我们感谢德克萨斯大学西南医学中心的Eric Olson博士团队慷慨地分享动物模型。我们感谢德克萨斯大学达拉斯分校的D-incubator成员提供的所有建设性意见。这项工作得到了 NIH R00HL148493 （Y.D.）、R01HL162635 （Y.D.） 和 UT Dallas STARS 计划 （Y.D.） 的支持。

动物协议和实验已在德克萨斯大学达拉斯分校机构动物护理和使用委员会 （IACUC #21-03） 的监督下获得批准和进行。本研究使用C57BL6小鼠，包括出生后第1天（P1）的新生儿和8周大的成人。男性和女性之间没有观察到差异。所有数据采集和图像后处理均使用具有研究或教育许可的开源软件或平台进行。这些资源可根据作者的合理要求提供。
1. 样品制备和组织清理（6 - 10 天）
<ol><li>在开始组织透明化程序之前，准备组织透明化方法所需的 材料表 中的所有化学溶液。 注意： 在通风橱中执行所有程序，同时穿戴适当的个人防护设备，尤其是在处理有毒化学品时。</li>
	<li>通过在所需的时间点（例如P1和8周）将它们置于CO2室中，用CO2对动物实施安乐死，以进行全心收集，并将新鲜分离的心脏在室温（RT）下浸入0.2M KCl中以停滞在舒张期16。</li>
	<li>通过将4%多聚甲醛（PFA）浸入1x磷酸盐缓冲盐水（PBS）中，在摇床上轻轻搅拌过夜来固定心脏，以保持心脏解剖结构在4°C，见 表1。 注意：PFA 是一种有毒化学物质，应在通风橱下进行。</li>
	<li>在室温下用 1x PBS 清洗心脏 30 分钟，3 次。 注：可选：将心脏嵌入琼脂糖凝胶中，以在步骤 2 中为定制的 LSM 系统创建心脏组件，如下所述。<ol><li>通过将琼脂糖溶解在PBS中来制备1%琼脂糖溶液，并在微波炉中加热混合物直至完全溶解。将溶液倒入模具中以形成底层，直到溶液冷却至40°C。</li>
		<li>将心脏放入模具中，并用琼脂糖凝胶填充模具，直至其凝固。消除琼脂糖凝胶中的任何气泡，以最大限度地减少成像中的伪影。 注意：嵌入样本可降低安装过程中对心脏解剖结构造成潜在损害的风险。 注意：使用低熔点琼脂糖，避免将心脏直接置于加热的琼脂糖中，以降低将样品暴露在高温下的风险。</li>
	</ol></li>
	<li>使用甲醇与去离子水混合的梯度对心脏进行脱水（图1B），通过20%、40%、60%、80%和100%的顺序浓度进行处理，如 表1所示。用新鲜的 100% 甲醇重复 1 次，以确保完全脱水。在每种甲醇混合物中将新生小鼠心脏脱水1小时，振荡。将8周龄小鼠心脏和大鼠心脏的孵育时间调整为2小时。 注意：甲醇是一种挥发性、刺激性和易燃的化学物质。</li>
	<li>在4°C下用新鲜的100%甲醇替换并冷却心脏10分钟。</li>
	<li>更换溶液并用5%过氧化氢（H2O2）在甲醇中的溶液，体积比为1：5（30%H2O2 至100%甲醇）在4°C下过夜以除去色素。 注意：去除色素可以最大限度地减少光子吸收，从而提高图像质量并减少伪影。</li>
	<li>替换溶液并在室温下将心脏在100%甲醇中孵育1小时。</li>
	<li>用含有66%二氯甲烷（DCM）和33%甲醇的溶液将心脏脱落3小时，振荡。确保在此步骤结束时，心脏沉入小瓶底部。如果没有，则更新溶液（66% DCM / 33% 甲醇）并延长孵育时间（例如，再 1 小时）以实现完全脱脂。快速将心脏转移到新鲜溶液中以防止干燥，因为 DCM 具有高度挥发性。 注意：使用聚丙烯或玻璃器皿进行实验，因为 DCM 与聚苯乙烯不相容。</li>
	<li>利用二苄基醚 （DBE） 进行折射率匹配 （RI = 1.56）。对于新生小鼠心脏，折射率匹配的过程需要2天，而对于8周龄的小鼠心脏，轻轻摇晃需要5天。用新鲜的 DBE 替换它并延长孵育时间，直到心脏达到完全透明。 注意：DBE 是危险的。避免与皮肤接触。</li>
</ol>2. 样品安装（1天）
注意：如果使用商业 LSM 系统，请按照公司提供的特定协议修复心脏并跳过步骤 2.1 - 2.9。
<ol><li>使用 3D 打印机和 Onyx 材料定制一个耐折射率匹配溶液的腔室和支架（例如，采用改编的 iDISCO 方法中的 DBE）（图 2A）16。</li>
	<li>将磁铁固定在腔室底部，以便于在LSM系统下进行快速连接和准确定位（图2B）。</li>
	<li>使用透明硅胶将 25 mm x 50 mm x 0.17 mm 盖板眼镜连接到腔室，并通过填充 DBE 并在 1 天后检查是否有泄漏来验证腔室的防水完整性。</li>
	<li>使用3D打印机定制夹具支架，并固定磁铁以将心脏组件安装在腔室内（图2C），或将针头插入琼脂糖凝胶以安装心脏组件。</li>
