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.

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.

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.

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.

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.

Perspectives for microbeam irradiation at the SYRMEP beamline.

It has been shown previously both in vitro and in vivo that microbeam irradiation (MBI) can control malignant tumour cells more effectively than the clinically established concepts of broad beam irradiation. With the aim to extend the international capacity for microbeam research, the first MBI experiment at the biomedical beamline SYRMEP of the Italian synchrotron facility ELETTRA has been conducted. Using a multislit collimator produced by the company TECOMET, arrays of quasi-parallel microbeams were successfully generated with a beam width of 50 µm and a centre-to-centre distance of 400 µm. Murine melanoma cell cultures were irradiated with a target dose of approximately 65 Gy at a mean photon energy of ∼30 keV with a dose rate of 70 Gy s-1 and a peak-to-valley dose of ∼123. This work demonstrated a melanoma cell reduction of approximately 80% after MBI. It is suggested that, while a high energy is essential to achieve high dose rates in order to deposit high treatment doses in a short time in a deep-seated target, for in vitro studies and for the treatment of superficial tumours a spectrum in the lower energy range might be equally suitable or even advantageous.

Chemical inhibition of xylem cellular activity impedes the removal of drought-induced embolisms in poplar stems - new insights from micro-CT analysis.

In drought-stressed plants a coordinated cascade of chemical and transcriptional adjustments occurs at the same time as embolism formation. While these processes do not affect embolism formation during stress, they may prime stems for recovery during rehydration by modifying apoplast pH and increasing sugar concentration in the xylem sap. Here we show that in vivo treatments modifying apoplastic pH (stem infiltration with a pH buffer) or reducing stem metabolic activity (infiltration with sodium vanadate and sodium cyanide; plant exposure to carbon monoxide) can reduce sugar accumulation, thus disrupting or delaying the recovery process. Application of the vanadate treatment (NaVO3, an inhibitor of many ATPases) completely halted recovery from drought-induced embolism for up to 24 h after re-irrigation, while partial recovery was observed in vivo in control plants using X-ray microcomputed tomography. Our results suggest that stem hydraulic recovery in poplar is a biological, energy-dependent process that coincides with accumulation of sugars in the apoplast during stress. Recovery and damage are spatially coordinated, with embolism formation occurring from the inside out and refilling from the outside in. The outside-in pattern highlights the importance of xylem proximity to the sugars within the phloem to the embolism recovery process.

Comparison of propagation-based CT using synchrotron radiation and conventional cone-beam CT for breast imaging.

OBJECTIVES: To evaluate and compare the image quality of propagation-based phase-contrast computed tomography (PB-CT) using synchrotron radiation and conventional cone-beam breast computed tomography (CBBCT) based on various radiological image quality criteria. METHODS: Eight excised breast tissue samples of various sizes and containing different lesion types were scanned using PB-CT at a synchrotron facility and using CBBCT at a university-affiliated breast imaging centre. PB-CT scans were performed at two different mean glandular dose (MGD) levels: standard (5.8 mGy) and low (1.5 mGy), for comparison with CBBCT scans at the standard MGD (5.8 mGy). Image quality assessment was carried out using six quality criteria and six independent medical imaging experts in a reading room with mammography workstations. The interobserver agreement between readers was evaluated using intraclass correlation coefficient (ICC), and image quality was compared between the two breast imaging modalities using the area under the visual grading characteristic curve (AUCVGC). RESULTS: Interobserver agreement between the readers showed moderate reliability for five image criteria (ICC: ranging from 0.488 to 0.633) and low reliability for one criterion (image noise) (ICC 0.307). For five image quality criteria (overall quality, perceptible contrast, lesion sharpness, normal tissue interfaces, and calcification visibility), both standard-dose PB-CT images (AUCVGC 0.958 to 1, p ≤ .05) and low dose PB-CT images (AUCVGC 0.785 to 0.834, p ≤ .05) were of significantly higher image quality than standard-dose CBBCT images. CONCLUSIONS: Synchrotron-based PB-CT can achieve a significantly higher radiological image quality at a substantially lower radiation dose compared with conventional CBBCT. KEY POINTS: • PB-CT using synchrotron radiation results in higher image quality than conventional CBBCT for breast imaging. • PB-CT using synchrotron radiation requires a lower radiation dose than conventional CBBCT for breast imaging. • PB-CT can help clinicians diagnose patients with breast cancer.

