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.

Preparation of large biological samples for high-resolution, hierarchical, synchrotron phase-contrast tomography with multimodal imaging compatibility.

Imaging across different scales is essential for understanding healthy organ morphology and pathophysiological changes. The macro- and microscale three-dimensional morphology of large samples, including intact human organs, is possible with X-ray microtomography (using laboratory or synchrotron sources). Preparation of large samples for high-resolution imaging, however, is challenging due to limitations such as sample shrinkage, insufficient contrast, movement of the sample and bubble formation during mounting or scanning. Here, we describe the preparation, stabilization, dehydration and mounting of large soft-tissue samples for X-ray microtomography. We detail the protocol applied to whole human organs and hierarchical phase-contrast tomography at the European Synchrotron Radiation Facility, yet it is applicable to a range of biological samples, including complete organisms. The protocol enhances the contrast when using X-ray imaging, while preventing sample motion during the scan, even with different sample orientations. Bubbles trapped during mounting and those formed during scanning (in the case of synchrotron X-ray imaging) are mitigated by multiple degassing steps. The sample preparation is also compatible with magnetic resonance imaging, computed tomography and histological observation. The sample preparation and mounting require 24-36 d for a large organ such as a whole human brain or heart. The preparation time varies depending on the composition, size and fragility of the tissue. Use of the protocol enables scanning of intact organs with a diameter of 150 mm with a local voxel size of 1 µm. The protocol requires users with expertise in handling human or animal organs, laboratory operation and X-ray imaging.

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).

Co-expression of transcription factor AP-2beta (TFAP2B) and GATA3 in human mammary epithelial cells with intense, apicobasal immunoreactivity for CK8/18.

AP-2ß is a new mammary epithelial differentiation marker and its expression is preferentially retained and enhanced in lobular carcinoma in situ and invasive lobular breast cancer. In normal breast epithelium AP-2ß is expressed in a scattered subpopulation of luminal cells. So far, these cells have not been further characterized. Co-expression of AP-2ß protein and luminal epithelium markers (GATA3, CK8/18), hormone receptors [estrogen receptor (ER), androgen receptor (AR)] and candidate stem cells markers (CK5/14, CD44) were assessed by double-immunofluorescence staining in normal mammary gland epithelium. The subpopulation of AP-2ß-positive mammary epithelial cells showed an almost complete, superimposable co-expression with GATA3 and a peculiar intense, ring-like appearing immunoreactivity for CK8/18. Confocal immunofluorescence microscopy revealed an apicobasal staining for CK8/18 in AP-2ß-positive cells, which was not seen in in AP-2ß-negative cells. Furthermore, AP-2ß-positive displayed a partial co-expression with ER and AR, but lacked expression of candidate stem cell markers CK5/14 and CD44. In summary, AP-2ß is a new luminal mammary epithelial differentiation marker, which is expressed in the GATA3-positive subpopulation of luminal epithelial cells. These AP-2ß-positive/GATA3-positive cells also show a peculiar CK8/18-expression which may indicate a previously unknown functionally specialized mammary epithelial cell population.

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.

[Mesothelial proliferation of the tunica vaginalis testis]. / Mesotheliale Proliferate der Tunica vaginalis testis.

Proliferative changes seen in reactive mesothelial hyperplasia of a hydrocele sac may mimic malignant mesothelioma. There is no immunohistochemical staining that reliably separates benign from malignant mesothelial proliferations. However, the combined analysis of BAP1 by immunohistochemistry and CDKN2A by FISH has been reported to yield both a high specificity and sensitivity in this differential diagnosis. In addition, the evaluation of risk factors such as asbestos exposure or prior traumata may be helpful for the correct diagnosis. Exclusion of stromal invasion, which is diagnostic for malign mesothelioma, is of utmost importance. Therefore, extended histological workup is essential.