Synchrotron X-ray Phase Contrast Imaging Research Articles

Correction: Corrigendum: Non-invasive airway health assessment: Synchrotron imaging reveals effects of rehydrating treatments on mucociliary transit in-vivo

To determine the efficacy of potential cystic fibrosis (CF) therapies we have developed a novel mucociliary transit (MCT) measurement that uses synchrotron phase contrast X-ray imaging (PCXI) to non-invasively measure the transit rate of individual micron-sized particles deposited into the airways of live mice. The aim of this study was to image changes in MCT produced by a rehydrating treatment based on hypertonic saline (HS), a current CF clinical treatment. Live mice received HS containing a long acting epithelial sodium channel blocker (P308); isotonic saline; or no treatment, using a nebuliser integrated within a small-animal ventilator circuit. Marker particle motion was tracked for 20 minutes using PCXI. There were statistically significant increases in MCT in the isotonic and HS-P308 groups. The ability to quantify in vivo changes in MCT may have utility in pre-clinical research studies designed to bring new genetic and pharmaceutical treatments for respiratory diseases into clinical trials.

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.

Non-invasive airway health assessment: Synchrotron imaging reveals effects of rehydrating treatments on mucociliary transit in-vivo

To determine the efficacy of potential cystic fibrosis (CF) therapies we have developed a novel mucociliary transit (MCT) measurement that uses synchrotron phase contrast X-ray imaging (PCXI) to non-invasively measure the transit rate of individual micron-sized particles deposited into the airways of live mice. The aim of this study was to image changes in MCT produced by a rehydrating treatment based on hypertonic saline (HS), a current CF clinical treatment. Live mice received HS containing a long acting epithelial sodium channel blocker (P308); isotonic saline; or no treatment, using a nebuliser integrated within a small-animal ventilator circuit. Marker particle motion was tracked for 20 minutes using PCXI. There were statistically significant increases in MCT in the isotonic and HS-P308 groups. The ability to quantify in vivo changes in MCT may have utility in pre-clinical research studies designed to bring new genetic and pharmaceutical treatments for respiratory diseases into clinical trials.

Dynamics of tracheal compression in the horned passalus beetle

Rhythmic patterns of compression and reinflation of the thin-walled hollow tubes of the insect tracheal system have been observed in a number of insects. These movements may be important for facilitating the transport and exchange of respiratory gases, but observing and characterizing the dynamics of internal physiological systems within live insects can be challenging due to their size and exoskeleton. Using synchrotron X-ray phase-contrast imaging, we observed dynamical behavior in the tracheal system of the beetle, Odontotaenius disjunctus. Similar to observations of tracheal compression in other insects, specific regions of tracheae in the thorax of O. disjunctus exhibit rhythmic collapse and reinflation. During tracheal compression, the opposing sides of a tracheal tube converge, causing the effective diameter of the tube to decrease. However, a unique characteristic of tracheal compression in this species is that certain tracheae collapse and reinflate with a wavelike motion. In the dorsal cephalic tracheae, compression begins anteriorly and continues until the tube is uniformly flattened; reinflation takes place in the reverse direction, starting with the posterior end of the tube and continuing until the tube is fully reinflated. We report the detailed kinematics of this pattern as well as additional observations that show tracheal compression coordinated with spiracle opening and closing. These findings suggest that tracheal compression may function to drive flow within the body, facilitating internal mixing of respiratory gases and ventilation of distal regions of the tracheal system.

High speed synchrotron x-ray phase contrast imaging of dynamic material response to split Hopkinson bar loading

The successful process of amalgamating both the time-resolved imaging capabilities present at the Advanced Photon Source beamline 32ID-B and the proficiency of high-rate loading offered by the split Hopkinson or Kolsky compression/tension bar apparatus is discussed and verification of system effectiveness is expressed via dynamic experiments on various material systems. Single particle sand interaction along with glass cracking during dynamic compression, and fiber-epoxy interfacial failure, ligament-bone debonding, and single-crystal silicon fragmentation due to dynamic tension, were imaged with 0.5 μs temporal resolution and μm-level spatial resolution. Synchrotron x-ray phase contrast imaging of said material systems being loaded with the Kolsky bar apparatus demonstratively depicts the effectiveness of the novel union between these two powerful techniques, thereby allowing for in situ analysis of the interior of the material system during high-rate loading for a variety of applications.

