The authors would like to thank Dr. Lars Philipsen for help with optimizing the intravital movies, Dr. Jonathan Lindquist for carefully reading the manuscript, and all members of the Gunzer laboratory for helpful discussions and comments during the development of the method. This work was funded by grants from the Deutsche Forschungsgemeinschaft (DFG, SFB 854) to M.G.

1. Infection
<ol>
	<li>Swollen conidia of the mold Aspergillus fumigatus (strain ATCC 46645) are generated by pre-incubation in RPMI 1640 (Biochrom AG, Berlin, Germany) 
	supplemented with 5% FCS (v/v). 2x107 resting conidia are resuspended in 5 ml medium and incubated in a 6-well tissue culture plate (TPP AG; Trasadingen, 
	Switzerland, catalog # 92006) for 7h at 37&deg;C.</li>
	<li>Following the swelling period, the spores are fluorescently labeled by adding 10 &mu;l of calcofluor white stock solution (Sigma Aldrich, Deisenhofen, Germany, 
	catalog # F3543; [25 mg/ml] in DMSO) to each Aspergillus containing well in the 6-well plate, reaching a final calcofluor concentration of 50 &mu;g/ml RPMI. 
	Following gentle mixing, the spores are incubated for an additional 15 min at 37 &deg;C. </li>
	<li>In the next step, the spores are harvested by filtration through a 70 &mu;m cell strainer (BD Biosciences, Heidelberg, Germany). After washing once 
	with 10 ml PBS (centrifuge at 900xg for 5 min at room temperature) the pellet is resuspended in 2 ml PBS and the total number of swollen conidia 
	determined by counting with a Neubauer chamber. Finally the spore suspension is spun down at 900xg for 5 min at room temperature, the 
	supernatant is carefully removed, and the pellet resuspended to the final concentration of 1x107 spores per 100 &mu;l PBS.</li>
	<li>For infection, 100 &mu;l of spore solution is applied intratracheally into female mice (C57/BL6, 8&#x2013;10 weeks old, Harlan, Germany). If neutrophils are to be 
	observed as well, the use of Lys-EGFP mice, carrying EGFP under the lysozyme promotor1 is recommended. Begin by anesthetizing the animal with a single i.p. 
	injection of 150 &mu;l Ketamin/Rompun solution (Inresa Arzneimittel GmbH, Freiburg, Germany (Ketamin) and Bayer Vital GmbH, Leverkusen, Germany (Rompun), 1 ml 
	Ketamin [50 mg/ml] + 0.5 ml Rompun [2 %] + 3.5 ml sterile NaCl [0.9 %]). 5 minutes later the anesthetized mouse can be fixed with an elastic band by its teeth 
	to the ramp (i.e. a sloped surface) to facilitate the intubation (supplemental figure 1). Using forceps, the tongue is pulled to one side and a 22G indwelling 
	venous catheter (B. Braun AG, Melsungen, Germany, Vasofix Braun&uuml;le) can be gently inserted into the trachea under permanent illumination with a goose neck 
	lamp. The successful insertion of the catheter is verified by observing regular movement of the animal's thorax with the frequency of the mechanical 
	ventilator. When the successful intubation has been confirmed, 100 &mu;l spore suspension is applied using a 100 &mu;l micropipette. This volume should be 
	inhaled by the animal without any additional help in 1-2 s. An enhanced distribution of fungal particles inside the lung is achieved by mechanically 
	ventilating the infected animal for 2 minutes with a small animal respirator (MiniVent, Hugo Sachs, March-Hugstetten, Germany) at rate of 250 breaths 
	per minute and an inhalation volume of 300 &mu;l per breath (supplemental figure 2). After the infection animals are returned to their cage and observed every 5 minutes until ambulatory again. Mice do not show any evidence of pain or distress during the following incubation period.</li>
</ol>
2. Lung preparation
<ol>
	<li>7 h after infection with the spores, the animal is euthanized by an overdose of isoflurane (Baxter GmbH, Unterschlei&beta;heim, Germany) until movements and 
	breathing have ceased for more than 30 seconds. This is then followed by thoracotomy as a secondary method to assure clinical death and allow for visualization 
