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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.

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.

Purpose: The Eiger 2X CdTe 1 M−W (Dectris ltd, Baden, Switzerland) single photon counting detector was characterized for imaging applications at the biomedical beamline ID17 of the European Synchrotron Radiation Facility. Methods: Linearity, Modulation Transfer Function, Noise Power Spectrum and Detective Quantum Efficiency were measured as a function of photon energy and flux in the range 26–80 keV. Results: The linearity was confirmed in the flux range specified by Dectris and a detection efficiency higher than 60 % was measured for energies up to 80 keV. The spatial resolution was inferred from the Modulation Transfer Function and was found to be compatible with the pixel size of the detector (75 μm), except at energies just above the K-edge of Cd and Te where it reached 150 μm. The study of the Noise Power Spectrum showed a time-dependency in the response of the sensor, which is mitigated at low photon fluxes (

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.

Conventional radiotherapy is a widely used non-invasive form of treatment for many types of cancer. However,due to a low threshold in the lung for radiation-induced normal tissue damage, it is of less utility in treating lung cancer. For this reason, surgery is the preferred treatment for lung cancer, which has the detriment of being highly invasive. Non-conventional ultra-high dose rate (FLASH) radiotherapy is currently of great interest in the radiotherapy community due to demonstrations of reduced normal tissue toxicity in lung and other anatomy. This study investigates the effects of FLASH microbeam radiotherapy, which in addition to ultra-high dose rate incorporates a spatial segmentation of the radiation field, on the normal lung tissue of rats. With a focus on fibrotic damage, this work demonstrates that FLASH microbeam radiotherapy provides an order of magnitude increase in normal tissue radio-resistance compared to FLASH radiotherapy. This result suggests FLASH microbeam radiotherapy holds promise for much improved non-invasive control of lung cancer.

The magnitude and distribution of strain imposed on the peripheral airspaces by mechanical ventilation at the microscopic level and the consequent deformations are unknown despite their importance for understanding the mechanisms occurring at the onset of ventilator-induced lung injury. Here a 4-Dimensional (3D + time) image acquisition and processing technique is developed to assess pulmonary acinar biomechanics at microscopic resolution. Synchrotron radiation phase contrast CT with an isotropic voxel size of 6 µm3 is applied in live anesthetized rats under controlled mechanical ventilation. Video animations of regional acinar and vascular strain are acquired in vivo. Maps of strain distribution due to positive-pressure breaths and cardiovascular activity in lung acini and blood vessels are derived based on CT images. Regional strain within the lung peripheral airspaces takes average values of 0.09 ± 0.02. Fitting the expression S = kVn, to the changes in peripheral airspace area (S) and volume (V) during a positive pressure breath yields an exponent n = 0.82 ± 0.03, suggesting predominant alveolar expansion rather than ductal expansion or alveolar recruitment. We conclude that this methodology can be used to assess acinar conformational changes during positive pressure breaths in intact peripheral lung airspaces.

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. Abstract 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.

Ex vivo X-ray phase contrast tomography of amyotrophic lateral sclerosis of a SOD1G93A mouse model is presented. Quantification of neuronal and vascular alteration in the central nervous system is described., 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.

Purpose This study provides the first experimental application of multiscale three-dimensional (3D) X-ray Phase Contrast Imaging Computed Tomography (XPCI-CT) virtual histology for the inspection and quantitative assessment of the late stage effects of radio-induced lesions on lungs in a small animal model. Methods and Materials Healthy male Fischer rats were irradiated with X-ray standard broad beams and Microbeam Radiation Therapy (MRT), a high dose rate (14 kGy/s), FLASH spatially-fractionated X-ray therapy to avoid the beamlets smearing due to cardiosynchronous movements of the organs during the irradiation. After organ dissection, ex-vivo XPCI-CT was applied to all the samples and the results were quantitatively analysed and correlated to histologic data. Results XPCI-CT enables the 3D visualization of lung tissues with unprecedented contrast and sensitivity allowing alveoli, vessels and bronchi hierarchical visualization. XPCI-CT discriminates in 3D radio-induced lesions such as fibrotic scars, Ca/Fe deposits and, in addition, allows a full-organ accurate quantification of the fibrotic tissue within the irradiated organs. The radiation-induced fibrotic tissue content is less than 10% of the analyzed volume for all the MRT treated organs while it reaches the 34% in the case of irradiations with 50 Gy using a broad beam. Conclusions XPCI-CT is an effective imaging technique able to provide detailed 3D information for the assessment of lung pathology and treatment efficacy in a small animal model.

