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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 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.

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.

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.

Computed tomography (CT) with hard X-rays is a mature technique that is in regular use to depict the interior of opaque specimens with spatial resolutions up to the micrometre range (microtomography or &micro, CT). Short acquisition times and sophisticated contrast modes are accessible when synchrotron light sources are combined with microtomography&mdash, SR-&micro, CT. Both features render SR-&micro, CT as excellent probe to study delicate samples in situ, for example under mechanical load by deploying corresponding sample environments. The so-called TomoPress is such a device available within the public user programme of tomography beamline ID19 of the European Synchrotron Radiation Facility (ESRF). It allows one to study samples under high axial load (up to 500 N) with high spatial resolution up to the micrometer range. Different gauges are installed to allow online monitoring of the applied force. Constant humidity, temperature and wetting are routinely available as well. The article shall outline basic design principles of the press as well as parameters for its utilisation in a descriptive manner. Selected examples underline the potential of the device for such diverse fields as biomedical research, life sciences and materials research.

Chronic pain (CP) represents a complex pathology profoundly involving both neural and glial compartments of the central nervous system. While most CP studies have also investigated the macroscopic brain vascular system, its microstructural architecture still remains largely unexplored. Further, the adaptive modifications of the vascular microstructure as consequence of diseases or pathological insults did not receive adequate attention. Here we show microtomographic signs of diffuse and conspicuous microvascular neogenesis in somatosensory cortex of CP animal models already peaking at 15 days from the model instantiation. Progressive fading of this microvessel neogenesis then ensued in the next six months yet maintaining higher vascular density with a preserved small fraction of them. Due to the important consequences on the neuron-glial-vessel arrangements and on the resulting metabolic and functional disorders of the local networks, novel additional scenarios of CP are thus conceivable with profound consequences of potential future CP diagnostic and therapeutic appraisals. Significance Statement Chronic pain (CP) is an excruciating condition with various clinical presentations. Major signs are characterized by a rich repertoire of pain quality, intensity and length. While neuronal and glial anomalies have been associated to CP less attention has been granted to the micro-vessel and capillary compartments of the involved brain regions. Our research illustrates how the microvessel compartments of the somatosensory cortex in experimental animals present profound and long-lasting signs of extensive neogenesis (about fourfold) early within the first two weeks from the CP start, undergoing a slow and incomplete decline within six months. These results may change the CP clinical picture with potential novel therapeutic approaches to it.

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.

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.

Objectives The purpose of our study was to evaluate the potential of x-ray propagation-based phase-contrast imaging (PCI) computed tomography (CT) for the detection and characterization of early changes after ischemia-reperfusion (IR) in a standardized rat liver transplantation (LTx) model. Materials and Methods Syngeneic orthotopic liver transplantation was performed in male Lewis rats. Ischemia-reperfusion injury (IRI)-induced changes of liver parenchyma were investigated in a time-dependent manner (2, 16, 24, and 32 hours). X-ray phase-contrast images of formalin-fixated liver specimens were acquired in CT mode by using a voxel size of 8 x 8 x 8 mu m(3). Necrapoptotic cell death was visualized with the TdT-mediated dUTP-biotin nick end labeling technique, and alterations of liver graft microhemodynamics, that is, acinar and sinusoidal perfusion failure, were evaluated by in vivo fluorescence microscopy. Results Acquired and reconstructed PCI-CT images showed an increase in necrotic liver parenchyma dependent on cold storage time, measuring 5.7% +/- 1.6% after 2 hours (comparable to 2.6% +/- 0.4% for sham livers), 11.5% +/- 2.1% (16 hours;P < 0.05 vs control), 23.0% +/- 0.5% (24 hours;P < 0.001 vs control), and 31.3% +/- 2.2% (32 hours;P < 0.001 vs control). There were a significant lower number of perfused acini in dependence on increasing cold storage time. The acinar perfusion index reached 0.970 +/- 0.006 after 2 hours of cold ischemia (comparable to 0.960 +/- 0.009 for sham livers) and declined continuously after 16, 24, and 32 hours cold ischemia (0.58 +/- 0.03, 0.49 +/- 0.02, 0.41 +/- 0.03, each P < 0.0001 vs controls). Comparable results were found for sinusoidal perfusion, reaching 1.8% +/- 0.4% of nonperfused sinusoids for 2 hours of cold ischemia and 8.2% +/- 0.8% after 16 hours, 18.8% +/- 1.4% after 24 hours, and 39.0% +/- 2.4% after 32 hours (each P < 0.0001 vs controls). Prolonged cold ischemia was associated with an increasing number of TdT-mediated dUTP-biotin nick end labeling-positive cells (hepatocytes and sinusoidal lining cells), reaching 0.4 +/- 0.1 (sham), 0.7 +/- 0.4 (2 hours), 6.4 +/- 1.1 (16 hours), 2.1 +/- 0.3 (24 hours), and 14.7 +/- 3.5 (32 hours;P = 0.002) for hepatocytes. Conclusions X-ray PCI of histological liver specimens can detect IR-induced tissue necrosis and can provide detailed complementary 3-dimensional information to standard histopathologic findings.

