The spatial distribution of 100 nanometer-sized respiratory pathogens (such as virus particles) in the lungs at different phases of inflammation is an important but challenging research question. We developed a high-resolution CLSM-based technique for imaging of 100 nanometer-sized FluoSpheres in the optically cleared whole murine lungs and conducting airway specimens. Airways in the whole-lung specimens were fluorescently labeled with Streptavidin-Alexa488 (Previously published in: Bogorodskiy AO et al. (2020) Front Immunol 11:298.). Ce3D protocol (Previously published in: Li W et al. (2019) Nat Protoc 14, 1708–1733.) was used for optical clearing the lung and to prevent FluoSpheres degradation. For visualization of large blood vessels in the whole lung specimens we used second-harmonic generation (SHG) imaging. FluoSpheres were detected in the murine lungs up to 72 hours after application in both bronchial branches and the alveolar space. FluoSpheres were detected either as free particles or particle agglomerates 4 hours after application. After 24 hours, particles were mostly internalized by immune cells and were detected as agglomerates both in the luminal side of the airway epithelium and in the lung parenchyma. At all investigated time-points FluoSpheres were not detected in the large blood vessels. Using high-resolution imaging of conducting airways we identified CD169-positive macrophages and CD11c-positive dendritic cells contributing to FluoSpheres internalization. Our technique can be useful for investigations of the mechanisms of immune cell-virus particle interactions in the respiratory tract.