Purpose Image processing plays a fundamental role in the study of central nervous system, for example in the analysis of the vascular network in neurodegenerative diseases. Synchrotron X-ray Phase-contrast micro-Tomography (SXPCT) is a very attractive method to study weakly absorbing samples and features, such as the vascular network in the spinal cord (SC). However, the identification and segmentation of vascular structures in SXPCT images is seriously hampered by the presence of image noise and strong contrast inhomogeneities, due to the sensitivity of the technique to small electronic density variations. In order to help with these tasks, we implemented a user-friendly ImageJ plugin based on a 3D Gaussian steerable filter, tuned up for the enhancement of tubular structures in SXPCT images. Methods The developed 3D Gaussian steerable filter plugin for ImageJ is based on the steerability properties of Gaussian derivatives. We applied it to SXPCT images of ex-vivo mouse SCs acquired at different experimental conditions. Results The filter response shows a strong amplification of the source image contrast-to-background ratio (CBR), independently of structures orientation. We found that after the filter application, the CBR ratio increases by a factor ranging from ~6 to ~60. In addition, we also observed an increase of 35% of the contrast to noise ratio in the case of injured mouse SC. Conclusion The developed tool can generally facilitate the detection/segmentation of capillaries, veins and arteries that were not clearly observable in non-filtered SXPCT images. Its systematic application could allow obtaining quantitative information from pre-clinical and clinical images.

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.

We present data from an implementation of Edge Illumination (EI) that uses a detector aperture designed for increasing dynamic range, suitable for clinically relevant X-ray energies and demonstrated here using synchrotron radiation. By utilising a sufficiently large crosstalk between pixels, this implementation enables single-scan imaging for phase and absorption, and double-scan for phase, absorption and dark field imaging. The presence of the detector mask enables a direct comparison between conventional EI and beam tracking (BT), which we conduct through Monte Carlo and analytical modelling in the case of a single-scan being used for the retrieval of all three contrasts. In the present case, where the X-ray beam width is comparable to the pixel size, we provide an analysis on best-positioning of the beam on the detector for accurate signal retrieval. Further, we demonstrate an application of this method by distinguishing different concentrations of microbubbles via their dark field signals at high energy using an EI system.

Several complementary methods able to visualize the internal structures of eyes are used in the clinical practice in the diagnosis of pathologies affecting a specific zone of the eye. Despite the significant technological progress, the visualization of the entire eyeball at micrometric resolution is yet an unsolved task both in clinical diagnostics and in laboratory research. With this respect, high resolution 3D images of the eyeball would be extremely useful, in the study of various pathologies of the retina, the lens, and of the optic nerve. In this work we combined the state-of-the-art of micro computed tomography technology with phase-contrast imaging, a recent highly sensitive technique well adapted to investigate soft tissues without the use of contrast agents; we applied the technique in the post-mortem analysis of monkey eyes, which share several similitudes with the human organ. We reported here vascular, nervous and anatomical details of monkey eyes imaged with a 3.1 × 3.1 × 3.1 µm3 voxel size as well as the first 3D visualisation of the entire globe of Macaca’s fascicularis eye. Results have also been compared with, and validated by, histological analysis.

[Cu(thp)4]PF6, [Cu(PTA)4]PF6, [Au(thp)4]PF6 and [Au(PTA)4]PF6 are phosphane (thp = tris(hydroxymethyl)phosphane; PTA = 1,3,5-triaza-7-phosphaadamantane) copper(I) and gold(I) water-soluble complexes characterized by high anticancer activity in a wide range of solid tumors, often able to overcome drug resistance of platinum-based compounds. For these reasons, they have been proposed as a valid alternative to platinum-based chemotherapeutic drugs (e.g., cisplatin and oxaliplatin). In vitro experiments performed on organotypic cultures of dorsal root ganglia (DRG) from 15-day-old rat embryos revealed that copper-based compounds were not neurotoxic even at concentrations higher than the IC50 obtained in human cancer cells while [Au(PTA)4]PF6 was neurotoxic at lower concentration than IC50 in cancer cell lines. The ability of these compounds to hinder the proteasome machinery in DRG neurons was tested by fluorimetric assay showing that the non-neurotoxic copper-based complexes do not inhibit proteasome activity in DRG primary neuron cultures. On the contrary, the neurotoxic complex [Au(PTA)4]PF6, induced a significant inhibition of proteasome activity even at concentrations lower than the IC50 in cancer cells. The proteasome inhibition induced by [Au(PTA)4]PF6 was associated with a significant increase in α-tubulin polymerization that was not observed following the treatment with copper-based compounds. Uptake experiments performed by atomic absorption spectrometry showed that both copper-based complexes and [Au(PTA)4]PF6 are internalized in neuron cultures. In vitro and in vivo preliminary data confirmed copper-based complexes as the most promising compounds, not only for their anticancer activity but also concerning the peripheral neurotoxicity profile.

