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 test tube for 2 h on a shaker at room temperature.</li>
	<li>Wash conidia with 20 mL of phosphate buffer saline (PBS): centrifuge at 1,000 x g for 15 min, gently remove the supernatant, and add&#160;fresh PBS in a volume of 20 mL. Repeat.</li>
	<li>Dissolve the conidia in 900 &#181;L of 0.1 M NaHCO3.</li>
	<li>Dissolve the succinimidyl ester of the 594 nm fluorescent dye in 100 &#181;L of dimethyl sulfoxide and add to conidia.</li>
	<li>Incubate the conidia with the dye for 1 h on a shaker at 150 rpm at room temperature.</li>
	<li>Wash the conidia twice with 20 mL of PBS in the centrifuge at 1,000 x g for 15 min.</li>
	<li>Dilute the conidia to a concentration of 1 &#215; 108 conidia/mL in PBS and store at 4 &#176;C.</li>
	<li>Anesthetize the mouse with 0.5-3% isoflurane vapor. Put the mouse on the holder, fix the tongue with smooth forceps, and hold the nares. Take a single channel pipette and apply 50 &#181;L of conidia suspension to the mouse pharynx. Wait until the suspension is inhaled.</li>
</ol>
2. Specimen preparation
<ol>
	<li>Prepare the 50 mL test tube and fill it with 15 mL of fresh 2% paraformaldehyde. Place the medical instruments (15 cm toothed dissecting forceps, 8 cm fine-tipped forceps, and 10 cm scissors with blunt ends) into 70% ethanol solution.</li>
	<li>Euthanize the mouse in accordance with the IACUC protocol. Then place the mouse in the dorsal position and fix the mouse paws with needles. 
	NOTE: If using cervical dislocation for euthanasia, ensure the integrity of the trachea.
	<ol>
		<li>Treat the mouse with 70% ethanol using a sprayer.</li>
		<li>Make a median longitudinal cut in the ventral skin from the hind paws to the forepaws and the chin.</li>
		<li>Separate the skin from the subcutaneous tissue using toothed forceps and closed scissors. Fix the upper ends of the skin with needles.</li>
		<li>Make an incision in the abdominal wall and separate the liver from the diaphragm. Carefully pick the diaphragm with closed scissors and then cut the diaphragm.</li>
		<li>Make a medial cut of the thorax and neck connective tissues until the trachea is visible.</li>
	</ol>
	</li>
	<li>Run a silk thread beneath the trachea and make a surgical knot using two forceps. 
	NOTE: Alternatively, use dental floss instead of silk thread.
	<ol>
		<li>Carefully pull the thread and cut off the lungs from the connective tissue with scissors. Hold the scissors perpendicular to the table.</li>
		<li>Put the lungs in the 50 mL test tube with 2% paraformaldehyde. Leave thread ends outside the tube, put the cover tight, and turn over the tube to cover the lungs with paraformaldehyde. Hold the lungs overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Dissect the lung lobes from the heart and each other with a scalpel.
	<ol>
		<li>Put the lung lobes in a 24 well plate, with each lobe in a separate well. Wash the lung lobes in 1 mL of Tris-buffered saline (TBS) pH 7.4, 5 times for 1 h each on a shaker at 150 rpm.</li>
		<li>Replace 1 mL of TBS with 1 mL of the blocking buffer (1% Triton X 100, 5% powdered milk in TBS) and leave the specimen overnight at room temperature on a shaker.</li>
		<li>Replace the blocking buffer with 1 mL of streptavidin-488- nm fluorchrome conjugate diluted 1:30 in TBS. Leave the specimen for at least 72 h at room temperature on a shaker (150 rpm).</li>
	</ol>
	</li>
	<li>Wash the specimen 5 times for 1 h each in 1 mL of TBS at room temperature on a shaker (150 rpm). Transfer the specimen to the new wells and cover it with 2% paraformaldehyde overnight at 4 &#176;C for post-fixation.</li>
</ol>
3. Mouse lung lobe optical clearing
<ol>
	<li>Place the specimen in the 5 mL glass bottle filled with 3 mL of 50% methanol-water solution and put it on to the sample mixer at room temperature for 1 h.</li>
	<li>Replace 3 mL of 50% methanol with 3 mL of 100% methanol and put it on to the sample mixer for 2 h. Prepare a 1:2 v/v mixture of benzyl alcohol and benzyl benzoate (BABB) in a total volume of 1 mL.</li>
	<li>Transfer the specimen to a 24 well plate and cover with 1 mL of BABB mixture for at least 30 min. Do not leave the specimen in BABB for a long time; BABB can damage the plastic plate and make the specimen too rigid.</li>
	<li>Put the specimen in the cell imaging coverglass-bottom chamber. The sample is ready for imaging.</li>
</ol>
4. Mouse lung lobe imaging with CLSM
<ol>
	<li>Turn the microscope system on. Open the microscope software. Turn the transmission light on in the Locate tab. Select the 10x objective. 
	NOTE: For more detailed analysis, use the 20x objective, but it increases the time of the experiment and the image file size.</li>
	<li>Put the chamber with the specimen in the cover slide holder. Put the holder on the XY stage over the objective. Center the specimen using the XY controls of the stage on top of the objective. Use the transmission light and eyepiece to manually find the specimen Z-plane.</li>
	<li>Switch the software to the Acquisition tab. Select CLSM &#955;-mode. Turn the 488 nm and 561 nm lasers on. Select the 488/561 nm dichroic mirror. Change the spectral range of the detector to 490-695 nm. Adjust the laser power to the appropriate range (10-50 &#181;W). Adjust the Detector Gain between 750-900 range. 
