Detection of fungal spores and airway-associated large vessels using two-photon microscopy

Two-photon microscopy allows the imaging of biological objects at a wide scale of resolution, from cells to organs, both with fluorescent labels and label-free. We applied this method to investigate the pathological conditions under which spores of A. fumigatus, an opportunistic fungus, can penetrate blood vessels. In the present study, using second harmonic generations in the optically cleared mouse lungs, we visualized airways and lung vessels. We developed an approach of image processing that allows us to distinguish the images of vessels and airways. Using the approach previously developed by us to investigate the distribution of fungal spores in the airways, we estimated the number of in advance fluorescently labeled spores of A. fumigatus in the lung vessels of immunocompetent mice 48 hours after the application. The study demonstrates that using two-photon microscopy for fluorescently labeled specimens in combination with second harmonic generation can be used for imaging microscale objects such as cells or pathogens in the lung vessels.