YW和PK非常感谢来自新加坡国家研究基金会，授予PK（NRF-NRFF-2011-04和NRF2012NRF-CRP001-084）的资金支持。我们也感谢对基础设施的支持MBI开放实验室，显微镜的核心设施。

1.成像试样制备<ol><li>因为背景的荧光信号与来自荧光团标签荧光干扰，通过在去离子水漂洗它们（DDH 2 O的），然后使用压缩空气空气干燥它们清洗盖玻片。接着，在等离子体清洁器执行等离子体蚀刻15秒，或更长的时间，如果必要的。 </li><li>为使漂移校正和iPALM校准，使用＃1.5圆（22毫米直径）嵌入荧光纳米粒子作为基准标记，供应可靠校准和漂移校正的高度光基准预清洁盖玻片。由于积累足够的荧光密度需要很长的采集时间（&gt; 15 - 30分钟），样品漂移是不可避免的。 </li><li>将每个fiducialed玻璃罩到6孔组织培养板。通过紫外线（UV）辐射在层流罩15分钟消毒他们。 </li><li>在无菌层流罩，准备成纤维10微克/毫升 - 通过稀释在无菌的Dulbecco磷酸盐缓冲盐水（DPBS）1mg / ml的纤连蛋白储备溶液至最终浓度2玻璃罩涂层的Nectin溶液。冲洗每个用DPBS玻璃罩三次，并在4℃下与2ml纤连蛋白溶液孵育过夜。接着，吸出纤连蛋白溶液并用DPBS冲洗一次。 </li><li>冲洗细胞简要DPBS。用1孵育 - 2 ml的胰蛋白酶的几分钟，在37℃，直到细胞分离并用约10毫升新鲜含血清细胞培养基的淬灭。例如，对于人脐静脉内皮细胞（HUVEC），用补充有大血管内皮因子和青霉素或链霉素大血管内皮培养基。 <ol><li> Replate细胞到纤连蛋白包被的盖玻片（为稀疏密度，板&lt;每玻璃罩50,000个细胞），并维持培养中设定为95％的湿度，5％CO 2的培养箱，和37＆＃176℃。 </li></ol></li><li>为F-肌动蛋白细胞骨架的精细结构的适当的保存，使用基于良好的缓冲试剂27缓冲溶液。 <ol><li>例如，制备PHEM缓冲液作为2×储液（120毫摩尔PIPES，50mM的HEPES，20mM的EGTA，4mM的MgCl 2的，pH为7.0，用KOH）溶解6.5克的HEPES，3.8克的EGTA，和190毫克的的MgCl 2〜300毫升DDH 2 O的，具有通过逐滴加入浓KOH溶液调节至7.0的pH值;然后，加入DDH 2 O的使体积为500毫升。使用0.22微米的过滤消毒的缓冲区，它保存在4℃，稀释1:使用前DDH 2 O的1。 </li></ol></li><li>用于与F-肌动蛋白的高密度标记最好的结果，使用鬼笔环肽缀合与有机荧光团，如的Alexa Fluor 647在细胞replating后所需的时间点，固定细胞如下: <ol><li>吸从每个文化以及包含CE媒体LL标本。轻轻，但很快免除2ml含在PHEM缓冲0.25％戊二醛与0.25％的Triton X-100温水（37℃）提取固定剂。 2分钟 - 在室温下搅拌1孵育它。对于随后的步骤中，使用2毫升固定液或骤冷缓冲每玻璃罩，除非另有说明。 </li><li>替换在PHEM缓冲2.5％戊二醛固定液中提取固定剂，让样品孵育10 - 12分钟。这和以下步骤在室温下都进行。 </li><li>吸出固定液及轻轻地用PHEM缓冲替换它们。倾斜和旋转轻轻地，然后用PHEM洗一遍。重复两次。 </li><li>以猝灭从戊二醛自发荧光，其可以压倒从所需荧光团信号，以在0.1％在PHEM质量含有浓度加入NaBH 4新鲜制备淬火缓冲器孵育标本。