	NOTE: Higher gain settings are undesirable due to noise.</li>
	<li>Narrow the pinhole to 1 Airy unit by clicking 1 AU button. Set pixel resolution to 512 &#215; 512 pixels.</li>
	<li>Switch on the Z-stack mode. Start Live Imaging. Select the focal plane in which both dyes are visible. 
	NOTE: If necessary, adjust the laser power to normalize the brightness of the two fluorescent dyes. Use Gallery and Single-channel mode, to avoid clipping and to precisely match fluorescence intensity.</li>
	<li>Expand the appeared Z-stack pane. Use the focusing wheel and find the lowest plane of the sample. Use the First button in the Z-stack pane. Move the focal plane upwards to find the top boundary of the specimen and save the position by using the Last button. Check the representation of the sample depth in the Z-stack pane. 
	NOTE: A representation of the sample depth will appear in the Z-stack pane after the First and the Last positions are chosen.</li>
	<li>Position the objective using the focusing wheel near the bottom of the specimen, by looking at the Z-stack pane. This is approximately 20 &#181;m from the bottom of the specimen.</li>
	<li>Turn the Z-stack mode off and turn the Tile Scan mode on. Acquire the image with an appropriate number of tiles for the size of the specimen. 5 &#215; 5 tiles are a good starting point.</li>
	<li>Adjust the number of tiles and the specimen XY position until the whole lung fits inside the tiled image. Recheck the correctness of the Z-stack positions with the newly obtained XY position of the center of the sample.</li>
	<li>Turn both Z-stack and Tile Scan modes on. Set Z step (in Z-stack pane) to 5 &#181;m. Set Scanning Speed to 6. Turn the Autosave function on. Turn the option Saving Separate Tiles on. Name the file. Press the Start Experiment button.</li>
	<li>Verify that the experiment does not exceed the allocated time on the microscope; if so - adjust the speed. 
	&#8203;NOTE: The approximate file size is more than 10 Gb; be sure that there is enough space on the hard drive.</li>
</ol>
5. Spectral unmixing and stitching
<ol>
	<li>Use the software for the initial image processing. For spectral unmixing, select the Unmixing option. Select two regions corresponding to the streptavidin/airways and conidia to acquire the spectra from the image. Click Start Unmixing. 
	&#8203;NOTE: Alternatively, use the existing spectra for 488 and 594 nm fluorochromes.</li>
	<li>Open the file for the image processing and select the Processing tab. In the Methods section, select Geometric and Stitching. In the Parameter section, select the New Output and mark the Fuse Tiles option. Use the reference mode with the selected channel corresponding to the airway fluorescence. Apply the Stitching by clicking Apply.</li>
</ol>
6. Image processing: surface rendering
<ol>
	<li>Open the image as a Surpass 3D View. Create an airway surface using the option Surface for the channel that was used for the airway visualization. Choose the Smoothing parameter of 10 &#181;m. 
	NOTE: Choose the automatic threshold value as well.</li>
	<li>Visually inspect the surface. Choose the thresholds to reduce the outlying signal. Remove the surfaces of pleura and vessels.</li>
	<li>Create a mask for the airway surface using options Edit and Mask All. Select the airway channel and set Voxels outside the surface to 0.001.</li>
	<li>Save the file with the airway mask as a TIFF series to the folder. Save the file with the conidia channel as a TIFF series to the separate folder.</li>
</ol>
7. Image processing: mask correction
<ol>
	<li>Open the file with the airway mask in an open-source platform for biological-image analysis17 as an 8-bit image. Make the image binary by clicking Process | Binary | Make Binary. 
	NOTE: If necessary, correct the mask: remove the excessive surfaces using the Polygon selection and Delete tab. Alternatively, use the Flood Fill Tool.</li>
	<li>Draw the missing surfaces using several times Dilate (3D): click Plugins | Process | Dilate (3D). Fill the holes by clicking Process | Binary | Fill Holes. Use selection and ROI manager interpolation to fill the residual holes in the mask manually. Apply several Erode (3D) options (Plugins | Process | Erode (3D)) to resample the mask thickness. 
	NOTE: Create macros by using Plugins | Macros options for multiple Dilate (3D) and Erode (3D) repeats. 
	Ensure the number of Erode (3D) is equal to the number of Dilate (3D) applications.</li>
	<li>Save the mask in a new folder as a TIFF series.</li>
</ol>
8. Conidia quantitative analysis
<ol>
	<li>Open the app in the programming and numeric computing platform: https://www.mathworks.com/matlabcentral/fileexchange/84525-conidia_counter.</li>
	<li>Press the Add files button. Select the airway mask folder and the conidia folder.</li>
	<li>Set a custom threshold between 0 and 1. Press the OK button.</li>
	<li>Save the output table to the custom .xls file.</li>
	<li>Analyze the data with software for statistical analysis.</li>
</ol>

