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.

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.

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'.

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.

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.

X-ray micro-computed tomography (µCT): an emerging opportunity in parasite imaging.

X-ray micro-computed tomography (µCT) is a technique which can obtain three-dimensional images of a sample, including its internal structure, without the need for destructive sectioning. Here, we review the capability of the technique and examine its potential to provide novel insights into the lifestyles of parasites embedded within host tissue. The current capabilities and limitations of the technology in producing contrast in soft tissues are discussed, as well as the potential solutions for parasitologists looking to apply this technique. We present example images of the mouse whipworm Trichuris muris and discuss the application of µCT to provide unique insights into parasite behaviour and pathology, which are inaccessible to other imaging modalities.

Temporal sparsity exploiting nonlocal regularization for 4D computed tomography reconstruction.

X-ray imaging applications in medical and material sciences are frequently limited by the number of tomographic projections collected. The inversion of the limited projection data is an ill-posed problem and needs regularization. Traditional spatial regularization is not well adapted to the dynamic nature of time-lapse tomography since it discards the redundancy of the temporal information. In this paper, we propose a novel iterative reconstruction algorithm with a nonlocal regularization term to account for time-evolving datasets. The aim of the proposed nonlocal penalty is to collect the maximum relevant information in the spatial and temporal domains. With the proposed sparsity seeking approach in the temporal space, the computational complexity of the classical nonlocal regularizer is substantially reduced (at least by one order of magnitude). The presented reconstruction method can be directly applied to various big data 4D (x, y, z+time) tomographic experiments in many fields. We apply the proposed technique to modelled data and to real dynamic X-ray microtomography (XMT) data of high resolution. Compared to the classical spatio-temporal nonlocal regularization approach, the proposed method delivers reconstructed images of improved resolution and higher contrast while remaining significantly less computationally demanding.

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.

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.

MRI measurements of vessel calibre in tumour xenografts: comparison with vascular corrosion casting.

Vessel size index (R(v), µm) has been proposed as a quantitative magnetic resonance imaging (MRI) derived imaging biomarker in oncology, for the non-invasive assessment of tumour blood vessel architecture and vascular targeted therapies. Appropriate pre-clinical evaluation of R(v) in animal tumour models will improve the interpretation and guide the introduction of the biomarker into clinical studies. The objective of this study was to compare R(v) measured in vivo with vessel size measurements from high-resolution X-ray computed tomography (µCT) of vascular corrosion casts measured post mortem from the same tumours, with and without vascular targeted therapy. MRI measurements were first acquired from subcutaneous SW1222 colorectal xenografts in mice following treatment with 0 (n=6), 30 (n=6) or 200 mg/kg (n=3) of the vascular disrupting agent ZD6126. The mice were then immediately infused with a low viscosity resin and, following polymerisation and maceration of surrounding tissues, the resulting tumour vascular casts were dissected and subsequently imaged using an optimised µCT imaging approach. Vessel diameters were not measurable by µCT in the 200 mg/kg group as the high dose of ZD6126 precluded delivery of the resin to the tumour vascular bed. The mean R(v) for the three treatment groups was 24, 23 and 23.5 µm respectively; the corresponding µCT measurements from corrosion casts from the 0 and 30 mg/kg cohorts were 25 and 28 µm. The strong association between the in vivo MRI and post mortem µCT values supports the use of R(v) as an imaging biomarker in clinical trials of investigational vascular targeted therapies.

A minute fossil phoretic mite recovered by phase-contrast X-ray computed tomography.

High-resolution phase-contrast X-ray computed tomography (CT) reveals the phoretic deutonymph of a fossil astigmatid mite (Acariformes: Astigmata) attached to a spider's carapace (Araneae: Dysderidae) in Eocene (44-49 Myr ago) Baltic amber. Details of appendages and a sucker plate were resolved, and the resulting three-dimensional model demonstrates the potential of tomography to recover morphological characters of systematic significance from even the tiniest amber inclusions without the need for a synchrotron. Astigmatids have an extremely sparse palaeontological record. We confirm one of the few convincing fossils, potentially the oldest record of Histiostomatidae. At 176 µm long, we believe this to be the smallest arthropod in amber to be CT-scanned as a complete body fossil, extending the boundaries for what can be recovered using this technique. We also demonstrate a minimum age for the evolution of phoretic behaviour among their deutonymphs, an ecological trait used by extant species to disperse into favourable environments. The occurrence of the fossil on a spider is noteworthy, as modern histiostomatids tend to favour other arthropods as carriers.

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.

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.

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.

Suppression of ring artefacts when tomographing anisotropically attenuating samples.

There are many objects for which the attenuation varies significantly as they are rotated during computerized X-ray tomography, for example plate samples. This can lead to significant ring artefacts in the subsequent tomographic reconstructions. In this paper a new method is presented that can successfully suppress such ring artefacts and is applicable to both parallel and cone-beam geometries. Rapid correction is achieved via an analytical formula which involves only a matrix-vector multiplication, for which the matrix is known and depends on a regularization parameter. The efficacy of the method is demonstrated for a paleontological sample (calcified shark cartilage) and a carbon-carbon composite/Ti-SiC metal matrix composite test sample.

Computed tomography recovers data from historical amber: an example from huntsman spiders.

Computed tomography (CT) methods were applied to a problematic fossil spider (Arachnida: Araneae) from the historical Berendt collection of Eocene (ca. 44-49 Ma) Baltic amber. The original specimens of Ocypete crassipes Koch and Berendt 1854 are in dark, oxidised amber and the published descriptions lack detail. Despite this, they were subsequently assigned to the living Pantropical genus Heteropoda Latreille, 1804 and are ostensibly the oldest records of huntsman spiders (Sparassidae) in general. Given their normally large size, and presumptive ability to free themselves more easily from resin, it would be surprising to find a sparassid in amber and traditional (optical) methods of study would likely have left O. crassipes as an equivocal record--probably a nomen dubium. However, phase contrast enhanced X-ray CT revealed exquisite morphological detail and thus 'saved' this historical name by revealing characters which confirm that it's a bona fide member both of Sparassidae and the subfamily Eusparassinae. We demonstrate here that CT studies facilitate taxonomic equivalence even between recent spiders and unpromising fossils described in older monographs. In our case, fine structural details such as eye arrangement, cheliceral dentition, and leg characters like a trilobate membrane, spination and claws, allow a precise referral of this fossil to an extant genus as Eusparassus crassipes (Koch and Berendt 1854) comb. nov.

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.

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.

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.

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.