我们感谢波士顿儿童医院的 Caroline Burns 博士慷慨地分享转基因斑马鱼。我们感谢伊丽莎白·伊巴涅斯女士在德克萨斯大学达拉斯分校帮助饲养斑马鱼。我们也感谢德克萨斯大学达拉斯分校的D孵化器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。

这项研究的批准由德克萨斯大学达拉斯分校的机构动物护理和使用委员会 （IACUC） 批准，协议编号为 #20-07。本研究采用 Tg（myl7:nucGFP） 转基因斑马鱼幼虫12 。所有数据采集和图像后处理均使用具有研究或教育许可证的开源软件或平台进行。这些资源可根据作者的合理要求获得。
1. 斑马鱼繁殖和胚胎显微注射
定时:2天
<ol><li>通过标准护理和繁殖程序维护和繁殖成年斑马鱼。详细方法见上一份报告13。</li>
	<li>按照既定方案14进行显微注射。在本研究中，显微注射在 1-2 细胞（受精卵）阶段进行。 注意:准确识别此阶段对于有效的显微注射并确保发育一致性至关重要。在单细胞阶段，卵黄顶部形成一个小圆顶，该阶段通常持续约 12 分钟。在进展到双细胞阶段时，两个相邻的圆顶变得可见，并且该阶段在受精后持续约 45 分钟15。有关系统构建的更详细信息，请参阅其他报告或协议12,16,17。</li>
</ol>2. 斑马鱼胚胎/幼虫的制备和安装
时间:7天
<ol><li>在受精后1天（dpf）将胚胎转移到含有0.2mM 1-苯基-2-硫脲（PTU）的E3水中（参见 材料表）以防止色素形成。</li>
	<li>每天用 PTU 的新鲜 E3 水替换培养基，直到 LSM 成像。</li>
	<li>用体视显微镜对胚胎或幼虫进行成像，以记录它们在0至7 dpf之间的所需时间点的发育。</li>
	<li>准备内径为 2 mm 的分段氟化乙丙烯 （FEP） 管，用于将斑马鱼幼虫嵌入 LSM 系统12。 注意: FEP 管用于使用与周围介质（例如水）非常相似的折射率匹配材料来固定样品。</li>
	<li>从 3 dpf 开始，将鱼转移到 150 mg/L 三卡因 （MS-222） 溶液（参见 材料表）中以在成像前固定鱼。</li>
	<li>用150mg / L三卡因制备0.8%低熔点琼脂糖，并在冷却至室温18后使用移液管将麻醉的鱼移至琼脂糖中。</li>
	<li>使用另一个转移移液管将装有琼脂糖的鱼安装在FEP管中（图1）。 注意:为了确定发育中的胚胎中的最小压力，在显微镜观察下对心脏活动（例如心率）进行固定前和固定后检查。这些评估旨在确定三卡因麻醉前后的任何显着差异。固定后还可以对皮肤和肌肉组织进行心脏结构不规则（如水肿）的额外检查。三卡因的浓度在心脏成像中也起着至关重要的作用19，因此本项目将浓度为150 mg/L的三卡因溶液用于斑马鱼幼虫收缩的成像。虽然目前的回顾性同步使我们能够解决心律不齐的问题 20，但斑马鱼心律失常 4D 成像和长期成像的综合解决方案仍在开发中。</li>
</ol>3. 光片成像系统设置和配置
定时:3-14天
<ol><li>使用特定波长（如 473 nm 和 532 nm）的连续波二极管泵浦固态 （DPSS） 激光系统作为照明源，构建基于柱面透镜的内部 LSM 系统（图 2A）。</li>
	<li>自定义LabVIEW（参见 材料表）控制代码，用于同步平移样品台、光片照明和sCMOS相机的曝光，以捕获图像序列。 注:有关系统构建的更多详细信息，请参阅其他报告或协议12,16,17。我们鼓励研究小组在系统构建过程中寻求与在光学成像方面拥有成熟专业知识的实验室合作的机会。</li>
</ol>4. 斑马鱼成像准备和数据收集
定时:1天
<ol><li>通过测量荧光微珠来校准 LSM 系统的空间分辨率并最大限度地减少不同深度的不透明度变化。<ol><li>首先，使用0.8%低熔点琼脂糖将直径为0.53μm的荧光珠（见 材料表）稀释至浓度为1:1.5×105 ，并将溶液转移到分段的FEP管中。</li>
		<li>其次，使用 LSM 系统对磁珠进行成像并测量整个样品的点扩散函数 （PSF），验证空间分辨率和系统对齐12。 注意:在该系统中，对半最大全宽 （FWHM） 的代表性结果的分析表明，横向和轴向分辨率分别为 1.26 ± 0.15 和 2.48 ± 0.15 μm（n = 60 个珠子）。这表明整个成像深度的空间分辨率一致。</li>
	</ol></li>