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The authors report no conflicts of interest in this work.

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><tbody><tr><td>Alexa Fluor 594 NHS Ester</td><td>ThermoFisher</td><td>A20004</td><td></td></tr><tr><td>&lt;em&gt;Aspergillus fumigatus&lt;/em&gt; 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>&amp;ge;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 ground-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 available 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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Panreac</td><td>141638</td><td></td></tr><tr><td>Objective</td><td>ZEISS</td><td>420640-9800-000</td><td>&amp;nbsp;Plan-Apochromat, 10 &amp;times; (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&amp;Pi;</td><td></td></tr><tr><td>Pipette</td><td>ProLine</td><td>722020</td><td>5 to 50 &amp;mu;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>&amp;nbsp;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 ...

confocal-laser-scanning-microscopy-based-quantitative-analysis

Research

JoVE Journal

Immunology and Infection

3.3K Views.  Moscow Institute of Physics and Technology. Onze aanpak stelt ons in staat om de kwantitatieve analyse uit te voeren van een Aspergillus fumigatus Conidia-verdeling in de optisch ontruimde muislong in zijn geheel. Aspergillus fumigatus Conidia kan de luchtwegen binnendringen en levensbedreigende ziekten veroorzaken bij immuungecompromitteerde patiënten. De luchtwegen van zoogdieren is een systeem van luchtwegen van verschillende generaties.Elke generatie wordt gekenmerkt door de verschillende structuren van luchtwegwanden en im...

