Microstructural insight into inhalation powder blends through correlative multi-scale X-ray computed tomography.

Dry powder inhalers (DPI) are important for topical drug delivery to the lungs, but characterising the pre-aerosolised powder microstructure is a key initial step in understanding the post-aerosolised blend performance. In this work, we characterise the pre-aerosolised 3D microstructure of an inhalation blend using correlative multi-scale X-ray Computed Tomography (XCT), identifying lactose and drug-rich phases at multiple length scales on the same sample. The drug-rich phase distribution across the sample is shown to be homogeneous on a bulk scale but heterogeneous on a particulate scale, with individual clusters containing different amounts of drug-rich phase, and different parts of a carrier particle coated with different amounts of drug-rich phase. Simple scalings of the drug-rich phase thickness with carrier particle size are used to derive the drug-proportion to carrier particle size relationship. This work opens new doors to micro-structural assessment of inhalation powders that could be invaluable for bioequivalence assessment of dry powder inhalers.

Predicting the Strength of Cohesive and Adhesive Interparticle Interactions for Dry Powder Inhalation Blends of Terbutaline Sulfate with α-Lactose Monohydrate.

Grid-based systematic search methods are used to investigate molecule-molecule, molecule-surface, and surface-surface contributions to interparticle interactions in order to identify the crystal faces that most strongly affect particle behavior during powder blend formulation and delivery processes. The model system comprises terbutaline sulfate (TBS) as an active pharmaceutical ingredient (API) and α-form lactose monohydrate (LMH). A combination of systematic molecular modeling and X-ray computed tomography (XCT) is used to determine not only the adhesive and cohesive interparticle energies but, also the agglomeration behavior during manufacturing and de-agglomeration behavior during delivery after inhalation. This is achieved through a detailed examination of the balance between the adhesive and cohesive energies with the XCT results confirming the blend segregation tendencies, through the particle-particle de-agglomeration process. The results reveal that the cohesive interaction energies of TBS-TBS are higher than the adhesive energies between TBS and LMH, but that the cohesive energies of LMH-LMH are the smallest between molecule and molecule, molecule and surface, and surface and surface. This shows how systematic grid-search molecular modeling along with XCT can guide the digital formulation design of inhalation powders in order to achieve optimum aerosolization and efficacy for inhaled medicines. This will lead to faster pharmaceutical design with less variability, higher quality, and enhanced performance.

The role of internal defects on anisotropic tensile failure of L-PBF AlSi10Mg alloys.

This paper investigates the effects of defects on tensile failure of additive manufactured AlSi10Mg alloy focusing particularly on the role of large pancake shaped loss of fusion (LOF) defects lying perpendicular to the build direction (BD). Time-lapse in situ synchrotron radiation X-ray micro-computed tomography during straining reveals how, when tested parallel to the BD, the LOF defects extend laterally with straining connecting to other defects and giving rise to low plasticity and an essentially brittle failure mode. When they are aligned edge-on to the straining direction, failure is characterised by a ductile cup-cone failure with significant elongation of the defects axially and extensive necking prior to failure. The soft fish-scale melt pool boundaries were also found to affect the fracture path. These results highlight the anisotropic effect of loss of fusion defects in controlling tensile ductility and the need to minimize their size and aspect ratio. In cases where these cannot be fully eliminated the component should be fabricated such that the BD is not aligned with the dominant in-service loading direction.

Quantifying multiple stain distributions in bioimaging by hyperspectral X-ray tomography.

Chemical staining of biological specimens is commonly utilised to boost contrast in soft tissue structures, but unambiguous identification of staining location and distribution is difficult without confirmation of the elemental signature, especially for chemicals of similar density contrast. Hyperspectral X-ray computed tomography (XCT) enables the non-destructive identification, segmentation and mapping of elemental composition within a sample. With the availability of hundreds of narrow, high resolution (~ 1 keV) energy channels, the technique allows the simultaneous detection of multiple contrast agents across different tissue structures. Here we describe a hyperspectral imaging routine for distinguishing multiple chemical agents, regardless of contrast similarity. Using a set of elemental calibration phantoms, we perform a first instance of direct stain concentration measurement using spectral absorption edge markers. Applied to a set of double- and triple-stained biological specimens, the study analyses the extent of stain overlap and uptake regions for commonly used contrast markers. An improved understanding of stain concentration as a function of position, and the interaction between multiple stains, would help inform future studies on multi-staining procedures, as well as enable future exploration of heavy metal uptake across medical, agricultural and ecological fields.

