The authors would like to thank Dr. Pina Colarusso, who provided significant expertise in the editing and revising of this manuscript.

All the procedures described here were performed with prior approval by the Dalhousie University Committee on Laboratory Animals (UCLA).
1. Preparation
<ol>
	<li>Lung imaging system: To prepare the window, administer a thin layer of vacuum grease to the top of the outer ring while avoiding contamination of the vacuum channel. Place a clean 8 mm glass coverslip on the window and gently press down to create a seal.</li>
	<li>Widefield Fluorescence Microscope: Perform imaging w...

<ol>
	<li>Alizadeh-Tabrizi, N., Hall, S., Lehmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+pulmonary+immune+response+in+inflammation+and+infection.">Intravital imaging of pulmonary immune response in inflammation and infection.</a> Frontiers in Cell and Developmental Biology. 8, 620471 (2021).</li><li>Gaertner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+comprehensive+interpretation+of+intravital+microscopy+images+in+studies+of+lung+tissue+dynamics.">Toward a comprehensive interpretation of intravital microscopy images in studies of lung tissue dynamics.</a> Journal of Biomedical Optics. 20 (6), 066009 (2015).</li><li>Kreisel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+two-photon+imaging+reveals+monocyte-dependent+neutrophil+extravasation+during+pulmonary+inflammation.">In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (42), 18073-18078 (2010).</li><li>Looney, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilized+imaging+of+immune+surveillance+in+the+mouse+lung.">Stabilized imaging of immune surveillance in the mouse lung.</a> Nature Methods. 8 (2), 91-96 (2011).</li><li>Bennewitz, M. F., Watkins, S. C., Sundda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+intravital+two-photon+excitation+microscopy+reveals+absence+of+pulmonary+vasoocclusion+in+unchallenged+sickle+cell+disease+mice.">Quantitative intravital two-photon excitation microscopy reveals absence of pulmonary vasoocclusion in unchallenged sickle cell disease mice.</a> IntraVital. 3 (2), 29748 (2014).</li><li>Blueschke, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+measurement+of+microcirculatory+blood+flow+velocity+in+pulmonary+metastases+of+rats.">Automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats.</a> Journal of Visualized Experiments: JoVE. (93), e51630 (2014).</li><li>Tabuchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precapillary+oxygenation+contributes+relevantly+to+gas+exchange+in+the+intact+lung.">Precapillary oxygenation contributes relevantly to gas exchange in the intact lung.</a> American Journal of Respiratory and Critical Care Medicine. 188 (4), 474-481 (2013).</li><li>Rodriguez-Tirado, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+high-resolution+intravital+microscopy+in+the+lung+with+a+vacuum+stabilized+imaging+window.">Long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window.</a> Journal of Visualized Experiments: JoVE. (116), e54603 (2016).</li><li>Fiole, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+intravital+imaging+of+lungs+during+anthrax+infection+reveals+long-lasting+macrophage-dendritic+cell+contacts.">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>Thanabalasuriar, A., Neupane, A. S., Wang, J., Krummel, M. F., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iNKT+cell+emigration+out+of+the+lung+vasculature+requires+neutrophils+and+monocyte-derived+dendritic+cells+in+inflammation.">iNKT cell emigration out of the lung vasculature requires neutrophils and monocyte-derived dendritic cells in inflammation.</a> Cell Reports. 16 (12), 3260-3272 (2016).</li><li>Neupane, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patrolling+alveolar+macrophages+conceal+bacteria+from+the+immune+system+to+maintain+homeostasis.">Patrolling alveolar macrophages conceal bacteria from the immune system to maintain homeostasis.</a> Cell. 183 (1), 110-125 (2020).</li><li>Tschernig, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualisation+of+microparticles+in+the+living+lung.">Direct visualisation of microparticles in the living lung.</a> Experimental and Toxicologic Pathology. 65 (6), 883-886 (2013).</li><li>Mertens, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+dynamics+in+acute+lung+injury:+Heterogeneous+distension+rather+than+cyclic+opening+and+collapse.">Alveolar dynamics in acute lung injury: Heterogeneous distension rather than cyclic opening and collapse.</a> Critical Care Medicine. 37 (9), 2604-2611 (2009).</li><li>Matuszak, J., Tabuchi, A., Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilation+and+perfusion+at+the+alveolar+level:+Insights+from+lung+intravital+microscopy.">Ventilation and perfusion at the alveolar level: Insights from lung intravital microscopy.</a> Frontiers in Physiology. 11, 291 (2020).</li><li>Margraf, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=6%+Hydroxyethyl+starch+(HES+130/0.4)+diminishes+glycocalyx+degradation+and+decreases+vascular+permeability+during+systemic+and+pulmonary+inflammation+in+mice.">6% Hydroxyethyl starch (HES 130/0.4) diminishes glycocalyx degradation and decreases vascular permeability during systemic and pulmonary inflammation in mice.