This research was supported by the INSERM, the University of Aix-Marseille and a Career Development award from HFSPO obtained by CL Forestier.

Ethics statement: All the animal studies were performed in accordance with guidelines and protocols that were approved by the Institutional Animal Care and Use Committee of the Aix-Marseille Universit&#233;, France. Female C57Bl/6 mice at 8 - 10 weeks old were commercially obtained and handled according to the rules of D&#233;cret N&#176; 8 87&#8211;848, October 19, 1987, Paris. All the experiments using L. donovani LD1S parasites were conducted in accordance with biosafety regulations from the French and European Union legislation.
1. Mouse Infection with L. donovani Promastigote Parasites

<ol>
	<li>Prepare the culture medium for in vitro maintenance of L. donovani promastigote parasites, LD1S (MHOM/SD/62/1S-CL2D).
	<ol>
		<li>Use M199 medium. Supplement M199 medium with 10% fetal calf serum (heat-inactivated at 56 &#176;C for 30 min), 25 mM HEPES pH 6.9, 12 mM NaHCO3, 1 mM glutamine, RPMI 16040 vitamin mix, 10 mM folic acid, 100 mM adenosine, 7.6 mM hemin, 50 U/ml penicillin and 50 mg/ml streptomycin.</li>
	</ol>
	</li>
	<li>Culture LD1S parasites in 10 ml of complete M199 medium at 26 &#176;C in a 25 cm2 ventilated flask.</li>
	<li>Using successive differential centrifugation steps, prepare a population of promastigote parasites that are enriched in metacyclic forms.
	<ol>
		<li>Harvest a mid-log phase parasite culture. Count on a hemocytomer. Resuspend 5 x 105 parasites/ml in 10 ml of complete M199 medium. Grow the parasite culture in anaerobic conditions until they reach the late stationary phase (in approximately 5-6 days).</li>
		<li>Spin the LD1S culture at 1,200 x g for 10 min at 26 &#176;C to pellet non-metacyclic parasites, recover the supernatant and spin it again at 2,500 x g at 26 &#176;C for 10 min. 
		Note: This final pellet is highly enriched in metacyclic promastigote parasites.</li>
		<li>Resuspend the pellet in 100 &#181;l of PBS. Remove a small aliquot for fixation with 25% glutaraldehyde 1/100 v:v and count parasites using a hemocytometer.</li>
	</ol>
	</li>
	<li>Dilute the enriched-metacyclic parasite population to 1 x 107/100 &#181;l in PBS. Inject the parasite suspension into the tail vein of an anesthetized mouse (see below) using a 1 ml syringe with a 30 G needle. For control animals, inject 100 &#181;l PBS into the tail vein.</li>
</ol>
2. Surgical Procedures
<ol>
	<li>Disinfect the working space with a biocidal disinfectant spray and all the surgical instruments with 70% ethanol for 30 min.</li>
	<li>Prepare an anesthetic solution, according to the weight of the animal, containing 125 mg/kg of Ketamine and 12.5 mg/kg of Xylazine diluted in PBS.</li>
	<li>Weigh the mouse and intraperitoneally inject the appropriate dose of anesthetic using a 1 ml syringe with a 30 G needle.</li>
	<li>Keep the mouse warm and check that the mouse is completely anesthetized by pinching the foot pad before proceeding. Re-inject a half dose of the anesthetic solution in the mouse every 60 min.</li>
	<li>Shave the mouse&#8217;s abdomen and clean it with Vetedine. Make a small incision in the skin and musculature under the thoracic cage on the left side of the abdomen.</li>
	<li>Lay a piece of moist gauze on the abdomen just below the opened area. Expose the liver and place it on the gauze. Lay another piece of moist gauze above the liver.</li>
	<li>Place a 24 x 60 mm2 to 150 &#181;m thick coverslip, cyanoacrylate-bonded to a metal frame, on the liver for visualization under a microscope.</li>
	<li>Protect the eyes of the mouse with a wet gauze.</li>
	<li>After the imaging session, euthanize the anesthetized animal by cervical dislocation.</li>
</ol>
3. Intravital Microscopy Imaging of the Liver Architecture
<ol>
	<li>Carry out the intravital microscopy experiments on an inverted confocal microscope equipped with a thermostatic controlled chamber, an apochromatic 63X oil glycerol immersion objective (NA 1.4), Argon (488 nm) and HeNe (543 nm, 633 nm) lasers and a blue diode (405 nm).</li>
	<li>Place the mouse on the stage of the microscope with the coverslip covering the liver face down on the objective. Set the temperature of the chamber to 29 &#176;C. Adjust the focus using the liver autofluorescence to allow for visualization of the sinusoids.</li>
	<li>To visualize the vasculature, prepare a solution of 500 &#181;g BSA-Alexa 647 diluted in 100 &#181;l of PBS, for each mouse. Inject this solution intravenously into the tail vein of the anesthetized mouse using a 1 ml syringe with a 30 G needle.</li>
