P.E. is supported by the Deutsche Forschungsgemeinschaft (DFG) Research Training Group 1373 and the Graduate School of the Technische Universit&#228;t M&#252;nchen (TUM-GS). G.P. was supported by TUM-GS. L.T. is supported by an EMBO fellowship (EMBO ALTF 108-2013). D.P.'s work on zebrafish was supported by the DFG through the Sonderforschungsbereich "Molecular Mechanisms of Neurodegeneration" (SFB 596); the Center for Integrated Protein Sciences (Munich) and the European Community's Seventh Framework Programme (FP7/2007-2013) under Grant agreement no. 200611 (MEMOSAD). He is currently a New York Stem Cell Foundation-Druckenmiller Fellow and was supported by a fellowship from the German Academy of Sciences Leopoldina. L.G. is supported by funding from the DFG through SFB 870 "Assembly and Function of Neuronal Circuits", Project A11. 
We are grateful to Kristina Wullimann for maintaining our fish facility, Yvonne Hufnagel for technical support and Thomas Misgeld for comments on the manuscript. We are grateful to R. K&#246;ster (Technische Universit&#228;t Braunschweig) for providing the M1 Medusa vector (pSKmemmRFP:5xUAS:H2B-CFP:5xUAS:Centrin2-YFP) from which we cloned out Centrin-YFP and S.C. Suzuki and T. Yoshimatsu (University of Washington) for providing the 14xUAS:MA-cerulean cassette which we used to generate the reporter construct to make CentrinFish. We further thank S.C. Suzuki and T. Yoshimatsu (University of Washington) for the Otx2:Gal4 transgenic line, A. Sagasti for the Sensory:Gal4-VP16 construct (UCLA) and M. Nonet (Washington University in St. Louis) for the pCold Heart Tol2 vector. We acknowledge Bettina Schmid, Alexander Hruscha and Christian Haass (German Center for Neurodegenerative Diseases Munich - DZNE) for contributing to the development of MitoFish.

The authors have nothing to disclose.