Dry deposition of pollutant and marker particles onto live mouse airway surfaces enhances monitoring of individual particle mucociliary transit behaviour

Particles suspended in the air are inhaled during normal respiration and unless cleared by airway defences, such as the mucociliary transit (MCT) system, they can remain and affect lung and airway health. Synchrotron phase-contrast X-ray imaging (PCXI) methods have been developed to non-invasively monitor the behaviour of individual particles in live mouse airways and in previous studies the MCT behaviour of particles and fibres in the airways of live mice after deposition in a saline carrier fluid have been examined. In this study a range of common respirable pollutant particles (lead dust, quarry dust and fibreglass fibres) as well as marker particles (hollow glass micro-spheres) were delivered into the trachea of live mice using a dry powder insufflator to more accurately mimic normal environmental particulate exposure and deposition via inhalation. The behaviour of the particles once delivered onto the airway surface was tracked over a five minute period via PCXI. All particles were visible after deposition. Fibreglass fibres remained stationary throughout while all other particle types transited the tracheal surface throughout the imaging period. In all cases the majority of the particle deposition and any airway surface activity was located close to the dorsal tracheal wall. Both the individual and bulk motions of the glass bead marker particles were visible and their behaviour enabled otherwise hidden MCT patterns to be revealed. This study verified the value of PCXI for examining the post-deposition particulate MCT behaviour in the mouse trachea and highlighted that MCT is not a uniform process as suggested by radiolabel studies. It also directly revealed the advantages of dry particle delivery for establishing adequate particulate presence for visualizing MCT behaviour. The MCT behaviour and rate seen after dry particle delivery was different from that in previous carrier-fluid studies. It is proposed that dry particle delivery is essential for producing environmentally realistic particle deposition and studying how living airway surfaces handle different types of inhaled particles by MCT processes.

High-speed synchrotron X-ray phase contrast imaging for analysis of low-Z composite microstructure

The study of high performance composites such as plastic-bonded explosives under extreme conditions often requires innovative experimental techniques. Here, static synchrotron X-ray phase-contrast imaging (PCI) of simulated explosive materials has been performed at high speed in an effort to determine feasibility of imaging material response to dynamic, high-strain rate events (102–107s−1). The microstructure of pristine materials, idealized composites and simulated explosive composites has been characterized with synchrotron PCI at the Advanced Photon Source. High spatial resolution (2μm) of the microstructure was achieved with 5μs exposures, and features such as interfaces, cracks, voids, and bubbles were clearly observed. The likelihood of obtaining sufficient phase information at even faster exposures (e.g., 0.2–0.5μs) is shown to be high.

Synchrotron phase-contrast X-ray imaging reveals fluid dosing dynamics for gene transfer into mouse airways

Although airway gene transfer research in mouse models relies on bolus fluid dosing into the nose or trachea, the dynamics and immediate fate of delivered gene transfer agents are poorly understood. In particular, this is because there are no in vivo methods able to accurately visualize the movement of fluid in small airways of intact animals. Using synchrotron phase-contrast X-ray imaging, we show that the fate of surrogate fluid doses delivered into live mouse airways can now be accurately and non-invasively monitored with high spatial and temporal resolution. This new imaging approach can help explain the non-homogenous distributions of gene expression observed in nasal airway gene transfer studies, suggests that substantial dose losses may occur at deliver into mouse trachea via immediate retrograde fluid motion and shows the influence of the speed of bolus delivery on the relative targeting of conducting and deeper lung airways. These findings provide insight into some of the factors that can influence gene expression in vivo, and this method provides a new approach to documenting and analyzing dose delivery in small-animal models.

Measurement of red blood cell aggregation using X-ray phase contrast imaging

When a coherent beam illuminates spatially disordered particles, speckle patterns are formed due to interference of the scattered light waves. Speckle patterns from biological tissues using synchrotron phase contrast X-ray imaging can provide functional information about micro-scale morphological structures of the tissues. In this study, we investigated the size and contrast variations of the speckles of aggregated red blood cells (RBCs) suspensions with varying the degree of RBC aggregation. Results show that the degree of RBC aggregation is a governing parameter on the change of speckle characteristics. This blood speckle analysis method can be used as a novel modality for monitoring RBC aggregation.

