Application of ethyl cinnamate based optical tissue clearing and expansion microscopy combined with retrograde perfusion for 3D lung imaging

Purpose: 3D imaging of the lung is not a trivial undertaking as during preparation the lung may collapse. Also serial sections and scans followed by 3D reconstruction may lead to artifacts. The present study aims to figure out the best way to perform 3D imaging in lung research. Materials and Methods: We applied an optical tissue clearing (OTC) method, which uses ethyl cinnamate (ECi) as a fast, non-toxic and cheap clearing solvent, for 3D imaging of retrograde perfused lungs by laser confocal fluorescence microscopy and light sheet fluorescence microscopy. We also introduced expansion microscopy (ExM), a cutting-edge technique, in 3D imaging of lungs. We examined and compared the usefulness of these techniques for 3D lung imaging. The ExM protocol was further extended to paraffin-embedded lung metastases blocks. Results: The MHI148-PEI labeled lung vasculature was visualized by retrograde perfusion combined with trachea ligation and ECi based OTC. As compared with trans-cardiac perfusion, the retrograde perfusion results in a better maintenance of lung morphology. 3D structure of alveoli, vascular branches and cilia in lung were revealed by immunofluorescence staining after ExM. 3D distribution of microvasculature and neutrophil cells in 10 years old paraffin-embedded lung metastases were analyzed by ExM. Conclusions: The retrograde perfusion combined with trachea ligation technique could be applied in the lung research in mice. 3D structure of lung vasculature can be visualized by MHI148-PEI perfusion and ECi based OTC in an efficient way. ExM and immunofluorescence staining protocol is highly recommended to perform 3D imaging of fresh fixed lung as well as paraffin-embedded lung blocks.This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Actions, grant agreement No 813839, Innovative Training Network RenalToolBox