The effect of root hairs on root water uptake is determined by root-soil contact and root hair shrinkage.

The effect of root hairs on water uptake remains controversial. In particular, the key root hair and soil parameters that determine their importance have been elusive. We grew maize plants (Zea mays) in microcosms and scanned them using synchrotron-based X-ray computed microtomography. By means of image-based modelling, we investigated the parameters determining the effectiveness of root hairs in root water uptake. We explicitly accounted for rhizosphere features (e.g. root-soil contact and pore structure) and took root hair shrinkage of dehydrated root hairs into consideration. Our model suggests that > 85% of the variance in root water uptake is explained by the hair-induced increase in root-soil contact. In dry soil conditions, root hair shrinkage reduces the impact of hairs substantially. We conclude that the effectiveness of root hairs on root water uptake is determined by the hair-induced increase in root-soil contact and root hair shrinkage. Although the latter clearly reduces the effect of hairs on water uptake, our model still indicated facilitation of water uptake by root hairs at soil matric potentials from -1 to -0.1 MPa. Our findings provide new avenues towards a mechanistic understanding of the role of root hairs on water uptake.

Sub-micrometer morphology of human atherosclerotic plaque revealed by synchrotron radiation-based µCT-A comparison with histology.

Histology is a long standing and well-established gold standard for pathological characterizations. In recent years however, synchrotron radiation-based micro-computed tomography (SRµCT) has become a tool for extending the imaging of two-dimensional thin sections into three-dimensional imaging of tissue blocks, enabling so-called virtual histology with arbitrary clipping planes, volumetric rendering and automatic segmentation. In this study, we present a thorough characterization of human carotid plaques after endarterectomy of patients with stroke or transient ischemic attack (TIA), investigating several different pathologic structures using both SRµCT and histology. Phase-contrast SRµCT was performed with two different magnifications (voxel sizes 6.5 µm and 0.65 µm, respectively), and histology was performed with multiple different stainings (Alpha-actin, Glycophorin A, von Kossa, Movat, CD68). The 0.65 µm high-resolution SRµCT was performed on selected areas with plaque typical relevant morphology, identified on the 6.5 µm low-resolution SRµCT. The tomography datasets were reconstructed with additional 3D volume rendering and compared to histology. In total, nine different regions with typical pathologic structures were identified and imaged with high-resolution SRµCT. The results show many characteristics typical for advanced atherosclerotic plaques, clinically relevant, namely ruptures with thrombosis, neo-vascularization, inflammatory infiltrates in shoulder regions, lipid rich necrotic cores (LRNC), thin fibrous cap, calcifications, lumen irregularities, and changes in vessel wall structures such as the internal elastic membrane. This method's non-destructive nature renders details of micro-structures with an excellent visual likeness to histology, with the additional strength of multiplanar and 3D visualization and the possibility of multiple re-scans.

Multiscale X-ray phase contrast imaging of human cartilage for investigating osteoarthritis formation.

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.

Distinct types of plexiform lesions identified by synchrotron-based phase-contrast micro-CT.

In pulmonary arterial hypertension, plexiform lesions are associated with severe arterial obstruction and right ventricular failure. Exploring their structure and position is crucial for understanding the interplay between hemodynamics and vascular remodeling. The aim of this research was to use synchrotron-based phase-contrast micro-CT to study the three-dimensional structure of plexiform lesions. Archived paraffin-embedded tissue samples from 14 patients with pulmonary arterial hypertension (13 idiopathic, 1 with known BMPR2-mutation) were imaged. Clinical data showed high-median PVR (12.5 WU) and mPAP (68 mmHg). Vascular lesions with more than 1 lumen were defined as plexiform. Prior radiopaque dye injection in some samples facilitated 3-D rendering. Four distinct types of plexiform lesions were identified: 1) localized within or derived from monopodial branches (supernumerary arteries), often with a connection to the vasa vasorum; 2) localized between pulmonary arteries and larger airways as a tortuous transformation of intrapulmonary bronchopulmonary anastomoses; 3) as spherical structures at unexpected abrupt ends of distal pulmonary arteries; and 4) as occluded pulmonary arteries with recanalization. By appearance and localization, types 1-2 potentially relieve pressure via the bronchial circulation, as pulmonary arteries in these patients were almost invariably occluded distally. In addition, types 1-3 were often surrounded by dilated thin-walled vessels, often connected to pulmonary veins, peribronchial vessels, or the vasa vasorum. Collaterals, bypassing completely occluded pulmonary arteries, were also observed to originate within plexiform lesions. In conclusion, synchrotron-based imaging revealed significant plexiform lesion heterogeneity, resulting in a novel classification. The four types likely have different effects on hemodynamics and disease progression.