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

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

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 respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

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 ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

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 investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

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

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

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

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

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

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

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

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

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 cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

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

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

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

Preserving Fungal Spore Localization in Lungs for Fluorescent Microscopy Analysis

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and bronchioles, branch until terminating in the alveolar airspace where gas exchange occurs. Infections originating in the bronchioles or alveoli elicit distinct host responses and disease manifestations. Therefore, understanding precisely where spores naturally localize in the lungs, particularly soon after infection, expands opportunities for investigation of host-pathogen interactions. Herein, we detail an in-situ analysis of lungs from mice infected with Coccidioides posadasii cts2/ard1/cts3&#916; arthroconidia. Conventional methods for histological preservation involve liquid inflation of the airways with a fixative solution, which displaces the natural location of aspirated fungal particles, pushing spores from proximal bronchioles to terminal airspaces.
Conversely, this method of air-inflation with blood vasculature perfusion-fixation preserves the physiologic position of fungal spores within the bronchioles. Moreover, we describe a simple approach to cryopreserving, embedding, and imaging lung specimens. We also share high-throughput computational techniques via the open-source QuPath program to analyze the spatial distribution of fungal spores within the lung. The method presented here is simple and quick, requires minimal equipment to perform, and can be easily adapted for use with many respiratory fungal infection models.

We present a method for a fungal pathogenesis model that preserves the natural positioning of fungal spores in the lung airways for analysis via fluorescent microscopy.

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and ...

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically estimated on solidified media by comparing colony size. However, to quantitatively measure the growth rate of genetically different fungal strains or strains under different environmental/growth conditions (pH, temperature, carbon and nitrogen sources, antibiotics, etc.) is challenging. Thus, the pursuit of complementary approaches to quantify growth kinetics becomes mandatory in order to better understand fungal cell growth. Furthermore, it is well-known that filamentous fungi, including Aspergillus spp., have distinct modes of growth and differentiation under sub-aerial conditions on solid media or submerged cultures. Here, we detail a quantitative microscopic method for analyzing growth kinetics of&#160;the model fungus Aspergillus nidulans, using live imaging in both submerged cultures and solid media. We capture images, analyze, and quantify growth rates of different fungal strains in a reproducible and reliable manner using an open source, free software for bio-images (e.g., Fiji), in a way that does not require any prior image analysis expertise from the user.

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

We present a label-free live imaging protocol using transmitted light microscopy techniques to capture images, analyze and quantify growth kinetics of the filamentous fungus A. nidulans in both submerged cultures and solid media. This protocol can be used in conjunction with fluorescence microscopy.

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically ...

Biology

Confocal Fluorescence Microscopy: A Technique to Determine the Localization of Proteins in Mouse Fibroblasts

