Name: photobleaching enables super-resolution imaging of the ftsz ring in the cyanobacterium prochlorococcus
Uploaded: 2018-11-06T15:00:00
Description: Watch this Scientific Journal Video about Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus at JoVE.com

作者感谢徐黛英对手稿的技术援助和评论。这项研究得到了中国国家自然科学基金 (项目编号 41476147) 和中国香港特别行政区研究资助局 (项目编号689813和 16103414) 的资助。

1. 样品制备和固定
<ol><li>接种1毫升的轴突性普绿球菌med4 至5毫升的海水原基原发物12。在23°c 的光下生长红球菌培养, 强度为 35μmol photons/m m 2 s. 五天后, 将1毫升培养物收集成 1.5 ml 管。 注: 接种五天后,红球菌med4 将达到后期日志阶段, 约 108 细胞, 这适合 storm 成像。</li>
	<li>将25% 的甲醛 (pfa) (wv) 和50% 戊二醛的2μl 加入 1.5 ml 管中, 将新鲜制?...

<ol>
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在该协议中, 我们描述了一个程序, 以显著减少原蓝菌的自体荧光 (图 3c), 然后, 免疫抑制细胞中的蛋白质, 使我们能够利用 storm 来研究三维 ftsz在古氯球菌环形态(图 4)。该协议可用于其他光合生物的超分辨率成像。
以往对光合生物的研究主要是使用 sim 卡实现超分辨率成像, 因为 sim 不需要高强度的激光。然而, sim 的空...

超分辨率显微镜可以打破光的衍射极限, 并在亚衍射分辨率 (& lt; 200 nm) 内提供图像。它们已被广泛用于许多生物体的蛋白质定位和亚细胞结构的研究。主要的超分辨率显微镜方法包括结构照明显微镜 (sim)、受刺激的发射耗尽显微镜 (sted)、storm 和光激活定位显微镜 (palm)。这些超分辨率显微镜的机理和应用在其他地方已经审查了 1,2。
storm 可以通过空间分离3,4实现高达10纳米的分辨率。对于 storm, 只有一个分子在衍射有限的区域内被激活 ("开"), 其余的分子被保持灭活 ("关闭")。通过积累单个分子的快速开关, 可以生成一个 "衍射无限" 的图像 3.同时, storm 中还使用了多种有机染料和荧光蛋白, 使其易于从常规荧光显微镜升级到高分辨率显微镜5,6。
storm 在光合细胞中没有得到广泛的应用, 如蓝藻、藻类和叶绿体7, 8 的植物细胞, 这是因为 storm 需要较高的激光强度来驱动光致敏化。高强度激光在光合细胞中激发强自体荧光背景, 干扰 storm 成像中的单分子定位。为了利用 storm 研究光合细胞中的亚细胞结构或蛋白质相互作用, 我们开发了一种光漂白协议来抑制背景自体荧光信号9。在常规的免疫荧光染色过程中, 标本在封堵步骤中暴露在高强度的白光下, 从而降低光合细胞的自体荧光, 以满足 storm 的要求。因此, 该协议使得用 storm 研究色素生物成为可能。
在这里, 我们描述了使用 storm 来成像 ftsz 环组织在单细胞的五氯球菌的协议。ftsz 是一种高度保守的管状细胞骨架蛋白, 它聚合在细胞10的周长周围形成环状结构 (z 环), 对细胞分裂11至关重要.保鲜的红球菌细胞首先被光导, 以减少自荧光背景和免疫染色与初级抗 ftsz 抗体, 然后继发抗兔 igg (h + l) 抗体与荧光结合 (例如。, 亚历克沙·福鲁尔 750)。最终, storm 被用来观察不同细胞周期阶段的古氯球菌中的详细 ftsz 环组织。

