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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>optical table</td>
			<td>Newport, CA&#160;</td>
			<td>RS4000</td>
			<td>iPALM, installed on 4 Newport Stabilizer vibration isolators&#160;</td>
		</tr>
		<tr>
			<td>vibration isolator for optical table</td>
			<td>Newport, CA&#160;</td>
			<td>S-2000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>laser-642</td>
			<td>Newport, CA&#160;</td>
			<td>1185055</td>
			<td>output power=100mw</td>
		</tr>
		<tr>
			<td>laser-561</td>
			<td>Newport, CA&#160;</td>
			<td>1168931</td>
			<td>output power=200mw</td>
		</tr>
		<tr>
			<td>laser-488</td>
			<td>Newport, CA&#160;</td>
			<td>1137970</td>
			<td>output power=200mw</td>
		</tr>
		<tr>
			<td>laser-405</td>
			<td>Newport, CA&#160;</td>
			<td>1142279</td>
			<td>output power=100mw</td>
		</tr>
		<tr>
			<td>broadband dielectric mirrors&#160;</td>
			<td>Thorlabs, NJ</td>
			<td>BB1-E02</td>
			<td>laser combiner</td>
		</tr>
		<tr>
			<td>dichroic beamsplitter&#160;</td>
			<td>Semrock, NY</td>
			<td>LM01-427-25</td>
			<td></td>
		</tr>
		<tr>
			<td>acousto-optic tunable filter&#160;</td>
			<td>AA Opto-Electronic, France</td>
			<td>AOTFnC-VIS-TN</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear polarizer</td>
			<td>Newport, CA&#160;</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&#176;)</td>
			<td>Reynard Corpporation, CA</td>
			<td>customized</td>
			<td>22.5&#176; mirror</td>
		</tr>
		<tr>
			<td>motorized optic mounts&#160;</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/1.49oil</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&#160;</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&#160;</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&#160; available</td>
		</tr>
		<tr>
			<td>fibronectin&#160;</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&#160;</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&#39;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>MgCl2</td>
			<td>Millipore, MT</td>
			<td>5985</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Alexa Fluor 647 Phalloidin</td>
			<td>Invitrogen, CA</td>
			<td>A22287</td>
			<td>staining</td>
		</tr>
		<tr>
			<td>sodium borohydride (NaBH4)&#160;</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&#160;</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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil&#160;</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)

10.6K 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

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

纳米有限元分析:蛋白质定位的光激活定位显微镜和电子显微镜

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

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 ...

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

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM 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)

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 ...

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 temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

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

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

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

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

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 ...

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

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

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 ...

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

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

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 ...