	<li>如先前报道的那样，使用柱面透镜和连续波二极管泵浦固态 （DPSS） 激光器进行内部 LSM 系统的开发16,22。使用532nm的激发波长捕获心肌的自发荧光（图2D）。</li>
	<li>将腔室安装在 6 维载物台上，并找到腔室垂直于激光传播方向的最佳位置。</li>
	<li>使用磁性夹具将样品放置在腔室的中间，并将支架连接到 4 维电动载物台（X、Y、Z 和 Yaw; 图2D）。</li>
	<li>使用电动载物台将心脏组件浸入充满 DBE 的腔室中，并确定沿检测轴（Z 方向）的扫描范围。</li>
	<li>根据步长 （S） 和图像采集时间 （T） 确定电机沿 Z 方向的扫描速度 （Vz），使用以下公式： <img alt="Equation 1" src="/files/ftp_upload/66707/66707eq01.jpg" style="margin: auto;"> 注意：成像系统的步长和轴向分辨率之间的关系遵循奈奎斯特-香农采样定理。例如，鉴于轴向分辨率为2.91 μm，步长设定为1 μm，如上一次报告16所示。</li>
	<li>为了验证空间分辨率并最大限度地减少不同深度的潜在不透明度问题，通过对直径为 0.53 μm 的荧光珠进行成像来测量系统的点扩散函数 （PSF），该荧光珠稀释在浓度为 1：1.5 x 105 的 1% 低熔点琼脂糖凝胶中。通过测量半峰全宽 （FWHM）16 来计算整个样品的 PSF。</li>
</ol>3. 图像拼接（4-8 小时）
<ol><li>考虑到有限的 FOV，计算覆盖整个小鼠心脏所需的瓷砖数量。对于每个图块，尺寸对应于 LSM 系统的 FOV。例如，横截面尺寸为 3 x 3.6 mm2 的新生小鼠心脏需要 2 x 2 个瓦片才能在定制的 LSM 中覆盖（图 3A）。相比之下，一个 8 周龄的小鼠心脏尺寸为 5 x 8 mm2 需要 3 x 4 个瓷砖来覆盖整个心脏结构（图 3B）。 注意：如果传统的 LSM 用于对整个心脏进行成像的 FOV 有限，则需要进行图像拼接。</li>
	<li>从图块 1 开始捕获心脏图像，即心脏的左上角图块（图 3A-B）。</li>
	<li>按顺序捕获剩余瓦片的图像，连续瓦片之间有 10% 的重叠，直到覆盖整个心脏，如图 3C 所示。</li>
	<li>下载并安装 Fiji23 开源软件。下载并安装 Fiji 插件 BigStitcher24。</li>
	<li>导航到“帮助”菜单，选择“ 更新”，选择“ 管理更新站点”，然后选择“ BigStitcher”。随后，单击 “关闭”，然后单击 “应用更改”。完成插件下载后，重新启动斐济软件。</li>
	<li>打开 BigStitcher 插件并通过图像文件目录导入所有必要的瓦片。将数据集另存为 HDF5 文件。</li>
	<li>通过选择 “按规则网格移动图块”来组织图块，选择用于移动心形的图案，并选择每个图块之间 10% 的重叠。使用 Stitching Wizard 选项拼接瓷砖。</li>
	<li>使用 Image Fusion 导出拼接数据，以生成 tiff 文件。</li>
</ol>4. 多视图反卷积（5 天）
<ol><li>利用荧光珠进行沿心脏的多视图重建的图像配准（图3D）。<ol><li>通过将双酚-A二缩水甘油醚（D.E.R. 332），异佛尔酮二胺，5-氨基-1,3,3-三甲基环己烷甲胺（IPDA）和聚丙二醇二缩水甘油醚（D.E.R 736）以4：1：1的体积比混合来制备树脂25 。</li>
		<li>将 10 μL 荧光珠以 161 x g 离心 5 分钟，并加入 20 μL 甲醇以替换珠子的储存溶液。重复 3 次。</li>
		<li>通过将甲醇基微珠溶液混合到步骤 4.1.1 中制备的树脂中，制备 10 mL 溶液，珠子浓度为 1：1 x 105 。</li>
		<li>在真空室中将溶液脱气3小时以去除气穴和气泡。</li>
		<li>将溶液倒入硅胶模具或塑料吸管中，等待 2 至 3 天，直到其凝固。在树脂凝固前 1 天后，将玻璃管放入模具内，等待管子附着在树脂上。用玻璃管从模具中取出树脂（图3D）。</li>
		<li>将心脏放入玻璃管中，用 DBE 填充，然后将玻璃管安装在充满 DBE 的腔室中。</li>
	</ol></li>
	<li>通过扫描跨检测轴（Z轴;图3E-F）。 注意：在步骤 3 中实施图像拼接，如果心脏大小超过 FOV，则生成单独的图像堆栈。</li>
	<li>沿 Y 轴旋转鼠标心脏 60°，以从多个角度捕获后续堆栈，即 0°、60°、120°、180°、240° 和 300°（图 3E-F）。</li>
	<li>打开 BigStitcher 插件，通过图片文件目录导入所有图片。将数据集另存为 HDF5 文件。</li>
