Name: confocal laser scanning microscopy-based quantitative analysis of aspergillus fumigatus conidia distribution in whole-mount optically cleared mouse lung
Uploaded: 2021-09-18T13:00:00
Description: Watch this Scientific Journal Video about Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung at Jo

The authors thank Prof. Sven Krappmann (University Hospital Erlangen and FUA Erlangen-N&#252;rnberg, 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 &#8470; 19-75-00082. The work regarding airways imaging was supported by RFBR &#8470; 20-04-60311.

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
<ol>
	<li>To obtain fluorescently labeled A. fumigatus conidia, fix 5 &#215; 108 conidia by adding 1 mL of 3% paraformaldehyde to the conidia pellet. Incubate in a 50 mL te...

<ol>
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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...

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 penetrating alveolar space pathogens cannot be eliminated by the mucociliary clearance6. Moreover, each airway generation is a niche for multiple immune cell populations and subsets of these populations are unique for certain airway compartments. Thus, alveolar macrophages reside in the alveolar compartments, while both the trachea and conducting airways are lined with the intraepithelial dendritic cells7,8.
The approximate size of A. fumigatus conidia is 2-3.5 &#181;m9. Since the diameter of small airways in humans and even in mice exceeds 3.5 &#181;m, it was suggested that conidia can penetrate the alveolar space2,10,11. In fact, histological examination showed the fungal growth in the alveoli of the patients suffering from aspergillosis12. Conidia were also detected in the alveoli of infected mice using live imaging of the thick lung slices13. Simultaneously, conidia were detected in the luminal side of the bronchial epithelium of mice14.
Three-dimensional (3D) imaging of the optically cleared whole-mount mouse lungs permits morphometric analysis of the airways15. Particularly, the quantitative analysis of the visceral pleural nerve distribution was performed using optically cleared mouse lung specimens15. Recently, Amich et al.16 investigated the fungal growth after intranasal application of conidia to the immunocompromised mice using a light-sheet fluorescence microscopy of optically cleared mouse lung specimens. The precise location of the resting conidia in the airways at different time points after the infection is important for identifying the cell populations that can provide sufficient antifungal defense in certain phases of inflammation. However, due to the relatively small size, the spatio-temporal aspects of A. fumigatus conidia distribution in the airways are poorly characterized.
Here, we present an experimental setup for the quantitative analysis of A. fumigatus conidia distribution in the airways of infected mice. Using fluorescent confocal laser scanning microscopy (CLSM) of optically cleared lungs of mice that received an oropharyngeal application of the fluorescently labeled A. fumigatus conidia, we obtain 3D images and perform the image processing. Using 3D imaging of the whole-mount lung lobe, we have previously shown the distribution of A. fumigatus conidia in the conducting airway of mice 72 hours after conidia application8.

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	<tbody>
		<tr>
			<td>Alexa Fluor 594 NHS Ester</td>
			<td>ThermoFisher</td>
			<td>A20004</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspergillus fumigatus conidia</td>
			<td>ATCC</td>
			<td>46645</td>
			<td>The strain AfS150, a ATCC 46645 derivative</td>
		</tr>
		<tr>
			<td>Benzyl alcohol</td>
			<td>Panreac</td>
			<td>141081.1611</td>
			<td>98.0-100 %</td>
		</tr>
		<tr>
			<td>Benzyl benzoate</td>
			<td>Acros</td>
			<td>AC10586-0010</td>
			<td>99+%</td>
		</tr>
		<tr>
			<td>C57Bl/6 mice</td>
			<td>Pushchino Animal Breeding Centre (Russia)</td>
			<td></td>
			<td>Male. 12 - 30 week old.</td>
		</tr>
		<tr>
			<td>Catheter</td>
			<td>Venisystems</td>
			<td>G715-A01</td>
			<td>18G</td>
		</tr>
		<tr>
			<td>Cell imaging coverglass-bottom chamber</td>
			<td>Eppendorf</td>
			<td>30742028</td>
			<td>4 or 8 well chamber with coverglass bottom</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5804R</td>
			<td>Any centrifuge provided 1000 g can be used</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>ZEISS</td>
			<td>ZEISS LSM780</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td>&#8805;99.9%</td>
		</tr>
		<tr>
			<td>FIJI image processing package</td>
			<td>FIJI</td>
			<td></td>
			<td>Free software</td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>B. Braun Aesculap</td>
			<td>BD557R</td>
			<td>Toothed</td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>B. Braun Aesculap</td>
			<td>BD321R</td>
			<td>Fine-tipped</td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Bochem</td>
			<td>1727</td>
			<td>Smooth</td>
		</tr>
		<tr>
			<td>Glass bottle</td>
			<td>DURAN</td>
			<td>242101304</td>
			<td>With groung-in lid</td>
		</tr>
		<tr>
			<td>Graphic Editor Photoshop</td>
			<td>Adobe Inc</td>
			<td></td>
			<td>Adobe Photoshop CS</td>
		</tr>
		<tr>
			<td>GraphPad Software</td>
			<td>GraphPad</td>
			<td></td>
			<td>Prism 8</td>
		</tr>
		<tr>
			<td>Imaris Microscopy Imaging Software</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td>Free trial is avalable https://imaris.oxinst.com/microscopy-imaging-software-free-trial</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Karizoo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Panreac</td>
			<td>141638</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>ZEISS</td>
			<td>420640-9800-000</td>
			<td>&#160;Plan-Apochromat, 10 &#215; (NA = 0.3)</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Paneco</td>
			<td>P060&#928;</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>ProLine</td>
			<td>722020</td>
			<td>5 to 50 &#956;L</td>
		</tr>
		<tr>
			<td>Powdered milk</td>
			<td>Roth</td>
			<td>T145.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample mixer</td>
			<td>Dynal</td>
			<td>MXIC1</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>B. Braun</td>
			<td>BC257R</td>
			<td>Blunt</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Apexlab</td>
			<td>GS-20</td>
			<td>50-300 rpm</td>
		</tr>
		<tr>
			<td>Skalpel</td>
			<td>Bochem</td>
			<td>12646</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk thread</td>
			<td>B. Braun</td>
			<td></td>
			<td>3 USP</td>
		</tr>
		<tr>
			<td>Streptavidin, Alexa Fluor 488 conjugate</td>
			<td>ThermoFisher</td>
			<td>S11223</td>
			<td></td>
		</tr>
		<tr>
			<td>Test tube</td>
			<td>SPL Lifesciences</td>
			<td>50050</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Tris (hydroxymethyl aminomethane)</td>
			<td>Helicon</td>
			<td>H-1702-0.5</td>
			<td>&#160;Mr 121.14; CAS Number: 77-86-1</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Amresco</td>
			<td>Am-O694-0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN microscope software</td>
			<td>ZEISS</td>
			<td>ZEN2012 SP5</td>
			<td>https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html</td>
		</tr>
	</tbody>
</table>

confocal laser scanning microscopy-based quantitative analysis of aspergillus fumigatus conidia distribution in whole-mount optically cleared mouse lung

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&#160;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.

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...

Watch this Scientific Journal Video about Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung at Jo

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

We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 &#181;m 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.

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

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and ...

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