	<li>将装有斑马鱼幼虫的 FEP 管牢固地连接到 6 轴（x、y、z、俯仰、偏航和滚动）电动样品台，并将管浸入充满 E3 水的样品室中。为了避免卵黄囊的光散射并更好地定位心脏的位置，旋转斑马鱼幼虫以从腹侧拍摄图像（图2B）。在这种情况下，大多数捕获的图像将包括心室和心房（图2C）。 注意:鉴于目前的成像程序通常每条鱼持续不到一小时，并且是在受控室温环境中进行的，因此本项目认为腔室温度控制和氧气水平调节是可选的。然而，对于长期的延时成像研究，特别是那些专注于发育过程的研究，建议在样品室内连续监测和调节27°C的温度，以确保斑马鱼的生理环境并防止任何潜在的发育异常。</li>
	<li>将电动样品台连接到工作站并配置所有必要的参数，包括所需的移动模式。</li>
	<li>在sCMOS相机和工作站之间建立连接。调整所有基本设置，例如 5 毫秒的曝光时间和所需的感兴趣区域。</li>
	<li>在定制的LabVIEW控制程序中配置图像序列和帧的数量。</li>
	<li>打开激光器电源并启动样品的LSM成像。保持该过程，直到成功记录所有图像序列。</li>
	<li>将 300 帧记录为 2D 图像序列，以 200 帧/秒的帧速率覆盖幼虫的 3-5 个心动周期。记录在灯片照亮的选择性平面上捕获心脏收缩力。</li>
	<li>将幼虫以 1 μm 的步长移动到光片照明上，以虚拟切片样品。 注意:采样台的步长应根据光片系统的轴向分辨率设置，遵循奈奎斯特-香农采样定理12。</li>
	<li>重复步骤 7 记录新样品切片的另一个图像序列，直到覆盖整个心脏。通常，步长为1μm的100-200个图像序列可确保覆盖斑马鱼幼虫的整个心脏（图2D）。 注:步骤 4.7-4.9 由 LabVIEW 程序控制，并由 LSM 系统自动执行（图 3）。鉴于生成的图像数据量很大，建议对图像序列采用标准化的命名约定。例如，使用"z-1"、"z-2"等名称指定文件夹，以对各种图像序列中的数据进行分类。在每个序列中，图像应按顺序命名（例如，"1"、"2"等），以表示不同的时间点。为一只斑马鱼幼虫安装鱼、调整成像角度和收集所有数据的总时间约为 1 小时。</li>
</ol>5. 使用并行计算进行4D图像重建
定时:1天 注:我们小组开发的 4D 重建算法和样本数据可公开访问21.这种方法允许人们从前面步骤中收集的图像序列中重建 4D 心脏图像（表 1）。
<ol><li>在 MATLAB 中打开 文件 test_Parallel.m （参见 材料表）。在变量"baseDir"中指定原始图像序列的存储文件夹位置。</li>
	<li>为变量"numOfSlice"分配图像序列总数（通常为 100-150），为变量"numOfImage"分配每个序列中的图像数（通常为 300-500）。</li>
	<li>检查显示斑马鱼心脏中间平面的图像序列（例如，总共 101 个序列中的第 51个 序列）。确定此序列中第一个和第四个收缩期的帧号，并将它们分配给变量 systolicPoint_1st 和 systolicPoint_4th。</li>
	<li>单击 "运行 "以启动该过程。 注意:每个图像序列中心动周期的长度将首先由MATLAB程序通过比较不同时间点图像之间的相似性（平方差之和）来识别。随后，将使用所有图像序列的周期长度的平均值来估计心周期。接下来，程序将通过对从第一个图像序列到最后一个图像序列的图像相似度进行迭代比较，对不同轴向位置的图像序列的起点进行对齐。</li>
	<li>检查输出文件夹中的结果。该程序会将图像保存为带有时间戳的".tif"文件。每个".tif"文件都是特定时间点的 3D 心脏图像。两个 3D 图像之间的时间间隔等于设置的曝光时间。 注意:重建从前面步骤中获取的图像以描绘 4D（3D 空间 + 1D 时间）心脏收缩。为了提高回顾性同步期间高吞吐量调查的熟练程度，这种方法通过 MATLAB 并行计算工具箱利用多个 CPU 内核并将图像转换为 gpuArray 格式，从而在 GPU 上实现同步计算操作。</li>
</ol>6. 3D细胞分割和细胞追踪
定时:1天
<ol><li>下载 3DeeCellTracker 软件包（参见 材料表）并设置 Python 环境22.</li>
	<li>下载 ITK-SNAP 注释软件23 （参见 材料表），并使用它在两个选定的时间点（一个在心室舒张期，另一个在心室收缩期）手动标记 3D 心脏图像，以创建训练和验证数据集。 注意:其他标签软件也可能适用。</li>