丰富的气泡会被观察到。偶尔抽头的样本盘轻轻DISLodge气泡。让它孵育5 - 10分钟。 </li><li>吸出淬火缓冲区，并轻轻地用PHEM缓冲区替换它。冲洗几次，以清除气泡。吸移管在2ml PHEM缓冲液和让它在黑暗中孵育5分钟。重复两次，完成后让PHEM缓冲标本休息。 </li><li>准备一个湿度室使用大塑料培养皿填充用一张纸巾用5蘸慷慨鬼笔环肽孵化- 10毫升的DDH 2 O的将一大张干净的封口膜对湿纸巾之上。 </li><li>由于鬼笔标记的成本相对较高，使用小体积的每个标签。用于高密度标记，开始以0.3μM的浓度。每次准备在PHEM缓冲用鬼笔环肽的Alexa Fluor 647玻璃罩〜60微升。 </li><li>移液器55 - 60微升的鬼笔解决方案到封口膜板在潮湿室。用细镊子，轻轻取出试样coverg姑娘。要小心，要注意正确的含有细胞的脸。 </li><li>通过触摸与一张折叠微妙的吸水纸的玻璃罩的边缘快速轻轻拍打多余的缓冲，然后将面朝下到上的封口膜毒伞素溶液滴在盖玻片细胞的一面。确保没有被玻璃罩被困气泡。 </li><li>放置在湿度室盖。包裹室铝箔保护它不受环境光，让样品在4℃孵育过夜。可将样品保持在该状态下数天。确保如果长期储存计划湿度室保持湿润。 </li></ol></li><li>成像之前，轻轻玻璃罩细胞面朝上放置到含2ml PHEM缓冲液每孔新的6孔板中。 </li><li>使用下列储备溶液制备氧清除的基于硫醇成像缓冲器:1M的葡萄糖，1M半胱胺，以及100×葡萄糖氧化酶/过氧化氢酶mixtURE（4毫克过氧化氢酶，并在100μlPHEM缓冲液中，通过涡旋充分混合10毫克葡萄糖氧化酶）28。成像前右，混合75微升1M的葡萄糖溶液，1M的半胱胺溶液30微升，和3微升100倍的股票酶混合物中。将音量调节到300微升与PHEM缓冲液和混合装入样品后，立即使用。 </li><li>为iPALM，用预清洁＃1.5玻璃罩（直径22毫米）制备成像样品。 <ol><li>另外，使用玻璃载片（3"×1"）的双面粘合间隔件在组装以有助于如果基于散光-3D-STORM 12将被替代。 </li><li>组装成像样品，用细镊子轻轻取出从缓冲区标本玻璃罩好。然后，很快，轻轻触摸折叠吸水纸玻璃罩的边缘挖掘多余的缓冲区。 </li><li>放在一块干净的镜头纸朝上玻璃罩细胞的一面。冲洗样品通过将30 - 50微升成像缓冲器的样品上，并通过倾斜，并与折叠的吸收纸轻敲除去过量的缓冲液。 </li><li>重复该漂洗步骤几次，然后将30 - 50微升成像缓冲器的样品上。印迹干燥玻璃罩的边缘，并放置快速固化的环氧树脂的多个非常小的点到干燥区。 </li><li>慢慢放下另一预清洗＃1.5玻璃罩（平原，圆形玻璃罩，18毫米直径）到含有细胞的22毫米玻璃罩的中心。让成像缓冲器通过毛细作用润湿两个盖玻片。快速固化环氧树脂的小点要坚持两个盖玻片。 </li><li>轻轻按压在使用折吸水纸均匀地分散压力组装的样本。使用足够的压力以使样品细胞薄而均匀（&lt;15微米），但没有那么多，以粉碎细胞。通过观察牛顿环图案衡量适当的厚度。此外，确保执行此STEP轻轻地减少气泡。如果需要，实行空盖玻片几次事前。 </li></ol></li><li>融化的凡士林，羊毛脂，石蜡密封样品（VALAP，从100备现货G各自凡士林，羊毛脂，石蜡的，熔化在一起）29，冲洗与DDH 2 O的密封的样品，并吹干用压缩空气。样品现在准备安装到显微镜成像。 </li></ol> 2.