The authors thank the Central Animal facility of the University Hospital Aachen for technical support. This work was supported by the German Research Foundation (DFG Ta434/2-1, DFG SFB/TRR 57) and by the Interdisciplinary Center for Clinical Research (IZKF) Aachen. This work was further supported by the Core Facility &#8221;Two-Photon Imaging&#8221;, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University.

NOTE: The experiments were performed in accordance with the German legislation governing animal studies following the &#8216;Guide for the care and use of Laboratory Animals&#8217; (NIH publication, 8th edition, 2011) and the Directive 2010/63/EU on the protection of animals used for scientific purposes (Official Journal of the European Union, 2010). Official permission was granted from the governmental animal care and use office (LANUV Nordrhein-Westfalen, Recklinghausen, Germany).
NOTE: Steps that can be omitted for short term imaging (e.g. snap shots, 3-D stacks or also short duration time-lapse microscopy) are marked with asterisks (*) to reduce preparation time and simplify the surgical protocol. Imaging can also be performed without extensive monitoring and circulation control if necessary, however, survival time will be markedly reduced.
1. Microscope Setup and Pre-surgery Preparation (5-10 min)
<ol>
	<li>Switch system on (microscope, power Lab, laser, heating pad, climate chamber, web cam, syringe pump device, respirator).</li>
	<li>Connect O2 supply to Isoflurane Vapor and set Isoflurane level to 1.5 Vol% (v/v), connect to a small animal respirator.
	<ol>
		<li>For short term imaging, maintain the anesthesia using isoflurane only after the initial i.p. anesthesia induction (see step 2.2). Then use 2 Vol% (v/v) Isoflurane, and keep the imaging time below 2 hr to guarantee stable liver microcirculation.</li>
	</ol>
	</li>
	<li>Set respiration to 120-140 respiratory cycles/min&#160;with a volume of 150-200 &#181;l.</li>
	<li>Set up mouse agarose stage. Prepare 100 ml of 3% (w/v) agarose, pouring it into a dish (approximately 10 cm x 15 cm base) in an angle of 40&#176;.</li>
	<li>Set up surgical work area collecting the required instrumentation.&#160;To prevent microbial infection, disinfect instruments using a 1% solution of Sekusept Forte S (9.9% w/v), Glutaral 9.8% w/v, Formaldehyde for 5 min before the experiment (Figure 1A).</li>
	<li>Obtain remaining components: skin disinfectant (e.g., 10% povidone iodine solution ), liver stage (stacks &#38; bridge), agarose solution (3% (w/v) in PBS), syringe pump with 5% (w/v) glucose solution (G-5), syringe pump with anesthetic solution I (Ketamine 0.1 mg/ml, Xylazine 0.5 mg/ml, Fentanyl 5 &#181;g/ml), cotton swabs, n-Butyl-2-Cyanoacrylat, and 1x PBS. Put agarose stage dish into incubation chamber for preheating.</li>
</ol>
2. Tracheotomy (10-15 min)
<ol>
	<li>Use C57BL/6 mice from in house breeding weighing 25-28 g. House the mice under specific pathogen-free conditions in accordance with the FELASA guidelines, in a temperature- and humidity-controlled environment with a 12 hr light/12 hr dark cycle. Allow animals free access to standard mice diet (Sniff Standard Rodent Diet) and water ad libitum.</li>
	<li>Anesthetize the mouse by initial intraperitoneal (i.p.) injection of 250 &#181;l of anesthetic solution II (Ketamine 0.1 mg/ml, Xylazine 1 mg/ml, Buprenorphine 10 &#181;g/ml).
	<ol>
		<li>* For upkeep during intravital imaging, continuously administer anesthetic solution I by continuous i.p. injection (Ketamine 0.1 mg/ml, Xylazine 0.5 mg/ml, Fentanyl 5 &#181;g/ml) using a syringe pump device with a flow rate of 0.2 ml/hr&#160;in combination with inhalative isoflurane 0.5 Vol% (v/v).</li>
		<li>Check reflexes after 5 min (e.g. pinch foot pad with forceps), disinfect skin for tracheotomy, start preparation if mouse is in surgical tolerant anesthesia state.</li>
		<li>Apply eye protective cream (e.g., Dexpanthenol 50 mg/g) to prevent cornea from drying out (Figure 1B).</li>
	</ol>
	</li>
	<li>Fixate mouse on preparation table exposing ventral side using adhesive tape for the extremities; gently overstretch neck, fixate in this position, e.g., by using a rubber band hooked to the incisors.
	<ol>
		<li>Disinfect skin and fur using povidone iodine solution, by applying disinfectant with a cotton swab.</li>
	</ol>
	</li>
	<li>Perform initial skin cut (0.5-1 cm length) directly below chin (Figure 1C)
	<ol>
		<li>Carefully dissect connective tissue between salivary glands (Figure 1D).</li>
		<li>Carefully tear open muscular tube surrounding trachea (Figure 1E,F).</li>
	</ol>
	</li>
	<li>Place surgical thread underneath the trachea for later ventilation tube fixation (Figure 1G).
	<ol>
		<li>Open trachea with micro-scissors between cartilage rings performing a T-shaped incision (Figure 1H)</li>
	</ol>
	</li>
	<li>Place ventilation tube into trachea through the incision (~0.5 cm). To push the tube forward, grab caudal end with small anatomical forceps and gently advance the tube into the trachea (Figure 1I)</li>
