A high-throughput 3D X-ray histology facility for biomedical research and preclinical applications.

Background: The University of Southampton, in collaboration with the University Hospital Southampton (UHS) NHS Foundation Trust and industrial partners, has been at the forefront of developing three-dimensional (3D) imaging workflows using X-ray microfocus computed tomography (µCT) -based technology. This article presents the outcomes of these endeavours and highlights the distinctive characteristics of a µCT facility tailored explicitly for 3D X-ray Histology, with a primary focus on applications in biomedical research and preclinical and clinical studies. Methods: The UHS houses a unique 3D X-ray Histology (XRH) facility, offering a range of services to national and international clients. The facility employs specialised µCT equipment explicitly designed for histology applications, allowing whole-block XRH imaging of formalin-fixed and paraffin-embedded tissue specimens. It also enables correlative imaging by combining µCT imaging with other microscopy techniques, such as immunohistochemistry (IHC) and serial block-face scanning electron microscopy, as well as data visualisation, image quantification, and bespoke analysis. Results: Over the past seven years, the XRH facility has successfully completed over 120 projects in collaboration with researchers from 60 affiliations, resulting in numerous published manuscripts and conference proceedings. The facility has streamlined the µCT imaging process, improving productivity and enabling efficient acquisition of 3D datasets. Discussion & Conclusions: The 3D X-ray Histology (XRH) facility at UHS is a pioneering platform in the field of histology and biomedical imaging. To the best of our knowledge, it stands out as the world's first dedicated XRH facility, encompassing every aspect of the imaging process, from user support to data generation, analysis, training, archiving, and metadata generation. This article serves as a comprehensive guide for establishing similar XRH facilities, covering key aspects of facility setup and operation. Researchers and institutions interested in developing state-of-the-art histology and imaging facilities can utilise this resource to explore new frontiers in their research and discoveries.

On Krylov methods for large-scale CBCT reconstruction.

Krylov subspace methods are a powerful family of iterative solvers for linear systems of equations, which are commonly used for inverse problems due to their intrinsic regularization properties. Moreover, these methods are naturally suited to solve large-scale problems, as they only require matrix-vector products with the system matrix (and its adjoint) to compute approximate solutions, and they display a very fast convergence. Even if this class of methods has been widely researched and studied in the numerical linear algebra community, its use in applied medical physics and applied engineering is still very limited. e.g. in realistic large-scale computed tomography (CT) problems, and more specifically in cone beam CT (CBCT). This work attempts to breach this gap by providing a general framework for the most relevant Krylov subspace methods applied to 3D CT problems, including the most well-known Krylov solvers for non-square systems (CGLS, LSQR, LSMR), possibly in combination with Tikhonov regularization, and methods that incorporate total variation regularization. This is provided within an open source framework: the tomographic iterative GPU-based reconstruction toolbox, with the idea of promoting accessibility and reproducibility of the results for the algorithms presented. Finally, numerical results in synthetic and real-world 3D CT applications (medical CBCT andµ-CT datasets) are provided to showcase and compare the different Krylov subspace methods presented in the paper, as well as their suitability for different kinds of problems.

X-ray directional dark-field imaging using Unified Modulated Pattern Analysis.

X-ray directional dark-field imaging is a recent technique that can reveal a sample's small-scale structural properties which are otherwise invisible in a conventional imaging system. In particular, directional dark-field can detect and quantify the orientation of anisotropic structures. Here, we present an algorithm that allows for the extraction of a directional dark-field signal from X-ray speckle-based imaging data. The experimental setup is simple, as it requires only the addition of a diffuser to a full-field microscope setup. Sandpaper is an appropriate diffuser material in the hard x-ray regime. We propose an approach to extract the mean scattering width, directionality, and orientation from the recorded speckle images acquired with the technique. We demonstrate that our method can detect and quantify the orientation of fibres inside a carbon fibre reinforced polymer (CFRP) sample within one degree of accuracy and show how the accuracy depends on the number of included measurements. We show that the reconstruction parameters can be tuned to increase or decrease accuracy at the expense of spatial resolution.

Numerically robust tetrahedron-based tomographic forward and backward projectors on parallel architectures.