在这里，我们证明了GAL4-UAS表达系统的通用性，以荧光标记的线粒体，中心体和体内在斑马鱼胚胎特定细胞类型的细胞膜。该标签其他细胞器或亚细胞结构的许多荧光融合蛋白可以在出版的文献中找到，并且可以从相应的实验室，商业来源或非商业质粒保藏（ 例如，Addgene）获得。设计一个新的荧光融合蛋白，几个参数需要考虑，包括FP使用哪个以及是否在感兴趣蛋白质的氨基或羧基末端融合在FP。有关生成荧光融合蛋白进行更彻底的讨论，读者对上述49-51提到有深度的文章。 我们强调三种策略实现使用UAS驱动转基因融合蛋白的共表达。多，分隔符ËUAS记者结构允许组合不同现有报道构建，而不需要额外的克隆。记者的表达水平也可以独立滴定。然而，当使用任一倍数的UAS盒或在单个构建体的双向UAS盒得以实现共表达的水平较高。自的FP被用作标记相对容易验证共表达。应当注意的是，其他方法用于多顺反子表达也是存在的，包括使用内部核糖体进入位点40和病毒2A肽52,53。作为替代基于DNA构建体， 在体外转录加帽的mRNA可以用于表达荧光融合蛋白来标记亚细胞结构1,4,8。这种方法的需要注意的是但是该表达仅限于发展的最初几天，而不是细胞或组织特异性。不管选择了表达的融合蛋白的方法的，重要的是以确保表达水平不会导致非特异性标记和破坏细胞的生理状态。在这方面，在GAL4-UAS系统27固有报告基因表达的扩增可以是有问题的，表现为例如如即使当FP被定位到特定的细胞器胞质标记。当可用时，抗体应当用来验证由融合蛋白获得的表达模式反映了内源的情况。在一些情况下，低（更低）的DNA浓度的注入可以减轻融合蛋白的误定位的问题。可替代地，具有低数目的UAS重复载体可有助于滴定向下转基因表达水平的30。同样，对于活化剂的Gal4-VP16，修改后的版本存在，它被报告给驱动表达比较弱，因此毒性更小30,33。如果融合蛋白的误定位仍然存在尽管努力滴定陶氏Ñ​​转基因表达水平，这可能是必要的，以放弃的GAL4-UAS系统，并通过直接启动子元件驱动表达。 稳定的转基因品系，MitoFish 10和CentrinFish 12，我们描述在该原稿用14xUAS盒进行的。我们维持在Gal4驱动子行后台这些线以检测更改在表达几代中，由于具有多个UAS重复记者线已报告容易发生沉默54。我们还没有看到沉默比3我们世世代代传播的CentrinFish的证据。然而，我们已经看到杂色表达MitoFish，因此严格选择具有表达的&#39;全&#39;，强烈的层次胚胎后代传播。目前的文献表明，与5xUAS重复表达载体可以产生稳定的转基因株系替代 - 记者是带动足够高的水平30和UAS的相对低一些重复可能使其不容易转基因沉默，但不重复的重复UAS据报道，即使是不太敏感54。 FP的目前可用的标记细胞器或其他亚细胞结构的调色板是非常广泛的55。当选择一个特定的FP作为标记，应考虑以下参数:第一，在FP的激发和发射光谱，以确定特定的激光线，以及作为其可视所需要的激发和发射滤波器。第二，亮度和在FP的光稳定性。第三，速度与在FP成熟以下翻译。第四，无论是在FP具有在融合物聚集的倾向。关于最后一个参数，应谨慎和控制实验，应该做测试每个FP的适用于特定的实验。例如，当重ð的FP mCherry和DsRed的被广泛使用，据说它们形成一定的融合体56聚集体。我们已经发现TagRFP-T 57，TagRFP更光稳定变型中，当靶向线粒体和细胞膜（未发表的观测）表现良好。当多个细胞器需要被伴随地可视化，FP的非重叠光谱应该被使用。我们已经成功地使用青 ​​色FPS（CFP和蔚蓝）和YFP（ 图 2-4）的组合。