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?...

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><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(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt; Calcium nitrate hydrate, 99.996%&amp;nbsp;</td><td>Aldrich</td><td>202967-50g</td><td>To prepare 30x Danieau&#039;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>PharmaQ</td><td>Tricaine PharmaQ-25g</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>35 mm petri dish, 14 mm microwell, No. 0 coverglass</td></tr><tr><td>Glass needles</td><td>World Precision Instruments Inc.&amp;nbsp;</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&#039;s</td></tr><tr><td>High vacuum grease</td><td>Dow Corning&amp;nbsp;</td><td>DCC000001242 150g</td><td>Silicon dioxide grease</td></tr><tr><td>KCl 99%</td><td>Sigma-Aldrich</td><td>S7643-5kg</td><td>To prepare 30x Danieau&#039;s</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O &amp;nbsp;&amp;nbsp;Magnesium sulfate heptahydrate 98+% A.C.S reagent</td><td>Sigma-Aldrich</td><td>230291-500g</td><td>To prepare 30x Danieau&#039;s</td></tr><tr><td>Microinjector&amp;nbsp;</td><td>Eppendorf</td><td></td><td>FemtoJet</td></tr><tr><td>Microloader tips</td><td>Eppendorf</td><td>930001007</td><td>0.5-20 ul</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 &gt;99.5%</td><td>Sigma-Aldrich</td><td>P9541-1kg</td><td>To prepare 30x Danieau&#039;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&amp;nbsp;</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&amp;nbsp;</td><td>821472</td><td>92 x 16mm&amp;nbsp;</td></tr><tr><td>Plastic molds&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&lt;br/&gt; Stir vigorously at room temperature&amp;nbsp;&lt;br/&gt; Store at -20 &lt;sup&gt;o&lt;/sup&gt;C in 1 ml aliquots&lt;br/&gt; 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&lt;br/&gt; Add 2 ml 1M Tris base (pH9)&lt;br/&gt; Adjust to pH 7&amp;nbsp;&lt;br/&gt; Store at -20 &lt;sup&gt;o&lt;/sup&gt;C in 1 ml aliquots&lt;br/&gt; Use at 1x working solution</td></tr><tr><td>Danieau&#039;s Solution (30x Stock)</td><td></td><td></td><td>1,740 mM&amp;nbsp; NaCl&amp;nbsp;&lt;br/&gt; 21 mM&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; KCl&amp;nbsp;&lt;br/&gt; 12 mM&amp;nbsp; &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;&lt;br/&gt; 18 mM&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Ca (NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 150 mM&amp;nbsp;&amp;nbsp;&amp;nbsp; HEPES buffer&amp;nbsp;&lt;br/&gt; Distilled water upto 1 L&amp;nbsp;&lt;br/&gt; Store at 4 &lt;sup&gt;o&lt;/sup&gt;C&amp;nbsp;&lt;br/&gt; 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?...

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

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

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

在斑马鱼后脑细胞标记和成像

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

科研

神经科学

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

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

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 ...

发育生物学

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 ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

生物学

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

<meta charset="utf8"></head><body>斑马鱼胚胎的精细多视图光片显微镜实验方案

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

<meta charset="utf8"></head><body>作者聚焦:用于高分辨率多视图光片显微镜的斑马鱼胚胎安装策略 — 成像和图像重建技术

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

早期斑马鱼胚胎的光片显微镜成像和安装策略

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

成像和胚胎斑马鱼脑血管结构的三维重建

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

在活斑马鱼胚胎成像的亚细胞结构

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在斑马鱼后脑细胞标记和成像

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

观察有丝分裂和动力学在现场斑马鱼胚胎

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

现场共聚焦显微镜对斑马鱼的胚胎脑成像

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

早期斑马鱼胚胎的光片显微镜成像和安装策略

早期斑马鱼胚胎的光片显微镜成像和安装策略

早期斑马鱼胚胎的光片显微镜成像和安装策略

成像和胚胎斑马鱼脑血管结构的三维重建