	<li>选择 Detect Interest Points 并手动选择感兴趣的磁珠进行注册。</li>
	<li>选择 仿射 变换模型进行配准。选择 Point Spread Function 并将 PSF 分配给所有视图。</li>
	<li>单击“ 多视图反卷积 ”，然后选择“ 高效贝叶斯”（Efficient Bayesian）作为迭代类型。将迭代次数定义为 10 并执行26。</li>
	<li>单击 Image Fusion 以导出 tiff 文件。 注意：有关多视图反卷积的更多详细信息，请参阅其他报告或协议 24,27,28。</li>
</ol>5.轴向扫掠光片系统硬件（1天）
<ol><li>在柱面透镜 （CL）27 （图 2D） 的傅里叶平面上安装电动可调谐透镜 （ETL） 以轴向扫描激光束14。确保ETL中的液体界面是水平的，以防止重力引起的波前畸变。精确对准 ETL，以最大限度地减少光束偏向和高阶光学像差30.</li>
	<li>在工作站中安装DAQ卡，并将BNC电缆连接到DAQ卡上，以同步相机曝光和ETL扫描。</li>
	<li>打开 ETL 驱动器的外壳并取下盖子（图 4）。</li>
	<li>将 BNC 电缆的信号线焊接到 B 引脚的模拟输入引脚上，如 PCB 板底部标记的那样（图 4）。</li>
	<li>将BNC电缆的地线焊接到GND引脚上;之后，将BNC电缆的另一端连接到DAQ卡中的AO（模拟输出）（图4）。</li>
	<li>将 ETL 驱动程序插入工作站的 USB 端口，使用 hirose 线将驱动程序连接到 ETL，然后安装 Lens Driver Controller 软件。</li>
	<li>将镜头驱动控制器软件中的操作模式更改为模拟模式，以使用从DAQ卡生成的触发器控制ETL。</li>
	<li>在sCMOS相机的软件中，将捕获模式切换为外部（Light-sheet）。确保有源像素扫描方向垂直于激光扫描方向，以辅助同步。</li>
	<li>使用 BNC 电缆将 sCMOS 相机背面的外部触发端口连接到 DAQ 卡的 AO（图 4）。</li>
</ol>6.轴向扫掠光片系统同步（7天）
<ol><li>根据可接受的信噪比 （SBR） 定义暴露时间，对于拟议的研究，该比至少为 10。如果 SBR 低于 10，则增加曝光时间。 注意：曝光时间范围为 10 到 50 毫秒，在此项目中提供了可接受的 SBR。</li>
	<li>根据相机的曝光时间 （E） 和 ETL 频率 （f），使用以下公式定义图像的采集时间 （T） 和线间隔 （L）： <img alt="Equation 2" src="/files/ftp_upload/66707/66707eq02.jpg" style="margin: auto;"> <img alt="Equation 3" src="/files/ftp_upload/66707/66707eq03.jpg" style="margin: auto;"> 其中 P 表示相机传感器上的像素列数。基准电压源的代表性参数分别为 f = 1 Hz、 P = 2048、 E = 10 ms 和 L = 243 μs。</li>
	<li>在LabVIEW程序触发器 Generator.vi 中，生成用于同步的方形和三角形触发器（图5）。 注意：定制的LabVIEW程序可从作者处获得。</li>
	<li>将荧光珠用浓度为1：1×10 3稀释在1%低熔点琼脂糖凝胶16 中稀释，以定位 图像中束腰的位置（图6A）。</li>
	<li>在程序中，通过沿扫描方向改变电压来识别 FOV 下激光束的起点和终点。</li>
	<li>要使sCMOS相机和ETL同步，请沿X轴扫描激光束，沿Y轴扫描有源像素以生成2D图像（图6B）。图像中沿对角线的珠子更明亮、更锐利，表明sCMOS和ETL之间的精确同步。 注意：ETL和sCMOS像素激活的扫描速度同步对于图像质量至关重要。图 6C-F 总结了各种常见的异步错误。</li>
	<li>确定最优同步参数进行同步后，将sCMOS相机绕Z轴旋转90°，使有源像素方向与激光扫描方向对齐。</li>
	<li>重复步骤2.10，并测量PSF的FWHM。 使用与上一次报告16中所示相同的方法，ASLM的平均横向和轴向分辨率分别为2.18μm和2.88μm（图7）。 注意：在理想条件下，整个 FOV 的均匀照明和分辨率是可行的（图 7A-B）。ETL与sCMOS相机的异步操作，以及照明和检测的错位，都可能导致空间分辨率不均匀（图7C-D）。</li>
	<li>对于心脏成像，在对准系统并确定系统同步的最佳参数后，继续执行步骤 2.6 至 2.9。</li>
</ol>

