This work was supported by the Swiss National Foundation (SNF) grants (176124, 145038, and 148183), the European Commission Marie Curie Reintegration Grant (612742), and the SystemsX.ch for a grant to D.U.P. (2013/124).

<ol><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+tissue+two-photon+microscopy%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Deep tissue two-photon microscopy.</a> Nature Methods. 2 (12), 932-940 (2005).</li>
<li>Zipfel, W. R., Williams, R. M., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonlinear+magic%3A+multiphoton+microscopy+in+the+biosciences%5BTitle%5D)+AND+%22Nature+biotechnology%22%5BJournal%5D)">Nonlinear magic: multiphoton microscopy in the biosciences.</a> Nature biotechnology. 21 (11), 1369-1377 (2003).</li>
<li>Fein, M. R., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caught+in+the+act%3A+revealing+the+metastatic+process+by+live+imaging%5BTitle%5D)+AND+%22Disease+Models+%26+Mechanisms%22%5BJournal%5D)">Caught in the act: revealing the metastatic process by live imaging.</a> Disease Models & Mechanisms. 6 (3), 580-593 (2013).</li>
<li>Dombeck, D. A., Harvey, C. D., Tian, L., Looger, L. L., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+imaging+of+hippocampal+place+cells+at+cellular+resolution+during+virtual+navigation%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Functional imaging of hippocampal place cells at cellular resolution during virtual navigation.</a> Nature Neuroscience. 13 (11), 1433-1440 (2010).</li>
<li>Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Choreography+of+cell+motility+and+interaction+dynamics+imaged+by+two-photon+microscopy+in+lymphoid+organs%5BTitle%5D)+AND+%22Annual+review+of+immunology%22%5BJournal%5D)">Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.</a> Annual review of immunology. 26, 585-626 (2008).</li>
<li>Germain, R. N., Robey, E. A., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Decade+of+Imaging+Cellular+Motility+and+Interaction+Dynamics+in+the+Immune+System%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A Decade of Imaging Cellular Motility and Interaction Dynamics in the Immune System.</a> Science. 336 (6089), 1676-1681 (2012).</li>
<li>Coombes, J. L., Robey, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+imaging+of+host-pathogen+interactions+in+vivo%5BTitle%5D)+AND+%22Nature+Reviews+Immunology%22%5BJournal%5D)">Dynamic imaging of host-pathogen interactions in vivo.</a> Nature Reviews Immunology. 10 (5), 353-364 (2010).</li>
<li>Pulendran, B., Maddur, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Innate+Immune+Sensing+and+Response+to+Influenza%5BTitle%5D)+AND+%22Life+Science+Journal%22%5BJournal%5D)">Innate Immune Sensing and Response to Influenza.</a> Life Science Journal. 6 (4), 23-71 (2014).</li>
<li>Lim, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+trails+guide+influenza-+specific+CD8+%2B+T+cells+in+the+airways%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Neutrophil trails guide influenza- specific CD8 + T cells in the airways.</a> Science. 349 (6252), (2015).</li>
<li>Kim, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+tracheal+epithelial+cells+in+mice+during+airway+regeneration%5BTitle%5D)+AND+%22American+journal+of+respiratory+cell+and+molecular+biology%22%5BJournal%5D)">In vivo imaging of tracheal epithelial cells in mice during airway regeneration.</a> American journal of respiratory cell and molecular biology. 47 (6), 864-868 (2012).</li>
