Drought impacts trees in varied temporal and spatial patterns, suggesting that heterogeneity of below-ground water stores influences the fate of trees under water stress. Karst ecosystems rely on shallow soil overlying bedrock that can store available water in primary pores. A contribution of rock moisture to tree water status has been previously demonstrated, but actual mechanisms and rates of rock-to-root water delivery remain unknown. We report accurate measurements of hydraulic properties of two rock types (Breccia and Dolostone), of typical Karst red soil, and of roots of a common Karst tree species grown under different rock-soil combinations. Experimental data were used to build a water exchange model that supported the hypothesis that roots can extract water from porous and highly conductive rocks (Breccia), but not from more compact ones (Dolostone), especially when plants grow in rocky substrate or experience water stress, and thus have low root hydraulic conductivity and low rates of water extraction from rocks. Our data support the hypothesis that rocks represent important water stores for plants growing in rock-dominated habitats. Heterogeneous rock properties translate into different rates of water delivery to root systems, underlying complex patterns of tree mortality under severe drought stress.

Synchrotron radiation propagation-based phase-contrast micro-computed tomography (SRμCT) has been used increasingly for detailed characterization of tissue specimens, alternative to classical histology. It allows rapid and non-destructive three-dimensional (3D) virtual histology of unstained specimens. Although many studies show that standard protocols for formalin-fixation and paraffin-embedded (FFPE) tissue specimens are well suited for propagation based phase-contrast imaging (PBI), refinement of sample preparation protocols for this relatively recent development is still in its early stage. There are several limitations using FFPE blocks including air-bubble inclusion, and crack formation, which affect the quality of the micro-CT scans. In this study we demonstrate that optical clearing of colon cancer specimen followed by embedding in Phytagel, as an alternative sample preparation protocol yields comparable PBI micro-CT image quality and mitigates the aforementioned drawbacks of FFPE specimens. Moreover, since single-distance phase retrieval algorithms for PBI image reconstruction expect scanned materials to be either weakly-absorbing or having a fixed ratio between absorption and phase shift, we hypothesize that optical clearing will result in specimens that are well in line with these mathematical assumption of the applied phase retrieval algorithm (homogeneous form of the Transport-of-Intensity equation TIE-HOM). In addition, we show that classical histological analysis is still possible after re-embedding of the specimen in paraffin. Thus, the proposed novel workflow to scan optical cleared specimens using SRμCT can be used in combination with light sheet microscopy and can be integrated in a classical pipeline for pathological tissue characterization.

Glycyrrhiza glabra extract is widely known for its antioxidant and anti-inflammatory properties and can improve the wound healing process. The aim of this work was to shorten the time of the healing process by using an ecosustainable wound dressing based on Spanish broom flexible cellulosic fabric by impregnation with G. glabra extract-loaded ethosomes. Chemical analysis of G. glabra extract was performed by LC-DAD-MS/MS and its encapsulation into ethosomes was obtained using the ethanol injection method. Lipid vesicles were characterized in terms of size, polydispersity index, entrapment efficiency, zeta potential, and stability. In vitro release studies, biocompatibility, and scratch test on 3T3 fibroblasts were performed. Moreover, the structure of Spanish broom dressing and its ability to absorb wound exudate was characterized by Synchrotron X-ray phase contrast microtomography (SR-PCmicroCT). Ethosomes showed a good entrapment efficiency, nanometric size, good stability over time and a slow release of polyphenols compared to the free extract, and were not cytotoxic. Lastly, the results revealed that Spanish broom wound dressing loaded with G. glabra ethosomes is able to accelerate wound closure by reducing wound healing time. To sum up, Spanish broom wound dressing could be a potential new green tool for biomedical applications.

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.

X-ray phase-contrast micro computed tomography using synchrotron radiation (SR PhC-µCT) offers unique 3D imaging capabilities for visualizing microstructure of the human brain. Its applicability for unstained soft tissue is an area of active research. Acquiring images from a tissue block without needing to section it into thin slices, as required in routine histology, allows for investigating the microstructure in its natural 3D space. This paper presents a detailed step-by-step guideline for imaging unstained human brain tissue at resolutions of a few micrometers with SR PhC-µCT implemented at SYRMEP, the hard X-ray imaging beamline of Elettra, the Italian synchrotron facility. We present examples of how blood vessels and neurons appear in the images acquired with isotropic 5 μm and 1 µm voxel sizes. Furthermore, the proposed protocol can be used to investigate important biological substrates such as neuromelanin or corpora amylacea. Their spatial distribution can be studied using specifically tailored segmentation tools that are validated by classical histology methods. In conclusion, SR PhC-µCT using the proposed protocols, including data acquisition and image processing, offers viable means of obtaining information about the anatomy of the human brain at the cellular level in 3D.