	<li>To visualize the liver cell nuclei, prepare a solution of Hoechst 33342 in PBS. Inject this solution at 8 mg/kg of mouse intraperitoneally using a 1 ml syringe with a 30 G needle.</li>
	<li>Use the normal sequential mode, the 63X immersion objective and the scanner at 400 Hz to image the hepatic cell nuclei and the liver vasculature. Turn on the 405 nm blue diode for Hoechst excitation and the 633 nm laser for BSA-Alexa 647 excitation. Turn on the Argon 488 nm laser and define a large acquisition band to visualize the liver autofluorescence.</li>
</ol>
4. Intravital Microscopy Imaging of the Liver for Blood Flow Speed Measurement
<ol>
	<li>Open the LAS software of the microscope (LAS-AF viewer Version 3.1.0 build 8587). Select the resonance scanner mode when opening the microscope software.</li>
	<li>Click on the configuration tab to set the hardware. Click on laser and select the HeNe 633 laser. Click on settings and select 12 bits resolution.</li>
	<li>Click on the acquisition menu. In the beam pathway settings window, select the objective 63X and set up the 633 laser power to 100%. Activate the signal gain and off set parameters by selecting Alexa-633 from the PM1-3 drop down menu.</li>
	<li>In the acquire tab, select the &#39;xyt&#39; acquisition mode. Set format width at 1,024 x 1,024. Select speed at 8,000 Hz. Select the pinhole of 3 Airy units. Select a zoom factor of 3. Do not select the bidirectional mode.</li>
</ol>
5. Quantitative Analysis of RBC Velocity Using the xyt Images (Method 1)
<ol>
	<li>Click on &#39;Live&#39; icon to visualize the blood flow on a live mode. Choose the area of interest and select a specific blood vessel for analysis. Set the vessel of interest to horizontal position by adjusting the &#39;X&#39;, &#39;Y&#39; and scan field rotation co-ordinates on USB control panel.</li>
	<li>Stop the &#39;Live&#39; mode once the vessel of interest is set. Change the format set width to 1,024 x 256 in the acquisition mode setting window, line average = 1, frame average = 1, select &#8220;stacks&#8221; 150. Click on &#39;start&#39; to acquire images.</li>
	<li>For quantitative analysis of RBC velocity using the &#8220;xyt&#8221; images open the ImageJ software. Select the &#39;File&#39; tab in ImageJ, click &#8220;Open&#8221; then choose the file of interest.</li>
	<li>Go to the &#8220;plugin&#8221; menu in ImageJ, select LOCI and Bio-formats import options. In this window, select the stack viewing parameters as &#39;hyperstack&#39;. In the stack order option, select the option: &#8220;xyzct&#8221;. Note: This opens a window with all the series of acquisitions.</li>
	<li>Click on &#39;Plugins&#39; on the ImageJ menu and select MTrackJ option. Click on &#39;add&#39; icon on the small window and click on the RBC to track. Click on the same RBC in each following frame and click on &#39;measure&#39; icon to measure the speed. 
	Note: The file generated can be saved in .exl format.</li>
	<li>Track a minimum of five RBCs per vessel and analyze a minimum of three individual vessels of the same size.</li>
</ol>
6. Quantitative Analysis of RBC Velocity Using the xt Line Images (Method 2)
<ol>
	<li>Click on &#39;Live&#39; to visualize the blood flow in live mode. Select a specific blood vessel for analysis. Set the vessel of interest to horizontal position. Stop the &#39;Live&#39; mode once the vessel of interest is set.</li>
	<li>Select the &#39;xt&#39; scanning mode and set the central lumen of the selected vessel along the line scan. Set format width at 1,024 x 512, speed = 8,000Hz, line average = 32, time = 512. Click on &#39;start&#39; and acquire the xt line images.</li>
	<li>Generate streaks for quantitative analysis of RBC velocity by opening the .lif file and opening the xt line image of interest. In LAS-AF software, select &#8220;experiments&#8221;, open the .lif and select the image. Note: It will automatically open a kymograph.</li>
	<li>Measure the time (t, obtained on the Y axis) required for a particle streak (showing dark reflection) to travel a certain distance (d, obtained on the X axis) on this kymograph. Select the tool &#8220;draw line&#8221; and draw a line horizontally to the streak. Note the distance in &#181;m and the time in seconds generated in the result tab at the bottom of the image.</li>
	<li>Calculate velocity as V = distance/time using the values obtained from the kymograph.</li>
	<li>Quantify a minimum of five particles streaks per xt image and a minimum of three individual vessels of the same size.</li>
</ol>

