This study was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korea government (MSIP; grant no. 2016R1A2B4008199 to Y-M. H.), and the Ministry of Health &amp; Welfare, Republic of Korea (grant number: HI14C1324).

All procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Yonsei University College of Medicine, Korea. LysM-green fluorescent protein (GFP; gfp/+) mice8 and CX3Cr1-GFP (gfp/+)9 mice at 8-12 weeks of age were used for intravital imaging.&#160;All surgical tools were autoclaved and disinfected with 70 % alcohol.
1. Custom-designed mouse liver imaging chamber with supporting tools
<ol>
	<li>Mouse liver imaging chamber design
	<ol>
		<li>Make a chamber of the following size for mouse liver imaging (Figure 1): inner chamber (120 mm length &#215; 160 mm width &#215; 30 mm height), outer chamber (100 mm length &#215; 100 mm width &#215; 24 mm height), bolts (&#1060; 3 mm &#215; 30 mm height), nuts (&#1060; 4 mm), and wing-shaped nuts (&#1060; 4 mm).</li>
		<li>Measure the curing agent and elastomer base in 1:9 ratio to make a total of 120 g of silicone.</li>
		<li>Put the liquid silicone mixture in a 250 mL Erlenmeyer flask and mix it well. Then put the flask in a desiccator and remove air bubbles for about 1 h under vacuum through the pump.</li>
		<li>Pour the liquid silicone mix into a custom-designed mouse chamber as high as 10 mm height and let it cure for a couple of days (Figure 1A).</li>
	</ol>
	</li>
	<li>Preparation of cover slides and the liver fixing bed
	<ol>
		<li>On the metal frame, attach the cover glass using silicone glue. Let it dry for 1 day (Figure 1B).</li>
		<li>In order to effectively hold the distilled water for the water immersion lens, a semipermanent water-blocking structure is essential. Make 1 mm rounding silicone glue and harden it overnight.</li>
		<li>Mix the curing agent and elastomer base and then pour it into a 6-well plate at a height of about 8-10 mm. Perform the hardening process as described in 1.1.2-1.1.4.</li>
	</ol>
	</li>
</ol>
2. Preparation of LPS
<ol>
	<li>Prepare sterile 1&#215; phosphate-buffered saline (PBS) to dilute the LPS powder from Salmonella enterica serotype enteritidis. (1&#215; PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4).</li>
	<li>Pre-heat PBS in water bath at 37 &#176;C, and open the LPS powder bottle (25 mg) carefully.</li>
	<li>Using a pipet aid, pour the sterile PBS (5 mL) into the LPS powder bottle and gently stir or suspend the solution to prepare the stock solution. 
	NOTE: After LPS powder is liquefied, harsh vortexing can cause bubbles. Avoid making bubbles to minimize loss when making aliquots in 1.5-mL tubes.</li>
	<li>Store the stock LPS solution at &#8722;20 &#176;C, and dilute it into a 1 mg/mL LPS solution for injection.</li>
	<li>Inject the 1 mg/mL LPS solution (20 mg/kg) into the peritoneum of a non-anesthetized mouse using a 1-mL syringe. For the control animal, inject the same volume of PBS.</li>
	<li>Wait for 2 h for sufficient inflammatory response.</li>
</ol>
3. Preparation of Texas-Red Dextran to stain blood vessels
<ol>
	<li>Prepare two 15-mL conical tubes and 10 mL of sterile PBS.</li>
	<li>Dilute Texas-Red Dextran powder (25 mg) in 3 mL of PBS and mix it well. Transfer the mixture to an empty 15-mL conical tube.</li>
	<li>Transfer the remaining PBS (7 mL) to the 15-mL conical tube containing the dextran mixture (3 mL).</li>
	<li>Connect a 0.45 &#181;m syringe filter to the tip of a 10-mL syringe. The syringe filter should replace the needle.</li>
	<li>Filter the dextran mixture (10 mL). Press the syringe evenly in order to avoid spilling.</li>
	<li>Dispense 500 &#181;L of 2.5 mg/mL dextran solution in the light-blocked 1.5-mL tube and store it at &#8722;20 &#176;C. Once thawed, store it at 4&#176;C.</li>
</ol>
