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 as their high fragility compared to other solid organs imposed several challenges for stable long term intravital imaging. Over recent years, many experimental studies in mice have revealed the highly dynamic nature of immune cell infiltration in injured liver28, with major contributions from resident or infiltrating monocytes/macrophages30-32, neutrophils33 or various lymphocyte subsets34,35. However, most of these data were derived from end-point analyses (e.g., multicolor flow cytometry after sacrificing the animals). Up to now only few studies exist describing the dynamics of cellular traffic during liver inflammation using multicolor intravital imaging. Using inverted microscopes circumvents the necessity for fixation and moisturizing of the liver due to the firm adhesion of the tissue to the glass bottom24. However, since many multiphoton microscopes are built as upright imaging systems, more elaborate preparation and fixation methods are needed to stabilize the tissue for the time of imaging. Commonly, custom made staging systems have been described for tissue fixation6, 36,37, however with these it is crucial to validate the liver microcirculation, since even minimal pressure on the tissue affects sinusoidal blood flow with even more severe effects on diseased liver38.
Therefore, the following points were addressed in the setup of this method to optimize the results obtained by in vivo TPLSM imaging: Enhancing the quality of the preparation by minimized sample perturbation and disruption of the microcirculation due to handling issues and increased animal survival; intensive care monitoring and subsequent adaptation of anesthesia, respiration and stabilization of the circulation further enhanced animal survival and minimized physiological artifacts, e.g., caused by imbalances in blood pH. Controlled respiration in combination with triggered imaging was essential to eliminate movement artifacts due to synchronization of the microscope with the respiratory cycles. The embedding of the organ in agarose containing physiological salt concentrations was beneficial to gently fixate the organ after preparation without any further mechanical manipulation and prevented also dehydration of the sample. Most protocols used for liver microscopy provide only insufficient methods for stable, long term anesthesia. However, the survival of the animals could be significantly enhanced with the anesthesia protocol provided here and in combination with the monitoring was sufficient to guarantee survival up to 8 hr. Alternative methods are generally suitable to visualize processes within a time period of up to 3 hours such as neutrophil invasion39, but lack the possibility to image longer lasting processes such as monocyte or lymphocyte invasion22 or even cell proliferation6. Understanding the dynamics of recruitment, infiltration and cellular interaction during acute and chronic liver inflammation can provide more detailed insight into the development and progression of liver disease. By using this enhanced understanding of the immunopathology it will be possible to design therapies much more refined in terms of addressing the target cells of interest rather than using e.g. non-selective immunosuppressive approaches.
To address the positioning, transmigration and interaction of leukocytes, high quality images are needed for accurate 4D tracking of the cells within the liver vasculature and liver tissue18,20,40.
The method we provide here can be applied using common laboratory reagents and equipment, making it both easy to handle and cost efficient with minimal perturbation of the sample. To maintain the physiological conditions of the mouse for as long as possible, it is necessary to fine-adjust the respiratory volume and frequency to control oxygenation and blood CO2 levels. Although due to the continuous infusion of liquids into the peritoneum there is at least a tentative supply to stabilize the circulation, ECG and heart frequency measurements only allow a partial control regarding stabilization of the circulation. By implementing direct blood pressure control and central venous liquid application it is likely that the vital parameters can be stabilized even more, leading to further enhancement of stability and survival.
Even under highly controlled conditions, imaging of animals which have been subjected to liver damage models commonly used in mice such as acetaminophen induced liver damage, CCl4 induced liver damage or dietary induced fatty liver disease remains challenging. Due to the essential function of the liver in metabolic state control and its central position in the circulation, alterations of the liver functionality or microcirculation directly impacts long-term survival of the animals, therefore limiting the possibility to obtain long TPLSM video-sequences of heavily damaged organs, e.g. with severe hemorrhages or destroyed microvasculature. Also, in severely altered liver microanatomy as in models with extensive necroses, it remains difficult to study cell-cell-interactions and local compartmentalization of inflammatory cell aggregates, because no standardized &#8220;counter-staining&#8221; for anatomical structures (such as hematoxylin-staining in conventional microscopy or DAPI staining in immunofluorescence) is available for intravital TPLSM.The quality of the data obtained by the imaging process further depends on the labeling intensity of the cells and the microscopic setup.
Several approaches exist to visualize GFP+ cells in liver. The highest fluorescence with very low background fluorescence is obtained between 900 and 920 nm excitation wavelength. The almost complete loss of liver background fluorescence does not allow investigating the positioning of the cells within the liver, when not using additional vessel trackers such as Dextran or Lectin. Therefore, we decided to image the migrating leukocytes at a wavelength of 840 nm, giving the best ratio between the GFP signal and detailed but not too bright liver background fluorescence.
Taken together, the intravital TPLSM imaging system introduced in this study can be applied to a broad variety of liver specific intravital microscopy questions. With this approach, TPLSM imaging in other solid organs such as kidney, liver, bladder, gut or pancreas is feasible with only minor modifications of the surgical procedures and tissue staging, therefore providing a highly flexible approach to investigate long term migratory processes of cells in vivo.

