Home › Microscopy & Imaging › Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy
Microscopy & Imaging JoVE (Open Access) Citable · DOI

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

DOI: 10.3791/2659-v
What you'll learn
  • ✓ Prepare Aspergillus fumigatus spores for controlled lung infection
  • ✓ Perform intratracheal infection to establish fungal pneumonia in mice
  • ✓ Acquire 2-photon microscopy images of neutrophil dynamics in infected lung tissue
  • ✓ Distinguish phagocytosis from NET-formation in real-time neutrophil responses
Protocol

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

Difficulty
advanced
Total time
~4–6 hours per mouse (including infection, lung preparation, and imaging acquisition)
Model organism
Mouse (strain not specified, likely C57BL/6J or similar)
Biosafety
BSL-2

Steps

1
Prepare Aspergillus fumigatus spores for infection

Culture and harvest A. fumigatus spores, quantify concentration, and prepare inoculum for controlled intratracheal infection. Ensure sterility and viability of spore suspension.

▶ 01:29
2
Perform intratracheal infection of mouse lungs

Anesthetize mouse and instill A. fumigatus spore suspension directly into the trachea to establish reproducible fungal pneumonia.

▶ 02:54
3
Isolate and prepare infected lung tissue

Excise lungs from infected mouse, remove excess tissue, and prepare thin slices or whole-mount specimens suitable for 2-photon microscopy imaging.

▶ 04:05
4
Acquire 2-photon microscopy images of neutrophil responses

Use 2-photon laser microscopy to visualize neutrophil granulocytes in real-time, capturing phagocytosis of pathogens and neutrophil extracellular trap (NET) formation in infected versus control lung tissue.

▶ 06:33
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