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

这项工作得到了中国国家自然科学基金委员会（81772133） 的支持。 81902444）、广东省自然科学基金（2020A1515010269、2020A15150111367）、广州市公民健康科技研究项目（201803010034、201903010072）和军事医学创新项目（17CXZ008）。

所有动物护理和程序均符合中国南方医院健康福祉政策（申请号：NFYY-2019-73）。
1. 鼠标准备
<ol><li>麻醉鼠标。<ol>
<li>在注射器中准备五巴比妥钠（50毫克/千克）。</li>
		<li>用左手抓住鼠标（8周大的雄性C57BL/6），使其腹部朝上，头部低于尾巴。用75%的酒精消毒腹部皮肤。</li>
		<li>右手拿着注射器，用针刺穿腹部白线，稍微刺穿皮肤右侧3-5毫米。确保注射针和皮肤处于 45° 角?...

<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. 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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. 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(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>

观察特定的活组织是了解组织内物质变化、定位和生物影响的有效手段。在这个实验中，重要的步骤是用器官成像固定装置固定肝脏，它可以解决呼吸和心跳导致的运动神器问题，并使用多光子显微镜进行观察。使用这种方法，通过多光子显微镜观察肝脏体内的内部组织，并荧光标记血管，以清楚地观察其位置和三维结构。这种方法可用于观察特定效果，通过荧光标记物质和动?...

血管可以为人体的各种器官组织提供营养，并交换物质。同时，许多细胞因子、激素、药物和细胞也通过血管输送到特定位置而发挥作用。观察肝组织血管变化有助于了解肝组织中血流的分布和物质的输送，有助于分析某些血管相关疾病1、2。
有许多方法可以观察小鼠肝脏的血管。其中，光学显微镜在观察不透明血管组织3方面有许多局限性。多光子显微镜可用于用非侵入性高分辨率4对活体肝脏的血管进行成像。不仅可以获得血管的三维图像，而且该技术还可用于组织组织观察其中的生物效应：此外，整个组织可以成像，而不仅仅是微维塞尔在计算断层扫描和磁共振成像5。
多光子显微镜可用于有效检测深层活组织中零散的荧光信号，光毒性6较小。因此，可以保证活组织的活动，减少损伤。多光子显微镜比共聚焦显微镜具有更好的穿透力，可观察到更深的层7，提供独特的3D成像。多光子显微镜现在常用于成像颅神经8，并已扩展到活小鼠9，10，11的神经元动力学研究。
在这个实验中，在老鼠血管的荧光标记后，肝脏固定在一个框架中，使用多光谱显微镜可以看到活体肝组织中血管的动力学。本实验演示了如何标记特定物质，使用多光子显微镜帮助观察组织内的位置，观察细胞间组织的细胞事件，进行光化学测量12，13，14，并观察活组织15内的物质动力学。例如，肿瘤内皮标记1（TEM1）已被确定为一种新的表面标记，在血管和频闪在许多实体肿瘤，标记单链变量片段（scFv）78对TEM1，然后多光子显微镜可用于小鼠血管瘤的位置和肿瘤16的评估。

<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

观察小鼠肝组织的血管内动力学，使我们能够对小鼠肝脏组织相关疾病进行进一步的深入观察和研究。老鼠被注射一种染料，可以染色血管。要观察小鼠肝脏的体内，它被暴露和固定在一个框架中。使用多光子显微镜获取肝脏组织中血管的二维和三维图像。不断获取选定地点的组织图像，以观察长期变化：还观察到肝组织血管的动态变化。多光显微镜是观察深层组织部分或器官细胞和细胞功能的一种方法。多光子显微镜对组织微观结构具有敏感性，能够在体内以高空间分辨率成像生物组织，从而能够捕获组织的生化信息。多光子显微镜用于观察肝脏的一部分，但修复肝脏，使图像更稳定是有问题的。在这个实验中，一种特殊的真空吸盘用于修复肝脏，并在显微镜下获得更稳定的肝脏图像。此外，该方法还可用于通过用染料标记此类物质来观察肝脏中特定物质的动态变化。

肝脏血管的分布可以在 图1中看到，该图是利用多光子显微镜获得的。血管被分成多个分支，从树干中流出，并分布到周围的空间。血管的外周是红色的，内腔是暗的，里面有很多东西。图像越清晰，离观察平面越近。周围也有一些红点，可能是因为染料穿透周围的组织来染色其他物质。在 视频1中，有非红色的东西，可能是细胞在红血管中移动。在视频的结尾?...

