Name: deep and spatially controlled volume ablations using a two-photon microscope in the zebrafish gastrula
Uploaded: 2021-07-15T15:00:00
Description: Watch this Scientific Journal Video about Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula at JoVE.com

我们感谢Emilie Menant的鱼类护理，Polytechnique Bioimaging Facility，特别是Pierre Mahou，感谢在法兰西岛地区（interDIM）和国家研究机构（ANR-11-EQPX-0029 Morphoscope2，ANR-10-INBS-04 France BioImaging）部分支持的设备上提供实时成像。这项工作得到了ANR拨款15-CE13-0016-1，18-CE13-0024，20-CE13-0016以及欧盟地平线2020研究和创新计划的支持，该计划根据Marie Skłodowska-Curie赠款协议No 840201，高级和研究部和国家科学研究中心。

所有动物工作均由伦理委员会N 59和国家教育部批准，文件编号为APAFIS#15859-2018051710341011v3。下面描述的一些步骤特定于我们的设备和软件，但可以很容易地适应不同的设备。
1. 注射准备
<ol><li>在胚胎培养基（EM）中制备75毫升1%琼脂糖溶液。</li>
	<li>将注射模具放入90毫米培养皿中，倒入约50毫升琼脂糖，足以让模具漂浮。让琼脂糖凝固并除去注射模具。</li>
	<li>通过将1 mL琼脂?...

<ol>
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P., Tada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine-1-phosphate+receptors+regulate+individual+cell+behaviours+underlying+the+directed+migration+of+prechordal+plate+progenitor+cells+during+zebrafish+gastrulation.">Sphingosine-1-phosphate receptors regulate individual cell behaviours underlying the directed migration of prechordal plate progenitor cells during zebrafish gastrulation.</a> Development. 135 (18), 3043-3051 (2008).</li><li>Smutny, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friction+forces+position+the+neural+anlage.">Friction forces position the neural anlage.</a> Nature Cell Biology. 19 (4), 306-317 (2017).</li><li>Johansson, M., Giger, F. 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C., Aigouy, B., Eaton, S., J&#252;licher, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+cell+mechanics,+cell-cell+interactions,+and+proliferation+on+epithelial+packing.">The influence of cell mechanics, cell-cell interactions, and proliferation on epithelial packing.</a> Current Biology. 17 (24), 2095-2104 (2007).</li><li>Willier, B. H., Oppenheimer, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Foundations of Experimental Embryology. , (1964).</li><li>Ashby, W. 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在这里，我们描述了一种协议，该协议使用非线性光学来执行深度和空间上定义良好的体积烧蚀。该协议最关键的一步是找到提供足够能量以允许消融的处理条件，但不能过多的能量，以避免过多的碎片或空化。目标部位传递的能量主要取决于：（1）激光退出功率，（2）激光对准的质量，（3）光通过到达烧蚀平面的组织的性质，（4）烧蚀平面的深度。因此，在每次实验之前，测量激光退出功率...