A new technique to examine individual pollutant particle and fibre deposition and transit behaviour in live mouse trachea

During respiration, particles suspended in the air are inhaled and unless cleared by airway defences they can remain and affect lung health. Their size precludes the use of standard imaging modalities so we have developed synchrotron phase-contrast X-ray imaging (PCXI) methods to non-invasively monitor the behaviour of individual particles in live mouse airways. In this study we used these techniques to examine post-deposition particle behaviour in the trachea. PCXI was used to monitor the deposition and subsequent behaviour of particles of quarry dust and lead ore; fibres of asbestos and fibreglass; and hollow glass micro-spheres. Visibility was examined in vitro and ex vivo to avoid the complicating effects of surrounding tissue and respiratory or cardiac motion. Particle behaviour was then examined after deposition onto the tracheal airway surfaces of live mice. Each particle and fibre looked and behaved differently on the airway surface. Particles lodged on the airway shortly after deposition, and the rate at which this occurred was dependent on the particle type and size. After the live-imaging experiments, excised airway samples were examined using light and electron microscopy. Evidence of particle capture into the airway surface fluids and the epithelial cell layer was found. PCXI is a valuable tool for examining post-deposition particulate behaviour in the tracheal airway. These first indications that the interaction between airways and individual particles may depend on the particle type and size should provide a novel approach to studying the early effects of respired particles on airway health.

Assisted walking in Malagasy dwarf chamaeleons

Chamaeleons are well known for their unique suite of morphological adaptations. Whereas most chamaeleons are arboreal and have long tails, which are used during arboreal acrobatic manoeuvres, Malagasy dwarf chamaeleons (Brookesia) are small terrestrial lizards with relatively short tails. Like other chamaeleons, Brookesia have grasping feet and use these to hold on to narrow substrates. However, in contrast to other chamaeleons, Brookesia place the tail on the substrate when walking on broad substrates, thus improving stability. Using three-dimensional synchrotron X-ray phase-contrast imaging, we demonstrate a set of unique specializations in the tail associated with the use of the tail during locomotion. Additionally, our imaging demonstrates specializations of the inner ear that may allow these animals to detect small accelerations typical of their slow, terrestrial mode of locomotion. These data suggest that the evolution of a terrestrial lifestyle in Brookesia has gone hand-in-hand with the evolution of a unique mode of locomotion and a suite of morphological adaptations allowing for stable locomotion on a wide array of substrates.

Real-time non-invasive detection of inhalable particulates delivered into live mouse airways

Fine non-biological particles small enough to be suspended in the air are continually inhaled as we breathe. These particles deposit on airway surfaces where they are either cleared by airway defences or can remain and affect lung health. Pollutant particles from vehicles, building processes and mineral and industrial dusts have the potential to cause both immediate and delayed health problems. Because of their small size, it has not been possible to non-invasively examine how individual particles deposit on live airways, or to consider how they behave on the airway surface after deposition. In this study, synchrotron phase-contrast X-ray imaging (PCXI) has been utilized to detect and monitor individual particle deposition. The in vitro detectability of a range of potentially respirable particulates was first determined. Of the particulates tested, only asbestos, quarry dust, fibreglass and galena (lead sulfate) were visible in vitro. These particulates were then examined after delivery into the nasal airway of live anaesthetized mice; all were detectable in vivo but each exhibited different surface appearances and behaviour along the airway surface. The two fibrous particulates appeared as agglomerations enveloped by fluid, while the non-fibrous particulates were present as individual particles. Synchrotron PCXI provides the unique ability to non-invasively detect and track deposition of individual particulates in live mouse airways. With further refinement of particulate sizing and delivery techniques, PCXI should provide a novel approach for live animal monitoring of airway particulates relevant to lung health.

Direct visualization of hemolymph flow in the heart of a grasshopper (Schistocerca americana)

BackgroundHemolymph flow patterns in opaque insects have never been directly visualized due to the lack of an appropriate imaging technique. The required spatial and temporal resolutions, together with the lack of contrast between the hemolymph and the surrounding soft tissue, are major challenges. Previously, indirect techniques have been used to infer insect heart motion and hemolymph flow, but such methods fail to reveal fine-scale kinematics of heartbeat and details of intra-heart flow patterns.ResultsWith the use of microbubbles as high contrast tracer particles, we directly visualized hemolymph flow in a grasshopper (Schistocerca americana) using synchrotron x-ray phase-contrast imaging. In-vivo intra-heart flow patterns and the relationship between respiratory (tracheae and air sacs) and circulatory (heart) systems were directly observed for the first time.ConclusionSynchrotron x-ray phase contrast imaging is the only generally applicable technique that has the necessary spatial, temporal resolutions and sensitivity to directly visualize heart dynamics and flow patterns inside opaque animals. This technique has the potential to illuminate many long-standing questions regarding small animal circulation, encompassing topics such as retrograde heart flow in some insects and the development of flow in embryonic vertebrates.