样品放置和iPALM对齐<ol><li>向上移动至弹簧加载顶物镜，以允许用于去除样品架的。在步骤1.10中制备的密封样品放置到样本保持器，并与几个小稀土类磁铁固定。在成像样品的两侧施加浸油。将样品架回光路，轻轻地降低顶物镜。 </li><li>打开激发激光器。打开电子倍增电荷耦合器件（EMCCD）相机帧传输模式。 <ol><li>旋转在适当的发射滤光片。激活机械快门来阻断顶部光束路径（ 图1A），并打开底部光束路径。通过翻译使用压电致动器小增量带来底部物镜成为关注的焦点。 </li><li>一旦该基准是在焦点，打开顶部光束路径而阻断底部束路径而带来的顶部物镜聚焦以类似的方式。监视计算机显示为最佳聚焦于基准的宽度。 </li></ol></li><li>为适当的中心定位，开放的顶部和底部光束路径。手动调整顶部物镜而底部目标是使用一对微细螺钉保持恒定，直到基准图像被尽可能重叠，理想中的一个像素。 <ol><li>接着，进行精细的调整，使在步骤基准图像2.3的EMCCD像素的十分之一内重叠。调整前通过控制软件2轴压电式后视镜，而同时持有物镜和底部反射镜不变。经由计算机显示屏比较的顶部的基准点和底客观的观点的中心引导过程。 </li></ol></li></ol> 3. iPALM设置校准 注意:由于荧光发射是不连贯的，用于向在iPALM被观测到的干扰，路径长度通过在顶部和底部的目标必须是相互靠近，在几微米之内。这可以实现如下: <ol><li>与上，摄像机连续流，并且两个顶部和底部光束路径开放的激光器，使用由控制软件用于连续z轴振荡产生的正弦电压波形超过400纳米的数量级振荡样品架z-压电。 </li><li>服用的事实，即，出于最佳比对时，基准强度变化不大用叔他振荡，手动由于所需的单光子干涉效应向上或向下，直到基准振荡的强度翻译机动束器组件。这表示的光路长度的紧密匹配。 &gt; 10的峰-谷比可以在最佳的情况下（ 图1D）来实现。 </li><li>确保两个在每个表面上的幅度和相位是尽可能均匀穿过田野，调整光束分离器组件的底部反射镜中的小步骤进行微调的间隙和倾斜角度。超过800纳米转换8纳米Z-步骤样品进行分束器精细对准。 <ol><li>监视中的相机＃1的基准强度 - 3.调整高度，位置，和倾斜的底部反射镜的小步分光镜组件内，以使得相机＃的振荡相位1相对于摄像机＃2被最大化，理想地在120℃（ 图1B-D）。 </li><li>一旦初始调整完成后，翻译样本扫描包含两个邻近的细胞图像和多基准点的视场适宜。将机箱系统周围阻止杂散光和环境扰动。一旦成像区域被发现，进行中的过程步骤3.4再次，并使用命令"获取校准扫描VS样本压电位置"在主界面记录用于后续z坐标提取使用校准曲线。 </li></ol></li></ol> 4.数据采集<ol><li>一旦所希望的区域被发现，并且获得校正曲线，输入相应的文件名转换成该软件。开的顶部和底部光束路径。增加642-nm激光最大的激励力量。对的Alexa Fluor 647，可能需要荧光团的关闭的初始阶段（ 图2A）。 <ol><li>具有恒定642-nm激发5分钟，或者如果需要的话更长暴露，直到SIngle分子闪烁被观察到（ 图2B）。该软件允许在收购过程中的405纳米的光激活一个自动增加。采集帧大批通常都需要对丝状功能清晰可见（&gt; 50000帧）。准备就绪后，在主界面中使用命令"开始iPALM收购"开始采集原始图像集。 </li></ol></li><li>在采集，调通过调整405-nm激光的强度的光活化水平根据需要（参见图2B中的实施例），以保持适当的闪烁密度。 