4. Surgical process
<ol>
	<li>Disinfect the surgery table and all instruments using 70% alcohol prior to the surgery. Then, set the tools for the liver surgery and adjust the heating plate to 37 &#176;C (Figure 2A, B).</li>
	<li>For anesthesia, intraperitoneally inject the mice with 30 mg/kg of tiletamine/zolazepam&#160; and 10 mg/kg of xylazine. These solutions can be mixed and diluted in PBS for simultaneous injection. Confirm anesthetization by gently pressing the toe of the mouse.&#160;&#160;Proceed next step when there is no reflex.</li>
	<li>Apply small amount of eye ointment to prevent the dryness of mouse&#39;s retina.</li>
	<li>Remove body hair around the abdomen area using&#160;hair removal cream (Figure 2C).</li>
	<li>Inject 200 &#181;L of Texas-Red Dextran solution (concentration of 2.5 mg/mL) via the tail vein or retro-orbital injection using an insulin syringe (Figure 2D).</li>
	<li>Mark the right subcostal area with a marker. The line should be drawn from the xiphoid process to the end of the rib. Cut the epidermis with forceps and scissors (Figure 2E, F).</li>
	<li>Open the abdominal cavity using micro-scissors and forceps, and expose the liver carefully. The left lateral lobe should be rolled out using cotton swabs (Figure 2G, H).</li>
	<li>Pour 500 &#181;L of sterile PBS on the abdominal cavity to prevent dryness of the liver.</li>
	<li>Place the mouse in the right-decubitus position in a custom-designed chamber, and apply a small amount of tissue glue to the silicon bed. Then, carefully attach the liver using cotton swabs (Figure 2I). 
	NOTE: Because the liver tissue is extremely friable, do not mechanically pull out the liver. Gently roll it out with cotton swabs.</li>
	<li>A few seconds later, wet the liver again with 500 &#181;L of sterile PBS to avoid drying out of the liver. Place the metal frame on the liver and fix it firmly with nuts (Figure 2J).</li>
</ol>
5. Intravital imaging of the liver with two-photon microscopy
<ol>
	<li>Run ZEN software (Carl Zeiss) to turn on the laser, set the wavelength to 880 nm, and set green, and red channels. Adjust laser power according to fluorescence intensity (Figure 3A). Emission filter information is as follows: Green/Alexa488, 500-550 nm and Red/Alexa 555, 575-610 nm.</li>
	<li>Place the chamber on the imaging stage and place the 20X water-immersion objective lens close to the cover glass.</li>
	<li>Fix the silicone rubber heater under the mouse body to maintain its body temperature (Figure 3B).</li>
	<li>Drop 500 &#181;L of distilled water on the metal frame, and then pull the objective lens close to the liver (Figure 3C).</li>
	<li>Adjust the focus and determine the depth and time of the imaging area.</li>
	<li>Conduct imaging.</li>
	<li>For long-term imaging, inject half a dose of tiletamine/zolazepam and xylazine mixture&#160; each hour during imaging.&#160;Maintaining the body temperature of the mice at 37 &#176;C is essential during this stage. Always keep on eye to the mouse, since anesthetization time differs from every mouse. 
	NOTE: This method is not suitable for recovery after surgery and imaging. We ethically sacrifice the mouse immediately after surgery.</li>
	<li>Check the pedal reflexes for every 30 min for stable anesthetization.</li>
</ol>
6. Data analysis
<ol>
	<li>Open the data analysis software Volocity and then import the raw data in the library into the software.</li>
	<li>Remove noise from the image to suit the situation and take a snapshot. 
	NOTE: To obtain smooth images, use the &#39;Fine&#39; or &#39;Median&#39; filter. Furthermore, use the &#39;Contrast enhancement&#39; feature to make dark or blurry images clearer.</li>
	<li>Fit the edited items to 100% and export as JPEG or TIFF files for snapshot images and AVI files for movies.</li>
</ol>

