3d Imaging Of Whole-Mount Optically Cleared Organs As A Tool To Estimate Pathogen Distribution In The Lungs

Daily, people inhale corpuscular pathogens of different origin and sizes. Among them are 3 um conidia of Aspergillus fumigatus fungi. Precise location of the pathogen in the airways can be helpful in indicating the potential cells that provide sufficient antifungal defense.The aim of the study was to build a 3D model of the mouse lung using fluorescence confocal laser scanning microscopy. Using the model we traced the location of A. fumigatus conidia in the different airway generations of immunocompetent mice and mice with induced immunosuppression. A. fumigatus conidia were labeled with Alexa594-NHS and applied to mice via the oropharyngeal route. At different time points mouse lungs were fixed, airways were labeled with streptavidin-Alexa488. Specimens were delipidated and optically cleared as whole-mounts. Specimens were subjected to confocal microscopy.Based on the fluorescence signal, the binary mask for the airways in the entire lung was built. We identified A. fumigatus conidia inside of bronchial branches and outside of them – in the alveolar space. At 6 hours after A. fumigatus conidia application, the percentage of conidia in the alveolar space of mice with immunosuppression was significantly decreased compared to immunocompetent mice, while in the bronchial branches the percentage of conidia was elevated in mice with immunosuppression. Thus using 3D imaging of whole-mount optically cleared lungs we showed the difference in A. fumigatus conidia distribution in bronchial branches and alveolar space in mice with induced immunosuppression compared to immunocompetent mice. The work was supported by RSF No 19-75-00082.