Name: mesoscopic optical imaging of whole mouse heart
Uploaded: 2021-10-14T14:15:00
Description: Watch this Scientific Journal Video about Mesoscopic Optical Imaging of Whole Mouse Heart at JoVE.com

该项目已获得欧盟地平线2020研究和创新计划的资金，根据赠款协议No 952166（REPAIR），FISR计划下的MUR，项目FISR2019_00320和托斯卡纳地区，Bando Ricerca Salute 2018，PERCARE项目。

所有动物处理和程序均按照欧洲议会关于保护用于科学目的的动物的指令2010/63 / EU的指导方针进行，并符合意大利卫生部的原则和法规。该实验方案已获得意大利卫生部批准（实验方案编号647/2015-PR）。所有动物均由意大利ENVIGO提供。对于这些实验，使用5只6个月大的雄性C57BL / 6J小鼠。
1. 溶液制备
<ol><li>在化学罩中的磷酸盐缓冲盐水（PBS）（pH 7.6）中制备4%多聚甲醛（PFA）?...

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在这项工作中，介绍了一种以高分辨率清除、染色和成像整个小鼠心脏的成功方法。首先，优化并执行组织转化方案（CLARITY），并针对其在心脏组织中的应用进行了轻微修改。事实上，为了获得整个心脏的3D有效重建，必须防止光散射现象。CLARITY方法使我们能够获得高度透明的完整心脏，但是被动进行时需要较长的潜伏时间（约5个月）。关于大脑，心脏组织不适合利用电场的主动清除。即使在?...

与心脏病相关的结构重塑会影响电传导并引入器官的机电功能障碍1，2。目前用于预测功能改变的方法通常采用MRI和DT-MRI来获得纤维化沉积，血管树和心脏纤维分布的整体重建，并且它们用于模拟跨器官的优先动作电位传播（APP）路径3，4。这些策略可以提供心脏组织的美丽概述。然而，它们的空间分辨率不足以在细胞水平上研究结构重塑对心脏功能的影响。
将这个框架放在不同的数量级，其中单个细胞可以在动作电位传播中发挥个体作用，这是具有挑战性的。主要限制是在大块（厘米大小）组织中执行高分辨率成像（微米分辨率）的标准成像方法效率低下。事实上，由于组织不透明，以高分辨率对生物组织进行3D成像非常复杂。在整个器官中进行3D重建的最常见方法是准备薄切片。然而，精确的切片、组装和成像需要大量的精力和时间。一种不需要切割样品的替代方法是生成透明组织。在过去几年中，已经提出了几种澄清组织的方法5，6，7，8。最近通过开发真正的组织转化方法（CLARITY9，SHIELD10）实现了生产大块，透明和荧光标记组织的挑战。特别是，CLARITY方法基于将完整组织转化为纳米多孔，水凝胶杂交，无脂形式，能够通过选择性去除膜脂质双层来赋予高透明度。值得注意的是，该方法在心脏制备中也被发现成功11，12，13，14。然而，由于心脏太脆弱而不适合主动清除，因此必须使用被动方法清除它，这需要很长时间才能赋予完全透明。
结合光片显微镜等先进的成像技术，CLARITY有可能以微米分辨率对3D大块心脏组织进行成像。在光片显微镜中，样品的照明是用限制在检测物镜焦平面上的薄片光进行的。荧光发射沿垂直于照明平面15的轴收集。该检测架构类似于宽场显微镜，使采集速度比激光扫描显微镜快得多。将样品穿过光片可以获得大样本的完整断层扫描，最大可达厘米大小的样品。然而，由于高斯光束的内在特性，只有在有限的空间扩展下，才有可能获得非常薄（几微米量级）的光片，从而极大地限制了视场（FoV）。最近，已经引入了一种新的激发方案来克服这一限制并应用于脑成像，允许以各向同性分辨率16进行3D重建。
在本文中，提出了一种被动清除方法，可以显着减少CLARITY协议所需的清除时间。这里描述的方法框架允许在单次断层扫描中以微米分辨率重建整个小鼠心脏，采集时间为几分钟。