通过利用从蓝色到近红外的整个光谱范围，可以极大地扩展了可同时可视化2亚细胞结构的数量。除了 ​​常规的FP，光活化的FP是特别有用的探测细胞器动力学， 例如，使用在FP枫的跟踪单个线粒体58的命运。 其中显微技术 - 宽视场或共聚焦 - 最适当成像取决于许多因素，包括细胞的位置进行成像，并且与特定的亚细胞或细胞事件预期发生的速度。宽视野显微镜是肤浅的地点和人烟稀少标记的样品的首选方式。它提供了在高速低光毒性和图像以合理的价格。然而，如果成像需要在斑马鱼的非浅表部位要执行，密集的标记的样品中或获得三维信息，共焦或者光学切片显微镜的另一种形式成为选择的方法。 不论其是被监测细胞或亚结构，经常会有获得最佳的图像和保持样本活着并且在重复时间推移成像良好的生理状态之间的权衡。光毒性可以表现为在细胞器的行为或变化，细胞的异常形态学甚至细胞死亡。键减少光漂白和光毒性为宽视场和共聚焦显微镜是使用光的尽可能低的水平，以获得图像。在这方面，具有最高可能的NA目标的关键是最大限度地提高可收集的荧光信号的量。对于共焦显微镜，一些参数可以调整，以补偿使用较低的激光功率。具有较高的量子效率检测器相对于传统的光电倍增管， 例如，磷砷化镓探测器，可用于确保发射的光子更容易被检测到。或者或另外，更高倍增极电压可以在PMT被应用于提高检测器的灵敏度。共焦孔（针孔）可以被打开以允许更多的荧光信号的采集，尽管轴向分辨率的损失。以暴露样品到尽可能少的光成为可能，扫描应该以高速进行，使得的停留时间每个象素的激光是低的。扫描以较低的空间分辨率也具有增加扫描的速度的效果。成像较少有样品暴露于较少的光的额外的好处，但是，如果它不危及调查过程的全面采样只应被考虑。 作为替代，旋转盘共聚焦显微镜和共焦显微镜，配备一个所谓的"共振"扫描仪提供快速扫描的能力。两种模式的优点在于它们是更快和更光毒性比"古典"，点扫描共聚焦显微镜。然而，旋转盘共焦显微镜在Z轴的分辨率较为有限，不能深深渗透到组织中的点扫描共焦显微镜。同样地，谐振扫描器共焦显微镜中的应用可以被限制为极短的像素停留时间会导致较差的图像质量可能，反过来，排除亚细胞事件（ 例如，二进制图像具有减少的信号-噪声）的检测。 最后，虽然宽视场和共焦成像在这里强调，其他形式的光学显微镜如双光子59和光片镜60的可能更适合于特定的问题。双光子显微镜是基于荧光团的两个光子的光谱的红外范围内的同时吸收的激发。在用共聚焦显微镜常见，它使用点扫描来获取从样本图像和已经凭借双光子激发的非线性的光学切片的能力。对于荧光激发使用长波长光的成像允许在深度从表面几百微米。此外激发到一个小的成像体积的限制可以防止光致漂白和光毒性焦平面之外。然而，并非所有的FP■找高多光子吸收横截面，一个问题，即在红色的FP特别普遍。此外，发现单一红外波长来同时激励多个不同的FP是困难的，使得多通道成像繁琐。光片显微镜采用激光光线的薄&#39;片&#39;（通常厚度为2至10微米），以激发样品，并在用一个灵敏的相机以垂直角度检测从这个照射焦平面荧光信号​​。光学切片是通过在一个时刻照亮单一平面来实现的。激发光的这种限制降低了光毒性的发生。由于从整个照亮平面荧光信号​​同时采集，图像采集比点扫描共聚焦和多光子显微镜显著更快。所有这些特点使光表镜特别适合于成像高时空分辨率的动态细胞活动大量的现场样品。事实上，在整个斑马鱼胚胎使用光片显微细胞的命运发展61的前24个小时的过程中进行了全面跟踪。随着持续改进，以更好的成像渗透62,63和空间分辨率64，可以想到的是光片镜将被用来探测动态不仅在细胞，而且在整个斑马鱼胚胎的亚细胞水平。

