Name: correlative light and electron microscopy (clem) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets
Uploaded: 2012-05-04T12:00:00
Description: Watch this Scientific Journal Video about Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets at JoVE.com

我们感谢的Alto实验室的有益讨论。我们也感谢德州大学西南医学中心，特别是克里斯·吉尔平博士，汤姆雅努谢夫斯基，和劳里·穆勒的分子和细胞成像设备在专业知识和咨询。我们也感谢Xionan董批判性阅读的手稿。支持这项工作是由美国国立卫生研究院授予AI083359 NMA

1。哺乳动物细胞培养<ol><li>正常大鼠肾（NRK），永生化宫颈癌（HeLa细胞），或其他适当的哺乳动物细胞系进行培养，在37℃下，5％CO 2，贴住细胞菜肴（ 见表1的任何试剂或光刻的玻璃屏栅盖玻片装置，在该协议中使用）在DMEM +10％FBS和1％青/链霉素。应采取预防措施，以维持一个无菌的环境中工作时，与培养的哺乳动物细胞。 </li><li>细胞的汇合根据您的实验时间线（0-5小时...

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这里介绍的方法能够直接提供纯化的蛋白质，核酸，小分子真核细胞的细胞质中，通过荧光显微镜和电子显微镜的相关性，并能提供超高分辨率分析。使用这种方法是简单而强大的，可以在内部进行，与许多现有的核心设施和/或适当配备的电子显微镜实验室。该技术也适合于活细胞，允许用户监视的动态实时荧光标记的分子，从而可以使用的时间推移显微镜和相关的断层电子显微镜。 <p class="...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>光刻盖玻片和活细胞板</td><td> MatTek </td><td> P35G-2-14-CGR​​D </td><td></td></tr><tr><td>德州红</td><td> Invitrogen公司</td><td> D3329 </td><td></td></tr><tr><td>级联蓝</td><td> Invitrogen公司</td><td> D7132 </td><td></td></tr><tr><td>罗丹明标记试剂盒</td><td> Thermo Scientific的</td><td> 53002 </td><td></td></tr><tr><td> 0.22微米离心过滤单元</td><td>密理博</td><td> UFC30GV00 </td><td></td></tr><tr><td>高硼硅玻璃吸管</td><td>萨特仪器</td><td> BF100-50-10 </td><td></td></tr><tr><td>微管牵引机</td><td>萨特仪器</td><td>型号：P-97 </td><td>在执行斜坡测试使用程序＃4 </td></tr><tr><td> FemtoJet显微注射系统</td><td> Eppendorf公司</td><td> Injectman NI2 </td><td></td></tr><tr><td>盖玻片切削液</td><td> MatTek </td><td> PDCF OS 30 </td><td></td></tr><tr><td> Technai透射电子显微镜</td><td> FEI </td><td> Technai G2 BioTWIN </td><td></td></tr><tr><td> Ultramicrotombe </td><td>徕卡</td><td> EM UC6 </td><td></td></tr></tbody></table>

correlative light and electron microscopy (clem) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets

依赖于复杂的真核细胞中，高稳定，功能不同的膜结合隔间保持必要的生化极性适当的细胞功能。因此，了解的酶，蛋白质和细胞骨架，治理和维护这种生化分离是非常重要的。使用荧光标记的分子定位和/或扰乱细胞内的车厢已经取得了丰富的知识和先进的细胞调控的认识。荧光和共聚焦显微镜成像技术，如确定一个荧光标记的小分子相对简单的位置，但是分辨率非常小的结构受限情况说明1。 另一方面，电子显微镜揭示了亚细胞形态的细节，以非常高的分辨率，但它的静态特性使得它难以衡量的高度动态的亲精度2,3 cesses用。因此，光显微镜和电子显微镜对同一样品，被称为的相关光学和电子显微镜（CLEM）4,5的组合，能提供超快荧光成像的双重优点，高分辨率的电子显微镜6。这种强大的技术已经实施，以研究细胞生物学的许多方面的5,7。公司自成立以来，这个过程增加了我们的辨别能力，在高分辨率下的亚细胞结构和形态。 在这里，我们提出了一种简化的方法进行快速显微注射，其次是CLEM（ 图1）。显微注射的CLEM程序可以使用，引入特定数量的小分子和/或蛋白直接进入真核细胞的细胞质中，研究了从毫米到纳米多分辨率（ 图2）。该技术是基于显微注射细胞生长的激光蚀刻玻璃网格底部的活细胞菜肴和成像，共聚焦荧光和电子显微镜的盖玻片贴。容易的格子图案，这是很容易转移，以及所关注的细胞，EPON用于固定样品的树脂切片前的电子显微镜分析（ 图3）的细胞的本地化（s）的权益。叠加的荧光灯和EM的图像允许用户确定的亚细胞定位，以及任何形态和/或超微结构的变化引起的显微注射分子的利益（ 图4）。此技术是适合从≤5 s到几个小时，取决于的显微注射样品的性质上的时间点。

Watch this Scientific Journal Video about Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets at JoVE.com

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

CLEM技术已被用于分析超微结构形态的细胞膜，细胞器，通过显微注射的分子和亚细胞结构的影响。这种方法结合了强大的技术，的显微/显微注射，共聚焦荧光显微镜，电子显微镜，允许毫米多纳米的分辨率。此技术是适合于各种各样的应用。


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Research

JoVE Journal

Biology

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

作为寄主植物挥发物的筛选工具electroantennographic生物活性

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

科研

生物学

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

一个具体的感觉细胞的共聚焦相关和3D电子显微镜

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

扫描浸软组织的电子显微镜可视化的细胞外基质

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

用化学干燥进行扫描电镜 (sem) 形态分析的原核生物和真核生物的制备

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

使用新型机器人系统 Spotiton 进行时间解决研究的低温电子微观网格准备

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy

内源性LC3的超微结构定位-截面相关光电子显微镜

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal ...

Novel Protocol for Simultaneous Fluorescence and Ultrastructure Preservation

Epon Post Embedding Correlative Light and Electron Microscopy

Author Spotlight: Advancements in Correlative Light and Electron Microscopy with Fluorescent Protein Preservation

Epon Post 嵌入相关光和电子显微镜

Correlative light and electron microscopy (CLEM) is a comprehensive microscopy that combines the localization information provided by fluorescence ...

相关的光学和电子显微镜（CLEM），作为工具来可视化显微注射分子和他们的真核细胞的亚细胞目标