这项工作得到了美国国立卫生研究院（K25HD097288，R21HD112663）尤尼斯·肯尼迪·施莱弗国家儿童健康与人类发展研究所的支持。

该协议已获得韦恩州立大学机构动物护理和使用委员会的批准。
1.实验准备
<ol><li>使用70%乙醇溶液清洁和消毒剪刀和镊子。此外，准备一次性移液器和注射器。</li>
	<li>通过向 495 mL 去离子水中加入 3.595 g NaCl 来制备洗涤介质。然后，向培养基中加入5ml青霉素 - 链霉素（5U / mL）。用洗涤介质填充100毫米培养皿并将其加热至37°C。</li>
	<li>根据 图 1 准备培养皿，图 1 说明了整体配置。<ol><li>准备一张滤纸，将其切成大约 17 毫米× 20 毫米的矩形，然后去掉四个角。在滤纸上创建一个中空中心，大小约为7毫米×10毫米，用于固定胚胎（图1，左）。 注意：胚胎的收集在步骤2中描述。</li>
		<li>将市售环（Φ = 1 英寸，参见 材料表）与柔性薄膜（即石蜡薄膜）层连接，确保柔性薄膜也具有空心。这个带有薄膜的环将用于固定带有胚胎的滤纸。</li>
		<li>最后，将准备好的环放入35毫米培养皿中（图1，右）。 注意：滤纸用于通过将滤纸放在膜上并将胚胎留在中心空心区域（将在步骤 2 中详细介绍）来固定和保持提取的胚胎。四个角被切割成适合外环的尺寸（如果已经安装，则不需要）。35 mm 玻璃底培养皿用于更好的激光束传播。培养皿有一个内孔 （Φ = 23 mm），可以填充蛋白进行 卵外 培养。环的直径必须大于内孔的直径，以便孔可以被环上的柔性薄膜覆盖（图1，插图）。滤纸的尺寸不受限制，可以根据所选环和培养皿的尺寸进行修改。或者，培养皿的底部可以预涂琼脂糖层（厚度：~1mm）。通过使用刀片从层中心去除琼脂糖，可以创建一个与柔性薄膜的空心中心形状相似的孔，用于装载蛋白。一个关键点是空心滤纸的四个侧面需要有足够的宽度（> 5 毫米）以确保胚胎的附着。</li>
	</ol></li>
</ol>2.鸡胚的提取和卵培养
注：此步骤是从以前发布的报告40,41修改而来的。
<ol><li>预培养后，从孵化器中取出鸡蛋并将其放在鸡蛋纸盒托盘上的长轴上。用70%乙醇清洗蛋壳，确保覆盖整个表面，然后静置15分钟。</li>
	<li>将鸡蛋放在短轴上，然后在底部打碎。在干净的 100 毫米培养皿上打开鸡蛋以提取其内容物，如 图 2 所示。确保在握住和打开鸡蛋时不要旋转鸡蛋。</li>
	<li>使用移液管，转移并收集约10mL稀蛋白（即液体蛋白42）到15mL管中，用于 卵外 培养。用收集的稀蛋白（约0.9mL）填充培养皿的中心孔，如 图3所示。填充过程应缓慢，避免在井内形成任何气泡。确保将含有蛋白的培养皿加热至37°C。 注意：这道菜将用于 培养胚胎。在布里渊成像期间，将使用充满洗涤介质的新培养皿（参见步骤 3）。</li>
	<li>使用薄纸，通过小心地将蛋白与卵黄膜分离，轻轻地去除附着在胚胎上的浓稠蛋白（即粘性蛋白），如 图4所示。避免直接接触卵黄膜。</li>
	<li>去除所有浓稠的蛋白后，小心地将滤纸贴在卵黄膜上。确保胚胎的身体轴与滤纸上中心矩形的长轴对齐。用剪刀剪开滤纸周围的膜。</li>
	<li>用镊子轻轻地将分离的滤纸倾斜地从蛋黄上拉开。将滤纸倒置，使胚胎背面朝下（用于倒置显微镜配置）。小心地将整个滤纸从长轴的一侧浸入100毫米培养皿中，并用洗涤介质以倾斜的方式浸入培养皿中。</li>
	<li>使用干净的移液管将洗涤介质平行于滤纸轻轻喷洒，洗掉任何残留的蛋黄。避免将洗涤介质直接喷洒到膜上。</li>
	<li>清除所有蛋黄后，小心地从洗涤介质中取出滤纸，并用薄纸吸收边缘多余的培养基。然后，将装有胚胎的滤纸放在培养皿上，确保胚胎的背面朝下，如 图5所示。为了保持湿度，请在盘子中放一张湿纸巾。</li>
	<li>将培养皿转移到舞台上的培养箱中进行 卵外 培养。</li>
</ol>3. 胚胎的布里渊测量
<ol><li>准备另一套培养皿并用洗涤介质填充它们。确保洗涤介质加热至37°C。</li>
	<li>当胚胎达到所需的发育阶段时，将带有胚胎的滤纸转移到装有洗涤介质的培养皿中。将培养皿放入布里渊显微镜的载物台培养箱中（参见 材料表）。<ol><li>在进行布里渊测量之前，保存整个胚胎的明场图像以供参考。布里渊显微镜的详细配置在前面已经描述过30 ，并在结果部分进行了总结（图6）。</li>
	</ol></li>
	<li>测量水和甲醇的布里渊信号，该信号将用于步骤4的校准过程。</li>
	<li>调整入射激光功率和电子倍增电荷耦合器件（EMCCD，见 材料表）相机曝光时间，以实现至少 10,000 次布里渊信号计数。以明场图像为指导，设置扫描范围和步长。通过使用 2D 翻译阶段扫描胚胎来获取感兴趣区域的布里渊图像。 注意：水平扫描范围可以根据明场图像确定，垂直（即深度）扫描范围可以根据快速粗略扫描获得的信号强度确定。为防止对胚胎造成任何光损伤，请将入射功率限制为 25 mW，并将 EMCCD 相机的曝光时间设置为 50 ms。在水平方向上选择 2 μm 的步长，在垂直方向上选择 1 μm 的步长，以平衡成像质量和采集时间。</li>
	<li>完成扫描后，小心地取出带有胚胎的滤纸，并用薄纸吸收多余的洗涤介质。然后，将滤纸放回装有稀蛋白的培养皿中，以便在载物台培养箱中连续培养。</li>
	<li>以固定的时间间隔（例如，1.5小时）重复步骤3.2-3.5，以在胚胎发育时捕获延时布里渊图像。</li>
	<li>按照步骤 4 重建 2D 布里渊图像。</li>
</ol>4. 布里渊图像重建
<ol><li>使用水和甲醇的布里渊信号校准布里渊光谱仪，计算光谱仪30 的自由光谱范围 （FSR） 和像素频率转换比 （PR）。FSR 和 PR 的校准值将用于计算胚胎在每个像素处的布里渊位移。 注： 图7A 显示了EMCCD相机捕获的原始布里渊光谱。通过对光谱进行垂直求和，然后进行洛伦兹拟合，可以得到两个点的峰距离Δd（图7B）。通过得到水Δd水 和甲醇Δd甲醇的峰距离，可以根据方程30计算FSR和PR：PR = 2·（ω甲醇-ω 水）/（Δd水 - Δd甲醇），FSR = 2·ω水 + PR·Δd水，已知水 ω水 = 6.01 GHz 和甲醇 ω甲醇 = 4.49 GHz 在 660 nm 处的布里渊位移。</li>
	<li>获得样品每个像素处布里渊信号的峰距离。根据校准的 FSR 和 PR 计算布里渊位移：ω样本 = 0.5 （FSR - PR x Δd样本）30，其中 ω样本 是样品的布里渊位移，Δd样本 是布里渊信号的峰距离。</li>
	<li>根据所有像素的布里渊位移重建二维布里渊图像。 注意：要进行局部分析，可以从采集的布里渊图像中选择感兴趣的区域（例如，神经板）并量化其机械性能。一种常用的方法是计算所选区域的平均布里渊位移。</li>
</ol>