体内成像提供在最生理上下文细胞行为的直接可视化。斑马鱼的胚胎，其快速的外部发展和丰富的允许荧光标记的遗传工具阵列的透明度，都促使越来越多地使用在体内显微镜阐明关键发育事件的动态。在斑马鱼神经系统发育的成像研究，例如大大扩展了我们的神经祖细胞的行为和他们的后代，包括其后续迁移，分化和电路集成1-8命运的知识。 舞台现在设置调查亚细胞动力学这些细胞行为背后。事实上，斑马鱼已经被利用作为用于体内细胞生物学的工具。它现在是可能的可视化线粒体9-11，中心体2,8,12-14，高尔基15中，微管4和肌动蛋白16细胞骨架，内涵体17和顶膜的组件复杂1,18，在体内斑马鱼胚胎其他亚细胞结构中。到目前为止，大部分的所谓对这些细胞器的功能来自于培养细胞研究他们的行为。而体外研究已经取得了巨大的洞察细胞生物学，细胞培养并不完全代表的体内情况的复杂性和因此不一定反映功能和体内亚细胞器的动力学。斑马鱼的胚胎提供体内替代一个可行的检查亚动态。 作为脊椎动物，斑马鱼拥有的同源那些在哺乳动物中发现许多器官系统（ 例如，视网膜神经）。此外，斑马鱼的胚胎被越来越多地用于人类疾病19,20建模</s向上&gt;，包括与中心体功能（ 例如，小头畸形21和勒伯尔先天性黑蒙22）和线粒体功能（ 例如，帕金森氏病23，τ蛋白病10,24和巴特综合征25）， 在体内在细胞和亚细胞水平的成像在这些情况下，将允许更好地了解细胞生物学的这些病理状态的根本。 这里描述的方法的总的目标是提供一种全面的指导来调查在使用体内光学显微镜斑马鱼胚胎细胞器和其它亚细胞结构。 参与体内可视化和跟踪亚细胞结构，整个工作流程描述-遗传标记的方法，以产生瞬时表达和稳定的转基因鱼，最后采用宽视场和共聚焦显微镜成像。虽然这些PROC的edures用于由众多斑马鱼实验室，中所述的协议进行了优化和简化调查亚细胞结构的动态。此处所描述的工作的两个特定方面值得提及:首先，使用在多个配置的GAL4-UAS表达系统在特定细胞类型的基因标签的细胞器。第二，宽视场和共聚焦显微镜的体内直接比较对图像亚细胞结构。 目前的策略，以转基因标签的细胞器和其他亚细胞结构的斑马鱼要么利用皑皑的mRNA 1,4,8或基于DNA的结构，其中子元件直接驱动融合蛋白9,14,15。 体外转录加帽的RNA结果的表达快速和广泛的表达，这不是组织特异性但是。此外，表达水平降低随着时间的推移加帽的RNA稀释或退化。因此，使用RNA的基于构造来检查在发育后期阶段的细胞器动力学是有限的（通常为3天受精后）。 这些限制可通过使用DNA构建，其中表达的空间和时间控制由特定的启动子元件决定来克服。当基于DNA构建中，以转基因表达水平的GAL4-UAS系统显著改善的上下文被用于观察26,27。在此二分表达系统，细胞类型特异性启动子元件驱动的转录激活的Gal4的表达，而报道基因被克隆的Gal4的结合上游激活序列（UAS）的下游。由UAS记者用适当的Gal4的驱动相结合，表达可以被限制到特定的细胞类型，绕过需要每一个特定的表达模式所需时间来克隆的报告基因的不同启动子的后面。此外，多个UAS报告基因的表达可以是由一个单一的Gal4的激活驱动。与GAL4-UAS系统，从而为亚细胞标记的一种多功能灵活的遗传方式。 宽视场和共聚焦显微镜是大多数实验室的工作母机。宽视场系统通常使用弧光灯作为光源，并使用被放置在光路的端部的敏感照相机检测所发射的光。此成像模态典型地限制为薄的样品作为失焦光掩盖较厚样品在焦信息。共焦显微镜之处在于它们是建立有利于从焦平面比那些源于失焦（ 即 "光学切片"）28日发起信号，宽领域的系统有所不同。以实现光学切片的针孔被放置在发射路径中的共轭位置以所述点光源。激光器被用作光源和信号与光电倍增管（PMT）来检测。实际上，激光束在样品刷卡逐点和在各点（像素）的荧光发射由光电倍增管检测到。 在这里，我们的图像中同时使用宽视场和共聚焦显微镜以提供镜方式的直接比较活的斑马鱼胚胎非常相同的亚细胞结构。提供这种比较的基础的目的是提供选择适合手边的特定问题的最合适的显微技术的指导方针。 使用这里介绍我们证明线粒体和中心体的GAL4-UAS的遗传标记的方法。这些细胞器是使用宽视场和共聚焦显微镜来证明每个成像模态的适用性在不同细胞类型中的肌肉细胞的神经系统和成像。这里描述的方法可以容易地适于在活斑马鱼胚胎调查其他细胞器和亚细胞结构。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose (2-hydroxyethylagarose)</td>
			<td>Sigma-Aldrich</td>
			<td>A4018-10G</td>
			<td>Low-gelling temperature Type VII</td>
		</tr>
		<tr>
			<td>Block heater</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Thermomixer compact</td>
		</tr>
		<tr>
			<td>Ca(NO3)2 Calcium nitrate hydrate, 99.996%&#160;</td>
			<td>Aldrich</td>
			<td>202967-50g</td>
			<td>To prepare 30x Danieau&#39;s</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Qimaging</td>
			<td>Retiga Exi Fast 1394</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic Coated Dumont #5 Forceps</td>
			<td>Dumont - Fine Science Tools</td>