Inhibiting weld cracking in high-strength aluminium alloys.

Cracking from a fine equiaxed zone (FQZ), often just tens of microns across, plagues the welding of 7000 series aluminum alloys. Using a multiscale correlative methodology, from the millimeter scale to the nanoscale, we shed light on the strengthening mechanisms and the resulting intergranular failure at the FQZ. We show that intergranular AlCuMg phases give rise to cracking by micro-void nucleation and subsequent link-up due to the plastic incompatibility between the hard phases and soft (low precipitate density) grain interiors in the FQZ. To mitigate this, we propose a hybrid welding strategy exploiting laser beam oscillation and a pulsed magnetic field. This achieves a wavy and interrupted FQZ along with a higher precipitate density, thereby considerably increasing tensile strength over conventionally hybrid welded butt joints, and even friction stir welds.

Tailoring the Microstructure of Lamellar Ti3C2Tx MXene Aerogel by Compressive Straining.

Aerogels are attracting increasing interest due to their functional properties, such as lightweight and high porosity, which make them promising materials for energy storage and advanced composites. Compressive deformation allows the nano- and microstructure of lamellar freeze-cast aerogels to be tailored toward the aforementioned applications, where a 3D nanostructure of closely spaced, aligned sheets is desired. Quantitatively characterizing their microstructural evolution during compression is needed to allow optimization of manufacturing, understand in-service structural changes, and determine how aerogel structure relates to functional properties. Herein we have developed methods to quantitatively analyze lamellar aerogel domains, sheet spacing, and sheet orientation in 3D and to track their evolution as a function of increasing compression through synchrotron phase contrast X-ray microcomputed tomography (µCT). The as-cast domains are predominantly aligned with the freezing direction with random orientation in the orthogonal plane. Generally the sheets rotate toward flat and their spacing narrows progressively with increasing compression with negligible lateral strain (zero Poisson's ratio). This is with the exception of sheets close to parallel with the loading direction (Z), which maintain their orientation and sheet spacing until ∼60% compression, beyond which they exhibit buckling. These data suggest that a single-domain, fully aligned as-cast aerogel is not necessary to produce a post-compression aligned lamellar structure and indicate how the spacing can be tailored as a function of compressive strain. The analysis methods presented herein are applicable to optimizing freeze-casting process and quantifying lamellar microdomain structures generally.

Enhanced hyperspectral tomography for bioimaging by spatiospectral reconstruction.

Here we apply hyperspectral bright field imaging to collect computed tomographic images with excellent energy resolution (~ 1 keV), applying it for the first time to map the distribution of stain in a fixed biological sample through its characteristic K-edge. Conventionally, because the photons detected at each pixel are distributed across as many as 200 energy channels, energy-selective images are characterised by low count-rates and poor signal-to-noise ratio. This means high X-ray exposures, long scan times and high doses are required to image unique spectral markers. Here, we achieve high quality energy-dispersive tomograms from low dose, noisy datasets using a dedicated iterative reconstruction algorithm. This exploits the spatial smoothness and inter-channel structural correlation in the spectral domain using two carefully chosen regularisation terms. For a multi-phase phantom, a reduction in scan time of 36 times is demonstrated. Spectral analysis methods including K-edge subtraction and absorption step-size fitting are evaluated for an ex vivo, single (iodine)-stained biological sample, where low chemical concentration and inhomogeneous distribution can affect soft tissue segmentation and visualisation. The reconstruction algorithms are available through the open-source Core Imaging Library. Taken together, these tools offer new capabilities for visualisation and elemental mapping, with promising applications for multiply-stained biological specimens.

Core Imaging Library - Part II: multichannel reconstruction for dynamic and spectral tomography.