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.

OBJECTIVE. The aim of this study was to quantitatively assess hyaline cartilage and subchondral bone conditions in a fully preserved cadaveric human knee joint using high-resolution x-ray propagation-based phase-contrast imaging (PBI) CT and to compare the performance of the new technique with conventional CT and MRI. MATERIALS AND METHODS. A cadaveric human knee was examined using an x-ray beam of 60 keV, a detector with a 90-mm 2 FOV, and a pixel size of 46 × 46 µm 2 . PBI CT images were reconstructed with both the filtered back projection algorithm and the equally sloped tomography method. Conventional 3-T MRI and CT were also performed. Measurements of cartilage thickness, cartilage lesions, International Cartilage Repair Society scoring, and detection of subchondral bone changes were evaluated. Visual inspection of the specimen akin to arthroscopy was conducted and served as a standard of reference for lesion detection. RESULTS. Loss of cartilage height was visible on PBI CT and MRI. Quantification of cartilage thickness showed a strong correlation between the two modalities. Cartilage lesions appeared darker than the adjacent cartilage on PBI CT. PBI CT showed similar agreement to MRI for depicting cartilage substance defects or lesions compared with the visual inspection. The assessment of subchondral bone cysts showed moderate to strong agreement between PBI CT and CT. CONCLUSION. In contrast to the standard clinical methods of MRI and CT, PBI CT is able to simultaneously depict cartilage and bony changes at high resolution. Though still an experimental technique, PBI CT is a promising high-resolution imaging method to evaluate comprehensive changes of osteoarthritic disease in a clinical setting.

CONTENTS 50.1 Introduction 50.2 General Considerations 50.3 Analyzer-Based Imaging 50.3.1 The Rocking Curve 50.3.2 A Simple Model of AB Image Formation 50.3.2.1 The Sample Introduces Refraction, But No USAXS 50.3.2.2 The Sample Introduces USAXS, But No Refraction 50.3.2.3 The Sample Introduces Both Refraction and USAXS 50.3.3 Phase Retrieval 50.3.3.1 The Sample Introduces Refraction, But No USAXS 50.3.3.2 The Sample Introduces USAXS, But No Refraction 50.3.3.3 The Sample Introduces Both Refraction and USAXS 50.3.3.4 Other Approaches to Phase Retrieval 50.3.4 Requirements 50.4 Edge Illumination 50.4.1 The Illumination Curve 50.4.2 Phase Retrieval and Sensitivity 50.4.3 Coherence Requirements 50.5 Computed Tomography 50.6 Applications to Biomedical Imaging 50.6.1 Breast 50.6.2 Musculoskeletal Phase-Contrast Imaging 50.6.3 Lung 50.6.4 Neuroimaging 50.6.5 Vasculature, Circulation, and Other Tissues 50.7 Conclusion References