			<td>11252-50</td>
			<td>#5 Forceps</td>
		</tr>
		<tr>
			<td>Confocal laser-scanning microscope</td>
			<td>Olympus</td>
			<td>FV1000 Fluoview</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture dish heater</td>
			<td>Warner Instrument Corporation</td>
			<td>DH-35</td>
			<td>Heating ring</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfate salt</td>
			<td>Fluka Analytical</td>
			<td>A5040-100G</td>
			<td>Tricaine (anesthetic)</td>
		</tr>
		<tr>
			<td>Fluorescence dissecting microscope</td>
			<td>Leica</td>
			<td>M205 FA</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneClean kit</td>
			<td>MP Biomedicals</td>
			<td>111001200</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Culture Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-14-C</td>
			<td>35mm petri dish, 14mm microwell, No. 0 coverglass</td>
		</tr>
		<tr>
			<td>Glass needles</td>
			<td>World Precision Instruments Inc.&#160;</td>
			<td>TW100F-4</td>
			<td>For microinjections</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375-250g</td>
			<td>To prepare 30x Danieau&#39;s</td>
		</tr>
		<tr>
			<td>High vacuum grease</td>
			<td>Dow Corning&#160;</td>
			<td>DCC000001242 150g</td>
			<td>Silicon dioxide grease</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td>Heraeus</td>
			<td>To maintain zebrafish embryos at 28.5&#8304; C</td>
		</tr>
		<tr>
			<td>KCl 99%</td>
			<td>Sigma-Aldrich</td>
			<td>S7643-5kg</td>
			<td>To prepare 30x Danieau&#39;s</td>
		</tr>
		<tr>
			<td>MgSO4.7H2O &#160;&#160;Magnesium sulfate heptahydrate 98+% A.C.S reagent</td>
			<td>Sigma-Aldrich</td>
			<td>230291-500g</td>
			<td>To prepare 30x Danieau&#39;s</td>
		</tr>
		<tr>
			<td>Microinjector&#160;</td>
			<td>Eppendorf</td>
			<td></td>
			<td>FemtoJet</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td>0.5-20uL</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Maerzhaeuser Wetzlar</td>
			<td>MM33 Rechts/00-42-101-0000/M3301R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Intracel</td>
			<td>P/N 50-00XX-130-1</td>
			<td></td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE SP6 Transcription Kit</td>
			<td>Ambion</td>
			<td>AM1340</td>
			<td>To transcribe PCS-Transposase</td>
		</tr>
		<tr>
			<td>NaCl BioXtra &#62;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-1kg</td>
			<td>To prepare 30x Danieau&#39;s</td>
		</tr>
		<tr>
			<td>Nanophotometer</td>
			<td></td>
			<td></td>
