3D imaging of SARS-CoV-2 infected hamster lungs by X-ray phase contrast tomography enables drug testing.

X-ray Phase Contrast Tomography (XPCT) based on wavefield propagation has been established as a high resolution three-dimensional (3D) imaging modality, suitable to reconstruct the intricate structure of soft tissues, and the corresponding pathological alterations. However, for biomedical research, more is needed than 3D visualisation and rendering of the cytoarchitecture in a few selected cases. First, the throughput needs to be increased to cover a statistically relevant number of samples. Second, the cytoarchitecture has to be quantified in terms of morphometric parameters, independent of visual impression. Third, dimensionality reduction and classification are required for identification of effects and interpretation of results. To address these challenges, we here design and implement a novel integrated and high throughput XPCT imaging and analysis workflow for 3D histology, pathohistology and drug testing. Our approach uses semi-automated data acquisition, reconstruction and statistical quantification. We demonstrate its capability for the example of lung pathohistology in Covid-19. Using a small animal model, different Covid-19 drug candidates are administered after infection and tested in view of restoration of the physiological cytoarchitecture, specifically the alveolar morphology. To this end, we then use morphometric parameter determination followed by a dimensionality reduction and classification based on optimal transport. This approach allows efficient discrimination between physiological and pathological lung structure, thereby providing quantitative insights into the pathological progression and partial recovery due to drug treatment. Finally, we stress that the XPCT image chain implemented here only used synchrotron radiation for validation, while the data used for analysis was recorded with laboratory µ CT radiation, more easily accessible for pre-clinical research.

Multilamellated Basement Membranes in the Capillary Network of Alveolar Capillary Dysplasia.

A minimal diffusion barrier is key to the pulmonary gas exchange. In alveolar capillary dysplasia (ACD), a rare genetically driven disease of early infancy, this crucial fibrovascular interface is compromised while the underlying pathophysiology is insufficiently understood. Recent in-depth analyses of vascular alterations in adult lung disease encouraged researchers to extend these studies to ACD and compare the changes of the microvasculature. Lung tissue samples of children with ACD (n = 12), adults with non-specific interstitial pneumonia (n = 12), and controls (n = 20) were studied using transmission electron microscopy, single-gene sequencing, immunostaining, exome sequencing, and broad transcriptome profiling. In ACD, pulmonary capillary basement membranes were hypertrophied, thickened, and multilamellated. Transcriptome profiling revealed increased CDH5, COL4A1, COL15A1, PTK2B, and FN1 and decreased VIT expression, confirmed by immunohistochemistry. In contrast, non-specific interstitial pneumonia samples showed a regular basement membrane architecture with preserved VIT expression but also increased COL15A1+ vessels. This study provides insight into the ultrastructure and pathophysiology of ACD. The lack of normally developed lung capillaries appeared to cause a replacement by COL15A1+ vessels, a mechanism recently described in interstitial lung disease. The VIT loss and FN1 overexpression might contribute to the unique appearance of basement membranes in ACD. Future studies are needed to explore the therapeutic potential of down-regulating the expression of FN1 and balancing VIT deficiency.

Neodymium acetate as a contrast agent for X-ray phase-contrast tomography.

Purpose: X-ray phase-contrast tomography (XPCT) is a non-destructive, three-dimensional imaging modality that provides higher contrast in soft tissue than absorption-based CT and allows one to cover the cytoarchitecture from the centi- and millimeter scale down to the nanoscale. To further increase contrast and resolution of XPCT, for example, in view of addressing connectivity issues in the central nervous system (CNS), metal staining is indispensable. However, currently used protocols, for example, based on osmium and/or uranium are less suited for XPCT, due to an excessive ß/δ-ratio. In this work, we explore the suitability of different staining agents for XPCT. Particularly, neodymium(III)-acetate (NdAc), which has recently been proposed as a non-toxic, non-radioactive easy to use alternative contrast agent for uranyl acetate (UAc) in electron microscopy, is investigated. Due to its vertical proximity to UAc in the periodic table, similar chemical but better suited optical properties for phase contrast can be expected. Approach: Differently stained whole eye samples of wild type mouse and tissues of the CNS are embedded into EPON epoxy resin and scanned using synchrotron as well as with laboratory radiation. Phase retrieval is performed on the projection images, followed by tomographic reconstruction, which enables a quantitative analysis based on the reconstructed electron densities. Segmentation techniques and rendering software is used to visualize structures of interest in the sample. Results: We show that staining neuronal samples with NdAc enhances contrast, in particular for laboratory scans, allowing high-resolution imaging of biological soft tissue in-house. For the example of murine retina, specifically rods and cones as well as the sclera and the Ganglion cell layer seem to be targeted by the stain. A comparison of electron density by the evaluation of histograms allowed to determine quantitative measures to describe the difference between the examined stains. Conclusion: The results suggest NdAc to be an effective stain for XPCT, with a preferential binding to anionic groups, such as phosphate and carboxyl groups at cell surfaces, targeting certain layers of the retina with a stronger selectivity compared to other staining agents. Due to the advantageous X-ray optical properties, the stain seems particularly well-suited for phase contrast, with a comparably small number density and an overall superior image quality at laboratory sources.

Human lung virtual histology by multi-scale x-ray phase-contrast computed tomography.

Objectives.As the central organ of the respiratory system, the human lung is responsible for supplying oxygen to the blood, which reaches the erythrocytes by diffusion through the alveolar walls and is then distributed throughout the body. By exploiting the difference in electron density detected by a phase shift in soft tissue, high-resolution x-ray phase-contrast computed tomography (XPCT) can resolve biological structures in a sub-µm range, shedding new light on the three-dimensional structure of the lungs, physiological functions and pathological mechanisms.Approach.This work presents both synchrotron and laboratory XPCT results of postmortem tissue from autopsies and biopsies embedded with various preparation protocols such as precision-cut lung slices, cryogenically fixed lung tissue, as well as paraffin and alcohol fixed tissue. The selection of pathological abnormalities includes channel of Lambert, bronchus-associated lymphoid tissue and alveolar capillary dysplasia with misalignment of pulmonary veins. Subsequently, quantification and visualization approaches are presented.Main results.The overall high image quality even of in-house XPCT scans for the case of FFPE biopsies can be exploited for a wide range of pulmonary pathologies and translated to dedicated and optimized instrumentation which could be operated in clinical setting. By using synchrotron radiation, contrast can be further increased to resolve sub-µm sized features down to the sub-cellular level. The results demonstrate that a wide range of preparation protocols including sample mounting in liquids can be used.Significance.With XPCT, poorly understood 3D structures can be identified in larger volume overview and subsequently studied in more detail at higher resolution. With the full 3D structure, the respective physiological functions of airways or vascular networks, and the different pathophysiologic mechanisms can be elucidated or at least underpinned with structural data. Moreover, synchrotron data can be used to validate laboratory protocols and provide ground truth for standardizing the method.