</a> Critical Care. 22 (1), 1-12 (2018).</li><li>Roller, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+in+vivo+observations+of+P-selectin+glycoprotein+ligand-1-mediated+leukocyte-endothelial+cell+interactions+in+the+pulmonary+microvasculature+in+abdominal+sepsis+in+mice.">Direct in vivo observations of P-selectin glycoprotein ligand-1-mediated leukocyte-endothelial cell interactions in the pulmonary microvasculature in abdominal sepsis in mice.</a> Inflammation Research. 62 (3), 275-282 (2012).</li><li>Marques, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+increases+endothelial+CXCL16-leukocyte+CXCR6+adhesion+in+vitro+and+in+vivo.+Potential+consequences+in+chronic+obstructive+pulmonary+disease.">Cigarette smoke increases endothelial CXCL16-leukocyte CXCR6 adhesion in vitro and in vivo. Potential consequences in chronic obstructive pulmonary disease.</a> Frontiers in Immunology. 8, 1766 (2017).</li><li>Condon, M. R., Kim, J. E., Deitch, E. A., Machiedo, G. W., Spolarics, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+an+erythrocyte+population+with+decreased+deformability+and+hemoglobin+content+following+sepsis.">Appearance of an erythrocyte population with decreased deformability and hemoglobin content following sepsis.</a> American Journal of Physiology-Heart and Circulatory Physiology. 284 (6), 2177-2184 (2003).</li><li>Trzeciak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+microcirculatory+perfusion+derangements+in+patients+with+severe+sepsis+and+septic+shock:+Relationship+to+hemodynamics,+oxygen+transport,+and+survival.">Early microcirculatory perfusion derangements in patients with severe sepsis and septic shock: Relationship to hemodynamics, oxygen transport, and survival.</a> Annals of Emergency Medicine. 49 (1), 88-98 (2007).</li><li>Kim, Y. M., Jeong, S., Choe, Y. H., Hyun, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+intravital+imaging+of+leukocyte+migration+during+inflammation+in+the+respiratory+system.">Two-photon intravital imaging of leukocyte migration during inflammation in the respiratory system.</a> Acute and Critical Care. 34 (2), 101-107 (2019).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <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 (2), 719-726 (2000).</li><li>Orthgiess, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+exhibit+Lyz2+promoter+activity+in+vivo:+Implications+for+using+LysM-Cre+mice+in+myeloid+cell+research.">Neurons exhibit Lyz2 promoter activity in vivo: Implications for using LysM-Cre mice in myeloid cell research.</a> European Journal of Immunology. 46 (6), 1529-1532 (2016).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+murine+pulmonary+microcirculation.">Intravital microscopy of the murine pulmonary microcirculation.</a> Journal of Applied Physiology. 104 (2), 338-346 (2008).</li><li>Butt, Y., Kurdowska, A., Allen, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+lung+injury:+A+clinical+and+molecular+review.">Acute lung injury: A clinical and molecular review.</a> Archives of Pathology and Laboratory Medicine. 140 (4), 345-350 (2016).</li><li>Park, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+disturb+pulmonary+microcirculation+in+sepsis-induced+acute+lung+injury.">Neutrophils disturb pulmonary microcirculation in sepsis-induced acute lung injury.</a> European Respiratory Journal. 53 (3), 1800786 (2019).</li><li>Kim, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+tracheal+epithelial+cells+in+mice+during+airway+regeneration.">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>Pieper, M., Schulz-Hildebrandt, H., Mall, M. A., H&#252;ttmann, G., K&#246;nig, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopic+optical+coherence+tomography+imaging+to+assess+mucus-mobilizing+interventions+for+muco-obstructive+lung+disease+in+mice.">Intravital microscopic optical coherence tomography imaging to assess mucus-mobilizing interventions for muco-obstructive lung disease in mice.</a> American Journal of Physiology - Lung Cellular and Molecular Physiology. 318 (3), 518-524 (2020).</li><li>Lamm, W. J. E., Bernard, S. L., Wiltz, W., Wagner, J., Glenny, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopic+observations+of+15-&#181;m+microspheres+lodging+in+the+pulmonary+microcirculation.">Intravital microscopic observations of 15-&#181;m microspheres lodging in the pulmonary microcirculation.</a> Journal of Applied Physiology. 98 (6), 2242-2248 (2005).</li><li>Entenberg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+permanent+window+for+the+murine+lung+enables+high-resolution+imaging+of+cancer+metastasis.">A permanent window for the murine lung enables high-resolution imaging of cancer metastasis.</a> Nature Methods. 15 (1), 73-80 (2018).</li><li>Amato, M. B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+protective-ventilation+strategy+on+mortality+in+the+acute+respiratory+distress+syndrome.">Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome.</a> New England Journal of Medicine. 338 (6), 347-354 (2009).</li><li>Looney, M. R., Bhattacharya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+lung.">Live imaging of the lung.</a> Annual Review of Physiology. 76, 431-445 (2013).</li></ol>