用于全面心脏成像的光片显微镜和组织透明化

作者没有要披露的利益冲突。

成像、计算和组织透明化方法的进步为广泛研究心脏结构和功能提供了无与伦比的机会。这对于使用完整的啮齿动物心脏模型加深我们对心脏形态发生和发病机制的理解具有很大的潜力。与使用类似方法对斑马鱼心脏进行体内研究相比 40,41,42,43，先进的 LSM 技术和组织透明化方法的集成使我们能够克服在对啮齿?...

心力衰竭仍然是全球死亡的主要原因，主要是由于成熟心肌细胞缺乏再生能力1。心脏错综复杂的结构在其功能中起着至关重要的作用，并提供了对发育过程的见解。对心脏结构的深刻理解对于阐明心肌梗死后心脏形态发生和重塑的基本过程至关重要。最近的进展表明，新生小鼠可以在受伤后恢复心脏功能，而成年小鼠则缺乏这种再生能力2。这为研究小鼠模型中与结构和功能异常相关的线索奠定了基础。传统的成像方法，如共聚焦显微镜，具有技术局限性，包括穿透深度受限、点扫描方案缓慢以及长时间暴露在激光下造成的照片损伤。这些阻碍了完整心脏的全面三维 （3D） 成像。在这种情况下，光片显微镜 （LSM） 作为一种强大的解决方案出现，具有高速成像、减少光损伤和卓越的光学切片能力等优势 3,4,5。LSM 的独特功能使其成为一种有前途的方法，可以克服传统技术的局限性，为心脏发育和重塑过程提供前所未有的见解 6,7,8。
在该协议中，我们引入了一种成像策略，该策略将先进的LSM与适应的组织透明化方法9相结合，允许对整个小鼠心脏进行成像，而无需特定的标记和机械切片。我们进一步提出，传统的LSM成像可以通过多视图反卷积10或轴向扫描光片显微镜（ASLM）技术11,12,13,14,15来增强，以提高轴向分辨率。此外，将图像拼接与这两种方法中的任何一种相结合都可以有效地克服空间分辨率和视场（FOV）之间的权衡，从而推进成年小鼠心脏的成像。结合多种组织透明化方法，包括疏水性、亲水性和基于水凝胶的方法，可以更深入地穿透光来捕获整个心脏的形态 16,17,18,19。
虽然多种透明化方法与当前的 LSM 系统兼容，但目标是通过用与其折射率非常匹配的介质替换脂质来最大限度地减少光子散射并增强光在组织（如心脏）中的穿透力。iDISCO被选为代表20,21，并因其快速处理和高透明度而适用于该协议中的自发荧光成像（图1A）。总的来说，先进的LSM方法与组织透明化技术的整合为揭示啮齿动物心脏中复杂的心脏解剖结构提供了一个有前途的框架，为推进我们对心脏形态发生和发病机制的理解具有巨大的潜力。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% Agarose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting point agarose</td>
			<td>Thermo Fisher</td>
			<td>16520050</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals for tissue clearing&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-Amino-1,3,3-trimethylcyclohexanemethylamine, mixture of cis and trans</td>
			<td>Sigma-Aldrich</td>
			<td>118184</td>
			<td></td>
		</tr>
		<tr>
			<td>D.E.R.&#8482; 332</td>
			<td>Sigma-Aldrich</td>
			<td>31185</td>
			<td></td>
		</tr>
		<tr>
			<td>D.E.R.&#8482; 736</td>
			<td>Sigma-Aldrich</td>
			<td>31191</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibenzyl ether (DBE)</td>
			<td>Sigma-Aldrich</td>
			<td>33630</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent beads</td>
			<td>Spherotech</td>
			<td>FP-0556-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2)</td>
			<td>Sigma-Aldrich</td>
			<td>216736</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>439193</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Thermo Fisher</td>
			<td>47392</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>79383</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Software and algorithms</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amira</td>
			<td>Thermo Fisher Scientific</td>
			<td>2021.2</td>
			<td></td>
		</tr>
		<tr>
			<td>BigStitcher</td>
			<td>H&#246;rl et al.22</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji-ImageJ</td>
			<td>Schindelin et al.20</td>
			<td>1.54f</td>
			<td></td>
		</tr>
		<tr>
			<td>HCImage Live</td>
			<td>Hamamatsu Photonics</td>
			<td>4.6.1.2</td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW</td>