SYRMEP is the hard X-ray imaging beamline of Elettra synchrotron offering X-ray full-field techniques, micro-computed tomography (microCT) and phase-contrast modality in the energy range 10–40 keV. The beamline operates in a multidisciplinary research context spanning from biomedical applications to botany, from zoology to food technology and cultural heritage, from materials engineering to geology and earth science. Thanks to the flexibility of SYRMEP setup, in situ experiments can be performed as well, novel imaging methods can be developed and implemented in a synergical manner with interested users and collaborators. SYRMEP peculiar wide beam together with the long sample-to-detector distance enables multiscale phase-contrast studies with optimized contrast and spatial resolution on rather large specimens, such as human lung phantoms. This is particularly relevant in view of future clinical lung imaging foreseen in the framework of Elettra 2.0 program. Here, the current beamline features and recent upgrades are illustrated, an overview of the imaging methods routinely offered to SYRMEP users’ community is presented, and the outlook for the new beamline SYRMEP-Life Science (SYRMEP-LS) is reported.

IntroductionSynchrotron-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.MethodsHere, 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.ResultsAt 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.DiscussionThus, we believe that our approach will permit longitudinal small animal lung disease studies, closely following the mice over longer time spans.

Lung diseases continue to present a major burden on public health. Therefore, improving the process of diagnosis by the development of novel imaging techniques is of great importance. In this perspective, phase sensitive CT imaging techniques such as propagation based imaging (PBI) might play an important role as they allow increasing the spatial resolution at very low x-ray dose levels that are comparable to clinical CT. However, the development of such methods is not only hindered by technological problems but also by the lack of precise validation strategies. We adapted formaldehyde (FA) vapor fixation to demonstrate that fresh porcine lungs that have been investigated by PBI can be fixed in their physiological shape and studied by multi-scale microCT imaging as well as classical histology. In addition, we show that FA vapor fixed pig lungs can be scanned by PBI without visible deterioration of image quality compared to fresh tissue. This opens the possibility of fixing and storing, for instance, human lung tissue before performing a PBI experiment, which in turn allows to study pathological changes in human lungs without questioning the translate-ability of findings in pig lung. The setup can be used by any interested researchers. ### Competing Interest Statement The authors have declared no competing interest.

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.

Precise morphological analysis of tumour tissue samples is crucial for accurate diagnosis and staging of colorectal cancer (CRC), but remains limited by the 2D nature of conventional histology. Our aim is to offer a 3D representation of tissue samples by means of X-ray-based imaging to facilitate the evaluation of clinically relevant features in cancer tissue, a process that is currently subject to various restrictions. In this study, we show that propagation-based synchrotron radiation-based free propagation phase-contrast microcomputed tomography (SRµCT) is suitable for the generation of 3D tumour volumes with 2-µm voxel size using standard formalin-fixed, paraffin-embedded tissue from CRC patients and provides sufficient contrast for virtual histology. We demonstrate that, using an existing registration pipeline, a 2D histologic haematoxylin–eosin slice can be placed in the context of the 3D µCT volume. The precisely registered histologic section can then be used as a “seed point” for the segmentation and depiction of major histologic features. This approach allows for a more comprehensive understanding of the organisation of the tumour in space with respect to other structures such as vessels, fat, and lymph nodes, and has the potential to improve patients’ prognostic outcomes.

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.

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.

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.

Hydroxyapatite is one of the materials of choice for tissue engineering bone scaffolds manufacturing. Vat photopolymerization (VPP) is a promising Additive Manufacturing (AM) technology capable of producing scaffolds with high resolution micro-architecture and complex shapes. However, mechanical reliability of ceramic scaffolds can be achieved if a high fidelity printing process is obtained and if knowledge of the intrinsic mechanical properties of the constituent material is available. As the hydroxyapatite (HAP) obtained from VPP is subjected to a sintering process, the mechanical properties of the material should be assessed with specific reference to the process parameters (e.g. sintering temperature) and to the specific characteristic size of the microscopic features in the scaffolds. In order to tackle this challenge the HAP solid matrix of the scaffold was mimicked in the form of miniaturized samples suitable for ad hoc mechanical characterization, which is an unprecedented approach. To this purpose small scale HAP samples, having a simple geometry and size similar to that of the scaffolds, were produced through VPP. The samples were subjected to geometric characterization and to mechanical laboratory tests. Confocal laser scanning and Computed micro-Tomography (micro-CT) were used for geometric characterization; while, micro-bending and nanoindentation were used for mechanical testing. Micro-CT analyses have shown a highly dense material with negligible intrinsic micro-porosity. The imaging process allowed quantifying the variation of geometry with respect to the nominal size showing high accuracy of the printing process and identifying printing defects on one specific sample type, depending on the printing direction. The mechanical tests have shown that the VPP produces HAP with an elastic modulus as high as approximately 100GPa and flexural strength of approximately 100MPa. The results of this study have shown that vat photopolymerization is a promising technology capable of producing high quality HAP with reliable geometric fidelity.