The protocol presented here requires practice and attention to a few critical steps. First, it is important to prepare the imaging window prior to initiating intubation and surgery. Use a minimal amount of vacuum grease to coat the outer ring of the imaging window, apply the cover glass, and test suction with a drop of distilled water. Preparing this in advance will prevent the exposed lung from drying out during the setup otherwise. While it is possible to flush with warm saline, doing so may risk damaging the fragile p...

Intravital microscopy (IVM) is a useful imaging tool for visualizing and studying various biophysical processes in vivo. The lung is highly challenging to image in vivo due to its enclosed location, the fragile nature of its tissue, and motion artifacts induced by respiration and heartbeat1,2. Various intravital microscopy (IVM) setups have been developed for real-time imaging of leukocyte-endothelial interactions in pulmonary microcirculation to overcome these challenges. Such approaches are based on surgically exposing and stabilizing the lung for imaging.
Animals are typically prepared for lung IVM by surgical procedures. First, animals are intubated and ventilated, which permits surgical excision of a thoracic window and subsequent interventions to stabilize the lung for imaging. One technique involves gluing the parenchyma onto a glass coverslip3, a procedure that risks significant physical trauma to the imaged tissue. More advanced is the utilization of a vacuum system to stabilize the lung under a glass window4. This setup facilitates loose adherence of the lung surface to the coverslip via a reversible vacuum spread over a large local area and expands the lung while still limiting the movement in x, y, and z dimensions4. The vacuum is applied evenly through a channel surrounding the imaging area of the setup and pulls the tissue into a shallow conical region facing the imaging-grade coverslip4. Through this viewing window, the lung microcirculation can be studied using various optical imaging modalities.
Lung IVM enables quantitative imaging of a multitude of microcirculatory parameters. These include measurements such as leukocyte track speed and length5, red blood cell flow velocity6 and oxygenation7, tumor metastases8, the distinction of immune cell subpopulations9,10,11, visualization of microparticles12, alveolar dynamics13,14, vascular permeability15, and capillary function16. The focus here is on leukocyte recruitment and capillary function. Initiation of leukocyte recruitment in the pulmonary microcirculation involves transient rolling interactions and firm adhesive interactions between leukocytes and endothelial cells, both of which are increased under inflammatory conditions16,17. Typically, rolling is quantified by the number of leukocytes that pass an operator-defined reference line, while adhesion is quantified by the number of leukocytes that are immobile on the endothelium16. Capillary function may also be affected in inflammatory states, often resulting in decreased perfusion. This can be attributed to several factors, including a reduction of red blood cell deformability18 and variegated expression of inducible NO synthase by endothelial cells resulting in pathological shunting19. Typically, the aggregate length of perfused capillaries per area is measured and reported as functional capillary density (FCD).
Studying leukocyte recruitment in the lungs in real-time requires labeling biological targets with fluorescent dyes or fluorescent-labeled antibodies20. Alternatively, various transgenic mouse strains such as lysozyme M-green fluorescent protein (LysM-GFP) mice can be utilized to image specific immune cell subsets such as neutrophils21,22. The fluorescent-labeled leukocytes can then be visualized using widefield fluorescence microscopy, confocal microscopy, or multiphoton microscopy. These techniques achieve contrast by utilizing specific excitation wavelengths and detecting emitted fluorescence while simultaneously blocking the detection of the excitation wavelength, thus highlighting the labeled object.
