Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

Area of Science:

• Immunology
• Microscopy
• Respiratory Science

Background:

• In vivo analysis of cell-cell and cell-pathogen interactions is crucial for understanding immune responses during infection.
• Two-photon intravital microscopy (2P-IVM) enables deep-tissue imaging in living animals, minimizing photobleaching.
• Imaging respiratory organs presents challenges due to breathing motion, limiting current 2P-IVM applications.

Purpose of the Study:

• To develop and describe a protocol for visualizing in vivo immune cell interactions within the mouse trachea using 2P-IVM.
• To overcome the limitations of respiratory organ imaging caused by breathing motion.
• To enable detailed analysis of immune cell dynamics during influenza virus infection.

Main Methods:

• Developed a custom imaging platform for surgical exposure and intubation of the mouse trachea.
• Acquired dynamic 4D images of neutrophils and dendritic cells (DCs) in the tracheal mucosal epithelium.
• Detailed influenza intranasal infection and flow cytometric analysis of tracheal immune cells.

Main Results:

• Successfully generated stable and bright 4D images of immune cell interactions in the trachea.
• Enabled visualization of neutrophil and DC motility and interactions during infection.
• Provided a robust method for studying cellular dynamics in the respiratory tract.

Conclusions:

• The described protocol effectively visualizes in vivo immune cell interactions in the mouse trachea using 2P-IVM.
• This method overcomes previous imaging challenges in respiratory organs.
• Facilitates the assessment of cell-cell interactions and immune responses in the trachea during infection.