Edge-illumination spectral phase-contrast tomography.

Following the rapid, but independent, diffusion of x-ray spectral and phase-contrast systems, this work demonstrates the first combination of spectral and phase-contrast computed tomography (CT) obtained by using the edge-illumination technique and a CdTe small-pixel (62µm) spectral detector. A theoretical model is introduced, starting from a standard attenuation-based spectral decomposition and leading to spectral phase-contrast material decomposition. Each step of the model is followed by quantification of accuracy and sensitivity on experimental data of a test phantom containing different solutions with known concentrations. An example of a micro CT application (20µm voxel size) on an iodine-perfusedex vivomurine model is reported. The work demonstrates that spectral-phase contrast combines the advantages of spectral imaging, i.e. high-Zmaterial discrimination capability, and phase-contrast imaging, i.e. soft tissue sensitivity, yielding simultaneously mass density maps of water, calcium, and iodine with an accuracy of 1.1%, 3.5%, and 1.9% (root mean square errors), respectively. Results also show a 9-fold increase in the signal-to-noise ratio of the water channel when compared to standard spectral decomposition. The application to the murine model revealed the potential of the technique in the simultaneous 3D visualization of soft tissue, bone, and vasculature. While being implemented by using a broad spectrum (pink beam) at a synchrotron radiation facility (Elettra, Trieste, Italy), the proposed experimental setup can be readily translated to compact laboratory systems including conventional x-ray tubes.

Integrating X-ray phase-contrast imaging and histology for comparative evaluation of breast tissue malignancies in virtual histology analysis.

Detecting breast tissue alterations is essential for cancer diagnosis. However, inherent bidimensionality limits histological procedures' effectiveness in identifying these changes. Our study applies a 3D virtual histology method based on X-ray phase-contrast microtomography (PhC µ CT), performed at a synchrotron facility, to investigate breast tissue samples including different types of lesions, namely intraductal papilloma, micropapillary intracystic carcinoma, and invasive lobular carcinoma. One-to-one comparisons of X-ray and histological images explore the clinical potential of 3D X-ray virtual histology. Results show that PhC µ CT technique provides high spatial resolution and soft tissue sensitivity, while being non-destructive, not requiring a dedicated sample processing and being compatible with conventional histology. PhC µ CT can enhance the visualization of morphological characteristics such as stromal tissue, fibrovascular core, terminal duct lobular unit, stromal/epithelium interface, basement membrane, and adipocytes. Despite not reaching the (sub) cellular level, the three-dimensionality of PhC µ CT images allows to depict in-depth alterations of the breast tissues, potentially revealing pathologically relevant details missed by a single histological section. Compared to serial sectioning, PhC µ CT allows the virtual investigation of the sample volume along any orientation, possibly guiding the pathologist in the choice of the most suitable cutting plane. Overall, PhC µ CT virtual histology holds great promise as a tool adding to conventional histology for improving efficiency, accessibility, and diagnostic accuracy of pathological evaluation.

Non-destructive 3D exploration of silicate glass corrosion: a combined multiscale approach from the macro to the nanoscale.

In this research, a comprehensive multi-technique analysis, including synchrotron-based X-ray micro-computed tomography, is used to visualize the microstructure of alteration in a very particular Roman glass fragment, in which millennia of corrosion history have not significantly impacted the integrity of the fragment itself. This exceptionally rare occurrence has allowed for the maximization of meaningful data acquisition, by examining the alteration structures from the macro to the nanoscale. This study elucidates the intricate mechanisms underlying glass corrosion when in contact with soil, providing quantitative data and phase correlations in the alteration structures. These findings validate and refine existing predictive corrosion models.

In vivo low-dose phase-contrast CT for quantification of functional and anatomical alterations in lungs of an experimental allergic airway disease mouse model.