Imaging atelectrauma in Ventilator-Induced Lung Injury using 4D X-ray microscopy.

Mechanical ventilation can damage the lungs, a condition called Ventilator-Induced Lung Injury (VILI). However, the mechanisms leading to VILI at the microscopic scale remain poorly understood. Here we investigated the within-tidal dynamics of cyclic recruitment/derecruitment (R/D) using synchrotron radiation phase-contrast imaging (PCI), and the relation between R/D and cell infiltration, in a model of Acute Respiratory Distress Syndrome in 6 anaesthetized and mechanically ventilated New-Zealand White rabbits. Dynamic PCI was performed at 22.6 µm voxel size, under protective mechanical ventilation [tidal volume: 6 ml/kg; positive end-expiratory pressure (PEEP): 5 cmH2O]. Videos and quantitative maps of within-tidal R/D showed that injury propagated outwards from non-aerated regions towards adjacent regions where cyclic R/D was present. R/D of peripheral airspaces was both pressure and time-dependent, occurring throughout the respiratory cycle with significant scatter of opening/closing pressures. There was a significant association between R/D and regional lung cellular infiltration (p = 0.04) suggesting that tidal R/D of the lung parenchyma may contribute to regional lung inflammation or capillary-alveolar barrier dysfunction and to the progression of lung injury. PEEP may not fully mitigate this phenomenon even at high levels. Ventilation strategies utilizing the time-dependence of R/D may be helpful in reducing R/D and associated injury.

Micrometer-resolution X-ray tomographic full-volume reconstruction of an intact post-mortem juvenile rat lung.

In this article, we present an X-ray tomographic imaging method that is well suited for pulmonary disease studies in animal models to resolve the full pathway from gas intake to gas exchange. Current state-of-the-art synchrotron-based tomographic phase-contrast imaging methods allow for three-dimensional microscopic imaging data to be acquired non-destructively in scan times of the order of seconds with good soft tissue contrast. However, when studying multi-scale hierarchically structured objects, such as the mammalian lung, the overall sample size typically exceeds the field of view illuminated by the X-rays in a single scan and the necessity for achieving a high spatial resolution conflicts with the need to image the whole sample. Several image stitching and calibration techniques to achieve extended high-resolution fields of view have been reported, but those approaches tend to fail when imaging non-stable samples, thus precluding tomographic measurements of large biological samples, which are prone to degradation and motion during extended scan times. In this work, we demonstrate a full-volume three-dimensional reconstruction of an intact rat lung under immediate post-mortem conditions and at an isotropic voxel size of (2.75 µm)3. We present the methodology for collecting multiple local tomographies with 360° extended field of view scans followed by locally non-rigid volumetric stitching. Applied to the lung, it allows to resolve the entire pulmonary structure from the trachea down to the parenchyma in a single dataset. The complete dataset is available online ( https://doi.org/10.16907/7eb141d3-11f1-47a6-9d0e-76f8832ed1b2 ).

Early Morphofunctional Changes in AngII-Infused Mice Contribute to Regional Onset of Aortic Aneurysm and Dissection.

Aortic aneurysms and dissections are silent and lethal conditions, whose pathogenesis remains incompletely understood. Although angiotensin II (AngII)-infused ApoE-/- mice have been widely used to study aortic aneurysm and dissection, early morphofunctional alterations preceding the onset of these conditions remain unknown. The goal of this study was to unveil early morphofunctional changes underlying the onset of aneurysm and dissection. At 3 days post-AngII infusion, suprarenal abdominal aorta presented significant volumetric dilatation and microstructural damage. Ex vivo assessment of vascular reactivity of the suprarenal dissection-prone aorta and its side branches, showed an endothelial and contractile dysfunctions that were severe in the suprarenal aorta, moderate distally, and absent in the side branches, mirroring the susceptibility to dissection of these different vascular segments. Early and specific morphofunctional changes of the suprarenal aorta may contribute to the regional onset of aortic aneurysm and dissection by exacerbating the biomechanical burden arising from its side branches.

Sub-trabecular strain evolution in human trabecular bone.

