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).

Multiorgan tropism of SARS-CoV-2 has previously been shown for several major organs. We have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n = 8), using histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA in situ hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. SARS-CoV-2 RNA was mainly localized in epithelial cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. These findings were validated using in situ hybridization on external COVID-19 autopsy samples (n = 9). Apart from the lung, correlation of viral detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2. In summary, SARS-CoV-2 and its replication could be observed across all organ systems, which co-localizes with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection.

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.

COVID-19 is characterized by a heterogeneous clinical presentation, ranging from mild symptoms to severe courses of disease. 9–20% of hospitalized patients with severe lung disease die from COVID-19 and a substantial number of survivors develop long-COVID. Our objective was to provide comprehensive insights into the pathophysiology of severe COVID-19 and to identify liquid biomarkers for disease severity and therapy response. We studied a total of 85 lungs (n = 31 COVID autopsy samples; n = 7 influenza A autopsy samples; n = 18 interstitial lung disease explants; n = 24 healthy controls) using the highest resolution Synchrotron radiation-based hierarchical phase-contrast tomography, scanning electron microscopy of microvascular corrosion casts, immunohistochemistry, matrix-assisted laser desorption ionization mass spectrometry imaging, and analysis of mRNA expression and biological pathways. Plasma samples from all disease groups were used for liquid biomarker determination using ELISA. The anatomic/molecular data were analyzed as a function of patients’ hospitalization time. The observed patchy/mosaic appearance of COVID-19 in conventional lung imaging resulted from microvascular occlusion and secondary lobular ischemia. The length of hospitalization was associated with increased intussusceptive angiogenesis. This was associated with enhanced angiogenic, and fibrotic gene expression demonstrated by molecular profiling and metabolomic analysis. Increased plasma fibrosis markers correlated with their pulmonary tissue transcript levels and predicted disease severity. Plasma analysis confirmed distinct fibrosis biomarkers (TSP2, GDF15, IGFBP7, Pro-C3) that predicted the fatal trajectory in COVID-19. Pulmonary severe COVID-19 is a consequence of secondary lobular microischemia and fibrotic remodelling, resulting in a distinctive form of fibrotic interstitial lung disease that contributes to long-COVID. This project was made possible by a number of funders. The full list can be found within the Declaration of interests / Acknowledgements section at the end of the manuscript.

Hierarchical phase-contrast tomography (HiP-CT) enables ex vivo virtual autopsy cardiac imaging with high spatial resolution, providing nondestructive, three-dimensional, multiscale analysis of intact healthy and diseased adult human hearts without contrast agents.

For the first time, we have used phase-contrast X-ray tomography to characterize the three-dimensional (3d) structure of cardiac tissue from patients who succumbed to Covid-19. By extending conventional histopathological examination by a third dimension, the delicate pathological changes of the vascular system of severe Covid-19 progressions can be analyzed, fully quantified and compared to other types of viral myocarditis and controls. To this end, cardiac samples with a cross-section of 3.5mm were scanned at a laboratory setup as well as at a parallel beam setup at a synchrotron radiation facility the synchrotron in a parallel beam configuration. The vascular network was segmented by a deep learning architecture suitable for 3d datasets (V-net), trained by sparse manual annotations. Pathological alterations of vessels, concerning the variation of diameters and the amount of small holes, were observed, indicative of elevated occurrence of intussusceptive angiogenesis, also confirmed by high-resolution cone beam X-ray tomography and scanning electron microscopy. Furthermore, we implemented a fully automated analysis of the tissue structure in the form of shape measures based on the structure tensor. The corresponding distributions show that the histopathology of Covid-19 differs from both influenza and typical coxsackie virus myocarditis.