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			<td>2-2&#8217; Thiodiethanol</td>
			<td>Sigma-Aldrich</td>
			<td>166782</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Bio-Rad</td>
			<td>61-0140</td>
			<td></td>
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			<td>AV-044 Initiator</td>
			<td>Wako Chemicals</td>
			<td>AVP5874</td>
			<td></td>
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		<tr>
			<td>Bis-Acrylamide</td>
			<td>Bio-Rad</td>
			<td>161-042</td>
			<td></td>
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			<td>Boric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>B7901</td>
			<td></td>
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			<td>Camera</td>
			<td>Hamamatsu</td>
			<td>Orca flash 4.0 v3</td>
			<td></td>
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			<td>Camera software</td>
			<td>Hamamatsu</td>
			<td>HC Image</td>
			<td></td>
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			<td>Collimating lens</td>
			<td>Thorlabs</td>
			<td>AC254-050-A-ML</td>
			<td></td>
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			<td>Detection arm</td>
			<td>Integrated optics</td>
			<td>0638L-15A-NI-PT-NF</td>
			<td></td>
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			<td>Excitation lens</td>
			<td>Nikon</td>
			<td>91863</td>
			<td></td>
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			<td>Extera&#236;nal quartz cuvette</td>
			<td>Portmann Instruments</td>
			<td>UQ-753</td>
			<td></td>
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			<td>Fold mirrors</td>
			<td>Thorlabs</td>
			<td>BBE1-E02</td>
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			<td>Galvanometric mirror</td>
			<td>Thorlabs</td>
			<td>GVS211/M</td>
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			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
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			<td>HCImage Live</td>
			<td>Hamamatsu</td>
			<td>4.6.1.19</td>
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			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
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			<td>Internal quartz cuvette</td>
			<td>Portmann Instruments</td>
			<td>UQ-204</td>
			<td></td>
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			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P4504</td>
			<td></td>
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			<td>Laser source</td>
			<td>Integrated Optics</td>
			<td>0638L-15A-NI-PT-NF</td>
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			<td>Long-pass filter</td>
			<td>Thorlabs</td>
			<td>FELH0650</td>
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			<td>Magnetic base</td>
			<td>Thorlabs</td>
			<td>KB25/M</td>
			<td></td>
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		<tr>
			<td>MgCl2</td>
			<td>Chem-Lab</td>
			<td>CI-1316-0250</td>
			<td></td>
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		<tr>
			<td>Motorized traslator</td>
			<td>Physisk Instrument</td>
			<td>M-122.2DD</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>59888</td>
			<td></td>
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		<tr>
			<td>Objective</td>
			<td>Thorlabs</td>
			<td>TL2X-SAP</td>
			<td></td>
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			<td>Paraformaldehyde</td>
			<td>Agar Scientific</td>
			<td>R1018</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
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			<td>Polycap AS</td>
			<td>Whatman</td>
			<td>2606T</td>
			<td></td>
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			<td>Relay lens</td>
			<td>Qioptiq</td>
			<td>G063200000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube lens</td>
			<td>Thorlabs</td>
			<td>ACT508-200-A-ML</td>
			<td></td>
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			<td>Tunable lens</td>
			<td>Optotune</td>
			<td>EL-16-40-TC-VIS-5D-1-C</td>
			<td></td>
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		<tr>
			<td>Vacuum pump</td>
			<td>KNF Neuberger Inc</td>
			<td>N86KT.18</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Memmert</td>
			<td>WTB</td>
			<td></td>
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mesoscopic optical imaging of whole mouse heart

遗传性和非遗传性心脏病都可能导致心脏发生严重的重塑过程。结构重塑，如胶原沉积（纤维化）和细胞错位，会影响电传导，引入机电功能障碍，最终导致心律失常。目前这些功能改变的预测模型基于非整合和低分辨率的结构信息。由于标准成像方法在大块组织中执行高分辨率成像时效率低下，因此将该框架置于不同的数量级上具有挑战性。在这项工作中，我们描述了一种方法框架，该方法框架允许以微米分辨率对整个小鼠心脏进行成像。实现这一目标需要技术努力，将组织转化和成像方法的进步结合起来。首先，我们描述了一种优化的CLARITY方案，能够将完整的心脏转化为纳米多孔，水凝胶杂交，无脂质形式，从而实现高透明度和深度染色。然后，描述了一种能够以微米级分辨率快速获取介观视场（mm尺度）图像的荧光光片显微镜。在mesoSPIM项目之后，构思的显微镜允许在单次断层扫描中以微米分辨率重建整个小鼠心脏。我们相信，这种方法框架将允许澄清细胞结构紊乱在电功能障碍中的参与，并为考虑功能和结构数据的综合模型铺平道路，从而能够统一研究导致组织重塑后电气和机械改变的结构原因。

开发的被动清除设置允许在大约3个月内获得清除的成年小鼠心脏（尺寸为10mm x 6 mm x 6 mm）。安装的所有组件均如图 1 所示。每个清除室之间的温度梯度可忽略不计（大约 3°C），允许所有腔室的温度保持在适当的范围内。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62795/62795fig01.jpg"> 图 1：被动清算...

Watch this Scientific Journal Video about Mesoscopic Optical Imaging of Whole Mouse Heart at JoVE.com

Mesoscopic Optical Imaging of Whole Mouse Heart

我们报告了一种将组织转化和染色的新进展与轴向扫描光片显微镜的开发相结合的整个小鼠心脏的介观重建方法。

全小鼠心脏的介观光学成像

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...

