Name: a custom multiphoton microscopy platform for live imaging of mouse cornea and conjunctiva
Uploaded: 2020-05-17T14:00:00
Description: Watch this Scientific Journal Video about A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva at JoVE.com

我们感谢台湾科技部（106-2627-M-002-034，107-2314-B-182A-089）的赠款支持， 108-2628-B-002-023，108-2628-B-002-023），台湾国立大学医院（NTUH108-T17）和台湾长荣纪念医院（CMRPG3G1621，CMRPG3G1622，CMRPG3G1623）。

所有动物实验都是按照台湾国立大学动物护理与使用委员会（IACUC）和长贡纪念医院批准的程序进行的。
1. 多光度显微镜设置
<ol><li>构建一个基于直立显微镜的系统，具有浸入水 20x 1.00 NA 目标（图 1A）。</li>
	<li>使用 Ti：蓝宝石激光（可调波长）作为激励源。将EGFP的激光输出波长设置为 880 nm，为 tdTomato 将激光输出波长设置为 940 nm（图...

<ol>
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这个定制的MPM成像平台与控制软件用于小鼠上皮器官的病毒内成像，包括皮肤10，毛囊10和眼表面9，10（109图1A）。自项目开始以来，定制系统一直用于灵活地更改各种实验的光学元件。这种成像方法适用于商用 MPM 产品。该协议描述了通过MPM成像平台对小鼠眼表面进行病毒内成像的详细方法。使...

眼表面结构，包括角膜和结膜，保护其他更深的眼组织免受外部干扰。角膜是眼睛的透明前部，既充当折射透镜，可将光线引导到眼睛中，也作为保护屏障。角膜上皮是角膜的最外层，由不同的表面细胞层、翼细胞和基底细胞组成。角膜频闪由嵌入角细胞的复杂包装胶原蛋白组成。角膜内皮是一层扁平六角细胞，通过泵送功能1保持角膜频闪，在保持角膜的透明度方面具有重要作用。林巴斯形成角膜和结膜之间的边界，是角膜上皮干细胞的蓄水池2。高度血管化的结膜有助于润滑眼睛产生粘液和眼泪3。
角膜表面结构的细胞动力学由组织学分析或体外细胞培养学进行常规研究，可能不足以模拟体内细胞动力学。因此，非侵入性实时成像方法可以弥补这种差距。由于其具有高分辨率、最小光损和深成像深度等优点，MPM已成为生物研究4、5、6、7、8,等不同,6,7领域的有力模式。对于角膜成像，MPM 提供来自细胞内NAD（P）H的固有自荧光的细胞信息。第二次谐波生成（SHG）信号来自非分色类型我胶原纤维在飞秒激光扫描下提供胶原蛋白生成结构，无需额外的染色程序9。此前，我们和其他团体利用MPM对动物和人类角膜进行成像，9、10、11、12、13、14、15。,10,11,12,13,14,15
在特定细胞群中表现出荧光蛋白的转基因小鼠系已广泛应用于细胞生物学的各种研究，包括发育、组织平衡、组织再生和致癌。我们使用标有荧光蛋白的转基因小鼠菌株，通过MPM对角膜9、10、,10毛囊10和表皮10进行体内成像。双荧光小鼠株与细胞膜标记与tDTomato和细胞核标记与EGFP是由两个小鼠株培育：R26R-GR（B6;129-Gt（ROSA）26Sortm1Ytchn/J，#021847）16和16tm1Ytchn/JmT-mG（Gt（ROSA26）ACTB-ttomato-EGFP，#007676ACTB-tdTomato-EGFP17R26R-GR转基因小鼠线包含双荧光蛋白报告器结构，包括H2B-EGFP融合基因和mCherry-GPI锚点信号融合基因，插入Gt（ROSA）26Sor位点。mT-mG转基因菌株是一种细胞膜靶向的tdTomato和EGFP荧光Cre-reporter小鼠。在Cre重组之前，具有tdTomato荧光表达的细胞膜蛋白广泛存在于各种细胞中。这种转基因小鼠菌株使我们能够可视化核-EGFP和膜与tdTomato没有Cre激发。两只雌性（R26R-GR+/+）和一只雄性（mT-mG+/+）转基因小鼠一起繁殖，以产生足够的小鼠进行实验。+/+他们的后代与R26R-GR+/--;mT-mG+/-基因型，双荧光小鼠株，在这项研究中被使用。+/-+/-与前面描述的9、10号荧光记者鼠标线相比，10这种双荧光测量仪的成像时间缩短了50%。
在这项工作中，我们描述了使用我们的成像平台和双荧光转基因小鼠逐步进行眼表面体内成像的详细技术方案。

