Name: multiphoton microscopic observation of vessels in mouse liver tissue
Uploaded: 2021-05-17T12:30:00
Description: Watch this Scientific Journal Video about Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue at JoVE.com

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 ha...

<ol>
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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...

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&#8211;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 surr...

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 ...

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