雏鸡胚胎发育中神经板力学的纵向监测

作者声明他们没有利益冲突。

胚胎的早期发育很容易受到外界干扰的影响。因此，在样品提取和转移过程中需要格外小心。一个潜在的问题是胚胎从滤纸上脱落，这可能导致卵黄膜收缩，并导致布里渊成像中神经板的倾斜伪影。此外，这种收缩可能会阻止胚胎的发育。应注意防止分离的几个关键步骤。首先，在步骤2.4中，确保从膜表面彻底去除蛋白至关重要。任何残留的蛋白都会阻碍膜与滤纸46,47的正确粘附。此外，在洗涤过程中，沿平行于膜的方向喷洒洗涤介质也很重要。用液体流直接撞击膜可能会导致膜脱落甚至撕裂。此外，整个洗涤过程应在短时间内完成（例如，<3分钟），因为长时间浸泡也会导致胚胎从滤纸上脱落。
在进行布里渊测量之前，有必要将胚胎转移到带有洗涤介质的培养皿中。这是因为蛋白的布里渊位移非常接近神经板组织，导致图像对比度差。布里渊成像是通过点扫描获得的，这可能很耗时，尤其是在处理许多点时。在该协议中，扫描区域水平方向约为400μm，垂直方向小于100μm。为了避免干扰胚胎的发育，总采集时间限制在30分钟以内。这是通过水平方向 2 μm 和垂直方向 1 μm 的步长来实现的。如果只对特定区域的平均布里渊位移感兴趣，则可以实施快速粗略的扫描（例如，水平和垂直的步长均为 4 μm），这将花费不到 5 分钟的时间，并且可以减轻对胚胎发育的负面影响。
布里渊显微镜为研究胚胎发育的生物力学提供了一种实时和非侵入性的成像方法。这种方法的一个局限性在于其穿透深度，根据胚胎的发育阶段，大约为100-200μm。虽然增加EMCCD的激光功率和/或曝光时间可以部分解决这个问题，但它的代价是潜在的光毒性和/或较长的采集时间。或者，现有的光学技术，如自适应光学，可以潜在地用于改善穿透深度48。
总之，我们已经建立了一种使用布里渊显微镜探测活鸡胚胎机械性能的协议。这种非接触式方法可以满足当前未满足的需求，并且是对其他用于研究胚胎发育中生物力学的现有工具的补充。

神经管缺陷 （NTD） 是胚胎发育过程中神经管闭合 （NTC） 失败引起的严重中枢神经系统出生缺陷1。被忽视的热带病的病因很复杂。研究表明，NTC 涉及一系列形态发生过程，包括收敛延伸、神经板弯曲（例如，顶端收缩）、神经皱襞抬高以及最终神经襞的粘附。这些过程受多种分子和遗传机制的调节2,3，这些过程中的任何故障都可能导致NTDs4,5,6。随着越来越多的证据表明，机械线索在 NTC3、7、8、9、10、11 中也起着至关重要的作用，并且已经发现了基因和机械线索之间的关系 12,13,14，因此研究神经形成过程中的组织生物力学变得势在必行。
已经开发了几种用于测量胚胎组织机械性能的技术，包括激光消融 （LA）15、组织解剖和松弛 （TDR）16,17、微量移液器抽吸 （MA）18、基于原子力显微镜 （AFM） 的纳米压痕19、微压痕器 （MI） 和微孔板 （MP）20、使用光学/磁镊的微流变学 （MR）21、22、23和基于液滴的传感器24.现有方法可以在从亚细胞到组织尺度的空间分辨率下测量机械性能。然而，这些方法大多是侵入性的，因为它们需要与样品接触（例如，MA、AFM、MI 和 MP）、外部材料注射（例如，MR 和基于液滴的传感器）或组织解剖（例如，LA 和 TDR）。因此，现有方法在原位监测神经板组织的机械演化具有挑战性25。最近，混响光学相干弹性成像在具有高空间分辨率的非接触式机械映射方面显示出前景26。
共聚焦布里渊显微镜是一种新兴的光学模式，能够以27、28、29、30 亚细胞分辨率对组织生物力学进行非接触式定量。布里渊显微镜基于自发布里渊光散射原理，即入射激光与材料内部热波动引起的声波之间的相互作用。因此，散射光会经历频率偏移，称为布里渊频移 ωR，公式为31：
<img alt="Equation 1" src="/files/ftp_upload/66117/66117eq01.jpg" style="margin: auto;"> (1)
这里，<img alt="Equation 2" src="/files/ftp_upload/66117/66117eq02.jpg" style="margin: auto;">是材料的折射率，λ是入射光的波长，M'是纵向模量，ρ是质量密度，θ是入射光与散射光之间的角度。对于同类型的生物材料，折射率和密度<img alt="Equation 3" src="/files/ftp_upload/66117/66117eq03.jpg" style="margin: auto;">之比近似恒定28,32,33,34,35,36。因此，布里渊位移可以直接用于估计生理过程中的相对机械变化。布里渊显微镜的可行性已在各种生物样品中得到验证29,37,38。最近，通过将布里渊显微镜与载物台孵化系统相结合，演示了活雏鸡胚胎的延时机械成像39。该协议提供了样品制备、实验实施以及数据后处理和分析的详细说明。我们希望这项工作将促进非接触式布里渊技术的广泛采用，以研究胚胎发育和出生缺陷中的生物力学调节。

