Name: visualization of cortex organization and dynamics in microorganisms, using total internal reflection fluorescence microscopy
Uploaded: 2012-05-01T12:03:00
Description: Watch this Scientific Journal Video about Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy at JoVE.com

这项工作是由马克斯 - 普朗克协会。

1。样品制备<ol class="lalpha"><li>准备盖玻片 盖玻片应清洗尘粒的TIRF从盖玻片（ 图1A，电影1）产生的背景信号是敏感。 <ol><li>在有盖的陶瓷人用钳子盖玻片到位。 </li><li> 1 M氢氧化钠（可重复使用多次）填补玻璃容器。 </li><li>缓慢连续旋转（轨道摇床）下孵育2小时。过多的潜伏期（&gt; 8小时），将创建不透明的盖玻片。 </li><li>下缓慢连续旋转5分钟，用蒸馏水清洗两次。 </li><li>?...

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	<li>Axelrod, D., Thompson, N. L., Burghardt, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+inflection+fluorescent+microscopy.">Total internal inflection fluorescent microscopy.</a> J. Microsc. 129, 19-28 (1983).</li><li>Axelrod, D., Omann, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+microscopy.">Combinatorial microscopy.</a> Nat. Rev. Mol. Cell Biol. 7, 944-952 (2006).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+surface+fluorescence+with+very+high+aperture+microscope+objectives.">Selective imaging of surface fluorescence with very high aperture microscope objectives.</a> J. Biomed. Opt. 6, 6-13 (2001).</li><li>Dominguez-Escobar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processive+movement+of+MreB-associated+cell+wall+biosynthetic+complexes+in+bacteria.">Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria.</a> Science. 333, 225-228 (2011).</li><li>Sparkes, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleach+it,+switch+it,+bounce+it,+pull+it:+using+lasers+to+reveal+plant+cell+dynamics.">Bleach it, switch it, bounce it, pull it: using lasers to reveal plant cell dynamics.</a> J. Exp. Bot. 62, 1-7 (2011).</li><li>Uchida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+synthesis+and+absolute+configuration+of+avenolide,+extracellular+factor+in+Streptomyces+avermitilis.">Total synthesis and absolute configuration of avenolide, extracellular factor in Streptomyces avermitilis.</a> J. Antibiot. (Tokyo). , (2011).</li><li>Yu, H., Wedlich-Soldner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+actin+dynamics:+Generating+randomness+by+formin(g)+and+moving.">Cortical actin dynamics: Generating randomness by formin(g) and moving.</a> Bioarchitecture. 1, 165-168 (2011).</li><li>Yu, J. H., Crevenna, A. H., Bettenbuhl, M., Freisinger, T., Wedlich-Soldner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+actin+dynamics+driven+by+formins+and+myosin+V.">Cortical actin dynamics driven by formins and myosin V.</a> J. Cell. Sci. 124, 1533-1541 (2011).</li><li>Berchtold, D., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TORC2+plasma+membrane+localization+is+essential+for+cell+viability+and+restricted+to+a+distinct+domain.">TORC2 plasma membrane localization is essential for cell viability and restricted to a distinct domain.</a> Mol. Biol. Cell. 20, 1565-1575 (2009).</li><li>Vizcay-Barrena, G., Webb, S. E., Martin-Fernandez, M. L., Wilson, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+and+single-molecule+imaging+of+plant+fluorescent+proteins+using+total+internal+reflection+fluorescence+microscopy+(TIRFM).">Subcellular and single-molecule imaging of plant fluorescent proteins using total internal reflection fluorescence microscopy (TIRFM).</a> J. Exp. Bot. 62, 5419-5428 (2011).</li></ol>

TIRF显微镜图像周边蛋白的首选技术。渐逝场的低渗透深度，最大限度地减少了光的焦点，这导致了一个非常好的信噪比，并允许数据采集，高帧频，或非常弱表达的蛋白质成像。高对比度和高帧率相结合，使TIRF显微镜的成像时空外皮本地化的蛋白质动力学的完美的工具。有趣的是，厚厚的细胞壁周围的许多微生物不妨碍周边蛋白的TIRF成像。事实上，渐逝的实际发电量很可能发生在细胞壁和细胞...