注意:一旦收购完成后，软件会自动将图像文件转换成适当的二进制格式。在计算服务器中的数据存储库被安装作为网络驱动器，允许数据被直接复制有用于进一步处理。 </li></ol> 5.数据处理与分析<ol><li>使用定制开发的软件中提取最佳拟合参数为所有单分子以及用于基准11,15,23进行局部化分析。这产生不仅在x，y坐标，同时也被用于校准曲线分析的强度。 <ol><li>导入使用命令在步骤3.5获得的原始校准数据"提取高峰多个标签"中的"文件"菜单下进行单分子定位。在初始定位分析，坐标从摄像机＃得到的1 - 3存在于红色，绿色和蓝色通道，分别与可使用的指挥下保存用于进一步分析"（的.sav）除耗时为IDL"该"文件"菜单。 </li></ol></li><li>若要从相机＃1把数据- 3到使用嵌入在盖玻片荧光基准注册，选择几个明亮基准为中央图像覆盖（ 例如，图1B）使用命令"锚点基准点"的"图像转换"菜单下。使用三重套定位从摄像机＃1坐标 - 在步骤5.1从明亮基准得到3来计算旋转和缩放矩阵，将带相机＃1和＃3到寄存器与照相机＃2。具有足够大的数目的基准点，可更好地适合来执行高阶多项式翘曲。 </li><li>一旦变换矩阵计算，变换的相机＃1的原始数据 - 3一起，以获得求和的原始数据。执行另一轮定位分析的，以产生一个更精确的x，y坐标，并确定每个摄像机信道向求和原始数据的相对贡献;使用"将原始，保存和另存总和（.DAT）"的指挥下，"图像转换"菜单。此强度比包含z坐标信息。 </li><li>选择一个明亮基准，进行Z-校准使用在弹出的对话框功能"测试风穴3D"通过点击"Z坐标操作"的"特殊功能"菜单下的拟合。这将适合3-正弦函数到3相机通道的强度，以确定校准曲线。然后校准文件被保存用于在主数据集进一步使用。 </li><li>为了测试校准曲线的质量，执行z坐标提取，如前面23所描述。一种用于在井校准系统乖基准，z坐标应线性扩展，因为校准数据集采取在z轴位置的线性扫描。此外，所有的基准点的z位置应与类似的斜率比例。 </li><li>一旦获得满意的校正，对于在步骤4以下步骤5.1-5.3中概述相同的方法获得的原始图像数据集执行定位和变换分析。完成后，加载步骤5中的校准文件。4，进行使用功能"匹克WND文件"和"提取Z坐标"通过点击"Z坐标操作"的"特殊功能"菜单下，在弹出的对话框z坐标提取。 注:由于采集时间&gt; 15分钟，机械漂移是可以预料的。 </li><li>要执行以x漂移校正，Y，请从定位坐标的明亮基准。这个基准应存在于所有的帧。然后，使用X，Y坐标基准漂移在同一框架内对齐所有其他坐标回登记（ 图3A-B）使用功能"测试/写指南之星"的"图像转换"菜单下。 Z坐标登记由同样通过点击"Z坐标操作"的"特殊功能"菜单下访问"测试指南之星"和"写入指南之星"，在弹出的对话框功能来执行。 </li><li>作为样品可以表现出轻微的倾斜，通过点击提供的x，y，3参考点z坐标使用功能在弹出对话框"删除XYZ倾斜"定义应设置电平的平面进行倾斜校正" Z坐标操作特殊功能"菜单中的"下"。 </li><li>继漂移和倾斜校正，节省定位坐标。通过主界面上的"渲染"命令重建作进一步的分析（ 图4A-D）的超级分辨率的图像。颜色可以用来指示z坐标。可替代地，所选择的区域的侧视图也可以呈现。本地化坐标可以导出为文本文件，进一步量化，或者重建图像可以保存为.tif文件。 </li></ol>

