eSoil: A low-power bioelectronic growth scaffold that enhances crop seedling growth.

Active hydroponic substrates that stimulate on demand the plant growth have not been demonstrated so far. Here, we developed the eSoil, a low-power bioelectronic growth scaffold that can provide electrical stimulation to the plants' root system and growth environment in hydroponics settings. eSoil's active material is an organic mixed ionic electronic conductor while its main structural component is cellulose, the most abundant biopolymer. We demonstrate that barley seedlings that are widely used for fodder grow within the eSoil with the root system integrated within its porous matrix. Simply by polarizing the eSoil, seedling growth is accelerated resulting in increase of dry weight on average by 50% after 15 d of growth. The effect is evident both on root and shoot development and occurs during the growth period after the stimulation. The stimulated plants reduce and assimilate NO3- more efficiently than controls, a finding that may have implications on minimizing fertilizer use. However, more studies are required to provide a mechanistic understanding of the physical and biological processes involved. eSoil opens the pathway for the development of active hydroponic scaffolds that may increase crop yield in a sustainable manner.

Optimization of phase contrast imaging with a nano-focus x-ray tube.

Propagation-based phase contrast imaging with a laboratory x-ray source is a valuable tool for studying samples that show only low absorption contrast, either because of low density, elemental composition, or small feature size. If a propagation distance between sample and detector is introduced and the illumination is sufficiently coherent, the phase shift in the sample will cause additional contrast around interfaces, known as edge enhancement fringes. The strength of this effect depends not only on sample parameters and energy but also on the experimental geometry, which can be optimized accordingly. Recently, x-ray lab sources using transmission targets have become available, which provide very small source sizes in the few hundred nanometer range. This allows the use of a high-magnification geometry with a very short source-sample distance, while still achieving sufficient spatial coherence at the sample position. Moreover, the high geometrical magnification makes it possible to use detectors with a larger pixel size without reducing the image resolution. Here, we explore the influence of magnification on the edge enhancement fringes in such a geometry. We find experimentally and theoretically that the fringes become maximal at a magnification that is independent of the total source-detector distance. This optimal magnification only depends on the source size, the steepness of the sample feature, and the detector resolution. A stronger influence of the sample feature on the optimal magnification compared to low-magnification geometries is observed.

Axon morphology is modulated by the local environment and impacts the noninvasive investigation of its structure-function relationship.

Axonal conduction velocity, which ensures efficient function of the brain network, is related to axon diameter. Noninvasive, in vivo axon diameter estimates can be made with diffusion magnetic resonance imaging, but the technique requires three-dimensional (3D) validation. Here, high-resolution, 3D synchrotron X-ray nano-holotomography images of white matter samples from the corpus callosum of a monkey brain reveal that blood vessels, cells, and vacuoles affect axonal diameter and trajectory. Within single axons, we find that the variation in diameter and conduction velocity correlates with the mean diameter, contesting the value of precise diameter determination in larger axons. These complex 3D axon morphologies drive previously reported 2D trends in axon diameter and g-ratio. Furthermore, we find that these morphologies bias the estimates of axon diameter with diffusion magnetic resonance imaging and, ultimately, impact the investigation and formulation of the axon structure-function relationship.

Dose-efficient multimodal microscopy of human tissue at a hard X-ray nanoprobe beamline.

X-ray fluorescence microscopy performed at nanofocusing synchrotron beamlines produces quantitative elemental distribution maps at unprecedented resolution (down to a few tens of nanometres), at the expense of relatively long measuring times and high absorbed doses. In this work, a method was implemented in which fast low-dose in-line holography was used to produce quantitative electron density maps at the mesoscale prior to nanoscale X-ray fluorescence acquisition. These maps ensure more efficient fluorescence scans and the reduction of the total absorbed dose, often relevant for radiation-sensitive (e.g. biological) samples. This multimodal microscopy approach was demonstrated on human sural nerve tissue. The two imaging modes provide complementary information at a comparable resolution, ultimately limited by the focal spot size. The experimental setup presented allows the user to swap between them in a flexible and reproducible fashion, as well as to easily adapt the scanning parameters during an experiment to fine-tune resolution and field of view.