The newly developed core imaging library (CIL) is a flexible plug and play library for tomographic imaging with a specific focus on iterative reconstruction. CIL provides building blocks for tailored regularized reconstruction algorithms and explicitly supports multichannel tomographic data. In the first part of this two-part publication, we introduced the fundamentals of CIL. This paper focuses on applications of CIL for multichannel data, e.g. dynamic and spectral. We formalize different optimization problems for colour processing, dynamic and hyperspectral tomography and demonstrate CIL's capabilities for designing state-of-the-art reconstruction methods through case studies and code snapshots. This article is part of the theme issue 'Synergistic tomographic image reconstruction: part 2'.

Morphological variability in the mucosal attachment site of Trichuris muris revealed by X-ray microcomputed tomography.

Parasitic infections can be challenging to study because two dimensional light and electron microscopy are often limited in visualising complex and inaccessible attachment sites. Exemplifying this, Trichuris spp. inhabit a tunnel of epithelial cells within the host caecum and colon. A significant global burden of this infection persists, partly because available anthelminthics lack efficacy, although the mechanisms underlying this remain unknown. Consequently, there is a need to pioneer new approaches to better characterize the parasite niche within the host and investigate how variation in its morphology and integrity may contribute to resistance to therapeutic intervention. To address these aims, we exploited three-dimensional X-ray micro-computed tomography (microCT) to image the mouse whipworm, Trichuris muris, in caeca of wild-type C57BL/6 and SCID mice ex vivo. Using osmium tetroxide staining to effectively enhance the contrast of worms, we found that a subset exhibited preferential positioning towards the bases of the intestinal crypts. Moreover, in one rare event, we demonstrated whipworm traversal of the lamina propria. This morphological variability contradicts widely accepted conclusions from conventional microscopy of the parasite niche, showing Trichuris in close contact with the host proliferative and immune compartments that may facilitate immunomodulation. Furthermore, by using a skeletonization-based approach we demonstrate considerable variation in tunnel length and integrity. The qualitative and quantitative observations provide a new morphological point of reference for future in vitro study of host-Trichuris interactions, and highlight the potential of microCT to characterise enigmatic host-parasite interactions more accurately.

Complementary time-lapse datasets of x-ray computed tomography and real-time strain mapping for an ex-situ study of non-crimp glass fibre composites under fatigue loading.

Data published in this paper corresponds to a time-lapse ex-situ experiment aimed at analyzing the tension-tension fatigue damage in non-crimp glass-epoxy composites by multi-scale x-ray computed tomography (XCT) of the damage features and their timeline. This is then correlated with the strain fields obtained through digital image correlation (DIC). The XCT - DIC datasets by is acquired by interrupting mechanical fatigue tests at three time-steps, after the material has undergone 0 cycles, 70,000 cycles, 80,000 cycles, and 120,000 cycles. This is one of the first multi-modally correlated datasets available for these types of non-crimp glass fibre composites, which explore the structure-property relationship in a time-dependent behavior. This dataset can be used to explore glass-fibre composites microstructure under a progressive damage scheme and can be used to test and train a plethora of image processing and analysis techniques. This dataset can also be used as an attempt to model the fatigue behavior of quasi-unidirectional non-crimp fibre composites by image-based simulations.

Size segregation of irregular granular materials captured by time-resolved 3D imaging.

When opening a box of mixed nuts, a common experience is to find the largest nuts at the top. This well-known effect is the result of size-segregation where differently sized 'particles' sort themselves into distinct layers when shaken, vibrated or sheared. Colloquially this is known as the 'Brazil-nut effect'. While there have been many studies into the phenomena, difficulties observing granular materials mean that we still know relatively little about the process by which irregular larger particles (the Brazil nuts) reach the top. Here, for the first time, we capture the complex dynamics of Brazil nut motion within a sheared nut mixture through time-lapse X-ray Computed Tomography (CT). We have found that the Brazil nuts do not start to rise until they have first rotated sufficiently towards the vertical axis and then ultimately return to a flat orientation when they reach the surface. We also consider why certain Brazil nuts do not rise through the pack. This study highlights the important role of particle shape and orientation in segregation. Further, this ability to track the motion in 3D will pave the way for new experimental studies of segregating mixtures and will open the door to even more realistic simulations and powerful predictive models. Understanding the effect of size and shape on segregation has implications far beyond food products including various anti-mixing behaviors critical to many industries such as pharmaceuticals and mining.

