Imaging of Murine Whole Lung Fibrosis by Large Scale 3D Microscopy aided by Tissue Optical Clearing

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.

[1]  Ralph Weissleder,et al.  Heterogeneity of macrophage infiltration and therapeutic response in lung carcinoma revealed by 3D organ imaging , 2017, Nature Communications.

[2]  V. Sergienko,et al.  Current rates and mechanisms of subsea permafrost degradation in the East Siberian Arctic Shelf , 2017, Nature Communications.

[3]  Andrew E. Gelman,et al.  Two-photon microscopy in pulmonary research , 2010, Seminars in Immunopathology.

[4]  A. Fryer,et al.  Tissue optical clearing, three-dimensional imaging, and computer morphometry in whole mouse lungs and human airways. , 2014, American journal of respiratory cell and molecular biology.

[5]  Ali Ertürk,et al.  Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO , 2014, Journal of visualized experiments : JoVE.

[6]  C. Cluzel,et al.  The Fibrillar Collagen Family , 2010, International journal of molecular sciences.

[7]  M. Neeman,et al.  Whole Organ Blood and Lymphatic Vessels Imaging (WOBLI) , 2018, Scientific Reports.

[8]  Incheol Seo,et al.  Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine , 2014, BMC Developmental Biology.

[9]  T. Oury,et al.  Animal models of fibrotic lung disease. , 2013, American journal of respiratory cell and molecular biology.

[10]  Ulrich Pohl,et al.  Signal improvement in multiphoton microscopy by reflection with simple mirrors near the sample. , 2010, Journal of biomedical optics.

[11]  G. Rossi,et al.  The role of histology in idiopathic pulmonary fibrosis: an update. , 2010, Respiratory medicine.

[12]  E. White,et al.  Lysyl oxidases regulate fibrillar collagen remodelling in idiopathic pulmonary fibrosis , 2017, Disease Models & Mechanisms.

[13]  B. Ameredes,et al.  NF‐&kgr;B Mediates Mesenchymal Transition, Remodeling, and Pulmonary Fibrosis in Response to Chronic Inflammation by Viral RNA Patterns , 2017, American journal of respiratory cell and molecular biology.

[14]  M. Levene,et al.  Multiphoton microscopy of cleared mouse organs. , 2010, Journal of biomedical optics.

[15]  K. Johnson.  An Update. , 1984, Journal of food protection.

[16]  W. Webb,et al.  Multiphoton microscopy and microspectroscopy for diagnostics of inflammatory and neoplastic lung. , 2012, Journal of biomedical optics.

[17]  H. Collard,et al.  Non-invasive Imaging of Idiopathic Pulmonary Fibrosis Using Cathepsin Protease Probes , 2016, Scientific Reports.

[18]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[19]  S. Perry,et al.  Second harmonic generation microscopy reveals altered collagen microstructure in usual interstitial pneumonia versus healthy lung , 2015, Respiratory Research.

[20]  Bruce McManus,et al.  Collagen matrix remodeling in 3-dimensional cellular space resolved using second harmonic generation and multiphoton excitation fluorescence. , 2010, Journal of structural biology.

[21]  Reinhold Förster,et al.  Application of light sheet microscopy for qualitative and quantitative analysis of bronchus-associated lymphoid tissue in mice , 2018, Cellular & Molecular Immunology.

[22]  W. Webb,et al.  Multiphoton microscopy: a potential "optical biopsy" tool for real-time evaluation of lung tumors without the need for exogenous contrast agents. , 2014, Archives of pathology & laboratory medicine.

[23]  H. Jones,et al.  Novel imaging approaches for small animal models of lung disease (2017 Grover Conference series) , 2018, Pulmonary circulation.

[24]  Vikas Singh,et al.  Second harmonic generation microscopy analysis of extracellular matrix changes in human idiopathic pulmonary fibrosis. , 2014, Journal of biomedical optics.

[25]  R. Homer,et al.  Airway remodeling in asthma. , 1999, The Journal of clinical investigation.

[26]  T. Dresselaers,et al.  Magnetic Resonance Imaging for Noninvasive Assessment of Lung Fibrosis Onset and Progression: Cross-Validation and Comparison of Different Magnetic Resonance Imaging Protocols With Micro–Computed Tomography and Histology in the Bleomycin-Induced Mouse Model , 2014, Investigative radiology.

[27]  Aaron S. Andalman,et al.  Structural and molecular interrogation of intact biological systems , 2013, Nature.

[28]  Paul Xu,et al.  Quantification of collagen I in airway tissues using second harmonic generation , 2014, Journal of biomedical optics.

[29]  Hiroki R Ueda,et al.  Whole-body and Whole-Organ Clearing and Imaging Techniques with Single-Cell Resolution: Toward Organism-Level Systems Biology in Mammals. , 2016, Cell chemical biology.

[30]  Georgios C. Antonopoulos,et al.  A combined method for correlative 3D imaging of biological samples from macro to nano scale , 2016, Scientific Reports.

[31]  W. Webb,et al.  Nonlinear magic: multiphoton microscopy in the biosciences , 2003, Nature Biotechnology.

[32]  D. Johns,et al.  Airway inflammation, basement membrane thickening and bronchial hyperresponsiveness in asthma , 2002, Thorax.

[33]  Franz Pfeiffer,et al.  X-ray dark-field radiography facilitates the diagnosis of pulmonary fibrosis in a mouse model , 2017, Scientific Reports.

[34]  B. Wollenberg,et al.  Autofluorescence multiphoton microscopy for visualization of tissue morphology and cellular dynamics in murine and human airways , 2016, Laboratory Investigation.

[35]  Stephan Saalfeld,et al.  Globally optimal stitching of tiled 3D microscopic image acquisitions , 2009, Bioinform..

[36]  Thomas Abraham,et al.  Minimally invasive multiphoton and harmonic generation imaging of extracellular matrix structures in lung airway and related diseases. , 2011, Pulmonary pharmacology & therapeutics.