<table><tbody><tr><td>100 mm Petri dish&amp;nbsp;</td><td>Fisherbrand</td><td>FB0875713</td><td></td></tr><tr><td>2D motorized stage&amp;nbsp;</td><td>Prior Scientific</td><td>H117E2</td><td></td></tr><tr><td>35 mm Petri dish</td><td>World Precision Instruments</td><td>FD35-100</td><td></td></tr><tr><td>Brillouin Microscope with on-stage incubator</td><td>N/A</td><td>N/A</td><td>This is a custom-built Brillouin Microscope system based on Ref. 30</td></tr><tr><td>Chicken eggs</td><td>University of Connecticut</td><td>N/A</td><td></td></tr><tr><td>CMOS camera</td><td>Thorlabs</td><td>CS2100M-USB</td><td></td></tr><tr><td>EMCCD camera</td><td>Andor</td><td>iXon</td><td></td></tr><tr><td>Ethanol</td><td>Decon Laboratories, Inc.</td><td>#2701</td><td></td></tr><tr><td>Filter paper</td><td>Whatman</td><td>1004-070</td><td></td></tr><tr><td>Incubator for in ovo culture</td><td>GQF Manufacturing Company Inc.&amp;nbsp;</td><td>GQF 1502&amp;nbsp;</td><td></td></tr><tr><td>Ring</td><td>Thorlabs</td><td>SM1RR</td><td></td></tr><tr><td>Microscope body</td><td>Olympus</td><td>IX73</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>On-stage incubator</td><td>Oko labs</td><td>OKO-H301-PRIOR-H117</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15070-063</td><td></td></tr><tr><td>Pipettes</td><td>Fisherbrand</td><td>13-711-6M</td><td></td></tr><tr><td>Scissors</td><td>Artman instruments</td><td>N/A</td><td>3pc Micro Scissors 5</td></tr><tr><td>Syringe</td><td>BD</td><td>305482</td><td></td></tr><tr><td>Tissue paper</td><td>Kimwipes</td><td>N/A</td><td></td></tr><tr><td>Tube</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>Tweezers</td><td>DR Instruments</td><td>N/A</td><td>Microdissection Forceps Set&amp;nbsp;</td></tr></tbody></table>

monitoring the mechanical evolution of tissue during neural tube closure of chick embryo

神经管闭合（NTC）是胚胎发育过程中的关键过程。这一过程的失败会导致神经管缺陷，导致先天性畸形甚至死亡。NTC涉及遗传、分子和机械水平的一系列机制。虽然近年来机械调节已成为一个越来越有吸引力的话题，但由于缺乏合适的技术来 对原位进行 3D 胚胎组织的机械测试，它在很大程度上仍未得到探索。作为回应，我们开发了一种以非接触式和非侵入性方式量化鸡胚胎组织的机械性能的方案。这是通过将共聚焦布里渊显微镜与载物台孵育系统集成来实现的。为了探究组织力学，收集预培养的胚胎并将其转移到载物台培养箱中进行 卵外 培养。同时，布里渊显微镜在发育过程中的不同时间点采集神经板组织的机械图像。该协议包括样品制备的详细说明、布里渊显微镜实验的实施以及数据后处理和分析。通过遵循该协议，研究人员可以纵向研究胚胎组织在发育过程中的机械进化。