			<td>To measure DNA/RNA concentration</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>Flaming/Brown</td>
		</tr>
		<tr>
			<td>NIR Apo 40x/0.80W&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td>Water-dipping-cone objective</td>
		</tr>
		<tr>
			<td>N-Phenylthiourea Grade I, approx. 98%</td>
			<td>Sigma</td>
			<td>P7629-10G</td>
			<td>PTU (prevents pigmentation)</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Sarstedt AG&#160;</td>
			<td>821472</td>
			<td>92 x 16mm&#160;</td>
		</tr>
		<tr>
			<td>Plastic molds&#160;</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td>For microinjections</td>
		</tr>
		<tr>
			<td>Plexiglas cover-with a hole</td>
			<td>Custom-made</td>
			<td></td>
			<td>The hole in the Plexiglas cover should be 3 mm larger than the diameter of the water-dipping-cone objective</td>
		</tr>
		<tr>
			<td>Tea-strainer (Plastic)</td>
			<td></td>
			<td></td>
			<td>To collect zebrafish eggs</td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Warner Instrument Corporation</td>
			<td>TC-344B</td>
			<td>Dual Automatic Temperature Controller</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Sarstedt AG&#160;</td>
			<td>86.1171</td>
			<td>3.5mL plastic transfer pipettes</td>
		</tr>
		<tr>
			<td>UMPlanFI 100x/1.00W</td>
			<td>Olympus</td>
			<td></td>
			<td>Water-dipping-cone objective</td>
		</tr>
		<tr>
			<td>UMPlanFLN 20x/0.50W&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Water-dipping-cone objective</td>
		</tr>
		<tr>
			<td>Widefield microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>PTU (50x Stock)</td>
			<td></td>
			<td></td>
			<td>Dissolve 76 mg PTU in 50 ml distilled water 
			Stir vigorously at room temperature&#160; 
			Store at -20 oC in 1 ml aliquots 
			Use at 1x working solution</td>
		</tr>
		<tr>
			<td>Tricaine (20x Stock)</td>
			<td></td>
			<td></td>
			<td>Dissolve 200 mg Tricaine in 48 ml distilled water 
			Add 2 ml 1M Tris base (pH9) 
			Adjust to pH 7&#160; 
			Store at -20 oC in 1 ml aliquots 
			Use at 1x working solution</td>
		</tr>
		<tr>
			<td>Danieau&#39;s Solution (30x Stock)</td>
			<td></td>
			<td></td>
			<td>1740 mM&#160; NaCl&#160; 
			&#60;21 mM&#160;&#160;&#160;&#160;&#160; KCl&#160; 
			12 mM&#160; &#160;&#160;&#160;&#160;MgSO4.7H2O&#160; 
			18 mM&#160;&#160;&#160;&#160;&#160; Ca (NO3)2 
			150 mM&#160;&#160;&#160; HEPES buffer&#160; 
			Distilled water upto 1 L&#160; 
			Store at 4 oC&#160; 
			Use at 0.3x working solution</td>
		</tr>
	</tbody>
</table>

