X-ray phase and dark-field computed tomography without optical elements.

X-ray diffusive dark-field imaging, which allows spatially unresolved microstructure to be mapped across a sample, is an increasingly popular tool in an array of settings. Here, we present a new algorithm for phase and dark-field computed tomography based on the x-ray Fokker-Planck equation. Needing only a coherent x-ray source, sample, and detector, our propagation-based algorithm can map the sample density and dark-field/diffusion properties of the sample in 3D. Importantly, incorporating dark-field information in the density reconstruction process enables a higher spatial resolution reconstruction than possible with previous propagation-based approaches. Two sample exposures at each projection angle are sufficient for the successful reconstruction of both the sample density and dark-field Fokker-Planck diffusion coefficients. We anticipate that the proposed algorithm may be of benefit in biomedical imaging and industrial settings.

Directional dark-field retrieval with single-grid x-ray imaging.

Directional dark-field imaging is an emerging x-ray modality that is sensitive to unresolved anisotropic scattering from sub-pixel sample microstructures. A single-grid imaging setup can be used to capture dark-field images by looking at changes in a grid pattern projected upon the sample. By creating analytical models for the experiment, we have developed a single-grid directional dark-field retrieval algorithm that can extract dark-field parameters such as the dominant scattering direction, and the semi-major and -minor scattering angles. We show that this method is effective even in the presence of high image noise, allowing for low-dose and time-sequence imaging.

Quantifying the x-ray dark-field signal in single-grid imaging.

X-ray dark-field imaging reveals the sample microstructure that is unresolved when using conventional methods of x-ray imaging. In this paper, we derive a new method to extract and quantify the x-ray dark-field signal collected using a single-grid imaging set-up, and relate the signal strength to the number of sample microstructures, N. This was achieved by modelling sample-induced changes to the shadow of the upstream grid, and fitting experimental data to this model. Our results suggested that the dark-field scattering angle from our spherical microstructures deviates slightly from the theoretical model of N, which was consistent with results from other experimental methods. We believe the approach outlined here can equip quantitative dark-field imaging of small samples, particularly in cases where only one sample exposure is possible, either due to sample movement or radiation dose limitations. Future directions include an extension into directional dark-field imaging.

Methods for dynamic synchrotron X-ray respiratory imaging in live animals.

Small-animal physiology studies are typically complicated, but the level of complexity is greatly increased when performing live-animal X-ray imaging studies at synchrotron and compact light sources. This group has extensive experience in these types of studies at the SPring-8 and Australian synchrotrons, as well as the Munich Compact Light Source. These experimental settings produce unique challenges. Experiments are always performed in an isolated radiation enclosure not specifically designed for live-animal imaging. This requires equipment adapted to physiological monitoring and test-substance delivery, as well as shuttering to reduce the radiation dose. Experiment designs must also take into account the fixed location, size and orientation of the X-ray beam. This article describes the techniques developed to overcome the challenges involved in respiratory X-ray imaging of live animals at synchrotrons, now enabling increasingly sophisticated imaging protocols.

Photon-counting, energy-resolving and super-resolution phase contrast X-ray imaging using an integrating detector.

This work demonstrates the use of a scientific-CMOS (sCMOS) energy-integrating detector as a photon-counting detector, thereby eliminating dark current and read-out noise issues, that simultaneously provides both energy resolution and sub-pixel spatial resolution for X-ray imaging. These capabilities are obtained by analyzing visible light photon clouds that result when X-ray photons produce fluorescence from a scintillator in front of the visible light sensor. Using low-fluence monochromatic X-ray projections to avoid overlapping photon clouds, the centroid of individual X-ray photon interactions was identified. This enabled a tripling of the spatial resolution of the detector to 6.71 ± 0.04 µm. By calculating the total charge deposited by this interaction, an energy resolution of 61.2 ± 0.1% at 17 keV was obtained. When combined with propagation-based phase contrast imaging and phase retrieval, a signal-to-noise ratio of up to 15 ± 3 was achieved for an X-ray fluence of less than 3 photons/mm2.