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><tbody><tr><td>Anesthetics</td><td></td><td></td><td></td></tr><tr><td>Buprenorphine</td><td>Essex Pharma</td><td>997.00.00</td><td>Analgeticum, 0.1 mg/kg</td></tr><tr><td>Fentanyl</td><td>Rotex Medica</td><td>charge: 30819</td><td></td></tr><tr><td>Fluovac anesthesia system</td><td>Harvard Apparatus</td><td>34-1030</td><td></td></tr><tr><td>Glucose 5%</td><td>Braun</td><td></td><td></td></tr><tr><td>ISOFLO (Isoflurane Vapor) vaporiser</td><td>Eickemeyer</td><td>4802885</td><td></td></tr><tr><td>Isoflurane</td><td>Forene Abbott</td><td>B 506</td><td></td></tr><tr><td>Isotonic (0.9%) NaCl solution</td><td>DeltaSelect GmbH</td><td>PZN 00765145</td><td></td></tr><tr><td>Ketamin 10%</td><td>ceva</td><td>Charge: 36217/09</td><td></td></tr><tr><td>Xylazin 2%</td><td>medistar</td><td>Charge: 04-03-9338/23</td><td></td></tr><tr><td>Consumable supplies</td><td></td><td></td><td></td></tr><tr><td>20 ml Syringe</td><td>BD Plastipak</td><td></td><td></td></tr><tr><td>250 ml Erlenmeyer flask</td><td>Schott Duran</td><td>21 226 36</td><td></td></tr><tr><td>25 ml Beaker 2x</td><td>Schott Duran</td><td>50-1150</td><td></td></tr><tr><td>2 ml syringe</td><td>BD Plastipak</td><td></td><td></td></tr><tr><td>4-0 Vicryl suture</td><td>Ethicon</td><td>V7980</td><td></td></tr><tr><td>Agarose</td><td>commercially available</td><td></td><td></td></tr><tr><td>Bepanthen Eye and Nose ointment</td><td>Bayer Vital GmbH</td><td>6029009.00.00</td><td></td></tr><tr><td>Change-A-Tip Deluxe High-Temp Cautery Kit</td><td>Fine Science Tools Inc.</td><td>18010-00</td><td></td></tr><tr><td>Cotton Gauze swabs</td><td>Fuhrmann GmbH</td><td>32014</td><td></td></tr><tr><td>Cover Slip 24x50 mm</td><td>ROTH</td><td>1871</td><td></td></tr><tr><td>Durapore silk tape</td><td>3M</td><td>1538-1</td><td></td></tr><tr><td>Feather disposable scalpel</td><td>Feather</td><td>02.001.30.011</td><td></td></tr><tr><td>Fine Bore Polythene Tubing 0.58 mm ID</td><td>Smiths medical</td><td>800/100/200</td><td></td></tr><tr><td>Histoacryl</td><td>Braun</td><td>1050052</td><td>5x 0.5 ml</td></tr><tr><td>Leukoplast</td><td>BSN Medical Inc.</td><td></td><td></td></tr><tr><td>Microscope Slides</td><td>ROTH</td><td>1879</td><td></td></tr><tr><td>Poly-Alcohol Haut&amp;hellip;farblos Antisepticum</td><td>Antiseptica GmbH</td><td>72PAH200</td><td></td></tr><tr><td>Sterican needle 18 G x 1</td><td>B. Braun</td><td>304622</td><td></td></tr><tr><td>Sterican needle 27 3/4 G x 1</td><td>B. Braun</td><td>4657705</td><td></td></tr><tr><td>Tissue paper</td><td>commercially available</td><td></td><td></td></tr><tr><td>Surgical Instruments</td><td></td><td></td><td></td></tr><tr><td>Amalgam burnisher 3PL</td><td>Gatz</td><td>0110?</td><td></td></tr><tr><td>Blair retractors (4 pronged (blunt)) x2</td><td>Storz&amp;amp;Klein</td><td>S-01134</td><td></td></tr><tr><td>Dumont No.7 forceps</td><td>Fine Science Tools Inc.</td><td>91197-00</td><td></td></tr><tr><td>Graefe forceps curved x1</td><td>Fine Science Tools Inc.</td><td>11151-10</td><td></td></tr><tr><td>Graefe forceps straight x2</td><td>Fine Science Tools Inc.</td><td>11050-10</td><td></td></tr><tr><td>Heidemann spatula HD2</td><td>Stoma</td><td>2030.00</td><td></td></tr><tr><td>Needle holder Mathieu</td><td>Fine Science Tools Inc.</td><td>12010-14</td><td></td></tr><tr><td>Scissor</td><td>Fine Science Tools Inc.</td><td>14074-11</td><td></td></tr><tr><td>Semken forceps</td><td>Fine Science Tools Inc.</td><td>11008-13</td><td></td></tr><tr><td>Small surgical scissors curved</td><td>Fine Science Tools Inc.</td><td>14029-10</td><td></td></tr><tr><td>Small surgical scissors straight</td><td>Fine Science Tools Inc.</td><td>14028-10</td><td></td></tr><tr><td>Standard pattern forceps</td><td>Fine Science Tools Inc.</td><td>11000-12</td><td></td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools Inc.</td><td>15000-08</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>ECG Trigger Unit</td><td>Rapid Biomedical</td><td>3000003686</td><td></td></tr><tr><td>MICROCAPSTAR End-Tidal Carbon Dioxide Analyzer</td><td>AD Instruments</td><td></td><td></td></tr><tr><td>Minivent Typ 845</td><td>Harvard Apparatus</td><td>73-0043</td><td></td></tr><tr><td>Multiphoton microscope Trimscope I</td><td>LaVision</td><td></td><td></td></tr><tr><td>Perfusor Compact</td><td>B. Braun</td><td></td><td></td></tr><tr><td>PowerLab 8/30 8 channel recorder</td><td>AD Instruments</td><td>PL3508</td><td></td></tr><tr><td>Temperature controlled heating pad</td><td>Sygonix</td><td>26857617</td><td></td></tr><tr><td>Temperature sensor</td><td>comercially available</td><td></td><td></td></tr><tr><td>Temperature controlled System for Microscopes -Cube&amp;amp;Box</td><td>Life Imaging Services</td><td></td><td></td></tr></tbody></table>