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作者什么都没有透露。

iPALM的光学系统是基于一个4π双相对目标设计， 如图1A所示 。设置同时使用定制加工和商业光学机械部件构成，如表 1中所述前面23和列出。除了我们的设置中，霍华德休斯医学研究所（HHMI）举办一个系统，是在高级影像中心，科学界在珍利亚农场研究园区访问。对于整个机械图纸，控制原理图和软件，我们鼓励读者在HHMI与哈拉尔赫斯询问进一步的?...

复杂的细胞结构的可视化一直是不可或缺的生物的见解和发现。虽然荧光显微镜可以形象细胞高分子特异性，其分辨能力被衍射限定在图像平面〜200纳米（X，Y，或横向尺寸）和&gt; 500nm的沿着光轴（z或轴向尺寸） 1,2。因此，超微结构特征观察历来限于电子显微镜（EM）。幸运的是，最近的超分辨率显微镜的发展已经绕过这个限制，使得在10空间分辨率- 100纳米范围1-6。具体地，超分辨率方法的基础上的单分子的定位，由缩略词如PALM（光敏定位显微镜）4，FPALM（荧光光敏定位显微镜） 图5（d）风暴（直接随机光学重构显微术）6,7，涂料（已知宝成像纳米形貌）8 INT积累，GSDIM（基态耗尽显微术，随后由个别分子返程）9，或SMACM（单分子有源控制显微镜）10，以及它们的3维（3D）实现方式中，如干涉PALM（iPALM）11或3D风暴12日 ，在揭示新的见解无数的生物结构的纳米级组织，包括神经元轴突已经有价值和突触13，粘着斑14,15，细胞间的连接处16，核孔17和中心体18-20，仅举几例。 在这超分辨率显微镜是潜在有用的细胞超微结构的另一个特点是肌动蛋白骨架。丝状（F）肌动蛋白在细胞皮质复杂小梁在细胞形状和机械性能21的控制中起重要作用。该组织为OF F肌动蛋白积极并动态调节尽管这强烈地影响聚合，交联，周转率，稳定性，和网络拓扑22众多调节蛋白。然而，尽管F-肌动蛋白小梁结构的特征为机械见解重要成不同范围的细胞过程中，小尺寸（〜8纳米）的f微丝阻碍通过常规衍射限制光镜的观察;因此，肌动蛋白的精细结构的可视化迄今完全由EM执行。在这里，我们描述了可视化的贴壁哺乳动物细胞中的F-肌动蛋白细胞骨架，使用iPALM超分辨率显微镜技术，以利用其精度非常高的能力，在3D 11,23协议。虽然iPALM仪器高度专业化，对设立这样一台仪器指令已经描述了最近23，同时获得由何主办的iPALM显微镜病房休斯医学研究所也已提供给以最小的成本研究团体。此外，本文描述的样品制备方法是直接适用于替代三维超分辨率方法中，如根据点扩散函数（PSF）的象散散焦那些12或双平面检测24，这是更广泛地可用。 我们注意到，在一般的基于定位单分子超分辨率显微镜的必要成分是光开关荧光团25，其允许基于本地化单分子超分辨率显微镜三个重要的要求必须满足:ⅰ）高的单分子亮度和对比度相对于背景信号; II）在给定的图像帧单分子分布稀疏;和iii）高贴标足以捕获底层结构（也称为奈奎斯特沙的轮廓空间密度n非取样标准）26。因此，对于满意的结果，应强调同样在两个试样的适当准备放置优化荧光光控和维护底层超微结构，以及在实验的仪器和采集方面。