<li>Kretschmer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autofluorescence+multiphoton+microscopy+for+visualization+of+tissue+morphology+and+cellular+dynamics+in+murine+and+human+airways%5BTitle%5D)+AND+%22Laboratory+investigation%3B+a+journal+of+technical+methods+and+pathology%22%5BJournal%5D)">Autofluorescence multiphoton microscopy for visualization of tissue morphology and cellular dynamics in murine and human airways.</a> Laboratory investigation; a journal of technical methods and pathology. 96 (8), 918-931 (2016).</li>
<li>Veres, T. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intubation-free+in+vivo+imaging+of+the+tracheal+mucosa+using+two-photon+microscopy%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Intubation-free in vivo imaging of the tracheal mucosa using two-photon microscopy.</a> Scientific Reports. 7 (1), 694(2017).</li>
<li>Looney, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stabilized+imaging+of+immune+surveillance+in+the+mouse+lung%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Stabilized imaging of immune surveillance in the mouse lung.</a> Nature. 8 (1), 91-96 (2011).</li>
<li>Thornton, E. E., Krummel, M. F., Looney, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+Imaging+of+the+Lung%5BTitle%5D)+AND+%22Current+Protocols+in+Cytometry%22%5BJournal%5D)">Live Imaging of the Lung.</a> Current Protocols in Cytometry. 60 (1), (2012).</li>
<li>Tabuchi, A., Mertens, M., Kuppe, H., Pries, A. R., Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+microscopy+of+the+murine+pulmonary+microcirculation%5BTitle%5D)+AND+%22Journal+of+Applied+Physiology%22%5BJournal%5D)">Intravital microscopy of the murine pulmonary microcirculation.</a> Journal of Applied Physiology. 104 (2), 338-346 (2008).</li>
<li>Fiole, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-photon+intravital+imaging+of+lungs+during+anthrax+infection+reveals+long-lasting+macrophage-dendritic+cell+contacts%5BTitle%5D)+AND+%22Infection+and+immunity%22%5BJournal%5D)">Two-photon intravital imaging of lungs during anthrax infection reveals long-lasting macrophage-dendritic cell contacts.</a> Infection and immunity. 82 (2), 864-872 (2014).</li>
<li>Secklehner, J., Lo Celso, C., Carlin, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+microscopy+in+historic+and+contemporary+immunology%5BTitle%5D)+AND+%22Immunology+and+Cell+Biology%22%5BJournal%5D)">Intravital microscopy in historic and contemporary immunology.</a> Immunology and Cell Biology. 95 (6), 506-513 (2017).</li>
<li>Lambrecht, B. N., Hammad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lung+Dendritic+Cells+in+Respiratory+Viral+Infection+and+Asthma%3A+From+Protection+to+Immunopathology%5BTitle%5D)+AND+%22Annual+Review+of+Immunology%22%5BJournal%5D)">Lung Dendritic Cells in Respiratory Viral Infection and Asthma: From Protection to Immunopathology.</a> Annual Review of Immunology. 30 (1), 243-270 (2012).</li>
<li>Camp, J. V., Jonsson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+for+neutrophils+in+viral+respiratory+disease%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">A role for neutrophils in viral respiratory disease.</a> Frontiers in Immunology. 8, (2017).</li>
<li>van Gisbergen, K. P. J. M., Sanchez-Hernandez, M., Geijtenbeek, T. B. H., van Kooyk, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophils+mediate+immune+modulation+of+dendritic+cells+through+glycosylation-dependent+interactions+between+Mac-1+and+DC-SIGN%5BTitle%5D)+AND+%22The+Journal+of+experimental+medicine%22%5BJournal%5D)">Neutrophils mediate immune modulation of dendritic cells through glycosylation-dependent interactions between Mac-1 and DC-SIGN.</a> The Journal of experimental medicine. 201 (8), 1281-1292 (2005).</li>
<li>Gonzalez, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capture+of+influenza+by+medullary+dendritic+cells+via+SIGN-R1+is+essential+for+humoral+immunity+in+draining+lymph+nodes%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes.</a> Nature Immunology. 11 (5), 427-434 (2010).</li>
