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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 ...

Our approach allows us to perform the quantitative analysis of a Aspergillus fumigatus Conidia distribution in whole-mount optically cleared mouse lung. Aspergillus fumigatus Conidia can penetrate the respiratory tract and cause life-threatening diseases in immunocompromised patients. The respiratory tract of mammalians is a system of airways of different generations.Each generation is characterized by the different structures of airway walls and immune cell populations. In the bronchial branches, aspergillus fumigatus Conidia be eliminated by mucociliary clearance. However, in available space, the mucociliary clearance doesn't work and Conidia must be cleared by the immune cells, such as alveolar macrophages and neutrophils.As is the spatial temporal aspects of Conidia distribution in the airways are important. However, not properly investigated question in understanding of the disease mechanisms. Here we present an experimental setup for the quantitative analysis of Aspergillus fumigatus Conidia distribution in the airways of infected mice.I apply 50 microliters of labeled Aspergillus fumigatus Conidia to mouse. Six hours later, harvest the lung. Fix overnight in 2%formaldehyde and stain with labeled Streptavidin conjugate.Then subject the specimen to the optical clearing. Place the specimen in the glass bottle filled with 50%methanol water solution and place it onto the sample mixer at room temperature for one hour. Replace 50%methanol with 100%methanol and put it under the sample mixer for two hours.Prepare a mixture consisting of one part benzyl alcohol, and two parts of benzyl-benzoate. Transfer the specimen to it 24 well plate and cover it with the benzyl-benzoate mixture for at least 30 minutes. The sample is ready for imaging.Lay the sample on the sample holder. And place the holder in the microscope. After turning the microscope system on and opening the software, Turn the transmission light on and select an X objective.Visually locate your sample in the transmission light and proceed to the acquisition tab. Select confocal laser microscopy Lambda mode. Switch the appropriate lasers on.Set the spectral range of the detector and select the dichroic mirror. Adjust the detector gain and narrow the pinhole to one area unit. Set pixel resolution to 512 by 512.Switch on the zed-stack mode and start live imaging. Find the focal plane where both dice are visible. Expand the zed-stack panel.Using the focusing wheel, find and select the lowest and the highest planes of the sample. Position the focal plane next to the bottom of the sample. Turn the zed-stack mode off and dial scan mode on.If needed, adjust X, Y position and the number of tiles. Turn zed-stack and dial scan modes on. Set zed-sectioning step to five microns.Set the scanning speed. Start the experiment. Imaging typically takes several hours dependent on the land size and the scanning speed.The obtained image is then spectrally unmixed. For spectral unmixing, use linear unmixing option. Select the regions corresponding to the land areas labeled with streptavidin and Conidia.Start unmixing. After unmixing save the unmixed file. To stich tiles from the obtained image, open the image.Select methods, geometric stitching. Select the image in the input window. In stitching options, select new output and fuse tiles option.Use reference mode with selected channel corresponding to the airway fluorescence. Open the image in a surplus 3D view. If needed, change colors used for their presentation.Here we show airways channel in gray shades and Conidia channel in purple. To create an airway mask, use add new surface tool. Select airway channel and choose the smoothing parameter of 10 microns.Various filters can be used to exclude out of airway signal. First, visually inspect the surface and set the intensity threshold. Additional filters, like surface area can also be applied to exclusively select airways, but not pleura or blood vessels.Next, manually delete the selected surface fragment that do not belong to the airways. Now you can observe the created surface and investigate the missing parts to correct them later on. Create a mask for the airway surface using options edit and mask all.Select the airway channel and set work cells outside the surface to 0.001. Here we showed the resulting mask in orange. Save Conidia channel and the airway mask in two separate folders, as T of F series.Open the file with the airway mask image. Make the image binary. Follow process binary, make binary.To resemble the mask thickness, apply several dilate 3D functions. Follow plugins, process, dilate 3D. You can also use macros functionality to do this.After several cycles of dilate process, use the fill holes function. Follow process, binary, fill holes. To fill the residual holes in the mask manually, open the region of interest manager.follow analyze, tools, ROI manager. Using polygon selection tool select an area that should be filled at one particular projection. Do this several times for few projections in zed-stack so that you can interpolate shapes of the regions of interest.To interpolate the select shapes, select all of them and press more, interpolate ROIs. Then fill all the ROIs with white. Use the fill holes procedure once again.To reassemble the mask thickness back after filling the holes, apply the erode 3D function. Follow plugins, process, erode 3D. You can also use micros for this.The number of iterations in erode 3D and LA 3D should be equal. Launch the Conidia count application. Press the add files button and select the folders with DIFF files with the prepared airway mask and the Conidia fluorescence images.Set a custom threshold between zero and one. Press OK and see the results. Using this approach, we perform the quantitative analysis of the Conidia inside and outside the bronchial tree of mice six hours after Conidia application.The data suggests that upon our original application, the majority of Conidia penetrate the alveola space and allocated there at the beginning of the inflammatory immune response. Three-dimensional imaging of the mouse lungs with confocal laser scanning microscopy allows identification of Aspergillus fumigatus Conidia in the airways. Processing of three-dimensional images using this protocol permits to perform quantitative analysis of Conidia distribution inside and outside the bronchial branches.Our approach can also be utilized to estimate the kinetics of the Conidia elimination from the aways of mice and to compare anatomical Conidia distribution in the immunocompetent and immunocompromised mice. Additionally, using this approach, one can analyze the distribution of the microparticles or nano particles agglomerates in the airways.

Research

JoVE Journal

Immunology and Infection

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...