X-ray in-line holography and holotomography at the NanoMAX beamline.

Coherent X-ray imaging techniques, such as in-line holography, exploit the high brilliance provided by diffraction-limited storage rings to perform imaging sensitive to the electron density through contrast due to the phase shift, rather than conventional attenuation contrast. Thus, coherent X-ray imaging techniques enable high-sensitivity and low-dose imaging, especially for low-atomic-number (Z) chemical elements and materials with similar attenuation contrast. Here, the first implementation of in-line holography at the NanoMAX beamline is presented, which benefits from the exceptional focusing capabilities and the high brilliance provided by MAX IV, the first operational diffraction-limited storage ring up to approximately 300 eV. It is demonstrated that in-line holography at NanoMAX can provide 2D diffraction-limited images, where the achievable resolution is only limited by the 70 nm focal spot at 13 keV X-ray energy. Also, the 3D capabilities of this instrument are demonstrated by performing holotomography on a chalk sample at a mesoscale resolution of around 155 nm. It is foreseen that in-line holography will broaden the spectra of capabilities of MAX IV by providing fast 2D and 3D electron density images from mesoscale down to nanoscale resolution.

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.

Six-dimensional real and reciprocal space small-angle X-ray scattering tomography.

When used in combination with raster scanning, small-angle X-ray scattering (SAXS) has proven to be a valuable imaging technique of the nanoscale, for example of bone, teeth and brain matter. Although two-dimensional projection imaging has been used to characterize various materials successfully, its three-dimensional extension, SAXS computed tomography, poses substantial challenges, which have yet to be overcome. Previous work using SAXS computed tomography was unable to preserve oriented SAXS signals during reconstruction. Here we present a solution to this problem and obtain a complete SAXS computed tomography, which preserves oriented scattering information. By introducing virtual tomography axes, we take advantage of the two-dimensional SAXS information recorded on an area detector and use it to reconstruct the full three-dimensional scattering distribution in reciprocal space for each voxel of the three-dimensional object in real space. The presented method could be of interest for a combined six-dimensional real and reciprocal space characterization of mesoscopic materials with hierarchically structured features with length scales ranging from a few nanometres to a few millimetres--for example, biomaterials such as bone or teeth, or functional materials such as fuel-cell or battery components.

Improved resolution in x-ray tomography by super-resolution.

In this paper, super-resolution imaging is described and evaluated for x-ray tomography and is compared with standard tomography and upscaling during reconstruction. Blurring is minimized due to the negligible point spread of photon counting detectors and an electromagnetically movable micro-focus x-ray spot. Scans are acquired in high and low magnification geometry, where the latter is used to minimize penumbral blurring from the x-ray source. Sharpness and level of detail can be significantly increased in reconstructed slices to the point where the source size becomes the limiting factor. The achieved resolution of the different methods is quantified and compared using biological samples via the edge spread function, modulation transfer function, and Fourier ring correlation.

3d phase-contrast nanotomography of unstained human skin biopsies may identify morphological differences in the dermis and epidermis between subjects.

BACKGROUND: Enteric neuropathy is described in most patients with gastrointestinal dysmotility and may be found together with reduced intraepidermal nerve fiber density (IENFD). The aim of this pilot study was to assess whether three-dimensional (3d) imaging of skin biopsies could be used to examine various tissue components in patients with gastrointestinal dysmotility. MATERIAL AND METHODS: Four dysmotility patients of different etiology and two healthy volunteers were included. From each subject, two 3-mm punch skin biopsies were stained with antibodies against protein gene product 9.5 or evaluated as a whole with two X-ray phase-contrast computed tomography (CT) setups, a laboratory µCT setup and a dedicated synchrotron radiation nanoCT end-station. RESULTS: Two patients had reduced IENFD, and two normal IENFD, compared with controls. µCT and X-ray phase-contrast holographic nanotomography scanned whole tissue specimens, with optional high-resolution scans revealing delicate structures, without differentiation of various fibers and cells. Irregular architecture of dermal fibers was observed in the patient with Ehlers-Danlos syndrome and the patient with idiopathic dysmotility showed an abundance of mesenchymal ground substance. CONCLUSIONS: 3d phase-contrast tomographic imaging may be useful to illustrate traits of connective tissue dysfunction in various organs and to demonstrate whether disorganized dermal fibers could explain organ dysfunction.