<li>Lindquist, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+dendritic+cell+networks+in+vivo%5BTitle%5D)+AND+%22Nature+immunology%22%5BJournal%5D)">Visualizing dendritic cell networks in vivo.</a> Nature immunology. 5 (12), 1243-1250 (2004).</li>
<li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+V%CE%B39V%CE%B42-T+cells+efficiently+kill+influenza+virus-infected+lung+alveolar+epithelial+cells%5BTitle%5D)+AND+%22Cellular+and+Molecular+Immunology%22%5BJournal%5D)">Human Vγ9Vδ2-T cells efficiently kill influenza virus-infected lung alveolar epithelial cells.</a> Cellular and Molecular Immunology. 10 (2), 159-164 (2013).</li>
<li>Tran Cao, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+the+transgenic+cyan+fluorescent+protein+%28CFP%29-expressing+nude+mouse+for+%26%2334%3Btechnicolor%26%2334%3B+cancer+imaging%5BTitle%5D)+AND+%22Journal+of+Cellular+Biochemistry%22%5BJournal%5D)">Development of the transgenic cyan fluorescent protein (CFP)-expressing nude mouse for &#34;technicolor&#34; cancer imaging.</a> Journal of Cellular Biochemistry. 107 (2), 328-334 (2009).</li>
<li>Jaber, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dose+regimens%2C+variability%2C+and+complications+associated+with+using+repeat-bolus+dosing+to+extend+a+surgical+plane+of+anesthesia+in+laboratory+mice%5BTitle%5D)+AND+%22Journal+of+the+American+Association+for+Laboratory+Animal+Science+JAALAS%22%5BJournal%5D)">Dose regimens, variability, and complications associated with using repeat-bolus dosing to extend a surgical plane of anesthesia in laboratory mice.</a> Journal of the American Association for Laboratory Animal Science JAALAS. 53 (6), 684-691 (2014).</li>
<li>Pizzagalli, D. U., et al. Leukocyte Tracking Database, a collection of immune cell tracks from intravital 2-photon microscopy videos. Scientific Data. , In press (2018).</li>
<li>Sommer, C., Straehle, C., Kothe, U., Hamprecht, F. A. <a target="_blank" href="https://ieeexplore.ieee.org/document/5872394/">Ilastik: Interactive learning and segmentation toolkit.</a> 2011 IEEE International Symposium on Biomedical Imaging: From Nano to Macro. , 230-233 (2011).</li>
<li>Beltman, J. B., Marée, A. F. M., De Boer, R. J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/19834485">Analysing immune cell migration.</a> Nature Reviews Immunology. 9 (11), 789-798 (2009).</li>
<li>Keller, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motility%2C+cell+shape%2C+and+locomotion+of+neutrophil+granulocytes%5BTitle%5D)+AND+%22Cell+motility%22%5BJournal%5D)">Motility, cell shape, and locomotion of neutrophil granulocytes.</a> Cell motility. 3 (1), 47-60 (1983).</li>
<li>Sumen, C., Mempel, T. R., Mazo, I. B., von Andrian, U. H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/15357943">Intravital Microscopy.</a> Immunity. 21 (3), 315-329 (2004).</li>
<li>Lambert Emo, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+Imaging+of+Influenza+Infection+of+the+Trachea+Reveals+Dynamic+Regulation+of+CD8%2B+T+Cell+Motility+by+Antigen%5BTitle%5D)+AND+%22PLOS+Pathogens%22%5BJournal%5D)">Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen.</a> PLOS Pathogens. 12 (9), e1005881(2016).</li>
<li>Kjos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bright+fluorescent+Streptococcus+pneumoniae+for+live-cell+imaging+of+host-pathogen+interactions%5BTitle%5D)+AND+%22Journal+of+bacteriology%22%5BJournal%5D)">Bright fluorescent Streptococcus pneumoniae for live-cell imaging of host-pathogen interactions.</a> Journal of bacteriology. 197 (5), 807-818 (2015).</li>
</ol>