Introduction: Synchrotron-based propagation-based imaging (PBI) is ideally suited for lung imaging and has successfully been applied in a variety of in vivo small animal studies. Virtually all these experiments were tailored to achieve extremely high spatial resolution close to the alveolar level while delivering high x-ray doses that would not permit longitudinal studies. However, the main rationale for performing lung imaging studies in vivo in small animal models is the ability to follow disease progression or monitor treatment response in the same animal over time. Thus, an in vivo imaging strategy should ideally allow performing longitudinal studies. Methods: Here, we demonstrate our findings of using PBI-based planar and CT imaging with two different detectors-MÖNCH 0.3 direct conversion detector and a complementary metal-oxide-semiconductor (CMOS) detector (Photonics Science)-in an Ovalbumin induced experimental allergic airway disease mouse model in comparison with healthy controls. The mice were imaged free breathing under isoflurane anesthesia. Results: At x-ray dose levels below those once used by commercial small animal CT devices at similar spatial resolutions, we were able to resolve structural changes at a pixel size down to 25 µm and demonstrate the reduction in elastic recoil in the asthmatic mice in cinematic planar x-ray imaging with a frame rate of up to 100 fps. Discussion: Thus, we believe that our approach will permit longitudinal small animal lung disease studies, closely following the mice over longer time spans.

Characterization of transient and progressive pulmonary fibrosis by spatially correlated phase contrast microCT, classical histopathology and atomic force microscopy.

Pulmonary fibrosis (PF) is a severe and progressive condition in which the lung becomes scarred over time resulting in pulmonary function impairment. Classical histopathology remains an important tool for micro-structural tissue assessment in the diagnosis of PF. A novel workflow based on spatial correlated propagation-based phase-contrast micro computed tomography (PBI-microCT), atomic force microscopy (AFM) and histopathology was developed and applied to two different preclinical mouse models of PF - the commonly used and well characterized Bleomycin-induced PF and a novel mouse model for progressive PF caused by conditional Nedd4-2 KO. The aim was to integrate structural and mechanical features from hallmarks of fibrotic lung tissue remodeling. PBI-microCT was used to assess structural alteration in whole fixed and paraffin embedded lungs, allowing for identification of fibrotic foci within the 3D context of the entire organ and facilitating targeted microtome sectioning of planes of interest for subsequent histopathology. Subsequently, these sections of interest were subjected to AFM to assess changes in the local tissue stiffness of previously identified structures of interest. 3D whole organ analysis showed clear morphological differences in 3D tissue porosity between transient and progressive PF and control lungs. By integrating the results obtained from targeted AFM analysis, it was possible to discriminate between the Bleomycin model and the novel conditional Nedd4-2 KO model using agglomerative cluster analysis. As our workflow for 3D spatial correlation of PBI, targeted histopathology and subsequent AFM is tailored around the standard procedure of formalin-fixed paraffin-embedded (FFPE) tissue specimens, it may be a powerful tool for the comprehensive tissue assessment beyond the scope of PF and preclinical research.

Detecting and Quantifying Xylem Embolism by Synchrotron-Based X-Ray Micro-CT.

The vulnerability to xylem embolism is a key trait underlying species-specific drought tolerance of plants, and hence is critical for screening climate-resilient crops and understanding vegetation responses to drought and heat waves. Yet, accurate determination of embolism in plant's xylem is challenging, because most traditional hydraulic techniques are destructive and prone to artefacts. Hence, direct and in vivo synchrotron-based X-ray micro-CT observation of xylem conduits has emerged as a key reference technique for accurate quantification of vulnerability to xylem embolism. Micro-CT is nowadays a fundamental tool for studies of plant hydraulic architecture, and this chapter describes the fundamentals of acquisition and processing of micro-CT images of plant xylem.

Distribution of corpora amylacea in the human midbrain: using synchrotron radiation phase-contrast microtomography, high-field magnetic resonance imaging, and histology.

Corpora amylacea (CA) are polyglucosan aggregated granules that accumulate in the human body throughout aging. In the cerebrum, CA have been found in proximity to ventricular walls, pial surfaces, and blood vessels. However, studies showing their three-dimensional spatial distribution are sparse. In this study, volumetric images of four human brain stems were obtained with MRI and phase-contrast X-ray microtomography, followed up by Periodic acid Schiff stain for validation. CA appeared as hyperintense spheroid structures with diameters up to 30 µm. An automatic pipeline was developed to segment the CA, and the spatial distribution of over 200,000 individual corpora amylacea could be investigated. A threefold-or higher-density of CA was detected in the dorsomedial column of the periaqueductal gray (860-4,200 CA count/mm3) than in the superior colliculus (150-340 CA count/mm3). We estimated that about 2% of the CA were located in the immediate vicinity of the vessels or in the peri-vascular space. While CA in the ependymal lining of the cerebral aqueduct was rare, the sub-pial tissue of the anterior and posterior midbrain contained several CA. In the sample with the highest CA density, quantitative maps obtained with MRI revealed high R2* values and a diamagnetic shift in a region which spatially coincided with the CA dense region.