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

在这个实验中，一只老鼠的尾静脉中注射了可染色血管的罗达明B异氰酸酯。肝脏暴露和固定后，可以选择肝脏的特定部分使用多光子显微镜观察活体的深层组织。

老鼠肝脏组织血管多光子微观观测

Observing the intravascular dynamics of mouse liver tissue allows us to conduct further in-depth observations and studies on tissue-related diseases of ...

这种方法可以帮助我们想象小鼠肝脏内血管的运动，以及肝脏多维动态的演变。使用这种方法，我们可以从三个维度来可视化肝脏内血管的动态。我们已经证明了使用血管染料，但你可以执行容器染色与你感兴趣的物质之前，微观可视化。在确认麻醉的八周大男性 C 57 黑六只老鼠缺乏正确的反射后。用75%的酒精擦拭老鼠的尾巴，使用装有30根量度针的微升注射器，将每毫升新鲜制备的罗达明B异氰酸酯-德克斯特兰注射到100微升的罗达明B异氰酸酯-德克斯特兰中，注入牛皮尾静脉。当所有溶液都送达后，使用棉签对穿刺部位施加压力，并用无菌水将纱布湿润，浸泡腹部毛皮。用剃须刀剃腹部，朝毛皮方向冲压，将鼠标放在37摄氏度的酒精消毒加热垫上。要用身体器官成像框架固定小鼠肝脏，首先在固定位置放置一个直径为五毫米的清洁吸盘，用75%的酒精擦拭加热垫和吸盘。将吸盘连接到真空泵软管，然后打开泵。在75%的酒精消毒表上，使用手术剪刀从小鼠的下胸围切下两厘米的皮肤，并露出肝脏。将鼠标和加热垫放在身体框架的支架底座上，并调整器官成像夹具，使吸盘能够容纳肝脏。然后使用30至35千帕的负压进行吸气，使肝脏附着在杯子上。设置多光子激光扫描显微镜打开显微镜并选择 60 X 目标。修复目标下方的框架和鼠标，并在杯子中加入一滴普通盐水，以覆盖镜头。调整目标，使镜头只接触正常的盐水，打开激光软件。打开激光并按下电源按钮并按住快门三秒钟。然后启动显微镜操作软件，将激光设置为 800 纳米。要设置图像采集控制，请单击荧光开关并关闭房间和设备灯。打开显微镜上的光路径快门，将荧光滤镜旋转到第四档。拉光学开关的两个杠杆，透过眼片看，使用路线和精细对焦准螺旋来调整焦距。然后使用 X Y 轴调整视场以定位目标区域。对于成像，关闭软件中的荧光开关，并将荧光滤镜切换到第二档。推动光学开关的两个杠杆，点击对焦时间二，预览目标区域调整采集设置和图像采集控制参数，并按控加C调整高压增益和偏移。单击"停止"停止预览，单击 X Y 按钮以两个维度进行扫描，然后单击"保存"。选择区域并单击深度并预览以选择显微镜设置中的结束和启动集。扫描 3D 图像并保存。扫描后，选择区域单击时间并调整其他采集设置和图像采集控制参数。然后扫描不同的切片并保存以获取生成的图像电影。这里使用多光纳米显微镜可视化肝脏内血管的分布，如所示，可以观察到。这种血管被分成多个分支，从树干中流出，并分布到周围的空间。血管的外周可以以红色观察，而内腔是暗的，正如视频中观察到的，在血管内移动的非红色物体可能是细胞。在这段视频中观察到的组织的黑暗可能是由于肝脏没有随着时间的推移被很好地修复。重要的是要记住，如何无疤痕肝脏是固定的，影响图像的稳定性。这种方法也可应用于肝脏内其他结构的位置，以便直接观察特定利益劳动结构的分布或变化。

Research

JoVE Journal

Bioengineering

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

在芽殖酵母连续高分辨率显微观察复制性衰老

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

科研

生物工程

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

组织工程血管在小鼠模型的生成和嫁接

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

在小型组织培养体系培养老鼠心脏瓣膜

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

三明治般的微环境来驾驭细胞/材料相互作用

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

倒胶体晶体聚（乙二醇）脚手架制作：肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

小鼠模型中生物合成组织工程气管移植的育苗与植入

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

使用微流体芯片以确定性方式建立基于单细胞的共培养物

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...