The authors have nothing to disclose.

This work presents a detailed protocol for the generation of 4D images showing the migration of adoptively transferred neutrophils and their interactions with DC during an influenza infection in the mouse trachea. The described 2P-IVM model will be relevant to study immune cell dynamics during an infection in the airways.
Recently, several models based on the visualization of cell dynamics in the airways have been developed9,10,</...

Two-photon intravital microscopy (2P-IVM) is an effective technique for real time imaging of cell-to-cell interactions as they occur in their natural environment1. One of the main advantages of this method is that it allows the study of cellular processes at a greater specimen depth (500 &#181;m to 1 mm) compared with other traditional imaging techniques2. At the same time, the use of two low-energy photons generated by the two-photon laser minimizes the tissue photo-damage typically associated with the image acquisition process2. During the last decade, 2P-IVM has been applied to study different types of cell-cell interactions in several disciplines3,4,5. These studies have been especially relevant to investigate immune cells, which are characterized by their high dynamism and the formation of prominent contacts following the signals generated by other cells and the environment. 2P-IVM has been also applied to study the interactions between pathogen and host6. Indeed, it has been previously shown that some pathogens can alter the type and duration of the contacts between immune cells, hampering, as a result, the immune response7.
The airway mucosa is the first site in which the immune response against airborne pathogens is generated8. Therefore, in vivo analysis of pathogen-host interactions in this tissue is critical to understand the initiation of the host defense mechanisms during infection. However, 2P-IVM of the airways is challenging mainly due to the artifacts produced by the breathing cycle of the animal, which compromises the process of image acquisition. Recently, different surgical models have been described for imaging murine trachea9,10,11,12 and lungs13,14,15,16. Tracheal 2P-IVM models represent an excellent set-up to visualize the initial phase of the immune reaction in the upper airways, while lung-alveoli 2P-IVM models are more suitable to study the late phase of infections. The developed lung models present a limitation associated with the presence of air-filled alveoli, which restrict the optical penetration of the laser and make the mucosal layer of the intrapulmonary airways inaccessible for in vivo imaging17. Conversely, the structure of the trachea, formed by a continuous epithelium, facilitates image acquisition.
Here, we present a protocol that includes a detailed description of the steps required to perform influenza infection, surgical preparation of the animals, and 2P-IVM of the trachea. In addition, we describe a specific experimental set-up for the visualization of neutrophils and dendritic cells (DC), two immune cell types that play an important role as mediators of the defense mechanism against influenza virus18,19. Finally, we describe a procedure to analyze neutrophil-DC interactions. These contacts have been shown to modulate DC activation and, subsequently, to affect the immune responses against pathogens20.