The optical method based on gas injection overestimates leaf vulnerability to xylem embolism in three woody species.

Plant hydraulic traits related to leaf drought tolerance, like the water potential at turgor loss point (TLP) and the water potential inducing 50% loss of hydraulic conductance (P50), are extremely useful to predict the potential impacts of drought on plants. While novel techniques have allowed the inclusion of TLP in studies targeting a large group of species, fast and reliable protocols to measure leaf P50 are still lacking. Recently, the optical method coupled with the gas injection (GI) technique has been proposed as a possibility to speed up the P50 estimation. Here, we present a comparison of leaf optical vulnerability curves (OVcs) measured in three woody species, namely Acer campestre (Ac), Ostrya carpinifolia (Oc) and Populus nigra (Pn), based on bench dehydration (BD) or GI of detached branches. For Pn, we also compared optical data with direct micro-computed tomography (micro-CT) imaging in both intact saplings and cut shoots subjected to BD. Based on the BD procedure, Ac, Oc and Pn had P50 values of -2.87, -2.47 and -2.11 MPa, respectively, while the GI procedure overestimated the leaf vulnerability (-2.68, -2.04 and -1.54 MPa for Ac, Oc and Pn, respectively). The overestimation was higher for Oc and Pn than for Ac, likely reflecting the species-specific vessel lengths. According to micro-CT observations performed on Pn, the leaf midrib showed none or very few embolized conduits at -1.2 MPa, consistent with the OVcs obtained with the BD procedure but at odds with that derived on the basis of GI. Overall, our data suggest that coupling the optical method with GI might not be a reliable technique to quantify leaf hydraulic vulnerability since it could be affected by the 'open-vessel' artifact. Accurate detection of xylem embolism in the leaf vein network should be based on BD, preferably of intact up-rooted plants.

Contrasting Responses of Two Grapevine Cultivars to Drought: The Role of Non-structural Carbohydrates in Xylem Hydraulic Recovery.

Xylem embolism is one of the possible outcomes of decreasing xylem pressure when plants face drought. Recent studies have proposed a role for non-structural carbohydrates (NSCs) in osmotic pressure generation, required for refilling embolized conduits. Potted cuttings of grapevine Grenache and Barbera, selected for their adaptation to different climatic conditions, were subjected to a drought stress followed by re-irrigation. Stem embolism rate and its recovery were monitored in vivo by X-ray micro-computed tomography (micro-CT). The same plants were further analyzed for xylem conduit dimension and NSC content. Both cultivars significantly decreased Ψpd in response to drought and recovered from xylem embolism after re-irrigation. However, although the mean vessel diameter was similar between the cultivars, Barbera was more prone to embolism. Surprisingly, vessel diameter was apparently reduced during recovery in this cultivar. Hydraulic recovery was linked to sugar content in both cultivars, showing a positive relationship between soluble NSCs and the degree of xylem embolism. However, when starch and sucrose concentrations were considered separately, the relationships showed cultivar-specific and contrasting trends. We showed that the two cultivars adopted different NSC-use strategies in response to drought, suggesting two possible scenarios driving conduit refilling. In Grenache, sucrose accumulation seems to be directly linked to embolism formation and possibly sustains refilling. In Barbera, maltose/maltodextrins could be involved in a conduit recovery strategy via the formation of cell-wall hydrogels, likely responsible for the reduction of conduit lumen detected by micro-CT.

Osteoporosis and Covid-19: Detected similarities in bone lacunar-level alterations via combined AI and advanced synchrotron testing.

