Name: super-resolution imaging of the bacterial division machinery
Uploaded: 2013-01-21T13:00:00
Description: Watch this Scientific Journal Video about Super-resolution Imaging of the Bacterial Division Machinery at JoVE.com

格兰特：5RO1GM086447 02。

1。样品制备<ol><li>应变JB281的单菌落接种LB培养基[BW25113 / pJB042（P 紫胶 ：的FtsZ的mEos2）]。在振动机中生长过夜，在37℃下</li><li>文化1:1,000稀释到M9 +最小的媒体M9盐，0.4％葡萄糖，2毫米硫酸镁 ，0.1毫米氯化钙 ，MEM氨基酸和维生素]和成长中旬至对数生长期（OD 600 = 0.2-0.3）氯霉素（150微克/毫升）的存在下，在室温（RT）。 </li><li>诱导文化与20μMIPTG 2小时（...

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	<li>Buddelmeijer, N., Beckwith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+cell+division+proteins+at+the+E.+coli+cell+center.">Assembly of cell division proteins at the E. coli cell center.</a> Curr. Opin. Microbiol. 5, 553-557 (2002).</li><li>de Boer, P., Crossley, R., Rothfield, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+bacterial+cell-division+protein+FtsZ+is+a+GTPase.">The essential bacterial cell-division protein FtsZ is a GTPase.</a> Nature. 359, 254-256 (1992).</li><li>Lutkenhaus, J. F., Wolf-Watz, H., Donachie, W. 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PALM图像包含有关分子计数和在小区内的位置的信息，允许的靶蛋白分子的分布和排列是难以通过其他方式实现的详细分析。下面，我们列出了应采取的预防措施，提取准确的定量信息，同时保持生物PALM图像的相关性。我们还探索能最好地利用活细胞与固定的信息。最后，我们建议的渠道，获得额外的超分辨率细胞分裂机械信息。 上面描述的方法进行了优化，以产生精确的PA...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 50×MEM氨基酸</td><td>西格玛</td><td> M5550 </td><td></td></tr><tr><td> 100×MEM维生素</td><td>西格玛</td><td> M6895 </td><td></td></tr><tr><td> IPTG </td><td>敏达</td><td> 46-102-RF </td><td></td></tr><tr><td> 16％的多聚甲醛</td><td>电子Micrsocopy </td><td> 15710-S </td><td></td></tr><tr><td> SeaPlaque GTG琼脂糖</td><td> Lonzo </td><td> 50111 </td><td></td></tr><tr><td> 50纳米的黄金珠</td><td>微球的纳米微球的制备</td><td> 790113-010 &lt;/ TD&gt; <td></td></tr><tr><td> FCS2成像室</td><td> Bioptechs </td><td></td><td></td></tr><tr><td>舞台适配器</td><td> ASI </td><td> I-3017 </td><td></td></tr><tr><td>倒置显微镜</td><td>奥林巴斯</td><td> IX71 </td><td></td></tr><tr><td> 1.45 NA，60倍的目标</td><td>奥林巴斯</td><td></td><td></td></tr><tr><td> IXON EMCCD相机</td><td>安道​​尔技术</td><td> DU897E </td><td></td></tr><tr><td> 488纳米蓝宝石激光</td><td>连贯</td><td></td><td></td></tr><tr><td> 561-nm的蓝宝石激光</td><td>连贯</td><td></td><td></td></tr><tr><td> 405纳米的CUBE激光</td><td>连贯</td><td></td><td></td>
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 </tbody>
 </table>

super-resolution imaging of the bacterial division machinery

细菌细胞分裂需要协调的装配超过10种必需的蛋白质，在midcell 1,2。这个过程的核心是形成一个环形的表壳构造（Z-环）的FtsZ蛋白的划分方案3,4。的Z-环由多个单链FtsZ的原丝，并理解的Z-环内的原丝的配置将提供洞察作为力发生器5,6 Z-环组件和它的功能的机制。此信息一直难以实现，由于目前的限制，在传统的荧光显微镜和电子显微镜。无法提供一个高分辨率图像的Z-环，由于光的衍射极限（〜200 nm）的常规的荧光显微镜。电子cryotomographic成像检测散FtsZ的原丝在小C. crescentus的电池7，但很难适用于较大的细胞，例如大肠杆菌或 B。 枯草芽孢杆菌 。在这里，我们描述了一个超高分辨率荧光显微镜方法的应用，光敏定位显微镜（PALM），定量表征的组织结构的E.大肠杆菌 Z-环8。 PALM成像可以同时提供高的空间分辨率（〜35纳米）和特定的标签，使靶蛋白的明确识别。我们标记FtsZ的可光活化的荧光蛋白mEos2，切换到绿色荧光（激发波长= 488纳米）的红色荧光（激发波长= 561纳米）激活后在405 nm 9。在一个PALM试验，单的FtsZ，mEos2分子，随机激活，相应的单分子质心的位置确定精度&lt;20纳米。甲的超分辨率图像的Z-环然后重建通过叠加的质心位置的所有检测到的FtsZ-mEos2分子。 &lt;p类=的“jove_content”&gt;使用这种方法，我们发现，在Z-环具有一个固定的宽度为100 nm，并组成一个松散捆FtsZ的原丝，在三维空间中彼此重叠。这些数据提供了进一步调查的跳板的Z形环10的细胞周期依赖性变化，并可以应用到其他感兴趣的蛋白质。

在图3Aiv是示出一个两维的，超分辨率渲染从PALM的成像方法，如上所述，产生的Z-环。下面，我们总结可以由它们得到的定性和定量信息。 定性地说，我们观察到的Z形环是一个不规则的结构，该结构采用在常规的荧光图像（比较图3A-DII和iv）是无法区分的多个配置（单一条带或螺旋形的弧）。如这些观察可以被用来确定该细胞群的百分比，显示一个特...

Watch this Scientific Journal Video about Super-resolution Imaging of the Bacterial Division Machinery at JoVE.com

Super-resolution Imaging of the Bacterial Division Machinery

我们描述了超分辨率成像方法的细菌FtsZ的环探测的组织结构，细胞分裂的重要设备。这种方法是基于光活化的定位显微镜（PALM）图像的定量分析，并可以应用到其他的细菌的细胞骨架蛋白。

超分辨率成像细菌科机械

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Research

JoVE Journal

Biology

Test Samples for Optimizing STORM Super-Resolution Microscopy

试验样品为优化风暴超高分辨率显微镜

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

科研

生物学

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

在Cytokineticž圈的活细菌超分辨率成像使用快速三维结构照明显微镜（F3D-SIM卡）

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

准备，成像和定量的细菌表面运动性测定

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

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三维超F-微丝高分辨率显微镜通过干涉光敏定位显微镜（iPALM）

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

高速超分辨速度显微镜在活原纤毛中的应用

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

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使用色马格龙的超分辨率生物成像时代3D色变的高精度校正

Quantitative multicolor fluorescence microscopy relies on the careful spatial matching of color channels acquired at different wavelengths. Due to ...

Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response

细菌群的延时成像与集体压力反应

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations ...