Multidimensional Analysis of the Adult Human Heart in Health and Disease using Hierarchical Phase-Contrast Tomography (HiP-CT).

Cardiovascular diseases (CVDs) are a leading cause of death worldwide. Current clinical imaging modalities provide resolution adequate for diagnosis but are unable to provide detail of structural changes in the heart, across length-scales, necessary for understanding underlying pathophysiology of disease. Hierarchical Phase-Contrast Tomography (HiP-CT), using new (4th) generation synchrotron sources, potentially overcomes this limitation, allowing micron resolution imaging of intact adult organs with unprecedented detail. In this proof of principle study (n=2), we show the utility of HiP-CT to image whole adult human hearts ex-vivo: one 'control' without known cardiac disease and one with multiple known cardiopulmonary pathologies. The resulting multiscale imaging was able to demonstrate exemplars of anatomy in each cardiac segment along with novel findings in the cardiac conduction system, from gross (20 um/voxel) to cellular scale (2.2 um/voxel), non-destructively, thereby bridging the gap between macroscopic and microscopic investigations. We propose that the technique represents a significant step in virtual autopsy methods for studying structural heart disease, facilitating research into abnormalities across scales and age-groups. It opens up possibilities for understanding and treating disease; and provides a cardiac 'blueprint' with potential for in-silico simulation, device design, virtual surgical training, and bioengineered heart in the future.

Imaging intact human organs with local resolution of cellular structures using hierarchical phase-contrast tomography.

Imaging intact human organs from the organ to the cellular scale in three dimensions is a goal of biomedical imaging. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique using the European Synchrotron Radiation Facility (ESRF)'s Extremely Brilliant Source (EBS). The spatial coherence of the ESRF-EBS combined with our beamline equipment, sample preparation and scanning developments enabled us to perform non-destructive, three-dimensional (3D) scans with hierarchically increasing resolution at any location in whole human organs. We applied HiP-CT to image five intact human organ types: brain, lung, heart, kidney and spleen. HiP-CT provided a structural overview of each whole organ followed by multiple higher-resolution volumes of interest, capturing organotypic functional units and certain individual specialized cells within intact human organs. We demonstrate the potential applications of HiP-CT through quantification and morphometry of glomeruli in an intact human kidney and identification of regional changes in the tissue architecture in a lung from a deceased donor with coronavirus disease 2019 (COVID-19).

Multiscale three-dimensional imaging of intact human organs down to the cellular scale using hierarchical phase-contrast tomography.

Human organs are complex, three-dimensional and multiscale systems. Spatially mapping the human body down through its hierarchy, from entire organs to their individual functional units and specialised cells, is a major obstacle to fully understanding health and disease. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique utilising the European Synchrotron Radiation Facility's Extremely Brilliant Source: the world's first high-energy 4 th generation X-ray source. HiP-CT enabled three-dimensional and non-destructive imaging at near-micron resolution in soft tissues at one hundred thousand times the voxel size whilst maintaining the organ's structure. We applied HiP-CT to image five intact human parenchymal organs: brain, lung, heart, kidney and spleen. These were hierarchically assessed with HiP-CT, providing a structural overview of the whole organ alongside detail of the organ's individual functional units and cells. The potential applications of HiP-CT were demonstrated through quantification and morphometry of glomeruli in an intact human kidney, and identification of regional changes to the architecture of the air-tissue interface and alveolar morphology in the lung of a deceased COVID-19 patient. Overall, we show that HiP-CT is a powerful tool which can provide a comprehensive picture of structural information for whole intact human organs, encompassing precise details on functional units and their constituent cells to better understand human health and disease.

The humerus of Eusthenopteron: a puzzling organization presaging the establishment of tetrapod limb bone marrow.

Because of its close relationship to tetrapods, Eusthenopteron is an important taxon for understanding the establishment of the tetrapod body plan. Notably, it is one of the earliest sarcopterygians in which the humerus of the pectoral fin skeleton is preserved. The microanatomical and histological organization of this humerus provides important data for understanding the evolutionary steps that built up the distinctive architecture of tetrapod limb bones. Previous histological studies showed that Eusthenopteron's long-bone organization was established through typical tetrapod ossification modalities. Based on a three-dimensional reconstruction of the inner microstructure of Eusthenopteron's humerus, obtained from propagation phase-contrast X-ray synchrotron microtomography, we are now able to show that, despite ossification mechanisms and growth patterns similar to those of tetrapods, it also retains plesiomorphic characters such as a large medullary cavity, partly resulting from the perichondral ossification around a large cartilaginous bud as in actinopterygians. It also exhibits a distinctive tubular organization of bone-marrow processes. The connection between these processes and epiphyseal structures highlights their close functional relationship, suggesting that either bone marrow played a crucial role in the long-bone elongation processes or that trabecular bone resulting from the erosion of hypertrophied cartilage created a microenvironment for haematopoietic stem cell niches.

A mathematical model for mechanotransduction at the early steps of suture formation.