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 procedure were plotted as a superimposed image (Figure 6A). While cells under baseline conditions showed long distance migration along the liver sinusoids towards the central vein, 18 hr after CCl4 treatment the CXCR6+/gfp&#160;cells showed already partially impaired movement patterns. Although some fast moving cells were still present in the liver sinusoids, several focal areas were visible with cells that had changed from random migration to local scanning, indicating a chemotactic response recruiting the cells to the area of injury. The effect was even more pronounced after 36 hr, showing an almost complete arrest of CXCR6+ cells with virtually no active migration. While under baseline conditions most migrating cells had variable migration speeds with random directional changes, CCl4 treated animals showed cells that were only slowly crawling as well as some freely motile cells patrolling the sinusoids after 18 hr but not after 36 hr. Using color-coded migration tracks, cell speed could be visualized directly to assess the effect of the CCl4 treatment. The impaired migration was also visible in the normalized track diagram (Figure 6B), showing that 18 hr after CCl4 the amount of long distance tracks as well as the average track length was markedly reduced, while after 36 hr no motile cells were detectable any more. In line with these findings, the cellular displacement, which describes the ability of a cell to freely patrol the vasculature, decreased over time, indicating a trapping of the cells 36 hr after CCl4 treatment at the site of hepatocellular damage (Figure 6C).
To follow the migratory behavior of CXCR6+/gfp&#160;cells in liver in more detail, the migration speed of representative individual cells was plotted to visualize their level of motility over time. We and others previously described several distinct motion patterns of CXCR6+/gfp&#160;cells: Active random crawling with a sticking rolling pattern, showing phases of cellular motility and temporal quiescent states; directed cellular recruitment with cells scanning an area of interest but still capable of active crawling; total cellular arrest accompanied by immune cell activation18,20. While mice without treatment mainly showed cells that were freely motile with speeds up to 15 &#181;m/sec, CXCR6+/gfp&#160;cells in mice treated with CCl4 displayed markedly reduced cell migration speed and partial arrest already after 18 hr and full migratory arrest after 36 hr (Figure 7A). In line with the single cell analysis, the statistical data of all cells within a sequence revealed decreasing overall migration speed after CCl4 treatment (Figure 7B), which was also resembled by average track speeds (Figure 7C) and track maximum migration speeds (Figure 7D), showing that the approach was suitable to describe differences in cell migration and positioning in developing liver disease.
<img alt="Figure 1" src="/files/ftp_upload/52607/52607fig1.jpg" /> 
Figure 1: Tracheotomy of the mouse. (A) Surgical equipment used for preparation. 1x standard pattern forceps, 1x Semken forceps, 1x Dumont No.7 forceps, Graefe forceps (serrated, 2x straight / 1x curved), 2x Blair retractors (4 pronged (blunt)), double ball stylus probe (e.g., Amalgam burnisher 3PL), Heidemann spatula HD2, needle holder (Mathieu or Halsey), 1x small surgical scissors (sharp/blunt, 1xcurved, 1x straight), micro-scissors (e.g., Vannas Spring Scissors), 1x small animal cauter, cotton swabs, eye protective salve, surgical thread, n-Butyl-2-Cyanoacrylatglue. (B) eyes are protected using an eye protective ointment. (C) Initial skin cut for trachea preparation. (D) Dissection of connective tissue between submaxillary salivary glands. (E) Exposition of the trachea. (F) Dissection of the muscular tissue surrounding trachea. (G) Placement of surgical thread underneath trachea for fixation of tube. (H) Tracheal incision using microscissors between upper cartilage rings. (I) Insertion of ventilation tube into trachea. (J) Placement of cranial surgical thread to fix tube to the skin. (K) Double fixation of the tube. (I) Sealing surgical incision using n-Butyl-2-Cyanoacrylat. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52607/52607fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52607/52607fig2.jpg" /> 