In this work a double-crystal setup is employed to study compound refractive lenses made of single-crystal diamond. The point spread function of the lens is calculated taking into account the lens transmission, the wavefront aberrations, and the ultra-small-angle broadening of the X-ray beam. It is shown that, similarly to the wavefront aberrations, the ultra-small-angle scattering effects can significantly reduce the intensity gain and increase the focal spot size. The suggested approach can be particularly useful for the characterization of refractive X-ray lenses composed of many tens of unit lenses.A double-crystal setup is used to quantify aberrations and to assess the influence of ultra-small-angle X-ray scattering on the optical properties of a single-crystal diamond compound refractive lens.

K-edge subtraction (KES) imaging is a technique able to map a specific element such as e.g. a contrast agent within the tissues, by exploiting the sharp rise of its absorption coefficient at the K-edge energy. Whereas mainly explored at synchrotron radiation sources, the energy discrimination properties of modern x-ray photon counting detectors (XPCDs) pave the way for an implementation of single-shot KES imaging with conventional polychromatic sources. In this work we present an x-ray CT imaging system based on the innovative Pixie-III detector and discrete reconstruction. The results reported here show that a reliable automatic localization of Barium (above a certain concentration) is possible with a few dozens of tomographic projections for a volume having an axial slice of 512 [Formula: see text] 512 pixels. The final application is a routine high-fidelity 3D mapping of a specific element ready for further morphological quantification by means of x-ray CT with potential promising applications in vivo.

X-ray Photon Counting Detectors (XPCDs) with thick semiconductor sensors and small pixel sizes suffer from a charge-sharing effect which can induce multiple counts from a single interacting photon. This issue degrades not only the energy resolution, but it also worsens the spatial resolution. Multiple counts can be removed by acting on the discriminator threshold or they can be corrected by means of specific acquisition modalities implemented in the readout electronics. In this paper we present the results of the characterization of the Pixirad-1/Pixie-III device, a XPCD carrying a 650 μ m thick CdTe sensor with a small pixel ( 62 × 62 μ m 2 ). The Pixie-III readout system includes programmable energy thresholds and it implements three acquisition modes: a pure photon counting mode and two modes specially designed to correct charge-sharing effects. The measured energy resolution of the three acquisition modes for different energies are here reported. Moreover, we characterize the imaging performance for different combinations of acquisition modes and thresholds by measuring the presampling Modulation Transfer Function (MTF), the Normalized Noise Power Spectrum (NNPS) and by assessing the Noise Equivalent number of Quanta (NEQ).

A crucial issue in the development of therapies to treat pathologies of the central nervous system is represented by the availability of non-invasive methods to study the threedimensional morphology of spinal cord, with a resolution able to characterize its complex vascular and neuronal organization. X-ray phase contrast micro-tomography enables a highquality, 3D visualization of both the vascular and neuronal network simultaneously without the need of contrast agents, destructive sample preparations or sectioning. Until now, high resolution investigations of the post-mortem spinal cord in murine models have mostly been performed in spinal cords removed from the spinal canal. We present here post-mortem phase contrast micro-tomography images reconstructed using advanced computational tools to obtain high-resolution and high-contrast 3D images of the fixed spinal cord without removing the bones and preserving the richness of micro-details available when measuring exposed spinal cords. We believe that it represents a significant step toward the in-vivo application. (C) 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

An efficient nano-sized delivery system is presented here allowing the immobilized, picolinium-tethered organic ligand to be released by X-ray irradiation. A marked difference was observed in the fragmentation efficiency by using conventional Cs-137

Modern ventilatory strategies are based on the assumption that lung terminal airspaces act as isotropic balloons that progressively accommodate gas. Phase contrast synchrotron radiation computed tomography (PCSRCT) has recently challenged this concept, showing that in healthy lungs, deflation mechanisms are based on the sequential de-recruitment of airspaces. Using PCSRCT scans in an animal model of acute respiratory distress syndrome (ARDS), this study examined whether the numerosity (ASnum) and dimension (ASdim) of lung airspaces change during a deflation maneuver at decreasing levels of positive end-expiratory pressure (PEEP) at 12, 9, 6, 3, and 0 cmH2O. Deflation was associated with significant reduction of ASdim both in the whole lung section (passing from from 13.1 &plusmn, 2.0 at PEEP 12 to 7.6 &plusmn, 4.2 voxels at PEEP 0) and in single concentric regions of interest (ROIs). However, the regression between applied PEEP and ASnum was significant in the whole slice (ranging from 188 &plusmn, 52 at PEEP 12 to 146.4 &plusmn, 96.7 at PEEP 0) but not in the single ROIs. This mechanism of deflation in which reduction of ASdim is predominant, differs from the one observed in healthy conditions, suggesting that the peculiar alveolar micromechanics of ARDS might play a role in the deflation process.