<table>
	<tbody>
		<tr>
			<td>AVIZO Lite software</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Version: 2019.3.0</td>
		</tr>
		<tr>
			<td>Bandpass filters</td>
			<td>Semrock</td>
			<td>FF01-434/17 
			FF01-500/24 
			FF01-585/40</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichroic mirrors</td>
			<td>Semrock</td>
			<td>FF495-Di01-25x36 FF580-Di01-25x36</td>
			<td></td>
		</tr>
		<tr>
			<td>Galvano</td>
			<td>Thorlabs</td>
			<td>GVS002</td>
			<td></td>
		</tr>
		<tr>
			<td>Jade BIO control software</td>
			<td>SouthPort Corporation</td>
			<td>Jade BIO</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxybuprocaine hydrochloride</td>
			<td>Sigma</td>
			<td>O0270000</td>
			<td></td>
		</tr>
		<tr>
			<td>PMT</td>
			<td>Hamamatsu</td>
			<td>H7422A-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyesthylene Tube</td>
			<td>BECTON DICKINSON</td>
			<td>427401</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic mouse holder</td>
			<td>Step Technology Co.,Ltd</td>
			<td>000111</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti: Sapphire laser</td>
			<td>Spectra-Physics</td>
			<td>Mai-Tai DeepSee</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscopy</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Vidisic Gel</td>
			<td>Dr. Gerhard Mann Chem-pharm. Fabrik GmbH</td>
			<td>D13581</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac</td>
			<td>VR-2831</td>
			<td></td>
		</tr>
	</tbody>
</table>

a custom multiphoton microscopy platform for live imaging of mouse cornea and conjunctiva

传统的组织分析和细胞培养系统不足以完全模拟体内的生理和病理动力学。多光子显微镜 （MPM） 已成为体内细胞水平上生物医学研究最流行的成像模式之一，其优点包括高分辨率、深层组织渗透和极小光毒性。我们设计了一个 MPM 成像平台，具有定制的鼠标眼架和用于体内眼表面成像的立体轴舞台。双荧光蛋白报告鼠标能够可视化细胞核、细胞膜、神经纤维和眼表面的毛细血管。除了多光子荧光信号外，获得第二谐波生成 （SHG） 可同时进行胶原蛋白生成结构。该平台可用于病毒内成像，可准确定位整个眼表面，包括角膜和结膜。

使用这个实时成像平台，鼠标眼表面可以在细胞水平上可视化。为了可视化眼表面的单个细胞，我们采用了双荧光转基因小鼠，其EGFP在细胞核中表达，tdTomato在细胞膜中表达。富含胶原蛋白的角膜频闪被SHG信号突出显示。
在角膜上皮，表面细胞，翼细胞和基底细胞（图2）被可视化。在双荧光转基因小鼠中，我们能够将单个细胞从基底层映射到角膜和边?...

Watch this Scientific Journal Video about A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva at JoVE.com

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

这里展示的是一个多光子显微平台，用于实时小鼠眼表面成像。荧光转基因小鼠能够可视化细胞核、细胞膜、神经纤维和眼表面的毛细血管。从胶原结构派生的非线性二谐波生成信号为频闪结构提供了无标签成像。

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. ...