			<td>National Instruments Corporation</td>
			<td>2017 SP1</td>
			<td></td>
		</tr>
		<tr>
			<td>Key components of the customized light-sheet system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.63 - 6.3X Zoom body</td>
			<td>Olympus</td>
			<td>MVX-ZB10&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Illumination objective</td>
			<td>Nikon</td>
			<td>MRH00105</td>
			<td></td>
		</tr>
		<tr>
			<td>1X detection objective</td>
			<td>Olympus</td>
			<td>MV PLAPO 1X/0.25&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>473nm DPSS Laser</td>
			<td>Laserglow Technologies</td>
			<td>LRS-0473-PFM-00100-05</td>
			<td></td>
		</tr>
		<tr>
			<td>532nm DPSS laser</td>
			<td>Laserglow Technologies</td>
			<td>LRS-0532-PFM-00100-05</td>
			<td></td>
		</tr>
		<tr>
			<td>589&#8201;nm DPSS laser</td>
			<td>Laserglow Technologies</td>
			<td>LRS-0589-GFF-00100-05</td>
			<td></td>
		</tr>
		<tr>
			<td>BNC connector</td>
			<td>National Instrument</td>
			<td>BNC-2110</td>
			<td></td>
		</tr>
		<tr>
			<td>Cylindrical lens</td>
			<td>Thorlabs</td>
			<td>ACY254-050-A</td>
			<td></td>
		</tr>
		<tr>
			<td>DC-Motor Controller, 4 axes</td>
			<td>Physik Instrumente</td>
			<td>C-884.4DC</td>
			<td></td>
		</tr>
		<tr>
			<td>ETL</td>
			<td>Optotune</td>
			<td>EL-16-40-TC-VIS-5D-1-C</td>
			<td></td>
		</tr>
		<tr>
			<td>ETL Cable</td>
			<td>Optotune</td>
			<td>CAB-6-300</td>
			<td></td>
		</tr>
		<tr>
			<td>ETL Lens Driver</td>
			<td>Optotune</td>
			<td>EL-E-4i</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Chroma</td>
			<td>ET525/30</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Chroma</td>
			<td>ET585-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Chroma</td>
			<td>ET645-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter wheel&#160;</td>
			<td>Shutter Instrument</td>
			<td>LAMBDA 10-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized translation stage</td>
			<td>Physik Instrumente</td>
			<td>L-406.20DG10</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized translation stage</td>
			<td>Physik Instrumente</td>
			<td>L-406.40DG10</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized translation stage</td>
			<td>Physik Instrumente</td>
			<td>M-403.4PD</td>
			<td></td>
		</tr>
		<tr>
			<td>NI multifunction I/O</td>
			<td>National Instrument</td>
			<td>PCIe-6363</td>
			<td></td>
		</tr>
		<tr>
			<td>sCMOS camera</td>
			<td>Hamamatsu</td>
			<td>C13440-20CU</td>
			<td></td>
		</tr>
		<tr>
			<td>Stepper motor</td>
			<td>Pololu</td>
			<td>1474</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube lens</td>
			<td>Olympus</td>
			<td>MVX-TLU</td>
			<td></td>
		</tr>
	</tbody>
</table>

