Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in
Ivan V Maslov1, Andrey O Bogorodskiy1, Mariia V Pavelchenko1
1Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology.
Abstract:
Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and immunological tolerance, while in immunocompromised patients, conidia can colonize airways and cause severe invasive respiratory disorders. Various cells in different airway compartments are involved in the immune response that prevents fungal invasion; however, the spatio-temporal aspects of pathogen elimination are still not completely understood. Three-dimensional (3D) imaging of optically cleared whole-mount organs, particularly the lungs of experimental mice, permits detection of fluorescently labeled pathogens in the airways at different time points after infection. In the present study, we describe an experimental setup to perform a quantitative analysis of A. fumigatus conidia distribution in the airways. Using fluorescent confocal laser scanning microscopy (CLSM), we traced the location of fluorescently labeled conidia in the bronchial branches and the alveolar compartment 6 hours after oropharyngeal application to mice. The approach described here was previously used for detection of the precise pathogen location and identification of the pathogen-interacting cells at different phases of the immune response. The experimental setup can be used to estimate the kinetics of the pathogen elimination in different pathological conditions.
Insights
This study introduces a novel 3D imaging method to track Aspergillus fumigatus conidia in mouse lungs. The technique quantifies fungal distribution, aiding understanding of immune responses to airborne pathogens.
Area of Science:
- Medical Mycology
- Immunology
- Respiratory Medicine
Background:
- Aspergillus fumigatus conidia are airborne pathogens that cause invasive respiratory diseases, especially in immunocompromised individuals.
- The immune response to fungal invasion involves various airway cells, but the precise timing and location of pathogen elimination remain unclear.
- Understanding fungal pathogen distribution is crucial for developing effective treatments.
Purpose of the Study:
- To develop and validate a quantitative method for analyzing Aspergillus fumigatus conidia distribution in the airways of experimental mice.
- To utilize advanced imaging techniques for precise pathogen localization within the respiratory system.
Main Methods:
- Development of a 3D imaging experimental setup using optically cleared whole-mount mouse lungs.
- Application of fluorescent confocal laser scanning microscopy (CLSM) to trace labeled A. fumigatus conidia.
- Quantitative analysis of conidia distribution in bronchial and alveolar compartments 6 hours post-infection.
Main Results:
- Successfully traced the spatial distribution of fluorescently labeled A. fumigatus conidia in mouse airways.
- Demonstrated the capability to quantify conidia location within bronchial branches and the alveolar compartment.
- Established a methodology for assessing pathogen elimination kinetics.
Conclusions:
- The described 3D imaging approach enables quantitative analysis of airborne fungal pathogen distribution in the respiratory tract.
- This method facilitates the study of immune responses and pathogen clearance in various pathological conditions.
- The technique provides insights into the spatio-temporal dynamics of fungal infections in the lungs.
