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>
	<li>Wu, Z., 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=Multi-photon+microscopy+in+cardiovascular+research.">Multi-photon microscopy in cardiovascular research.</a> Methods. 130, 79-89 (2017).</li><li>Zhou, M., Ling, W., Luo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrahepatic+mass-forming+cholangiocarcinoma+growing+in+a+giant+hepatic+hemangioma:+A+case+report.">Intrahepatic mass-forming cholangiocarcinoma growing in a giant hepatic hemangioma: A case report.</a> Medicine (Baltimore). 98 (27), 16410 (2019).</li><li>Werkmeister, 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=Multiphoton+microscopy+for+blood+vessel+imaging:+new+non-invasive+tools+(Spectral+SHG,+FLIM).">Multiphoton microscopy for blood vessel imaging: new non-invasive tools (Spectral SHG, FLIM).</a> Clinical Hemorheology and Microcirculation. 37 (1-2), 77 (2007).</li><li>Wang, H., 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=Does+optical+microangiography+provide+accurate+imaging+of+capillary+vessels?:+validation+using+multiphoton+microscopy.">Does optical microangiography provide accurate imaging of capillary vessels?: validation using multiphoton microscopy.</a> Journal of Biomedical Optics. 19 (10), 1-5 (2014).</li><li>Upputuri, P. 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. J., 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+of+amyloid-&#946;+deposits+in+brains+of+living+mice+permits+direct+observation+of+clearance+of+plaques+with+immunotherapy.">Imaging of amyloid-&#946; deposits in brains of living mice permits direct observation of clearance of plaques with immunotherapy.</a> Nature Medicine. 7 (3), 369-372 (2001).</li><li>Lendvai, B., Stern, E. A., Chen, B., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+plasticity+of+dendritic+spines+in+the+developing+rat+barrel+cortex+in+vivo.">Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo.</a> Nature. 404 (6780), 876-881 (2000).</li><li>Svoboda, K., Denk, W., Kleinfeld, D., Tank, 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=In+vivo+dendritic+calcium+dynamics+in+neocortical+pyramidal+neurons.">In vivo dendritic calcium dynamics in neocortical pyramidal neurons.</a> Nature. 385 (6612), 161-165 (1997).</li><li>Liu, H., 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+Deep-Brain+Structural+and+Hemodynamic+Multiphoton+Microscopy+Enabled+by+Quantum+Dots.">In vivo Deep-Brain Structural and Hemodynamic Multiphoton Microscopy Enabled by Quantum Dots.</a> Nano Letters. , (2019).</li><li>Sandoval, R. M., Molitoris, B. 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+multiphoton+microscopy+as+a+tool+for+studying+renal+physiology+and+pathophysiology.">Intravital multiphoton microscopy as a tool for studying renal physiology and pathophysiology.</a> Methods. 128, 20-32 (2017).</li><li>Shear, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peer+Reviewed:+Multiphoton-Excited+Fluorescence+in+Bioanalytical+Chemistry.">Peer Reviewed: Multiphoton-Excited Fluorescence in Bioanalytical Chemistry.</a> Analytical Chemistry. 71 (17), 598-605 (1999).</li><li>Heymann, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+intravital+multiphoton+microscopy+imaging+of+immune+cells+in+healthy+and+diseased+liver+using+CXCR6.Gfp+reporter+mice.">Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.</a> Journal of Visualized Experiments. (97), (2015).</li><li>Yuan, X., 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=Characterization+of+the+first+fully+human+anti-TEM1+scFv+in+models+of+solid+tumor+imaging+and+immunotoxin-based+therapy.">Characterization of the first fully human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy.</a> Cancer Immunology &amp; Immunotherapy. 66 (3), 367-378 (2017).</li><li>Williams, R. M., Zipfel, W. R., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+microscopy+in+biological+research.">Multiphoton microscopy in biological research.</a> Current Opinion in Chemical Biology. 5 (5), 603-608 (2001).</li></ol>

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...

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 ...