Material decomposition from a single x-ray projection via single-grid phase contrast imaging.

This study describes a new approach for material decomposition in x-ray imaging, utilizing phase contrast both to increase sensitivity to weakly attenuating samples and to act as a complementary measurement to attenuation, therefore allowing two overlaid materials to be separated. The measurements are captured using the single-exposure, single-grid x-ray phase contrast imaging technique, with a novel correction that aims to remove propagation-based phase effects seen at sharp edges in the attenuation image. The use of a single-exposure technique means that images can be collected in a high-speed sequence. Results are shown for both a known two-material sample and for a biological specimen.

Multimodal Precision Imaging of Pulmonary Nanoparticle Delivery in Mice: Dynamics of Application, Spatial Distribution, and Dosimetry.

Targeted delivery of nanomedicine/nanoparticles (NM/NPs) to the site of disease (e.g., the tumor or lung injury) is of vital importance for improved therapeutic efficacy. Multimodal imaging platforms provide powerful tools for monitoring delivery and tissue distribution of drugs and NM/NPs. This study introduces a preclinical imaging platform combining X-ray (two modes) and fluorescence imaging (three modes) techniques for time-resolved in vivo and spatially resolved ex vivo visualization of mouse lungs during pulmonary NP delivery. Liquid mixtures of iodine (contrast agent for X-ray) and/or (nano)particles (X-ray absorbing and/or fluorescent) are delivered to different regions of the lung via intratracheal instillation, nasal aspiration, and ventilator-assisted aerosol inhalation. It is demonstrated that in vivo propagation-based phase-contrast X-ray imaging elucidates the dynamic process of pulmonary NP delivery, while ex vivo fluorescence imaging (e.g., tissue-cleared light sheet fluorescence microscopy) reveals the quantitative 3D drug/particle distribution throughout the entire lung with cellular resolution. The novel and complementary information from this imaging platform unveils the dynamics and mechanisms of pulmonary NM/NP delivery and deposition for each of the delivery routes, which provides guidance on optimizing pulmonary delivery techniques and novel-designed NM for targeting and efficacy.

Live-pig-airway surface imaging and whole-pig CT at the Australian Synchrotron Imaging and Medical Beamline.

The Australian Synchrotron Imaging and Medical Beamline (IMBL) was designed to be the world's widest synchrotron X-ray beam, partly to enable clinical imaging and therapeutic applications for humans, as well as for imaging large-animal models. Our group is currently interested in imaging the airways of newly developed cystic fibrosis (CF) animal models that display human-like lung disease, such as the CF pig. One key outcome measure for assessing the effectiveness of CF airway therapies is the ability of the lung to clear inhaled particulates by mucociliary transit (MCT). This study extends the ex vivo sheep and pig tracheal-tissue studies previously performed by the authors at the IMBL. In the present study, attempts were made to determine whether the design of the IMBL is suitable for imaging tracheal MCT in live pigs. The movement of 200 µm-diameter high-refractive-index (HRI) glass-bead marker particles deposited onto the tracheal airway surface of eight live piglets was tracked and quantified and the MCT response to aerosol delivery was examined. A high-resolution computed tomographic (CT) whole-animal post-mortem scan of one pig was also performed to verify the large sample CT capabilities of the IMBL. MCT tracking particles were visible in all animals, and the automated MCT tracking algorithms used were able to identify and track many particles, but accuracy was reduced when particles moved faster than ∼6 mm min-1 (50 pixels between exposures), or when the particles touched or overlapped. Renderings were successfully made from the CT data set. Technical issues prevented use of reliable shuttering and hence radiation doses were variable. Since dose must be carefully controlled in future studies, estimates of the minimum achievable radiation doses using this experiment design are shown. In summary, this study demonstrated the suitability of the IMBL for large-animal tracheal MCT imaging, and for whole-animal CT.

Ring artifact suppression in X-ray computed tomography using a simple, pixel-wise response correction.