Comparing Xe+ pFIB and Ga+ FIB for TEM sample preparation of Al alloys: Minimising FIB-induced artefacts.

Recently, the dual beam Xe+ plasma focused ion beam (Xe+ pFIB) instrument has attracted increasing interest for site-specific transmission electron microscopy (TEM) sample preparation for a local region of interest as it shows several potential benefits compared to conventional Ga+ FIB milling. Nevertheless, challenges and questions remain especially in terms of FIB-induced artefacts, which hinder reliable S/TEM microstructural and compositional analysis. Here we examine the efficacy of using Xe+ pFIB as compared with conventional Ga+ FIB for TEM sample preparation of Al alloys. Three potential source of specimen preparation artefacts were examined, namely: (1) implantation-induced defects such as amophisation, dislocations, or 'bubble' formation in the near-surface region resulting from ion bombardment of the sample by the incident beam; (2) compositional artefacts due to implantation of the source ions and (3) material redeposition due to the milling process. It is shown that Xe+ pFIB milling is able to produce improved STEM/TEM samples compared to those produced by Ga+ milling, and is therefore the preferred specimen preparation route. Strategies for minimising the artefacts induced by Xe+ pFIB and Ga+ FIB are also proposed. LAY DESCRIPTION: FIB (focused ion beam) instruments have become one of the most important systems in the preparation of site-specific TEM specimens, which are typically 50-100 nm in thickness. TEM specimen preparation of Al alloys is particularly challenging, as convention Ga-ion FIB produces artefacts in these materials that make microstructural analysis difficult or impossible. Recently, the use of noble gas ion sources, such as Xe, has markedly improved milling speeds and is being used for the preparation of various materials. Hence, it is necessary to investigate the structural defects formed during FIB milling and assess the ion-induced chemical contamination in these TEM samples. Here we explore the feasibility and efficiency of using Xe+ PFIB as a TEM sample preparation route for Al alloys in comparison with the conventional Ga+FIB.

An in-situ method for protecting internal cracks/pores from ion beam damage and reducing curtaining for TEM sample preparation using FIB.

Focused ion beam (FIB) milling has evolved to be one of the most important Transmission Electron Microscope (TEM) site specific sample preparation techniques. However, this technique still poses challenges, such as the structural damage and potential curtaining issues often observed for thin TEM lamella. These artefacts can negatively affect the TEM analysis results. In particular, structures such as internal cracks and pores in FIB prepared TEM samples can often be damaged during sample preparation. This is commonly regarded as an unavoidable problem, even though microstructurally intact thin lamellae TEM samples are widely needed for the investigation of crack tips or pore morphologies in many different materials. This presents a strong driver for the development of innovative methods to overcome damage and curtaining issues during FIB sample preparation. Here we report on a new methodology developed to protect internal cracks and pores from ion beam damage. Our proposed method also mitigates curtaining issues, which often make TEM analysis more difficult. This method uses the FIB to sputter and redeposit material onto the edges of any cracks or pores in order to fill these features in-situ prior to lamella thinning. Case studies showcasing this method are presented, demonstrating the approach on a modular pure iron sample and on a porous laser treated Al/B4C composite sample. Our proposed 'filling' method has demonstrated a two key benefits; it preserves the integrity of the edges of any cracks and pores and it reducing curtaining. The results also demonstrate that this technique can be an alternative to conventional Gas Injection System (GIS) deposition for protecting the external top surface.

X-ray Micro-Computed Tomography: An Emerging Technology to Analyze Vascular Calcification in Animal Models.

