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Immunology and Infection

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Published: September 18th, 2021

DOI:

10.3791/62436

1Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 2Department of Immunology, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 3Institute of Biological Information Processing (IBI-7: Structural Biochemistry), Forschungszentrum Jülich

We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 µm in size) in the airways of mice. The method also can be used for the analysis of microparticles and nanoparticle agglomerate distribution in the airways in various pathological condition models.

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.

On a daily basis, people inhale airborne pathogens, including spores of opportunistic fungi Aspergillus fumigatus (A. fumigatus conidia) that can penetrate the respiratory tract1. The respiratory tract of mammals is a system of airways of different generations that are characterized by the different structures of the airway walls2,3,4. Tracheobronchial walls consist of several cell types among which are ciliated cells that provide the mucociliary clearance5. In the alveoli, there are no ciliated cells and the p....

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All methods concerning laboratory animals described here have been approved by the Institutional Animal Care and Use Committee (IACUC) at the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences (protocol number 226/2017).

1. A. fumigatus conidia application

  1. To obtain fluorescently labeled A. fumigatus conidia, fix 5 × 108 conidia by adding 1 mL of 3% paraformaldehyde to the conidia pellet. Incubate in a 50 mL te.......

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Following the protocol above, the 3D image showing the airways and A. fumigatus conidia in the lung lobe of a mouse was obtained (Figure 1A). Streptavidin (that was used for airway visualization) labeled bronchi and bronchioles15. Additionally, the large vessels, which are easily distinguishable from the airways by their morphology, and pleura are visualized in the airway channel (Figure 1A-C). The creation of th.......

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Whole-organ 3D imaging permits obtaining of the data without dissection of the specimen, which is of great importance for investigating the spatial aspects of the anatomical distribution of the pathogen in the organism. There are several techniques and modifications of tissue optical clearing that help to overcome the laser light scattering and allow whole-organ imaging15,16,18,19. One of the c.......

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The authors thank Prof. Sven Krappmann (University Hospital Erlangen and FUA Erlangen-Nürnberg, Germany) for providing the Aspergillus fumigatus conidia strain AfS150. The authors thank MIPT Press Office. V.B. acknowledges the Ministry of Science and Higher Education of the Russian Federation (#075-00337-20-03, project FSMG-2020-0003). The work regarding A. fumigatus conidia imaging and quantification was supported by RSF № 19-75-00082. The work regarding airways imaging was supported by RFBR № 20-04-60311.

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Name Company Catalog Number Comments
Alexa Fluor 594 NHS Ester ThermoFisher A20004
Aspergillus fumigatus conidia ATCC 46645 The strain AfS150, a ATCC 46645 derivative
Benzyl alcohol Panreac 141081.1611 98.0-100 %
Benzyl benzoate Acros AC10586-0010 99+%
C57Bl/6 mice Pushchino Animal Breeding Centre (Russia) Male. 12 - 30 week old.
Catheter Venisystems G715-A01 18G
Cell imaging coverglass-bottom chamber Eppendorf 30742028 4 or 8 well chamber with coverglass bottom
Centrifuge Eppendorf 5804R Any centrifuge provided 1000 g can be used
Confocal laser scanning microscope ZEISS ZEISS LSM780
Dimethyl sulfoxide Sigma-Aldrich 276855 ≥99.9%
FIJI image processing package FIJI Free software
Forcep B. Braun Aesculap BD557R Toothed
Forcep B. Braun Aesculap BD321R Fine-tipped
Forcep Bochem 1727 Smooth
Glass bottle DURAN 242101304 With groung-in lid
Graphic Editor Photoshop Adobe Inc Adobe Photoshop CS
GraphPad Software GraphPad Prism 8
Imaris Microscopy Imaging Software Oxford Instruments Free trial is avalable https://imaris.oxinst.com/microscopy-imaging-software-free-trial
Isoflurane Karizoo
NaHCO3 Panreac 141638
Objective ZEISS 420640-9800-000  Plan-Apochromat, 10 × (NA = 0.3)
Paraformaldehyde Sigma-Aldrich 158127
PBS Paneco P060Π
Pipette ProLine 722020 5 to 50 μL
Powdered milk Roth T145.2
Sample mixer Dynal MXIC1
Scissors B. Braun BC257R Blunt
Shaker Apexlab GS-20 50-300 rpm
Skalpel Bochem 12646
Silk thread B. Braun 3 USP
Streptavidin, Alexa Fluor 488 conjugate ThermoFisher S11223
Test tube SPL Lifesciences 50050 50 mL
Tris (hydroxymethyl aminomethane) Helicon H-1702-0.5  Mr 121.14; CAS Number: 77-86-1
Triton X-100 Amresco Am-O694-0.1
ZEN microscope software ZEISS ZEN2012 SP5 https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html

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