Hard and soft X-ray imaging to resolve human ovarian cortical structures.

Laboratory and synchrotron X-ray tomography are powerful tools for non-invasive studies of biological samples at micrometric resolution. In particular, the development of phase contrast imaging is enabling the visualization of sample details with a small range of attenuation coefficients, thus allowing in-depth analyses of anatomical and histological structures. Reproductive medicine is starting to profit from these techniques, mainly applied to animal models. This study reports the first imaging of human ovarian tissue where the samples consisted of surgically obtained millimetre fragments, properly fixed, stained with osmium tetroxide and included in epoxydic resin. Samples were imaged by the use of propagation phase contrast synchrotron radiation micro-computed tomography (microCT), obtained at the SYRMEP beamline of Elettra light source (Trieste, Italy), and X-ray absorption microCT at the Theoretical Biology MicroCT Imaging Laboratory in Vienna, Austria. The reconstructed microCT images were compared with the soft X-ray absorption and phase contrast images acquired at the TwinMic beamline of Elettra in order to help with the identification of structures. The resulting images allow the regions of the cortex and medulla of the ovary to be distinguished, identifying early-stage follicles and visualizing the distribution of blood vessels. The study opens to further application of micro-resolved 3D imaging to improve the understanding of human ovary's structure and support diagnostics as well as advances in reproductive technologies.

Advancements towards the implementation of clinical phase-contrast breast computed tomography at Elettra.

Breast computed tomography (BCT) is an emerging application of X-ray tomography in radiological practice. A few clinical prototypes are under evaluation in hospitals and new systems are under development aiming at improving spatial and contrast resolution and reducing delivered dose. At the same time, synchrotron-radiation phase-contrast mammography has been demonstrated to offer substantial advantages when compared with conventional mammography. At Elettra, the Italian synchrotron radiation facility, a clinical program of phase-contrast BCT based on the free-space propagation approach is under development. In this paper, full-volume breast samples imaged with a beam energy of 32 keV delivering a mean glandular dose of 5 mGy are presented. The whole acquisition setup mimics a clinical study in order to evaluate its feasibility in terms of acquisition time and image quality. Acquisitions are performed using a high-resolution CdTe photon-counting detector and the projection data are processed via a phase-retrieval algorithm. Tomographic reconstructions are compared with conventional mammographic images acquired prior to surgery and with histologic examinations. Results indicate that BCT with monochromatic beam and free-space propagation phase-contrast imaging provide relevant three-dimensional insights of breast morphology at clinically acceptable doses and scan times.

Insights from in vivo micro-CT analysis: testing the hydraulic vulnerability segmentation in Acer pseudoplatanus and Fagus sylvatica seedlings.

The seedling stage is the most susceptible one during a tree's life. Water relations may be crucial for seedlings due to their small roots, limited water buffers and the effects of drought on water transport. Despite obvious relevance, studies on seedling xylem hydraulics are scarce as respective methodical approaches are limited. Micro-CT scans of intact Acer pseudoplatanus and Fagus sylvatica seedlings dehydrated to different water potentials (Ψ) allowed the simultaneous observation of gas-filled versus water-filled conduits and the calculation of percentage loss of conductivity (PLC) in stems, roots and leaves (petioles or main veins). Additionally, anatomical analyses were performed and stem PLC measured with hydraulic techniques. In A. pseudoplatanus, petioles showed a higher Ψ at 50% PLC (Ψ50 -1.13MPa) than stems (-2.51 MPa) and roots (-1.78 MPa). The main leaf veins of F. sylvatica had similar Ψ50 values (-2.26 MPa) to stems (-2.74 MPa) and roots (-2.75 MPa). In both species, no difference between root and stems was observed. Hydraulic measurements on stems closely matched the micro-CT based PLC calculations. Micro-CT analyses indicated a species-specific hydraulic architecture. Vulnerability segmentation, enabling a disconnection of the hydraulic pathway upon drought, was observed in A. pseudoplatanus but not in the especially shade-tolerant F. sylvatica. Hydraulic patterns could partly be related to xylem anatomical traits.

Synchrotron inline phase contrast µCT enables detailed virtual histology of embedded soft-tissue samples with and without staining.

