This work was supported by the National Natural Science Foundation of China (81772133, 81902444), the Guangdong Natural Science Fund (2020A1515010269, 2020A1515011367), the Guangzhou Citizen Health Science and Technology Research Project (201803010034, 201903010072), and the Military Medical Innovation Project (17CXZ008).

All animal care and procedures were in accordance with China Nanfang Hospital policies for heath and well-being (application No: NFYY-2019-73).
1. Mouse preparation
<ol>
	<li>Anesthetize the mouse.
	<ol>
		<li>Prepare sodium pentobarbital (50 mg/kg) in a syringe.</li>
		<li>Grab the mouse (8-week-old male C57BL/6) with the left hand so that its belly is facing up and its head is lower than its tail. Disinfect the abdominal skin with 75% alcohol.</li>
		<li>Holding the syringe in the right hand, pierce the abdomen white line with the needle slightly to the right side of the skin by 3-5 mm. Ensure that the syringe needle and the skin are at a 45&#176; angle, inject the pentobarbital slowly into the abdominal cavity.&#160;&#160;After shaving, disinfects around the mouse&#8217;s incision site 3 times alternately with 75% alcohol and Chlorhexidine solution.</li>
		<li>Check whether the anesthesia was successful by the lack of a righting reflex.</li>
	</ol>
	</li>
	<li>Inject Rhodamine B isothiocyanate&#8211;dextran into the caudal vein.
	<ol>
		<li>Prepare 100 &#181;L of 10 mg/mL Rhodamine B isothiocyanate&#8211;dextran in a syringe.</li>
		<li>Wipe the mouse&#8217;s tail with 75% alcohol.</li>
		<li>Hold the tail with the left hand, hold the syringe in the right hand with the needle parallel to the vein, and inject the 100 &#181;L dye.</li>
		<li>Stop the bleeding with a cotton swab after the injection.</li>
	</ol>
	</li>
	<li>Soak the mouse&#8217;s abdominal fur with gauze wetted with sterile water, and shave the mouse&#8217;s abdomen using a razor, making strokes in the direction of the fur.After shaving, disinfects around the mouse&#8217;s incision site 3 times alternately with 75% alcohol and Chlorhexidine solution.</li>
	<li>After wiping a heating pad with 75% alcohol, open the heating pad, and place the mouse on its back in the heating pad to maintain its body temperature at 37 &#176;C.</li>
</ol>
2. Fixing the mouse liver with the body organ imaging frame
NOTE: The commercial organ imaging frame has not been released yet.
<ol>
	<li>Place a clean suction cup in a fixed position.</li>
	<li>Install an organ imaging fixture (Supplementary Figure 1) and wipe the heating pad and suction cup with 75% alcohol.</li>
	<li>Connect the suction cup (5 mm diameter) to the vacuum pump hose and turn on the vacuum pump.</li>
	<li>On a sterile table sterilized with 75% alcohol, disinfects around the mouse&#8217;s incision site 3 times alternately with 75% alcohol and Chlorhexidine solution, then disinfects around the mouse&#8217;s incision site 3 times alternately with 75% alcohol and Chlorhexidine solution, then use surgical scissors to cut 2 cm of skin off the lower sternal border of the mouse and expose the liver.</li>
	<li>Place the mouse together with the heating pad on the holder base.</li>
	<li>Adjust the organ imaging fixture so that the suction cup holds the liver. Then use a negative pressure of 30-35 kPa for suction so that the liver attaches to the suction cup (Supplementary Figure 2).</li>
</ol>
3. Adjusting the multiphoton laser scanning microscope
<ol>
	<li>Turn on the multiphoton microscope and select the 60x/1.00 W objective.</li>
	<li>Fix the frame and mouse under the objective lens.</li>
	<li>Add a drop of normal saline that can cover the entire lens, which is the center of the suction cup, and adjust the objective lens to just touch the normal saline.</li>
	<li>Double-click on the computer icon to turn on the laser software and turn on the switch. Then press and hold the power button and shutter for 3 s.</li>
	<li>Set the wavelength to 800 nm.</li>
	<li>Start the microscope operating software using the computer.</li>
	<li>For the Acquisition Setting, set Laser 800.</li>
</ol>
4. Observation using multiphoton laser scanning microscope
<ol>
	<li>For Image Acquisition Control, click the Fluorescent switch.</li>
	<li>Ensure that the room and equipment lights are switched off. To reduce noise levels, use the microscope in a darkened environment.</li>
	<li>Open the light path shutter on the microscope.</li>
	<li>Rotate the fluorescence filter to the fourth gear and pull the two levers of the optical switch.</li>
	<li>Looking through the eyepiece, use the coarse and fine focusing quasispirals to adjust the focal length, and use the X/Y axes to adjust the field of view to find the target area.</li>
</ol>
5. Multiphoton laser scanning micrography
<ol>
	<li>Turn off the fluorescent switch on the software, switch the fluorescent filter to the second gear (second gear is RXD1), and push the two levers of the optical switch.</li>
	<li>Click on Focus &#215;2 to preview the target area and adjust the acquisition setting and image acquisition control parameters (image resolution: 1024).</li>
	<li>Press Control + C and adjust the high voltage, gain, and offset (to reduce noise levels).</li>
	<li>Click Stop to stop preview, click the XY button to scan in two dimensions, and save after completion (Figure 1).</li>
	<li>Select a region, click on Depth, and then Preview to select the end set and start set (see the complete required part of the blood vessel) in the Microscope setting. Scan 3D images and save after completion (Figure 2).</li>
	<li>Select a region, click on Time, and adjust other acquisition setting and image acquisition control parameters (Step Size: 1 &#956;m; Slices: 10; Time Scan Number: 40). Scan different slices and save after completion to obtain moving images (Video 1). 
	NOTE: Have someone care for the mice during the scan and add anesthesia accordingly.</li>
	<li>Sacrifice the mouse by neck dissection.</li>
</ol>

