Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue

Summary

In this experiment, a mouse is injected in its tail vein with Rhodamine B isothiocyanate–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.

Abstract

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.

Introduction

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 diseases^1,2.

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 mice^9,10,11.

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 measurements^12,13,14, 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.

Protocol

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

1. Anesthetize the mouse.
   1. Prepare sodium pentobarbital (50 mg/kg) in a syringe.
   2. 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.
   3. 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° angle, inject the pentobarbital slowly into the abdominal cavity.  After shaving, disinfects around the mouse’s incision site 3 times alternately with 75% alcohol and Chlorhexidine solution.
   4. Check whether the anesthesia was successful by the lack of a righting reflex.
2. Inject Rhodamine B isothiocyanate–dextran into the caudal vein.
   1. Prepare 100 µL of 10 mg/mL Rhodamine B isothiocyanate–dextran in a syringe.
   2. Wipe the mouse’s tail with 75% alcohol.
   3. 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 µL dye.
   4. Stop the bleeding with a cotton swab after the injection.
3. Soak the mouse’s abdominal fur with gauze wetted with sterile water, and shave the mouse’s abdomen using a razor, making strokes in the direction of the fur.After shaving, disinfects around the mouse’s incision site 3 times alternately with 75% alcohol and Chlorhexidine solution.
4. 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 °C.

2. Fixing the mouse liver with the body organ imaging frame

NOTE: The commercial organ imaging frame has not been released yet.

1. Place a clean suction cup in a fixed position.
2. Install an organ imaging fixture (Supplementary Figure 1) and wipe the heating pad and suction cup with 75% alcohol.
3. Connect the suction cup (5 mm diameter) to the vacuum pump hose and turn on the vacuum pump.
4. On a sterile table sterilized with 75% alcohol, disinfects around the mouse’s incision site 3 times alternately with 75% alcohol and Chlorhexidine solution, then disinfects around the mouse’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.
5. Place the mouse together with the heating pad on the holder base.
6. 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).

3. Adjusting the multiphoton laser scanning microscope

1. Turn on the multiphoton microscope and select the 60x/1.00 W objective.
2. Fix the frame and mouse under the objective lens.
3. 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.
4. 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.
5. Set the wavelength to 800 nm.
6. Start the microscope operating software using the computer.
7. For the Acquisition Setting, set Laser 800.

4. Observation using multiphoton laser scanning microscope

1. For Image Acquisition Control, click the Fluorescent switch.
2. Ensure that the room and equipment lights are switched off. To reduce noise levels, use the microscope in a darkened environment.
3. Open the light path shutter on the microscope.
4. Rotate the fluorescence filter to the fourth gear and pull the two levers of the optical switch.
5. 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.

5. Multiphoton laser scanning micrography

1. 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.
2. Click on Focus ×2 to preview the target area and adjust the acquisition setting and image acquisition control parameters (image resolution: 1024).
3. Press Control + C and adjust the high voltage, gain, and offset (to reduce noise levels).
4. Click Stop to stop preview, click the XY button to scan in two dimensions, and save after completion (Figure 1).
5. 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).
6. Select a region, click on Time, and adjust other acquisition setting and image acquisition control parameters (Step Size: 1 μm; 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.
7. Sacrifice the mouse by neck dissection.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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