我们感谢蒂尔·贝克宁博士为原来的单分子制备协议。
这项工作得到了全国社保基金赠款1121972（SS）的支持。

1. 化学制备
<ol><li>PLL-g-PEG-生物素和阿维丁的制备和储存:<ol>
<li>根据制造商的指示，在浓度为 1.0 毫克/毫升时，在 PBS 缓冲区中溶解聚合物 PLL-g-PEG-生物素。</li>
		<li>阿里报价根据盖片大小（见 表1），并存储在-80°C长达一年。</li>
	</ol>
</li>
	<li>阿维丁/纽特拉丁的准备和储存<ol>
<li>根据制造商的指示，在浓度为 1.0 毫克/毫升的情况下，在 PBS 缓冲区中溶解未贴标签的阿维丁/纽特拉丁。</li>
		<li>阿里报价根据盖滑大小（见 表1），并存储在-20°C长达3年。</li>
	</ol>
</li>
	<li>病毒样本准备:在此检测之前，根据实验需要和实验室协议准备病毒样本。方法 5A 使用步骤 1.3.1，方法 5B 使用 1.3.2。<ol>
<li>在第 2 阶段之前为方法 5A 准备病毒样本。在制备后1-2天内利用准备好的病毒样本，或根据盖滑尺寸（见 表1）引用，然后在液氮中闪烁冻结，储存在-80°C长达一年。解冻后，立即使用。</li>
		<li>为类似于 1.3.1 的方法 5B 准备病毒样本。在5B之前解冻病毒，在与抗体混合之前，在4°C下储存不超过24小时。</li>
	</ol>
</li>
	<li>为实验准备适当的生物素化抗体。</li>
</ol>2. 化学处理准备中的盖滑清洁
<ol><li>将盖片放在一个适当大小的特氟隆盖滑架中，将它们保持垂直方向，并将其浸入装满过滤过的（0.2 μm）乙醇（190证明）的烧嘴中。</li>
	<li>将过滤过的乙醇（190证明）中的盖片酸化30分钟。</li>
	<li>从酒精中取出盖片，用超纯水广泛冲洗盖片。</li>
	<li>将冲洗盖片放在一个单独的清洁特氟隆架和装满 1 M NaOH 的烧嘴中。</li>
	<li>在1 M NaOH中将盖片索尼酸盐30分钟。</li>
	<li>从 NaOH 中取出盖片，用超纯水广泛冲洗。</li>
	<li>用氮流干燥冲洗盖片，并将它们放在干净干燥的特氟隆架中。</li>
	<li>检查盖片上是否存在任何可见膜或颗粒。如果仍有任何可见的材料，盖片尚未得到适当清洁和冲洗。如果未正确清洁重复步骤 2.1-2.7。</li>
	<li>用氧气等离子体清洁盖片2分钟。此步骤是可选的，但建议。</li>
</ol>3. PLL-g-PEG 薄膜层的形成
<ol><li>氮流（或可选的氧等离子体）干燥后，立即将一个盖片水平放置在无菌的培养皿中。在盖子的中心，管道可以适当解冻 PBS 缓冲 PLL-g-PEG-生物素（见表 1）。</li>
	<li>用三明治方法将另一个清洁的盖子放在液体上。请注意，此方法包括让两个盖片通过将化学处理的"活性面"定向相互孵化其化学薄膜，仅由当前的化学层分离。确保有足够的液体，使两个盖片之间的体积完全充满液体，但不得从盖片之间泄漏液体（见表1）。此后，"活动面"指定与夹心式配置中的流体接触的盖唇面。</li>
	<li>将培养皿的盖子放在盖唇三明治上，在 RT 孵化 45-60 分钟。</li>
	<li>取下培养皿的盖子，用一对钳子拿起三明治。小心不要捏出多余的液体。使用拇指和食指轻轻捏三明治，并水平滑动封面，彼此朝相反的方向滑动，然后将两个盖片彼此分开，而无需触摸活动面。</li>
	<li>用超纯水冲洗两个活动面，将 25 毫升超纯水管道输送到活动面 2-4 倍，然后用氮流擦干盖片。请务必注意哪些遮盖面是活跃的。</li>
	<li>立即使用盖片，或将 O/N 与活动面朝上存放在无菌的 Petri 菜中，置于无尘低湿度环境中。</li>
</ol>4. 阿维丁绑定增强
<ol><li>将经过处理的 PLL-g-PEG 盖唇与主动面朝上放在无菌的培养皿中。</li>
	<li>在缓冲区（以步骤 1.2 制备）中适当数量的解冻阿维丁到活动面的中间。</li>
	<li>在三明治方法中的液体上放置另一个盖子（向下活动面）。注意盖片的活跃面。在孵化过程中，活动面必须与流体接触（参见步骤 3.2）。</li>
	<li>将培养皿的盖子放在盖唇三明治上，在 RT 孵化 25-30 分钟。</li>
	<li>取下Petri盘盖，并按步骤 3.4 描述的两个盖子唇部分开，确保跟踪并不触摸活动面。</li>
	<li>用超纯水冲洗两个活动面，将 25 毫升超纯水输送到活动面 2-4 倍，然后用氮流擦干盖片。注意盖片的活跃面。立即用于下一阶段。</li>
</ol>重要提示:根据实验需要的检测类型，有两种不同的方法可以完成样品准备。实验者应进行第5A阶段，在添加超分辨率成像所需的任何额外的抗体治疗之前，将病毒锚定在玻璃上。5B 描述了一种替代方法，在将病毒固定在玻璃上之前，在溶液中标记病毒。本手稿中的代表性数据使用 5A 编写。应根据实验分析类型选择适当的方法。
5A. 活性面部抗体系绳
<ol><li>在阶段 4.6 中用氮流干燥后，立即将经过 Avidin 处理的盖唇活面放在无菌的培养皿中。</li>
	<li>在缓冲器中将适量解冻的生物基化抗体放在活动面的中间（见表1）。</li>
	<li>用三明治方法将另一个盖子（向下活动面）放在液体上。注意盖片的活跃面，重要的是活动面与培养液接触（参见步骤 3.2.）。</li>
	<li>将培养皿的盖子放在盖唇三明治上，在 RT 孵化 45-60 分钟。</li>
	<li>取下Petri盘盖，并按步骤 3.4 描述的两个盖子唇部分开，确保跟踪并不触摸活动面。</li>
	<li>用 PBS 冲洗两个活动面，通过在活动面上管道 25 毫升 PBS 2-4 倍，不要干燥并立即使用。请务必跟踪哪些遮盖面处于活动状态。</li>
	<li>将经过抗体处理的盖唇活面放在无菌的Petry盘中，并将适量解冻的病毒放在活性面的中间。</li>
	<li>用三明治方法将另一个盖子（向下活动面）放在液体上。注意活动面，因为活动面与流体接触进行孵化非常重要（参见步骤 3.2.）。</li>
	<li>将培养皿的盖子放在盖唇三明治上，在 RT 孵化 45-60 分钟。</li>
	<li>取下Petri盘盖，并按步骤 3.4 描述的两个盖子唇部分开，确保跟踪并不触摸活动面。</li>
	<li>用 PBS 冲洗两个活动面，在活动面上管道 25 毫升 PBS 2-4 倍。不要擦干盖片，而是将它们放在特氟隆架和装满 PBS 的烧嘴中。请务必跟踪哪些遮盖面处于活动状态。</li>
	<li>立即使用准备好的样品或在 4 °C 的适当缓冲区存储长达三天，而不会显著丧失完整性。 重要注:如果样品要用于 AFM 检测，则制备已完成，可立即使用或存储步骤 5A.20 - 5A.21 中描述的样本。如果样品要用于超分辨率荧光成像检测，则使用步骤 5A.12 - 5A.19 中描述的抗体标记。</li>
	<li>将两种经过病毒处理的盖片活面朝上放在无菌的Petry盘中，并在盖片上吹出足够的蛋白质阻塞缓冲液，以达到足够的液体体积，覆盖盖片中央95%的部位，而不会将任何阻塞缓冲液从盖片上流出（通常为100- 200 μl）。在 RT 孵化 60-90 分钟。</li>
	<li>小心地去除阻塞缓冲区，每次从培养皿中取出一个盖片，并将盖片上的液体倒入废烧杯中。小心不要掉盖子。</li>
	<li>用 PBS 冲洗两个活动面，通过在活动面上管道 25 毫升 PBS 2-4 倍，不要干燥并立即使用。请务必跟踪哪些遮盖面处于活动状态。</li>
	<li>将单个阻塞缓冲区处理的盖片活面放在无菌的Petry盘中，并在缓冲液中将适量的荧光标记病毒抗体放在活动面的中间。</li>
	<li>将另一个盖滑（活性面向下）放在三明治方法的液体顶部。请注意哪个面部是活跃的，重要的是活动面与流体接触进行孵化（参见步骤 3.2）。</li>
	<li>将培养皿的盖子放在盖唇三明治上，在 RT 孵化 30 分钟。</li>
	<li>取下Petri盘盖，并按步骤 3.4 描述的两个盖子唇部分开，确保跟踪并不触摸活动面。</li>
	<li>用 PBS 冲洗两个活动面，通过在活动面上管道 25 毫升 PBS 2-4 倍，不要干燥并立即使用。请务必跟踪哪些遮盖面处于活动状态。</li>
	<li>不要擦干盖片，而是将其放在装满 PBS 的烧嘴内的特氟隆架中。请务必跟踪哪些遮盖面处于活动状态。</li>
	<li>立即使用准备好的样品或在 4 °C 的适当缓冲区存储长达三天，而不会显著丧失完整性。</li>
</ol>5B.在溶液抗体攻击中
此方法可选增强超分辨率荧光成像的标记策略。通过应用溶液攻击，可以在病毒表面实现更好的抗体涂层。
<ol><li>根据实验需要将抗体溶液按比例混合。示例 1:4 0.01 毫克/毫升生物基化抗体与 0.01 毫克/毫升荧光标记抗体的比例将具有良好的结合亲和力，并允许在超分辨率成像检测中恢复良好的信封。</li>