这种方法将清除协议的改进与Mesos PIM技术的眼睛切片能力相结合，使我们能够以微米分辨率进行细观重建。它提供了在一次扫描中对大块心脏组织或整个小鼠心脏整个溶液进行成像的可能性，而不会丢失原始的三维组织组织。在用泰罗溶液洗涤近端主动脉并固定在0.01摩尔PBS中的4%副形醛中后，就可以开始清除方案了。在 0.01 摩尔 PBS 中以 4 摄氏度洗涤心脏 3 次，持续 15 分钟。完成此步骤后，心脏可以在PBS和0.01%叠氮化钠中以4摄氏度储存数月。将心脏在20毫升水凝胶溶液中以15 RPM振荡状态在4摄氏度下孵育三天。使用干燥器、真空泵和将干燥器连接到泵带和氮气管道的管系统在室温下对样品进行脱气。将样品放入干燥器中并打开小瓶，保持盖子。关闭干燥器，打开氮气管道从管中除去氧气。打开真空泵，从干燥机中除去氧气 10 分钟。关闭泵并打开干燥机的旋钮连接到氮气管道。然后打开氮气水龙头。一旦压力等于大气压，请小心打开干燥器并快速关闭小瓶。将心脏保持在37摄氏度的脱气水凝胶溶液中约三个小时。当水凝胶适当聚合并呈现完全凝胶状时，小心地从中取出心脏并将其放入带有透明溶液的小瓶中的透明溶液中。每三天更换一次小瓶中的澄清溶液，以加快澄清过程。一旦心脏完全澄清，将其从小瓶中取出，并在50毫升加热的PBS中在摇晃条件下洗涤24小时。在50毫升一个X PBS T中再次洗涤24小时，摇动状态。将样品在每毫升0.01毫克小麦胚芽凝集素Alexa地板633中在三毫升一个X PBS T中在室温下以50RPM振荡状态孵育7天。孵育七天后，将样品在室温下在50毫升一个X PBS T中振荡洗涤24小时。将样品在 4% PFA 中孵育 15 分钟，然后在 0.01 摩尔 PBS 中洗涤三次，每次 5 分钟。将心脏在 20 和 47% TDE 中在 0.01 摩尔 PBS 中依次孵育 8 小时。最后在0.01摩尔PBS中以68%TDE提供所需的折射率1.46。用折射率介质轻轻填充约80%的外部石英Quvet。用相同的折射率介质填充内部石英Quvet。将样品浸入内部Quvet中。使用细镊子轻轻地将样品移动到Quvet的底部，并排列心脏的纵轴平行于Quvet的主轴，以最小化扫描过程中穿过组织的激发光路。用两个螺钉小心地将定制的插头固定在内部 quvet 上方。使用磁铁将样品安装到显微镜载物台上。手动平移垂直样品台，将内部Quvet浸入外部样品台中。打开波长为638纳米的激发光源，并将其设置为三毫瓦量级的低功率。使用电动翻译器移动样品以照亮组织的内板。打开HC图像直播相机软件，将相机触发器设置为外部边缘触发光片模式，以通过控制整个设置的自定义软件驱动相机的采集触发。在相机软件中启用自动保存，并设置需要保存图像的输出文件夹。使用线性转换器手动调整X Y平面中的样品位置，将样品移动到相机传感器视场的中心。使用线性电动转换器沿 Z 轴移动样品，以识别心脏边界以进行断层扫描重建。在开始成像会话之前，将激光功率增加到大约 20 毫瓦。通过单击成像软件捕获面板中的开始按钮开始断层扫描采集，同时使用电动转换器开始以每秒 6 微米的恒定速度沿 Z 轴移动样品。澄清度方法与TDE作为折射率介质相结合不会显着改变样品的最终体积，也不会导致样品的各向同性变形。激发光学器件产生的光片最小浪费约为6微米，在视场边缘发散至175微米。相机卷帘快门与光片废料的轴向扫描同步，确保仅在激发的样品部分收集发射信号，从而在整个视场上产生约6.7微米的平均F W H M。显微镜的Z点扩散函数也通过荧光纳米球的断层扫描重建来估计，并通过拟合估计6.4微米的F W H M。还证实了该系统在整个器官中以足够的信噪比解析三维单细胞膜的潜力。脱气程序是协议中最关键的一步。如果操作不当，组织可能会在清洁溶液中孵育期间遇到损坏。所提出的方案可以与多染色方案相结合，实现整合不同生物结构的全器官重建，并可用于研究病理模型。

Research

JoVE Journal

Biology

Proper Care and Cleaning of the Microscope

适当的照顾和清洁的显微镜

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

科研

生物学

Major Components of the Light Microscope

光镜下的主要组成部分

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase Contrast and Differential Interference Contrast DIC Microscopy

相衬，微分干涉对比（DIC）显微镜

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

生于正常化荧光整个心脏成像的光学投影层析成像

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

为介观荧光体层摄影术在体内成像的发展果蝇</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

全安装胚胎肢体皮肤血管的免疫组织化学分析：一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

的内肌浆网钙光学标测<sup&gt; 2+</sup&gt;和的Langendorff灌注兔心脏跨膜电位

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...