	<li>在 Python 中，运行 3DeeCellTracker 训练程序，并在 TrainingUNet3D 函数中初始化 noise_level（在本例中为 100）、folder_path和模型参数，以设置预定义的 3D U-Net 模型。</li>
	<li>在 MATLAB 中，使用 imageDimConverter.m 程序将训练和验证数据集转换并重命名为正确的格式以进行加载。</li>
	<li>在 Python 中，使用 trainer.load_dataset（） 和 trainer.draw_dataset（） 函数加载训练和验证数据集。</li>
	<li>在 Python 中，运行 3DeeCellTracker 跟踪程序的第一部分并初始化参数。 注意:这包括定义图像参数以表征图像尺寸，分割参数以选择应用于细胞分割的机器学习模型，以及跟踪参数以选择用于细胞配准的预训练深度神经网络模型。首次使用时，建议使用 U-Net 模型进行细胞分割，并使用 FFN+ PR-GLS 进行细胞配准。需要将数据、模型和结果存储在将在此步骤中自动创建的文件夹中。</li>
	<li>在MATLAB中，使用 imageDimConverter.m 程序将所有3D心形图像转换并重命名为正确的格式，并将它们传输到最后一步创建的数据文件夹中。</li>
	<li>在 Python 中，运行 3DeeCellTracker 程序的第二部分以开始分割。</li>
	<li>分割第一张 3D 图像后，将分割结果与原始图像进行比较，如果发现任何不正确的分割，请执行手动校正。将更正后的分段移动到创建的"/manual_vol1"文件夹。</li>
	<li>在 Python 中，运行 3DeeCellTracker 程序的第三部分来分割所有图像。</li>
	<li>使用 Amira（参见 材料表）通过将跟踪单元的位置与其相应的原始图像进行比较，对跟踪结果进行可视化评估。如果测试结果不令人满意，请从步骤 6.6 重新开始该程序，使用替代机器学习模型进行分割或细胞配准，然后重新启动跟踪过程。 注意:分割后，细胞标签的数据默认存储为 8 位图像（0 到 255），这意味着仅提供 256 个尺度作为细胞分类的标签。当需要其他类时，有两种解决方案可以解决此问题。一种解决方案是在跟踪程序中将单元格标签的数据格式设置为16位图像，这将导致处理时间呈指数级增加，在本例中超过两天。另一种解决方案是使用"separateTrackingResults.py"程序对单元格标签进行后处理，这将根据其物理位置将具有相同标签的单元格分开，并为每个单元格分配不同的标签。在这种情况下，此过程大约需要 10 分钟。</li>
</ol>7. 虚拟现实模式下的心脏收缩力分析
定时:1天
<ol><li>获取前面步骤的细胞跟踪结果。</li>
	<li>手动验证数据，并在所有体积中选择具有一致图像强度的细胞（大约 600 个细胞中的约 500 个细胞）。</li>
	<li>使用 cellLabelsToObj.ipynb 脚本21 通过 3D Slicer 软件24 为每个单独的单元生成曲面网格（参见 材料表），并为每个单元分配唯一的颜色代码。</li>
	<li>将每个 3D 模型（由单个时间点中的所有单元格组成）导出为由多个子对象组成的单个.obj文件，并附有一个 .mtl 文件来描述单元格标签。</li>
	<li>使用教育许可证将模型导入 Unity，教育许可证是用于扩展现实的开发引擎。</li>
	<li>将包含用 C# 编写的函数的自定义脚本应用于模型和用户界面元素，以实现 4D 可视化和交互式分析。按照步骤 7.7 执行 VR 交互。</li>
	<li>使用以下功能在虚拟现实 （VR） 中与模型进行交互（图 4）:<ol><li>单元格选择:一次最多选择两个单元格，并查看它们的轨迹、速度、体积、表面积和相对距离。</li>
		<li>时间点选择:在数据中选择一个特定的时间点来分析所选细胞的输出，例如舒张期的t = 0 ms，或收缩期的t = 200 ms。</li>
		<li>暂停时间:冻结动态模型以专注于所选单元格及其输出。</li>
		<li>对比度:调制模型中所选像元与周围像元之间的对比度。</li>
	</ol></li>
</ol>