Existing research concerning the quantification of leukocyte rolling, adhesion, and functional capillary density in the murine lung has relied primarily on manual video analysis. This is made possible through open-source software such as Fiji6,23, proprietary software such as CapImage12, or custom-made image processing systems24. Conversely, various proprietary software platforms (e.g., NIS Element, Imaris, Volocity, MetaMorph) enable automated measurement of a wide array of other physiological parameters, including many of those previously mentioned here5,6,7,8,9,10,11,12,13,15.
Important observations have been made regarding the pathology of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) using lung IVM. ARDS is characterized by a host of pathophysiological processes in the lung, including pulmonary edema and alveolar damage caused by dysfunction of the endothelium and epithelial barrier25. Using a murine model, it has been found that sepsis-induced ALI is associated with significant detrimental changes in immune cell trafficking in the lung environment26. Neutrophils recruited to the capillaries of mice with sepsis-induced ALI were found to impede microcirculation, thereby increasing hypoxia in ALI26. Additionally, IVM has been used to gain insights into the underlying mechanism of repair following the onset of ARDS27. Lung IVM has also been a valuable tool in understanding pathophysiological changes in various obstructive lung diseases. For example, visualization of mucus transport in diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) has facilitated the study of novel and existing treatments for mucous clearance28. Leukocyte trafficking under these conditions has been analyzed as well17.
This protocol expands on the approach initially described by Lamm et al.29 to study leukocyte-endothelial interactions using conventional fluorescence microscopy. The described procedures employ an in vivo lung imaging system, which includes a 16.5 cm x 12.7 cm metal base, micromanipulator, and vacuum imaging window (Figure 1). The system is mounted in a 20 cm x 23.5 cm 3-D printed platform (Supplemental File 1) to provide secure attachment for the ventilator tubing and heating pad. This method offers reproducible and quantifiable imaging of murine pulmonary microcirculation in vivo. Important aspects of the surgical preparation as well as proper utilization of a vacuum-stabilized lung imaging system are explained in detail. Finally, an experimental model of ALI is used to provide representative imaging and analysis of altered leukocyte rolling, leukocyte adhesion, and capillary perfusion associated with inflammation. The use of this protocol should facilitate further important investigations into pathophysiological changes in pulmonary microcirculation during acute disease states.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL BD Luer Slip Tip Syringe sterile, single use</td>
			<td>Becton, Dickinson and Company</td>
			<td>309659</td>
			<td>1 mL syringe</td>
		</tr>
		<tr>
			<td>ADSON Dressing Forceps, Tip width 0.6 mm, teeth length 11.5 mm, 12 cm</td>
			<td>RWD Life Science Co.</td>
			<td>F12002-12</td>
			<td>Blunt forceps</td>
		</tr>
		<tr>
			<td>Albumin-Fluorescein Isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td>A9771-1G</td>
			<td>FITC-albumin</td>
		</tr>
		<tr>
			<td>Alcohol Swab Isopropyl Alcohol 70% v/v</td>
			<td>Canadian Custom Packaging Company</td>
			<td>80002455</td>
			<td>Alcohol wipe</td>
		</tr>
		<tr>
			<td>AVDC110 Advanced Digital Video Converter</td>
			<td>Canopus</td>
			<td>00631069602029</td>
			<td>Digital video converter</td>
		</tr>
		<tr>
			<td>B/W - CCD - Camera</td>
			<td>Horn Imaging</td>
			<td>BC-71</td>
			<td>Camera</td>
		</tr>
		<tr>
			<td>Bovie Deluxe High Temperature Cautery Kit</td>
			<td>Fine Science Tools</td>
			<td>18010-00</td>
			<td>Cauterizer</td>
		</tr>
		<tr>
			<td>C57BL/6 Mice</td>
			<td>Charles River Laboratories International</td>
			<td>C57BL/6NCrl</td>
			<td>C57BL/6 Mice</td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicators</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td>Cotton applicator</td>
		</tr>
		<tr>
			<td>CS-8R 8mm Round Glass Coverslip</td>
			<td>Warner Instruments</td>
			<td>64-0701</td>
			<td>Glass coverslip</td>
		</tr>
		<tr>
			<td>Digital Pressure Gauge</td>