light-sheet imaging to reveal cardiac structure in rodent hearts

光片显微镜 （LSM） 在理解心脏复杂的三维 （3D） 结构方面发挥着关键作用，为基本的心脏生理学和病理反应提供了重要的见解。在此，我们深入研究了LSM技术的开发和实施，以阐明小鼠模型中心脏的微结构。该方法将定制的 LSM 系统与组织透明化技术相结合，可减轻心脏组织内的光散射以进行体积成像。将传统的 LSM 与图像拼接和多视图反卷积方法相结合，可以捕获整个心脏。为了解决轴向分辨率和视场 （FOV） 之间固有的权衡问题，我们进一步引入了轴向扫描光片显微镜 （ASLM） 方法，以最大限度地减少离焦光并在整个传播方向上均匀地照亮心脏。同时，iDISCO等组织透明化方法增强了光的穿透力，促进了深层结构的可视化，并确保了对整个心脏心肌的全面检查。所提出的LSM与组织透明化方法的结合为研究人员解析啮齿动物心脏心脏结构提供了一个有前途的平台，为理解心脏形态发生和重塑具有巨大的潜力。

LSM 已被证明可以促进心脏研究 31,32,33,34,35,36,37，因为与其他光学成像方法（如明场和点扫描技术）相比，LSM 具有最小的光损伤风险、高空间分辨率和光学切片 6,8,38,39,40 </su...