imaging subcellular structures in the living zebrafish embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

这里使用宽视场和共聚焦显微镜图像线粒体和在活斑马鱼胚胎中心体的直接比较和对比。这取决于细胞器动力学是细胞的位置要被检查和特定的亚细胞事件的固有频率，通常无论是宽视场或共聚焦显微镜是更好的选择。我们在位于胚胎的表面上的RB的神经元和在位于更深的视网膜细胞成像的细胞器。 RB神经元与其二维几何在一起的表观位置使它们的良好候选用于通过两个宽视场和共聚焦显微镜（ 图 2）被成像。使用共聚焦显微镜获取图像然而显著较慢，并可能导致特定的亚细胞事件（ 如，线粒体的运动， 图2C，D的动力学的低估</strong&gt;的）。通过宽视场显微镜获取的视网膜细胞的图像从对比度差遭受从大体积的组织的荧光信号在焦平面掩盖细节。这里，在显着改善的对比度共焦显微镜的结果光学切片能力（ 图3）。 以使细胞器和细胞膜的并发可视我们使用了GAL4-UAS系统在三种不同的配置。首先，UAS MitoFish记者行的Tg（UAS-E1B:mYFP，mitoCFP）用于mde6。在这里，双向UAS允许线粒体靶向CFP和膜针对性YFP伴随表达。在用适当的Gal4驱动子行组合，MitoFish（ 图3A，B）和CFP和YFPř标记的RB的感觉神经元的线粒体和细胞膜（ 图 2A-C）或视网膜细胞<html> espectively。第二，两个UA构造（UAS:mitoCFP和UAS:MA-YFP）用Gal4驱动子构建体合并（感官:GAL4-VP16，从胰岛-1基因的感觉神经元特异性增强子元件）在短暂注射7。镶嵌表达式通常导致从这些注射允许各个单元的跟踪过天（ 图2E）。第三种方法使用两种UAS盒的使用中，每个驱动不同的融合蛋白的表达，在一个单一的连续结构。这种策略用于生成CentrinFish的Tg（UAS:cetn4-YFP，UAS:MA-蔚蓝）tum1，其中centrin4-YFP融合标签中心体和蔚蓝定位到细胞膜。结合特定Gal4驱动子线组合的中心体和视网膜细胞（ 图 3C，D）或肌纤维（ 图4）的细胞膜标记。 ENT"FO: - together.within页保留="1"&gt; <img alt="图2" src="/files/ftp_upload/53456/53456fig2.jpg" /> 图2: 在胚胎斑马鱼的RB感觉神经元 成像线粒体 体内 。 在2翅片折叠的尾部的RB的感觉神经元的dpf MitoFish用一个复消色差透镜40倍的水浸渍锥目标具有0.80的NA成像，或者通过宽视场（A）或共焦（B）的显微镜。面板的区域概述。C的宽视场时间推移在RB神经元的2外围乔木DPF MitoFish感兴趣的小区域的图像的右侧显示详细信息。单个线粒体（0箭头&#39;&#39;）这一运动的跟踪超过100秒;显示六个时间点。在前面的时间点的线粒体的位置在洋红色概述d。在C中的移动线粒体的位置</strong&gt;的标绘在以1赫兹（淡粉红色）的采样频率的100秒的时间窗。非常相同的线粒体的位置被加0.05赫兹的一个人工向下抽样的时间分辨率，以模仿共聚焦显微镜（品红色）的典型采集速度作图。品红kymograph不会在2和3旦揭示跟踪线粒体的细的非定向运动，这将导致其动力学的低估。的RB感觉神经电子共聚焦图像。个别的RB细胞及其线粒体标记物所达到共注射感觉神经元特异性Gal4驱动子具有两个UAS报道构建，UAS一起构造:mitoCFP和UAS:MA-YFP，在单细胞阶段和筛选分离的双标记的细胞。图像对比度反转和个别线粒体示意性地描绘为品红色点。比例尺A，B 20微米，C 2微米，Ë50微米。靶向线粒体CFP（mitoCFP），膜有针对性的YFP（MA-YFP）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53456/53456fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53456/53456fig3.jpg" /> 图3:宽视场和共聚焦显微镜的 胚胎斑马鱼视网膜 对比图像细胞器 体内 。 OTX2启动子元件驱动在细胞膜（A和B）或YFP中在2视网膜的dpf斑马鱼的胚胎细胞膜（C和D）中心体和蔚蓝线粒体和YFP CFP的表达。为了实现这个标签模式，OTX2:Gal4的转基因鱼要么越过MitoFish（A和B）O- [R CentrinFish（C和D）。 40倍的水浸泡锥复消色差透镜的目标（NA 0.80）用于获取使用宽视场（A，C）和共聚焦（B，D）显微镜在每个视网膜上的同一区域的图像。 所述的插图和内核层的区域的B示出详细地，在C和D的插图显示在M相的细胞。比例尺为20μm。靶向线粒体CFP（mitoCFP），膜有针对性的YFP（MA-YFP），centrin4-YFP（cetn4-YFP），膜有针对性的蔚蓝（MA-蔚蓝）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53456/53456fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53456/53456fig4.jpg" /> 图4:宽视场和共聚焦microsc的比较OPY 体内图像中心体。 采用宽视场（A）和共聚焦（B）显微镜成像;:用荧光单肌纤维标记的细胞膜和中心体（CentrinFish Gal4的OTX2）。在这两种情况下使用了40倍的水浸泡锥复消色差透镜的目标（NA 0.80）。比例尺为10μm。 Centrin4-YFP（cetn4-YFP），膜有针对性的蔚蓝（MA-蔚蓝） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53456/53456fig4large.jpg" target="_blank">点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Imaging Subcellular Structures in the Living Zebrafish Embryo at JoVE.com

Imaging Subcellular Structures in the Living Zebrafish Embryo

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

imaging-subcellular-structures-in-the-living-zebrafish-embryo

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.5K 次观看.  Technische Universität München. Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy. 

视频: Imaging Subcellular Structures in the Living Zebrafish Embryo

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

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

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

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

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

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 conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform 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

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

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

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model 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 ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

观察有丝分裂和动力学在现场斑马鱼胚胎

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

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 zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

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 ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

利用 Immunolabeling 分析斑马鱼胚胎中的稳定、动态和初生的微管

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...