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K., Sivasubramanian, K., Mark, C. S., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+vascular+imaging+techniques+in+tissue+engineering+and+regenerative+medicine.">Recent developments in vascular imaging techniques in tissue engineering and regenerative medicine.</a> Biomed Research International. 2015, 783983 (2015).</li><li>Ustione, A., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+introduction+to+multiphoton+microscopy.">A simple introduction to multiphoton microscopy.</a> Journal of Microscopy. 243 (3), 221-226 (2011).</li><li>Centonze, V. E., White, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+excitation+provides+optical+sections+from+deeper+within+scattering+specimens+than+confocal+imaging.">Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging.</a> Biophys Journal. 75 (4), 2015-2024 (1998).</li><li>Vogt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatic+multiphoton+imaging+of+the+whole+brain.">Chromatic multiphoton imaging of the whole brain.</a> Nature Methods. 16 (6), 459 (2019).</li><li>Bacskai, B. 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The authors have nothing to disclose.

Observing a specific living tissue is an effective means of understanding the changes, localization, and biological effects of the material inside the tissue17. In this experiment, the important steps are fixing the liver with an organ imaging fixture, which can solve the problem of motion artifacts due to breathing and heartbeats, and the use of a multiphoton microscope for observation. Using this method, the internal tissues of the liver in vivo are observed through a multiphoton microscope, and the blood vessels are fluorescently labeled to clearly observe their position and three-dimensional structure. This method can be used to observe specific effects by fluorescently labeling a substance and dynamically following the substance in the living tissue to provide further visualizations.
Multiphoton microscopy has evolved from being a novel technique to become an indispensable tool for collecting information from cellular events in an organized tissue environment. However, the organ must be fixed in place to produce a stable image so that the method can be effective in observing certain tissues and organs. Movement artifacts from breathing and the heartbeat can prevent the liver from being directly imaged, so a body organ fixture is used, which uses the principle of vacuum adsorption to secure the organ and prevent image shake. The vacuum chuck can also fix a variety of body organs. However, there is a risk of tissue rupture if the appropriate negative pressure is not used or the imaging time extends beyond 2 h.
Although the resolution of multiphoton microscopy is slightly less than that of confocal microscopy, the depth of penetration is increased to 9-10 &#181;m, enabling the provision of images for deeper tissue observation, cellular events, and material effects.