图6 显示了布里渊显微镜的原理图。该系统采用660nm激光作为光源。在激光头的正后放置一个隔离器以抑制任何背向反射光，并使用中性密度 （ND） 滤光片来调节激光功率。一对焦距分别为 f1 = 16 mm 和 f2 = 100 mm 的透镜 L1 和 L2 用于扩展激光束。在偏振分束器（PBS）之后，使用半波板（HWP）和线性偏振器（偏振器1）来调节照射在样品或校准材料（即水和甲醇）上的光束功率。为了将激光聚焦到样品中并收集向后散射光，使用了数值孔径 （NA） 为 0.6、放大倍率为 40 的物镜 （OBJ2）。入射光和向后散射光在使用四分之一波板 （QWP2） 进行偏振调整后由相同的 PBS 分离。同样，OBJ1 和 QWP1 用于将激光束引导至校准材料。布里渊信号通过单模光纤传送到布里渊光谱仪43 进行分析。在光谱仪内部，柱面透镜 （C1） 将布里渊信号耦合到第一个 VIPA（虚拟成像相控阵）标准具中。第一个VIPA标准枪的输出由另一个柱面透镜（C2）重塑，并聚焦到掩模1上，以抑制非散射激光。然后，布里渊信号通过球面透镜 （SL1） 耦合到第二个 VIPA 标准具中，由另一个球面透镜 （SL2） 重塑，并投射到掩模 2 上。然后，生成的光谱图案通过4-f单元进行噪声抑制，并投影到EMCCD上进行记录。我们使用了商用显微镜主体（见 材料表）。一般来说，任何不同品牌的显微镜机身都可以使用，只要它有一个可用的端口来传递外部光束。台上的培养箱是一个金属室，具有温度、气体和气流控制功能。 样品的位置通过 2D电动载物台进行调整。互补金属氧化物半导体（CMOS）相机用于采集明场图像。
图 8 显示了具有代表性的鸡胚胎的延时明场和布里渊图像。胚胎在卵培养29 h后提取，在29 h、30.5 h和32 h用卵外培养物拍摄图像，分别对应体节数4、5和8，分别为44。通常，胚胎可以在载物台培养箱中培养至少24小时。明场图像中的红线表示布里渊成像的位置。通过采集的布里渊图像，选择神经板区域（由图 8 中的黑色虚线突出显示）并计算该区域的平均布里渊位移。如图 9 所示，在神经管闭合期间，组织的平均布里渊位移增加。纵向模量也是根据公式（1）计算的，其中= 1.3330的值<img alt="Equation 3" src="/files/ftp_upload/66117/66117eq03.jpg" style="margin: auto;">是根据使用斑马鱼胚胎45的文献估计的，假设该值对于相似种类的生物组织28,33相对恒定。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66117/66117fig01.jpg"> 图1：胚胎发育的整个培养皿配置示意图。该图左侧显示了用于连接胚胎的滤纸，右侧显示了一个 35 毫米的培养皿，该培养皿带有一个带有柔性薄膜层的环。还提供了横截面示意图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig01large.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/66117/66117fig02.jpg"> 图 2：在干净的 100 毫米培养皿上方打开蛋壳以将鸡蛋提取到培养皿中的过程。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig02large.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/66117/66117fig03.jpg"> 图 3：用稀蛋白填充培养皿的中心孔。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig03large.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/66117/66117fig04.jpg"> 图 4：使用一张薄纸沿红色箭头指示的方向拖走浓稠的蛋白。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66117/66117fig05.jpg"> 图 5：将滤纸放入培养皿中，胚胎位于滤纸的上方。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66117/66117fig06.jpg"> 图6：布里渊显微镜示意图。 红色箭头表示激光束的光路，橙色箭头表示布里渊信号的光路。L1、L2、SL1-SL4：球面透镜;SF： 空间滤波器;C1、C2：柱面透镜，HWP：半波片;PBS：偏振分束器;QWP1、QWP2：四分之一波板;OBJ1、OBJ2：物镜;LP：镜头对。VIPA：虚拟成像相控阵。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66117/66117fig07.jpg"> 图 7：代表性布里渊信号。 （A） EMCCD捕获的代表性布里渊光谱。（B） 布里渊谱的垂直求和和相应的洛伦兹拟合结果。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig07large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/66117/66117fig08v2.jpg"> 图 8：胚胎在 29 小时、30.5 小时和 32 小时的明场图像和相应的布里渊横截面图像，具有 4、5 和 8 个体节。 红色实线表示布里渊成像的位置，黑色虚线表示神经板区域。32 h（8 体节）图像中的灰色区域是由于布里渊信号较弱而导致的曲线拟合伪影，在计算平均偏移时被排除在外。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig08largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/66117/66117fig09.jpg"> 图 9：布里渊位移和神经板的估计纵向模量与体节数量和孵育时间的关系。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig09large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo at JoVE.com

作者聚焦：使用布里渊显微镜对活鸡胚胎的组织力学进行非接触式测量

该协议旨在纵向监测雏鸡胚胎神经形成过程中神经板组织的机械性能。它基于布里渊显微镜和载物台孵化系统的集成，能够对 卵 培养雏鸡胚胎中的神经板组织进行实时机械成像。

监测雏鸡胚胎神经管闭合过程中组织的机械进化

Neural tube closure (NTC) is a critical process during embryonic development. Failure in this process can lead to neural tube defects, causing congenital ...

