3D XPCI-CT datasets enabled also the quantification of tumor volumes and Ca/Fe deposits and their full-organ visualization. The multi-scale and multi-technique approach enabled a detailed visualization and classification in 3D of the radio-induced effects on brain tissues bringing new essential information towards the clinical implementation of the MRT and MB radiation therapy techniques. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mechanical ventilation can damage the lungs, a condition called Ventilator-Induced Lung Injury (VILI). However, the mechanisms leading to VILI at the microscopic scale remain poorly understood. Here we investigated the within-tidal dynamics of cyclic recruitment/derecruitment (R/D) using synchrotron radiation phase-contrast imaging (PCI), and the relation between R/D and cell infiltration, in a model of Acute Respiratory Distress Syndrome in 6 anaesthetized and mechanically ventilated New-Zealand White rabbits. Dynamic PCI was performed at 22.6 µm voxel size, under protective mechanical ventilation [tidal volume: 6 ml/kg; positive end-expiratory pressure (PEEP): 5 cmH2O]. Videos and quantitative maps of within-tidal R/D showed that injury propagated outwards from non-aerated regions towards adjacent regions where cyclic R/D was present. R/D of peripheral airspaces was both pressure and time-dependent, occurring throughout the respiratory cycle with significant scatter of opening/closing pressures. There was a significant association between R/D and regional lung cellular infiltration (p = 0.04) suggesting that tidal R/D of the lung parenchyma may contribute to regional lung inflammation or capillary-alveolar barrier dysfunction and to the progression of lung injury. PEEP may not fully mitigate this phenomenon even at high levels. Ventilation strategies utilizing the time-dependence of R/D may be helpful in reducing R/D and associated injury. [ABSTRACT FROM AUTHOR]

• X-ray phase-contrast CT (X-PCI-CT) enables label-free 3D nervous-tissue microscopy. • Multiscale X-PCI-CT captures full-organ to intra-cellular spinal cord neuroanatomy. • Combination of aldehyde fixatives optimizes visualizations of deep micro-vasculature. • Osmium impregnation highlights white-matter and individual motor neuron perikarya. • Post-mortem X-PCI-CT resolving power can exceed that of post-mortem high-field MRI. Dense and unbiased cellular-resolution representations of extended volumetric central nervous system soft-tissue anatomy are difficult to obtain, even in experimental post-mortem settings. Interestingly, X-ray phase-contrast computed tomography (X-PCI-CT), an emerging soft-tissue-sensitive volumetric imaging technique, can provide multiscale organ- to cellular-level morphological visualizations of neuroanatomical structure. Here, we tested different nervous-tissue fixation procedures, conventionally used for transmission electron microscopy, to better establish X-PCI-CT-specific sample-preparation protocols. Extracted rat spinal medullas were alternatively fixed with a standard paraformaldehyde-only aldehyde-based protocol, or in combination with glutaraldehyde. Some specimens were additionally post-fixed with osmium tetroxide. Multiscale X-PCI-CT datasets were collected at several synchrotron radiation facilities, using state-of-the-art setups with effective image voxel sizes of 3.03 to 0.33 μm3, and compared to high-field magnetic resonance imaging, histology and vascular fluorescence microscopy data. Multiscale X-PCI-CT of aldehyde-fixed spinal cord specimens resulted in dense histology-like volumetric representations and quantifications of extended deep spinal micro-vascular networks and of intra-medullary cell populations. Osmium post-fixation increased intra-medullary contrast between white and gray-matter tissues, and enhanced delineation of intra-medullary cellular structure, e.g. axon fibers and motor neuron perikarya. Volumetric X-PCI-CT provides complementary contrast and higher spatial resolution compared to 9.4 T MRI. X-PCI-CT's advantage over planar histology is the volumetric nature of the cellular-level data obtained, using samples much larger than those fit for volumetric vascular fluorescence microscopy. Deliberately choosing (post-)fixation protocols tailored for optimal nervous-tissue structural preservation is of paramount importance in achieving effective and targeted neuroimaging via the X-PCI-CT technique. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Purpose: Experimental neuroimaging provides a wide range of methods for the visualization of brain anatomic morphology down to subcellular detail. Still, each technique-specific detection mechanism presents compromises among the achievable field-of-view size, spatial resolution, and nervous tissue sensitivity, leading to partial sample coverage, unresolved morphologic structures, or sparse labeling of neuronal populations and often also to obligatory sample dissection or other sample invasive manipulations. X-ray phase-contrast imaging computed tomography (PCI-CT) is an experimental imaging method that simultaneously provides micrometric spatial resolution, high soft-tissue sensitivity, and ex vivo full organ rodent brain coverage without any need for sample dissection, staining or labeling, or contrast agent injection. In the present study, we explored the benefits and limitations of PCI-CT use for in vitro imaging of normal and cancerous brain neuromorphology after in vivo treatment with synchrotron-generated x-ray microbeam radiation therapy (MRT), a spatially fractionated experimental high-dose radiosurgery. The goals were visualization of the MRT effects on nervous tissue and a qualitative comparison of the results to the histologic and high-field magnetic resonance imaging findings.Methods and Materials: MRT was administered in vivo to the brain of both healthy and cancer-bearing rats. At 45 days after treatment, the brain was dissected out and imaged ex vivo using propagation-based PCI-CT.Results: PCI-CT visualizes the brain anatomy and microvasculature in 3 dimensions and distinguishes cancerous tissue morphology, necrosis, and intratumor accumulation of iron and calcium deposits. Moreover, PCI-CT detects the effects of MRT throughout the treatment target areas (eg, the formation of micrometer-thick radiation-induced tissue ablation). The observed neurostructures were confirmed by histologic and immunohistochemistry examination and related to the micro-magnetic resonance imaging data.Conclusions: PCI-CT enabled a unique 3D neuroimaging approach for ex vivo studies on small animal models in that it concurrently delivers high-resolution insight of local brain tissue morphology in both normal and cancerous micro-milieu, localizes radiosurgical damage, and highlights the deep microvasculature. This method could assist experimental small animal neurology studies in the postmortem evaluation of neuropathology or treatment effects. [ABSTRACT FROM AUTHOR]