该协议使用多滤显微镜对双荧光转基因小鼠模型中小鼠眼表面的整个结构进行实时成像。定制多光子显微平台与双荧光转基因小鼠相结合，实现了眼表面和结构的实时可视化。要设置多光子显微镜，请选择浸入式 20X、1.00 NA 目标，并在直立显微镜上将钛蓝宝石激光器设置为激励源。将 EGFP 的激光输出波长设置为 880 纳米，将 tdTomato 的激光输出波长设置为 940 纳米。包括两个双色镜，用于分离SHG EGFP和EGFP tdTomato，并使用434-17、510-84和585-40纳米带通滤波器，以光谱方式分离SHG EGFP和tDTomato信号。要设置眼架，在确认麻醉的8-12周大小鼠对踏板反射反应缺乏反应后，将鼠标放在加热显微镜阶段，并插入温度监测探头。将鼠标放入自定义的立体鼠标支架中，并将耳条插入外听觉肉质中。使用三点固定固定固定固定头架，并在眼表面涂抹 0.4% 盐酸氧丙酸和盐水溶液。用 PE 管回路盖住一对五号 Dumont 钳子的尖端。三分钟后，执行手动眼睑缩回，以确认眼球突出，并小心地沿眼睑边缘放置一个PE支架管环，以暴露眼表面。并使用支架的旋钮用钳子稳定眼球。然后，用眼凝胶覆盖角膜表面，折射率为 1.338。对于角膜表面的 Z 串行成像，使用汞光源对靶向场进行成像并打开显微镜软件。选择适当的照片乘数和数字增益，以可视化眼表面的细胞结构，并设置第一张和最后一张幻灯片，以便采集图像堆栈。将图像分辨率设置为 512 x 512 像素，将 Z 步长设置为 1 微米。然后，单击"开始"收集 Z 串行图像，获取一个带 880 纳米的实时图像，用于 SHG EGFP 信号采集，并在每个区域内以 940 纳米的激发获取一个实时图像。在整个图像中，使用步进电动舞台支架旋转眼球，以便在整个角膜表面进行成像。获取所有图像后，将 Z 串行图像加载到斐济并选择中位数 3D 滤镜插件。消除背景噪音后，选择取消锐化蒙版滤镜包斐济锐化图像，然后单击自动亮度对比度自动优化图像质量。处理后，将图像保存为串行图像序列，然后将图像序列导出到适当的 3D 重建软件程序中。在所有多光子显微镜图像中，在通过快照捕获 3D 结构图像之前，分别以伪绿色、红色和青色呈现 EGFP tdTomato 和 SHG 信号。多光子显微镜允许在双荧光转基因小鼠的角膜上皮上对表面翼和下层细胞进行可视化。基底层的单个细胞可以映射到表面层以及单个六边形表面细胞。正如tdTomato信号的细胞质表达所揭示的，富含细胞内细胞内的膜蛋白系统，包括高尔吉装置和内质神经细胞分散在翼细胞内。在胶原状频闪中，这些小鼠的EGFP荧光膜勾勒出硬质形状的皮质状。嵌入在大学频闪中的皮质体比内皮细胞内更松散的空间。此外，角膜频闪内的薄分支神经也可以通过膜瞄准tdTomato信号和角膜内皮细胞单层显示一个相对均匀的六边形形状连接在蜂窝图案。林皮上皮由一到两层上皮细胞组成。双荧光报告器转基因菌株还能够成像结膜内的毛细血管，促进毛细血管的3D结构的重建，勾勒出血管内皮。在不伤害的中等压力下稳定眼球对于获得高质量的图像至关重要，因为压力不足或过大会损害图像质量。此NVivo图像成像平台可用于眼表面以下结构的可视化，并可应用于各种眼科疾病的原位研究。

Research

JoVE Journal

Bioengineering

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

刀口扫描显微镜的样品制备，图像和分析协议

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

科研

生物工程

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

多显微镜下的血管生成术在基因修饰的 3 d-PLGA/nHAp 支架上进行颅骨临界骨缺损修复

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

影像学在两层微流控平台拟南芥幼苗的根头发形态

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...