We present a pixel-specific, measurement-driven correction that effectively reduces errors in detector response that give rise to the ring artifacts commonly seen in X-ray computed tomography (CT) scans. This correction is easy to implement, suppresses CT artifacts significantly, and is effective enough for use with both absorption and phase contrast imaging. It can be used as a standalone correction or in conjunction with existing ring artifact removal algorithms to further improve image quality. We validate this method using two X-ray CT data sets acquired using monochromatic sources, showing post-correction signal-to-noise increases of up to 55%, and we define an image quality metric to use specifically for the assessment of ring artifact suppression.

High-resolution mucociliary transport measurement in live excised large animal trachea using synchrotron X-ray imaging.

BACKGROUND: The Australian Synchrotron Imaging and Medical Beamline (IMBL) was designed as the world's widest synchrotron X-ray beam, enabling both clinical imaging and therapeutic applications for humans as well as the imaging of large animal models. Our group is developing methods for imaging the airways of newly developed CF animal models that display human-like lung disease, such as the CF pig, and we expect that the IMBL can be utilised to image airways in animals of this size. METHODS: This study utilised samples of excised tracheal tissue to assess the feasibility, logistics and protocols required for airway imaging in large animal models such as pigs and sheep at the IMBL. We designed an image processing algorithm to automatically track and quantify the tracheal mucociliary transport (MCT) behaviour of 103 µm diameter high refractive index (HRI) glass bead marker particles deposited onto the surface of freshly-excised normal sheep and pig tracheae, and assessed the effects of airway rehydrating aerosols. RESULTS: We successfully accessed and used scavenged tracheal tissue, identified the minimum bead size that is visible using our chosen imaging setup, verified that MCT could be visualised, and that our automated tracking algorithm could quantify particle motion. The imaging sequences show particles propelled by cilia, against gravity, up the airway surface, within a well-defined range of clearance speeds and with examples of 'clumping' behaviour that is consistent with the in vivo capture and mucus-driven transport of particles. CONCLUSION: This study demonstrated that the wide beam at the IMBL is suitable for imaging MCT in ex vivo tissue samples. We are now transitioning to in vivo imaging of MCT in live pigs, utilising higher X-ray energies and shorter exposures to minimise motion blur.

Capturing and visualizing transient X-ray wavefront topological features by single-grid phase imaging.

The detection, localisation and characterisation of stationary and singular points in the phase of an X-ray wavefield is a challenge, particularly given a time-evolving field. In this paper, the associated difficulties are met by the single-grid, single-exposure X-ray phase contrast imaging technique, enabling direct measurement of phase maxima, minima, saddle points and vortices, in both slowly varying fields and as a means to visualise weakly-attenuating samples that introduce detailed phase variations to the X-ray wavefield. We examine how these high-resolution vector measurements can be visualised, using branch cuts in the phase gradient angle to characterise phase features. The phase gradient angle is proposed as a useful modality for the localisation and tracking of sample features and the magnitude of the phase gradient for improved visualization of samples in projection, capturing edges and bulk structure while avoiding a directional bias. In addition, we describe an advanced two-stage approach to single-grid phase retrieval.

Singularimetry of local phase gradients using vortex lattices and in-line holography.

We have developed a differential form of singularimetry, which utilizes phase vortices or intensity gradient singularities as topological fiducial markers in a structured illumination context. This approach analytically measures phase gradients imparted by refracting specimens, yielding quantitative information that is both local and deterministic. We have quantified our phase gradient experiments to demonstrate that lattices of wave field singularities can be used to detect subtle phase gradients imparted by a spherical specimen and fiber optic cylinders.

Exploring experimental parameter choice for rapid speckle-tracking phase-contrast X-ray imaging with a paper analyzer.