<table><tbody><tr><td>optical table</td><td>Newport, CA</td><td>RS4000</td><td>iPALM, installed on 4 Newport Stabilizer vibration isolators</td></tr><tr><td>vibration isolator for optical table</td><td>Newport, CA</td><td>S-2000</td><td></td></tr><tr><td>laser-642</td><td>Newport, CA</td><td>1185055</td><td>output power=100 mw</td></tr><tr><td>laser-561</td><td>Newport, CA</td><td>1168931</td><td>output power=200 mw</td></tr><tr><td>laser-488</td><td>Newport, CA</td><td>1137970</td><td>output power=200 mw</td></tr><tr><td>laser-405</td><td>Newport, CA</td><td>1142279</td><td>output power=100 mw</td></tr><tr><td>broadband dielectric mirrors</td><td>Thorlabs, NJ</td><td>BB1-E02</td><td>laser combiner</td></tr><tr><td>dichroic beamsplitter</td><td>Semrock, NY</td><td>LM01-427-25</td><td></td></tr><tr><td>acousto-optic tunable filter</td><td>AA Opto-Electronic, France</td><td>AOTFnC-VIS-TN</td><td></td></tr><tr><td>Linear polarizer</td><td>Newport, CA</td><td>05LP-VIS-B</td><td></td></tr><tr><td>baseplate</td><td>local workshop</td><td>customized</td><td></td></tr><tr><td>turning mirror (22.5&amp;deg;)</td><td>Reynard Corpporation, CA</td><td>customized</td><td>22.5&amp;deg; mirror</td></tr><tr><td>motorized optic mounts</td><td>New Focus, CA</td><td>8816</td><td></td></tr><tr><td>motorized XYZ translation stage</td><td>Thorlabs, NJ</td><td>MT3/M-Z6</td><td>sample holder</td></tr><tr><td>T-Cube DC servo motor controller</td><td>Thorlabs, NJ</td><td>TDC001</td><td></td></tr><tr><td>Piezo Phase Shifter</td><td>Physik Instrumente, Germany</td><td>S-303.CD</td><td></td></tr><tr><td>objective lens</td><td>Nikon, Japan</td><td>MRD01691</td><td>objective. Apo TIRF 60X/1.49 oil</td></tr><tr><td>translation stage</td><td>New Focus, CA</td><td>9062-COM-M</td><td></td></tr><tr><td>Pico Motor Actuator</td><td>New Focus, CA</td><td>8301</td><td></td></tr><tr><td>rotary Solenoid/Shutter</td><td>DACO Instruments, CT</td><td>5423-458</td><td></td></tr><tr><td>3-way beam splitter</td><td>Rocky Mountain Instruments, CO</td><td>customized</td><td>beamsplitter</td></tr><tr><td>Piezo Z/Tip/Tilt scanner</td><td>Physik Instrumente, Germany</td><td>S-316.10</td><td></td></tr><tr><td>motorized five-axis tilt aligner</td><td>New Focus, CA</td><td>8081</td><td></td></tr><tr><td>Picmotor ethernet controller</td><td>New Focus, CA</td><td>8752</td><td></td></tr><tr><td>Piezo controllers/amplifier/digital operation module</td><td>Physik Instrumente, Germany</td><td>E-509/E-503/E-517</td><td></td></tr><tr><td>band-pass filter</td><td>Semrock, NY</td><td>FF01-523/20</td><td>filters</td></tr><tr><td>band-pass filter</td><td>Semrock, NY</td><td>FF01-588/21</td><td></td></tr><tr><td>band-pass filter</td><td>Semrock, NY</td><td>FF01-607/30</td><td></td></tr><tr><td>band-pass filter</td><td>Semrock, NY</td><td>FF01-676/37</td><td></td></tr><tr><td>notch filter</td><td>Semrock, NY</td><td>NF01-405/488/561/635</td><td></td></tr><tr><td>motorized filter wheel with controllter</td><td>Thorlabs, NJ</td><td>FW103H</td><td></td></tr><tr><td>EMCCD</td><td>Andor, UK</td><td>DU-897U-CSO-#BV</td><td>3 sets</td></tr><tr><td>Desktop computers for controlling cameras and synchronization</td><td>Dell</td><td>Precision T3500</td><td>PC, 4 sets</td></tr><tr><td>coverslips with fiducial</td><td>Hestzig, VA</td><td>600-100AuF</td><td>sample preparation. fiducial marks with various density and spectra available</td></tr><tr><td>fibronectin</td><td>Millipore, MT</td><td>FC010</td><td></td></tr><tr><td>paraformaldehyde</td><td>Electron Microscopy Sciences, PA</td><td>15710</td><td>fixation. 16%</td></tr><tr><td>glutaraldehyde</td><td>Electron Microscopy Sciences, PA</td><td>16220</td><td>25%</td></tr><tr><td>triton X-100</td><td>Sigma aldrich, MO</td><td>T8787</td><td></td></tr><tr><td>HUVEC cells</td><td>Life Technologies, CA</td><td>C-015-10C</td><td></td></tr><tr><td>Medium 200</td><td>Life Technologies, CA</td><td>M-200-500</td><td></td></tr><tr><td>Large Vessel Endothelial Factors</td><td>Life Technologies, CA</td><td>A14608-01</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td></td><td>14190367</td><td></td></tr><tr><td>Pennicillin/Streptomycin</td><td></td><td>15140122</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life Technologies, CA</td><td>25200056</td><td></td></tr><tr><td>PIPES</td><td>Sigma aldrich, MO</td><td>P1851</td><td>PHEM</td></tr><tr><td>HEPES</td><td>1st base, Malaysia</td><td>BIO-1825</td><td></td></tr><tr><td>EGTA</td><td>Sigma aldrich, MO</td><td>E3889</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Millipore, MT</td><td>5985</td><td></td></tr><tr><td>Alexa Fluor 647 Phalloidin</td><td>Invitrogen, CA</td><td>A22287</td><td>staining</td></tr><tr><td>sodium borohydride (NaBH&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma aldrich, MO</td><td>480886</td><td>quenching</td></tr><tr><td>glucose</td><td>1st base, Malaysia</td><td>BIO-1101</td><td>imaging buffer</td></tr><tr><td>glucose oxidase</td><td>Sigma aldrich, MO</td><td>G2133</td><td></td></tr><tr><td>catalase</td><td>Sigma aldrich, MO</td><td>C9322</td><td></td></tr><tr><td>cysteamine</td><td>Sigma aldrich, MO</td><td>30070</td><td></td></tr><tr><td>Epoxy</td><td>Thorlabs, NJ</td><td>G14250</td><td></td></tr><tr><td>vaseline</td><td>Sigma aldrich, MO</td><td>16415</td><td>sample sealing</td></tr><tr><td>lanolin</td><td>Sigma aldrich, MO</td><td>L7387</td><td></td></tr><tr><td>parafin wax</td><td>Sigma aldrich, MO</td><td>327204</td><td></td></tr><tr><td>Immersion oil</td><td>Electron Microscopy Sciences, PA</td><td>16915-04</td><td>imaging. Cargille Type HF</td></tr></tbody></table>