	<li>将抗体和病毒溶液的等份量分别混合在 （5B.1） 和 （1.3） 中。通过在别名中上下吹几次轻轻混合。此由此产生的解决方案可以存储，直到需要长达 24 小时，在 4 °C。</li>
	<li>完成第 4.6 步后，将经过艾维丁处理的盖唇与活面向上放在无菌的培养皿中。</li>
	<li>在活动面的中间，用抗体溶液（在步骤 5B.2 中制备）的病毒量适中。</li>
	<li>用三明治方法将另一个盖子（向下活动面）放在液体上。注意活动面，因为活动面与流体接触进行孵化非常重要（参见步骤 3.2）。</li>
	<li>将培养皿的盖子放在盖唇三明治上，在 RT 孵化 45-60 分钟。</li>
	<li>取下Petri盘盖，并按步骤 3.4 描述的两个盖子唇部分开，确保跟踪并不触摸活动面。</li>
	<li>用 PBS 冲洗两个活动面，在活动面上管道 25 毫升 PBS 2-4 倍。不要擦干盖片，而是将它们放在特氟隆架和装满 PBS 的烧嘴中。请务必跟踪哪些遮盖面处于活动状态。</li>
	<li>立即使用准备好的样品或在 4 °C 中储存长达三天，而不会严重丧失完整性。</li>
</ol>6. 原子力显微镜材料属性测量
<ol><li>打开自动对焦激光并打开自动对焦软件。从打开的对话中选择适当的扫描模式，以进行所需的检测。默认情况下，所有必要的窗口都应打开:如果不咨询用户手册以打开所需的窗口。</li>
	<li>选择适合所需测量的悬臂，并根据制造商指南将其插入悬臂外壳中。有各种各样的条件和实验类型，有各种各样的罐装机。实验者应该根据他们的需要研究坎蒂勒弗。</li>
	<li>根据制造商的指南，将悬臂外壳插入 AFM 头。</li>
	<li>如果样品在潮湿条件下使用，则从存储缓冲区中取出样品，然后继续步骤 6.7。</li>
	<li>如果样品在环境（干燥）条件下使用，则从其存储缓冲区中取出样品，并浸入超纯水烧嘴中短暂冲洗。注意:这将在样品上留下一个薄的水化层，这可能会诱发毛细管的吸引力与花冠具有低弹簧常数。</li>
	<li>如果样品要在环境（干燥）条件下使用，则用氮流轻轻干燥样品。</li>
	<li>用无尘滤纸轻轻干燥盖片的背面，确保不接触样品的主动面。</li>
	<li>将样品放在适当的 AFM 样品支架中，然后将样品放在 AFM 阶段。</li>
	<li>如果样品要在潮湿条件下进行成像，则将少量缓冲液移到样品中心（~10 μl）。</li>
	<li>将自动对焦头放在样品上。</li>
	<li>如果样品是湿的，请确保悬臂和 AFM 尖端穿透表面层，并且悬臂和悬臂外壳之间没有气泡。</li>
	<li>根据制造商的建议，将 AFM 激光器与悬臂对齐，以获得最佳信号。</li>
	<li>测量悬臂共振频率，并根据实验类型设置扫描频率。</li>
	<li>将自动对焦器尖端降低到表面并优化信号设置。</li>
	<li>以您选择的方式执行 20μm 方形 AFM 扫描。在生成代表性数据时使用了点击 （AC） 模式。按大致高度识别病毒候选者，这通常可视为玻璃表面的尖刺。</li>
	<li>选择病毒候选者并将其上方的扫描头放在中心位置。</li>
	<li>对所选病毒进行 250 nm 方形扫描（或适当的超分辨率扫描），以获取其拓扑学和方向。</li>
	<li>选择病毒点上的材料属性测量（例如 球形病毒的中心为杨的模组测量），并根据制造商的方向进行测量。</li>
</ol>7. 超分辨率成像方法
<ol><li>打开超分辨率成像软件，并根据制造商的指南校准软件。</li>
	<li>使用荧光珠根据制造商的指示校准超分辨率成像设备。</li>
	<li>通过将 50 μl 的 1 M MEA 和 100 μl 的 10 倍 Gloxy 组合到 850 μl 的库存缓冲器，从成像前的库存中组装样品成像缓冲器。在实验期间保持冰上的成像缓冲。</li>
	<li>从存储缓冲区取出带有样品的预制盖滑，通过浸入超纯水烧嘴中冲洗 5 倍。</li>
	<li>使用无灰尘滤纸干燥非活性盖唇面。确保不要触摸主动面。</li>
	<li>插入成像系统示例支架，并将 250 μl 的成像缓冲器添加到示例支架中。每 2 小时交换一次样品上的成像缓冲器以保持一致的结果是合理的。</li>
	<li>用胶片覆盖样品架，以减少与房间环境的大气相互作用。</li>
	<li>将样品支架放入成像设备中进行成像。</li>
	<li>利用制造商的指示将样品集中到焦点中。</li>