这项研究的范围是开发一种可以测量活胚胎组织力学的新工具。使用这个工具，我们想了解当胚胎经历神经元管闭合时组织力学是如何进化的。这可以帮助我们了解胚胎发育中的机械规律。为了测量组织刚度，目前的方法，如原子力显微镜、纳米鉴定和微量移液器抽吸，需要物理接触样品并施加力使其变形。这对于原位测量活胚胎的组织力学极具挑战性。在这里，我们采用了一种名为布里渊显微镜的光学技术。与其他技术相比，布里渊显微镜仅使用聚焦光束来测量组织力学。因此，这是一种非接触式和非侵入性方法，并且具有一定的微空间分辨率。这使我们能够在原位捕获活胚胎的延时机械图像。未来，我们希望彻底了解组织力学在胚胎发育中的作用。具体来说，我们想研究遗传因素、生化信号通路和组织力学在形态发生中如何相互协调。这可以为我们提供提供出生疾病（如神经元管缺陷）的新见解。

monitoring-mechanical-evolution-tissue-during-neural-tube-closure

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo

620 次观看.  Wayne State University. 这项研究的范围是开发一种可以测量活胚胎组织力学的新工具。使用这个工具，我们想了解当胚胎经历神经元管闭合时组织力学是如何进化的。这可以帮助我们了解胚胎发育中的机械规律。为了测量组织刚度，目前的方法，如原子力显微镜、纳米鉴定和微量移液器抽吸，需要物理接触样品并施加力使其变形。这对于原位测量活胚胎的组织力学极具挑战性。在这里，我们?...

视频: 作者聚焦：使用布里渊显微镜对活鸡胚胎的组织力学进行非接触式测量

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

优化<em&gt;前OVO</em&gt;鸡胚培养，以发展的高级阶段

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

科研

发育生物学

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

配售早期鸡胚生长因子包被的磁珠

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

生物学

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

水下滤纸三明治长期<em&gt;防爆大毛</em&gt;早期鸡胚时间推移成像

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

在小鼠全胚胎培养的神经管封闭

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

神经科学

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

在鸡胚神经诱导的含量为

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

鸡颅神经嵴细胞培养物的制备及形态学分析

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

Embryonic development is traditionally studied from the perspective of biomolecular genetics, but the fundamental importance of mechanics in morphogenesis is becoming increasingly recognized. In particular, the embryonic chick heart and brain tube, which undergo drastic morphological changes as they develop, are among the prime candidates to study the role of physical forces in morphogenesis. Progressive ventral bending&#160;and rightward torsion of the tubular embryonic chick brain happen at the earliest stage of organ-level left-right asymmetry in chick embryonic development. The vitelline membrane (VM) constrains the dorsal side of the embryo and has been implicated in providing the force necessary to induce torsion of the developing brain. Here we present a combination of new ex-ovo experiments and physical modeling to identify the mechanics of brain torsion. At Hamburger-Hamilton stage 11, embryos are harvested and cultured ex ovo (in media). The VM is subsequently removed using a pulled capillary tube. By controlling the level of the fluid and subjecting the embryo to a fluid-air interface, the fluid surface tension of the media can be used to replace the mechanical role of the VM. Microsurgery experiments were also performed to alter the position of the heart to find the resultant change in the chirality of brain torsion. Results from this protocol illustrate the fundamental roles of mechanics in driving morphogenesis.

Here, we present a protocol introducing a set of new ex-ovo experiments and physical modeling approaches for studying the mechanics of morphogenesis during early chick embryonic brain torsion.

早期雏鸡胚胎形态发生中物理力的作用探讨

Embryonic development is traditionally studied from the perspective of biomolecular genetics, but the fundamental importance of mechanics in morphogenesis ...

生物工程

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo 

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...