细胞 - 细胞相互作用在发育中起着至关重要的作用。细胞提供信号，它们的直接邻居或更远的细胞可以感知，从而影响它们的命运和/或行为。其中许多信号本质上是化学性质的。例如，在表征良好的诱导事件中，一个细胞群产生可扩散的分子，影响另一个细胞群的命运1。然而，其他信号是机械的。细胞对邻居施加力和约束，邻居感知并做出反应2。
研究这些细胞 - 细胞 相互作用在体内 的重要性的一种方法是消除一些细胞并观察随后的发展。不幸的是，去除或破坏细胞的可用技术是有限的。细胞可以通过手术切除3，4，使用针头或小线，但这种治疗是侵入性的，不是很精确，通常在立体显微镜下进行，阻止在显微镜下立即成像。此外，靶向深层细胞意味着在覆盖的组织中刺穿一个洞，从而产生不必要的扰动。基因编码的光敏剂，如KillerRed，已被用于通过光照诱导细胞死亡5。光敏剂是在光照射下产生活性氧的发色团。它们的主要局限性是它们需要长光照明（约15分钟），如果细胞移动，这可能很难实现，并且它们通过细胞凋亡诱导细胞死亡，这不是立即的。
最后，激光烧蚀在过去15年中得到了发展和广泛的应用6，7，8，9，10，11，12。激光束聚焦在目标细胞/组织上。它通过加热，光消融或血浆诱导的消融诱导其消融;所涉及的过程取决于功率密度和曝光时间13。大多数烧蚀方案使用紫外激光器来实现其高能量。然而，紫外线既被生物组织吸收又被散射。因此，靶向深层细胞需要高激光功率，然后在更浅表的，平面外的组织中诱导损伤。这限制了紫外激光器在浅表结构上的使用，并解释了它们相对较低的轴向分辨率。非线性光学（所谓的双光子显微镜）使用光的非线性特性来激发荧光团，荧光团在红外域中具有大约一半能量的两个光子。当应用于消融术时，这有三个主要优点。首先，与紫外光相比，红外光的散射更少，被生物组织吸收更少14，从而可以在不增加所需激光功率的情况下到达更深的结构。其次，使用飞秒脉冲激光器可提供非常高的功率密度，通过等离子体感应产生烧蚀，这与加热相反，不会在空间上扩散15。第三，诱导等离子体形成的功率密度仅在焦点处达到。由于这些特性，双光子激光烧蚀可用于精确靶向深层细胞，而不会影响周围的组织环境。
集体迁移是发育过程的一个很好的例子，其中细胞 - 细胞相互作用是根本的。集体迁移被定义为相邻细胞影响一个细胞行为的细胞迁移16。这些相互作用的性质（化学或机械）以及它们如何影响细胞迁移可能会有很大差异，并且通常不完全理解。移除细胞并观察其如何影响其他细胞的能力对于进一步解开这些集体过程至关重要。几年前，我们使用手术方法确定，斑马鱼原肠胚形成过程中的洄游是集体迁徙17。Polster是一组细胞，构成胚胎背侧的第一个内化细胞18。这些细胞在 Tg（gsc：GFP） 转基因系中以绿色标记，位于胚胎深处，在几层外胚层细胞的下方。在原肠胚形成过程中，该组导致轴向中胚层的延伸，从胚胎组织者迁移到动物极19，20，21，22，23 （图1A）。我们确定细胞需要与邻居接触，以将其迁徙定位在动物极点的方向上。然而，更好地了解这种集体迁移的细胞和分子基础涉及去除一些细胞，看看这如何影响其余的细胞。因此，我们使用双光子显微镜设置开发了大而深体积的烧蚀。在这里，我们演示了使用该协议来切断中间的polster，并通过跟踪用Histone2B-mCherry标记的细胞核来观察对细胞迁移的影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>25x water immersion objective</td>
			<td>Olympus</td>
			<td>XLPLN25XWMP2</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>PanReac AppliChem</td>
			<td>A8963,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Data analysis software : Matlab</td>
			<td>Math Works</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electro-optic modulator (EOM)</td>
			<td>ConOptics</td>
			<td>350-80LA</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo Medium (EM) solution</td>
			<td></td>
			<td></td>
			<td>Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 5th Edition. University of Oregon Press, Eugene (Book). (2000).</td>
		</tr>
		<tr>
			<td>Environmental chamber chamber</td>
			<td>Okolab</td>
			<td>H201-T-UNIT-BL</td>
			<td></td>
		</tr>
		<tr>
			<td>EOM driver</td>
			<td>ConOptics</td>
			<td>302RM</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence source</td>
			<td>Lumencor</td>
			<td>SOLA</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-0-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>300085</td>
			<td>Outside diameter 1.0 mm, inside diameter 0.58&#160;mm</td>
		</tr>
		<tr>
			<td>Glass pipettes</td>
			<td>Volac</td>
			<td>D810</td>
			<td>Tip should be fire polished</td>
		</tr>
		<tr>
			<td>Green/ablation laser</td>
			<td>Spectra Physics</td>
			<td>Mai Tai HP DeepSee</td>
			<td></td>
		</tr>
		<tr>
			<td>Histone2B-mCherry mRNA</td>
			<td></td>
			<td></td>
			<td>Synthesized from pCS2-H2B-mCherry plasmid (Dumortier&#38; al. 2012)</td>
		</tr>
		<tr>
			<td>Image analysis software: IMARIS</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImSpector software</td>
			<td>Abberior Instruments Development Team</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Injection mold</td>
			<td>Adapative Science Tools</td>
			<td>I-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>MN-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE SP6 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermofisher</td>
			<td>15140-122</td>
			<td>10 000 units penicillin and 10 mgstreptomycin per ml</td>
		</tr>
		<tr>
			<td>Photomultiplier tube (PMT)</td>
			<td>Hammamatsu</td>
			<td>H7422-40</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoPump (Air injector)</td>
			<td>World Precision Instrument</td>
			<td>PV820</td>
			<td></td>
		</tr>
		<tr>
			<td>Red laser</td>
			<td>Spectra Physics</td>
			<td>OPO/Insight DeepSee</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse free water for injection</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet software: Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ18</td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(gsc:GFP) zebrafish line</td>
			<td></td>
			<td></td>
			<td>Doitsidou, M. et al. Guidance of primordial germ cell migration by the chemokine SDF-1. Cell. 111 (5), 647&#8211;59, doi: doi.org/10.1016/S0092-8674(02)01135-2 (2002).</td>
		</tr>
		<tr>
			<td>TriM Scope II microscope</td>
			<td>La Vision Biotech</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

