Rationale. Influenza is potentially fatal infectious disease that is difficult to both treat and prevent due to the natural range, and unpredictable nature of host immune responses. Studies evaluate immune reaction on quantitative and functional data, but anatomical in only a few cases with histological analysis. Objectives. Could two-photon excitation microscopy (TPEM) improve anatomical description at cell level of pulmonary Influenzavirus infection? Methods. Pulmonary sections from transgenic C57BL/6 mice that had CD11c+ cells expressing yellow fluorescent protein were examined following experimental infection with a red fluorescent protein-expressing Influenza A virus. Measurements. Two-photon lung micrographs were taken on days 1, 2, 3 and 4 post-infections. Flow cytometry analysis and whole-lung cytokine assays were also performed at the same post-infections time points. Results. TPEM showed how the infection resulted in bronchial and bronchiolar epithelial destruction. Initially located in the bronchial and bronchiolar epithelium, virus diffused homogeneously into the alveolar compartment. In response, the host secreted proinflammatory cytokines and rapidly recruited macrophages into the lungs from the perivascular compartment. On day 4 post infection, macrophages were homogeneously distributed throughout the entire pulmonary parenchyma, starting to scavenge cellular debris. Three-dimensional reconstructions of TPEM images provided quantifiable map that traced infection through the lungs. Conclusions. This study demonstrated the added value of using TPEM to map pulmonary viral infections, epithelial lesions, macrophages recruitment and cellular interactions with more precision than histological analysis. Finally, TPEM coupled to cytometry could provide a more precise picture of the cell interactions after a lung infection by influenza virus.