Abstract Pulmonary fibrosis, characterized by excessive collagen deposition in the lungs, comprises a key and debilitating component of chronic lung diseases. Methods are lacking for the direct visualization of fibrillar collagen throughout the whole murine lung, a capability that would aid the understanding of lung fibrosis. We combined an optimized organ-level optical clearing (OC) approach with large-scale, label-free multiphoton microscopy (MPM) and second harmonic generation microscopy (SHGM) to reveal the complete network of fibrillar collagen in whole murine lungs. An innate inflammation-driven model based on repetitive poly(I:C) challenge was evaluated. Following OC, mosaic MPM/SHGM imaging with 3D reconstruction and whole organ quantitative analysis revealed significant differences in collagen deposition between PBS and poly(I:C) treated lungs. Airway specific analysis in whole lung acquisitions revealed significant sub-epithelial fibrosis evident throughout the proximal conductive and distal airways with higher collagen deposition in the poly(I:C) group vs PBS group. This study establishes a new, powerful approach based on OC and MPM/SHGM imaging for 3D analysis of lung fibrosis with macroscopic views of lung pathology based on microscopy and providing a new way to analyze the whole lung while avoiding regional sampling bias.

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3d analysis of lung fibrosis organlevel optical clearing oc approach repetitive polyic challenge regional sampling chronic lung macroscopic views of lung pathology of fibrillar collagen largescale labelfree multiphoton microscopy second harmonic generation microscopy powerful approach oc mosaic mpmshgm imaging with 3d reconstruction inflammationdriven subepithelial fibrosis

Lorenzo F. Ochoa,Alexander Kholodnykh,Paula Villarreal,Bing Tian,Rahul Pal,Alexander N. Freiberg,Allan R. Brasier,Massoud Motamedi,Gracie Vargas,.Imaging of Murine Whole Lung Fibrosis by Large Scale 3D Microscopy aided by Tissue Optical Clearing. 8 (1),.

Lorenzo F. Ochoa,Alexander Kholodnykh,Paula Villarreal,Bing Tian,Rahul Pal,Alexander N. Freiberg,Allan R. Brasier,Massoud Motamedi,Gracie Vargas,"Imaging of Murine Whole Lung Fibrosis by Large Scale 3D Microscopy aided by Tissue Optical Clearing" 8

Lorenzo F. Ochoa,Alexander Kholodnykh,Paula Villarreal,Bing Tian,Rahul Pal,Alexander N. Freiberg,Allan R. Brasier,Massoud Motamedi,Gracie Vargas,"Imaging of Murine Whole Lung Fibrosis by Large Scale 3D Microscopy aided by Tissue Optical Clearing"