	<li>Fixate tube with surgical thread (e.g., 5-0) with thread placed in Step 2.5 tying a knot around tube inside trachea; perform second cranial fixation of the tube at the skin to avoid accidental depositioning (Figure 1J,K).</li>
	<li>Seal cut with tissue glue (e.g., n-Butyl-2-Cyanoacrylat) (Figure 1L).</li>
	<li>Fixate tube at the head using adhesive tape .</li>
</ol>
3. Laparatomy (15-20 min)
<ol>
	<li>Place mouse on heating pad to prevent hypothermia. Shave abdomen, carefully remove hair from the skin. Disinfect shaved skin and fur by using povidone iodine solution.</li>
	<li>Perform a small skin cut below the sternum using surgical scissors (Figure 2A). Extend the cut laterally below the ribs to both sides, cauterizing all visible blood vessels to prevent bleeding.</li>
	<li>Carefully perform a small cut at the linea alba below the sternum, opening the peritoneum (Figure 2B). Extend the cut to both sides using cauterization to prevent bleeding (Figure 2C, D).</li>
	<li>Place mouse into agarose stage dish (Figure 2E). Place stacks of appropriate height on both sides of the mouse (usually 12-14 cover slips) (Figure 2F).</li>
	<li>Place respiratory trigger sensor underneath upper back to synchronize the microscope with the respiration. Activate trigger unit (Figure 2G).</li>
	<li>Place surgical thread (5-0) through the sternum to retract ribs (Figure 2I).</li>
	<li>Carefully cut ligament connecting liver and diaphragm (falciform ligament) as well as liver and gastrointestinal tract using curved surgical scissors. Cut the falciform ligament down to the aorta (Figure 2J).</li>
</ol>
4. Sample Setup (10-15 min)
<ol>
	<li>Place mouse on the right side with an angle of 45&#176; for easy access to large liver lobe.</li>
	<li>Add staging bridge below the ribs, covering the abdominal cavity. Manufacture a bridge by using a standard cover slip with adhesive tape coating to cover sharp edges (Figure 2K).</li>
	<li>Place large liver lobe on stage. Carefully slip double ball stylus probe or a padded spatula below the liver and hold the top of the organ using a wet cotton swab or wet tissue. Lift lobe onto the slide and apply gentle drag. Lobe can be bent or folded. Provide extra care to only gentle manipulation of the liver (Figure 2L).</li>
	<li>Place lateral supporting stakes, e.g., piles of small cover slips (20 mm x 20 mm), of approximately the same height of the liver lobe next to it to support cover slip.</li>
	<li>Place large cover slip (24 mm x 50 mm) on the liver lobe. Ensure that cover slip is oriented as horizontal as possible (Figure 2M). 
	NOTE: The cover slip should be in contact with the tissue without squeezing it. Check for visible signs of impaired blood flow (white tissue). If microcirculation is disrupted, add further supporting stakes.</li>
	<li>* Place two independent intraperitoneal catheters (Figure 2N) for long term anesthesia and G-5 application. Install catheters laterally in the lower abdomen next to the hind limbs. 
	NOTE: The intraperitoneal catheters can be self-made by connecting 27 G needles with flexible silicone tubing (Figure 3).</li>
	<li>* Fixate needle with a loop of 5-0 surgical thread to the skin to avoid accidental displacement.</li>
	<li>* Attach syringe pump with anesthesia solution I to catheter 1, set flow rate to 0.2 ml/hr; attach syringe pump with G-5 to catheter 2, set flow rate to 0.1 ml/hr.</li>
</ol>
5. Mouse Monitoring
<ol>
	<li>* Place ECG electrodes into front and hind extremities (Figure 4A).</li>
	<li>* Attach expiration tubing to CO2 level sensor.</li>
	<li>Add external temperature sensor connected to heat pad (Figure 4C).</li>
</ol>
6. Embedding and Tissue Fixation (5-10 min)
<ol>
	<li>Prepare 100 ml of 3% (w/v) agarose in 1X PBS. Embed liver when temperature is at 41 &#176;C using a 5 ml syringe and an 18 G needle (Figure 5A).</li>
	<li>Pour remaining agarose on coverslip and around the mouse (Figure 5B, C). Wait until agarose is fully gelatinized.</li>
	<li>Remove excess agarose using the Heidemann spatula, preparing a viewfield large enough to scan the prepared liver lobe (Figure 5D, E).</li>
</ol>
7. Imaging
<ol>
	<li>Transfer the mouse into microscope climate chamber.</li>
	<li>Add 50-100 ml of 1x PBS (preheated on 37 &#176;C).</li>
	<li>Cover sample dish to prevent evaporation (Figure 5F).</li>
	<li>* Decrease inhalative isoflurane to 0.5 Vol% (v/v).</li>
	<li>* Start monitoring software, recording ECG, heart rate and expiratory CO2.</li>
	<li>Start imaging: open laser shutters, identify view fields of interest, define upper and lower boundary for Z-stacks, and start time lapse recording. Readjust Z-stacks if necessary to correct Z-drift (e.g. due to changes in blood pressure or temperature fluctuation, Figure (5G). 
	NOTE: After completion of the imaging period or if the circulation or anesthesia of the animals becomes unstable, sacrifice mice by cervical dislocation (without awakening from the anesthesia).</li>
</ol>