The current approach to evaluating cartilage degeneration at the knee joint requires visualization of the joint space on radiographic images where indirect cues such as joint space narrowing serve as markers for osteoarthritis. A recent novel approach to visualizing the knee cartilage matrix using phase contrast imaging (PCI) with computed tomography (CT) was shown to allow direct examination of chondrocyte patterns and their subsequent correlation to osteoarthritis. This study aims to characterize chondrocyte cell patterns in the radial zone of the knee cartilage matrix in the presence and absence of osteoarthritic damage through texture analysis. Statistical features derived from gray-level co-occurrence matrices (GLCM) and geometric features derived from the Scaling Index Method (SIM) were extracted from 404 regions of interest (ROI) annotated on PCI images of healthy and osteoarthritic specimens of knee cartilage. These texture features were then used in a machine learning task to classify ROIs as healthy or osteoarthritic. A fuzzy k-nearest neighbor classifier was used and its performance was evaluated using the area under the Receiver Operating Characteristic (ROC) curve (AUC). The best classification performance was observed with high-dimensional geometrical feature vectors derived from SIM and GLCM correlation features. With the experimental conditions used in this study, both SIM and GLCM achieved a high classification performance (AUC value of 0.98) in the task of distinguishing between healthy and osteoarthritic ROIs. These results show that such quantitative analysis of chondrocyte patterns in the knee cartilage matrix can distinguish between healthy and osteoarthritic tissue with high accuracy.

Phase contrast X-ray computed tomography (PCI-CT) has been demonstrated to be effective for visualization of the human cartilage matrix at micrometer resolution, thereby capturing osteoarthritis induced changes to chondrocyte organization. This study aims to systematically assess the efficacy of deep transfer learning methods for classifying between healthy and diseased tissue patterns. We extracted features from two different convolutional neural network architectures, CaffeNet and Inception-v3 for characterizing such patterns. These features were quantitatively evaluated in a classification task measured by the area (AUC) under the Receiver Operating Characteristic (ROC) curve as well as qualitative visualization through a dimension reduction approach t-Distributed Stochastic Neighbor Embedding (t-SNE). The best classification performance, for CaffeNet, was observed when using features from the last convolutional layer and the last fully connected layer (AUCs > 0.91 ). Meanwhile, off-the-shelf features from Inception-v3 produced similar classification performance (AUC > 0.95 ). Visualization of features from these layers further confirmed adequate characterization of chondrocyte patterns for reliably distinguishing between healthy and osteoarthritic tissue classes. Such techniques, can be potentially used for detecting the presence of osteoarthritis related changes in the human patellar cartilage.

Phase contrast X-ray computed tomography (PCI-CT) has recently emerged as a novel imaging technique that allows visualization of cartilage soft tissue, subsequent examination of chondrocyte patterns, and their correlation to osteoarthritis. Previous studies have shown that 2D texture features are effective at distinguishing between healthy and osteoarthritic regions of interest annotated in the radial zone of cartilage matrix on PCI-CT images. In this study, we further extend the texture analysis to 3D and investigate the ability of volumetric texture features at characterizing chondrocyte patterns in the cartilage matrix for purposes of classification. Here, we extracted volumetric texture features derived from Minkowski Functionals and gray-level co-occurrence matrices (GLCM) from 496 volumes of interest (VOI) annotated on PCI-CT images of human patellar cartilage specimens. The extracted features were then used in a machine-learning task involving support vector regression to classify ROIs as healthy or osteoarthritic. Classification performance was evaluated using the area under the receiver operating characteristic (ROC) curve (AUC). The best classification performance was observed with GLCM features correlation (AUC = 0.83 ± 0.06) and homogeneity (AUC = 0.82 ± 0.07), which significantly outperformed all Minkowski Functionals (p < 0.05). These results suggest that such quantitative analysis of chondrocyte patterns in human patellar cartilage matrix involving GLCM-derived statistical features can distinguish between healthy and osteoarthritic tissue with high accuracy.