<ol>
	<li>Girard, J. R., 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=Fuels,+hormones,+and+liver+metabolism+at+term+and+during+the+early+postnatal+period+in+the+rat.">Fuels, hormones, and liver metabolism at term and during the early postnatal period in the rat.</a> J Clin Invest. 52 (12), 3190-3200 (1973).</li><li>Marques, P. E., 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=Imaging+liver+biology+in+vivo+using+conventional+confocal+microscopy.">Imaging liver biology in vivo using conventional confocal microscopy.</a> Nat Protoc. 10 (2), 258-268 (2015).</li><li>Dasari, S., Weber, P., Makhloufi, C., Lopez, E., Forestier, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Microscopy+Imaging+of+the+Liver+following+Leishmania+Infection:+An+Assessment+of+Hepatic+Hemodynamics.">Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics.</a> J Vis Exp. (101), e52303 (2015).</li><li>Honda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+neutrophil+recruitment+in+hepatic+ischemia-reperfusion+injury+in+mice.">Intravital imaging of neutrophil recruitment in hepatic ischemia-reperfusion injury in mice.</a> Transplantation. 95 (4), 551-558 (2013).</li><li>Jenne, C. N., Wong, C. H., Petri, B., 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+use+of+spinning-disk+confocal+microscopy+for+the+intravital+analysis+of+platelet+dynamics+in+response+to+systemic+and+local+inflammation.">The use of spinning-disk confocal microscopy for the intravital analysis of platelet dynamics in response to systemic and local inflammation.</a> PLoS One. 6 (9), e25109 (2011).</li><li>Ritsma, L., 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+microscopy+through+an+abdominal+imaging+window+reveals+a+pre-micrometastasis+stage+during+liver+metastasis.">Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis.</a> Sci Transl Med. 4 (158), 158ra145 (2012).</li><li>Sierro, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Liver+Capsular+Network+of+Monocyte-Derived+Macrophages+Restricts+Hepatic+Dissemination+of+Intraperitoneal+Bacteria+by+Neutrophil+Recruitment.">A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment.</a> Immunity. 47 (2), 374-388 (2017).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96 (2), 719-726 (2000).</li><li>Jung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20 (11), 4106-4114 (2000).</li><li>Heymann, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+intravital+multiphoton+microscopy+imaging+of+immune+cells+in+healthy+and+diseased+liver+using+CXCR6.Gfp+reporter+mice.">Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.</a> J Vis Exp. (97), (2015).</li></ol>

The authors declare no competing financial interests.

The liver has been actively studied by many groups3,10, due to importance of its function. For instance, Heymann et al. suggested a delicate method using agarose. Although this method was found to be highly precise, the agarose setting takes time. However, with our methodology, all procedures could be carried out within 30 min, minimizing other confounding factors. Second, stabilizing the liver is extremely important because the heartbeat can disturb the video. T...

The liver is the major metabolic organ in mammals; it acts as a control tower for energy, hormones, and detoxification1. Due to its importance, researchers have conducted many in vitro and in vivo experiments to elucidate the changes in the liver during inflammation. Intravital microscopy has been used to capture live images from the liver2 . Indeed, various liver imaging methods have been introduced2,3,4,5,6. However, in order to develop a more simplified surgical approach and achieve stabilization of the target organ and the immune cells, we designed a simple protocol that can guide researchers to minimize their effort for intravital imaging.
In this study, we introduced a stable and novel imaging chamber and surgical technique that could be used to minimize bleeding and possible infections. We confirmed that the liver remained intact for more than 2 h. With this method, we successfully identified morphologies of LCMs7 and neutrophils under septic condition. Since this method minimizes physical and biological confounding factors such as trembling and unexpected inflammation during surgical procedure, we anticipate that this method could be used to observe acute reactions of immune cells in the liver in response to reagents like LPS.

