Multiscale 3-dimensional pathology findings of COVID-19 diseased lung using high-resolution cleared tissue microscopy

Guang Li; Sharon E. Fox; Brian Summa; Carola Wenk; Aibek Akmatbekov; Jack L. Harbert; Richard S. Vander Heide; J. Quincy Brown

result of COVID-19 infection. Consent for autopsy was non-restricted by the next of kin, and these studies on autopsy tissues were determined to be exempt by the IRB at LSU Health Sciences Center and Tulane University. We demonstrate near-teravoxel volumetric images of optically-cleared lung tissue over cubic millimeter volumes with virtual H&E contrast and at submicron resolution. Our work builds on the prior autopsy case-series report from LSU-HSC, confirming and expanding novel findings of widespread pulmonary microangiopathy involving activation of mature megakaryocytes in the lung. Figure 1 contains reconstructed volumes, selected optical slices, and 3D renderings from a 7.8 mm × 5.9 mm × 0.9 mm formalin-fixed block of lung tissue sampled from the left peripheral upper lobe. Tissue blocks were stained with TO-PRO-3 for nuclear contrast and Eosin-Y for cytoplasmic/stromal contrast, followed by optical clearing with ethyl-cinnamate, according to the method of Reder et al. 7 Blocks were then imaged using one side of a dual-inverted selective plane illumination microscope 12 customized for large cleared tissue imaging, described previously. 8, 11 Two grayscale images were collected corresponding to each fluorescent label, which were reconstructed independently, and synthesized into a single pseudo-colored RGB image replicating the color palette and appearance of H&E staining using the approach described by Giacomelli et al. (Supplemental Figure 1) . 5 The total raw volume in Figure 1a comprised 929 gigavoxels, with a voxel size of 0.363 μm × 0.363 μm × 0.363 μm. A smaller volume (1.2 mm × 0.6 mm × 0.3 mm comprising 4.5 gigavoxels) was selected from the larger volume for detailed visualization. Supplemental Video 1 shows all slices from all orthogonal orientations in the volume. Figure 1b shows a single XY slice from the smaller subvolume -the location of the slice in the volume is shown in the inset of Figure 1a . Prominent features observed in this virtual H&E slice included a megakaryocyte in a branching small vessel, apparent multi-nucleated or grouped cells demonstrating viral cytopathic changes including prominent nucleoli in the alveolar space, and scattered hyaline-fibrin aggregates in the alveolar spaces. Further analysis of these areas in 3D (Figure 1c ) provided additional value in interpretation, confirming that the megakaryocyte displayed the expected lobular, fused multinuclear shape (Supplemental Video 2), enabling determination that the apparent multi-nucleated cell with viral cytopathic effect in Figure 1b was in fact a cluster of independent cells, and showing a spherical hyaline-fibrin aggregate surrounded by apparent inflammatory cells in the alveolar space, consistent with diffuse alveolar damage. author/funder. All rights reserved. No reuse allowed without permission.