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. 2018 Sep 6;8:13348. doi: 10.1038/s41598-018-31182-2

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Optical Clearing and Large Scale Microscopy of Whole Lung. (a) Native murine lung following extraction (b) Intact lung following whole lung optical clearing. In this photograph, the lung is held in air following perfusion and immersion in BABB. To capture the contrast of the airway against the parenchyma, a cannula is used to perfuse the lung with fluid of slightly different refractive index to the BABB OC fluid. (c) Optically cleared lung infused with and immersed in BABB in a sample imaging chamber. A dotted line outlines the borders of the lung and the arrow points to area of cardiac structures that appear dark. (d) Volumetric (3D) representation of the entire murine lung obtained by mosaicking and stitching of MPM-SHGM image stacks using the 4 × 0.16 NA objective. Imaging was through the full depth of the lung with mosaicking (r x r arrangement) to acquire the entire organ, with MPM tissue autofluorescence shown in red and collagen SHG shown in green. The right lung shows multi- lobe structure (arrow). To create the cutout shown in (d), cropped areas of the red channel were removed from the left lung to reveal underlying sub epithelial collagen network surrounding airways. 3D reconstruction was by Imaris 3D analysis software. Reconstructed volume represents n = ~25,000 images. (e) Representative high resolution 25x MPM of lung regions showing a segment of a pulmonary bronchus (shown in false color). (f) MPM (25x) showing a segment of lung tissue with a vessel (+) near two converging bronchi (*) and surrounded by alveoli (^). (Epithelial cells (*), alveoli (^), and blood vasculature distribution (+)).