three-dimensional super resolution microscopy of f-actin filaments by interferometric photoactivated localization microscopy (ipalm)

荧光显微镜使细胞内的特定生物分子的直接可视化。但是，对于以往的荧光显微镜，空间分辨率由衍射图像平面和&gt; 500nm的沿着光轴内限制为〜200纳米。其结果是，荧光显微镜早已严重中的超微结构特征的细胞内观察的限制。最近的超分辨率显微镜方法发展克服了这一限制。特别是，光开关的荧光团的到来使基于与定位超分辨率显微镜，其提供接近分子长度尺度的分辨率。在这里，我们描述了基于单分子定位显微镜和多相干涉三维超高分辨率显微镜方法，称为干涉光敏定位显微镜（iPALM）的应用程序。这种方法提供了几乎各向同性分辨率为20nm顺序在所有三个维度。可视化丝状肌动蛋白骨架，其中包括iPALM仪器的样品制备和操作方案，如下所述。这些协议也容易适应，并启发对其他超微结构细胞研究。

对于iPALM关键要求是对准，配准，并且光学系统的校准。这些是必要的，以确保3路分束器位于必要的适当的干扰为z坐标萃取。要启用连续监测，荧光不断的点源是必要的。这可以通过使用荧光Au或双金属纳米粒子23的光致发光从局域型表面等离子体共振（LSPR）出现而实现。它们作为在照明用的稳定的单偶极子和通常可以用5本地化 - 10纳米的精度。这些可商购的纳?...

Watch this Scientific Journal Video about Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM at JoVE.com

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

我们提出了一个协议，用于干涉式光活化的定位显微镜（iPALM），3维单分子定位超分辨率显微镜方法的应用，以便在贴壁哺乳动物细胞的肌动蛋白细胞骨架的成像。这种方法允许的纳米结构特征，否则将通过常规的衍射限制的光学显微镜仍未解决基于光的可视化。

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 ...

three-dimensional-super-resolution-microscopy-f-actin-filaments

Research

JoVE Journal

Biology

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

10.7K 次观看.  South University of Science and Technology of China, Shenzhen. 我们提出了一个协议，用于干涉式光活化的定位显微镜（iPALM），3维单分子定位超分辨率显微镜方法的应用，以便在贴壁哺乳动物细胞的肌动蛋白细胞骨架的成像。这种方法允许的纳米结构特征，否则将通过常规的衍射限制的光学显微镜仍未解决基于光的可视化。 

视频: Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

科研

生物学

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

免疫与感染

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

生物化学

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

利用微流体和荧光显微镜研究单根肌动蛋白丝和束的组装动力学

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

细菌细胞的三维成像，实现精确的细胞表现和精确的蛋白质定位

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

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 ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

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

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

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

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

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 ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells