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

Three-dimensional (3D) imaging with dual-view inverted selective plane microscopy (diSPIM). The system used to acquire 3D images in this work was a customized dual-view inverted selective plane microscopy (diSPIM) system, originally developed by Hari Shroff and co-workers and commercialized by Applied Scientific Instrumentation (ASI, Eugene, OR). 12 Our system has been modified for large cleared tissue imaging with adjustable illumination tube lenses and dual Cleared Tissue Objectives (CTO, ASI/Special Optics, 15.3X -17.9X, NA: 0.37-0.43 over RI range of 1.33 to 1.55, working distance: 12 mm) in order to adapt to diverse immersion mounting media, as described previously. 8 The system is fitted with a 488-nm laser source (Omicron, PhoxX; maximum power = 200 mW) for fluorescence excitation of Eosin and a 647-nm laser source (Omicron, PhoxX; maximum power = 140 mW) for fluorescence excitation of TO-PRO-3 iodide. During imaging, the lung sample was immersed in ECi in a custom sample chamber, and imaged from above using the standard diSPIM arrangement. 8, 11 No additional material or interface was introduced between the objective lens surfaces and the cleared tissue other than the ECi immersion media, and no compression was applied, enabling reconstruction of the natural tissue surface topography. Although the system can be used to acquire 2 orthogonal views of the sample for dual-view imaging and reconstruction, 8, 12, 14 in this work only one view was acquired. Captured light-sheet images from a scientific CMOS camera (Hamamatsu ORCA-Flash 4.0v2) were transferred to a controller workstation (Dell T3610) and stored in an 8TB SSD array set as RAID 0 mode. The raw data comprised a stack of 221,400 individual images with a single frame size of 2,048 pixels by 2,048 pixels, giving a total raw volume of 929 gigavoxels with a total file size of 1.69 TB.