Figure 2: Laparatomy and liver preparation. (A) Initial skin incision, blood vessels were cauterized to prevent excessive bleeding. (B) Opening of the peritoneum underneath the sternum. (C) Extension of the peritoneal cut using cautery unit. (D) Sealing of epigastric vessels. (E) Further extension of the peritoneal incision below ribs. (F) Placement of animal into agarose stage dish. (F) Placement of support stacks. (G) Placement of respiratory trigger sensor. (H) Setup of trigger threshold for respiration synchronized imaging. (I) Sternum fixation to retract ribs using surgical thread. (J) Disconnection of the gall bladder from the diaphragm and dissection of the falciform ligament. (K) Placement of staging cover slip. (L) Placement of liver lobe on stage. (M) Placement of coverslip on liver lobe. (N) Placement of i.p. catheter for long term anesthesia. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52607/52607fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52607/52607fig3.jpg" /> 
Figure 3: Intraperitoneal catheter. 27 G needles connected with flexible silicone tubing.
<img alt="Figure 4" src="/files/ftp_upload/52607/52607fig4.jpg" /> 
Figure 4: Monitoring of the mouse. (A) Placement of ECG electrodes (green, red, blue cables). Needles for ECG detection are placed subcutaneously as shown. (B) Monitoring of ECG (red), expiratory CO2 (blue) and heart rate (red) showing long term progression (left) and actual measurement (right). (C) Temperature controlled power unit for heat pad control. Temperature is set to 36 &#176;C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52607/52607fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52607/52607fig5.jpg" /> 
Figure 5: Liver embedding and imaging. (A) Embedding of liver lobe. 3% agarose in PBS is injected at 41 &#176;C under the cover slip using a 2 ml syringe with an 18G needle. (B) Liver lobe is fully embedded in agarose to minimize movement artifacts. (C) Sealing of peritoneum with remaining agarose to prevent dehydration. (D) Removal of agarose covering microscope field of view after full gelatinization. (E) Preparation of viewfield. F Adjustment of Z-level and evaluation of sample blood flow using light microscopy. (D) Cover dish to prevent excessive evaporation. Cover is lifted in the picture to show setup. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52607/52607fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52607/52607fig6.jpg" /> 
Figure 6: Tracking of CXCR6gfp/+ cells in chronic liver damage after CCl4 injection. Mice were injected i.p. with 0.6 ml/kg CCl4 dissolved in sunflower oil 18 hr or 36 hr before imaging. On the day of the experiment, mice were subjected to intravital TPLSM as described. (A) Still images of cell tracking. Tracks from all time points were superimposed to show cellular motility and displacement. Images were taken using a single beam Titan Saphire Laser at 840 nm and a TPLSM microscope. Areas were scanned taking 3-5 Z-stacks with a penetration depth of 70&#181;m with a sampling rate of approximately 30 seconds per scan cycle. Tracks were color coded to show migration speed (light blue: slow moving or sessile cells; purple: motile cells). Scale bars: 100 &#181;m. (B) XY-diagrams of paths normalized for their origin showing track directionality and track length (&#181;m). (C) Total displacement of cells from their origin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52607/52607fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52607/52607fig7.jpg" /> 
Figure 7: Migration statistics of CXCR6gfp/+ cells followed by intravital TPLSM in liver tissue in baseline and CCl4 treated mice. Cells were analyzed for their migration speed patrolling the liver. (A) Representative speed of single cells followed over time. Speed was measured at each time point and plotted for each frame individually. Lines represent individual cells. Y-axes are scaled according to maximum migration speed. (B) Statistical analysis of A. Frame specific speeds from all cells were grouped and analyzed in baseline and CCl4 treated mice. (C) Average track speed was calculated per track and data from all tracks were grouped for analysis. (D) Maximum speed of each track was plotted and analyzed. All experiments were performed in groups of 3 animals and results were confirmed in two independent experiments. ***P &#60;0.001 (two-tailed unpaired Student t-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52607/52607fig7highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.9K 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.

