Name: two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos
Uploaded: 2009-02-16T00:00:00
Description: Watch this Scientific Journal Video about Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos at JoVE.com

我们感谢马克寺崎初步意见，对利用双光子显微镜，创造局部组织损伤，凯西茹班安装时间推移成像的意见，并讨论Sagasti和Portera - Cailliau实验室。最初的实验中，只要在中，马伍兹霍尔海洋生物实验室的草基金会研究员。在Sagasti实验室的工作是支持由白厅基金会，该Klingenstein基金会和巴勒斯惠康生物科学基金事业奖补助。

第1部分：安装斑马鱼的胚胎长期成像<ol><li>准备嵌入1％低熔点琼脂糖溶液。离子水在微波炉，分装成小管和储存在摄氏42度加热块加热溶解琼脂糖。 </li><li>选择成像的胚胎，并用镊子轻轻拉出绒毛膜除了删除chorions。胚胎可以被放置在受精后22-24小时（HPF），抑制色素形成到5％的机动部队铃声的解决方案。这提高了成像的清晰度和表演的感觉神经轴突的双光子axotomies至关重要。如果允许形成的?...

<ol>
	<li>Sagasti, A., Guido, M. R., Raible, D. W., Schier, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repulsive+interactions+shape+the+morphologies+and+functional+arrangement+of+zebrafish+peripheral+sensory+arbors.">Repulsive interactions shape the morphologies and functional arrangement of zebrafish peripheral sensory arbors.</a> Curr Biol. 15, 804-814 (2005).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomed Eng Online. 2 (13), (2003).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li><li>Sacconi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+multiphoton+nanosurgery+on+cortical+neurons.">In vivo multiphoton nanosurgery on cortical neurons.</a> J Biomed Opt. 12, 050502-050502 (2007).</li><li>Galbraith, J. A., Terasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+damage+in+thick+specimens+by+multiphoton+excitation.">Controlled damage in thick specimens by multiphoton excitation.</a> Mol Biol Cell. 14, 1808-1817 (2003).</li><li>Yanik, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosurgery:+functional+regeneration+after+laser+axotomy.">Neurosurgery: functional regeneration after laser axotomy.</a> Nature. 432, 822-822 (2004).</li><li>Quinto-Su, P. A., Venugopalan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+laser+cellular+microsurgery.">Mechanisms of laser cellular microsurgery.</a> Methods Cell Biol. 82, 113-151 (2007).</li></ol>

我们用描述的正是axotomize外周感觉神经轴突和生活斑马鱼的胚胎中直接观察到再生的方法。长期时间推移在斑马鱼的共焦成像可用于观察在体内的发育过程。双光子干切断过程可以修改许多不同的实验目标。我们使用相同的一般程序消融整个三叉神经的感觉神经元胞体，放大细胞体内，而不是一个分支的末梢神经轴突。荧光识别任何类型的细胞，可以精确的损坏或集中功率飞秒激光烧蚀。?...

two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

斑马鱼长有被利用来研究时间推移成像的生活透明的胚胎发展的细胞和分子机制。在这里，我们描述的方法安装长期成像斑马鱼的胚胎，并演示如何自动捕获的时间推移，使用共聚焦显微镜图像。我们还描述了一种方法来创建控制，精确地破坏个人使用集中安装在双光子显微镜飞秒激光功率在斑马鱼的外周感觉轴突分支。成功的双光子干切断参数必须为每个显微镜进行了优化。定制的双光子显微镜和蔡司510焦/双光子提供​​了两个例子，我们将展示双光子干切断。 斑马鱼三叉神经感觉神经元1感官神经元特异性启动子驱动的转基因表达的GFP可以可视化。我们已经适应了这种斑马鱼三叉模式，以直接观察活的斑马鱼胚胎的感觉轴突再生。胚胎与tricaine麻醉和定位，因为它巩固了在琼脂糖下降。固定的胚胎被密封在一个充满成像与苯基硫脲嘧啶（PTU）林格室。我们已经发现胚胎可以在这些商会不断成像为12-48小时。一个单一的共聚焦图像，然后捕获，以确定所需的干切断的网站。双光子显微镜成像低，非破坏性的权力下的感觉轴突位于该地区的利益。放大所需的站点上的干切断后，功率增加，单一的，定义区域扫描足以切断轴突。然后设置多个位置的时间推移成像共聚焦显微镜直接观察到，从性轴索损伤恢复。

Watch this Scientific Journal Video about Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos at JoVE.com

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

在这里，我们描述了一个用于安装长期成像，双光子成像和组织损伤的技术，和时间推移共焦成像的斑马鱼胚胎的方法。

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Research

JoVE Journal

Biology

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

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 ...

科研

生物学

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

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 ...

Time-lapse Imaging of Mitosis After siRNA Transfection

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

多色成像定时转基因斑马鱼：可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

使用时间的推移显微镜可视化缺氧诱导假死<em&gt; C.线虫</em&gt;胚胎

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...