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 exvivo 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 invitro imaging of normal and cancerous brain neuromorphology after invivo 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 invivo to the brain of both healthy and cancer-bearing rats. At 45days after treatment, the brain was dissected out and imaged exvivo 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 exvivo 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. &nbsp

The degenerative effects of multiple sclerosis at the level of the vascular and neuronal networks in the central nervous system are currently the object of intensive investigation. Preclinical studies have demonstrated the efficacy of mesenchymal stem cell (MSC) therapy in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis, but the neuropathology of specific lesions in EAE and the effects of MSC treatment are under debate. Because conventional imaging techniques entail protocols that alter the tissues, limiting the reliability of the results, we have used non-invasive X-ray phase-contrast tomography to obtain an unprecedented direct 3D characterization of EAE lesions at micro-to-nano scales, with simultaneous imaging of the vascular and neuronal networks. We reveal EAE-mediated alterations down to the capillary network. Our findings shed light on how the disease and MSC treatment affect the tissues, and promote X-ray phase-contrast tomography as a powerful tool for studying neurovascular diseases and monitoring advanced therapies.

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 50keV) using both 2× and 1× optical magnification systems. The final pixel size resulted in 3.1 and 6.2μm, 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.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 refractive-index gradient vector field approach establishes a connection between a tomographic data set of differential phase contrast images and the distribution of the partial spatial derivatives of the refractive index in an object. The reconstruction of the refractive index in a plane requires the integration of its gradient field. This work shows how this integration can be efficiently performed by converting the problem to the Poisson equation, which can be accurately solved even in the case of noisy and large datasets. The performance of the suggested method is discussed and demonstrated experimentally by computing the refractive index distribution in both a simple plastic phantom and a complex biological sample. The quality of the reconstruction is evaluated through the direct comparison with other commonly used methods. To this end, the refractive index is retrieved from the same data set using also (1) the filtered backprojection algorithm for gradient projections, and (2) the regularized phase-retrieval procedure. Results show that the gradient vector field approach combined with the developed integration technique provides a very accurate depiction of the sample internal structure. Contrary to the two other techniques, the considered method does not require a preliminary phase-retrieval and can be implemented with any advanced computer tomography algorithm. In this work, analyzer-based phase contrast images are used for demonstration. Results, however, are generally valid and can be applied for processing differential phase-contrast tomographic data sets obtained with other phase-contrast imaging techniques.

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 codeGATE/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 inGATE/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 agreementversusboth 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.

A collection of more than 1800 carbonized papyri, discovered in the Roman ‘Villa dei Papiri’ at Herculaneum is the unique classical library survived from antiquity. These papyri were charred during 79 A.D. Vesuvius eruption, a circumstance which providentially preserved them until now. This magnificent collection contains an impressive amount of treatises by Greek philosophers and, especially, Philodemus of Gadara, an Epicurean thinker of 1st century BC. We read many portions of text hidden inside carbonized Herculaneum papyri using enhanced X-ray phase-contrast tomography non-destructive technique and a new set of numerical algorithms for ‘virtual-unrolling’. Our success lies in revealing the largest portion of Greek text ever detected so far inside unopened scrolls, with unprecedented spatial resolution and contrast, all without damaging these precious historical manuscripts. Parts of text have been decoded and the ‘voice’ of the Epicurean philosopher Philodemus is brought back again after 2000 years from Herculaneum papyri.

A collection of more than 1800 carbonized papyri, discovered in the Roman ‘Villa dei Papiri’ at Herculaneum is the unique classical library survived from antiquity. These papyri were charred during 79 A.D. Vesuvius eruption, a circumstance which providentially preserved them until now. This magnificent collection contains an impressive amount of treatises by Greek philosophers and, especially, Philodemus of Gadara, an Epicurean thinker of 1st century BC. We read many portions of text hidden inside carbonized Herculaneum papyri using enhanced X-ray phase-contrast tomography non-destructive technique and a new set of numerical algorithms for ‘virtual-unrolling’. Our success lies in revealing the largest portion of Greek text ever detected so far inside unopened scrolls, with unprecedented spatial resolution and contrast, all without damaging these precious historical manuscripts. Parts of text have been decoded and the ‘voice’ of the Epicurean philosopher Philodemus is brought back again after 2000 years from Herculaneum papyri.

