This study used X-ray Velocimetry (XV) to explore lung damage in a model of Acute Respiratory Distress Syndrome (ARDS). XV is a novel imaging technique that uses short X-ray videos recorded from 5 angles around the lung acquired with a fluoroscope during breathing to create detailed 3D maps showing how the lungs expand. Here, we introduced a lung injury in pigs to mimic damage from inhaling stomach contents, with XV images taken before and after a localised hydrochloric acid delivery. XV revealed localised ventilation changes at the injury site, as well as compensatory shifts in other lung regions. This pilot study provides a non-invasive, targeted approach to assess lung diseases, offering significant advantages over traditional methods. Our findings highlight the potential of XV to advance respiratory research and improve diagnostics and treatments for conditions like ARDS. This first demonstration of spatially resolved ventilation mapping in an ARDS model shows promise for both preclinical studies and clinical applications.

Propagation-based X-ray phase-contrast imaging (PBI) enables high-contrast visualization of lung structures and holds strong medical potential. However, safe translation to the clinic will require a substantial radiation dose reduction, which inevitably increases image noise. Supervised convolutional-neural-network-based denoising can restore image quality but depends on paired low- and high-dose datasets, which are rarely available in practice. Self-supervised methods avoid this limitation, yet most are not well adapted to the inverse problem of PBI computed tomography (CT). We introduce Neighbor2Inverse, a self-supervised denoising framework designed for low-dose PBI-CT that generalizes to clinical CT. Building on the Neighbor2Neighbor principle, each noisy projection is subsampled into two variants that preserve structural information but contain independent noise realizations. These are reconstructed separately, and the resulting pairs are used to train a denoising network directly in the image domain. We benchmark the proposed method against established analytical and self-supervised denoising approaches. In region-of-interest PBI CT experiments, Neighbor2Inverse achieves superior noise suppression while preserving fine structural details, as demonstrated by improved contrast-to-noise ratio, spatial resolution, and composite image quality metrics. Competitive performance is also observed on clinical CT data under simulated low-dose conditions. This work has been submitted to the IEEE for possible publication. Copyright may be transferred without notice, after which this version may no longer be accessible. Code, data, and interactive figures are available at https://github.com/J-3TO/Neighbor2Inverse.

AI-driven lung imaging: Advancing early diagnosis and monitoring Standard methods for diagnosing lung diseases often only detect problems once damage has already occurred. Learn how AI-driven lung imaging technologies can provide a way to identify disease earlier, allowing for more targeted interventions. Respiratory diseases remain a leading cause of morbidity and mortality worldwide, (1) yet our ability to detect and monitor lung disease at an early stage remains surprisingly limited. In many cases, clinically meaningful changes in lung function occur long before they are detectable using standard tools, delaying intervention and contributing to irreversible damage. This is particularly evident in chronic conditions such as cystic fibrosis, asthma, and bronchiectasis, where disease progression is often patchy and evolves silently over time.

X-ray dark-field imaging is well-suited to visualizing the health of the lungs because the alveoli create a strong dark-field signal. However, time-resolved and tomographic (i.e., 4D) dark-field imaging is challenging, since most x-ray dark-field techniques require multiple sample exposures, captured while scanning the position of crystals or gratings. Here, we present the first in vivo 4D x-ray dark-field lung imaging in mice. This was achieved by synchronizing the data acquisition process of a single-exposure grid-based imaging approach with the breath cycle. The short data acquisition time per dark-field projection made this approach feasible for 4D x-ray dark-field imaging by minimizing the motion-blurring effect and the total time required. Images were captured from a control mouse and from mouse models of muco-obstructive disease and lung cancer, where a change in the size of the alveoli was expected. This work demonstrates that the 4D dark-field signal provides complementary tomographic information that is inaccessible from conventional attenuation-based CT images, in particular, measurements that indicate changes in the size of the alveoli from different parts of the lungs during the breath cycle, with examples shown across the different models. By quantifying the dark-field signal and relating it to physical properties of the alveoli, this technique could be used to perform functional lung imaging that allows the assessment of both global and regional lung conditions where the size or expansion of the alveoli is affected.