X-ray tomographic reconstruction typically uses voxel basis functions to represent volumetric images. Due to the structure in voxel basis representations, efficient ray-tracing methods exist allowing fast, GPU accelerated implementations. Tetrahedral mesh basis functions are a valuable alternative to voxel based image representations as they provide flexible, inhomogeneous partitions which can be used to provide reconstructions with reduced numbers of elements or with arbitrarily fine object surface representations. We thus present a robust parallelizable ray-tracing method for volumetric tetrahedral domains developed specifically for Computed Tomography image reconstruction. Tomographic image reconstruction requires algorithms that are robust to numerical errors in floating point arithmetic whilst typical data sizes encountered in tomography require the algorithm to be parallelisable in GPUs which leads to additional constraints on algorithm choices. Based on these considerations, this article presents numerical solutions to the design of efficient ray-tracing algorithms for the projection and backprojection operations. Initial reconstruction results using CAD data to define a triangulation of the domain demonstrate the advantages of our method and contrast tetrahedral mesh based reconstructions to voxel based methods.

Imaging techniques for observing laminar geometry in the feather shaft cortex.

Bird feather shafts are light, stiff and strong, but the fine details of how their structure, mechanics and function relate to one another remains poorly understood. The missing piece in our understanding may be the various fibrous layers that make up the shaft's cortex. Detailed imaging techniques are needed to enable us to capture, analyse and quantify these layers before we can begin to unravel the relationship between their structure, mechanics and function. We show that Serial-Block-Face scanning electron microscopy, scanning confocal polarised microscopy and synchrotron-based computed tomography are three suitable techniques to investigate layer thickness and fibre orientation in the feather cortex. These techniques and other are discussed in terms of their ability to resolve the fibrous laminar structure of the feather cortex, on sample preparation, and on throughput. Annotated images are presented for each and less suitable techniques are presented in the Supplementary Material. LAY DESCRIPTION: Bird feathers have a light, stiff and strong central shaft. However, the fine details of how their structure, mechanics and function relate to one another remains poorly understood. The missing piece in our understanding may have to do with how fibrous layers within the shaft vary in thickness and alignment. Detailed imaging techniques are needed so that we can quantify some of this variation before we can revisit some long-unanswered questions about the feather shaft's structure, mechanics and function. We investigate a number of microscopy techniques and show that three techniques are suitable for the sort of investigation that is required. These techniques and others are discussed in terms of their ability to resolve the layers' thickness and alignment, on sample preparation, and on the sample sizes they are able to process. Annotated images are presented and discussed for each of the three techniques and unsuitable techniques receive the same examination in the Supplementary Material.

Recent Advances in X-ray Cone-beam Computed Laminography.

X-ray computed tomography is an established volume imaging technique used routinely in medical diagnosis, industrial non-destructive testing, and a wide range of scientific fields. Traditionally, computed tomography uses scanning geometries with a single axis of rotation together with reconstruction algorithms specifically designed for this setup. Recently there has however been increasing interest in more complex scanning geometries. These include so called X-ray computed laminography systems capable of imaging specimens with large lateral dimensions or large aspect ratios, neither of which are well suited to conventional CT scanning procedures. Developments throughout this field have thus been rapid, including the introduction of novel system trajectories, the application and refinement of various reconstruction methods, and the use of recently developed computational hardware and software techniques to accelerate reconstruction times. Here we examine the advances made in the last several years and consider their impact on the state of the art.

High-resolution computed tomography reconstructions of invertebrate burrow systems.

The architecture of biogenic structures can be highly influential in determining species contributions to major soil and sediment processes, but detailed 3-D characterisations are rare and descriptors of form and complexity are lacking. Here we provide replicate high-resolution micro-focus computed tomography (µ-CT) data for the complete burrow systems of three co-occurring, but functionally contrasting, sediment-dwelling inter-tidal invertebrates assembled alone, and in combination, in representative model aquaria. These data (≤ 2,000 raw image slices aquarium(-1), isotropic voxel resolution, 81 µm) provide reference models that can be used for the development of novel structural analysis routines that will be of value within the fields of ecology, pedology, geomorphology, palaeobiology, ichnology and mechanical engineering. We also envisage opportunity for those investigating transport networks, vascular systems, plant rooting systems, neuron connectivity patterns, or those developing image analysis or statistics related to pattern or shape recognition. The dataset will allow investigators to develop or test novel methodology and ideas without the need to generate a complete three-dimensional computation of exemplar architecture.