Synchrotron radiation micro-computed tomography (SRµCT) based virtual histology, in combination with dedicated ex vivo staining protocols and/or phase contrast, is an emerging technology that makes use of three-dimensional images to provide novel insights into the structure of tissue samples at microscopic resolution with short acquisition times of the order of minutes or seconds. However, the high radiation dose creates special demands on sample preparation and staining. As a result of the lack of specific staining in virtual histology, it can supplement but not replace classical histology. Therefore, the aim of this study was to establish and compare optimized ex vivo staining and acquisition protocols for SRµCT-based virtual histology of soft-tissue samples, which could be integrated into the standard workflow of classical histology. The high grade of coherence of synchrotron radiation allows the application of propagation-based phase contrast imaging (PBI). In this study, PBI yielded a strong increase in image quality even at lower radiation doses and consequently prevented any damage to the tissue samples or the embedding material. This work has demonstrated that the improvement in contrast-to-noise ratio by PBI enabled label-free virtual histology of soft-tissue specimens embedded in paraffin to a level of detail that exceeds that achieved with staining protocols.

MÖNCH detector enables fast and low-dose free-propagation phase-contrast computed tomography of in situ mouse lungs.

Due to the complexity of the underlying pathomechanism, in vivo mouse lung-disease models continue to be of great importance in preclinical respiratory research. Longitudinal studies following the cause of a disease or evaluating treatment efficacy are of particular interest but challenging due to the small size of the mouse lung and the fast breathing rate. Synchrotron-based in-line phase-contrast computed tomography imaging has been successfully applied in lung research in various applications, but mostly at dose levels that forbid longitudinal in vivo studies. Here, the novel charge-integrating hybrid detector MÖNCH is presented, which enables imaging of mouse lungs at a pixel size of 25 µm, in less than 10 s and with an entrance dose of about 70 mGy, which therefore will allow longitudinal lung disease studies to be performed in mouse models.

Towards synchrotron phase-contrast lung imaging in patients - a proof-of-concept study on porcine lungs in a human-scale chest phantom.

In-line free propagation phase-contrast synchrotron tomography of the lungs has been shown to provide superior image quality compared with attenuation-based computed tomography (CT) in small-animal studies. The present study was performed to prove the applicability on a human-patient scale using a chest phantom with ventilated fresh porcine lungs. Local areas of interest were imaged with a pixel size of 100 µm, yielding a high-resolution depiction of anatomical hallmarks of healthy lungs and artificial lung nodules. Details like fine spiculations into surrounding alveolar spaces were shown on a micrometre scale. Minor differences in artificial lung nodule density were detected by phase retrieval. Since we only applied a fraction of the X-ray dose used for clinical high-resolution CT scans, it is believed that this approach may become applicable to the detailed assessment of focal lung lesions in patients in the future.

Advantages of breast cancer visualization and characterization using synchrotron radiation phase-contrast tomography.

The aim of this study was to highlight the advantages that propagation-based phase-contrast computed tomography (PB-CT) with synchrotron radiation can provide in breast cancer diagnostics. For the first time, a fresh and intact mastectomy sample from a 60 year old patient was scanned on the IMBL beamline at the Australian Synchrotron in PB-CT mode and reconstructed. The clinical picture was described and characterized by an experienced breast radiologist, who underlined the advantages of providing diagnosis on a PB-CT volume rather than conventional two-dimensional modalities. Subsequently, the image quality was assessed by 11 breast radiologists and medical imaging experts using a radiological scoring system. The results indicate that, with the radiation dose delivered to the sample being equal, the accuracy of a diagnosis made on PB-CT images is significantly higher than one using conventional techniques.

The pitfalls of in vivo imaging techniques: evidence for cellular damage caused by synchrotron X-ray computed micro-tomography.

Synchrotron X-ray computed micro-tomography (microCT) has emerged as a promising noninvasive technique for in vivo monitoring of xylem function, including embolism build-up under drought and hydraulic recovery following re-irrigation. Yet, the possible harmful effects of ionizing radiation on plant tissues have never been quantified. We specifically investigated the eventual damage suffered by stem living cells of three different species exposed to repeated microCT scans. Stem samples exposed to one, two or three scans were used to measure cell membrane and RNA integrity, and compared to controls never exposed to X-rays. Samples exposed to microCT scans suffered serious alterations to cell membranes, as revealed by marked increase in relative electrolyte leakage, and also underwent severe damage to RNA integrity. The negative effects of X-rays were apparent in all species tested, but the magnitude of damage and the minimum number of scans inducing negative effects were species-specific. Our data show that multiple microCT scans lead to disruption of fundamental cellular functions and processes. Hence, microCT investigation of phenomena that depend on physiological activity of living cells may produce erroneous results and lead to incorrect conclusions.