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.

Purpose Edge illumination (EI) X-ray phase-contrast imaging (XPCI) has been under development at University College London in recent years, and has shown great potential for both laboratory and synchrotron applications. In this work, we propose a new acquisition and processing scheme. Contrary to existing retrieval methods for EI, which require as input two images acquired in different setup configurations, the proposed approach can retrieve an approximate map of the X-ray phase from a single image, thus significantly simplifying the acquisition procedure and reducing data collection times. Methods The retrieval method is analytically derived, based on the assumption of a quasi-homogeneous object, i.e. an object featuring a constant ratio between refractive index and absorption coefficient. The noise properties of the input and retrieved images are also theoretically analyzed under the developed formalism. The method is applied to experimental synchrotron images of a biological object. Results The experimental results show that the method can provide high-quality images, where the “edge” signal typical of XPCI images is transformed to an “area” contrast that enables an easier interpretation of the sample geometry. Moreover, the retrieved images confirm that the method is highly stable against noise. Conclusions We anticipate that the developed approach will become the method of choice for a variety of applications of EI XPCI, thanks to its ability to simplify the acquisition procedure and reduce acquisitions time and dose to the sample. Future work will focus on the adaptation of the method to computed tomography and to polychromatic radiation from X-ray tubes.

We report results of the computed tomography reconstruction of the index of refraction in a whole rabbit knee joint examined at the photon energy of 51 keV. Refraction based images make it possible to delineate the bone, cartilage, and soft tissues without adjusting the contrast window width and level. Density variations, which are related to tissue composition and are not visible in absorption X-ray images, are detected in the obtained refraction based images. We discuss why refraction-based images provide better detectability of low contrast features than absorption images.

The application of x-ray phase contrast computed tomography (PCT) to the field of tissue engineering is dis- cussed. Specific focus is on the edge illumination PCT method, which can be adapted to weakly coherent x-ray sources, permitting PCT imaging in standard (non-synchrotron) laboratory environments. The method was applied to a prominent research topic in tissue engineering, namely the development of effective and reliable decellularization protocols to derive scaffolds from native tissue. Results show that edge illumination PCT provides sufficient image quality to evaluate the microstructural integrity of scaffolds and, thus, to assess the performance of the used decellularization technique. In order to highlight that edge illumination PCT can ultimately comply with demands on a high specimen throughput and low doses of radiation, recently developed strategies for scan time and dose reduction are discussed.

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.

Diffraction enhanced imaging (DEI), also called analyzer-based imaging (ABI), cultivated within the synchrotron community, has the potential to revolutionize medical imaging through reduced dose and enhanced contrast imaging.

Over the last two decades phase contrast x-ray imaging techniques have been extensively studied for applications in the biomedical field. Published results demonstrate the high capability of these imaging modalities of improving the image contrast of biological samples with respect to standard absorption-based radiography and routinely used clinical imaging techniques. A clear depiction of the anatomic structures and a more accurate disease diagnosis may be provided by using radiation doses comparable to or lower than those used in current clinical methods. In the literature many works show images of phantoms and excised biological samples proving the high sensitivity of the phase contrast imaging methods for in vitro investigations. In this scenario, the applications of the so-called analyzer-based x-ray imaging (ABI) phase contrast technique are particularly noteworthy. The objective of this work is to demonstrate the feasibility of in vivo x-ray ABI phase contrast imaging for biomedical applications and in particular with respect to joint anatomic depiction and osteoarthritis detection. ABI in planar and tomographic modes was performed in vivo on articular joints of guinea pigs in order to investigate the animals with respect to osteoarthritis by using highlymonochromatic x-rays of 52 keV and a low noise detector with a pixel size of 47 × 47 μm2. Images give strong evidence of the ability of ABI in depicting both anatomic structures in complex systems as living organisms and all known signs of osteoarthritis with high contrast, high spatial resolution and with an acceptable radiation dose. This paper presents the first proof of principle study of in vivo application ofABI. The technical challenges encountered when imaging an animal in vivo are discussed. This experimental study is an important step toward the study of clinical applications of phase contrast x-ray imaging techniques. © 2010 Institute of Physics and Engineering in Medicine.