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.

3D analysis of the myenteric plexus of the human bowel by X-ray phase-contrast tomography - a future method?

OBJECTIVES: Light microscopical analysis in two dimensions, combined with immunohistochemistry, is presently the gold standard to describe the enteric nervous system (ENS). Our aim was to assess the usefulness of three-dimensional (3D) imaging by X-ray phase-contrast tomography in evaluating the ENS of the human bowel. MATERIAL AND METHODS: Myenteric ganglia were identified in full-thickness biopsies of the ileum and colon by hematoxylin & eosin staining. A1-mm biopsy punch was taken from the paraffin blocks and placed into a Kapton® tube for subsequent tomographic investigation. The samples were scanned, without further preparation, using phase-contrast tomography at two different scales: overview scans (performed with laboratory setups), which allowed localization of the nervous tissue (∼1µm effective voxel size); and high-resolution scans (performed with a synchrotron endstation), which imaged localized regions of 320x320x320 µm3 (176 nm effective voxel size). RESULTS: The contrast allowed us to follow the shape and the size changes of the ganglia, as well as to study their cellular components together with the cells and cellular projections of the periganglional space. Furthermore, it was possible to show the 3D network of the myenteric plexus and to quantify its volume within the samples. CONCLUSIONS: Phase-contrast X-ray tomography can be applied for volume analyses of the human ENS and to study tissue components in unstained paraffin-embedded tissue biopsies. This technique could potentially be used to study disease mechanisms, and to compare healthy and diseased tissues in clinical research.

Validation strategies for the interpretation of microstructure imaging using diffusion MRI.

Extracting microanatomical information beyond the image resolution of MRI would provide valuable tools for diagnostics and neuroscientific research. A number of mathematical models already suggest microstructural interpretations of diffusion MRI (dMRI) data. Examples of such microstructural features could be cell bodies and neurites, e.g. the axon's diameter or their orientational distribution for global connectivity analysis using tractography, and have previously only been possible to access through conventional histology of post mortem tissue or invasive biopsies. The prospect of gaining the same knowledge non-invasively from the whole living human brain could push the frontiers for the diagnosis of neurological and psychiatric diseases. It could also provide a general understanding of the development and natural variability in the healthy brain across a population. However, due to a limited image resolution, most of the dMRI measures are indirect estimations and may depend on the whole chain from experimental parameter settings to model assumptions and implementation. Here, we review current literature in this field and highlight the integrative work across anatomical length scales that is needed to validate and trust a new dMRI method. We encourage interdisciplinary collaborations and data sharing in regards to applying and developing new validation techniques to improve the specificity of future dMRI methods.

X-ray phase-contrast tomography with a compact laser-driven synchrotron source.

Between X-ray tubes and large-scale synchrotron sources, a large gap in performance exists with respect to the monochromaticity and brilliance of the X-ray beam. However, due to their size and cost, large-scale synchrotrons are not available for more routine applications in small and medium-sized academic or industrial laboratories. This gap could be closed by laser-driven compact synchrotron light sources (CLS), which use an infrared (IR) laser cavity in combination with a small electron storage ring. Hard X-rays are produced through the process of inverse Compton scattering upon the intersection of the electron bunch with the focused laser beam. The produced X-ray beam is intrinsically monochromatic and highly collimated. This makes a CLS well-suited for applications of more advanced--and more challenging--X-ray imaging approaches, such as X-ray multimodal tomography. Here we present, to our knowledge, the first results of a first successful demonstration experiment in which a monochromatic X-ray beam from a CLS was used for multimodal, i.e., phase-, dark-field, and attenuation-contrast, X-ray tomography. We show results from a fluid phantom with different liquids and a biomedical application example in the form of a multimodal CT scan of a small animal (mouse, ex vivo). The results highlight particularly that quantitative multimodal CT has become feasible with laser-driven CLS, and that the results outperform more conventional approaches.