Phase-contrast X-ray imaging using a paper analyzer enables the visualization of X-ray transparent biological structures using the refractive properties of the sample. The technique measures the sample-induced distortions of a spatially random reference pattern to retrieve quantitative sample information. This phase-contrast method is promising for biomedical application due to both a simple experimental set-up and a capability for real-time imaging. The authors explore the experimental configuration required to achieve robustness and accuracy in terms of (i) the paper analyzer feature size, (ii) the sample-to-detector distance, and (iii) the exposure time. Results using a synchrotron source confirm that the technique achieves accurate phase retrieval with a range of paper analyzers and at exposures as short as 0.5 ms. These exposure times are sufficiently short relative to characteristic physiological timescales to enable real-time dynamic imaging of living samples. A theoretical guide to the choice of sample-to-detector distance is also derived. While the measurements are specific to the set-up, these guidelines, the example speckle images, the strategies for analysis in the presence of noise and the experimental considerations and discussion will be of value to those who wish to use the speckle-tracking paper analyzer technique.

Live small-animal X-ray lung velocimetry and lung micro-tomography at the Australian Synchrotron Imaging and Medical Beamline.

The high flux and coherence produced at long synchrotron beamlines makes them well suited to performing phase-contrast X-ray imaging of the airways and lungs of live small animals. Here, findings of the first live-animal imaging on the Imaging and Medical Beamline (IMBL) at the Australian Synchrotron are reported, demonstrating the feasibility of performing dynamic lung motion measurement and high-resolution micro-tomography. Live anaesthetized mice were imaged using 30 keV monochromatic X-rays at a range of sample-to-detector propagation distances. A frame rate of 100 frames s(-1) allowed lung motion to be determined using X-ray velocimetry. A separate group of humanely killed mice and rats were imaged by computed tomography at high resolution. Images were reconstructed and rendered to demonstrate the capacity for detailed, user-directed display of relevant respiratory anatomy. The ability to perform X-ray velocimetry on live mice at the IMBL was successfully demonstrated. High-quality renderings of the head and lungs visualized both large structures and fine details of the nasal and respiratory anatomy. The effect of sample-to-detector propagation distance on contrast and resolution was also investigated, demonstrating that soft tissue contrast increases, and resolution decreases, with increasing propagation distance. This new capability to perform live-animal imaging and high-resolution micro-tomography at the IMBL enhances the capability for investigation of respiratory diseases and the acceleration of treatment development in Australia.

Feasibility study of propagation-based phase-contrast X-ray lung imaging on the Imaging and Medical beamline at the Australian Synchrotron.

Propagation-based phase-contrast X-ray imaging (PB-PCXI) using synchrotron radiation has achieved high-resolution imaging of the lungs of small animals both in real time and in vivo. Current studies are applying such imaging techniques to lung disease models to aid in diagnosis and treatment development. At the Australian Synchrotron, the Imaging and Medical beamline (IMBL) is well equipped for PB-PCXI, combining high flux and coherence with a beam size sufficient to image large animals, such as sheep, due to a wiggler source and source-to-sample distances of over 137 m. This study aimed to measure the capabilities of PB-PCXI on IMBL for imaging small animal lungs to study lung disease. The feasibility of combining this technique with computed tomography for three-dimensional imaging and X-ray velocimetry for studies of airflow and non-invasive lung function testing was also investigated. Detailed analysis of the role of the effective source size and sample-to-detector distance on lung image contrast was undertaken as well as phase retrieval for sample volume analysis. Results showed that PB-PCXI of lung phantoms and mouse lungs produced high-contrast images, with successful computed tomography and velocimetry also being carried out, suggesting that live animal lung imaging will also be feasible at the IMBL.

Tracking extended mucociliary transport activity of individual deposited particles: longitudinal synchrotron X-ray imaging in live mice.