In x-ray phase-contrast analyzer-based imaging, the contrast is provided by a combination of absorption, refraction and scattering effects. Several extraction algorithms, which attempt to separate and quantify these different physical contributions, have been proposed and applied. In a previous work, we presented a quantitative comparison of five among the most well-known extraction algorithms based on the geometrical optics approximation applied to planar images: diffraction-enhanced imaging (DEI), extended diffraction-enhanced imaging (E-DEI), generalized diffraction-enhanced imaging (G-DEI), multiple-image radiography (MIR) and Gaussian curve fitting (GCF). In this paper, we compare these algorithms in the case of the computed tomography (CT) modality. The extraction algorithms are applied to analyzer-based CT images of both plastic phantoms and biological samples (cartilage-on-bone cylinders). Absorption, refraction and scattering signals are derived. Results obtained with the different algorithms may vary greatly, especially in the case of large refraction angles. We show that ABI-CT extraction algorithms can provide an excellent tool to enhance the visualization of cartilage internal structures, which may find applications in a clinical context. Besides, by using the refraction images, the refractive index decrements for both the cartilage matrix and the cartilage cells have been estimated.

X-ray computed tomography (CT) is a powerful and routinely used clinical diagnostic technique, which is well tolerated by patients, and which provides high-resolution images and volumetric information about the body. However, two important limitations still affect this examination procedure: (1) its low sensitivity with respect to soft tissues, and (2) the hazards associated with x-ray exposure. Conventional radiology is based on the detection of the different photon absorption properties that characterize biological tissues, and thus the obtainable image contrast from soft and/or similar tissues is intrinsically limited. In this scenario, x-ray phase contrast imaging (XPCI) has been extensively tested and proven to overcome some of the main issues surrounding standard x-ray imaging. In addition to the absorption signal, XPCI relies on detecting the phase shifts induced by an object. Interestingly, as the order of magnitude of the phase contrast is higher than that of absorption, XPCI can, in principle, offer higher sensitivity at lower radiation doses. However, other technical aspects may counterbalance this gain, and an optimized setup and image processing solutions need to be implemented. The work presented here describes the strategies and developments we have realized, with the aim of controlling the radiation dose for the highly sensitive and quantitative XPCI-CT. Different algorithms for the phase retrieval and CT reconstruction of the XPCI data are presented. The CT algorithms we have implemented, namely the equally sloped tomography and the dictionary learning method, allow the image quality to be preserved while reducing the number of angular projections required by a factor of five. The results applied to breast imaging report accurate reconstructions at clinically compatible doses of the 3D distribution of the refractive properties of full human organs obtained by using three different phase retrieval methods. The described methodologies and the presented results have been validated by a team of clinical radiologists and represent an important step in the exploitation of XPCI-CT for in vivo and possible clinical applications.

Monitoring the progression of osteoarthritis (OA) and the effects of therapy during clinical trials is still a challenge for present clinical imaging techniques since they present intrinsic limitations and can be sensitive only in case of advanced OA stages. In very severe cases, partial or complete joint replacement surgery is the only solution for reducing pain and restoring the joint functions. Poor imaging quality in practically all medical imaging technologies with respect to joint surfaces and to metal implant imaging calls for the development of new techniques that are sensitive to stages preceding the point of irreversible damage of the cartilage tissue. In this scenario, X-ray phase contrast modalities could play an important role since they can provide improved contrast compared to conventional absorption radiography, with a similar or even reduced tissue radiation dose. In this study, the analyzer-based imaging (ABI), a technique sensitive to the X-ray refraction and permitting a high scatter rejection, has been successfully applied in vitro on excised human synovial joints and sheep implants. Pathological and healthy joints as well as metal implants have been imaged in projection and computed tomography ABI mode at high resolution and clinically compatible doses (

The first practical application of X-rays, more than 100 years ago, was for biomedical imaging. Although not central to the initial development of 3 rd generation synchrotron light sources, imaging techniques have today established themselves as a core element of the research program at all major light sources, including the ESRF, where X-ray imaging has been identified as one of five main themes for its Upgrade Programme [1].