While advanced imaging strategies have improved the diagnosis of bone-related pathologies, early signs of bone alterations remain difficult to detect. The Covid-19 pandemic has brought attention to the need for a better understanding of bone micro-scale toughening and weakening phenomena. This study used an artificial intelligence-based tool to automatically investigate and validate four clinical hypotheses by examining osteocyte lacunae on a large scale with synchrotron image-guided failure assessment. The findings indicate that trabecular bone features exhibit intrinsic variability related to external loading, micro-scale bone characteristics affect fracture initiation and propagation, osteoporosis signs can be detected at the micro-scale through changes in osteocyte lacunar features, and Covid-19 worsens micro-scale porosities in a statistically significant manner similar to the osteoporotic condition. Incorporating these findings with existing clinical and diagnostic tools could prevent micro-scale damages from progressing into critical fractures.

High resolution propagation-based lung imaging at clinically relevant X-ray dose levels.

Absorption-based clinical computed tomography (CT) is the current imaging method of choice in the diagnosis of lung diseases. Many pulmonary diseases are affecting microscopic structures of the lung, such as terminal bronchi, alveolar spaces, sublobular blood vessels or the pulmonary interstitial tissue. As spatial resolution in CT is limited by the clinically acceptable applied X-ray dose, a comprehensive diagnosis of conditions such as interstitial lung disease, idiopathic pulmonary fibrosis or the characterization of small pulmonary nodules is limited and may require additional validation by invasive lung biopsies. Propagation-based imaging (PBI) is a phase sensitive X-ray imaging technique capable of reaching high spatial resolutions at relatively low applied radiation dose levels. In this publication, we present technical refinements of PBI for the characterization of different artificial lung pathologies, mimicking clinically relevant patterns in ventilated fresh porcine lungs in a human-scale chest phantom. The combination of a very large propagation distance of 10.7 m and a photon counting detector with [Formula: see text] pixel size enabled high resolution PBI CT with significantly improved dose efficiency, measured by thermoluminescence detectors. Image quality was directly compared with state-of-the-art clinical CT. PBI with increased propagation distance was found to provide improved image quality at the same or even lower X-ray dose levels than clinical CT. By combining PBI with iodine k-edge subtraction imaging we further demonstrate that, the high quality of the calculated iodine concentration maps might be a potential tool for the analysis of lung perfusion in great detail. Our results indicate PBI to be of great value for accurate diagnosis of lung disease in patients as it allows to depict pathological lesions non-invasively at high resolution in 3D. This will especially benefit patients at high risk of complications from invasive lung biopsies such as in the setting of suspected idiopathic pulmonary fibrosis (IPF).

Novel setup for rapid phase contrast CT imaging of heavy and bulky specimens.

This work introduces a novel setup for computed tomography of heavy and bulky specimens at the SYRMEP beamline of the Italian synchrotron Elettra. All the key features of the setup are described and the first application to off-center computed tomography scanning of a human chest phantom (approximately 45 kg) as well as the first results for vertical helical acquisitions are discussed.

Three-Dimensional Study of Polymer Composite Destruction in the Early Stages.

The investigation of destruction processes in composite materials is a current problem for their structural application and the improvement of their functional properties. This work aimed to visualize structural changes induced in layered carbon fiber reinforced plastics (CFRP) with the help of synchrotron X-ray microtomography. This article presents the details of destructive processes in the early stages of the deformation of reinforced polymers under uniaxial stretching, investigated at the micro level. Individual structural elements of the composite-filaments, parallel fiber bundles, the nonuniformity of the polymer binder distribution, and continuity defects-were observed under an external load. We have considered the influence of the material architecture and technological defects on fracture evolution in cross-ply and quasi-isotropic fiber-reinforced plastics. The results indicate the sequence of irreversible structural changes before the destruction of the material.

Micromorphology of pineal gland calcification in age-related neurodegenerative diseases.

