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

Abstract

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.

Fig. 1. The laser path adjustment system.

a, Schematic image of the system for correcting the laser beam path. b, Layout of active mirrors to adjust the laser path. c, The window through which the laser connecting the inside and outside of the BSL3 facility passes. d, Arrangement of the two-photon excitation microscope inside the BSL3 facility and the laser unit outside the BSL3 facility. Some images provided courtesy of Coherent and Zeiss.

Fig. 2. The in vivo lung imaging system for virus-infected mouse.

a, Schematic image of the imaging system for virus-infected lungs. b, Placement of life support devices and lung stabilizer devices. c, Surgical stage. d, Anesthesia machine and mechanical ventilator. e–g, The operator wearing a Tyvek suit and a positive-pressure mask. All our animal care and experiments conformed to the guidelines for animal experiments of the University of Tokyo and were approved by the animal research committee of the University of Tokyo (PA17-31 and PA17-17). Some images in a provided courtesy of Zeiss.

Fig. 3. Devices to stabilize lungs.

a, Surgical tools. b, Thoracic suction window. c, Setup of thoracic suction window and the holding devices. d, Device layout pertaining to lung stabilization.

Fig. 4. Surgical procedure for lung imaging.

a, Place the mouse on its back and tape with adhesive tape. b, Cut the skin beneath the chin and expose the trachea. c, Insert a tracheal cannula. d, Place the mouse in the right lateral decubitus position. e, Make an incision in the skin at the left axilla. f, Expose the left lung lobe and keep it exposed by using retractors. g, Lower the thoracic suction window gently to immobilize the lungs of the mouse. h, Close-up of the thoracic suction window. i, Lower the objective lens to the thoracic suction window. All our animal care and experiments conformed to the guidelines for animal experiments of the University of Tokyo and were approved by the animal research committee of the University of Tokyo (PA17-31 and PA17-17).

Fig. 5. In vivo multicolor imaging of influenza virus–infected lungs.

a, Catchup^IVM-red mice were intranasally infected with 10⁵ PFU of MA-Venus-H5N1 or MA-Venus-PR8 virus and observed at 4 d post-infection. Fluorescent dextran (blue) was intravenously administered to visualize the lung architecture. Red and green indicate neutrophils and virus-infected cells, respectively. b, Ly6g^Cre/+;R26^mTFP1/+ mice were intranasally infected with 10⁵ PFU of MA-Venus-PR8 virus and observed at 7 d post-infection. PE-conjugated anti-mouse Ly-6G antibody (red) and fluorescent dextran (white) were intravenously administered to visualize the vascular neutrophils and lung architectures, respectively. Green indicates virus-infected cells. Blue indicates both infiltrating (arrowheads) and vascular neutrophils (arrow). c, Ly6g^Cre/+;R26^tdTomato/+;Cx3cr1^GFP/+ mice were intranasally infected with 10⁵ PFU of MA-Venus-PR8 virus and observed at 5 d post-infection. Fluorescent dextran (white) was intravenously administered to visualize the lung architecture. Red, green, and blue indicate neutrophils, monocytes, and virus-infected cells, respectively. The yellow arrowhead and arrow indicate a neutrophil and a monocyte, respectively, in contact. AB, antibody.

Fig. 6. Co-infection imaging of influenza virus–infected lungs.

B6 mice were intranasally infected with 10⁵ PFU of MA-Venus-H5N1 and MA-Cerulean-H5N1, or MA-Venus-PR8 and MA-Cerulean-PR8 viruses and observed at 3 d (H5N1) or 4 d (PR8) post-infection. Fluorescent dextran (white) was intravenously administered to visualize the lung architecture. Red and green indicate MA-Cerulean-virus-infected cells (yellow arrows) and MA-Venus-virus-infected cells (white arrows), respectively. The yellow arrowheads indicate cells co-infected with MA-Cerulean-virus and MA-Venus-virus. Scale bar, 50 µm.