Almost all patients with mucopolysaccharidosis (MPS) develop respiratory dysfunction of varying severity during disease progression. While respiratory disease in MPS has traditionally been attributed to upper airway obstruction caused by glycosaminoglycan (GAG) accumulation in the trachea and bronchi, involvement of the intrapulmonary conducting airways and lung parenchyma remains poorly defined. Here, we characterised lung disease in a mouse model of MPS I using a combination of non-invasive X-ray Velocimetry (XV) functional lung imaging and gold-standard flexiVent respiratory mechanics testing, complemented by lung volume measurements and histological analysis. XV provides regional ventilation information across the entire lung during tidal breathing. MPS I mice demonstrated reduced mean specific ventilation (the average regional expansion of lung tissue across the respiratory cycle), driven predominantly by reduced ventilation in the inner (mediastinal-adjacent) lung regions, with evidence of spatially heterogeneous ventilation distribution. Lung mechanics testing showed increased conducting airway resistance, increased respiratory system compliance and reduced tissue elastance, consistent with impaired elastic recoil and expiratory flow limitation. Lung volume analysis revealed reduced opening pressure following degassing together with increased residual volume, functional residual capacity and vital capacity. Histological analysis demonstrated heterogeneous parenchymal architecture with regions of enlarged airspaces. Together, these findings demonstrate that respiratory dysfunction in MPS I is not limited to upper airway obstruction but also involves intrinsic abnormalities of the intrapulmonary conducting airways and lung parenchyma. This intrinsic pulmonary pathology likely contributes to obstructive lung disease and may underlie the susceptibility to respiratory failure observed in patients with MPS I.

Recurrent bacterial infections with Pseudomonas aeruginosa result in chronic airway inflammation, lung damage and eventual respiratory failure, and are the major cause of morbidity and mortality in people with cystic fibrosis (CF). Animal models are essential for understanding disease progression and assessing potential treatments in the presence of infection. Previously reported P. aeruginosa lung infection rodent models for CF research have weaknesses that include being acute rather than chronic infections, high levels of associated mortality, use laboratory strains of P. aeruginosa, or do not utilise CF rodents. The aim of this study was to create a localised single-lung P. aeruginosa infection in wildtype and two CF rat models, by using a miniature bronchoscope to deliver bacteria embedded in agar beads generated from a clinical CF bacterial isolate. Cohorts of animals were assessed at days 7, 14, 21 and 63. The number of colony forming units were measured, along with bronchoalveolar lavage, flexiVent mechanics, X-ray Velocimetry (XV) ventilation analysis, and histopathology. The resulting infection was well tolerated by all animals of all genotypes with no mortality associated with the procedure or infection. The right-lung exhibited localised acute bronchopneumonia and lymphocytic vasculitis early, progressing to chronic interstitial pneumonia with fibrosis and emphysema. Bacteria persisted for 9 weeks (63 days) in all genotypes, with lung mechanics changes observed by day 63 of the infection. The precise delivery of bacterial laden beads using a miniature bronchoscope generated a controlled and reproducible infection that persisted for up to nine weeks, with minimal impact on animal health.

Barely visible impact damage (BVID) can cause serious issue for composite structures, due to sub-surface damage seriously reducing the strength of the material without showing easily detectable surface signs. Dark-field imaging measures ultra-small angle scattering caused by microscopic features within samples. It is sensitive to damage in composite materials which would otherwise be invisible in conventional radiography. Here we demonstrate BVID detection with speckle-based dark-field imaging, a technique requiring only sandpaper (to create the speckle-pattern) in addition to a conventional X-ray imaging setup to extract the dark-field imaging. We demonstrate that the technique is capable of detecting both matrix cracking and delaminations by imaging materials susceptible to these failure mechanisms.