	and access of the trachea and lungs. Then the chest is carefully opened to expose the lung and the upper respiratory tract. Then another 22G indwelling venous 
	catheter is inserted into the trachea starting from the exposed epiglottis. Through this tubule the lungs are filled with 1 ml pre-warmed low melting agarose 
	(2 % w/v, Promega, Mannheim, Germany) using a 1 ml Omnifix syringe (B.Braun). As soon as the lungs are filled the trachea is tied off with a short piece of 
	sewing thread and the whole animal is put into a refrigerator at 4 &deg;C. </li>
	<li>15 min later, when the agarose has solidified, the whole lung is excised beginning from the trachea. Using a pair of sharp pointed scissors, 
	the right lung lobe is subsequently removed, the bottom surface is briefly dried on a sheet of tissue paper and then the whole lobe is glued to the 
	preparation block of a vibratome (Campden Instruments, UK, 752M Vibroslice) using a drop of tissue adhesive for fixation (Carl Roth GmbH, Karlsruhe, 
	Germany, Roti-Coll 1). After 1 min the block is installed in the cutting chamber of the vibratome, which is filled with pre-chilled (4&deg;C) PBS. The 
	vibratome is set to a mid-range vibration (5 out of 10 max.) and also moderate speed (5 out of 10 max.) such that a horizontal cross section of the lung 
	is produced. The upper half of the organ is then transferred into a small plastic Petri dish (5 cm diameter) where it is inverted and fixed with a self-made 
	flat washer (1 cm inner diameter) that is covered by a set of parallel nylon threads, each 1 mm apart (supplemental figure 3). Finally the Petri dish is 
	filled with 10 ml PBS supplemented with 10 &mu;l of the DNA dye Sytox Orange [5 mM] (Invitrogen, Germany) resulting in a final dye concentration of 5 &mu;M.</li>
</ol>
3. 2-photon laser microscopy
<ol>
	<li>The Petri dish containing the lung sample is then installed under the microscope objective so that the buffer can be warmed to 37&deg;C by a temperature 
	sensor-controlled heater (supplemental figure 4). 2-photon microscopy is then carried out over the whole cut surface using a Zeiss LSM 710 NLO microscope 
	on an upright Axio Examiner stage equipped with a 20xNA1.0 water dipping lens (Zeiss, Jena, Germany). For imaging, different areas along the section are 
	scanned down to 400 &mu;m depth using an illumination wavelength of 800 nm detecting green (530 nm) and red (580 nm) fluorescence, as well as the second 
	harmonic generation (SHG)-signal and the blue calcofluor fluorescence (at 400&#x2013;470 nm emission) with external non descanned detectors (NDD). Besides 
	2-D images and time lapse movies (1 image every 5-10 seconds), single or repetitive 3-dimensional picture stacks can be subsequently rendered as 
	voxel renderings or single extended focus images or 4-D data (3D over time) using different software packages. This microscopic setup allows the 
	observation of cellular behavior in an almost intact environment that, as it constitutes the entry port for a variety of airborne pathogens, is 
	of enormous immunological relevance. Cell motility, phagocytosis and NET production by endogenous neutrophils following infection with the mold 
	Aspergillus fumigatus can be easily investigated under these in situ conditions.</li>
</ol>
4. Representative Results
If done properly the imaging will generate 2- or 3-color slides or movies. Although coloring is done in a post processing procedure and is thus freely adaptable, we generally select a coloring scheme that reflects the natural color of the dye/signal, which is detected in the relative channel. Thus, the Sytox dyes are depicted in red, the fungus as well as the SHG signal is shown in blue and, if present, EGFP-labeled cells are stained in green. In the first example (Fig. 1) the blue SHG signal depicts the tissue fibers of a non-infected lung and the red Sytox signal is produced by nuclei of lung-resident cells cut open by the vibratome. In the second example (Fig. 2) an infected lung is shown, where both, alveolar structures as well as fungal masses, appear in blue, while nuclei and NETs are stained red. The third example (Fig. 3) is from a Lys-EGFP transgenic animal where in addition to the blue and red structures the green neutrophils can also be seen. The migration of neutrophils and their phagocytosis of individual fungal elements in time lapse sequences is shown in the supplemental movie.