			<td>ITM Instruments Inc.</td>
			<td>DG2551L0NAM02L0IM&#38;V</td>
			<td>Digital Pressure Gauge</td>
		</tr>
		<tr>
			<td>Dr Mom Slimline Stainless LED Otoscope</td>
			<td>Dr. Mom Otoscopes</td>
			<td>1001</td>
			<td>Otoscope</td>
		</tr>
		<tr>
			<td>Ethyl Alchohol 95% Vol</td>
			<td>Commercial Alcohols</td>
			<td>P016EA95</td>
			<td>95% ethanol</td>
		</tr>
		<tr>
			<td>Fine Scissors - Martensitic Stainless Steel</td>
			<td>Fine Science Tools</td>
			<td>14094-11</td>
			<td>Scissors</td>
		</tr>
		<tr>
			<td>Fisherbrand Colored Labeling Tape</td>
			<td>Fisher Scientific</td>
			<td>1590110</td>
			<td>Labeling tape</td>
		</tr>
		<tr>
			<td>Gast DOA-P704-AA High-Capacity Vacuum Pump</td>
			<td>Cole-Parmer Canada Company</td>
			<td>ZA-07061-40</td>
			<td>Vacuum pump</td>
		</tr>
		<tr>
			<td>Hartman Hemostats</td>
			<td>Fine Science Tools</td>
			<td>13003-10</td>
			<td>Hemostatic forceps</td>
		</tr>
		<tr>
			<td>High Vacuum Grease</td>
			<td>Dow Corning</td>
			<td>DC976VF</td>
			<td>Vacuum grease</td>
		</tr>
		<tr>
			<td>Isoflurane USP</td>
			<td>Fresenius Kabi</td>
			<td>CP0406V2</td>
			<td>Isoflurane</td>
		</tr>
		<tr>
			<td>LIDOcaine HCl Injection 1% 50 mg/5 mL</td>
			<td>Teligent Canada</td>
			<td>0121AD01</td>
			<td>Lidocaine HCl 1%</td>
		</tr>
		<tr>
			<td>Lung SurgiBoard</td>
			<td>Luxidea, Inc.</td>
			<td>IMCH-0001</td>
			<td>Designed for intravital microscopy of the lung</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Teva Canada</td>
			<td>00485802</td>
			<td>Mineral oil</td>
		</tr>
		<tr>
			<td>Mouse Endotracheal Intubation Kit</td>
			<td>Kent Scientific Corporation</td>
			<td>ETI-MSE</td>
			<td>Intubation stand, anesthesia mask, 20 G endotracheal cannula, fibre optic cable</td>
		</tr>
		<tr>
			<td>MST49 Fluorescence Microscope</td>
			<td>Leica Microsystems</td>
			<td>10 450 022</td>
			<td>Fluorescence Microscope</td>
		</tr>
		<tr>
			<td>N Plan L 20x/0.40 Long Working Distance Microscope Objective</td>
			<td>Leica Microsystems</td>
			<td>566035</td>
			<td>20x objective</td>
		</tr>
		<tr>
			<td>Non-Woven Sponges 2&#34; x 2&#34;</td>
			<td>AMD-Ritmed</td>
			<td>A2101-CH</td>
			<td>Gauze</td>
		</tr>
		<tr>
			<td>Optixcare Eye Lube Plus</td>
			<td>Aventix</td>
			<td>5914322</td>
			<td>Tear gel</td>
		</tr>
		<tr>
			<td>Original Prusa i3 MK3S+ 3D Printer</td>
			<td>Prusa Research</td>
			<td>PRI-MK3S-KIT-ORG-PEI</td>
			<td>3D printer</td>
		</tr>
		<tr>
			<td>Oxygen, Compressed</td>
			<td>Linde Canada Inc.</td>
			<td></td>
			<td>Oxygen</td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 30 G x 1/2 (0.3 mm x 13 mm)</td>
			<td>Becton, Dickinson and Company</td>
			<td>305106</td>
			<td>30 G needle</td>
		</tr>
		<tr>
			<td>Pyrex 5340-2L 5340 Filtering Flasks, 2000 mL</td>
			<td>Cole-Parmer Canada Company</td>
			<td>5340-2L</td>
			<td>Vacuum flask</td>
		</tr>
		<tr>
			<td>Rhodamine 6 G</td>
			<td>Sigma-Aldrich</td>
			<td>252433</td>
			<td>Rhodamine 6G</td>
		</tr>
		<tr>
			<td>Secure Soft Cloth Medical Tape - 3&#34;</td>
			<td>Primed</td>
			<td>PM5-630709</td>
			<td>Cloth tape</td>
		</tr>
		<tr>
			<td>Silastic Medical Grade Tubing .040 in. ID x .085 in. OD</td>
			<td>Dow Corning</td>
			<td>602-205</td>
			<td>1.0 mm I.D. polyethylene tubing</td>
		</tr>
		<tr>
			<td>Somnosuite Low-Flow Anesthesia System</td>
			<td>Kent Scientific Corporation</td>
			<td>SS-01, SS-04-module</td>
			<td>Small rodent ventilator, Low-flow anesthesia system, Heating pad, Rectal temperature probe, Pulse oximeter</td>
		</tr>
		<tr>
			<td>Tissue Forceps, 12.5cm long, Curved, 1 x 2 Teeth</td>
			<td>World Precision Instruments</td>
			<td>501216</td>
			<td>Toothed forceps</td>
		</tr>
		<tr>
			<td>Transpore Medical Tape, 1527-1, 1 in x 10 yd (2.5 cm x 9.1 m)</td>
			<td>3M</td>
			<td>7000002795</td>
			<td>Medical tape</td>
		</tr>
		<tr>
			<td>Tubing,Clear,3/8 in Inside Dia.</td>
			<td>Grainger Canada</td>
			<td>USSZUSA-HT3314</td>
			<td>1.0 cm I.D. polyethylene tubing</td>
		</tr>
		<tr>
			<td>Whatman 6720-5002 50 mm In-Line Filters, PTFE, 0.2 &#181;m</td>
			<td>Cole-Parmer Canada Company</td>
			<td>6720-5002</td>
			<td>Inline 0.2&#181;m filter</td>
		</tr>
	</tbody>
</table>