Vascular calcification describes the formation of mineralized tissue within the blood vessel wall, and it is highly associated with increased cardiovascular morbidity and mortality in patients with chronic kidney disease, diabetes, and atherosclerosis. In this article, we briefly review different rodent models used to study vascular calcification in vivo, and critically assess the strengths and weaknesses of the current techniques used to analyze and quantify calcification in these models, namely 2-D histology and the o-cresolphthalein assay. In light of this, we examine X-ray micro-computed tomography (µCT) as an emerging complementary tool for the analysis of vascular calcification in animal models. We demonstrate that this non-destructive technique allows us to simultaneously quantify and localize calcification in an intact vessel in 3-D, and we consider recent advances in µCT sample preparation techniques. This review also discusses the potential to combine 3-D µCT analyses with subsequent 2-D histological, immunohistochemical, and proteomic approaches in correlative microscopy workflows to obtain rich, multifaceted information on calcification volume, calcification load, and signaling mechanisms from within the same arterial segment. In conclusion we briefly discuss the potential use of µCT to visualize and measure vascular calcification in vivo in real-time.

Coupled Broad Ion Beam-Scanning Electron Microscopy (BIB-SEM) for polishing and three dimensional (3D) serial section tomography (SST).

Here we describe the first automated fully integrated in-microscope broad ion beam (BIB) system. Ar+-BIB has several advantages over Ga+ focused ion beam (FIB) and Xe+ plasma-FIB (PFIB) methods inducing less beam damage, especially for ion beam sensitive materials. It can mill areas several orders of magnitude larger (up to millimetre scale), and is not confined to the edge of the sample with associated curtaining issues. BIB is shown to have sputter rates up to five times higher than comparable FIB techniques. This new coupled BIB-SEM system (commercial name 'iPrep™II') enables in-microscope surface polishing to remove contaminants or damage for two dimensional (2D) imaging, as well as automated serial section tomography (SST) by milling and imaging hundreds of slices, cost and time efficiently. The milled slice thickness can be controlled from a few nanometers up to a micrometre. A novel sample transfer, handling and interlock system allows automated and sequential BIB polishing, scanning electron microscopy (SEM) and analysis by secondary electron (SE) imaging, electron back scatter diffraction (EBSD) and energy dispersive spectroscopy (EDS) for 3D microstructure analysis. Furthermore, insulating surfaces can be sputter coated after milling each slice to reduce charging during SEM analysis. The performance of the instrument is demonstrated through a series of case studies across the materials, earth and life sciences exploiting the imaging, crystallographic and chemical mapping capabilities. These include the study of butterfly defects in bearing steels, meta-stable intermetallic phases in bronze bearings, shale gas rock, aluminium plasma electrolytic oxide (PEO) coatings as well as liver and mouse brain tissues.

Characterisation of cuticular inflation development and ultrastructure in Trichuris muris using correlative X-ray computed tomography and electron microscopy.

The parasitic nematode Trichuris trichiura is a significant burden on public health in developing countries, and currently available drugs exhibit a poor cure rate. Worms live within a specialised tunnel of host intestinal epithelial cells and have anterior-ventral projections of the cuticle termed "cuticular inflations", which are thought to be involved in host-parasite interactions. This work aimed to characterise structure and suggest a function of cuticular inflations in the most tractable and widely-used model of trichuriasis, Trichuris muris. Using scanning electron microscopy, we show for the first time that most cuticular inflations develop between the second and third larval moults. Correlative X-ray computed tomography (CT)-steered Serial Block Face Scanning Electron Microscopy (SBF-SEM) and transmission electron microscopy enabled ultrastructural imaging of cuticular inflations, and showed the presence of an additional, web-like layer of cuticle between the median and cortical layers of the inflation. Additionally, we characterised variation in inflation morphology, resolving debate as to the inflations' true shape in situ. Cells underlying the inflations had many mitochondria, and we highlight their potential capacity for active transport as an area for future investigation. Overall, insights from the powerful imaging techniques used provide an excellent basis for future study of cuticular inflation function.

Novel Methods for Recording Stress-Strain Curves in Proton Irradiated Material.