X-ray-based lung function measurement reveals persistent loss of lung tissue elasticity in mice recovered from allergic airway inflammation.

Chronic asthma patients experience difficulties even years after the inciting allergen. Although studies in small animal asthma models have enormously advanced progress in uncovering the mechanisms of inception and development of the disease, little is known about the processes involved in the persistence of asthma symptoms in the absence of allergen exposure. Long-term asthma mouse models have so far been scarce or not been able to reproduce the findings in patients. Here we used a common ovalbumin-induced acute allergic airway inflammation mouse model to study lung function and remodeling after a 4-mo recovery period. We show by X-ray-based lung function measurements that the recovered mice continue to show impaired lung function by displaying significant air trapping compared with controls. High-resolution synchrotron phase-contrast computed tomography of structural alterations and diaphragm motion analysis suggest that these changes in pulmonary function are the result of a pronounced loss in lung elasticity. Histology of lung sections confirmed that this is most likely caused by a decrease in elastic fibers, indicating that remodeling can develop or persist independent of acute inflammation and is closely related to a loss in lung function. Our findings demonstrate that this X-ray-based imaging platform has the potential to comprehensively and noninvasively unravel long-term effects in preclinical mouse models of allergic airway inflammation and thus benefits our understanding of chronic asthma.

X-ray microtomography observations of xylem embolism in stems of Laurus nobilis are consistent with hydraulic measurements of percentage loss of conductance.

Drought-induced xylem embolism is a serious threat to plant survival under future climate scenarios. Hence, accurate quantification of species-specific vulnerability to xylem embolism is a key to predict the impact of climate change on vegetation. Low-cost hydraulic measurements of embolism rate have been suggested to be prone to artefacts, thus requiring validation by direct visualization of the functional status of xylem conduits using nondestructive imaging techniques, such as X-ray microtomography (microCT). We measured the percentage loss of conductance (PLC) of excised stems of Laurus nobilis (laurel) dehydrated to different xylem pressures, and compared results with direct observation of gas-filled vs water-filled conduits at a synchrotron-based microCT facility using a phase contrast imaging modality. Theoretical PLC calculated on the basis of microCT observations in stems of laurel dehydrated to different xylem pressures overall were in agreement with hydraulic measurements, revealing that this species suffers a 50% loss of xylem hydraulic conductance at xylem pressures averaging -3.5 MPa. Our data support the validity of estimates of xylem vulnerability to embolism based on classical hydraulic techniques. We discuss possible causes of discrepancies between data gathered in this study and those of recent independent reports on laurel hydraulics.

Role of Cortico-Cancellous Heterologous Bone in Human Periodontal Ligament Stem Cell Xeno-Free Culture Studied by Synchrotron Radiation Phase-Contrast Microtomography.

This study was designed to quantitatively demonstrate via three-dimensional (3D) images, through the Synchrotron Radiation Phase-Contrast Microtomography (SR-PhC-MicroCT), the osteoinductive properties of a cortico-cancellous scaffold (Osteobiol Dual Block-DB) cultured with human Periodontal Ligament Stem Cells (hPDLSCs) in xeno-free media. In vitro cultures of hPDLSCs, obtained from alveolar crest and horizontal fibers of the periodontal ligament, were seeded onto DB scaffolds and cultured in xeno-free media for three weeks. 3D images were obtained by SR-PhC-microCT after one and three weeks from culture beginning. MicroCT data were successively processed with a phase-retrieval algorithm based on the Transport of Intensity Equation (TIE). The chosen experimental method, previously demonstratively applied for the 3D characterization of the same constructs in not xeno-free media, quantitatively monitored also in this case the early stages of bone formation in basal and differentiating conditions. Interestingly, it quantitatively showed in the xeno-free environment a significant acceleration of the mineralization process, regardless of the culture (basal/differentiating) medium. This work showed in 3D that the DB guides the osteogenic differentiation of hPDLSCs in xeno-free cultures, in agreement with 2D observations and functional studies previously performed by some of the authors. Indeed, here we fully proved in 3D that expanded hPDLSCs, using xeno-free media formulation, not only provide the basis for Good Manufacturing Practice (preserving the stem cells' morphological features and their ability to differentiate into mesenchymal lineage) but have to be considered, combined to DB scaffolds, as interesting candidates for potential clinical use in new custom made tissue-engineered constructs.