Blood vessels can provide nutrients for various organ tissues of the human body, and exchange substances. At the same time, many cytokines, hormones, drugs, and cells also function through vascular transport to specific locations. Observing vascular changes in liver tissue can help in understanding the distribution of blood flow in liver tissue and the transport of substances, and assist in the analysis of certain vascular-related diseases1,2.
There are many ways to observe the blood vessels of the liver in mice. Among them, optical microscopy has many limitations in observing opaque vascular tissue3. Multiphoton microscopy can be used to image the blood vessels of living livers with noninvasive high resolution4. Not only can three-dimensional images of blood vessels be obtained, but the technique can also be used to help organize the tissue to observe biological effects therein; furthermore, the whole tissue can be imaged rather than only the microvessels as in computed tomography and magnetic resonance imaging5.
Multiphoton microscopy can be used to effectively detect scattered fluorescent signals in deep living tissue, with less phototoxicity6. Therefore, the activity of living tissue can be ensured, and the amount of damage can be reduced. Multiphoton microscopy has better penetrating power than confocal microscopy, allowing deeper layers to be observed7, providing unique 3D imaging. Multiphoton microscopy is now often used in imaging cranial nerves8 and has been extended to the study of neuronal dynamics in live mice9,10,11.
In this experiment, after fluorescent labeling of mouse blood vessels, the liver is fixed in a frame, and the dynamics of blood vessels in living liver tissue can be seen using multiphoton microscopy. This experiment demonstrates how to mark specific substances, use multiphoton microscopy to help observe a location within the tissue, observe cellular events in the intercellular tissue, make photochemical measurements12,13,14, and observe the material dynamics inside the living tissue15. For example, tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors, marking single-chain variable fragment (scFv) 78 against TEM1, and then multiphoton microscopy can be used for mouse hemangioma location and evaluation of tumors16.

<table><tbody><tr><td>1 mL syringe x 2</td><td>Hunan Pinan Medical Devices Technology</td><td>YA0551</td><td></td></tr><tr><td>5 W heating pad</td><td>BiolinkOptics Technology</td><td>BL336</td><td></td></tr><tr><td>75% absolute ethanol</td><td>Guangdong Guanghua Sci-Tech</td><td>1.17113.023</td><td></td></tr><tr><td>Absorbent cotton ball</td><td>Healthy Sanitation Kingdom</td><td></td><td></td></tr><tr><td>Mouse surgical instrument</td><td>RWD Life Science</td><td>SP0001-G</td><td>Including scissors and tweezers</td></tr><tr><td>Multiphoton microscopy</td><td>Olympus</td><td>FV1200MPE</td><td></td></tr><tr><td>Organ imaging fixture</td><td>BiolinkOptics Technology</td><td>BL336</td><td>Including suction cup, hose, negative pressure pump and bracket</td></tr><tr><td>Rhodamine B isothiocyanate&amp;ndash;Dextran</td><td>Sigma</td><td>R9379</td><td></td></tr><tr><td>Shaving machine</td><td>Lei Wa</td><td>RE-3201</td><td></td></tr><tr><td>Sodium pentobarbital</td><td>Sigma</td><td>P3761-25G</td><td></td></tr></tbody></table>

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.

The distribution of blood vessels in the liver can be seen in Figure 1, obtained using multiphoton microscopy. The blood vessel is divided into a plurality of branches emanating from a trunk and distributed to the surrounding space. The outer circumference of the blood vessel is red, the inner cavity is dark, and there are many things inside. The clearer the image, the closer to the plane of observation it is. There are also some red spots around, probably because the dye penetrates the surrounding tissue to stain other substances. In Video 1, there are non-red things that could be cells moving in the red blood vessels. At the end of the video, some of these things are darkened. This is probably because the fixed liver has not been fixed well over time. This method will allow imaging for 2 hours.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60932/60932fig01.jpg" /> 
Figure 1: The blood vessels in the liver tissue under multiphoton microscopy.&#160;The blood vessels are red. A two-dimensional image of the blood vessel with a resolution of 2048 and an HV of 512, can clearly image the red blood vessels after staining. There are also some red spots around, and the dye may also penetrate the surrounding tissue to dye other substances. <a href="https://www.jove.com/files/ftp_upload/60932/60932fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60932/60932fig02.jpg" /> 
Figure 2: Three-dimensional image of living liver tissue in the same position.&#160;The thickness is 9 &#181;m, the morphology of the blood vessels is observed from different angles, and dynamic images are collected. After synthesis, the cells in the blood vessels (dark) are seen flowing. <a href="https://www.jove.com/files/ftp_upload/60932/60932fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Body organ imaging frame. (a) Heating pad: It maintains the mouse body temperature. (b) Bracket: It can provide a base to place the mouse and the adjustable suction cup position with knobs. (c) Suction cup: It is fixed by the bracket. The negative pressure provided by the negative pressure pump causes the liver to adsorb to the transparent lens in the center of the suction cup. (d) Flexible pipe: It connects the suction cup with the pressure pump. The pressure pump provides negative pressure to the suction cup. (e) Centrifuge tube: The centrifuge tube in the middle of the flexible pipe can hold the liquid, to prevent these liquids from being sucked into the vacuum pump. (f) Negative pressure pump: It is connected to the suction cup by the flexible pipe. The negative pressure pump can provide negative pressure to the suction cup to make the liver cling to the lens. <a href="https://cloudflare.jove.com/files/ftp_upload/60932/60932supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 2: A close-up image of the liver attached to transparent lens. The transparent lens is in the center of the suction cup. The pump provides negative pressure to the suction cup to make it suck the liver and the liver is attached to the transparent lens. Because the suction-faced liver is a bit away from the mouse, the effect of breathing and the heartbeat on artifacts is reduced. The transparent lens can provide a stable view for the multiphoton microscope. <a href="https://cloudflare.jove.com/files/ftp_upload/60932/60932supfig02.jpg" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue at JoVE.com

Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue

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 ...

multiphoton-microscopic-observation-of-vessels-in-mouse-liver-tissue

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

3.6K Views.  Southern Medical University. Deze methode kan ons helpen om de bewegingen van bloedvaten in de muizenlever te visualiseren langs de evolutie van de dynamiek van de lever in meerdere dimensies. Met deze methode kunnen we de dynamiek van bloedvaten in de lever in drie dimensies visualiseren. We hebben het gebruik van vasculaire kleurstof aangetoond, maar u kunt de vaatkleuring met uw stof van belang uitvoeren vóór microscopische visualisatie.Na het bevestigen van een gebrek aan het rechttrekken van reflex in een v...

Video: Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

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 ...

Immunology and Infection

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, ...

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 ...

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 ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Medicine

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Neuroscience

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

Ultrafast Doppler Ultrasound for Hepatic Microvasculature Analysis in Mice

Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the microvasculature using Doppler ultrasound methods, imaging modalities employing the concept of ultrafast imaging are preferred. By increasing the frame rate of the ultrasound scanner to thousands of frames per second, it becomes possible to improve the sensitivity of the blood flow down to 2 mm/s and to obtain functional information about the microcirculation in comparison to a sensitivity of around 1 cm/s in conventional Doppler modes. While Ultrafast Doppler ultrasound (UFUS) imaging has become adopted in neuroscience, where it can capture brain activity through neurovascular coupling, it presents greater challenges when imaging the vasculature of abdominal organs due to larger motions linked to breathing. The liver, positioned anatomically under the diaphragm, is particularly susceptible to out-of-plane movement and oscillating respiratory motion. These artifacts not only adversely affect Doppler imaging but also complicate the anatomical analysis of vascular structures and the computation of vascular parameters. Here, we present a qualitative and quantitative imaging analysis of the hepatic vasculature in mice by UFUS. We identify major anatomical vascular structures and provide graphical illustrations of the hepatic macroscopical anatomy, comparing it to an in-depth anatomical assessment of the hepatic vasculature based on Doppler readouts. Additionally, we have developed a quantification protocol for robust measurements of hepatic blood volume of the microvasculature over time. To contemplate further research, qualitative vascular analysis provides a comprehensive overview and suggests a standardized terminology for researchers working with mouse models of liver disease. Furthermore, it offers the opportunity to apply ultrasound as a non-invasive complementary method to inspect hepatic vascular defects in vivo and measure functional microvascular alterations deep within the organ before unraveling blood vessel anomalies at the micron scale levels using ex vivo staining on tissue sections.

Ultrafast Doppler ultrasound (UFUS) with high spatial resolution (100 &#956;m) and sensitivity represents a suitable noninvasive imaging modality for obtaining a comprehensive qualitative overview of the hepatic vasculature. UFUS also facilitates quantitative measurements of the microvasculature, aiming to enhance our understanding of vascular disease mechanisms.

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the ...

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the end pathway for both inflammation and thrombosis, it is not capable of showing the temporal changes that occur throughout the time course of these processes. Intravital microscopy (IVM) is the use of live-animal imaging to gain temporal insight into physiologic processes in vivo. This method is particularly powerful when assessing cellular and protein interactions within the circulation due to the rapid and sequential events that are often necessary for these interactions to occur. While IVM is an extremely powerful imaging methodology capable of viewing complex processes in vivo, there are a number of methodological factors that are important to consider when planning an IVM study. This paper outlines the process of conducting intravital imaging of the liver, identifying important considerations and potential pitfalls that may arise. Thus, this paper describes the use of IVM to study platelet-leukocyte-endothelial interactions in liver sinusoids to study the relative contributions of each in different models of acute liver injury.

Intravital microscopy is a powerful tool that provides insight into both the temporal and spatial relationships of rapid and/or sequential processes. Herein, we describe a protocol to assess both protein-protein interactions and platelet-neutrophil-endothelial interactions in liver sinusoids in a murine model of experimental sepsis (endotoxemia).

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the ...

Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue

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