BACKGROUND: The formation of concrements in human pineal gland (PG) is a physiological process and, according to many researchers, is associated with the involution of PG structures. The majority of scientific publications concern progressive calcification of PG, leaving out studies on the destruction of already formed calcified concrements. Our study fills the gap in knowledge about calcified zones destruction in PG in normal aging and neuropathological conditions, which has not been addressed until now. PURPOSE: Our objective is to gain insight into human PG tissue impairment in both normal aging and neurodegenerative conditions. X-ray phase-contrast tomography (XPCT) allowed us to study PG tissue degeneration at high spatial resolution and, for the first time, to examine the damaged PG concrements in detail. Our research finding could potentially enhance the understanding of the PG involvement in the process of aging as well as in Alzheimer's disease (AD) and vascular dementia (VD). METHODS: The research was carried out on human PG autopsy material in normal aging, VD, and AD conditions. Laboratory-based micro-computed tomography (micro-CT) was used to collect and evaluate samples of native, uncut, and unstained PG with different degrees of pineal calcification. The detailed high-resolution 3D images of the selected PGs were produced using synchrotron-based XPCT. Histology and immunohistochemistry of soft PG tissue confirmed XPCT results. RESULTS: We performed via micro-CT the evaluation of the morphometric parameters of PG such as total sample volume, calcified concrements volume, and percentage of concrements in the total volume of the sample. XPCT imaging revealed high-resolution details of age-related PG alteration. In particular, we noted signs of moderate degradation of concrements in some PGs from elderly donors. In addition, our analysis revealed noticeable degenerative change in both concrements and soft tissue of PGs with neuropathology. In particular, we observed a hollow core and separated layers as well as deep ragged cracks in PG concrements of AD and VD samples. In parenchyma of some samples, we detected wide pinealocyte-free fluid-filled areas adjacent to the calcified zones. CONCLUSION: The present work provides the basis for future scientific research focused on the dynamic nature of PG calcium deposits and PG soft tissue in normal aging and neurodegenerative diseases.

Microvascular imaging of the unstained human superior colliculus using synchrotron-radiation phase-contrast microtomography.

Characterizing the microvasculature of the human brain is critical to advance understanding of brain vascular function. Most methods rely on tissue staining and microscopy in two-dimensions, which pose several challenges to visualize the three-dimensional structure of microvessels. In this study, we used an edge-based segmentation method to extract the 3D vasculature from synchrotron radiation phase-contrast microtomography (PC-µCT) of two unstained, paraffin-embedded midbrain region of the human brain stem. Vascular structures identified in PC-µCT were validated with histology of the same specimen. Using the Deriche-Canny edge detector that was sensitive to the boundary between tissue and vascular space, we could segment the vessels independent of signal variations in PC-µCT images. From the segmented volumetric vasculature, we calculated vessel diameter, vessel length and volume fraction of the vasculature in the superior colliculi. From high resolution images, we found the most frequent vessel diameter to be between 8.6-10.2 µm. Our findings are consistent with the known anatomy showing two types of vessels with distinctive morphology: peripheral collicular vessels and central collicular vessels. The proposed method opens up new possibilities for vascular research of the central nervous system using synchrotron radiation PC-µCT of unstained human tissue.

No Evidence for Light-Induced Embolism Repair in Cut Stems of Drought-Resistant Mediterranean Species under Soaking.

(1) Recent studies suggested that stem photosynthesis could favor bark water uptake and embolism recovery when stem segments are soaked in water under light conditions, but evidence for this phenomenon in drought-resistant Mediterranean species with photosynthetic stems is missing. (2) Embolism recovery upon immersion in water for 2 h-4 h under light was assessed (i) via a classical hydraulic method in leafless Fraxinus ornus and Olea europaea branch segments stressed to xylem water potentials (Yxyl) inducing ca. 50% loss of hydraulic conductivity (PLC) and (ii) via X-ray micro-CT imaging of the stem segments of drought-stressed potted F. ornus saplings. Hydraulic recovery was also assessed in vivo in intact drought-stressed F. ornus saplings upon soil re-irrigation. (3) Intact F. ornus plants recovered hydraulic function through root water uptake. Conversely, the soaked stem segments of both species did not refill embolized conduits, although Yxyl recovered to pre-stress levels (between -0.5 MPa and -0.2 MPa). (4) We hypothesize that xylem embolism recovery through bark water uptake, even in light conditions, may not be a common phenomenon in woody plants and/or that wounds caused by cutting short stem segments might inhibit the refilling process upon soaking.