deep and spatially controlled volume ablations using a two-photon microscope in the zebrafish gastrula

形态发生涉及许多细胞运动，以将细胞组织成组织和器官。为了适当的发展，所有这些运动都需要紧密协调，积累的证据表明，这至少部分是通过机械相互作用实现的。在胚胎中测试这一点需要直接的身体扰动。激光烧蚀是一种日益常用的选择，可以缓解机械约束或物理隔离两个细胞群。然而，许多烧蚀都是用紫外线（UV）激光进行的，其提供的轴向分辨率和组织穿透有限。这里描述了一种使用双光子显微镜消融深度，显着和空间上明确定义的体积的方法。消融在表达轴向中端肠胚层中绿色荧光蛋白的转基因斑马鱼品系中表现出来，并用于切断轴向中胚层，而不影响上覆的外胚层或下面的卵黄细胞。在消融之前和之后通过实时成像监测细胞行为。消融方案可用于不同的发育阶段，任何细胞类型或组织，规模从几微米到超过一百微米不等。

为了切断中间的污染物，注射了Histone2B-mCherry mRNA的 Tg（gsc：GFP） 胚胎在70%的表观阶段安装，如步骤4所述。通过GFP表达鉴定出极点，并且将胚胎安装，以使极点的平面垂直于光轴（图1B）。将胚胎倾斜远离这个位置将使程序复杂化。光必须穿过更多的组织才能到达消融平面，并且消融平面将相对于胚胎轴倾斜。在验证了所有细胞核都正确标记后，记录了10分钟的消融前...

Watch this Scientific Journal Video about Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula at JoVE.com

Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula

胚胎发育需要细胞运动的大规模协调。双光子激发介导的激光烧蚀允许对大群深层细胞进行空间控制的三维烧蚀。此外，该技术可以探测 体内 集体迁移细胞对其机械环境中的扰动的反应。

在斑马鱼胃中使用双光子显微镜进行深度和空间控制的体积消融

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly ...

