Name: two-photon-based photoactivation in live zebrafish embryos
Uploaded: 2010-12-24T15:00:00
Description: Watch this Scientific Journal Video about Two-Photon-Based Photoactivation in Live Zebrafish Embryos at JoVE.com

感谢由于热尼亚图图形布罗德斯基;请提供neurogenin1记者一行乌韦Strahle和维亚切斯拉Kalchenko，道格拉斯卢茨和列昂尼德Roitman两个光子uncaging的技术咨询和援助; Maayan Tahor提供技术援助和Suliman Elsadin阿莫斯Gutnick本书稿的意见。在Levkowitz实验室的研究是由德国和以色列基金会（批准数二千〇七分之一百八十三）;以色列科学基金会（批准号928/08）和哈里特马塞尔德克尔基金会的支持。 GL Tauro的职业生涯中的生物医学研究的发展主席是现任。

我们描述了一个协议使用笼荧光标记的细胞，但是，其他照片激活的染料和蛋白质可以同样使用。 1。注射笼荧光<ol><li>准备5％的股票，共轭葡聚糖4,5 - 二甲氧基- 2 - 硝基苯（DMNB）笼荧光素（10,000兆瓦右旋糖酐，阴离子，Invitrogen公司，分子探针，卡尔斯巴德，加利福尼亚的解决方案（5毫克笼荧光素/ 100μL0.2M氯化钾）猫没有D - 3310）。分装和储存在-20 ° C。注意DMNB笼荧光是?...

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光激活的化合物分子，其功能被屏蔽，直到它们与特定波长（通常是紫外线）诱导光化学反应转换成一种生物或化学活性状态的分子，照亮。这些探头在细胞生物学研究提供了非常强大的工具，因为激活可精确控制时间和空间限制了其暴露在光线下。 多光子显微镜的显着优点是比较深的光学穿透和减少非特异性光毒性。激活过程中，必须吸收低能量的两个光子的光激活化合?...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Dextran-conjugated 4,5-dimethoxy-2-nitrobenzyl (DMNB) caged fluorescein (10,000 MW dextran, anionic)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D-3310</td>
<td>molecular probes</td>
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<tr>
<td>Agarose for injection trough and coated plates</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9539</td>
<td>&nbsp;</td>
<tr>
<td>Thin Wall Glass Capillaries with filament</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW100F-6</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
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<td>Microloader tip</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5242 956.003</td>
<td>&nbsp;</td>
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<td>Pneumatic picopump</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>PV820</td>
<td>&nbsp;</td>
<tr>
<td>Phenylthiourea (PTU)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>22290-9</td>
<td>&nbsp;</td>
<tr>
<td>Low melting point agarose for embryo mounting</td>
<td>&nbsp;</td>
<td>Ultra Pure LMP agarose</td>
<td>16520100</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Fluorescein- alkaline phosphatase (AP) Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11426338910</td>
<td>&nbsp;</td>
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<td>Fast Red</td>
<td>&nbsp;</td>
<td>Roche</td>
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two-photon-based photoactivation in live zebrafish embryos

Photoactivati​​on一个活的有机体中的目标化合物已被证明是一个有价值的方法，调查各种生物，如胚胎发育，细胞信号和成人的生理过程。在这方面，多光子显微镜的使用，使深部组织的一个给定的光反应剂在单细胞分辨率定量photoactivati​​on。由于斑马鱼的胚胎是透明的，其发展可以监测体内。这些特点使斑马鱼的一个完美的模式生物，用于控制各种化学制剂和聚焦光线的蛋白质的活动。在这里，我们描述双光子显微镜的使用，以诱导荧光，化学笼激活，从而允许我们按照活的斑马鱼胚胎细胞的命运。我们使用表达一个活的遗传具有里程碑意义的（GFP）的定位和精确瞄准任何利益细胞的胚胎。此过程同样可以用于精确的光诱导活化蛋白质，激素类，小分子和其他笼化合物。

Watch this Scientific Journal Video about Two-Photon-Based Photoactivation in Live Zebrafish Embryos at JoVE.com

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

多光子显微镜允许控制与深厚的光学渗透和减少光毒性低能量的光子。我们描述了使用这项技术在斑马鱼胚胎活细胞标记。该协议可以很容易地适应各种光反应的分子的光感应。

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 ...

Research

JoVE Journal

Biology

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

科研

生物学

Microinjection of Zebrafish Embryos to Analyze Gene Function

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

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

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.  ...

Generating Chimeric Zebrafish Embryos by Transplantation

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

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 ...

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 ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Metabolic Profile Analysis of Zebrafish Embryos

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Facial Transplants in Xenopus laevis Embryos

Facial Transplants in Xenopus laevis Embryos

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Analysis of Oxidative Stress in Zebrafish Embryos

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

在现场的斑马鱼胚胎的基于双光子Photoactivation