	<li>根据采用的超分辨率技术对样品进行成像。请务必调整成像条件以优化信号，同时减少可光开关荧光素的同步激活。 重要注:成像条件取决于样品，并根据实验需要而变化。然后，利用 Alexa 647 在成像缓冲区高效初始化到其黑暗状态的典型实验可以通过 405 nm UV 光的应用进行照片激活。图像通过在密集标记的样品中激发Alexa 647分子的稀疏子集获得，并定位每个氟磷，其精度主要受收集光子数量的限制。此程序由软件反复重复，直到出现所需的收购周期数。实验者应该将此设置与样本中已进行照片漂白的每个氟磷相关。</li>
	<li>成像完成后，根据制造商的指示关闭成像设备。该软件可能留待数据分析。</li>
	<li>数据分析在很大程度上依赖于实验:然而，在所有情况下，通过最大宽度一半来识别病毒，这与已知的病毒维度进行比较，确保考虑到荧光标签的额外大小。</li>
	<li>为了更好地可视化，使用 Isosurface 渲染选项或体积渲染选项查看样本，这两种选项通常都位于超分辨率成像软件中。</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-High+Resolution+Imaging+by+Fluorescence+Photoactivation+Localization+Microscopy.">Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy.</a> Biophys. J. 91, 4258-4272 (2006).</li><li>Juette, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+sub-100+nm+resolution+fluorescence+microscopy+of+thick+samples.">Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples.</a> Nat. Methods. 5, 527-529 (2008).</li><li>Xu, K., Zhong, G., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin,+Spectrin,+and+Associated+Proteins+Form+a+Periodic+Cytoskeletal+Structure+in+Axons.">Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in Axons.</a> Science. 339, 452-456 (2013).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Super-Resolution+Imaging+by+Stochastic+Optical+Reconstruction+Microscopy.">Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy.</a> Science. 319, 810-813 (2008).</li><li>Hodges, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+packaging+of+polymerases+within+vesicular+stomatitis+virus.">Asymmetric packaging of polymerases within vesicular stomatitis virus.</a> Biochemical and Biophysical Research Communications. 440, 271-276 (2013).</li><li>Lawson, N. D., Stillman, E. A., Whitt, M. A., Rose, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+vesicular+stomatitis+viruses+from+DNA.">Recombinant vesicular stomatitis viruses from DNA.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 4477-4481 (1995).</li></ol>

作者在本文中没有相互竞争的经济利益。

采用 AFM 和高分辨率荧光成像的单病毒成像可用作 CryoEM 断层扫描的替代方法。这些方法中的每一个都有其特定的优势。例如，AFM 可以在 WT 病毒上完成，无需标记样本或内部病毒蛋白。病毒结构的位置与它们对病毒弹性特性的贡献不一致。
单病毒超分辨率成像与新开发的病毒反向遗传方法相结合，成为本地化低拷贝数病毒蛋白20的有力技术。重组病毒，用与荧光蛋白融合的蛋白质取代蛋白质，可以利用这些反向遗传方法制造和纯化。虽然超分辨率成像具有特异性，部分由于遗传标记，它需要使用突变病毒。
AFM 和超分辨率成像是利用非常相似的样品制剂的免费方法。本文概述的方法，采用早期单分子成像检测的形式，允许锚定单病毒，对病毒的拓扑学影响甚微。

基于电荷的非特异性相互作用通常用于原子力显微镜1，2中的病毒粘附。这些技术特别工作得很好，当用于非封顶的病毒与非常僵硬的辣椒3-6。虽然这些技术非常有效地固定样品，但它们不能防止蛋白质与表面的非特异性结合。当尝试使用 AFM 和超分辨率荧光技术对病毒进行成像时，非特定结合可能会产生问题，这些技术需要用各种抗体孵化样品。在这里，我们概述了特定固定病毒的样品准备方法。
聚（乙二醇）（PEG）嫁接到聚（L-赖氨酸）（PLL）和吸收到玻璃表面提供了一个重要的块蛋白质与玻璃7静电相互作用。单分子检测需要在玻璃表面固定单分子，利用这一特性，并用它来创建一个特定的PEG为基础的单分子固定技术8-10。这种制备还用于固定在玻璃表面11的克拉斯林笼，以及创建一个同质纤维素涂层控制细胞粘附12。
我们采用了基于PLL-g-PEG吸附到单分子成像方法的玻璃表面的样品制备方法，并将其应用于血管口腔炎病毒（VSV）的单病毒成像。这些单一的病毒是使用原子力显微镜（AFM）成像的。类似的实验也进行了功能化珠作为控制。病毒还通过超分辨率荧光显微镜成像，这是一种标有Alexa 647的VSV-G抗体，用于创建单病毒信封的图像。高分辨率荧光成像利用单分子的本地化来创建图像13-15。fPALM双平面成像允许在平面上定位20纳米和沿光轴15，16分辨率为50纳米的单分子。这种双平面技术用于对本研究中存在的超分辨率荧光图像进行成像。另一种具有类似结果的技术是 STORM13，17，18。
通过本文概述的程序锚定在玻璃上的 VSV 的 AFM 和超分辨率荧光图像都显示了具有最小非特定相互作用的玻璃具有特定的粘性。在这里，我们介绍自动对焦和超分辨率荧光成像实验的样品准备方案。简言之:清洁玻璃盖通过吸食PLL-g-PEG和PLL-g-PEG-生物素的混合物而功能化。这种薄膜在涂层表面上提供15-25%的功能化生物素是典型的。盖片进一步孵育四聚丁。然后，生物基化病毒抗体用于为病毒创建一个独特的结合点。抗体的结合可以通过两种方式进行。
第一种方法是优化为自动对焦，但它仍然适合超分辨率荧光成像。在这种方法中，在病毒粘附之前，用生物素化抗体处理阿维丁涂层表面。固定的病毒用Alexa 647标记的抗体处理，以覆盖信封，并允许病毒的超分辨率荧光成像。
第二种方法是在将病毒粘附到所处理表面之前，用生物基化抗体和Alexa 647标记抗体在溶液中攻击病毒:该方法用于超级分辨率荧光成像实验进行优化，其中病毒包络的恢复非常重要。这种方法的优点是允许在病毒表面涂上均匀的抗体涂层。生物素化抗体可能与 Alexa 647 标记的病毒抗体混合，其比例允许将病毒充分吸附到所处理的 avidin 表面，同时在病毒包络外部保持高浓度的荧光标签。这些Alexa 647标记的病毒抗体不生物素化，其多余的可以冲洗掉，以减少背景噪音。

<table><tbody><tr><td>Glass coverslips (fPALM)</td><td>Electron Microscopy Services</td><td>72225-01</td><td>25 mm Coverslips</td></tr><tr><td>PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-Biotin (20%)&amp;nbsp;</td><td>SuSoS</td><td>-</td><td>Stock: 0.5 mg/ml in PBS</td></tr><tr><td>NeutrAvidin biotin-binding protein</td><td>Invitrogen</td><td>A2666</td><td>Stock: 0.25 mg/ml in PBS</td></tr><tr><td>Alexa Fluor 647 goat anti-rabbit IgG (H+L)&amp;nbsp;</td><td>Invitrogen</td><td>A21245</td><td>1:200 in 0.2 M KPO&lt;sub&gt;4&lt;/sub&gt;, 0.15 M NaCl, 10% Glycol, pH 7.2 buffer</td></tr><tr><td>&amp;alpha;-VSV-G&amp;nbsp;</td><td>Invitrogen</td><td>ab34774</td><td>1:200 in 0.2 M KPO&lt;sub&gt;4&lt;/sub&gt;, 0.15 M NaCl, 10% Glycol, pH 7.2 buffer</td></tr><tr><td>Gluox</td><td>Sigma</td><td>G2133-250KU</td><td>Glucose oxidase type seven from Aspergillius&amp;nbsp;</td></tr><tr><td>Catalase</td><td>Sigma</td><td>C40-100mg</td><td>Catalase from Bovine liver&amp;nbsp;</td></tr><tr><td>MEA</td><td>Sigma</td><td>30070-10G</td><td>Cysteamine (MEA</td></tr><tr><td>Stock Buffer</td><td></td><td></td><td>50 mM Tris-HCl (pH 8.0) + 10 mM NaCl + 10% glucose</td></tr><tr><td>NTE</td><td></td><td></td><td>10 mM Tris pH 7.4, 100 mM NaCl, 66 mM EDTA</td></tr><tr><td>Slide-A-Lyzer, mini dialysis unit</td><td>Thermo Scientific</td><td></td><td>10,000 MW</td></tr><tr><td>Microscope Cover Glass: 35 CIRCLE #1</td><td>Fisherbrand</td><td>35 CIRCLE #1</td><td></td></tr></tbody></table>

sample preparation for single virion atomic force microscopy and super-resolution fluorescence imaging

将病毒固定到玻璃表面是单一病毒成像的关键步骤。在这里，我们介绍了一种从单分子成像检测中采用的技术，该技术允许单个病毒粘附到具有特异性的玻璃表面。这种制备基于用PLL-g-PEG和PLL-g-PEG-生物素的混合物嫁接玻璃表面，加入一层阿维丁，最后通过附着生物基化病毒特异性抗体产生病毒锚。我们在一系列实验中应用了这项技术，包括原子力显微镜 （AFM） 和超分辨率荧光成像。此样品制备方法导致病毒对表面的控制粘附。

使用 AFM 的单病毒成像:
上面概述的样品制备方案用于将野生型病毒锚定到玻璃表面。VSV 病毒是子弹形状的 180 nm 长和直径 80 nm。由于有多种病毒，这种技术可以应用，这个概念也在这里证明在生物基化36纳米珠。由此产生的AFM实验显示在 图1中。值得注意的是，与非病毒 （GPa） 相比，VSV 病毒的杨氏模式 （100 MPa） 较低。单病毒 VSV 的特定图像是在具有僵硬悬臂的环境条件下在攻丝模式下获得的。其目的是使病毒变形，使病毒内额外的蛋白质密度在 AFM 图像中作为凹凸可见。这种方法用于检测病毒腔19内的额外蛋白质密度。
单病毒超分辨率成像:
Alexa 647 标记 VSV-G 抗体用于使用方法 5A 涂覆单个 VSV 病毒的信封，用于超分辨率实验。为了演示系绳密度和低非特定绑定，在 图 2A中显示样本的大扫描。具有代表性的病毒信封的恢复显示在 图 2B（ 蓝色等面）中。
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51366/51366fig1highres.jpg" src="/files/ftp_upload/51366/51366fig1.jpg" style="opacity: 0.9; font-size: 14.399999618530273px; line-height: 28px;">
图1。在功能化的PEGG表面上对珠子和VSV进行自动对焦成像。AFM扫描36纳米生物基化珠（A， B） 和 VSV 病毒（C）锚定在表面与生物基化 VSVG 抗体.AFM 是在交流空气地形扫描模式下完成的。尖端半径为<25纳米，测量是在力调制和轻敲下进行的。尖端的力恒定为3N/m，谐振频率为75 kHz，不确定性为15 kHz。虽然珠子在 AFM 扫描期间保持了高度，但 VSV 病毒具有明显较小的年轻人的模组，并且高度显著变形。在这张图像中，病毒被专门用一个僵硬的悬臂在攻丝模式下成像，产生一个小的xy卷积（用于确定病毒的尖端与钝端），病毒尖端和钝端之间的高度差异用于检测病毒19的钝端额外的蛋白质密度。
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51366/51366fig2highres.jpg" src="/files/ftp_upload/51366/51366fig2.jpg"> 图2。基于荧光的 VSV 病毒成像在 PEGG 功能化表面上。 A） 使用在广域荧光中成像的生物基化抗VSVG抗体在PEGG表面固定的重组VSV病毒。 B） 通过生物基化抗 VSVG 抗体，高分辨率荧光重建连接到 PEGG 表面的 VSV 信封，并饰有 Alexa 647 标记的反 VSVG 抗体（方法 5A 用于创建这些图像）。蓝色表面是 2D 等面投影。左图为子弹形病毒模型。
<table border="0" cellpadding="0" cellspacing="0" width="212"><colgroup>
<col>
<col>
</colgroup>
<tbody>
<tr height="27">
<td height="27" style="height:27px;width:119px;">盖滑尺寸</td>
			<td style="width:93px;">流体</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">40 毫米</td>
			<td>65 微升</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">35 毫米</td>
			<td>50 微升</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">30 毫米</td>
			<td>36 微升</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">25 毫米</td>
			<td>25 微升</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">20 毫米</td>
			<td>16 微升</td>
		</tr>
</tbody></table>表1。根据盖片大小，流体引用。

Watch this Scientific Journal Video about Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging at JoVE.com

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

通过超分辨率荧光成像或原子力显微镜 （AFM） 将病毒附着在表面上是单一病毒成像的要求。在这里，我们演示了一种样品制备方法，用于控制将病毒粘附到适合用于 AFM 和超分辨率荧光成像的玻璃表面。

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 ...

sample-preparation-for-single-virion-atomic-force-microscopy-super

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.6K 次观看.  University of Utah. 通过超分辨率荧光成像或原子力显微镜 （AFM） 将病毒附着在表面上是单一病毒成像的要求。在这里，我们演示了一种样品制备方法，用于控制将病毒粘附到适合用于 AFM 和超分辨率荧光成像的玻璃表面。

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

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or &#8220;tapping mode&#8221; AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.
PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.
The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.

A method for investigating the structure of a protein photoreceptor using atomic force microscopy (AFM) is described in this paper. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) reveals intact protein dimers on a mica surface.

冲红灯光感受器原子力显微镜使用PeakForce定量纳米机械性能映射

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be ...

科研

工程学

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

使用"单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

免疫与感染

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

发育生物学

Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and chemical composition of sample with nanoscale resolution. By combining AFM with IR, the spatial resolution limitation of conventional IR is overcome, enabling a resolution of 20&#8211;100 nm to be achieved. This opens the door for a broad array of new applications of IR toward probing samples smaller than several micrometers, previously unachievable by means of conventional IR microscopy. AFM-IR is eminently suited for bacterial research, providing both spectral and spatial information at the single cell and intracellular level. The increasing global health concerns and unfavorable future prediction regarding bacterial infections, and especially, rapid development of antimicrobial resistance, has created an urgent need for a research tool capable of phenotypic probing at the single cell and subcellular level. AFM-IR offers the potential to address this need, by enabling detail characterization of chemical composition of a single bacterium. Here, we provide a complete protocol for sample preparation and data acquisition of single spectra and mapping modality, for the application of AFM-IR toward bacterial studies.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) provides a powerful platform for bacterial studies, enabling to achieve nanoscale resolution. Both, mapping of subcellular changes (e.g., upon cell division) as well as comparative studies of chemical composition (e.g., arising from drug resistance) can be conducted at a single cell level in bacteria.

原子力显微镜与红外光谱相结合，作为探测单一细菌化学的工具

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and ...

化学

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

推进液体和冰中病毒组件的高分辨率成像

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

生物化学

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

原子力显微镜罐体功能化,具有单T细胞或单粒子免疫单细胞力光谱

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

生物学

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 ...

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 ...

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

通过 3D 相关聚焦离子束铣削制备样品，用于高分辨率冷冻电子断层扫描

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, ...