<img src="/files/ftp_upload/2659/2659fig1.jpg" alt="Figure 1" /> 
Figure 1. The appearance of a non-infected lung slice in 2-color 2-photon microscopy. A lung slice was prepared from a non-infected C57/BL6 mouse and imaged as described in the protocol. Presented here are the SHG signal of the alveolar tissue structure (A), the Sytox signal of cell nuclei cut open during the preparation of the lung slice (B), and an overlay of the two channels (C). The boxed area in (C) is seen enlarged in (D). Please note the fibrous tissue at the bottom of the lung slice as compared to the clearly alveolar organization within the breathing-active areas of the lung above.
<img src="/files/ftp_upload/2659/2659fig2.jpg" alt="Figure 2" /> 
Figure 2. The appearance of a lung slice of an Aspergillus infected wild type animal in 2-color 2-photon microscopy. A lung slice from a C57/BL6 mouse infected 7 h before with A. fumigatus was prepared and imaged as described in the protocol. Shown is the combined fungal structure and SHG of the alveolar tissue (A), the Sytox signal of DNA NETs as well as cell nuclei cut open during the preparation of the lung slice (B), and an overlay of the two channels (C). The boxed area in (C) is seen enlarged in (D). Please note, the clearly distinct areas of fungal masses, alveoli, and NET-structures are marked with the letters F, A and N, respectively.
<img src="/files/ftp_upload/2659/2659fig3.jpg" alt="Figure 3" /> 
Figure 3. The appearance of a lung slice of an Aspergillus infected Lys-EGFP animal in 3-color 2-photon microscopy. A lung slice of a Lys-EGFP mouse 
infected 7 h before with A. fumigatus was prepared and imaged as described in the protocol. Shown is the combined fungal structure and SHG of the alveolar tissue (blue), the Sytox signal of cell nuclei and NETs (red), as well as well as numerous neutrophils (green).
<img src="/files/ftp_upload/2659/2659supfig1.jpg" alt="Figure 4" /> 
Supplemental figure 1. Mouse fixation for intubation. Under Ketamin/Rompun anesthesia the animal is fixed with an elastic band at its teeth in order to 
facilitate the intubation with a 22G indwelling venous catheter.
<img src="/files/ftp_upload/2659/2659supfig2.jpg" alt="Figure 5" /> 
Supplemental figure 2. Mechanical mouse ventilation. The intubated mouse is infected by an i.t. application of 1x107 spores resuspended in 
100 &mu;l PBS. An enhanced distribution of fungal particles inside the lung is achieved by mechanically ventilating the infected mouse with a small animal respirator.
<img src="/files/ftp_upload/2659/2659supfig3.jpg" alt="Figure 6" /> 
Supplemental figure 3. Lung lobe fixation. After preparation of the right lung lobe the organ is fixed in a Petri dish by use of a laboratory-made 
flat washer that is covered by a set of parallel nylon threads.
<img src="/files/ftp_upload/2659/2659supfig4.jpg" alt="Figure 6" /> 
Supplemental figure 4. 2-photon imaging setup. The Petri dish containing the right lung lobe is installed on a heating mat under the 2-photon microscope after addition of the DNA dye Sytox Orange.
Supplemental movie: Neutrophils migrating and phagocytosing fungal elements in Aspergillus infected lungs as seen by time lapse 2-photon microscopy of a 
living lung slice 7 h after infection with the fungus. Neutrophils are green, fungal elements and SHG are blue, and cell nuclei as well as NET-structures are depicted in red. The real time of the experiment is shown on the lower right. The scale bar depicts 50 &mu;m.<a href="https://www.jove.com/files/ftp_upload/2659/5_9_1 - supplemental.mpg">Click here to watch video</a>

<ol>
	<li>Faust, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96, 719-719 (2000).</li><li>Gunzer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spectrum+of+biophysical+interaction+modes+between+T+cells+and+different+antigen+presenting+cells+during+priming+in+3-D+collagen+and+in+vivo.">A spectrum of biophysical interaction modes between T cells and different antigen presenting cells during priming in 3-D collagen and in vivo.</a> Blood. 104, 2801-2801 (2004).</li><li>Mempel, T. R., Henrickson, S. E., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+priming+by+dendritic+cells+in+lymph+nodes+occurs+in+three+distinct+phases.">T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases.</a> Nature. 427, 154-154 (2004).</li><li>Bousso, P., Robey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+CD8(+)+T+cell+priming+by+dendritic+cells+in+intact+lymph+nodes.">Dynamics of CD8(+) T cell priming by dendritic cells in intact lymph nodes.</a> Nat. Immunol. 4, 579-579 (2003).</li><li>Beuneu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+functional+diversification+of+CD8(+)+T+cell+responses+in+lymph+nodes.">Visualizing the functional diversification of CD8(+) T cell responses in lymph nodes.</a> Immunity. 33, 412-412 (2010).</li><li>Aimanianda, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+hydrophobin+prevents+immune+recognition+of+airborne+fungal+spores.">Surface hydrophobin prevents immune recognition of airborne fungal spores.</a> Nature. 460, 1117-1117 (2009).</li><li>Snelgrove, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Critical+Role+for+LTA4H+in+Limiting+Chronic+Pulmonary+Neutrophilic+Inflammation.">A Critical Role for LTA4H in Limiting Chronic Pulmonary Neutrophilic Inflammation.</a> Science. 330, 90-90 (2010).</li><li>Chou, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-Cytokine-Chemokine+Cascade+Drives+Neutrophil+Recruitment+in+a+Murine+Model+of+Inflammatory+Arthritis.">Lipid-Cytokine-Chemokine Cascade Drives Neutrophil Recruitment in a Murine Model of Inflammatory Arthritis.</a> Immunity. 33, 266-266 (2010).</li><li>Nitschke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+and+4-D+imaging+of+immune+cells+in+vitro+and+in+vivo.">3-D and 4-D imaging of immune cells in vitro and in vivo.</a> Histochem. Cell Biol. 130, 1053-1053 (2008).</li><li>Niesner, R. A., Andresen, V., Gunzer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+2-photon+microscopy+-+focus+on+speed+and+time+resolved+imaging+modalities.">Intravital 2-photon microscopy - focus on speed and time resolved imaging modalities.</a> Immunol Rev. 221, 7-7 (2008).</li><li>Bruns, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Extracellular+Traps+against+Aspergillus+fumigatus+in+vitro+and+in+Infected+Lung+Tissue+is+Dependent+on+Invading+Neutrophils+and+Influenced+by+Hydrophobin+RodA.">Production of Extracellular Traps against Aspergillus fumigatus in vitro and in Infected Lung Tissue is Dependent on Invading Neutrophils and Influenced by Hydrophobin RodA.</a> PLoS Pathog. 6, 1000873-1000873 (2010).</li><li>Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+T+cell+trafficking+examined+in+vivo+with+intravital+two-photon+microscopy.">Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 2604-2604 (2003).</li><li>Brinkmann, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Extracellular+Traps+Kill+Bacteria.">Neutrophil Extracellular Traps Kill Bacteria.</a> Science. 303, 1532-1532 (2004).</li><li>Marcos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR2+mediates+NADPH+oxidase-independent+neutrophil+extracellular+trap+formation+in+cystic+fibrosis+airway+inflammation.">CXCR2 mediates NADPH oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation.</a> Nat Med. 16, 1018-1018 (2010).</li><li>Clark, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+TLR4+activates+neutrophil+extracellular+traps+to+ensnare+bacteria+in+septic+blood.">Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood.</a> Nat Med. 13, 463-463 (2007).</li><li>Bianchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+NET+formation+by+gene+therapy+in+CGD+controls+aspergillosis.">Restoration of NET formation by gene therapy in CGD controls aspergillosis.</a> Blood. 114, 2619-2619 (2009).</li></ol>