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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=NKT-associated+hedgehog+and+osteopontin+drive+fibrogenesis+in+non-alcoholic+fatty+liver+disease.">NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease.</a> Gut. 61 (9), 1323-1329 (2012).</li><li>McDonald, B., 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=Interaction+of+CD44+and+hyaluronan+is+the+dominant+mechanism+for+neutrophil+sequestration+in+inflamed+liver+sinusoids.">Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids.</a> J Exp Med. 205 (4), 915-927 (2008).</li><li>Egen, J. 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=Intravital+imaging+reveals+limited+antigen+presentation+and+T+cell+effector+function+in+mycobacterial+granulomas.">Intravital imaging reveals limited antigen presentation and T cell effector function in mycobacterial granulomas.</a> Immunity. 34 (5), 807-819 (2011).</li><li>Singer, G., Stokes, K. Y., Granger, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+microcirculation+in+murine+sepsis:+role+of+lymphocytes.">Hepatic microcirculation in murine sepsis: role of lymphocytes.</a> Pediatr Surg Int. 24 (1), 13-20 (2008).</li><li>Phillipson, M., 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=The+neutrophil+in+vascular+inflammation.">The neutrophil in vascular inflammation.</a> Nat Med. 17 (11), 1381-1390 (2011).</li><li>Khandoga, A. 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=In+vivo+imaging+and+quantitative+analysis+of+leukocyte+directional+migration+and+polarization+in+inflamed+tissue.">In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue.</a> PLoS One. 4 (3), e4693 (2009).</li></ol>

The authors disclose no con&#64258;ict of interests.

The aim of our study was to develop a highly standardized, stable and reproducible method for intravital TPLSM imaging of the liver. Intravital imaging in general has given valuable insights into cellular behavior under real life conditions following homing and interaction of different leukocyte populations in development, homeostasis and disease. However, the somewhat challenging anatomical position of the liver, due to which respiratory and peristaltic intestinal movement directly are transmitted to the liver as well a...