This method can help us to visualize the movements of blood vessels within the mouse liver along evolution of the dynamics of the liver in multiple dimensions. Using this method, we can visualize the dynamics of blood vessels within the liver in three dimensions. We have demonstrated the use vascular dye, but you can perform the vessel staining with your substance of interest before microscopic visualization.After confirming a lack of righting reflex in an anesthetized eight week old male C 57 black six mouse. Wipe the mouse's tail with 75%alcohol and use a one microliter syringe equipped with a 30 gauge needle to inject 10 milligrams per milliliter of freshly prepared Rhodamine B Isothiocyanate-Dextran in 100 microliters of Rhodamine B Isothiocyanate-Dextran into the caudal tail vein. When all of the solution has been delivered use a cotton swab to apply pressure to the puncture site and use gauze wet with sterile water to soak the abdominal fur.Use a razor to shave the abdomen, making strokes in the direction of the fur, and place the mouse onto a 37 degrees Celsius, alcohol disinfected heating pad. To fix the mouse liver with a body organ imaging frame, First place a clean five millimeter diameter suction cup in a fixed position and wipe the heating pad and suction cup with 75%alcohol. Connect the suction cup to the vacuum pump hose and turn on the pump.On a 75%alcohol sterilized table use surgical scissors to cut two centimeters of skin from the lower sternal border of the mouse and expose the liver. Place the mouse and the heating pad onto the holder base of the body frame and adjust the organ imaging fixture so that the suction cup can hold the liver. Then use a negative pressure of 30 to 35 kilopascals for suction so that the liver attaches to the cup.To set up the Multiphoton Laser Scanning Microscope turn on the Microscope and select the 60 X objective. Fix the frame and the mouse under the objective and add a drop of normal saline to the cup large enough to cover the lens. Adjust the objective so the lens just touches the normal saline and turn on the laser software.Turn on the laser and press and hold the power button and shutter for three seconds. Then start the microscope operating software and set the laser to 800 nanometers. To set the image acquisition control click the fluorescent switch and turn off the room and equipment lights.Open the light path shutter on the microscope and rotate the fluorescence filter to the fourth gear. Pull the two levers of the optical switch and looking through the eye piece, use the course and fine focusing quasi spirals to adjust the focal length. Then use the X Y axes to adjust the field of view to locate the target area.For imaging, turn off the fluorescent switch in the software and switch the fluorescent filter to the second gear. Push the two levers of the optical switch and click focus times two to preview the target area adjust the acquisition settings and the image acquisition control parameters and press control plus C to adjust the high voltage gain and offset. Click stop to stop the preview, click the X Y button to scan in two dimensions and click save.Select a region and click depth and preview to select the end and start sets in the microscope settings. Scan 3D images and save. After scanning, select a region click time and adjust the other acquisition settings and image acquisition controlled parameters.Then scan through the different slices and save to obtain the resulting image movie. Here visualization of the distribution of blood vessels within the liver using multi photon microscopy as demonstrated, can be observed. This blood vessel is divided into a plurality of branches emanating from a trunk and distributed into the surrounding space.The outer circumference of the blood vessel can be observed in red while the inner cavity is dark, as observed in the video non red objects moving within the vessels could be cells. The darkness of the tissue observed in this video is likely due to the liver not having been fixed well over time. It is important to remember that how scar-less liver is fixed, affects the stability of the image.This methodology can also be applied to the position of other structures within the liver for the direct observation of the distribution or changes in specific labor structures of interest.

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

Research

JoVE Journal

Bioengineering

3.5K Görüntüleme.  Southern Medical University. Bu yöntem, karaciğerin dinamiklerinin evrimi boyunca fare karaciğeri içindeki kan damarlarının hareketlerini birden fazla boyutta görselleştirmemize yardımcı olabilir. Bu yöntemi kullanarak karaciğer içindeki kan damarlarının dinamiklerini üç boyutlu olarak görselleştirebiliriz. Damar boyası kullanımını gösterdik, ancak mikroskobik görselleştirmeden önce ilgi çekici maddenizle damar boyama işlemi yapabilirsiniz.Uyuşturulmış sekiz haftalık erkek C 57 siy...