Paleoarchean stromatolites are among the oldest compelling evidence for life. We present advanced two- and three-dimensional (2-D and 3-D) reconstructions of the morphology, mineralogy, trace element geochemistry, and taphonomy of permineralized stromatolites from the lowermost horizons of the ca. 3.5 Ga Dresser Formation, Pilbara, Western Australia. Rare earth element plus yttrium compositions suggest a restricted paleodepositional setting influenced by marine influxes; this contrasts with other Dresser stromatolites, which developed around terrestrial hot springs. Mineral phase relationships and positive Eu anomalies denote syndepositional hydrothermal influence and silicification promoting high-fidelity microstructural preservation. Although no primary kerogen is preserved, numerous 2-D and 3-D morphological characteristics denote a biogenic origin, including the onlap of sedimentary layers onto stromatolitic topography, fine-scale undulatory laminations, non-isopachous laminations with crestal thickening, laminoid fenestrae, and subvertical pillar-like fabrics interpreted as microbial palisade structure; these features suggest that the stromatolite eco-system was dominantly phototrophic. The deep iron-rich weathering profile of the Dresser stromatolites makes them pertinent analogues for potential microbialites in altered carbonates on Mars. Were similar putative biogenic macro-, meso- and micromorphologies identified in habitable Martian settings by rover imaging systems, such materials would be compelling targets for sample return.

The dramatic increase in fragility fractures and the related health and economic burden rise the urge of a cutting-edge perspective to anticipate catastrophic fracture propagation in human bones. Recent studies address the issue from a multi-scale perspective, elevating the micro-scale phenomena as the key for detecting early damage occurrence. However, several limitations arise specifically for defining a quantitative framework to assess the contribution of lacunar micro-pores to fracture initiation and propagation. Moreover, the need for high resolution imaging imposes time-demanding post-processing phases. Here, we exploit synchrotron scans in combination with micro-mechanical tests, to offer a fracture mechanics-based approach for quantifying the critical stress intensification in healthy and osteoporotic trabecular human bones. This is paired with a morphological and densitometric framework for capturing lacunar network differences in presence of pathological alterations. To address the current time-consuming and computationally expensive manual/semi-automatic segmenting steps, we implement convolutional neural network to detect the initiation and propagation of micro-scale damages. The results highlight the intimate cross talks between toughening and weakening phenomena at micro-scale as a fundamental aspect for fracture prevention.

(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.

Most tissues in clinical practice are formalin-fixed and paraffin-embedded for histological as well as molecular analyses. The reproducibility and uniformity of molecular analyses is strictly dependent on the quality of the biomolecules, which is highly influenced by pre-analytical processes. In this study, the effect of different fixatives was compared, including formalin, Bouin's solution, RCL2® and TAG-1™ fixatives, by stringent application of ISO standards in mouse liver tissue processing, including formalin-free transport of tissues and tissue grossing in a refrigerated environment. The effect of fixatives was studied in terms of nucleic acid quality at the time of tissue processing and after one year of tissue storage at room temperature in the dark. Furthermore, a microcomputed tomography (CT) scan analysis was applied to investigate the paraffin embedding. The results show that the application of ISO standards in tissue processing allows analysis of 400 bases amplicons from RNA and 1000 bases from DNA, even in extracts from formalin-fixed and paraffin-embedded tissues. However, after one year storage at room temperature in the dark, a degradation of the nucleic acids was observed. Nevertheless, extracts can still be analyzed, but for metachronous tests it is highly recommended to repeat the quantitation of housekeeping genes in order to standardize the extent of nucleic acid degradation.

The 3D complexity of biological tissues and intricate structural-functional connections call for state-of-the-art X-ray imaging approaches to overcome limitations of classical imaging. Unlike other imaging techniques, X-ray phase-contrast tomography (XPCT) offers a highly sensitive 3D imaging approach to investigate different disease-relevant networks at levels ranging from single cell through to intact organ. We present here a concomitant study of the evolution of tissue damage and inflammation in different organs affected by the disease in the murine model for multiple sclerosis, a demyelinating autoimmune disorder of the central nervous system. XPCT identifies and monitors structural and cellular alterations throughout the central nervous system, but also in the gut, and eye, of mice induced to develop multiple sclerosis-like disease and sacrificed at pre-symptomatic and symptomatic time points. This study details the sequential evolution of multi-organ damages in the murine multiple sclerosis model showing the disease development and progression which is of relevance for the human case.