To comprehend the most detrimental characteristics behind bone fractures, it is key to understand the material and tissue level strain limits and their relation to failure sites. The aim of this study was to investigate the three-dimensional strain distribution and its evolution during loading at the sub-trabecular level in trabecular bone tissue. Human cadaver trabecular bone samples were compressed in situ until failure, while imaging with high-resolution synchrotron radiation X-ray tomography. Digital volume correlation was used to determine the strains inside the trabeculae. Regions without emerging damage were compared to those about to crack. Local strains in close vicinity of developing cracks were higher than previously reported for a whole trabecular structure and similar to those reported for single isolated trabeculae. Early literature on bone fracture strain thresholds at the tissue level seem to underestimate the maximum strain magnitudes in trabecular bone. Furthermore, we found lower strain levels and a reduced ability to capture detailed crack-paths with increased image voxel size. This highlights the dependence between the observed strain levels and the voxel size and that high-resolution is needed to investigate behavior of individual trabeculae. Furthermore, low trabecular thickness appears to be one predictor of developing cracks. In summary, this study investigated the local strains in whole trabecular structure at sub-trabecular resolution in human bone and confirmed the high strain magnitudes reported for single trabeculae under loading and, importantly extends its translation to the whole trabecular structure.

Synchrotron MicroCT Reveals the Potential of the Dual Contrast Technique for Quantitative Assessment of Human Articular Cartilage Composition.

Dual contrast micro computed tomography (CT) shows potential for detecting articular cartilage degeneration. However, the performance of conventional CT systems is limited by beam hardening, low image resolution (full-body CT), and long acquisition times (conventional microCT). Therefore, to reveal the full potential of the dual contrast technique for imaging cartilage composition we employ the technique using synchrotron microCT. We hypothesize that the above-mentioned limitations are overcome with synchrotron microCT utilizing monochromatic X-ray beam and fast image acquisition. Human osteochondral samples (n = 41, four cadavers) were immersed in a contrast agent solution containing two agents (cationic CA4+ and non-ionic gadoteridol) and imaged with synchrotron microCT at an early diffusion time point (2 h) and at diffusion equilibrium (72 h) using two monochromatic X-ray energies (32 and 34 keV). The dual contrast technique enabled simultaneous determination of CA4+ (i.e., proteoglycan content) and gadoteridol (i.e., water content) partitions within cartilage. Cartilage proteoglycan content and biomechanical properties correlated significantly (0.327 < r < 0.736, p < 0.05) with CA4+ partition in superficial and middle zones at both diffusion time points. Normalization of the CA4+ partition with gadoteridol partition within the cartilage significantly (p < 0.05) improved the detection sensitivity for human osteoarthritic cartilage proteoglycan content, biomechanical properties, and overall condition (Mankin, Osteoarthritis Research Society International, and International Cartilage Repair Society grading systems). The dual energy technique combined with the dual contrast agent enables assessment of human articular cartilage proteoglycan content and biomechanical properties based on CA4+ partition determined using synchrotron microCT. Additionally, the dual contrast technique is not limited by the beam hardening artifact of conventional CT systems. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:563-573, 2020.

Synchrotron-based phase-contrast micro-CT as a tool for understanding pulmonary vascular pathobiology and the 3-D microanatomy of alveolar capillary dysplasia.

This study aimed to explore the value of synchrotron-based phase-contrast microcomputed tomography (micro-CT) in pulmonary vascular pathobiology. The microanatomy of the lung is complex with intricate branching patterns. Tissue sections are therefore difficult to interpret. Recruited intrapulmonary bronchopulmonary anastomoses (IBAs) have been described in several forms of pulmonary hypertension, including alveolar capillary dysplasia with misaligned pulmonary veins (ACD/MPV). Here, we examine paraffin-embedded tissue using this nondestructive method for high-resolution three-dimensional imaging. Blocks of healthy and ACD/MPV lung tissue were used. Pulmonary and bronchial arteries in the ACD/MPV block had been preinjected with dye. One section per block was stained, and areas of interest were marked to allow precise beam-alignment during image acquisition at the X02DA TOMCAT beamline (Swiss Light Source). A ×4 magnifying objective coupled to a 20-µm thick scintillating material and a sCMOS detector yielded the best trade-off between spatial resolution and field-of-view. A phase retrieval algorithm was applied and virtual tomographic slices and video clips of the imaged volumes were produced. Dye injections generated a distinct attenuation difference between vessels and surrounding tissue, facilitating segmentation and three-dimensional rendering. Histology and immunohistochemistry post-imaging offered complementary information. IBAs were confirmed in ACD/MPV, and the MPVs were positioned like bronchial veins/venules. We demonstrate the advantages of using synchrotron-based phase-contrast micro-CT for three-dimensional characterization of pulmonary microvascular anatomy in paraffin-embedded tissue. Vascular dye injections add additional value. We confirm intrapulmonary shunting in ACD/MPV and provide support for the hypothesis that MPVs are dilated bronchial veins/venules.