在胚胎中，细胞相互作用，交换化学和机械信息。探测这些相互作用的一种有效方法是杀死一些细胞并监测对邻近细胞的影响。我们描述的方法使我们能够准确地消融细胞，即使在胚胎内，也不会损坏邻近组织。通过将第一激光设置为烧蚀波长为820纳米，并将第二激光设置为mCherry成像波长1，160纳米来准备双光子显微镜。使用光路上的可移动反射镜，将两束激光束对准扫描头的入口和出口，然后测量第一个激光器在820纳米处的最大功率。将功率计放在物镜下方，关闭黑室，并将第一激光功率设置为100%开始在功率计上收集数据，打开激光快门，然后测量输出功率并计算达到300毫瓦所需的激光功率百分比。将第一个激光器设置为 920 纳米的 GFP 激发波长和 7% 将第二个激光器的功率调整为 15% 激活 PMT 探测器的绿线和红线，并将绿线和红线 PMT 灵敏度设置为 65。将视场调整为 400 x 400 微米，将图像分辨率调整为 512 x 512 像素，并将扫描频率调整为 800 赫兹。选择 3D 延时成像模式，然后创建一个文件夹并激活自动保存以在每次采集后保存数据。组装加热室并将其设置为28摄氏度。等待至少10分钟，使腔室和目标变暖。在荧光立体显微镜下，以70%的密度鉴定表达GFP的胚胎。使用塑料巴斯德移液器，将三到四个选定的胚胎转移到琼脂糖包被的培养皿中，并用细镊子小心地将它们去脉。在一个小玻璃小瓶中，加入一毫升含有2%琼脂糖的青霉素 - 链霉素胚胎培养基溶液，并将小瓶置于预热的42摄氏度的干燥块加热器中。使用火抛光玻璃移液器，将去壳的胚转移到小瓶中，注意不要向琼脂糖中添加过多的胚培养基。从移液器中丢弃剩余的胚胎培养基。将胚胎吸回，并吸出足够的琼脂糖以覆盖玻璃底皿的载玻片。将琼脂糖与胚胎一起吹在培养皿的载玻片上，确保胚胎不会接触空气或培养皿的塑料侧。然后，用琼脂糖填充载玻片周围的腔室。使用睫毛，定向胚胎，使目标区域位于顶部。五分钟后，当琼脂糖完全凝固时，向载玻片中加入几滴青霉素 - 链霉素胚胎培养基。将玻璃底盘放在加热室中物镜下方。在关闭腔室之前，将物镜浸入青霉素 - 链霉素胚胎培养基中。移动滑块以设置目镜的光路。打开荧光灯。找到一个胚胎，并将焦点放在它的表面。关闭荧光灯并将光路设置为PMT，然后再关闭黑室。开始实时成像并定位轴向中胚层。要获得良好的信号，请调整激光功率。使用红色通道将阶段移动到胚胎的最顶部，并将此位置指定为Z等于零。选择一分钟的时间步长和两微米的 Z 步长。将第一个切片设置在轴向中胚层上方 15 微米处，将最后一个切片设置在轴向中胚层下方 15 微米处。录制 10 到 15 分钟的消融前电影。在实时成像中，找到光栅轮廓，然后使用感兴趣的电光调制器区域或EOM-ROI工具，绘制一个跨越光栅宽度的20像素大矩形，并将矩形放在极格的中间。注意要烧蚀的最高和最低平面的轴向位置，确保ROI不会与任何平面上的蛋黄池重叠。将载物台放置在要消融的最低 Z 位置。将第一个激光波长设置为820纳米，并输入前面计算的功率百分比，以获得300毫瓦的退出功率。将成像频率设置为 200 赫兹，并将第一个激光成像 EOM 设置为零。选择 ROI 处理模式后，关闭 EOM，并将处理设置为在零帧后立即开始。将成像模式设置为延时摄影并停用自动保存。在"时间步长"中选择"快速模式"后，将处理帧数和帧数调整为与目标深度相对应的值。开始成像。采集应为黑色，因为在 EOM 处理期间 PMT 的快门关闭。接下来，将舞台上移到下一个Z位置，并同样执行消融。在每个 Z 位置重复此步骤，直到到达 polster 的顶部。烧蚀过程完成后，将第一激光器设置为920纳米，并调整到先前选择的成像功率。将第一个激光成像EOM设置为100，然后选择全场模式。成像频率应为800赫兹，在实时模式下检查整个堆栈之前，应关闭EOM，以检查每个平面是否已被烧蚀。确认烧蚀后，选择3D延时作为成像模式。重新激活自动保存，并开始录制消融后短片 40 到 60 分钟。在消融后电影中，检查靶细胞是否有效消融。由于整个桫椤的消融不应损害胚胎，因此可以在受精后24小时内观察到消融胚胎的正常形态。在激光消融的成功结果中，覆盖的组织不受底层结构消融的影响。太密集的治疗，或太少的治疗，导致激光消融失败。不成功消融的例子显示，桔体上方的细胞被消融，这从极光体上方焦平面的自发荧光碎片中可见一斑。在另一个无效的尝试中，细胞被漂白但未被消融，低荧光信号仍然显示完整的细胞轮廓。最后，由于激光处理不足，一些细胞甚至没有漂白。气泡的形成是由于过于强烈的激光处理引起的空化。在成功的消融中，每分钟捕获Z-堆栈40分钟，记录细胞质GFP和核mCherry信号。此外，属于栉水星前半部分的细胞核用洋红色点标记，并随着时间的推移，在消融之前和之后进行跟踪。作为迁移持久性的衡量标准，研究了消融前后细胞的方向自相关。属于桔梗前部的细胞在消融前表现出持续的运动，在消融后急剧减少，表明集体定向迁移的丧失。正确对准激光器和测量激光功率对于获得可重复的结果至关重要。检查第一张消融后图像上的消融效率也是关键。我们使用激光消融来质疑细胞 - 细胞相互作用在指导胚胎内细胞迁移中的作用。但该程序可以很容易地适应许多其他问题，其中细胞或组织需要精确消融，可能在生物体深处。

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

Microbead Implantation in the Zebrafish Embryo

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

采用触摸诱发反应和运动测定以评估在斑马鱼肌肉性能和功能

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

使用"彩虹"和"延时共聚焦成像"可视化活斑马鱼中发育中的脑

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...