<ol>
	<li>Vollmar, B., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hepatic+microcirculation:+mechanistic+contributions+and+therapeutic+targets+in+liver+injury+and+repair.">The hepatic microcirculation: mechanistic contributions and therapeutic targets in liver injury and repair.</a> Physiol. Rev. 89, 1269-1339 (2009).</li><li>Srinivasan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+blood+flow+measured+by+optical+methods.">Absolute blood flow measured by optical methods.</a> SPIE Newsroom. , (2011).</li><li>Seifalian, A. M., Stansby, G. P., Hobbs, K. E., Hawkes, D. J., Colchester, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+liver+blood+flow:+a+review.">Measurement of liver blood flow: a review.</a> HPB. Surg. 4, 171-186 (1991).</li><li>Engwerda, C. R., Ato, M., Kaye, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages,+pathology+and+parasite+persistence+in+experimental+visceral+leishmaniasis.">Macrophages, pathology and parasite persistence in experimental visceral leishmaniasis.</a> Trends Parasitol. 20, 524-530 (2004).</li><li>Beattie, 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=Dynamic+imaging+of+experimental+Leishmania+donovani-induced+hepatic+granulomas+detects+Kupffer+cell-restricted+antigen+presentation+to+antigen-specific+CD8+T+cells.">Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.</a> PLoS Pathog. 6, e1000805 (2010).</li><li>Lee, W. Y., 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=An+intravascular+immune+response+to+Borrelia+burgdorferi+involves+Kupffer+cells+and+iNKT+cells.">An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells.</a> Nat. Immunol. 11, 295-302 (2010).</li><li>Geissmann, 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=Intravascular+immune+surveillance+by+CXCR6++NKT+cells+patrolling+liver+sinusoids.">Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids.</a> PLoS Biol. 3, e113 (2005).</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. protocols. 10, 258-268 (2015).</li><li>Thiberge, 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=In+vivo+imaging+of+malaria+parasites+in+the+murine+liver.">In vivo imaging of malaria parasites in the murine liver.</a> Nat. protocols. 2, 1811-1818 (2007).</li><li>Vacchina, P., Morales, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+screening+test+using+Leishmania+promastigotes+stably+expressing+mCherry+protein.">In vitro screening test using Leishmania promastigotes stably expressing mCherry protein.</a> Antimicrob. Agents Chemother. 58, 1825-1828 (2014).</li><li>Ferrer, M., Martin-Jaular, L., Calvo, M., del Portillo, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+spleen:+quantitative+analysis+of+parasite+mobility+and+blood.">Intravital microscopy of the spleen: quantitative analysis of parasite mobility and blood.</a> J. Vis. Exp. , (2012).</li><li>Kamoun, W. 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=Simultaneous+measurement+of+RBC+velocity,+flux,+hematocrit+and+shear+rate+in+vascular+networks.">Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks.</a> Nat. Methods. 7, 655-660 (2010).</li><li>MacPhee, P. J., Schmidt, E. E., Groom, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittence+of+blood+flow+in+liver+sinusoids,+studied+by+high-resolution+in+vivo+microscopy.">Intermittence of blood flow in liver sinusoids, studied by high-resolution in vivo microscopy.</a> Am. J. Phys. 269, G692-G698 (1995).</li></ol>

The authors have nothing to disclose.

The recent development of intravital microscopy of the mouse liver opens up new possibilities for the investigation of physiological response to infection in vivo and&#160;in real time5,9,10. Organ blood flow is a critical physiological parameter that is often altered in many diseases.&#160;However, the status of liver hemodynamics under physiological and infectious conditions remains a poorly explored area. In this study, IVM-based methods, which were previously adapted for the study of the spleen an...