Proton irradiation is often used as a proxy for neutron irradiation but the irradiated layer is typically <50 µm deep; this presents a problem when trying to obtain mechanical test data as a function of irradiation level. Two novel methodologies have been developed to record stress-strain curves for thin proton-irradiated surface layers of SA-508-4N ferritic steel. In the first case, in-situ loading experiments are carried out using a combination of X-ray diffraction and digital image correlation on the near surface region in order to measure stress and strain, thereby eliminating the influence of the non-irradiated volume. The second approach is to manufacture small-scale tensile specimens containing only the proton irradiated volume but approaching the smallest representative volume of the material. This is achieved by high-speed focused ion beam (FIB) milling though the application of a Xe+ Plasma-FIB (PFIB). It is demonstrated that both techniques are capable of recording the early stage of uniaxial flow behaviour of the irradiated material with sufficient accuracy providing a measure of irradiation-induced shift of yield strength, strain hardening and tensile strength.

X-ray computed tomography in life sciences.

Recent developments within micro-computed tomography (µCT) imaging have combined to extend our capacity to image tissue in three (3D) and four (4D) dimensions at micron and sub-micron spatial resolutions, opening the way for virtual histology, live cell imaging, subcellular imaging and correlative microscopy. Pivotal to this has been the development of methods to extend the contrast achievable for soft tissue. Herein, we review the new capabilities within the field of life sciences imaging, and consider how future developments in this field could further benefit the life sciences community.

Anisotropic crack propagation and deformation in dentin observed by four-dimensional X-ray nano-computed tomography.

Understanding the cracking behaviour of biological composite materials is of practical importance. This paper presents the first study to track the interplay between crack initiation, microfracture and plastic deformation in three dimensions (3D) as a function of tubule and collagen fibril arrangement in elephant dentin using in situ X-ray nano-computed tomography (nano-CT). A nano-indenter with a conical tip has been used to incrementally indent three test-pieces oriented at 0°, 45° and 70° to the long axis of the tubules (i.e. radial to the tusk). For the 0° sample two significant cracks formed, one of which linked up with microcracks in the axial-radial plane of the tusk originating from the tubules and the other one occurred as a consequence of shear deformation at the tubules. The 70° test-piece was able to bear the greatest loads despite many small cracks forming around the indenter. These were diverted by the microstructure and did not propagate significantly. The 45° test-piece showed intermediate behaviour. In all cases strains obtained by digital volume correlation were well in excess of the yield strain (0.9%), indeed some plastic deformation could even be seen through bending of the tubules. The hoop strains around the conical indenter were anisotropic with the smallest strains correlating with the primary collagen orientation (axial to the tusk) and the largest strains aligned with the hoop direction of the tusk. STATEMENT OF SIGNIFICANCE: This paper presents the first comprehensive study of the anisotropic nature of microfracture, crack propagation and deformation in elephant dentin using time-lapse X-ray nano-computed tomography. To unravel the interplay of collagen fibrils and local deformation, digital volume correlation (DVC) has been applied to map the local strain field while the crack initiation and propagation is tracked in real time. Our results highlight the intrinsic and extrinsic shielding mechanisms and correlate the crack growth behavior in nature to the service requirement of dentin to resist catastrophic fracture. This is of wide interest not just in terms of understanding dentin fracture but also can extend beyond dentin to other anisotropic structural composite biomaterials such as bone, antler and chitin.

Experimental steering of electron microscopy studies using prior X-ray computed tomography.

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) can provide unrivalled high-resolution images of specific features and volumes of interest. However, the regions interrogated are typically very small, and sample preparation is both time-consuming and destructive. Here we consider how prior X-ray micro-computed tomography (microCT) presents an opportunity to increase the efficiency of electron microscopy in biology. We demonstrate how it can be used to; select the most promising samples and target site-specific locations; provide a wider context of the location being interrogated (multiscale correlative imaging); guide sample preparation and 3D imaging schemes; as well as quantify the effects of destructive sample preparation and staining procedures. We present a workflow utilising open source software in which microCT can be used either broadly, or precisely, to experimentally steer and inform subsequent electron microscopy studies. As automated sample registration procedures are developed to enable correlative microscopy, experimental steering by prior CT could be beneficially routinely incorporated into many experimental workflows.