Bone mineral crystal size and organization vary across mature rat bone cortex.

The macro- and micro-features of bone can be assessed by using imaging methods. However, nano- and molecular features require more detailed characterization, such as use of e.g., vibrational spectroscopy and X-ray scattering. Nano- and molecular features also affect the mechanical competence of bone tissue. The aim of the present study was to reveal the effects of mineralization and its alterations on the mineral crystal scale, by investigating the spatial variation of molecular composition and mineral crystal structure across the cross-section of femur diaphyses in young rats, and healthy and osteoporotic mature rats (N=5). Fourier transform infrared spectroscopy and scanning small- and wide-angle X-ray scattering (SAXS/WAXS) techniques with high spatial resolution were used at identical locations over the whole cross-section. This allowed quantification of point-by-point information about the spatial distribution of mineral crystal volume. All measured parameters (crystal dimensions, degree of orientation and predominant orientation) varied across the cortex. Specifically, the crystal dimensions were lower in the central cortex than in the endosteal and periosteal regions. Mineral crystal orientation followed the cortical circumference in the periosteal and endosteal regions, but was less well-oriented in the central regions. Central cortex is formed rapidly during development through endochondral ossification. Since rats possess no osteonal remodeling, this bone remains (until old age). Significant linear correlations were observed between the dimensional and organizational parameters, e.g., between crystal length and degree of orientation (R(2)=0.83, p<0.001). Application of SAXS/WAXS provides valuable information on bone nanostructure and its constituents, effects of diseases and, prospectively, mechanical competence.

Experimental results from a preclinical X-ray phase-contrast CT scanner.

To explore the future clinical potential of improved soft-tissue visibility with grating-based X-ray phase contrast (PC), we have developed a first preclinical computed tomography (CT) scanner featuring a rotating gantry. The main challenge in the transition from previous bench-top systems to a preclinical scanner are phase artifacts that are caused by minimal changes in the grating alignment during gantry rotation. In this paper, we present the first experimental results from the system together with an adaptive phase recovery method that corrects for these phase artifacts. Using this method, we show that the scanner can recover quantitatively accurate Hounsfield units in attenuation and phase. Moreover, we present a first tomography scan of biological tissue with complementary information in attenuation and phase contrast. The present study hence demonstrates the feasibility of grating-based phase contrast with a rotating gantry for the first time and paves the way for future in vivo studies on small animal disease models (in the mid-term future) and human diagnostics applications (in the long-term future).

Emphysema diagnosis using X-ray dark-field imaging at a laser-driven compact synchrotron light source.

In early stages of various pulmonary diseases, such as emphysema and fibrosis, the change in X-ray attenuation is not detectable with absorption-based radiography. To monitor the morphological changes that the alveoli network undergoes in the progression of these diseases, we propose using the dark-field signal, which is related to small-angle scattering in the sample. Combined with the absorption-based image, the dark-field signal enables better discrimination between healthy and emphysematous lung tissue in a mouse model. All measurements have been performed at 36 keV using a monochromatic laser-driven miniature synchrotron X-ray source (Compact Light Source). In this paper we present grating-based dark-field images of emphysematous vs. healthy lung tissue, where the strong dependence of the dark-field signal on mean alveolar size leads to improved diagnosis of emphysema in lung radiographs.

Non-binary phase gratings for x-ray imaging with a compact Talbot interferometer.

X-ray imaging using a Talbot-Lau interferometer, consisting of three binary gratings, is a well-established approach to acquire x-ray phase-contrast and dark-field images with a polychromatic source. However, challenges in the production of high aspect ratio gratings limit the construction of a compact setup for high x-ray energies. In this study we consider the use of phase gratings with triangular-shaped structures in an x-ray interferometer and show that such gratings can yield high visibilities for significantly shorter propagation distances than conventional gratings with binary structures. The findings are supported by simulation and experimental results for both cases of a monochromatic and a polychromatic source.

Pulmonary emphysema diagnosis with a preclinical small-animal X-ray dark-field scatter-contrast scanner.