Triple Contrast CT Method Enables Simultaneous Evaluation of Articular Cartilage Composition and Segmentation.

Early degenerative changes of articular cartilage are detected using contrast-enhanced computed tomography (CT) with a cationic contrast agent (CA). However, cationic CA diffusion into degenerated cartilage decreases with proteoglycan depletion and increases with elevated water content, thus hampering tissue evaluation at early diffusion time points. Furthermore, the contrast at synovial fluid-cartilage interface diminishes as a function of diffusion time hindering accurate cartilage segmentation. For the first time, we employ quantitative dual-energy CT (QDECT) imaging utilizing a mixture of three CAs (cationic CA4+ and non-ionic gadoteridol which are sensitive to proteoglycan and water contents, respectively, and bismuth nanoparticles which highlight the cartilage surface) to simultaneously segment the articulating surfaces and determine of the cartilage condition. Intact healthy, proteoglycan-depleted, and mechanically injured bovine cartilage samples (n = 27) were halved and imaged with synchrotron microCT 2-h post immersion in triple CA or in dual CA (CA4+ and gadoteridol). CA4+ and gadoteridol partitions were determined using QDECT, and pairwise evaluation of these partitions was conducted for samples immersed in dual and triple CAs. In conclusion, the triple CA method is sensitive to proteoglycan depletion while maintaining sufficient contrast at the articular surface to enable detection of cartilage lesions caused by mechanical impact.

Co-localization of microstructural damage and excessive mechanical strain at aortic branches in angiotensin-II-infused mice.

Animal models of aortic aneurysm and dissection can enhance our limited understanding of the etiology of these lethal conditions particularly because early-stage longitudinal data are scant in humans. Yet, the pathogenesis of often-studied mouse models and the potential contribution of aortic biomechanics therein remain elusive. In this work, we combined micro-CT and synchrotron-based imaging with computational biomechanics to estimate in vivo aortic strains in the abdominal aorta of angiotensin-II-infused ApoE-deficient mice, which were compared with mouse-specific aortic microstructural damage inferred from histopathology. Targeted histology showed that the 3D distribution of micro-CT contrast agent that had been injected in vivo co-localized with precursor vascular damage in the aortic wall at 3 days of hypertension, with damage predominantly near the ostia of the celiac and superior mesenteric arteries. Computations similarly revealed higher mechanical strain in branching relative to non-branching regions, thus resulting in a positive correlation between high strain and vascular damage in branching segments that included the celiac, superior mesenteric, and right renal arteries. These results suggest a mechanically driven initiation of damage at these locations, which was supported by 3D synchrotron imaging of load-induced ex vivo delaminations of angiotensin-II-infused suprarenal abdominal aortas. That is, the major intramural delamination plane in the ex vivo tested aortas was also near side branches and specifically around the celiac artery. Our findings thus support the hypothesis of an early mechanically mediated formation of microstructural defects at aortic branching sites that subsequently propagate into a macroscopic medial tear, giving rise to aortic dissection in angiotensin-II-infused mice.

Diffractive small angle X-ray scattering imaging for anisotropic structures.

Insights into the micro- and nano-architecture of materials is crucial for understanding and predicting their macroscopic behaviour. In particular, for emerging applications such as meta-materials, the micrometer scale becomes highly relevant. The micro-architecture of such materials can be tailored to exhibit specific mechanical, optical or electromagnetic behaviours. Consequently, quality control at micrometer scale must be guaranteed over extended areas. Mesoscale investigations over millimetre sized areas can be performed by scanning small angle X-ray scattering methods (SAXS). However, due to their long measurement times, real time or operando investigations are hindered. Here we present a method based on X-ray diffractive optics that enables the acquisition of SAXS signals in a single shot (few milliseconds) over extended areas. This method is applicable to a wide range of X-ray sources with varying levels of spatial coherence and monochromaticity, as demonstrated from the experimental results. This enables a scalable solution of spatially resolved SAXS.

Synchrotron-based visualization and segmentation of elastic lamellae in the mouse carotid artery during quasi-static pressure inflation.

In computational aortic biomechanics, aortic and arterial tissue are typically modelled as a homogeneous layer, making abstraction not only of the layered structure of intima, media and adventitia but also of the microstructure that exists within these layers. Here, we present a novel method to visualize the microstructure of the tunica media along the entire circumference of the vessel. To that end, we developed a pressure-inflation device that is compatible with synchrotron-based phase-contrast imaging. Using freshly excised left common carotid arteries from n = 12 mice, we visualized how the lamellae and interlamellar layers inflate as the luminal pressure is increased from 0 to 120 mm Hg in quasi-static steps. A graph-based segmentation algorithm subsequently allowed us to automatically segment each of the three lamellae, resulting in a three-dimensional geometry that represents lamellae, interlamellar layers and adventitia at nine different pressure levels. Our results demonstrate that the three elastic lamellae unfold and stretch simultaneously as luminal pressure is increased. In the long term, we believe that the results presented in this work can be a first step towards a better understanding of the mechanics of the arterial microstructure.