虚拟现实交互分析 Zebrafish 心脏收缩

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<li>Kaufmann, A., Mickoleit, M., Weber, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multilayer+mounting+enables+long-term+imaging+of+zebrafish+development+in+a+light+sheet+microscope%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.</a> Development. 139 (17), 3242-3247 (2012).</li>
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<li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((4D+light-sheet+imaging+and+interactive+analysis+of+cardiac+contractility+in+Zebrafish+larvae%5BTitle%5D)+AND+%22Zenodo%22%5BJournal%5D)">4D light-sheet imaging and interactive analysis of cardiac contractility in Zebrafish larvae.</a> Zenodo. , (2023).</li>
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<li>Ding, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiscale+light-sheet+for+rapid+imaging+of+cardiopulmonary+system%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Multiscale light-sheet for rapid imaging of cardiopulmonary system.</a> JCI Insight. 3 (16), e121396(2018).</li>
<li>Choe, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transgenic+fluorescent+zebrafish+lines+that+have+revolutionized+biomedical+research%5BTitle%5D)+AND+%22Lab.+Anim.+Res%22%5BJournal%5D)">Transgenic fluorescent zebrafish lines that have revolutionized biomedical research.</a> Lab. Anim. Res. 37 (1), 1-29 (2021).</li>
<li>Coelho-Filho, O. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+cardiomyocyte+hypertrophy+by+cardiac+magnetic+resonance%3A+implications+on+early+cardiac+remodeling%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Quantification of cardiomyocyte hypertrophy by cardiac magnetic resonance: implications on early cardiac remodeling.</a> Circulation. 128 (11), 1225(2013).</li>
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</ol>

作者没有要披露的利益冲突。

斑马鱼模型与工程方法的整合对于心肌梗塞、心律失常和先天性心脏缺陷的体内探索具有巨大的潜力。利用其光学透明度、再生能力以及与人类的遗传和生理相似性，斑马鱼胚胎和幼虫已被广泛用于研究 1,2,4。光片成像卓越的时空分辨率、最小的光损伤和光学切片能力使其在斑马鱼幼虫心脏形态和收缩功能的 4D 研究中脱颖而出 12,26,27,28。该协议引入了一种综合方法，该方法集成了光片成像、回顾性同步、3D 细胞跟踪和用于定量评估的交互式虚拟现实 （VR） 平台。它提供了一个切入点来捕获和重建心脏收缩，跟踪和量化细胞动力学，并评估心脏发育和再生过程中的结构、功能和机械变化。该方法还可以通过多通道荧光成像来跟踪各种细胞类型和谱系，以研究细胞异质性和细胞间相互作用。虽然斑马鱼模型与哺乳动物系统的二分法及其作为急性模型的局限性值得仔细考虑，但基因工程的进步可能允许开发更接近人类疾病状态的转基因斑马鱼品系，从而提高斑马鱼研究结果的转化价值29。
这种基于周期性心动周期假设的回顾性同步用于重建收缩斑马鱼心脏的光片图像。这种方法在光片成像中的实施使人们能够以每秒 200 多体积的速度可视化和分析收缩的心脏。为了应对当前分析中大量数据带来的挑战，使用了多核CPU和GPU的并行计算，使图像重建效率提高了10倍以上。在回顾性同步期间，假设心肌细胞在每次心跳时都会回到其基线位置。虽然考虑到斑马鱼26 的心跳变异性较低，如 图4C所示，这一假设相对安全，但它仍然是一种简化，可能无法解释所有生物学上的细微差别。成像方面的创新，如压缩荧光和光场显微镜，可以通过减少切片冗余来减轻这种假设产生的影响。
为了能够对复杂的4D心脏模型进行深入分析，我们开发了一个计算框架，包括基于3DeeCellTracker的细胞分割和用于用户指导研究的VR平台。该框架的固有功能，包括高效率和交互式操作，使人们能够量化细胞速度变化，研究细胞间相互作用，并评估区域和整体心肌力学，例如分数缩短、射血分数和区域应变30（图 4).注意到现有文献中缺乏对 7 dpf 时斑马鱼心脏功能的先前评估。然而，与其他研究人员提供的 3 dpf 数据相比，目前的结果与先前在 3 dpf 至 7 dpf 间隔期间射血分数 （EF） 和分数缩短 （FS） 下降的迹象一致25,31。这些分析方法在阐明斑马鱼心脏损伤模型中的心脏动力学方面具有巨大的潜力12,25。
由于VR技术和软件具有立体视觉和细胞位置自动配准等独特功能，因此该平台允许一种身临其境的简化方法来选择特定细胞并分析它们在整个心动周期中的轨迹。VR的使用提供了一种直观的方式来执行复杂的任务，如细胞分割和注释，具有更高的效率和准确性32,33,34。它还支持多个用户在处理大型复杂数据集时使用独特的工具34.虽然长时间使用VR可能会引起头晕等副作用，但这可以通过改进的人体工程学设计和不受束缚的耳机来缓解。此外，为了解决实时渲染复杂生物结构所需的大量计算能力，优化即时处理软件并利用基于云的计算解决方案可以增强系统管理复杂可视化的能力。
随着硬件设备和计算能力的不断进步，该策略可以扩展到在细胞和组织水平上研究心肌心律失常，最终有可能揭示心脏形态发生的潜在机制并推进治疗干预措施的发展。