Organ-specific hemodynamics are important physiological features of any mammalian organ. Abnormalities in blood flow may be the consequence of inflammation and a sign of organ dysfunction1. Thus, blood flow organization, structure and function appear as critical parameters for analysis under physiological and pathological conditions. The techniques that have been commonly used for analyzing blood flow in a specific organ contain several limitations, including the resolution limit of the technique itself (e.g.&#160;Doppler imaging of blood flow), the capacity for measurement of absolute blood flow only (volume of blood per unit serving an organ) (e.g.&#160;Optical Coherence tomography) and the measurement of average changes in speed in a large and heterogeneous population of blood vessels2,3. The liver&#8217;s circulatory system links different vessel subtypes that are heterogeneous in their size, structure and function. In this study, intravital microscopy (IVM) imaging technology is applied to evaluate liver hemodynamics in vivo, in real time, at high resolution and in parallel to uncover the characteristics of the individual blood vessels that comprise the hepatic organ. The recent development of this powerful optical imaging technique allows the researcher to collect dynamic data on living animals at a high spatial and temporal resolution. By allowing the direct visualization and real time monitoring of specific and rapid biological processes in vivo, IVM provides a unique opportunity to the researcher to image individual blood vessels, and measure and quantify the velocity of single red blood cells (RBC) within a specifically selected hepatic vessel.
In this study, we have implemented the IVM technique in the mouse liver to investigate the influence of mouse infection by the hepatotropic Leishmania parasite on liver hemodynamics. L. donovani is the agent responsible for visceral leishmaniasis, a severe disease characterized by acute-on-chronic inflammatory responses and a pathology that is present in multiple organs, including the spleen and the liver. In an experimental mouse model of visceral leishmaniasis, the liver infection is self-resolving whereas the splenic infection is progressive4. These outcomes of Leishmania infection with respect to the individual organs are still not completely understood. Investigation of liver and spleen hemodynamics under pathological conditions will shed new light on host-parasite interactions and disease pathogenesis.
Our experimental model system is based on exposing and imaging the liver of an anesthetized mouse that received intravenous injection of specific fluorescent dyes for the labeling of the hepatic intravasculature. The liver is a favorable organ for intra-vital microscopy. After performing a small incision in the abdomen, the liver is gently externalized and placed on wet gauze, then on&#160;a coverslip with the goal of reducing any motion artifacts due to heartbeat and respiration. The liver is then placed within the view of a microscope lens. As compared to the spleen and lymph node which require the use of two photon microscopy for IVM studies, the advantage of the liver lies in its homogenous 3D architecture/anatomy that allows for the use of a conventional confocal microscope, with a maximum penetration depth of approximately 50 &#956;m, for intravital microscopy imaging5-8.
This study describes two independent imaging methods for the quantitative measurement of RBC velocity and blood flow speed in individual blood vessels. In the first method, liver blood flow is acquired using a xy bi-dimensional mode over time. The resulting xyt data are analyzed using the MtrackJ plugin in the free ImageJ software, which allows for the tracking of individual RBCs over time. In the second method, a single blood vessel is selected and its corresponding blood flow is analyzed using the line scanning fast acquisition mode of the confocal laser-scanning microscope. The vessel of interest is scanned at high frequency along its central axis through an axial line. The blood flow velocity is then quantified based on the difference in contrast between unlabeled dark erythrocytes and fluorescently labeled plasma. The fluorescence intensities of RBCs and plasma acquired along the line scan are plotted against time to obtain streaks, the angles of which are proportional to the velocities of an individual RBC.
The goal of this article is to provide a simple and reproducible method for imaging and measuring blood flow velocity within individual blood vessels of the liver and to make available the basic tools for the successful performance of mouse surgery, IVM and quantitative analyses of the velocity of individual RBCs. This approach will allow researchers to gain new insights into blood velocity under pathological conditions.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-Alexa 647</td>
			<td>lifetechnologies</td>
			<td>A34785</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran-FITC 500 mol wt</td>
			<td>SIGMA</td>
			<td>46947</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>PanPharma</td>
			<td>20434</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>KP07KEU</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetedine</td>
			<td>Pharma Animal</td>
			<td>6869029</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate liquid</td>
			<td>Cyanolit</td>
			<td>5833300005</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip frame: Membrane slide for microdissection part N&#176;: 5013</td>
			<td>Molecular machines</td>
			<td>50103</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip DiaPath 24x60 ep: 1.6 mm</td>
			<td>DiaPath</td>
			<td>61061</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica&#160;</td>
			<td>TCS-SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS-AF viewer&#160;</td>
			<td>Leica software</td>
			<td>Version 3.1.0 buid 8587</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The specific architectural organization of the sinusoids in the liver can be visualized based on the autofluorescent property of this organ (Figure 1, panel B and C, green), the intraperitoneal injection of Hoechst for the labeling of hepatocyte nuclei (Figure 1B, blue) and the intravenous injection of fluorescent BSA for the staining of the hepatic circulatory system (Figure 1C, red). The liver is composed of several different vessel subtypes with different functions an...

Watch this Scientific Journal Video about Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics at JoVE.com

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 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 ...

intravital-microscopy-imaging-liver-following-leishmania-infection-an

Research

JoVE Journal

Immunology and Infection

11.4K Views.  University of Aix-Marseille. 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.

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

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

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

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

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

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

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, ...

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 ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...