Source: Dominique R. Bollino1, Eric A. Legenzov2, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201 
2 Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Confocal fluorescence microscopy is an imaging technique that enables increased optical resolution as compared to conventional &#34;wide-field&#34; epifluorescence microscopy. Confocal microscopes are able to achieve improved x-y optical resolution through &#34;laser scanning&#34;- typically a set of voltage-controlled mirrors (galvanometer or &#34;galvo&#34; mirrors) that direct laser-illumination to each pixel of the specimen at a time. More importantly, confocal microscopes achieve superior z-axial resolution by using a pinhole to remove out of focus light originating from locations that are not in the z-plane being scanned, thus enabling the detector to collect data from a specified z-plane. Because of the high z-resolution achievable in confocal microscopy, it is possible to collect images from a series of z-planes (also-called z-stack) and construct a 3D image through software.
Before discussing the mechanism of a confocal microscope, it is important to consider how a sample interacts with light. Light is composed of photons, packets of electromagnetic energy. A photon impinging on a biological sample can interact with the molecules comprising the sample in one of four ways: 1) the photon does not interact and passes through the sample; 2) the photon is reflected/scattered; 3) the photon is absorbed by a molecule and the absorbed energy is released as heat through processes collectively known as nonradiative decay; and 4) the photon is absorbed and the energy is then rapidly reemitted as a secondary photon through the process known as fluorescence. A molecule whose structure permits fluorescence emission is called a fluorophore. Most biological samples contain negligible endogenous fluorophores; therefore exogenous fluorophores must be used to highlight features of interest in the sample. During fluorescence microscopy, the sample is illuminated with light of the appropriate wavelength for absorption by the fluorophore. Upon absorbing a photon, a fluorophore is said to be &#34;excited&#34; and the process of absorption is referred to as &#34;excitation&#34;. When a fluorophore gives up energy in the form of a photon, the process is known as &#34;emission&#34;, and the emitted photon is called fluorescence.
The light beam used to excite a fluorophore is focused by the objective lens of a microscope and converges at a &#34;focal point&#34; where it is maximally focused. Beyond the focal point the light again diverges. The entering and exiting beams may be visualized as a pair of cones touching at the focal point (see Figure 1, left panel). The phenomenon of diffraction imposes a limit on how tightly a beam of light can be focused - the beam actually focuses to a spot of finite size. Two factors determine the size of the focal spot: 1) the wavelength of the light, and 2) the light-gathering ability of the objective lens, which is characterized by its numerical aperture (NA). The focal &#34;spot&#34; extends not only in the x-y plane, but also in the z direction, and is in reality a focal volume. The dimensions of this focal volume define the maximum resolution achievable by optical imaging. Although the number of photons is greatest within the focal volume, the conical light paths above and below the focus also contain a lower density of photons. Any fluorophore in the light path can thus be excited. In conventional (wide-field) epifluorescence microscopy, emission from fluorophores above and below the focal plane contribute out-of-focus fluorescence (a &#34;hazy background&#34;), which reduces image resolution and contrast, as demonstrated in Figure 1, with the red cube representing fluorophore emission above the focal plane (red star) that results in out-of-focus fluorescence (top right). This problem is ameliorated in confocal microscopy, due to the utilization of a pinhole. (Figure 2, bottom right). As depicted in Figure 3, the pinhole allows emissions originating from the focal place to reach the detector (left), while blocking the out-of-focus fluorescence (right) from reaching the detector, thus improving both resolution and contrast.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig1.jpg" /> 
Figure 1. Optical resolution of epifluorescence versus confocal microscopy. <a href="https://www.jove.com/files/ftp_upload/10501/10501fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The light beam used to excite a fluorophore is focused by the objective lens of a microscope and converges at a focal volume and then diverges (left). The red star represents the focal plane of a sample that is being imaged while the red square represents fluorophore emission above the focal plane. When capturing an image of this sample using an epifluorescent microscope, the emission from the out-of-focus red square will visible and contribute to a &#34;hazy background&#34; (top right). Confocal microscopes have a pinhole which prevents the detection of light emitted outside of the focal plane, eliminating the &#34;hazy background&#34; (bottom right).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig2.jpg" /> 
Figure 2. Pinhole effect in confocal microscopy. <a href="https://www.jove.com/files/ftp_upload/10501/10501fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although the highest intensity of the excitation light is at the focal point of the lens (left, red oval), other parts of the sample not in the focal point (right, red star) will get light and fluoresce. To prevent light emitted from these out-of-focus regions to reach the detector, a screen with a pinhole is present in front of the detector. Only the in-focus light (left) emitting from the focal plane is able to travel through the pinhole and reach the detector. The out-of-focus light (right) is blocked with the pinhole and fails to reach the detector.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig3.jpg" /> 