斑马鱼 （Danio rerio） 是一种广泛使用的模式生物，用于研究心脏发育、生理学和修复，因为它具有光学透明度、遗传可处理性和再生能力 1,2,3,4。心肌梗死后，虽然结构和功能变化会影响心脏射血和血流动力学，但技术限制继续阻碍以高时空分辨率研究心脏再生过程中动态过程的能力。例如，传统的成像方法，如共聚焦显微镜，在成像深度、时间分辨率或光毒性方面存在局限性，无法捕捉多个心动周期期间的动态变化和评估心脏收缩功能5。
光片显微镜代表了一种最先进的成像方法，它通过快速扫描激光扫过心脏的心室和心房，获得具有增强的时空分辨率和可忽略不计的光漂白和光毒性效应的详细图像，从而成功地解决了这些问题6,7,8,9,10,11。
该协议引入了一种全面的成像策略，包括 LSM 系统构建、4D 图像重建、3D 细胞跟踪和交互式分析，以捕获和分析多个心动周期期间整个心脏的心肌细胞动力学12。定制的成像系统和计算方法允许人们在转基因 Tg（myl7:nucGFP） 斑马鱼幼虫的单细胞水平上跟踪心肌微观结构和收缩功能。此外，使用显微注射将小分子化合物输送到胚胎中，以评估药物诱导的心脏损伤和随后的再生。这种整体 策略为在 心脏发育和再生过程中在单细胞水平上研究心肌的结构、功能和机械特性提供了一个切入点。

<table><tbody><tr><td>&lt;strong&gt;RESOURCE&lt;/strong&gt;</td><td>&lt;strong&gt;SOURCE/Reference&lt;/strong&gt;</td><td>&lt;strong&gt;IDENTIFIER&lt;/strong&gt;</td><td></td></tr><tr><td>&lt;strong&gt;Animal models&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Tg(myl7:nucGFP) transgenic zebrafish&lt;/em&gt;</td><td>Burns Lab in Boston Children&#039;s Hospital</td><td>ZDB-TGCONSTRCT-070117-49</td><td></td></tr><tr><td>Software and algorithms</td><td></td><td></td><td></td></tr><tr><td>MATLAB</td><td>The MathWorks Inc.</td><td>R2023a</td><td></td></tr><tr><td>LabVIEW</td><td>National Instruments Corporation</td><td>2017 SP1</td><td></td></tr><tr><td>HCImage Live</td><td>Hamamatsu Photonics</td><td>4.6.1.2</td><td></td></tr><tr><td>Python</td><td>The Python Software Foundation</td><td>3.9.0</td><td></td></tr><tr><td>Fiji-ImageJ</td><td>Schneider et al.&lt;sup&gt;18&lt;/sup&gt;</td><td>1.54f</td><td></td></tr><tr><td>3DeeCellTracker</td><td>Chentao Wen et al.&lt;sup&gt;15&lt;/sup&gt;</td><td>v0.5.2</td><td></td></tr><tr><td>Unity</td><td>Unity Software Inc.</td><td>2020.3.2f1</td><td></td></tr><tr><td>Amira</td><td>Thermo Fisher Scientific</td><td>2021.2</td><td></td></tr><tr><td>3D Slicer</td><td>Andriy Fedorov et al.&lt;sup&gt;17&lt;/sup&gt;</td><td>5.2.1</td><td></td></tr><tr><td>ITK SNAP</td><td>Paul A Yushkevich et al.&lt;sup&gt;16&lt;/sup&gt;</td><td>4</td><td></td></tr><tr><td>&lt;strong&gt;Light-sheet system&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cylindrical lens</td><td>Thorlabs</td><td>ACY254-050-A</td><td></td></tr><tr><td>4X Illumination objective</td><td>Nikon</td><td>MRH00045</td><td></td></tr><tr><td>20X Detection objective</td><td>Olympus</td><td>1-U2M585</td><td></td></tr><tr><td>sCMOS camera</td><td>Hamamatsu</td><td>C13440-20CU</td><td></td></tr><tr><td>Motorized XYZ stage</td><td>Thorlabs</td><td>PT3/M-Z8</td><td></td></tr><tr><td>Two-axis tilt stage</td><td>Thorlabs</td><td>GN2/M</td><td></td></tr><tr><td>Rotation stepper motor</td><td>Pololu</td><td>1474</td><td></td></tr><tr><td>Fluorescent beads</td><td>Spherotech</td><td>FP-0556-2</td><td></td></tr><tr><td>473nm DPSS Laser</td><td>Laserglow</td><td>R471003GX</td><td></td></tr><tr><td>532nm DPSS laser</td><td>Laserglow</td><td>R531003FX</td><td></td></tr><tr><td>&lt;strong&gt;Microinjector and vacuum pump&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microinjector</td><td>WPI</td><td>PV850</td><td></td></tr><tr><td>Vacuum pump</td><td>Welch</td><td>2522B-01</td><td></td></tr><tr><td>Pre-Pulled Glass Pipettes</td><td>WPI</td><td>TIP10LT</td><td></td></tr><tr><td>Capillary tip for gel loading</td><td>Bio-Rad</td><td>2239912</td><td></td></tr><tr><td>&lt;strong&gt;Virtual reality hardware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>VR headset</td><td>Meta</td><td>Quest 2</td><td></td></tr><tr><td>&lt;strong&gt;30mg/L PTU solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PTU</td><td>Sigma-Aldrich</td><td>P7629</td><td></td></tr><tr><td>1X E3 working solution</td><td>-</td><td>-</td><td></td></tr><tr><td>&lt;strong&gt;1% Agarose&lt;/strong&gt;</td><td>-</td><td>-</td><td></td></tr><tr><td>Low-melt 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>&lt;strong&gt;10g/L Tricaine stock solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tricaine</td><td>Syndel</td><td>SYNC-M-GR-US02</td><td></td></tr><tr><td>Deionized water</td><td>-</td><td>-</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>&lt;strong&gt;150mg/L Tricaine working solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10g/L Tricaine stock solution</td><td>-</td><td>-</td><td></td></tr><tr><td>Deionized water</td><td>-</td><td>-</td><td></td></tr><tr><td>&lt;strong&gt;60X E3 stock solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium Chloride</td><td>Lab Animal Resource Center (LARC), The University of Texas at Dallas</td><td>NaCl</td><td></td></tr><tr><td>Potassium Chloride</td><td>-</td><td>KCL</td><td></td></tr><tr><td>Calcium Chloride Dihydrate</td><td>-</td><td>CaCL&lt;sub&gt;2 &lt;/sub&gt;x 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td></tr><tr><td>Magnesium Sulfate Heptahydrate</td><td>-</td><td>MgSO&lt;sub&gt;4 &lt;/sub&gt;x 7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td></tr><tr><td>RO Water</td><td>-</td><td>-</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;1X E3 working solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>60X E3 stock solution</td><td>Lab Animal Resource Center (LARC), The University of Texas at Dallas</td><td>-</td><td></td></tr><tr><td>RO Water</td><td>-</td><td>-</td><td></td></tr><tr><td>1% Methylene Blue (optional)&amp;nbsp;</td><td>-</td><td>C&lt;sub&gt;16&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;ClN&lt;sub&gt;3&lt;/sub&gt;S</td><td></td></tr></tbody></table>