Growth and patterning of craniofacial sutures is subjected to the effects of mechanical stress. Mechanotransduction processes occurring at the margins of the sutures are not precisely understood. Here, we propose a simple theoretical model based on the orientation of collagen fibres within the suture in response to local stress. We demonstrate that fibre alignment generates an instability leading to the emergence of interdigitations. We confirm the appearance of this instability both analytically and numerically. To support our model, we use histology and synchrotron X-ray microtomography and reveal the fine structure of fibres within the sutural mesenchyme and their insertion into the bone. Furthermore, using a mouse model with impaired mechanotransduction, we show that the architecture of sutures is disturbed when forces are not interpreted properly. Finally, by studying the structure of sutures in the mouse, the rat, an actinopterygian (Polypterus bichir) and a placoderm (Compagopiscis croucheri), we show that bone deposition patterns during dermal bone growth are conserved within jawed vertebrates. In total, these results support the role of mechanical constraints in the growth and patterning of craniofacial sutures, a process that was probably effective at the emergence of gnathostomes, and provide new directions for the understanding of normal and pathological suture fusion.

High-resolution imaging of craniofacial sutures: new tools for understanding the origins of craniosynostoses.

AIM AND SCOPE: The developmental genetics and the biomechanics of sutures are well-studied topics, while their microanatomy is still imperfectly known. Here, we aim to investigate the structure of skull vault sutures using a high-resolution imaging device. MATERIAL AND METHODS: We used synchrotron X-ray microtomography in order to obtain high-resolution images of skull vault sutures from an extant mammal (the mouse Mus musculus) and from an extinct fish (the placoderm Compagopiscis croucheri). We used segmentation and 3D reconstruction softwares in order to reveal the microanatomy of sutures in these species. RESULTS: The high-resolution images allowed us to study the distribution of osteocytes, the organisation of vascular canals, the shapes of the suture borders, the insertion of Sharpey's fibres, the bone growth lines and the structure of the soft tissues surrounding the sutures. CONCLUSION: Synchrotron imaging provides new perspectives for the study of the normal microanatomy of sutures. The submicronic resolution of the synchrotron scans gives access to the 3D organisation of structures that were previously only known in 2D, even in normal sutures. The description of anatomical entities such as vascular canals and Sharpey's fibres in abnormally fused sutures would be of interest in the understanding of craniosynostoses.

Trimodal low-dose X-ray tomography.

X-ray grating interferometry is a coherent imaging technique that bears tremendous potential for three-dimensional tomographic imaging of soft biological tissue and other specimens whose details exhibit very weak absorption contrast. It is intrinsically trimodal, delivering phase contrast, absorption contrast, and scattering ("dark-field") contrast. Recently reported acquisition strategies for grating-interferometric phase tomography constitute a major improvement of dose efficiency and speed. In particular, some of these techniques eliminate the need for scanning of one of the gratings ("phase stepping"). This advantage, however, comes at the cost of other limitations. These can be a loss in spatial resolution, or the inability to fully separate the three imaging modalities. In the present paper we report a data acquisition and processing method that optimizes dose efficiency but does not share the main limitations of other recently reported methods. Although our method still relies on phase stepping, it effectively uses only down to a single detector frame per projection angle and yields images corresponding to all three contrast modalities. In particular, this means that dark-field imaging remains accessible. The method is also compliant with data acquisition over an angular range of only 180° and with a continuous rotation of the specimen.

Limb-bone histology of temnospondyls: implications for understanding the diversification of palaeoecologies and patterns of locomotion of Permo-Triassic tetrapods.

The locomotion of early tetrapods has long been a subject of great interest in the evolutionary history of vertebrates. However, we still do not have a precise understanding of the evolutionary radiation of their locomotory strategies. We present here the first palaeohistological study based on theoretical biomechanical considerations among a highly diversified group of early tetrapods, the temnospondyls. Based on the quantification of microanatomical and histological parameters in the humerus and femur of nine genera, this multivariate analysis provides new insights concerning the adaptations of temnospondyls to their palaeoenvironments during the Early Permian, and clearly after the Permo-Triassic crisis. This study therefore presents a methodology that, if based on a bigger sample, could contribute towards a characterization of the behaviour of species during great evolutionary events.

Sexual intercourse involving giant sperm in Cretaceous ostracode.

Reproduction with giant sperm occurs in distinct groups scattered over the animal kingdom. Although experiments in Drosophila assessed the influence of different selection pressures on this character, no information was available on its long-term stability. Sub-micrometer-resolution synchrotron quantitative phase tomography (holotomography) of exceptionally well-preserved three-dimensional Cretaceous ostracode fossils from the Brazilian Santana Formation indicates that ostracode reproduction with giant sperm persisted for at least over the past 100 million years. Remnants of the male sperm pumps as well as giant, inflated female sperm receptacles evidence that, despite high costs, reproduction with giant sperm can be an evolutionary successful strategy.