PURPOSE: To test the hypothesis that the joint distribution of x-ray transmission and dark-field signals obtained with a compact cone-beam preclinical scanner with a polychromatic source can be used to diagnose pulmonary emphysema in ex vivo murine lungs. MATERIALS AND METHODS: The animal care committee approved this study. Three excised murine lungs with pulmonary emphysema and three excised murine control lungs were imaged ex vivo by using a grating-based micro-computed tomographic (CT) scanner. To evaluate the diagnostic value, the natural logarithm of relative transmission and the natural logarithm of dark-field scatter signal were plotted on a per-pixel basis on a scatterplot. Probability density function was fit to the joint distribution by using principle component analysis. An emphysema map was calculated based on the fitted probability density function. RESULTS: The two-dimensional scatterplot showed a characteristic difference between control and emphysematous lungs. Control lungs had lower average median logarithmic transmission (-0.29 vs -0.18, P = .1) and lower average dark-field signal (-0.54 vs -0.37, P = .1) than emphysematous lungs. The angle to the vertical axis of the fitted regions also varied significantly (7.8° for control lungs vs 15.9° for emphysematous lungs). The calculated emphysema distribution map showed good agreement with histologic findings. CONCLUSION: X-ray dark-field scatter images of murine lungs obtained with a preclinical scanner can be used in the diagnosis of pulmonary emphysema. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.13122413/-/DC1.

Quantitative X-ray phase-contrast computed tomography at 82 keV.

Potential applications of grating-based X-ray phase-contrast imaging are investigated in various fields due to its compatibility with laboratory X-ray sources. So far the method was mainly restricted to X-ray energies below 40 keV, which is too low to examine dense or thick objects, but a routine operation at higher energies is on the brink of realisation. In this study, imaging results obtained at 82 keV are presented. These comprise a test object consisting of well-defined materials for a quantitative analysis and a tooth to translate the findings to a biomedical sample. Measured linear attenuation coefficients ? and electron densities ?e are in good agreement with theoretical values. Improved contrast-to-noise ratios were found in phase contrast compared to attenuation contrast. The combination of both contrast modalities further enables to simultaneously assess information on density and composition of materials with effective atomic numbers Z? > 8. In our biomedical example, we demonstrate the possibility to detect differences in mass density and calcium concentration within teeth.

Aggrecan accumulates at sites of increased pulmonary arterial pressure in idiopathic pulmonary arterial hypertension.

Expansion of extracellular matrix occurs in all stages of pulmonary angiopathy associated with pulmonary arterial hypertension (PAH). In systemic arteries, dysregulation and accumulation of the large chondroitin-sulfate proteoglycan aggrecan is associated with swelling and disruption of vessel wall homeostasis. Whether aggrecan is present in pulmonary arteries, and its potential roles in PAH, has not been thoroughly investigated. Here, lung tissue from 11 patients with idiopathic PAH was imaged using synchrotron radiation phase-contrast microcomputed tomography (TOMCAT beamline, Swiss Light Source). Immunohistochemistry for aggrecan core protein in subsequently sectioned lung tissue demonstrated accumulation in PAH compared with failed donor lung controls. RNAscope in situ hybridization indicated ACAN expression in vascular endothelium and smooth muscle cells. Based on qualitative histological analysis, aggrecan localizes to cellular, rather than fibrotic or collagenous, lesions. Interestingly, ADAMTS15, a potential aggrecanase, was upregulated in pulmonary arteries in PAH. Aligning traditional histological analysis with three-dimensional renderings of pulmonary arteries from synchrotron imaging identified aggrecan in lumen-reducing lesions containing loose, cell-rich connective tissue, at sites of intrapulmonary bronchopulmonary shunting, and at sites of presumed elevated pulmonary blood pressure. Our findings suggest that ACAN expression may be an early response to injury in pulmonary angiopathy and supports recent work showing that dysregulation of aggrecan turnover is a hallmark of arterial adaptations to altered hemodynamics. Whether cause or effect, aggrecan and aggrecanase regulation in PAH are potential therapeutic targets.