<table><tbody><tr><td>Gigasept instru AF</td><td>Sch&amp;uuml;lke &amp;amp; Mayr GmbH</td><td></td><td>4% solution</td></tr><tr><td>CD11c-YFP mice</td><td>Jackson Laboratories</td><td>008829</td><td>mice were bred in-house</td></tr><tr><td>CK6-ECFP mice</td><td>Jackson Laboratories</td><td>004218</td><td>mice were bred in-house</td></tr><tr><td>1x Dulbecco&#039;s Phosphate Buffered Saline modified without Calcium Choride and Magnesium Chloride</td><td>Sigma</td><td>D8537-500ML</td><td></td></tr><tr><td>10x Dulbecco&#039;s Phosphate Buffered Saline modified without Calcium Choride and Magnesium Chloride</td><td>Sigma</td><td>D1408-500ML</td><td></td></tr><tr><td>Percoll PLUS</td><td>Sigma</td><td>E0414-1L</td><td>Store at 4&amp;deg;C</td></tr><tr><td>Ketamin Labatec</td><td>Labatec Pharma</td><td>7680632310024</td><td>Store at RT, store at 4 &amp;deg;C when in solution of ket/xyl mixture</td></tr><tr><td>Rompun 2% (Xylazin)</td><td>Bayer</td><td>6293841.00.00</td><td>Store at RT, store at 4 &amp;deg;C when in solution of ket/xyl mixture</td></tr><tr><td>26 G 1 mL Sub-Q BD Plastipak</td><td>BD Plastipak</td><td>305501</td><td></td></tr><tr><td>30 G, 0.3 mL BD Micro-Fine Insulin Syringes</td><td>BD</td><td>324826</td><td></td></tr><tr><td>Falcon 40 &amp;micro;m Cell Strainer</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>2 mL Syringes</td><td>BD Plastipak</td><td>300185</td><td></td></tr><tr><td>Microlance 3x 18 G needles</td><td>BD</td><td>304622</td><td></td></tr><tr><td>Introcan Safety 20 G (catheter)</td><td>Braun</td><td>4251652.01</td><td></td></tr><tr><td>6 Well Cell Culture Cluster</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>RPMI medium 1640 + HEPES (1x)</td><td>ThermoFisher Scientific</td><td>42401-018</td><td>Store at 4&amp;deg;C</td></tr><tr><td>Liberase TL Research Grade</td><td>Roche</td><td>5401020001</td><td>Store at -20 &amp;deg;C / collagenase (I and II) mixture</td></tr><tr><td>DNAse I</td><td>Amresco (VWR)</td><td>0649-50KU</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>CellTrace Violet stain</td><td>ThermoFisher Scientific</td><td>C34557</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>EDTA</td><td>Sigma</td><td>EDS-500G</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10270-106</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>PE-10 Micro Medical Tubing</td><td>2Biological Instruments SNC</td><td>#BB31695-PE/1</td><td></td></tr><tr><td>Surgical Plastic Tape</td><td>M Plast</td><td></td><td></td></tr><tr><td>Viscotears</td><td>Bausch &amp;amp; Lomb</td><td></td><td>Store at RT</td></tr><tr><td>Plasticine</td><td>Ohropax</td><td></td><td></td></tr><tr><td>High Tolerance Glass Coverslip 15 mm Round</td><td>Warner Instruments</td><td>64-0733</td><td></td></tr><tr><td>SomnoSuite Portable Animal Anesthesia System</td><td>Kent Scientific</td><td>SS-01</td><td></td></tr><tr><td>Nuvo Lite mark 5</td><td>GCE medline</td><td>14111211</td><td></td></tr><tr><td>MiniTag (gaseous anesthesia and heating bench)</td><td>Tem Sega</td><td></td><td></td></tr><tr><td>SURGICAL BOARD</td><td>University of Bern</td><td></td><td></td></tr><tr><td>TrimScope II Two-photon microscope</td><td>LaVision Biotec</td><td></td><td></td></tr><tr><td>Chameleon Vision Ti:Sa lasers</td><td>Coherent Inc.</td><td></td><td></td></tr><tr><td>25X NA 1.05 water immersion objective</td><td>Olympus</td><td>XLPLN25XWMP2</td><td></td></tr><tr><td>The Cube&amp;amp;The Box incubation chamber and temperature controller</td><td>Life imaging Services</td><td></td><td></td></tr><tr><td>Imaris 9.1.0</td><td>Bitplane</td><td></td><td>Imaging software</td></tr><tr><td>GraphPad Prism 7</td><td>GraphPad</td><td></td><td>Statistical software</td></tr></tbody></table>

imaging cell interaction in tracheal mucosa during influenza virus infection using two-photon intravital microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this work, we described a detailed protocol to study in vivo the motility and the interactions between neutrophils and DC during influenza infection in murine trachea (Figure 3A). To this purpose, we isolated CFP+ neutrophils (92% purity; Figure 3B) from CK6-ECFP mice and we adoptively transferred them into a CD11c-YFP mouse infected with influenza. After that, we performed 2P-IVM of the trachea at day 3 p....

Watch this Scientific Journal Video about Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy at JoVE.com

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

imaging-cell-interaction-tracheal-mucosa-during-influenza-virus

Research

JoVE Journal

Immunology and Infection

8.3K Views.  Università della Svizzera italiana (USI). In deze studie presenteren we een protocol voor het uitvoeren van twee-foton intravital beeldvorming en cel interactie analyse in de lymfkliertest tracheale mucosa na infectie met influenzavirus. Dit protocol is relevant voor onderzoekers bestuderen immuun cel dynamiek tijdens infecties van de luchtwegen.

Video: Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Cancer Research

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Neuroscience

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

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 ...

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 ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Bioengineering

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Medicine

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

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 ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

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