Modern bony fishes display a high morphological diversity in their auditory structures. Yet, unraveling the sound-induced in situ interaction of the auditory structures has been challenging. Synchrotron radiation-based imaging techniques with high spatial and temporal resolutions now provide a powerful tool to study the functional morphology of such structures in a non-invasive way. We investigated two species representing two types of otophysic connections, namely, goldfish (Weberian apparatus) and Etroplus canarensis (swimbladder extensions contacting the inner ears) asking how different ancillary auditory structures affect otolith motion. Fishes were subjected to a 200 Hz pure tone in a 2L-standing wave tube-like setup while performing 2D radiography based on hard x-ray phase-contrast imaging. The shakers at each end of the tube were either driven 0° in-phase or 180° out-of-phase resulting in maximized sound pressure or sound-induced particle motion in the tube center, respectively. In both species, saccular otolith motion was more pronounced when the swimbladder walls oscillated under 0° in-phase condition and the motion patterns mainly matched the respective orientation patterns of ciliary bundles on the sensory epithelia. In future, we will quantify the motion patterns of fish auditory structures by applying “sound tomography” to cover the three-dimensional aspect of the moving structures.

Purpose: The intracranial three-dimensional (3D) visualization of the whole volume of the eyeball at micrometric resolution has not been achieved yet either in clinical nor in preclinical diagnostic research. Overcoming this limitation may provide a new tool for clinical and preclinical studies of different pathologies of the various sections of the eye. The aim of this work is to give the first insight of a volumetric visualization at the high resolution of the entire enucleated and intracranial postmortem rabbit eyeballs. Methods: X-ray computed tomography phase-contrast imaging was used to obtain 3D models of enucleated and intracranial rabbit eyes. Images were compared with the ones measured by using optical coherence tomography (OCT) images. The experiment was carried out at the European Synchrotron Radiation Facility. Results: Combining the unique possibilities offered by phase-contrast imaging, microtomography, and the properties of synchrotron radiation, the 3D visualization of the whole eyeball, at an isotropic voxel size of 3.1 μm3, is reported here for the first time. Conclusions: High image contrast is achieved without the necessity of injection of contrast agents, thanks to the superior performances, achieved by x-ray phase-contrast imaging with respect to the conventional radiographic imaging. The measurement protocol developed within this work opens the way for in vivo high-resolution visualization of the entire organ

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.

Background 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. New Method 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. Results 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. Comparison with Existing Methods 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. Conclusions 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.

3D breast modelling for 2D and 3D breast x-ray imaging would benefit from the availability of digital and physical phantoms that reproduce accurately the complexity of the breast anatomy. While a number of groups have produced digital phantoms with increasing level of complexity, physical phantoms reproducing that software approach have been scarcely developed. One possibility is offered by 3D printing technology. This implies the assessment of the energy dependent absorption index β of 3D printing materials for absorption based imaging, as well as the assessment of the refractive index decrement, δ, of the printing material, for phase contrast imaging studies, at the energies of interest for breast imaging. In this work we set-up a procedure and performed a series of measurements (at 30, 45 and 60 keV, at the European Synchrotron Radiation Facility) for assessing the relative value of δ with respect to that of breast tissues, for twelve 3D printing materials. The method included propagation based phase contrast 2D imaging and retrieval of the estimated phase shift map, using the Paganin's algorithm. Breast glandular, adipose and skin tissues were used as reference materials of known ratio δ/β. A percentage difference Δδ was introduced to assess the suitability of the printing materials as tissue substitutes. The accuracy of the method (about 4%) was assessed based on the properties of PMMA and Nylon, acting as gold standard. Results show that, for the above photon energies, ABS is a good substitute for adipose tissue, Hybrid as a substitute of the glandular tissue and PET-G for simulating the skin. We plan to realize a breast phantom manufactured by fused deposition modelling (FDM) technology using ABS, Hybrid and PET-G as substitutes of the glandular and skin tissue and a second phantom by stereolithography (SLA) technology with the resins Flex, Tough and Black.