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

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

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

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

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

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

Neuroscience

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

Cancer Research

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 ...

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 ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

Medicine

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue

Observing the intravascular dynamics of mouse liver tissue allows us to conduct further in-depth observations and studies on tissue-related diseases of the mouse liver. A mouse is injected with a dye that can stain blood vessels. To observe the mouse liver in vivo, it is exposed and fixed in a frame. Two and three-dimensional images of the blood vessels in the liver tissue are obtained using a multiphoton microscope. Images of the tissues at the selected sites are continuously acquired to observe long-term changes; the dynamic changes of blood vessels in the liver tissues are also observed. Multiphoton microscopy is a method for observing cell and cell function in deep tissue sections or organs. Multiphoton microscopy has sensitivity to tissue microstructure and enables imaging of biological tissues at high spatial resolution in vivo, providing the ability to capture the biochemical information of the organization. Multiphoton microscopy is used to observe part of the liver but fixing the liver to make the image more stable is problematic. In this experiment, a special vacuum suction cup is used to fix the liver and obtain a more stable image of the liver under the microscope. In addition, this method can be used to observe dynamic changes of specific substances in the liver by marking such substances with dyes.

In this experiment, a mouse is injected in its tail vein with Rhodamine B isothiocyanate&#8211;dextran that can stain blood vessels. After the liver is exposed and fixed, a specific part of the liver can be selected to observe the deep tissue in the living body using multiphoton microscopy.

Observing the intravascular dynamics of mouse liver tissue allows us to conduct further in-depth observations and studies on tissue-related diseases of ...

Bioengineering

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Real-Time Monitoring of Cellular Dynamics in Mouse Lung Transplantation

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

Noninvasive Intraocular In Vivo Imaging of Engrafted Liver Spheroids in Mice

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until now, optical imaging of the liver in situ is possible by intravital imaging, which offers high-resolution imaging at the cellular level but cannot be performed multiple times and, therefore, longitudinally in the same animal. Noninvasive imaging methods, such as bioluminescence, allow repeated imaging sessions on the same animal but do not achieve cell resolution. To address this methodology gap, we have developed a platform for noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. In the workflow described in this study, primary mouse liver spheroids are generated in vitro and transplanted into the anterior chamber of the eye of recipient mice, where they engraft on the iris. The cornea acts as a natural body window through which we can image the engrafted spheroids by conventional confocal microscopy. The spheroids survive for months in the eye, during which the cells can be studied in contexts of health and disease, as well as being monitored in response to different stimuli over repeated imaging sessions using appropriate fluorescent probes. In this protocol, we provide a breakdown of the necessary steps to implement this imaging system and explain how to best harness its potential.

Author Spotlight: Harnessing Mouse Eye Chambers for Noninvasive Liver Spheroid Studies

Here, we describe a platform that allows noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. The workflow spans from generating spheroids from primary liver cells to transplantation into the mouse eye and in vivo imaging at cellular resolution by confocal microscopy.

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until ...

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

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