The visualization of cells and cellular functions in whole organs or even whole organisms has been of great interest for more than 50 years, including virtually all parts of the body2. Therefore, some early studies already employed intravital imaging of the liver3,4. However, several limitations exist up to date regarding long term stable high-resolution imaging of liver tissue.
Due to the anatomical position of the liver in close contact with the diaphragm and the gastrointestinal tract5, the most common problem for microscopic intravital imaging is movement due to respiration and, to a lesser extent, peristaltic of the intestinal tract6. In comparison to other solid organs, liver surgery is particularly challenging. Due to the dense microvascular structure, surgical manipulation can lead to massive hemorrhagic lesions, impaired microcirculation7 and also activation of resident immune cells such as Kupffer cells8. Therefore, mechanical fixation of the tissue as published elsewhere6,9 is likely to interfere with the intravital microscopy imaging.
In a healthy liver, 10-15% of the total blood volume resides within the liver vasculature, and the organ receives around 25% of the overall cardiac output10, rendering the organ highly susceptible to changes in the circulation (e.g., blood pressure fluctuations). Therefore, disruptions in the hepatic blood flow due to e.g., shear stress, displacement, injury by excessive tissue handling or centralized circulation will lead to artificial alterations in leukocyte migratory behavior, impaired liver oxygenation and therefore further liver damage, affecting liver immune responses as well as organ preservation and overall life time of the animal.
Early microscopic&#160;studies were based on intravital epifluorescence microscopy, but several technical constraints such as photo bleaching and low penetration depth limit the use of this technique for long term liver imaging4,11,12. With the development of multiphoton microscopy in the 1990s, the limitations of photo bleaching or penetration depth were mainly solved, as this new method was technically capable to perform imaging studies in virtually all organs under real life situations13-15. However, the main remaining challenges with respect to liver imaging were: breath movements, autofluorescence of liver tissue, securing unaltered blood flow in the hepatic sinusoids, and especially stable imaging for longer periods of several hr16.
Although several studies addressed the function and migration of various leukocytes in the liver17, e.g., NKT-cells18-20, T cells21,22, liver macrophages23,24 or neutrophils25, long term multiphoton microscopy imaging had not yet been successfully established, a task even more challenging in animals with acute or chronic liver disease due to the existing damage and therefore higher susceptibility to further damage26. However, monitoring migratory behavior and cellular function of leukocytes in the liver in real time allows novel insights in their particular role in liver homeostasis and disease27.
The chemokine receptor CXCR6 is expressed on several lymphocyte subsets, including natural killer (NK) cells, NKT cells and some T cell populations18,28. Prior studies in mice have indicated that CXCR6 and its cognate ligand CXCL16 may control the patrolling of NKT cells on liver sinusoids during homeostasis. Consequently, the use of CXCR6.gfp mice (carrying a knock-in of green fluorescent protein [gfp] in the CXCR6 locus) has been described to investigate the migration of lymphocytes in various organs such as brain29 and also liver18,20, showing increased infiltration of CXCR6.gfp cells upon inflammation.
With the method provided in this study it was possible to follow these processes over a long period of time under stabilized conditions. The intravital multiphoton based procedure allowed imaging that was highly reproducible with minimal perturbation of the animal and the organ; optimized for long-term animal survival by extensive monitoring followed by close control of respiration and circulation; and highly flexible and easy to adopt also to other parenchymal organs such as kidney or spleen.