4d light-sheet imaging of zebrafish cardiac contraction

斑马鱼是一种有趣的模式生物，以其非凡的心脏再生能力而闻名。 在体内 研究收缩的心脏对于深入了解损伤引起的结构和功能变化至关重要。然而，获得斑马鱼心脏的高分辨率和高速 4 维（4D、3D 空间 + 1D 时间）图像以评估心脏结构和收缩力仍然具有挑战性。在这种情况下，使用内部光片显微镜（LSM）和定制的计算分析来克服这些技术限制。该策略涉及LSM系统构建、回顾性同步、单细胞跟踪和用户导向分析，使人们能够以单细胞分辨率研究转基因 Tg（myl7:nucGFP） 斑马鱼幼虫的整个心脏的微观结构和收缩功能。此外，我们能够进一步结合小分子化合物的显微注射，以精确和可控的方式诱导心脏损伤。总体而言，该框架允许人们跟踪生理和病理生理变化，以及心脏形态发生和再生过程中单细胞水平的区域力学。

目前的方案包括三个主要步骤:斑马鱼制备和显微注射、光片成像和 4D 图像重建，以及细胞跟踪和 VR 交互。允许成年斑马鱼交配，收集受精卵，并根据所提出的实验的需要进行显微注射（图1）。这一步为探索斑马鱼在心脏发育和再生研究中的应用提供了一个切入点，它在随后的成像和分析中也起着至关重要的作用。使用定制的 LSM 系统在不同阶段（从 3 dpf 到 7 dpf）对收缩心脏进行成像，并沿 z 轴对齐图像序列以重建 4D 斑马鱼心脏模型（图 2 和 图 3）。这些模型为深度表型表征提供了基础，对于揭示整个发育时间线的心脏形态和功能动态至关重要。对斑马鱼心脏中的单个细胞进行跟踪，并使用定制的VR平台量化它们的运动和相互作用。还比较了心房和心室中一个心动周期中所选细胞的速度和相对距离变化，以评估区域收缩力并研究局部应变（图4）。区域应变是根据感兴趣区域中两个相邻心肌细胞之间的位移变化确定的。使用已发表文献中描述的方法估计缩短分数 （FS） 和射血分数 （EF）25。FS 和 EF 的公式如下:
<img alt="Equation 1" src="/files/ftp_upload/66263/66263eq01.jpg" style="margin: auto;">
<img alt="Equation 2" src="/files/ftp_upload/66263/66263eq02.jpg" style="margin: auto;">
其中 Dd 和 Ds 分别是舒张末期和收缩末期的心室直径（短轴，通过不同心室细胞之间的距离测量），Vd 和 Vs 分别是舒张末期和收缩末期的心室容积（通过心室的长轴和短轴直径计算）。
总的来说，这些结果展示了一种整合生理学和工程学的新策略，以促进斑马鱼模型的体积成像和数据解释，为推进心脏形态发生和力学的探索带来了巨大的希望。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66263/66263fig01.jpg"> 图1:斑马鱼卵采集和显微注射过程。 该过程包括与成年斑马鱼交配、收集受精卵和进行可选的显微注射。在显微注射过程中，预拉式玻璃移液器中装有所需的材料。受精卵在琼脂糖霉菌狭缝内直行排列，并垂直于针头放置。显微注射是在对卵和针尖进行恒定显微镜观察的情况下进行的。 <a href="https://www.jove.com/files/ftp_upload/66263/66263fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66263/66236fig02v2.jpg"> 图 2:光片成像过程和 4D 图像重建。 （A） 内部光片成像系统结构示意图。CL:柱面透镜。EO:激发物镜。DO:检测目标。TL:镜筒透镜。FL:过滤器。CAM:sCMOS相机。（B） 安装在FEP管中并用琼脂糖包埋的斑马鱼的示意图。（C） 从 4 dpf 斑马鱼幼虫捕获的原始 2D 图像序列。每帧左上角的时钟表示从收缩末期开始的心形阶段。（D） 4D斑马鱼图像配准的回顾性同步图示。图像序列表示沿 z 轴在一定深度处的连续记录。图像序列中的每一帧都由一个红点表示，从舒张末期到收缩期末期的开始和结束阶段在黄色框中突出显示。（E） 根据 2D 图像序列重建的 4D 心脏模型。Dpf:受精后天数。该图改编自Zhang等人12。 <a href="https://www.jove.com/files/ftp_upload/66263/66236fig02largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66263/66263fig03.jpg"> 图3:LabVIEW控制面板。 （A） 控制面板中使用的设置包括激光、相机、图像路径和电机模式的配置。（B） LabVIEW 程序捕获的原始斑马鱼心脏图像示例。比例尺:30 μm。 <a href="https://www.jove.com/files/ftp_upload/66263/66263fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66263/66263fig04.jpg"> 图 4:细胞跟踪和 VR 交互有助于揭示斑马鱼的心脏收缩力。 （A） 3 dpf 下 Tg（myl7:nucGFP） 斑马鱼心脏的跟踪细胞。（B） VR平台为用户提供4D斑马鱼心脏模型的沉浸式观看和互动体验，允许用户通过用户定义的分析可视化心脏功能随时间的变化。（C） 从 VR 平台收集测量值后，比较 3 dpf 和 7 dpf 所选细胞在一个心动周期内的速度和相对距离变化。前两张图显示了五个心室细胞和五个心房细胞的平均速度变化，其他图显示了三组细胞（即两个心室细胞、一个心室细胞和一个心房细胞以及两个心房细胞）之间的相对距离变化。（D） 3 dpf 和 7 dpf 斑马鱼心脏的心脏功能评估。在斑马鱼心脏中以 3 dpf 追踪共有 370 个细胞，以 7 dpf 追踪 580 个细胞。该图改编自Zhang等人12。 <a href="https://www.jove.com/files/ftp_upload/66263/66263fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>名字</td>
			<td colspan="2">用法</td>
			<td>标识符</td>
		</tr><tr><td colspan="4">定制程序和算法</td>
		</tr><tr><td>PSF.m</td>
			<td colspan="2">从荧光珠的FWHM测量结果计算PSF。</td>
			<td>MATLAB程序</td>
		</tr><tr><td>4D斑马鱼 Imaging.vi</td>
			<td colspan="2">控制和同步 LSM 系统中的硬件。</td>
			<td>LabVIEW程序</td>
		</tr><tr><td>test_Parallel.m等</td>
			<td colspan="2">从 2D 图像序列重建 4D 图像。</td>
			<td>MATLAB程序</td>
		</tr><tr><td>imageDimConverter.m</td>
			<td colspan="2">将图像数据转换为指定格式以进行单元格跟踪。</td>
			<td>MATLAB程序</td>
		</tr><tr><td>separateTrackingResults.py</td>
			<td colspan="2">对具有相同标记的细胞进行分离的细胞跟踪结果的后处理。</td>
			<td>Python 程序</td>
		</tr><tr><td>cellTracking_All.py</td>
			<td colspan="2">选择在所有体积上具有一致图像强度的细胞。</td>
			<td>Python 程序</td>
		</tr><tr><td>cellLabelsToObj.ipynb</td>
			<td colspan="2">通过 3D 切片器为每个单元生成曲面网格。</td>
			<td>Python 程序</td>
		</tr><tr><td>DynamicHeartModel.cs等</td>
			<td colspan="2">创建 VR 环境并与对象交互。</td>
			<td>C# 程序</td>
		</tr><tr><td colspan="4">定制硬件设计</td>
		</tr><tr><td>3D打印样品室</td>
			<td colspan="2">与水浸物镜一起使用。</td>
			<td>SolidWorks 设计</td>
		</tr><tr><td>3D打印样品架</td>
			<td colspan="2">适应样品台并保持样品。</td>
			<td>SolidWorks 设计</td>
		</tr></tbody></table>表 1:自定义资源表。示例代码和数据上传到 Zenodo21。