Non-convexly constrained image reconstruction from nonlinear tomographic X-ray measurements.

The use of polychromatic X-ray sources in tomographic X-ray measurements leads to nonlinear X-ray transmission effects. As these nonlinearities are not normally taken into account in tomographic reconstruction, artefacts occur, which can be particularly severe when imaging objects with multiple materials of widely varying X-ray attenuation properties. In these settings, reconstruction algorithms based on a nonlinear X-ray transmission model become valuable. We here study the use of one such model and develop algorithms that impose additional non-convex constraints on the reconstruction. This allows us to reconstruct volumetric data even when limited measurements are available. We propose a nonlinear conjugate gradient iterative hard thresholding algorithm and show how many prior modelling assumptions can be imposed using a range of non-convex constraints.

Nanomechanical properties of bird feather rachises: exploring naturally occurring fibre reinforced laminar composites.

Flight feathers have evolved under selective pressures to be sufficiently light and strong enough to cope with the stresses of flight. The feather shaft (rachis) must resist these stresses and is fundamental to this mode of locomotion. Relatively little work has been done on rachis morphology, especially from a mechanical perspective and never at the nanoscale. Nano-indentation is a cornerstone technique in materials testing. Here we use this technique to make use of differentially oriented fibres and their resulting mechanical anisotropy. The rachis is established as a multi-layered fibrous composite material with varying laminar properties in three feathers of birds with markedly different flight styles; the Mute Swan (Cygnus olor), the Bald Eagle (Haliaeetus leucocephalus) and the partridge (Perdix perdix). These birds were chosen not just because they are from different clades and have different flight styles, but because they have feathers large enough to gain meaningful results from nano-indentation. Results from our initial datasets indicate that the proportions and orientation of the laminae are not fixed and may vary either in order to cope with the stresses of flight particular to the bird or with phylogenetic lineage.

High resolution synchrotron imaging of wheat root hairs growing in soil and image based modelling of phosphate uptake.

· Root hairs are known to be highly important for uptake of sparingly soluble nutrients, particularly in nutrient deficient soils. Development of increasingly sophisticated mathematical models has allowed uptake characteristics to be quantified. However, modelling has been constrained by a lack of methods for imaging live root hairs growing in real soils. · We developed a plant growth protocol and used Synchrotron Radiation X-ray Tomographic Microscopy (SRXTM) to uncover the three-dimensional (3D) interactions of root hairs in real soil. We developed a model of phosphate uptake by root hairs based directly on the geometry of hairs and associated soil pores as revealed by imaging. · Previous modelling studies found that root hairs dominate phosphate uptake. By contrast, our study suggests that hairs and roots contribute equally. We show that uptake by hairs is more localized than by roots and strongly dependent on root hair and aggregate orientation. · The ability to image hair-soil interactions enables a step change in modelling approaches, allowing a more realistic treatment of processes at the scale of individual root hairs in soil pores.

Three hydrolases and a transferase: comparative analysis of active-site dynamics via the BioSimGrid database.

Comparative molecular dynamics (MD) simulations enable us to explore the conformational dynamics of the active sites of distantly related enzymes. We have used the BioSimGrid (http://www.biosimgrid.org) database to facilitate such a comparison. Simulations of four enzymes were analyzed. These included three hydrolases and a transferase, namely acetylcholinesterase, outer-membrane phospholipase A, outer-membrane protease T, and PagP (an outer-membrane enzyme which transfers a palmitate chain from a phospholipid to lipid A). A set of 17 simulations were analyzed corresponding to a total of approximately 0.1 micros simulation time. A simple metric for active-site integrity was used to demonstrate the existence of clusters of dynamic conformational behaviour of the active sites. Small (i.e. within a cluster) fluctuations appear to be related to the function of an enzymatically active site. Larger fluctuations (i.e. between clusters) correlate with transitions between catalytically active and inactive states. Overall, these results demonstrate the potential of a comparative MD approach to analysis of enzyme function. This approach could be extended to a wider range of enzymes using current high throughput MD simulation and database methods.