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.

To illustrate results achievable through this protocol, acute lung injury (ALI) was induced 6 h prior to imaging using a model of intranasal bacterial lipopolysaccharide (LPS) instillation. Briefly, mice (n = 3) were anesthetized with isoflurane, and small droplets of LPS from Pseudomonas aeruginosa in sterile saline (10 mg/mL) were pipetted into the left naris at a dosage of 5 mg/kg. This was compared to na&#239;ve mice (n = 3; no intranasal administration).
Upon imaging, a successfu...

Watch this Scientific Journal Video about Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System at JoVE.

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

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 ...

This protocol describes in detail, the surgical preparation and use of a vacuum-stabilized system to image pulmonary leukocyte adhesion and capillary perfusion in vivo. The main advantage of this technique is that it allows stable high resolution intravital imaging of the intact mirroring lung with minimal physical trauma. To begin, prepare the imaging window by administering a thin layer of vacuum grease to the top of the outer ring while avoiding contamination of the vacuum channel.Place a clean 8-millimeter glass cover slip on the window and gently press down to create a seal. Connect the imaging window to a vacuum pump fitted with a digital pressure gauge and capable of providing 50 to 60 millimeters of mercury-constant suction. Pass a length of fiber optic cable through a 20-gauge endotracheal cannula and submerge the tip in lidocaine HCL.After anesthesia, insert a modified otoscope, such that the upper incisors fit within the gap in the speculum. Adjust the scope and tongue position until the epiglottis and the vocal cords are clearly visible. Insert the fiber optic cable through the gap in the speculum and use small circular movements to pass the cable between the vocal cords, then using the fiber optic cable as a guide, gently slide the cannula into the trachea to intubate the mouse.After starting ventilation with 1.5%isoflurane, confirm the depth of anesthesia by testing for pedal reflex. Secure the cannula to the snout with medical tape. Use labeling tape to secure the right forepaw to the heating pad at the nine o'clock position and extend the left hind paw caudally and secure it at the six o'clock position.Using a cloth tape, lightly stretch the left forepaw to the 12 o'clock position and secure the other end of the tape to the top of the intravital microscopy platform, then apply a rectal temperature probe and paw pulse oximeter to monitor the vital signs throughout the experiment. To prepare the mouse for surgery, sterilize the thorax and abdomen with a 70%alcohol wipe and apply light coat of mineral oil to dampen the hair on the left side of the mouse. Make a small incision near the bottom of the rib cage to expose the underlying muscle layer, then extend the initial incision and use blunt dissection to expose the rib cage.Using hemostatic forceps, grasp the dissected epithelial and adipose tissue and place clear of the surgical area. To fluorescently label leukocytes and blood flow, administer a 2.5-milliliter per kilogram bolus of Rhodamine 6G and FITC-albumin via tail vein. Using toothed forceps, grasp the rib immediately inferior to the position of the base of the lung at end inspiration.Slightly retract the forceps to pull the rib away from the lung and then cut the rib to induce a pneumothorax. Extend the incision laterally in both directions, taking care not to touch the lung. Grasp the next highest rib with blunt forceps and slightly retract to allow the lung to fall away from the chest wall.If the lung does not detach, press the chest wall lightly against the lung to cause the lung to adhere to the underlying pleura and thus fall away more easily. Continue the original incision eventually until the sternum and cranially until the apex of the lung is exposed. Use cotton applicators and gauze to attenuate any