Read this Scientific Article Protocol about Author Spotlight: Customized Light-Sheet Imaging for Investigating Myocardial Structures in Rodent Hearts at JoVE.com

作者聚焦：用于研究啮齿动物心脏心肌结构的定制光片成像

该协议利用先进的光片显微镜以及适应的组织透明化方法来研究啮齿动物心脏中复杂的心脏结构，为理解心脏形态发生和重塑具有巨大潜力。

光片成像揭示啮齿动物心脏的心脏结构

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights ...

我们的研究旨在定制一种光片成像系统，以在细胞水平上研究完整啮齿动物心脏的心肌结构。我们已经开发了一种工作流程，以微米级的各向同性分辨率探索从新生儿到成年啮齿动物心脏的完整心肌。我们的研究填补了研究完整心室内错综复杂的心肌微结构（例如小梁形成）的空白，具有高空间分辨率。这种成像策略提供了一个切入点，可以在没有物理切片的情况下评估微观结构，从而增强对心脏发育和重塑的理解。我们将专注于探索心肌在心脏损伤和重塑过程中的结构变化。

light-sheet-imaging-to-reveal-cardiac-structure-in-rodent-hearts

Research

JoVE Journal

Bioengineering

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

731 次观看.  The University of Texas at Dallas. 我们的研究旨在定制一种光片成像系统，以在细胞水平上研究完整啮齿动物心脏的心肌结构。我们已经开发了一种工作流程，以微米级的各向同性分辨率探索从新生儿到成年啮齿动物心脏的完整心肌。我们的研究填补了研究完整心室内错综复杂的心肌微结构（例如小梁形成）的空白，具有高空间分辨率。这种成像策略提供了一个切入点，可以在没有物理切片的情况下评?...

视频: 作者聚焦：用于研究啮齿动物心脏心肌结构的定制光片成像

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

冲击波应用细胞培养

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

在心肌结构，功能的体内定量评估，灌注与活力使用心脏微型计算机断层扫描

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

用超常光透射法定量测定人血清中的心脏标志物

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

光片荧光显微术捕捉4维图像调制剪切应力对斑马鱼心脏发育的影响

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

光片荧光显微术在小鼠心脏研究中的应用

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Creation of Patient-Specific Silicone Cardiac Models with Applications in Pre-surgical Plans and Hands-on Training

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In particular, 3D models can play an important role in the field of cardiology, where complex congenital heart diseases occur. While many 3D printers can provide anatomically correct and detailed models, existing 3D printing materials fail to replicate myocardial tissue properties and can be extremely costly. This protocol aims to develop a process for the creation of patient-specific models of complex congenital heart defects using a low-cost silicone that more closely matches cardiac muscle properties. With improved model fidelity, actual surgical procedural training could occur in advance of the procedure. Successful creation of cardiac models begins with the segmentation of radiologic images to generate a virtual blood pool (blood that fills the chambers of the heart) and myocardial tissue mold. The blood pool and myocardial mold are 3D printed in acrylonitrile butadiene styrene (ABS), a plastic dissolvable in acetone. The mold is assembled around the blood pool, creating a negative space simulating the myocardium. Silicone with a shore hardness of 2A is poured into the negative space and allowed to cure. The myocardial mold is removed, and the remaining silicone/blood pool model is submerged in acetone. The described process results in a physical model in which all cardiac features, including intra-cardiac defects, are represented with more realistic tissue properties and are more closely approximated than a direct 3D printing approach. The successful surgical correction of a model with a ventricular septal defect (VSD) using a GORE-TEX patch (standard surgical intervention for defect) demonstrates the utility of the method.

Patient-specific models improve surgeon and fellow confidence when developing or learning surgical plans. Three-dimensional (3D) printers generate adequate detail for surgical preparation, but fail to replicate tissue haptic fidelity. A protocol is presented detailing the creation of patient-specific, silicone cardiac models, combining 3D printing precision with simulated silicone tissue.

创建患者专用硅胶心脏模型，应用于术前计划和实践培训

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In ...