We have nothing to disclose.

Real-time 2-photon microscopy in vivo or in intact organs has gained profound importance in studies dealing with the physiology of immune cells over the past 10 years. It was with this technique that important events like the dynamics of T-cell activation within the lymph nodes first became visible2-4. More recently, researchers have also started to analyze specific cellular functions like the first steps in the generation of effector cells in lymphatic tissues using this approach5.
However, although a number of new biological concepts have been revealed using this method, there are still challenging and important 
questions for which no intravital visualization studies have been published thus far. Notably this applies to the mammalian lung. The interesting aspect of 
this organ, as entry port for a variety of airborne pathogens, makes it one of the most crucial surfaces at which immunological processes take place in the 
mammalian body. With every breath over the entire life span, unwanted particles are inhaled some of which have the potential to induce life threatening 
infections6. It is self-explanatory that at such a sensitive and endangered site a tight network of defense mechanisms needs to be present exhibiting 
the whole repertoire of immune responses. On the other hand it is very important that the induced immunological fight against potential pathogens at 
such a "dirty" place is tightly controlled. Exaggerated reactions of the immune system bear a high risk of harming the own body by massively injuring 
organ tissue upon stimulation of unspecific immune cell actions7,8.
In the light of these thoughts it would be extremely interesting and helpful to have the possibility to investigate cell behavior in 
mammalian lungs under true in vivo conditions. However, the fact that such a system has so far not been successfully implemented clearly points to the enormous 
difficulties that have to be solved in setting up a working protocol. The most demanding challenge probably is focus stability. The lung as the organ responsible 
for breathing is under constant movement in all three directions of space to realize inhalation. This circumstance alone causes severe imaging problems and can be 
considered an intravital imagers "nightmare". Already a slight motion to any dimension in space has a massive deteriorating effect on the microscopic view, which 
needs to be stable with micrometer precision in order to generate meaningful images9. Given its inherently tight local focus10, 2-photon microscopy is 
even more sensitive to focal instabilities, as a dislocation of a certain structure in the range of just a few micrometers in the Z-direction is 
equivalent to a complete loss of focus and thus a failed experiment.
The protocol presented in this study for the observation of immune cells within the murine lung is still not an in vivo 
application, but rather a close approximation to the situation in a functionally intact lung11. Ex vivo approaches for imaging lymphocytes, e.g. in 
explanted lymph nodes, have been shown to yield results equivalent to true in vivo observations12 and thus are highly relevant5. The in situ 
observations in lung slices, which are only possible with our approach, take place in an infected lung shortly after excision. The 3D integrity during the cutting 
process is ensured by the agarose matrix, an essential step to allow a precisely controlled cutting process of the lung. Although it is necessary to 
cool the explanted lung for a short period to allow a solidification of the agarose matrix, it is possible to return to near physiological conditions 
for cells after cutting and rewarming of the tissue. This is clearly shown by our data, which demonstrate that under these conditions neutrophils are 
highly active and exhibit their full potential as agile phagocytes, which are necessary for the effective clearance of an Aspergillus fumigatus 
infection. They patrol the lung tissue passing through epithelial barriers in order to reach the inner parts of alveoli and furthermore they 
actively take up fungal spores 11. A key finding of this work was the appearance of structures resembling Neutrophil extracellular traps (NETs) in 
the Aspergillus infected organ. NETs are a very recent finding of a novel defense mechanism in neutrophils13. However, since its initial description 
in 2004, the number of physiological or pathological conditions in animal models or humans where this phenomenon has been observed or is lacking has
been explosively increasing14-16. Interestingly, although so much work has been spent on these structures by so many different groups, most reports 
are still on a very descriptive level and not much is known about the mechanism of NET release and its regulation. With our protocol we were able 
for the first time to show NET fibers in an infected lung. Furthermore, we could demonstrate the importance of freshly recruited neutrophils as well 
as molecular fungal structures for their occurrence or inhibition11. This clearly shows the potential of our method to investigate the single 
steps of NET formation in more detail. One could think for example to use neutrophils from suitable knock-out mice to observe their ability of 
NET formation in adoptive transfer experiments.
Thus, although the direct observation of neutrophil immigration from the peripheral blood is not possible with this system due to the lack of 
blood supply after organ explantation, we still believe that our protocol is a valuable and relatively easy to handle approach that allows the imaging of early or 
late steps in the immune defense against lung infections. This is, therefore, an important step towards investigating this phenomenon within the breathing lung of 
live animals.

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.

Watch this Scientific Journal Video about Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy at JoVE.com

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

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 ...

direct-observation-phagocytosis-net-formation-neutrophils-infected

Research

JoVE Journal

Immunology and Infection

20.6K Aufrufe.  Otto-von-Guericke University Magdeburg. Wir zeigen, wie 2-Photonen-Mikroskopie für die Beobachtung der Dynamik der neutrophilen Granulozyten im infizierten Lungengewebe verwenden, während sie Krankheitserreger oder phagozytieren produzieren neutrophilen extrazellulären Traps (NETs).

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

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

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 ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.