Correlated patterns of tracheal compression and convective gas exchange in a carabid beetle

Rhythmic tracheal compression is a prominent feature of internal dynamics in multiple orders of insects. During compression parts of the tracheal system collapse, effecting a large change in volume, but the ultimate physiological significance of this phenomenon in gas exchange has not been determined. Possible functions of this mechanism include to convectively transport air within or out of the body, to increase the local pressure within the tracheae, or some combination thereof. To determine whether tracheal compressions are associated with excurrent gas exchange in the ground beetle Pterostichus stygicus, we used flow-through respirometry and synchrotron x-ray phase-contrast imaging to simultaneously record CO(2) emission and observe morphological changes in the major tracheae. Each observed tracheal compression (which occurred at a mean frequency and duration of 15.6+/-4.2 min(-1) and 2.5+/-0.8 s, respectively) was associated with a local peak in CO(2) emission, with the start of each compression occurring simultaneously with the start of the rise in CO(2) emission. No such pulses were observed during inter-compression periods. Most pulses occurred on top of an existing level of CO(2) release, indicating that at least one spiracle was open when compression began. This evidence demonstrates that tracheal compressions convectively pushed air out of the body with each stroke. The volume of CO(2) emitted per pulse was 14+/-4 nl, representing approximately 20% of the average CO(2) emission volume during x-ray irradiation, and 13% prior to it. CO(2) pulses with similar volume, duration and frequency were observed both prior to and after x-ray beam exposure, indicating that rhythmic tracheal compression was not a response to x-ray irradiation per se. This study suggests that intra-tracheal and trans-spiracular convection of air driven by active tracheal compression may be a major component of ventilation for many insects.

Similarity between the Primary and Secondary Air-Assisted Liquid Jet Breakup Mechanisms

We report an ultrafast synchrotron x-ray phase-contrast imaging study of the primary breakup mechanism of a coaxial air-assisted water jet. There exist great similarities between the primary (jet) and the secondary (drop) breakup, and in the primary breakup on different length scales. A transition from a ligament- to a membrane-mediated breakup is identified around an effective Weber number We' approximately 13. This observation reveals the critical role an effective Weber number plays in determining the atomization process and strongly supports the cascade breakup model.

High-resolution visualization of airspace structures in intact mice via synchrotron phase-contrast X-ray imaging (PCXI)

High-resolution visualization of airspace structures in intact mice via synchrotron phase-contrast X-ray imaging (PCXI)

Increase in tracheal investment with beetle size supports hypothesis of oxygen limitation on insect gigantism.

Recent studies have suggested that Paleozoic hyperoxia enabled animal gigantism, and the subsequent hypoxia drove a reduction in animal size. This evolutionary hypothesis depends on the argument that gas exchange in many invertebrates and skin-breathing vertebrates becomes compromised at large sizes because of distance effects on diffusion. In contrast to vertebrates, which use respiratory and circulatory systems in series, gas exchange in insects is almost exclusively determined by the tracheal system, providing a particularly suitable model to investigate possible limitations of oxygen delivery on size. In this study, we used synchrotron x-ray phase-contrast imaging to visualize the tracheal system and quantify its dimensions in four species of darkling beetles varying in mass by 3 orders of magnitude. We document that, in striking contrast to the pattern observed in vertebrates, larger insects devote a greater fraction of their body to the respiratory system, as tracheal volume scaled with mass1.29. The trend is greatest in the legs; the cross-sectional area of the trachea penetrating the leg orifice scaled with mass1.02, whereas the cross-sectional area of the leg orifice scaled with mass0.77. These trends suggest the space available for tracheae within the leg may ultimately limit the maximum size of extant beetles. Because the size of the tracheal system can be reduced when oxygen supply is increased, hyperoxia, as occurred during late Carboniferous and early Permian, may have facilitated the evolution of giant insects by allowing limbs to reach larger sizes before the tracheal system became limited by spatial constraints.

Quasi–static studies of the deformation and failure of PBX 9501

This paper examines the influence of microstructure on the quasi–static failure of PBX 9501, a polymer–bonded explosive (PBX) manufactured for the Los Alamos National Laboratory in America. Optical microscopy has been used to examine qualitatively cracked and pristine material. Consequent on the manufacturing process, the explosive crystals display angular features and natural facets. In addition, considerable growth twinning, internal defects and voidage has been observed. These defects are found significantly to alter the failure path. In common with other PBXs, failure paths tend to run around the long straight edges of the explosive filler and avoid regions of fine filler and binder. Explosive crystals were found to fracture due either to cracks propagating from another region or internal defects. These observations are confirmed by the use of high–resolution moire interferometry. This sensitive optical technique allows the deformation of the sample to be measured up to and including the point of failure. By taking white–light micrographs that are in exact registration with the measured displacement maps, the influence of the underlying microstructure can be seen.