To assess potential therapies for respiratory diseases in which mucociliary transit (MCT) is impaired, such as cystic fibrosis and primary ciliary dyskinesia, a novel and non-invasive MCT quantification method has been developed in which the transit rate and behaviour of individual micrometre-sized deposited particles are measured in live mice using synchrotron phase-contrast X-ray imaging. Particle clearance by MCT is known to be a two-phase process that occurs over a period of minutes to days. Previous studies have assessed MCT in the fast-clearance phase, ∼20 min after marker particle dosing. The aim of this study was to non-invasively image changes in particle presence and MCT during the slow-clearance phase, and simultaneously determine whether repeat synchrotron X-ray imaging of mice was feasible over periods of 3, 9 and 25 h. All mice tolerated the repeat imaging procedure with no adverse effects. Quantitative image analysis revealed that the particle MCT rate and the number of particles present in the airway both decreased with time. This study successfully demonstrated for the first time that longitudinal synchrotron X-ray imaging studies are possible in live small animals, provided appropriate animal handling techniques are used and care is taken to reduce the delivered radiation dose.

Dual scanning and full-field hard x-ray microscopy with a laboratory source.

We report on the experimental demonstration of a hard x-ray microscopy scheme achieving absorption and phase contrast imaging with a standard laboratory source. The x-ray optical system features two crossed planar waveguides coupled to the primary source. The dual waveguide acts as a secondary micron-sized source, enabling high imaging resolution. Both scanning and full-field imaging modes are demonstrated with the same experimental system, with a resolution of about 2 µm in scanning mode. Examples of absorption, differential phase and retrieved phase depth of thin metal grids and glass micro-spheres are reported as proof of concept of the technique.

Ultra-fastin vivodirectional dark-field x-ray imaging for visualising magnetic control of particles for airway gene delivery.

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 particlesin situ. Significance.This is the first evidence of the effectiveness ofin vivodark-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.

A sensitive x-ray phase contrast technique for rapid imaging using a single phase grid analyzer.

Phase contrast x-ray imaging (PCXI) is a promising imaging modality, capable of sensitively differentiating soft tissue structures at high spatial resolution. However, high sensitivity often comes at the cost of a long exposure time or multiple exposures per image, limiting the imaging speed and possibly increasing the radiation dose. Here, we demonstrate a PCXI method that uses a single short exposure to sensitively capture sample phase information, permitting high speed x-ray movies and live animal imaging. The method illuminates a checkerboard phase grid to produce a fine grid-like intensity reference pattern at the detector, then spatially maps sample-induced distortions of this pattern to recover differential phase images of the sample. The use of a phase grid is an improvement on our previous absorption grid work in two ways. There is minimal loss in x-ray flux, permitting faster imaging, and, a very fine pattern is produced for homogenous high spatial resolution. We describe how this pattern permits retrieval of five images from a single exposure; the sample phase gradient images in the horizontal and vertical directions, a projected phase depth image, an edge-enhanced image, and a type of scattering image. Finally, we describe how the reconstruction technique can achieve subpixel distortion retrieval and study the behavior of the technique in regard to analysis technique, Talbot distance, and exposure time.

On the quantification of sample microstructure using single-exposure x-ray dark-field imaging via a single-grid setup.

The size of the smallest detectable sample feature in an x-ray imaging system is usually restricted by the spatial resolution of the system. This limitation can now be overcome using the diffusive dark-field signal, which is generated by unresolved phase effects or the ultra-small-angle x-ray scattering from unresolved sample microstructures. A quantitative measure of this dark-field signal can be useful in revealing the microstructure size or material for medical diagnosis, security screening and materials science. Recently, we derived a new method to quantify the diffusive dark-field signal in terms of a scattering angle using a single-exposure grid-based approach. In this manuscript, we look at the problem of quantifying the sample microstructure size from this single-exposure dark-field signal. We do this by quantifying the diffusive dark-field signal produced by 5 different sizes of polystyrene microspheres, ranging from 1.0 to 10.8 µm, to investigate how the strength of the extracted dark-field signal changes with the sample microstructure size, [Formula: see text]. We also explore the feasibility of performing single-exposure dark-field imaging with a simple equation for the optimal propagation distance, given microstructure with a specific size and thickness, and show consistency between this model and experimental data. Our theoretical model predicts that the dark-field scattering angle is inversely proportional to [Formula: see text], which is also consistent with our experimental data.