<table border="0" cellpadding="0" cellspacing="0" width="1184">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Anesthetics</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td style="width:293px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Buprenorphine</td>
			<td style="width:263px;">Essex Pharma</td>
			<td style="width:219px;">997.00.00</td>
			<td>Analgeticum, 0.1 mg/kg</td>
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			<td height="21" style="height:21px;width:411px;">Fentanyl</td>
			<td style="width:263px;">Rotex Medica</td>
			<td style="width:219px;">charge: 30819</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fluovac anesthesia system</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td style="width:219px;">34-1030</td>
			<td></td>
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			<td height="21" style="height:21px;width:411px;">Glucose 5%</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">ISOFLO (Isoflurane Vapor) vaporiser</td>
			<td style="width:263px;">Eickemeyer</td>
			<td style="width:219px;">4802885</td>
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			<td height="21" style="height:21px;width:411px;">Isoflurane</td>
			<td style="width:263px;">Forene Abbott</td>
			<td style="width:219px;">B 506</td>
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			<td height="21" style="height:21px;width:411px;">Isotonic (0.9%) NaCl solution</td>
			<td style="width:263px;">DeltaSelect GmbH</td>
			<td style="width:219px;">PZN 00765145</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Ketamin 10%</td>
			<td style="width:263px;">ceva</td>
			<td style="width:219px;">Charge: 36217/09</td>
			<td></td>
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			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
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			<td></td>
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			<td height="21" style="height:21px;width:411px;">Xylazin 2%</td>
			<td style="width:263px;">medistar</td>
			<td style="width:219px;">Charge: 04-03-9338/23</td>
			<td></td>
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			<td style="width:263px;"></td>
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			<td height="21" style="height:21px;width:411px;">Consumable supplies</td>
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			<td style="width:219px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:411px;">20ml Syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">250ml Erlenmeyer flask</td>
			<td style="width:263px;">Schott Duran</td>
			<td>21 226 36</td>
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			<td height="21" style="height:21px;width:411px;">25mL Beaker 2x</td>
			<td style="width:263px;">Schott Duran</td>
			<td style="width:219px;">50-1150</td>
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			<td height="21" style="height:21px;width:411px;">2ml syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl suture</td>
			<td style="width:263px;">Ethicon</td>
			<td style="width:219px;">V7980</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Agarose</td>
			<td style="width:263px;">commercially available</td>
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			<td height="21" style="height:21px;width:411px;">Bepanthen Eye and Nose ointment</td>
			<td style="width:263px;">Bayer Vital GmbH</td>
			<td style="width:219px;">6029009.00.00</td>
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			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">18010-00</td>
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			<td height="21" style="height:21px;width:411px;">Cotton Gauze swabs</td>
			<td style="width:263px;">Fuhrmann GmbH</td>
			<td style="width:219px;">32014</td>
			<td></td>
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			<td height="21" style="height:21px;width:411px;">Cover Slip 24x50mm</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1871</td>
			<td></td>
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			<td height="21" style="height:21px;width:411px;">Durapore silk tape</td>
			<td style="width:263px;">3M</td>
			<td style="width:219px;">1538-1</td>
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			<td height="21" style="height:21px;width:411px;">Feather disposable scalpel</td>
			<td style="width:263px;">Feather</td>
			<td style="width:219px;">02.001.30.011</td>
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			<td height="21" style="height:21px;width:411px;">Fine Bore Polythene Tubing 0,58mm ID</td>
			<td style="width:263px;">Smiths medical</td>
			<td style="width:219px;">800/100/200</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Histoacryl</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;">1050052</td>
			<td>5x 0,5ml</td>
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			<td height="21" style="height:21px;width:411px;">Leukoplast</td>
			<td style="width:263px;">BSN Medical Inc.</td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Microscope Slides</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1879</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Poly-Alcohol Haut&#8230;farblos Antisepticum</td>
			<td style="width:263px;">Antiseptica GmbH</td>
			<td style="width:219px;">72PAH200</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 18 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">304622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 27 3/4 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">4657705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Tissue paper</td>
			<td style="width:263px;">commercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Surgical Instruments</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amalgam burnisher 3PL</td>
			<td style="width:263px;">Gatz</td>
			<td style="width:219px;">0110?</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blair retractors (4 pronged (blunt)) x2</td>
			<td style="width:263px;">Storz&#38;Klein</td>
			<td style="width:219px;">S-01134</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dumont No.7 forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">91197-00</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Graefe forceps curved x1</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11151-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Graefe forceps straight x2</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11050-10</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Heidemann spatula HD2</td>
			<td style="width:263px;">Stoma</td>
			<td style="width:219px;">2030.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Needle holder Mathieu</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">12010-14</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Scissor</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14074-11</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Semken forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11008-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors curved</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14029-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors straight</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14028-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Standard pattern forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11000-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas spring scissors</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">15000-08</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td style="width:263px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">ECG Trigger Unit</td>
			<td style="width:263px;">Rapid Biomedical</td>
			<td style="width:219px;">3000003686</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">MICROCAPSTAR End-Tidal Carbon Dioxide Analyzer</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Minivent Typ 845</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td>73-0043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Multiphoton microscope Trimscope I</td>
			<td style="width:263px;">LaVision</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Perfusor Compact</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">PowerLab 8/30 8 channel recorder</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;">PL3508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature controlled heating pad</td>
			<td style="width:263px;">Sygonix</td>
			<td style="width:219px;">26857617</td>
			<td></td>
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long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using cxcr6.gfp reporter mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

To validate our intravital TPLSM approach, we subjected CXCR6gfp/+ mice to intravital TPLSM imaging. Mice were either left untreated as baseline controls or subjected to a single intraperitoneal injection of carbon tetrachloride (CCl4) to induce acute liver damage20.
Video sequences were taken over a time period of 2-5 hr, and cells were traced over time due to their green fluorescence. To show general cellular motility, all tracks that were detected during the...

Watch this Scientific Journal Video about Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice at JoVE.com

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

long-term-intravital-multiphoton-microscopy-imaging-immune-cells

Research

JoVE Journal

Immunology and Infection

11.9K vues.  RWTH University-Hospital Aachen. Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Vidéo: Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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 Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

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 ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...