Chronic pain (CP) is a condition characterized by a wide spectrum of clinical signs and symptoms, missing a sound modelling at the neuronal network scale. Recently, we presented a general theory showing common electrophysiological traits in different CP rat models, i.e. a collapse of relevant functional connectivity network properties, such as modularity, in the somatosensory thalamo-cortical (TC) network. In this work, we preliminary investigated by anin silicoaccurate simulator of the six-layer mammalian cortical networks that evidenced the crucial collapse of network modularity in CP simulated conditions and the consequent reduction of network adaptive processes. On this track, in studies on CP experimental animals affected by sciatic nerve multiple ligature (Bennett-Xie model), by synchrotron-generated X-ray microbeam (MB) irradiations (7 parallel beams, 100um width), we targetedin vivothe CP involved hindlimb somatosensory projection cortex that, because of the doses radiation (360 Gy, peak at each beam), non-invasively produced fast and precise tissue destruction along the 7 beam projections. These parcellated the cortical tissue and restored the cortical network statistics related to modularity and information processing efficiency as evidenced from post irradiationin vivoelectrophysiological recordings. In addition, by MB treatment there was an ensuing removal of behavioral signs of allodynia and hyperalgesia accompanied by recovered normal gait schemes yet preserving the normal sensory thresholds of the experimental rats up to three months after the MB irradiation. Finally, novel and unprecedented therapeutic appraisals for CP are devised.Significance StatementChronic pain (CP) is an excruciating condition with severe effects on patients’ life. Apart from many clinical and experimental studies no current theory on CP is generally accepted. Recently, we proposed a general theory of CP in experimental animals as characterized by strong alteration of the connections among neurons in different brain regions. We show here onin silicosimulations that specific connectivity changes in the somatosensory cortex recover the lost functional integrity. Concurrently, in experimental animals, we re-modulated,in vivo, some anatomical connections of the somatosensory cortex by extremely thin synchrotron generated X-ray microbeam irradiations. The resulting behavioral and electrophysiological signs of CP disappeared yet maintaining normal sensory responses. No adverse or pathological effects on blank animals were observable.

International audience; OBJECTIVES:Airway closure is involved in adverse effects of mechanical ventilation under both general anesthesia and in acute respiratory distress syndrome patients. However, direct evidence and characterization of individual airway closure is lacking. Here, we studied the same individual peripheral airways in intact lungs of anesthetized and mechanically ventilated rabbits, at baseline and following lung injury, using high-resolution synchrotron phase-contrast CT.DESIGN:Laboratory animal investigation.SETTING:European synchrotron radiation facility.SUBJECTS:Six New-Zealand White rabbits.INTERVENTIONS:The animals were anesthetized, paralyzed, and mechanically ventilated in pressure-controlled mode (tidal volume, 6 mL/kg; respiratory rate, 40; FIO2, 0.6; inspiratory:expiratory, 1:2; and positive end-expiratory pressure, 3 cm H2O) at baseline. Imaging was performed with a 47.5 × 47.5 × 47.5 μm voxel size, at positive end-expiratory pressure 12, 9, 6, 3, and 0 cm H2O. The imaging sequence was repeated after lung injury induced by whole-lung lavage and injurious ventilation in four rabbits. Cross-sections of the same individual airways were measured.MEASUREMENTS AND MAIN RESULTS:The airways were measured at baseline (n = 48; radius, 1.7 to 0.21 mm) and after injury (n = 32). Closure was observed at 0 cm H2O in three of 48 airways (6.3%; radius, 0.35 ± 0.08 mm at positive end-expiratory pressure 12) at baseline and five of 32 (15.6%; radius, 0.28 ± 0.09 mm) airways after injury. Cross-section was significantly reduced at 3 and 0 cm H2O, after injury, with a significant relation between the relative change in cross-section and airway radius at 12 cm H2O in injured, but not in normal lung (R = 0.60; p < 0.001).CONCLUSIONS:Airway collapsibility increases in the injured lung with a significant dependence on airway caliber. We identify "compliant collapse" as the main mechanism of airway closure in initially patent airways, which can occur at more than one site in individual airways.

The aim of this study was to validate a Geant4 Monte Carlo application for dosimetry measurements at low energies when using water and PMMA phantoms. To measure the water-equivalence in plastic materials, relative dosimetry can be performed by deploying the calculation of depth dose profiles and output factors. We simulated a quasi-uniform monochromatic X-ray beam, with dimension of 2x2 cm2, perpendicularly impinging on a phantom, with energy ranging from 30 to 140 keV. A comparison of the attenuation of the beam after passing through slabs of PMMA and/or water is presented and validated against experimental measurement performed at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The use of PMMA as a substitute of water at low energies is not recommended for dosimetric studies as the depth dose profiles show discrepancies of at least 3%.