Watch this Scientific Journal Video about 4D Light-sheet Imaging of Zebrafish Cardiac Contraction at JoVE.com

作者聚焦:斑马鱼中的高分辨率 4D 光片成像和虚拟现实用于心脏功能的单细胞分析

该协议利用光片成像来研究斑马鱼幼虫的心脏收缩功能，并通过细胞跟踪和交互式分析深入了解心脏力学。

斑马鱼心脏收缩的 4D 光片成像

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...

我们的研究使用4D光片成像来探索斑马鱼的心脏收缩功能，旨在了解单细胞水平的心脏收缩动力学。最重要的实验挑战之一是在观察自然心律的同时实现高空间终端分辨率、心脏功能的动态成像。我们的研究填补了在单细胞分辨率下分析复杂 4D 心脏动力学的空白。我们利用基于 GPU 的并行计算、虚拟现实和先进的细胞跟踪来解释光片成像数据。我们新颖的虚拟现实平台提供了一种交互式方法来评估区域心脏收缩，为更深入的分析提供操作功能。通过促进用户导向的心脏收缩分析，我们的方案可以增强对心脏发育和疾病的理解。

4d-light-sheet-imaging-of-zebrafish-cardiac-contraction

Nanyang Technological University

Research

JoVE Journal

Bioengineering

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

1.2K 次观看.  The University of Texas at Dallas. 我们的研究使用4D光片成像来探索斑马鱼的心脏收缩功能，旨在了解单细胞水平的心脏收缩动力学。最重要的实验挑战之一是在观察自然心律的同时实现高空间终端分辨率、心脏功能的动态成像。我们的研究填补了在单细胞分辨率下分析复杂 4D 心脏动力学的空白。我们利用基于 GPU 的并行计算、虚拟现实和先进的细胞跟踪来解释光片成像数据。我们新颖的虚拟现实平台?...

视频: 作者聚焦:斑马鱼中的高分辨率 4D 光片成像和虚拟现实用于心脏功能的单细胞分析

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

投诉矩阵量化细胞收缩的准备工作

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

科研

生物工程

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture cellular behaviors involved in developmental processes.&#160; The ability to track and follow cell movements is limited by sample movements that occur due to drift of the sample or, in some cases, growth during image acquisition. Tracking cells in datasets affected by drift and/or growth will incorporate these movements into any analysis of cell position. This may result in the apparent movement of static structures within the sample. Therefore prior to cell tracking, any sample drift should be corrected. Using the open source Fiji distribution 1 &#160;of ImageJ 2,3 and the incorporated LOCI tools 4, we developed the Correct 3D drift plug-in to remove erroneous sample movement in confocal datasets. This protocol effectively compensates for sample translation or alterations in focal position by utilizing phase correlation to register each time-point of a four-dimensional confocal datasets while maintaining the ability to visualize and measure cell movements over extended time-lapse experiments.

Time-lapse microscopy allows the visualization of developmental processes. Growth or drift of samples during image acquisition reduces the ability to accurately follow and measure cell movements during development. We describe the use of open source image processing software to correct for three dimensional sample drift over time.

样品漂移校正继4D共焦时间推移成像

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

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 ...

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

心肌机械变形（DIAMOND）的位移分析揭示了胚胎斑马鱼心脏功能的分段异质性

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

发育生物学

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

人工程心脏组织的收缩自动分析心脏药物安全筛选

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

改善斑马鱼幼虫实验操作的长期活体成像装置

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

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. ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.