Simultaneous Quantitation of Cationic and Non-ionic Contrast Agents in Articular Cartilage Using Synchrotron MicroCT Imaging.

Early diagnosis of acute cartilage injuries enables monitoring of disease progression and improved treatment option planning to prevent post-traumatic osteoarthritis. In contrast-enhanced computed tomography (CECT), the changes in cationic agent diffusion within the tissue reflect cartilage degeneration. The diffusion in degenerated cartilage depends on proteoglycan (PG) content and water content, but each having an opposite effect on diffusion, thus compromising the diagnostic sensitivity. To overcome this limitation, we propose the simultaneous imaging of cationic (sensitive to PG and water contents) and non-ionic (sensitive to water content) agents. In this study, quantitative dual-energy CT (QDECT) imaging of two agents is reported for the first time at clinically feasible imaging time points. Furthermore, this is the first time synchrotron microCT with monochromatic X-rays is employed in cartilage CECT. Imaging was conducted at 1 and 2 h post contrast agent immersion. Intact, PG-depleted, and mechanically injured + PG-depleted cartilage samples (n = 33) were imaged in a mixture of cationic (iodine-based CA4+) and non-ionic (gadolinium-based gadoteridol) agents. Concurrent evaluation of CA4+ and gadoteridol partitions in cartilage is accomplished using QDECT. Subsequent normalization of the CA4+ partition with that of the gadoteridol affords CA4+ attenuations that significantly correlate with PG content - a key marker of OA.

A new bioinspired method for pressure and flow sensing based on the underwater air-retaining surface of the backswimmer Notonecta.

In technical systems, static pressure and pressure changes are usually measured with piezoelectric materials or solid membranes. In this paper, we suggest a new biomimetic principle based on thin air layers that can be used to measure underwater pressure changes. Submerged backswimmers (Notonecta sp.) are well known for their ability to retain air layers on the surface of their forewings (hemelytra). While analyzing the hemelytra of Notonecta, we found that the air layer on the hemelytra, in combination with various types of mechanosensitive hairs (clubs and pins), most likely serve a sensory function. We suggest that this predatory aquatic insect can detect pressure changes and water movements by sensing volume changes of the air layer under water. In the present study, we used a variety of microscopy techniques to investigate the fine structure of the hemelytra. Furthermore, we provide a biomimetic proof of principle to validate our hypothesis. The suggested sensory principle has never been documented before and is not only of interest for sensory biologists but can also be used for the development of highly sensitive underwater acoustic or seismographic sensory systems.

Tomographic in vivo microscopy for the study of lung physiology at the alveolar level.

Lungs represent the essential part of the mammalian respiratory system, which is reflected in the fact that lung failure still is one of the leading causes of morbidity and mortality worldwide. Establishing the connection between macroscopic observations of inspiration and expiration and the processes taking place at the microscopic scale remains crucial to understand fundamental physiological and pathological processes. Here we demonstrate for the first time in vivo synchrotron-based tomographic imaging of lungs with pixel sizes down to a micrometer, enabling first insights into high-resolution lung structure. We report the methodological ability to study lung inflation patterns at the alveolar scale and its potential in resolving still open questions in lung physiology. As a first application, we identified heterogeneous distension patterns at the alveolar level and assessed first comparisons of lungs between the in vivo and immediate post mortem states.

Automated computer-assisted quantitative analysis of intact murine lungs at the alveolar scale.

Using state-of-the-art X-ray tomographic microscopy we can image lung tissue in three dimensions in intact animals down to a micrometer precision. The structural complexity and hierarchical branching scheme of the lung at this level of details, however, renders the extraction of biologically relevant quantities particularly challenging. We have developed a methodology for a detailed description of lung inflation patterns by measuring the size and the local curvature of the parenchymal airspaces. These quantitative tools for morphological and topological analyses were applied to high-resolution murine 3D lung image data, inflated at different pressure levels under immediate post mortem conditions. We show for the first time direct indications of heterogeneous intra-lobar and inter-lobar distension patterns at the alveolar level. Furthermore, we did not find any indication that a cyclic opening-and-collapse (recruitment) of a large number of alveoli takes place.