Figure 3. Principal components of a confocal laser scanning microscope. <a href="https://www.jove.com/files/ftp_upload/10501/10501fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For the sake of simplicity, the mechanistic description of a confocal microscope will be limited to that of the Nikon Eclipse Ti A1R. Although there may be minor technical differences between different confocal microscopes, the A1R serves well as a good model for describing confocal microscope function. The excitation light beam, produced by an array of diode lasers, is reflected by the primary dichroic mirror into the objective, which focuses the light onto the specimen being imaged. The primary dichroic mirror selectively reflects the excitation light while allowing light at other wavelengths to pass through. The light then encounters the scanning mirrors that sweep the light beam across the specimen in an x-y manner, illuminating a single (x,y) pixel at a time. Fluorescence emitted by fluorophores at the illuminated pixel is collected by the objective lens and passes through the primary dichroic mirror to reach an array of photomultiplier tubes (PMTs). Secondary dichroic mirrors direct the emission light to the appropriate PMT. Excitation light scattered by the sample back into the objective is reflected by the primary dichroic mirror back towards the specimen, and thus prevented from entering the detection light path and reaching the PMTs (see Figure 3). This allows the relatively feeble fluorescence to be quantified without contamination by light scattered from the excitation light beam, which is typically orders of magnitude more intense than the fluorescence. Because the pinhole blocks light from outside of the focal volume, the light arriving at the detector comes from a narrow, selected z-plane. Therefore, images can be collected from a series of adjacent z-planes; this series of images is often referred to as a &#39;z-stack&#39;. By using the appropriate software, a z-stack can be processed to generate a 3D image of the specimen. A particular advantage of confocal microscopy is the ability to distinguish the subcellular localization of staining. For example, the differentiation between membrane staining from intracellular staining, which is very challenging with conventional epifluorescence microscopy (1, 2, 3).
Sample preparation is an important facet of confocal imaging. A strength of optical microscopy techniques is the flexibility to image live or fixed cells. When attempting to produce 3D images, because of the number of images that must be acquired for a z-stack, the difficulty of maintaining cell health, and the movement of live cells and their organelles, the use of fixed cells is typical. The procedure for fixing and staining cells for confocal fluorescence is similar to that conventionally used in immunofluorescence. After culture in chamber slides or on coverslips, cells are fixed using paraformaldehyde to preserve cellular morphology. Non-specific antibody binding is blocked using bovine serum albumin, milk, or normal serum. In order to maintain the specificity of the secondary antibodies, the solution used should not originate from the same species in which the primary antibodies were generated. The cells are incubated with primary antibodies that bind the antigen of interest. When labeling several cellular targets, the primary antibodies must each be derived from a different species. Antibodies tagging an antigen are then bound by fluorophore-conjugated secondary antibodies. Fluorophore-conjugated secondary antibodies should be selected so that they are compatible with the wavelengths of laser excitation available in the confocal microscope. When visualizing multiple antigens, the excitation/emission spectra of the fluorophores should differ enough so that their signals can be discriminated by microscopic analysis. The stained specimen is then mounted on a slide for imaging. A mounting medium is used to prevent photobleaching and specimen dehydration. If desired, a mounting medium containing a nuclear counterstain (e.g. DAPI or Hoechst) can be used (4).
In the following protocol, mouse fibroblasts transfected to express CD1d (LCD1) were stained with antibodies recognizing CD1d and CD107a (LAMP-1). CD1d is a major histocompatibility complex 1 (MHC 1)-like receptor present on the surface of antigen presenting cells that presents lipid antigens. LAMP-1 (lysosomal associated membrane protein-1) is a transmembrane protein primarily present in lysosomal membranes. For proper antigen presentation, CD1d is trafficked through the low pH lysosomal compartment, so LAMP-1 is being used as a marker of the lysosomal compartment for this protocol. By probing the LCD1 cells with anti-CD1d and anti- LAMP-1 that were produced in different species, secondary antibodies with unique fluorophores can be used to determine the localization of each protein in the cell and whether CD1d is present in the LAMP-1 positive lysosomal compartments.

Confocal Fluorescence Microscopy and Localization of Proteins

Source: Dominique R. Bollino1, Eric A. Legenzov2, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine ...

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

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