<table border="1"><tbody><tr><td> 该工具的名称/试剂 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>轨道摇床</td><td>工具</td><td> UniEquip </td><td> UNITWIST的3-D摇杆振动筛</td></tr><tr><td>的TIRF显微镜</td><td>直到</td><td></td><td>自定义安装</td></tr><tr><td>玻璃容器</td><td>工具</td><td> vitlab </td><td> 340-232880353 </td></tr><tr><td>陶瓷染色架</td><td>工具</td><td>托马斯科学。 </td><td> 8542E40 </td></tr><tr><td>刀豆蛋白A </td><td>试剂</td><td>西格玛</td><td> L7647 </td></tr><tr><td>的盖玻片＃1（18×18毫米） </td><td>显微镜</td><td>门泽尔格拉泽</td><td> BB018018A1 </td></tr><tr><td>显微镜载玻片</td><td>显微镜</td><td>门泽尔Ğ激光</td><td> AA00000102E </td></tr><tr><td>浸油</td><td>显微镜</td><td>蔡司</td><td> immersol 518F </td></tr><tr><td>琼脂糖</td><td>试剂</td><td> Invitrogen公司</td><td> 16500-500 </td></tr><tr><td> FluoSpheres </td><td>试剂</td><td> Invitrogen公司</td><td> F8795 </td></tr></tbody></table>

visualization of cortex organization and dynamics in microorganisms, using total internal reflection fluorescence microscopy

TIRF显微镜已成为一个强大的成像技术，研究时空动态的荧光分子在体外和活细胞1。全内反射光的现象时有发生，当光线从高折射率介质传递到与低折射率介质在一个角度大于特征的临界角（即接近边界平行）。尽管所有的光被反射回在这种情况下，创建渐逝波传播横跨和沿边界，距离指数衰减，只有穿透样本地区是100-200纳米界面附近的2。除了 ​​提供优越的轴向分辨率，减少了重点荧光激发创建了一个非常高的信号噪声比和减少漂白2,3破坏性影响。作为一个广角技术的TIRF还允许更快的图像交流quisition比扫描基于共聚焦设置。 乍一看，低的TIRF穿透深度，似乎是不符合，这往往是厚厚的细胞壁所包围的细菌和真菌细胞成像。相反，我们发现，酵母和细菌细胞的细胞壁，实际上提高可用性的TIRF和增加4-8观察到的结构范围内。许多细胞过程，因此可以直接访问的TIRF小，单细胞微生物，它往往提供了强有力的遗传操作技术。这使我们能够执行在体内的生化实验，可以直接在活细胞中进行评估蛋白质相互作用和活动的动力学。 在这里，我们描述了酵母或枯草芽孢杆菌细胞获得高品质的TIRF图像所需的各个步骤。我们指出的各种问题，可以affeCT的TIRF可视化细胞中的荧光探针和几个应用实例说明过程。最后，我们展示的TIRF图像可进一步提高使用既定的图像恢复技术。

Watch this Scientific Journal Video about Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy at JoVE.com

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

全内反射荧光显微镜（TIRF）是一个功能强大的方法来观察结构在细胞表面的高对比度和时间分辨率。我们展示的TIRF如何可受聘在皮层细胞壁封闭的细菌和真菌细胞的蛋白质动力学研究。

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Research

JoVE Journal

Bioengineering

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

重组DNA和蛋白质复合物，用原子力显微镜的可视化

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

科研

生物工程

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

Nanotopology细胞粘附在可变角度的全内反射荧光显微镜（VA-TIRFM）

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

3d轨道跟踪的改进双光子显微镜之应用：细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

用数字全息显微镜 (DHM) 定量化低浓度微生物

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

枯草芽孢杆菌中细菌体和内膜的可视化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...