<table><tbody><tr><td>Custom designed chamber</td><td>Live Cell Instruments</td><td></td><td>Custom-designed size</td></tr><tr><td>Metal cover slide</td><td>Live Cell Instruments</td><td></td><td>Custom-designed size</td></tr><tr><td>Cover glass</td><td>Electron Microscopy Sciences</td><td>#72204-03</td><td></td></tr><tr><td>Sylgard 184 Silicone elastomer kit</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Erlenmeyer flask</td><td>DURAN</td><td>21 216 36</td><td></td></tr><tr><td>Cell culture plate (Flat type)</td><td>HYUNDAI Micro Co.</td><td>H31006</td><td></td></tr><tr><td>Desiccator</td><td>ADARSH</td><td>55204</td><td></td></tr><tr><td>High Q BOND SE 5 mL</td><td>BJM LAB</td><td></td><td>To make semi-permanent dam</td></tr><tr><td>Flexible Silicone Rubber Heaters</td><td>Omega</td><td>SRFR/SRFG SERIES, M1250/0800</td><td></td></tr><tr><td>DC power supply</td><td>Toyotech</td><td>DP30-03A</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td></td><td></td><td>Home-made</td></tr><tr><td>Lipopolysaccharides (LPS) from Salmonella enteria serotype enteritidis</td><td>Sigma-Aldrich</td><td>L6011</td><td></td></tr><tr><td>Texas Red Dextran</td><td>Thermo Fisher Scientific</td><td>D1830</td><td></td></tr><tr><td>15 mL High-clarity polypropylene&lt;br/&gt; conical tube</td><td>FALCON</td><td>14-959-49B</td><td></td></tr><tr><td>0.45 &amp;mu;m filter (Minisart Syringe Filter)</td><td>Sartorius</td><td>16555k</td><td></td></tr><tr><td>1.5ml Micro Tube, Amber</td><td>Axygen</td><td>MCT-150-X</td><td>For light-blocking</td></tr><tr><td>1 mL syringe</td><td>Korea Vaccine</td><td>KV-S01</td><td></td></tr><tr><td>3 mL syringe</td><td>Korea Vaccine</td><td>KV-S03</td><td></td></tr><tr><td>10 mL syringe</td><td>Korea Vaccine</td><td>KV-S10</td><td></td></tr><tr><td>0.3 mL insulin syringe</td><td>Becton Dickinson</td><td>324900</td><td></td></tr><tr><td>Hair removal cream 100 mL</td><td>Body natur</td><td></td><td></td></tr><tr><td>Cotton swab</td><td>ABDI</td><td></td><td></td></tr><tr><td>Zoletil 50 5 mL</td><td>Virbac</td><td></td><td></td></tr><tr><td>Rompun 10 mL</td><td>Bayer</td><td></td><td></td></tr><tr><td>Heating plate</td><td>Live Cell Instruments</td><td>PH-S-10</td><td>Custom-designed size</td></tr><tr><td>DC controller</td><td>Live Cell Instruments</td><td>CU-301</td><td></td></tr><tr><td>Microdessection Scissors</td><td>Harvard Apparatus</td><td>72-5418</td><td></td></tr><tr><td>Scissors</td><td>Roboz</td><td>RS-5882</td><td></td></tr><tr><td>Forceps</td><td>Roboz</td><td>RS-5137</td><td></td></tr><tr><td>Vet bond</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>ZEN software (Black Edition)</td><td>Carl Zeiss</td><td></td><td>Program for LSM 7 MP</td></tr><tr><td>LSM 7 MP</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Volocity</td><td>PerkinElmer</td><td></td><td>Analysis program</td></tr></tbody></table>

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.

A simple and very stable imaging chamber was devised. The bottom of chamber was covered with silicone, which prevented slipping of the mouse body and organs. Additionally, because silicon has the proper amount of elasticity, it could prevent expected damage induced by the pressure of the cover slide (Figure 1A, C). Second, tissue-safe glue (Vet bond) was applied in order to fix the extracted liver on the circle-shaped silicone bed. This metho...

Watch this Scientific Journal Video about Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver at JoVE.com

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

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 ...

two-photon-intravital-imaging-leukocytes-during-immune-response

Research

JoVE Journal

Immunology and Infection

10.2K Views.  Yonsei University College of Medicine. 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. 

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

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 ...

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.

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, ...

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

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

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

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

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 Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Bioengineering

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 ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Medicine

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

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 ...

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

Summary

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