Mammography is the primary imaging tool for screening and diagnosis of human breast cancers, but ∼10-20% of palpable tumors are not detectable on mammograms and only about 40% of biopsied lesions are malignant. Here we report a high-resolution, low-dose phase contrast X-ray tomographic method for 3D diagnosis of human breast cancers. By combining phase contrast X-ray imaging with an image reconstruction method known as equally sloped tomography, we imaged a human breast in three dimensions and identified a malignant cancer with a pixel size of 92 μm and a radiation dose less than that of dual-view mammography. According to a blind evaluation by five independent radiologists, our method can reduce the radiation dose and acquisition time by ∼74% relative to conventional phase contrast X-ray tomography, while maintaining high image resolution and image contrast. These results demonstrate that high-resolution 3D diagnostic imaging of human breast cancers can, in principle, be performed at clinical compatible doses. [ABSTRACT FROM AUTHOR]

We applied transfer learning using Convolutional Neuronal Networks to high resolution X-ray phase contrast computed tomography datasets and tested the potential of the systems to accurately classify Computed Tomography images of different stages of two diseases, i.e. osteoarthritis and liver fibrosis. The purpose is to identify a time-effective and observer-independent methodology to identify pathological conditions. Propagation-based X-ray phase contrast imaging WAS used with polychromatic X-rays to obtain a 3D visualization of 4 human cartilage plugs and 6 rat liver samples with a voxel size of 0.7 × 0.7 × 0.7 µm3 and 2.2 × 2.2 × 2.2 µm3, respectively. Images with a size of 224 × 224 pixels are used to train three pre-trained convolutional neuronal networks for data classification, which are the VGG16, the Inception V3, and the Xception networks. We evaluated the performance of the three systems in terms of classification accuracy and studied the effect of the variation of the number of inputs, training images and of iterations. The VGG16 network provides the highest classification accuracy when the training and the validation-test of the network are performed using data from the same samples for both the cartilage (99.8%) and the liver (95.5%) datasets. The Inception V3 and Xception networks achieve an accuracy of 84.7% (43.1%) and of 72.6% (53.7%), respectively, for the cartilage (liver) images. By using data from different samples for the training and validation-test processes, the Xception network provided the highest test accuracy for the cartilage dataset (75.7%), while for the liver dataset the VGG16 network gave the best results (75.4%). By using convolutional neuronal networks we show that it is possible to classify large datasets of biomedical images in less than 25 min on a 8 CPU processor machine providing a precise, robust, fast and observer-independent method for the discrimination/classification of different stages of osteoarthritis and liver diseases. [ABSTRACT FROM AUTHOR]

X-ray phase contrast micro-tomography (μPCI-CT) is a high resolution technique that can be used to investigate vascular and neurodegenerative disorders overcoming the limitations of the conventional imaging modalities. In fact, currently available neuroimaging techniques are based on sample-invasive imaging protocols involving dissections, staining or labeling of nervous system structures. On the other hand, μPCI-CT permits to visualize the spinal cord micro-vasculature, to detect single neuronal cells in the vertebral column and even cells infiltrating the nervous system in pathological conditions. These properties make μPCI-CT a potential powerful instrument in the study of vascular and neurodegenerative disorders as well as in the patient evaluation during medical treatment. Moreover, it would be a powerful instrument to localize in preclinical model of immune mediated diseases ectopic cells infiltrating the nervous system in a multifocal and unpredictable way. To optimize tissue fixation protocols for μPCI-CT analysis, several attempts were performed combining different protein and lipid fixation procedures and time points. The high-resolution synchrotron μPCI-CT setup allowed recognition offull-organ spinal cord anatomy of healthy rats, including anterior/posterior gray horns, the dorsal/ventral roots and ganglions, the central canal and the meninges, was clearly depicted. Superficial and deep vessels were visualized without need of any contrastagent. At the highest resolution used, single neuronal cells perfused by surrounding vasculature were recognized allowing the detection of specific structure such as bundles of nerve fibers, single motor neurons and neuro-glial cells, cell bodies and axons as well as intra-cellular structure (i.e. cell nuclei and nucleoli). Moreover, in preclinical studies, the optimization of protocol for μPCI-CT allowed to localize ectopic infiltrating cells in nervous system organs in both mouse and rat models of Krabbe disease and Multiple Sclerosis which would allow a further accurate analysis of the areas and cell-parenchima fine interaction. [ABSTRACT FROM AUTHOR]