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

11.8K Views.  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.