Multicolor two-photon imaging of in vivo cellular pathophysiology upon influenza virus infection using the two-photon IMPRESS

• Published in: Nature protocols Vol. 15; no. 3; pp. 1041 - 1065
• Main Authors: Ueki, Hiroshi, Wang, I-Hsuan, Zhao, Dongming, Gunzer, Matthias, Kawaoka, Yoshihiro
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.03.2020; Nature Publishing Group
• Subjects: 631/1647/245/2186; 631/1647/767/1972; 631/250/2503; 631/326/596/1578; Analytical Chemistry; Animals; Antibodies; Biological Techniques; Biomedical and Life Sciences; Computational Biology/Bioinformatics; Development and progression; Dogs; Fluorescence microscopy; Fluorescent dyes; Fluorescent indicators; Gene Expression Regulation, Viral; Genes, Reporter; Host-virus relationships; Imaging; In vivo methods and tests; Inflammation; Influenza; Influenza A virus - genetics; Life Sciences; Luminescent Proteins - chemistry; Luminescent Proteins - metabolism; Lung - physiopathology; Lung - virology; Lung diseases; Lungs; Madin Darby Canine Kidney Cells; Medical research; Medicine, Experimental; Methods; Mice; Microarrays; Microscopy; Microscopy, Medical; Organic Chemistry; Orthomyxoviridae Infections - physiopathology; Orthomyxoviridae Infections - virology; Pathogenesis; Pathophysiology; Photons; Protocol; Respiratory diseases; Viruses; Japan
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/s41596-019-0275-y

In vivo two-photon imaging is a valuable technique for studies of viral pathogenesis and host responses to infection in vivo. In this protocol, we describe a methodology for analyzing influenza virus–infected lung in vivo by two-photon imaging microscopy. We describe the surgical procedure, how to stabilize the lung, and an approach to analyzing the data. Further, we provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (Color-flu) for multicolor analysis. Setup of this model typically takes ~30 min and enables the observation of influenza virus–infected lungs for >4 h during the acute phase of the inflammation and at least 1 h in the lethal phase. This imaging system, which we termed two-photon IMPRESS (imaging pathophysiology research system), is broadly applicable to analyses of other respiratory pathogens and reveals disease progression at the cellular level in vivo. Infected lung is analyzed by two-photon imaging microscopy and can be observed for >4 h during the acute phase of inflammation and at for least 1 h in the lethal phase.