<table>
	<tbody>
		<tr>
			<td>Polystyrene particles</td>
			<td>Spherotech</td>
			<td>PP-20-10</td>
			<td>2.0-2.4 &#181;m</td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Marienfeld</td>
			<td>0111580</td>
			<td>18 mm &#8709;, Thickness No. 1</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Scharlau</td>
			<td>ET00021000</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P9155</td>
			<td>mol wt 70,000-150,000</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde solution, 50%</td>
			<td>Sigma-Aldrich</td>
			<td>340855</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA Disodium Salt, 2-hydrate</td>
			<td>Gold biotechnology</td>
			<td>E-210-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Anabaena FtsZ antibody</td>
			<td>Agrisera</td>
			<td>AS07217</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody</td>
			<td>Life Technologies</td>
			<td>A-21039</td>
			<td>conjugated with Alexa Fluor 750</td>
		</tr>
		<tr>
			<td>D-Glucose Anhydrous</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A5960</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl Viologen</td>
			<td>Sigma-Aldrich</td>
			<td>856177</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclooctatetraene</td>
			<td>Sigma-Aldrich</td>
			<td>138924</td>
			<td></td>
		</tr>
		<tr>
			<td>tris(2-carboxyethyl)phosphine (TCEP)</td>
			<td>Sigma-Aldrich</td>
			<td>646547</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose Oxidase</td>
			<td>Sigma-Aldrich</td>
			<td>G2133</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma-Aldrich</td>
			<td>C9322</td>
			<td></td>
		</tr>
		<tr>
			<td>XD-300 Xenon light source</td>
			<td></td>
			<td></td>
			<td>250 W</td>
		</tr>
		<tr>
			<td>STORM microscope</td>
			<td>NBI</td>
			<td>SRiS microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Rohdea</td>
			<td>NBI</td>
			<td>SRiS 3.0</td>
			<td>software for imaging acquisition</td>
		</tr>
		<tr>
			<td>Luna</td>
			<td>NBI</td>
			<td>SRiS 3.0</td>
			<td>software for drifting correction</td>
		</tr>
		<tr>
			<td>QuickPALM</td>
			<td></td>
			<td></td>
			<td>https://code.google.com/archive/p/quickpalm/wikis</td>
		</tr>
		<tr>
			<td>3D Viewer</td>
			<td></td>
			<td></td>
			<td>http://132.187.25.13/ij3d/?page=Home&#38;category=Home</td>
		</tr>
	</tbody>
</table>

photobleaching enables super-resolution imaging of the ftsz ring in the cyanobacterium prochlorococcus

超分辨显微镜已被广泛用于研究许多生物体的蛋白质相互作用和亚细胞结构。然而, 在光合生物中, 超分辨率成像的横向分辨率仅为 ~ 100 纳米。低分辨率的主要原因是光合细胞的高自体荧光背景, 这是由高强度激光引起的, 而超强分辨率成像需要这些激光, 如随机光学重建显微镜 (storm)。在这里, 我们描述了一种光浸辅助 storm 方法, 该方法是最近开发的用于海洋皮亚星杆菌的成像。光漂白后, 有效地降低了红球菌的自体荧光, 使 storm 能够在横向分辨率 ~ 10 纳米的情况下进行。利用该方法, 我们获得了 ftsz 蛋白的体内三维 (三维) 组织, 并在红球菌的细胞周期中表征了四种不同的 ftsz 环形态。我们在这里描述的方法可以用于其他光合生物的超分辨率成像。

storm 通过随机激活单个可光敏荧光体来实现超分辨率成像。记录每个荧光体的位置, 然后根据这些位置构建一个超分辨率图像4。因此, 荧光体定位的精度对于超分辨率图像重建具有重要意义。在447 纳米和680纳米的峰值和在波长大于700纳米16时, 普罗氯球菌的吸收光谱具有最低吸收.然而, 当暴露在750纳米激光的极高强度 (<strong...

Watch this Scientific Journal Video about Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus at JoVE.com

Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus

在这里, 我们描述了一种光漂白方法, 以减少蓝藻的自体荧光。光漂白后, 采用随机光学重建显微镜获得蓝藻 ftsz 环的三维超分辨率图像。

光漂白可实现前列腺素球菌中 ftsz 环的超分辨率成像

Super-resolution microscopy has been widely used to study protein interactions and subcellular structures in many organisms. In photosynthetic organisms, ...