In vivo models that replicate and reproduce human lung cancer and its response to therapy are necessary for the development of new therapeutic strategies and understanding drug resistance. Imaging lung tumors in live animals to monitor tumor growth and response to therapy is challenging owing to the location of the lungs and their constant movement during breathing. X-ray velocimetry (XV) is a novel functional lung imaging technique that maps regional lung expansion during breathing, providing spatial information on where ventilation changes occur. The aim of this pilot study was to use XV and flexiVent lung mechanics assessments to determine the effect of tumor growth on lung function in mice at 2 or 3 weeks post tumor induction and to evaluate the efficacy of these two tools. Histological analysis showed that tumor growth was not uniform between animals. At 3 weeks post tumor induction, some XV ventilation and flexiVent lung mechanics parameters were significantly different from baseline. Both techniques gave metrics that correlated with the tumor counts from the histology. In some mice, XV revealed localized regions with altered expansion rates.

The lung is a complex organ with a hierarchical structure, containing four times more air than tissue. It is in constant contact with environmental factors such as pollution and pathogens, leading to pathological alterations at various hierarchical levels. Because of its intricate structure and continuous movement, lung imaging presents significant challenges for most existing techniques. Recent advancements in phase-contrast computed tomography and photon-counting detectors have greatly enhanced lung imaging capabilities. Specifically, propagation-based imaging (PBI), a phase-contrast method that does not require optical elements, has proven particularly effective at low X-ray dose rates due to the strong phase shifts between lung tissue and aerated regions. This study introduces an in situ imaging approach for large-scale lungs using PBI at the Imaging and Medical Beamline (IMBL) of the Australian Synchrotron. We investigated optimal conditions for PBI, including energy and propagation distance settings, and found that an X-ray beam energy of 70 keV combined with a 7 m propagation distance yields the highest image quality in terms of contrast-to-noise ratio while also delivering the lowest radiation dose. Furthermore, Monte Carlo simulations were performed on the reconstructed volume to calculate absorbed radiation doses in tissues. These findings provide valuable insights for designing future experiments aimed at minimizing radiation exposure and potentially enable in vivo applications in larger animals or even humans.

Objective. Endobronchial valves (EBVs) are a minimally invasive treatment for emphysema. After bronchoscopic placement the valves reduce the flow of air into targeted areas of the lung, causing collapse, and allowing the remainder of the lung to function more effectively. This pilot study aims to demonstrate the capability and potential of a new imaging modality-x-ray velocimetry (XV)-for detecting these changes to lung function. Approach. XV is a novel method that uses x-ray images taken during a breath to track lung motion, producing 3D maps of local ventilation. Healthy sheep received a computed tomography (CT) scan and underwent XV imaging before and after EBVs were placed in the lung. Sheep were imaged again when the EBVs were removed after 14 days. Main results. XV enabled visualisation and quantification of a reduction of airflow to the areas downstream of the EBVs, both in areas where collapse was and was not visible in CT. Changes to ventilation were also clearly visible in the remainder of the lungs. Significance. This preclinical pilot study has shown XV is capable of detecting changes to ventilation caused by EBV placement, paving the way towards use in patients.

BACKGROUND AND OBJECTIVE:β-ENaC-Tg mice serve as a relevant model of muco-obstructive lung disease and diffuse-type emphysema, with impaired mucociliary clearance, mucus obstruction, chronic airway inflammation, structural lung damage, and altered lung function. The aim of this study was to undertake a comprehensive analysis of lung function and mechanics of the adult β-ENaC-Tg model. METHODS:Adult β-ENaC-Tg and wild-type littermates underwent X-ray velocimetry (XV) scans using a Permetium XV scanner (4DMedical, Melbourne, Australia). For comparative lung mechanics, lung function assessments were conducted with a flexiVent system (SCIREQ, Montreal, Canada). RESULTS:XV imaging demonstrated elevated ventilation defect percentage, mean specific ventilation, and ventilation heterogeneity in β-ENaC-Tg mice. Spatial analysis of ventilation maps indicated increased ventilation variability in the peripheral lung regions, as well as an increased proportion of under-ventilated areas. The flexiVent analysis indicated that compared to wild types, β-ENaC-Tg mice have a significantly more compliant lungs with increased inspiratory capacity, reduced tissue elastance, and increased hysteresivity (heterogeneity), suggesting loss of parenchymal integrity. CONCLUSION:This research highlights the utility of XV imaging in evaluating ventilation defects in the β-ENaC-Tg model and provides a comprehensive lung function analysis.

This paper shows the internal evolution of wrinkles occurring in a single diaphragm forming process using time resolved in situ XCT scanning. Forming trials were carried out over a curved C-spar geometry using Non-Crimp Fabric (NCF) plies of 45 degrees/135 degrees, 0 degrees/90 degrees and 0 degrees/45 degrees orientations arranged in single-orientation and mixed orientation stacks. Results show most of the diaphragm movement and change in wrinkle size, shape, and location occurs in the early application (<0.1 bar) of vacuum. The further application of vacuum shows consolidation, with wrinkle size decreasing but shape and location remaining largely unchanged. Single orientation stacks of 45 degrees/135 degrees plies showed the closest resemblance between initial and final wrinkling, while mixed-orientation stacks showed complex wrinkling due to the 0 degrees plies creating internal ply separation due to excess length. Overall, the study shines a light on the internal interaction between layers that occurs during wrinkling, showing mechanisms only observable with XCT.

Soil compaction and increased global drought enhance soil mechanical strength and stiffness, which poses threat to soil health and ultimately adversely impacts society. Despite these restrictive conditions, some plant roots are able to grow under these harsh environments. This study was formed to develop a proof of concept measurement system that could be used to quaantify local plant biomechanical pressures during growth in in-situ assays. Using micro cone penetrometer root analogues, we tested the viability of combining X-ray computed tomography (XCT) with X-ray diffraction (XRD) measurements in order to understand whether we can characterize the mechanics during penetration associated with root growth. Cone penetrometers were used to penetrate into surrogate soil materials (e.g. gypsum), as these materials have porous and granular behaviour similar to natural soils but facilitate diffraction measurements due to their well defined atomic structures. Diffraction maps were generated across a sub region of the imaged space in order to quantify changes in the diffraction patterns. Diffraction maps are simplified representations by spectra peaks resulting from integrated Debeye-Scherrerr rings. Shifts in the spectra peaks are associated with deformations of the lattice crystals that comprise the surrogate soil material resulting from applied loads from the micro cone penetrometers. XCT images highlight compaction zones near the cone tip during penetration, with an increase in the compression zones radius as we push the cone deeper into the media. The XRD measurements peak shifts just outside of the local compacted zone of the cone. Qualitative finite element simulations illustrate that this is likely due to local inelastic deformations near the cone tip during penetration. However, the remote elastic deformations can be monitored as long as the inelastic zone remains local to the cone tip. While direct measurements near a root tip might be difficult due to the inelastic nature of the penetration process, the results show promise in terms of gaining insights into the locality of root growth biomechanical processes and the potential to elucidate root strategies under compacted soils in an in-situ test. ### Competing Interest Statement The authors have declared no competing interest.

Two cystic fibrosis (CF) rat models, one carrying the common Phe508del mutation and the other a nonsense cystic fibrosis transmembrane conductance regulator (CFTR) mutation (knockout) were previously characterised. Although relevant CFTR mRNA reductions were present in the lung, no overt CF lung disease was observed. This study used flexiVent lung mechanic assessment and regional ventilation assessment via X-ray velocimetry (XV) functional imaging to assess the lung phenotype in both models. To determine the sensitivity of XV regional ventilation imaging, the effect of a localised physical obstruction (delivery of agar beads to part of the lungs) on lung ventilation was examined. At baseline, Phe508del and knockout CF rats had a lower inspiratory capacity, total respiratory system compliance, and static compliance than wildtype rats. Following agar bead delivery all XV ventilation parameters were altered, with substantial increases in poorly ventilated regions and ventilation heterogeneity. XV ventilation maps accurately identified locations of bead-induced airflow changes. Despite unremarkable lung histopathology, this study indicated that CF rats display altered respiratory mechanics, with CF rats needing to exert additional effort to expand and deflate their lungs due to increased stiffness. This study demonstrated the utility of XV imaging providing spatial lung ventilation information.