Medical application of synchrotron radiation (SR) is a fast-growing field of research. Since the advent of the angiography studies at SSRL first and then at NSLS in the U.S. in the 1990s, preclinical and clinical research protocols have been developed at Hasylab (Germany), Photon Factory (Japan), ELETTRA (Italia) and at the ESRF (France). Despite the fact that there are only a few dedicated beamlines in the world (two new ones are under construction at the Australian and Canadian synchrotrons), medical research is carried out in almost all synchrotron facilities.

Acellular scaffolds obtained via decellularization are a key instrument in regenerative medicine both per se and to drive the development of future-generation synthetic scaffolds that could become available off-the-shelf. In this framework, imaging is key to the understanding of the scaffolds’ internal structure as well as their interaction with cells and other organs, including ideally post-implantation. Scaffolds of a wide range of intricate organs (esophagus, lung, liver and small intestine) were imaged with x-ray phase contrast computed tomography (PC-CT). Image quality was sufficiently high to visualize scaffold microarchitecture and to detect major anatomical features, such as the esophageal mucosal-submucosal separation, pulmonary alveoli and intestinal villi. These results are a long-sought step for the field of regenerative medicine; until now, histology and scanning electron microscopy have been the gold standard to study the scaffold structure. However, they are both destructive: hence, they are not suitable for imaging scaffolds prior to transplantation and have no prospect for post-transplantation use. PC-CT, on the other hand, is non-destructive, 3D and fully quantitative. Importantly, not only do we demonstrate achievement of high image quality at two different synchrotron facilities, but also with commercial x-ray equipment, which makes the method available to any research laboratory.

International audience; X-ray refraction-based computer tomography imaging is a well-established method for nondestructive investigations of various objects. In order to perform the 3D reconstruction of the index of refraction, two or more raw computed tomography phase-contrast images are usually acquired and combined to retrieve the refraction map (i.e. differential phase) signal within the sample. We suggest an approximate method to extract the refraction signal, which uses a single raw phase-contrast image. This method, here applied to analyzer-based phase-contrast imaging, is employed to retrieve the index of refraction map of a biological sample. The achieved accuracy in distinguishing the different tissues is comparable with the non-approximated approach. The suggested procedure can be used for precise refraction computer tomography with the advantage of a reduction of at least a factor of two of both the acquisition time and the dose delivered to the sample with respect to any of the other algorithms in the literature.

International audience; Phase contrast X-ray computed tomography (PCI-CT) has been demonstrated as a novel imaging technique that can visualize human cartilage with high spatial resolution and soft tissue contrast. Different textural approaches have been previously investigated for characterizing chondrocyte organization on PCI-CT to enable classification of healthy and osteoarthritic cartilage. However, the large size of feature sets extracted in such studies motivates an investigation into algorithmic feature reduction for computing efficient feature representations without compromising their discriminatory power. For this purpose, geometrical feature sets derived from the scaling index method (SIM) were extracted from 1392 volumes of interest (VOI) annotated on PCI-CT images of ex vivo human patellar cartilage specimens. The extracted feature sets were subject to linear and non-linear dimension reduction techniques as well as feature selection based on evaluation of mutual information criteria. The reduced feature set was subsequently used in a machine learning task with support vector regression to classify VOIs as healthy or osteoarthritic; classification performance was evaluated using the area under the receiver-operating characteristic (ROC) curve (AUC). Our results show that the classification performance achieved by 9-D SIM-derived geometric feature sets (AUC: 0.96 +/- 0.02) can be maintained with 2-D representations computed from both dimension reduction and feature selection (AUC values as high as 0.97 +/- 0.02). Thus, such feature reduction techniques can offer a high degree of compaction to large feature sets extracted from PCI-CT images while maintaining their ability to characterize the underlying chondrocyte patterns