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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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 border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Glass coverslips (fPALM)</td>
			<td style="width:71px;">Electron Microscopy Services</td>
			<td style="width:119px;">72225-01</td>
			<td style="width:167px;">25 mm Coverslips</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-Biotin (20%)&#160;</td>
			<td style="width:71px;">SuSoS&#160;</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Stock: 0.5 mg/ml in PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NeutrAvidin biotin-binding protein</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">A2666</td>
			<td style="width:167px;">Stock: 0.25 mg/ml in PBS</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Alexa Fluor 647 goat anti-rabbit IgG (H+L)&#160;</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">A21245</td>
			<td style="width:167px;">1:200 in 0.2 M KPO4, 0.15 M NaCl, 10% Glycol, pH 7.2 buffer</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">&#945;-VSV-G&#160;</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">ab34774</td>
			<td style="width:167px;">1:200 in 0.2 M KPO4, 0.15 M NaCl, 10% Glycol, pH 7.2 buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gluox</td>
			<td style="width:71px;">Sigma</td>
			<td>G2133-250KU&#160;</td>
			<td>Glucose oxidase type seven from Aspergillius&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Catalase</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C40-100mg&#160;</td>
			<td>Catalase from Bovine liver&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEA</td>
			<td style="width:71px;">Sigma</td>
			<td>30070-10G&#160;</td>
			<td>Cysteamine (MEA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stock Buffer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>50 mM Tris-HCl (pH 8.0) + 10 mM NaCl + 10% glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NTE</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>10 mM Tris pH 7.4, 100 mM NaCl, 66 mM EDTA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Slide-A-Lyzer, mini dialysis unit</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>10,000 MW</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Microscope Cover Glass: 35 CIRCLE #1</td>
			<td style="width:71px;">Fisherbrand</td>
			<td style="width:119px;">35 CIRCLE #1</td>
			<td style="width:167px;"></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 ...

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9.4K 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

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

科研

生物学

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 ...

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 ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

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 ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

哺乳动物细胞基态衰竭超分辨成像

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

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

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 ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

亚细胞结构的超分辨率活细胞成像

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...