Name: multiphoton microscopy of cleared mouse brain expressing yfp
Uploaded: 2012-09-23T14:00:00
Description: Watch this Scientific Journal Video about Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP at JoVE.com

我们要感谢他的援助，在视频编辑雅各索利斯。 这部分工作是由美国国家科学基金会职业奖DBI-0953902 MJ列文。

1。动物灌注5和整个小鼠脑结算<ol><li>的整个长度上的程序可以根据每脱水工序中使用的时间长度而变化，但总的整个过程可以在两天内进行。 </li><li>称重YFP的小鼠和然后深深麻醉腹膜内注射氯胺酮/赛拉嗪（100毫克/公斤10毫克/公斤）。 </li><li>确认的手术平面的深度麻醉，然后再进行手术。检查动物，每隔5分钟，看看它的反应到公司或脚趾尾捏。如果动物的反应，氯胺酮/赛拉嗪的补充?...

<ol>
	<li>Parra, S. G., Chia, T. H., Zinter, J. P., Levene, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+microscopy+of+cleared+mouse+organs.">Multiphoton microscopy of cleared mouse organs.</a> J. Biomed. Opt. 15, 036017 (2010).</li><li>Vesuna, S., Torres, R., Levene, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+fluorescence,+second+harmonic+generation,+and+fluorescence+lifetime+imaging+of+whole+cleared+mouse+organs.">Multiphoton fluorescence, second harmonic generation, and fluorescence lifetime imaging of whole cleared mouse organs.</a> J. Biomed. Opt. 16, 106009 (2011).</li><li>Zucker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+insect+and+mammalian+embryo+imaging+with+confocal+microscopy:+Morphology+and+apoptosis.">Whole insect and mammalian embryo imaging with confocal microscopy: Morphology and apoptosis.</a> Cytometry. A69, 1143-1152 (2006).</li><li>Sakhalkar, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+in+bulk+tissue+specimens+using+optical+emission+tomography:+Fluorescence+preservation+during+optical+clearing.">Functional imaging in bulk tissue specimens using optical emission tomography: Fluorescence preservation during optical clearing.</a> Phys. Med. Biol. 52, 2035-2054 (2007).</li><li>Dazai, J., Spring, S., Cahill, L. S., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple-mouse+Neuroanatomical+Magnetic+Resonance+Imaging.">Multiple-mouse Neuroanatomical Magnetic Resonance Imaging.</a> J. Vis. Exp. (48), e2497 (2011).</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, (2003).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2001).</li><li>Abramoff, M. D., Magelhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Feng, G., Mellor, R. H., Bernstein, M., Keller-Peck, C., Nguyen, Q. T., Wallace, M., Nerbonne, J. M., Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Neuronal+Subsets+in+Transgenic+Mice+Expressing+Multiple+Spectral+Variants+of+GFP.">Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Hama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat. Neuro. 14, 1481-1488 (2011).</li></ol>

虽然标准的有机染料是兼容的有机溶剂的范围内，因此，不清除协议构成特别的挑战，荧光蛋白往往是在溶剂中的改变的耐性低。4本工作的目的是克服一个严重的限制以前的光学结算协议XFPS荧光的丢失或严重退化。这里所呈现的图像表明，荧光YFP被保存。光透明这里描述的技术允许YFP标记的神经元在长达2毫米的整个鼠脑组织表面下的高清晰度成像。先前证明，使用乙醇脱水，也将保留?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td>磷酸盐缓冲液</td><td> Sigma-Aldrich公司</td><td> D8537 </td><td> 500毫升，pH 7.2的</td></tr><tr><td>多聚甲醛</td><td>电子显微镜科学</td><td> 15710 </td><td> 10×10毫升，16％多聚甲醛</td></tr><tr><td>乙醇</td><td>美国生物分析</td><td> AB00515-00500 </td><td> 500毫升，200证明</td></tr><tr><td>乙醇</td><td> Pharmco产品，Inc。 </td><td> 111000190 </td><td> 1加仑，190证明</td></tr><tr><td>苯甲醇</td><td> Sigma-Aldrich公司</td><td> 402834 </td><td> 500毫升，99 +％的</td></tr><tr><td>苯甲酸苄酯</td><td> Sigma-Aldrich公司</td><td> B6630-IL </td><td> 500毫升，≥99％的</td></tr><tr><td> 5X/0.5 NA目标</td><td>尼康</td><td> AZ计划面粉5倍</td><td> 15 WD </td></tr></tbody></table>

multiphoton microscopy of cleared mouse brain expressing yfp

多光子显微镜是通过光学成像之前清除器官的固有荧光和二次谐波产生（SHG）的整个鼠标机关。1,2然而，对于器官，包含荧光蛋白，如GFP和YFP，光使用甲醇的结算协议脱水和用苄醇苯甲酸苄酯（巴布），而未受保护的光3不保留的荧光信号。这里介绍的是一种新的方式，同时保留YFP的荧光信号在神经元中表达，在其中执行整个器官对小鼠脑组织的光透明的协议。改变光透明的协议，例如使用的乙醇梯度系列已经发现，减少损伤的荧光蛋白，并保持其多光子成像的荧光信号，器官是脱水。4使用一个优化的方法，光透明的基于乙醇的脱水和清除由巴布从光屏蔽时，我们显示高分辨率的多光子图像，黄色荧光蛋白（YFP）表达在神经元的小鼠脑组织表面下方超过2毫米。

Watch this Scientific Journal Video about Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP at JoVE.com

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

整个小鼠脏器的多光子显微镜能够通过光学成像之前清零器官，但并非所有协议保留的荧光蛋白的荧光信号。使用光学乙醇脱水和苯甲醇苯甲酸苄酯结算的结算方法，显示高分辨率的多光子图像的整个小鼠脑组织表达YFP。

多光子显微镜的清除小鼠脑组织表达YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Research

JoVE Journal

Neuroscience

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

制备小鼠脑组织中的免疫电镜

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

科研

神经科学

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

活体鼠脑微循环显微镜使用一个封闭的颅窗口

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

聚焦离子束铣削和脑组织的扫描电子显微镜

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Purification of Mouse Brain Vessels

对小鼠脑血管净化

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Peering at Brain Polysomes with Atomic Force Microscopy

在脑核糖凝视与原子力显微镜

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...