bleeding that arises.Raise the rib cage to expose the intercostal blood vessels on the dorsal aspect of the thoracic cavity, taking care not to damage the lung, cauterize the most inferior intercostal vessel near the spinal column, then cut the adjoining rib. Moving cranially, and eventually, use a repeated pattern of cauterization and cutting to excise an approximately one-by-two centimeter portion of the ribcage. To prepare for imaging, transfer the intravital microscopy platform to the microscope stage and position the imaging window directly above the exposed lung.Use the micro manipulator to carefully lower the imaging window until it adheres to and stabilizes the lung surface. To visualize the pulmonary microcirculation, use a wide field fluorescence microscope fitted with a 20x objective and standard FITC and Rhodamine filter sets. Use a black and white CCD camera to record the videos with optimal contrast.Using the FITC filter set, identify a pulmonary venue based on the convergent pattern of blood flow and record a 30-second video with the venue in focus, then repeat the recording in the same field of view using the Rhodamine filter set to visualize leukocytes. To locate a pulmonary arterial, use the FITC filter set to identify a vessel with a divergent pattern of blood flow and record a 30-second video. Repeat the recording in the same field of view using the Rhodamine filter set to visualize leukocytes.To locate the capillary regions of interest, use the FITC filter set to identify an area of alveoli and capillaries not intersected by larger vessels and record a 30-second video. Repeat the recording in the same field of view using the Rhodamine filter set to visualize leukocytes. This video depicts FITC imaging of blood flow within a pulmonary venue as indicated by the green arrow.Rhodamine imaging permits visualization of leukocytes in the same venue with two adherent leukocytes indicated by the red arrows. These methods were also applied to visualize the same phenomena and pulmonary arterials and capillary regions of interest. To demonstrate the quantification of microcirculatory parameters, mice were treated with intranasal LPS to induce pulmonary inflammation.Leukocyte trafficking in pulmonary venues is shown here with outlined areas representing the analyzed endothelial regions in naive and LPS-treated mice. Leukocyte adhesion and rolling were increased in LPS-treated mice. Leukocyte trafficking in pulmonary arterials is shown here with outlined areas representing the analyzed endothelial regions in naive and LPS-treated mice.Leukocyte adhesion was higher in LPS-treated mice. This figure shows leukocyte adhesion within pulmonary capillaries in naive and LPS-treated mice. Leukocyte adhesion was shown to be higher in LPS-treated mice versus naive.This figure shows perfusion of pulmonary capillaries in naive and LPS-treated mice. Functional capillary density was shown to be lower in LPS-treated mice versus naive. There are a lot of challenging aspects of this protocol.Optimizing the mouse's positioning on the platform is a simple step that can make the surgical and imaging steps much more reproducible. This procedure is adaptable to a wide range of study parameters and microscopy approaches. Therefore, it should facilitate further research of pulmonary microcirculation in both healthy and diseased states.

intravital-widefield-fluorescence-microscopy-pulmonary

Research

JoVE Journal

Immunology and Infection

2.6K vistas.  Dalhousie University. Este protocolo describe en detalle, la preparación quirúrgica y el uso de un sistema estabilizado al vacío para obtener imágenes de la adhesión de leucocitos pulmonares y la perfusión capilar in vivo. La principal ventaja de esta técnica es que permite imágenes intravitales estables de alta resolución del pulmón reflejado intacto con un trauma físico mínimo. Para comenzar, prepare la ventana de imágenes administrando una capa delgada de grasa al vacío en la parte superior del an...

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