Alpine dwarf shrubs show high proportions of nonfunctional xylem: Visualization and quantification of species-specific patterns.

Xylem conductive capacity is a key determinant of plant hydraulic function and intimately linked to photosynthesis and productivity, but can be impeded by temporary or permanent conduit dysfunctions. Here we show that persistent xylem dysfunctions in unstressed plants are frequent in Alpine dwarf shrubs and occur in various but species-specific cross-sectional patterns. Combined synchrotron micro-computed tomography (micro-CT) imaging, xylem staining, and flow measurements in saturated samples of six widespread Ericaceae species evidence a high proportion (19%-50%) of hydraulically nonfunctional xylem areas in the absence of drought stress, with regular distribution of dysfunctions between or within growth rings. Dysfunctions were only partly reversible and reduced the specific hydraulic conductivity to 1.38 to 3.57 ×10-4 m2 s-1 MPa-1 . Decommission of inner growth rings was clearly related to stem age and a higher vulnerability to cavitation of older rings, while the high proportion of nonfunctional conduits in each annual ring needs further investigations. The lower the xylem fraction contributing to the transport function, the higher was the hydraulic efficiency of conducting xylem areas. Improved understanding of the functional lifespan of xylem elements and the prevalence and nature of dysfunctions is critical to correctly assess structure-function relationships and whole-plant hydraulic strategies.

Motion artifacts assessment and correction using optical tracking in synchrotron radiation breast CT.

PURPOSE: The SYRMA-3D collaboration is setting up a breast computed tomography (bCT) clinical program at the Elettra synchrotron radiation facility in Trieste, Italy. Unlike the few dedicated scanners available at hospitals, synchrotron radiation bCT requires the patient's rotation, which in turn implies a long scan duration (from tens of seconds to few minutes). At the same time, it allows the achievement of high spatial resolution. These features make synchrotron radiation bCT prone to motion artifacts. This article aims at assessing and compensating for motion artifacts through an optical tracking approach. METHODS: In this study, patients' movements due to breathing have been first assessed on seven volunteers and then simulated during the CT scans of a breast phantom and a surgical specimen, by adding a periodic oscillatory motion (constant speed, 1 mm amplitude, 12 cycles/minute). CT scans were carried out at 28 keV with a mean glandular dose of 5 mGy. Motion artifacts were evaluated and a correction algorithm based on the optical tracking of fiducial marks was introduced. A quantitative analysis based on the structural similarity (SSIM) index and the normalized mean square error (nMSE) was performed on the reconstructed CT images. RESULTS: CT images reconstructed through the optical tracking procedure were found to be as good as the motionless reference image. Moreover, the analysis of SSIM and nMSE demonstrated that an uncorrected motion of the order of the system's point spread function (around 0.1 mm in the present case) can be tolerated. CONCLUSIONS: Results suggest that a motion correction procedure based on an optical tracking system would be beneficial in synchrotron radiation bCT.

Elastic transformation of histological slices allows precise co-registration with microCT data sets for a refined virtual histology approach.

Although X-ray based 3D virtual histology is an emerging tool for the analysis of biological tissue, it falls short in terms of specificity when compared to conventional histology. Thus, the aim was to establish a novel approach that combines 3D information provided by microCT with high specificity that only (immuno-)histochemistry can offer. For this purpose, we developed a software frontend, which utilises an elastic transformation technique to accurately co-register various histological and immunohistochemical stainings with free propagation phase contrast synchrotron radiation microCT. We demonstrate that the precision of the overlay of both imaging modalities is significantly improved by performing our elastic registration workflow, as evidenced by calculation of the displacement index. To illustrate the need for an elastic co-registration approach we examined specimens from a mouse model of breast cancer with injected metal-based nanoparticles. Using the elastic transformation pipeline, we were able to co-localise the nanoparticles to specifically stained cells or tissue structures into their three-dimensional anatomical context. Additionally, we performed a semi-automated tissue structure and cell classification. This workflow provides new insights on histopathological analysis by combining CT specific three-dimensional information with cell/tissue specific information provided by classical histology.