β-ENaC-Tg mice serve as a relevant model of muco-obstructive lung disease, with impaired mucociliary clearance, mucus obstruction, chronic airway inflammation, structural lung damage, and altered lung function. The aim of this study was to undertake a comprehensive lung function and mechanics analysis of the adult β-ENaC-Tg model. β-ENaC-Tg and wild-type littermates underwent X-ray Velocimetry (XV) scans using a Permetium XV scanner (4DMedical, Melbourne, Australia). For comparative lung mechanics, lung function assessments were conducted with a flexiVent system. XV imaging demonstrated elevated ventilation defect percentage, mean specific ventilation, and ventilation heterogeneity in β-ENaC-Tg mice. Spatial analysis of ventilation maps indicated increased ventilation variability in the peripheral lung regions, as well as an increased proportion of under-ventilated areas. The flexiVent analysis indicated that compared to wild-types, β-ENaC-Tg mice have a significantly more compliant lungs with increased inspiratory capacity, reduced tissue elastance and increased hysteresivity (heterogeneity), suggesting loss of parenchymal integrity. This research highlights the utility of XV imaging in evaluating ventilation defects in the β-ENaC-Tg model and provides a comprehensive lung function analysis. ### Competing Interest Statement MD and DP were involved in the research development and validation of the XV technology and have personally purchased shares in 4DMedical. NE and KN employed by 4DMedical.

Introduction:Phe508del is the most common cystic fibrosis transmembrane conductance regulator (CFTR) gene variant that results in the recessive genetic disorder cystic fibrosis (CF). The recent development of highly effective CFTR modulator therapies has led to significant health improvements in individuals with this mutation. While numerous animal models of CF exist, few have a CFTR mutation that is amenable to the triple combination therapy elexacaftor-tezacaftor-ivacaftor (ETI).Methods: To determine the responsiveness of Phe508del rats to ETI, a baseline nasal potential difference was measured. Subsequently, they received ETI daily for 14 days, after which post-treatment nasal potential difference, lung mechanics (via flexiVent) and lung ventilation (via X-ray Velocimetry) were assessed.Results: Chloride ion transport in nasal airways was restored in Phe508del rats treated with ETI, but neither lung mechanics nor ventilation were significantly altered.Discussion: These findings validate the usefulness of this rat model for future investigations of modulator therapy in CF.

Objective. Magnetic nanoparticles can be used as a targeted delivery vehicle for genetic therapies. Understanding how they can be manipulated within the complex environment of live airways is key to their application to cystic fibrosis and other respiratory diseases. Approach. Dark-field x-ray imaging provides sensitivity to scattering information, and allows the presence of structures smaller than the detector pixel size to be detected. In this study, ultra-fast directional dark-field synchrotron x-ray imaging was utlilised to understand how magnetic nanoparticles move within a live, anaesthetised, rat airway under the influence of static and moving magnetic fields. Main results. Magnetic nanoparticles emerging from an indwelling tracheal cannula were detectable during delivery, with dark-field imaging increasing the signal-to-noise ratio of this event by 3.5 times compared to the x-ray transmission signal. Particle movement as well as particle retention was evident. Dynamic magnetic fields could manipulate the magnetic particles in situ. Significance. This is the first evidence of the effectiveness of in vivo dark-field imaging operating at these spatial and temporal resolutions, used to detect magnetic nanoparticles. These findings provide the basis for further development toward the effective use of magnetic nanoparticles, and advance their potential as an effective delivery vehicle for genetic agents in the airways of live organisms.