该方法有助于回答蛋白质定位和光合作用生物动力学的关键问题。通过将光漂白方法与超分辨率显微镜相结合，我们能够显著地检测光合作用生物体的蛋白质动力学。这种方法不仅提供了对丙氯球菌蛋白质动力学的洞察。它也可以适用于许多其他含有放射性-重唱和细菌叶绿素的生物体。演示这个程序的将是我实验室的研究生刘燕欣。首先，在培养瓶中加入五毫升的基于海水的Pro99介质，用一毫升的Pro氯球菌MED4进行接种。在每平方米35微莫光子强度的光线下，在23摄氏度下生长丙氯球菌。五天后，将培养的一毫升收集到1.5毫升的管子中。在管子中加入100微升新鲜准备好的甲醛，以固定培养。在室温下在黑暗中孵育20分钟。然后，在 13，500 次 G 下旋转样品一分钟。取出上一代，在Pro99介质的100微升中重新暂停细胞。将样品储存在黑暗中四摄氏度，直到准备好进行免疫破坏。首先，涡流聚苯乙烯珠的小瓶。使用50%乙醇，准备一到2万稀释作为工作泥浆。打开加热板，将温度设置为 120 摄氏度。接下来，将盖玻片放在热板上。将工作泥浆的 100 微升加载到每个盖玻片上，并在加热板上孵化盖玻片 10 分钟。然后小心地将盖玻片转移到培养皿珠侧，在室温下存放。当准备涂上盖玻片时，取回一个，确保它是珠面向上。将每毫升聚L-莱辛溶液100微升添加到盖玻片的中心，让它在室温下坐30分钟。在此之后，使用移液器小心吸气任何未连接的聚L-莱辛，并转移到1.5毫升管。将这种聚L-lysine储存在4摄氏度，供以后使用。在60毫米培养皿中用10毫升的超滤水冲洗盖玻片，然后用纸巾短暂干燥。将涂层盖玻片转移到底部有 Parafilm 的新培养皿中，该盘将用作染色盘。将固定样品的 100 微升装到盖玻片上，让其坐 30 分钟，以便进行单元连接。接下来，使用纸巾吸收剩余的溶液，然后将盖玻片转移到12井板的井中清洗。向井中加入一毫升 PBS 缓冲液以清洗盖玻片。然后取出 PBS 并添加一毫升新鲜 PBS 以重新清洗盖玻片。使用移液器取出 PBS，并将其替换为一毫升新鲜准备的渗透缓冲液。在37摄氏度下孵育洗碗20分钟。然后删除渗透缓冲液。添加一毫升新鲜 PBS 缓冲液，开始洗涤过程。将洗碗盘放在摇床上，轻轻搅拌五分钟。在此之后，取出 PBS 并重复洗涤过程三次。将洗净的盖玻片转移到染色盘上。在盖玻片顶部添加 50 微升的阻塞缓冲器。将整个染色盘放在冰上，然后将放在 Xenon 光源下进行光漂白至少 60 分钟。光漂白和阻塞后，使用纸巾的边缘去除阻塞缓冲液。首先，将一微升抗 FtsZ 抗体稀释成 99 微升的阻尼缓冲液。将稀释原抗体的50微升放在盖玻片上，并在室温下孵育30分钟。将盖玻片转移到洗碗的井上。要开始洗涤，请加入一毫升 PBS。然后将洗碗盘转移到摇床中，轻轻摇动五分钟。取出 PBS 并重复整个洗涤过程三次。接下来，将盖玻片转移到染色盘。将稀释的二次抗体的50微升放在盖玻片上，在黑暗中和室温下孵育30分钟。将盖玻片转回洗碗盘。要开始洗涤，请加入一毫升 PBS。用铝箔包裹洗涤盘，防止其光线。将板转移到摇床，轻轻摇动五分钟。取出 PBS 并重复整个洗涤过程三次。在 STORM 成像之前，请准备文本协议中概述的一毫升成像缓冲区。将盖玻片装入装料室。加入新鲜准备好的成像缓冲液，温和地避免洗掉蓝藻细胞。在此之后，在成像缓冲器顶部放置一个矩形盖玻片，以防止其与空气中的氧气发生反应。打开相机、LED 灯和激光，然后打开 STORM 软件。在镜头顶部加入半滴浸入油。加载腔室，确保目标镜头与盖玻片接触。用750纳米激光检查信号。确定包含单元格和基准标记的样本区域。然后启动用于样品漂移校正的软件。获取一个宽图图像作为参考，相机电子乘法增益为300，曝光时间为30毫秒。将 750 纳米激励激光强度增加到每平方厘米约 4.5 千瓦。一旦荧光团过渡到稀疏的闪烁模式，通过收集1万帧33赫兹，获得一个超分辨率的图像。然后从文本协议中概述的原始数据重建超分辨率图像。在这项研究中，STORM通过随机激活单个光可切换荧光团来实现超分辨率成像。记录每个荧光团的位置，然后根据这些位置构建一个超分辨率图像。虽然Prochlorococcus的吸收光谱达到447和680纳米的峰值，最小吸收量超过700纳米，但Prochlorococcus MED4细胞在暴露于750纳米激光器的极高强度时仍会发出高自荧光，这是STRAM成像所需的极强强度。使用此处描述的光漂白方法30分钟后，细胞的自荧光量会减少，尽管仍然检测到几个具有自荧光的细胞。将光漂白时间拉长至60分钟会导致大多数细胞失去自荧光。结果表明，研制的光漂白法能够大大减少光合生物的自荧光。光漂白后，STORM用于可视化细胞中的细胞分裂蛋白FtSZ。使用 STORM，可以揭示 FtsZ 环的详细形态。通过旋转 3D STORM 图像，确定了四种不同类型的形态、聚类、不完整的环、完整的环和双环。重要的是要记住，光漂白的光强度和持续时间可能因不同的生物体而不同。该方法开发后，为研究光合作用细胞中的蛋白质组织和潜在功能的研究人员铺平了道路。

Research

JoVE Journal

Immunology and Infection

Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Real-time Live Imaging of T-cell Signaling Complex Formation

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

使用madSTORM进行高分辨率，超分辨率的T细胞成像

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...