我们感谢皮特鲁斯 Lelek, Orestis Faklaris 和尼古拉斯村镇为富有成果的讨论, 安德烈 Aristov 和埃琳娜 Rensen 帮助与超分辨率技术和 Shailaja Seetharaman 仔细阅读的手稿。我们感激地感谢巴斯德研究所 (巴黎、法国) 的 UtechS 光子 BioImaging (Imagopole) Citech 以及法国国家研究机构支持的法国 BioImaging 基础设施网络 (ANR-10-INSB-04;对未来的投资), 和 Région 法国 (计划酒庄 d ' Intérêt 不可抗力 Malinf) 使用 Elyra 显微镜。这项工作得到了反癌症和法国国家研究局 (ANR-16-CE13-0019) 的支持。

1. 盖玻片准备 (1 天, 30 分钟)
<ol><li>将18毫米 nº1.5H (170 µm +/5µm) 玻璃盖玻片放在塑料架上。</li>
	<li>将机架放在装有丙酮的100毫升烧杯中, 浸泡5分钟。</li>
	<li>将机架转到装有绝对乙醇的100毫升烧杯中, 浸泡5分钟。</li>
	<li>将机架转移到装有绝对乙醇的新100毫升烧杯中, 在室温下将烧杯放在超声波清洗装置中 (见材料表)。按 "on", 等待10分钟。</li>
	<li>将机架放在层流罩下, 让盖玻片干燥或干燥盖玻片与过滤空气流动。</li>
	<li>将机架与盖玻片在等离子清洗装置中。打开真空泵, 关上门, 等待3分钟, 使真空和开关在等离子上1分钟. 清洗后不久使用盖玻片。不要存储它们。</li>
</ol>2. 电池电镀 (1 天, 20 分钟)
<ol><li>用10% 胎牛血清、1% 青霉素-链霉素和1% 非必需氨基酸在10厘米直径的塑料培养皿中生长胶质细胞系 U373。</li>
	<li>将干净的玻璃盖玻片在层流罩下的12井板中, 并在每井中加入1毫升的预热细胞培养基。</li>
	<li>从细胞孵化器 (37 °c, 5% CO2) 中取一个包含细胞的盘子。</li>
	<li>卸下介质并添加10毫升 PBS。</li>
	<li>取出 PBS 并添加1毫升0.05% 胰蛋白酶-EDTA。</li>
	<li>把盘子放回孵化器里2-3 分钟。</li>
	<li>添加10毫升的暖介质预热在37摄氏度的盘子和并用重悬细胞。</li>
	<li>在含有盖玻片的12井板中, 每井 100 000 细胞 (150 µL 细胞) 种子。</li>
	<li>等待至少6小时 (或过夜), 以允许细胞传播。</li>
</ol>3. 细胞固定 (2 天, 2h)
<ol><li>用80毫米管子, 7 毫米氯化镁2, 1mM EGTA, 150 毫米氯化钠, 并将 pH 值调整为 6.9, 制备骨架缓冲器。</li>
	<li>用 PBS 洗一次细胞。</li>
	<li>去除 PBS, 并轻轻地添加1毫升每井提取/固定溶液 (0.25% 戊二醛, 0.3% 海卫 X-100 在骨架缓冲) 预热37摄氏度。</li>
	<li>在室温下孵化六十年代。</li>
	<li>删除解决方案, 并轻轻地添加1毫升每井预热和新鲜准备的4% 粉煤灰 (多聚甲醛) 溶液稀释在骨架缓冲。 警告:使用手套和工作在化学罩下。</li>
	<li>将12井板放回在37°c 的孵化器中10分钟。</li>
	<li>除去粉煤灰, 并增加3毫升的 PBS 每井。 注意:在化学罩下工作, 用手套把回收的粉煤灰放在一个特定的箱子里。</li>
	<li>用 PBS 洗两次。</li>
	<li>卸下 pbs 并添加新准备的 10 mM NaBH4解决方案稀释在 PBS 中, 以消除来自戊二醛的自体荧光。</li>
	<li>室温下孵育7分钟。</li>
	<li>将回收的 NaBH4放在特定的 bin 中。</li>
	<li>用 PBS 清洗三次5分钟。</li>
	<li>卸下 pbs 并添加阻塞解决方案 (pbs 中的 5% BSA 解决方案)。</li>
	<li>在室温下孵化1小时 (或隔夜在4摄氏度)。 注意:细胞骨架缓冲可以储存在室温下几个月。粉煤灰、NaBH4和戊二醛应特别小心处理 (罩、手套和特定的回收箱)。为了保持单元格的完整性, 应轻轻地添加和删除解决方案。重要的是不要让细胞干燥。</li>
</ol>4. 染色 (2 天, 5h)
<ol><li>用抗波形和抗蛋白, 在阻断溶液中进行1:500 稀释, 制备原发抗体的溶液。</li>
	<li>将100µL 滴稀释的原发抗体放在石蜡膜层上, 将盖玻片放在上面, 细胞朝下向下。</li>
	<li>孵化细胞的主要抗体为 2 h. 把盖玻片放回一个装满 PBS 的12井板里。每次在 PBS 的室温下在一个轨道振动筛上冲洗三次5分钟。</li>
	<li>同时, 用抗鼠 Alexa647 和抗大鼠 Alexa555, 在阻断溶液中稀释1:1、000制备二次抗体溶液。室温下培养1小时二级抗体的细胞。继续像4.2。保护细胞不受光线的侵害。</li>
	<li>把盖玻片放回一个装满 PBS 的12井盘子里。每次在 PBS 的室温下在一个轨道振动筛上冲洗三次5分钟。卸下 pbs 并添加0.5 毫升 pbs 混合1µL 0.1 µm tetraspeck 珠。</li>
	<li>将井放在轨道振动筛上, 用 PBS 冲洗30分钟。去除 pbs 和孵化细胞5分钟与4% 粉煤灰溶液在 PBS。在 PBS 冲洗三次。 注意:固定的细胞可以储存几个星期在 PBS 在4°c。染色应该在最后关头完成。</li>
</ol>5. 样品准备 (3 天, 5 分钟)
<ol><li>把整除数 (半胱胺, 1 米, pH 8.3), 葡萄糖氧化酶 (100x, 50 毫克/毫升), 过氧化氢酶 (500x, 20 毫克/毫升) 在一个冰篮。</li>
	<li>准备50毫升的三氯化钠缓冲器 (50 毫米三, 10 毫米氯化钠, pH = 8) 补充10% 葡萄糖 (100 毫克/毫升)。</li>
	<li>在与40葡萄糖的三氯化钠缓冲液中, 用10毫米, 0.5 毫克/毫升 (75 U/毫升) 葡萄糖氧化酶, 80 µg/毫升过氧化氢酶 (200-10% U/毫升) 成像前准备1.2 毫升成像缓冲器。</li>
	<li>将包含标记单元格的盖玻片装入磁性样品持有者 (如 chamlide 室), 并添加成像缓冲器以填满整个腔室。把盖子打开, 确保成像缓冲器和盖子之间没有气泡。</li>
	<li>用绝对乙醇清洁样品的底部, 并验证是否有泄漏。必要时, 使用油脂密封样品持有人。 注意:准备1米的溶液 (使用 HCl 调整 pH 值 8.3), 葡萄糖氧化酶在50毫克/毫升的库存和在水中20毫克/毫升的过氧化氢酶的库存, 使整除数和储存它们在20°c 一个月的时间为多边环境协定和葡萄糖氧化酶和过氧化氢酶的一年。不要冻结整除数。预先准备三氯化钠缓冲液, 并将其贮存在室温下几个月。在实验当天添加葡萄糖 (步骤 5.3)。</li>
</ol>6. 图像采集 (3 天, 每单元1小时)
<ol><li>打开显微镜。打开购置软件并按启动系统。</li>
	<li>选择购置选项卡. 展开 "安装管理器" 工具组中的图像设置工具。选择激光 WF (宽字段) 获取模式。选择100X NA 1.46目标。选择TIRF u HP模式, 它使用准直器减少被照亮的视野, 并将激光能量集中到较小的区域。使用 optovar透镜 1.6x , 其像素大小为100nm。</li>
	<li>选择时间序列工具, 并在帧之间设置 50 000 帧和0.0 毫秒的时间间隔。展开 "获取参数" 工具组中的通道工具, 然后选择 "全部显示"。</li>
	<li>设置 Alexa647 轨道: 检查642和405激光线路的励磁, 并接受将其打开。在成像设置中选择 "655 LP" 发射过滤器。重命名 "647"。</li>
	<li>在通道工具中单击 "+" 以添加另一个光通道, 并在成像设置工具中选择 "切换轨道每: 帧" 模式。检查561、488和405激光线并接受将其打开。选择 "BP 570-650 + LP750" 发射过滤器, 并使用 optovar 透镜1.6x。检查是否选择了 TIRF 超高压模式。重命名曲目 "555"。</li>
	<li>选择捕获模式工具, 并将帧大小设置为 200 x 200 像素。选择焦点设备和策略工具, 并将确定的焦点设置为 1 000 帧 (如果有焦点锁定系统)。</li>
	<li>把油放在目标上, 把样品放在上面。选择定位选项卡, 然后打开荧光灯的快门。找到焦点, 并寻找的细胞图像使用目镜。用操纵杆把它放在视野的中心。</li>
	<li>选择购置选项卡以返回到获取模式。选择 "647" 轨道, 将642激光功率设置为 0.2%, 将405激光功率设置为 0, 将曝光时间设置为50毫秒, 增益为50。选择连续捕获模式以观察屏幕上的单元格。调整焦点。确保在视野中至少有一个 tetraspeck 珠, 如果需要, 增加对比度以查看它们。使用自动缩放模式进行显示。通过单击对齐并保存该波形网络的宽字段荧光图像。要检查 TIRF 照明, 点击 TIRF, 调整照明和/或准直器的角度, 如果需要有一个均匀的照明领域, 并保存它。 注意: 在开始原始数据采集之前, TIRF 和 epifluorescent 图像的质量很重要。不连续的, 不均匀标记的细丝会导致质量较差的 dSTORM 图像。</li>
	<li>取消选中 "647" 轨道, 选择并检查 "555" 轨道。采取微管网络的荧光图像, 并保存它。采取 TIRF 的形象, 并保存它。</li>
	<li>取消选中 "555" 轨道, 选择并检查 "647" 轨道, 然后按连续。将增益提高到0和曝光时间为10毫秒. 将 642 nm 激光功率设置为 100%, 以将大部分 Alexa647 染料泵入到暗态 (~ 2 千瓦/厘米2)。一旦显影开始闪烁, 将曝光率提高到15毫秒, 并获得300。选择TIRF光照模式。使用Range 指示器模式而不自动缩放, 以验证单个分子信号不会使照相机饱和 (由红色表示)。在这种情况下, 减少增益, 直到饱和消失。Tetraspeck 珠子在收购开始时将保持饱和。按停止以停止对单元格的连续观察。</li>
	<li>展开联机处理工具, 并检查联机处理 PALM , 以便在获取过程中可视化风暴图像。使用以下参数: 9 为峰值掩码大小 (9 像素直径), 6 为峰值强度到噪声。这些参数将定义在处理过程中考虑的分子 (足够明亮且不重叠)。在PAL 统计工具中, 选择绘图类型:直方图和直方图源:精度。按开始购置。</li>
	<li>在购置过程中, 必要时重新对焦 (如果没有焦点锁定装置)。调整参数 (曝光, 激光功率) 和最终开关在 405 nm 激光线 (从0.001% 开始) 保持一个中等密度的显影与最小距离1µm 之间的单一分子 (> 9 像素100毫微米, 峰值掩码大小) (图 1AB)。如果分子密度有点太高, 使用希洛 (高度倾斜和层压光片) 模式的照明, 而不是 TIRF。这将使激光功率提高20%。确保在重构图像下的直方图的定位精度峰值在10毫微米以下。 注意:通常分子数随着时间的推移而减少, 405 nm 的激光功率也随之慢慢增加。其目标是尽可能减少 10 nm 以下的峰值 (图 1C), 以降低在超分辨率图像下显示的定位精度 (直方图)。如果需要, 增加手掌图像的对比度, 如果有太多的明亮的珠子存在的领域。</li>
	<li>当达到超过10个6定位 (通常在 40 000 帧之后) 时, 请按停止以停止影片。把激光器放回 0.2% (642 纳米) 和 0 (405 纳米)。用. czi 格式保存风暴采集的原始数据。在 "通道" 工具中取消选中 "647", 然后选择并检查 "555"。</li>
	<li>将曝光时间设置为25毫秒, 并增益为0。按连续按钮, 并将488和561激光器设置为 100%, 以将 Alexa555 染料泵入暗态 (~ 2 千瓦/厘米2 )。一旦显影开始闪烁, 将增益提高到 250, 请使用希洛光照模式, 并检查单个分子是否将相机与范围指示器模式饱和。如果是这样的话, 减少收益。按停止。将488激光功率降低到50%。</li>
	<li>按开始购置。在收购过程中, 逐渐增加增益, 始终处于饱和极限。逐步增加 405 nm 的激光功率, 以保持单一分子在视野中的中等密度 (见步骤 6.18)。当405激光器高于15% 时, 将488激光功率降低到20-30%。然后逐渐减少 488, 同时增加405激光线, 以保持分子的闪烁尽可能快。488激光线与561励磁激光器的最大强度同时增加了显影的上、外速率, 而405激光线只增加了速率。</li>
	<li>当超过 500 000 定位 (大约 20 000 帧) 或更多的分子没有闪烁时, 停止采集。用. czi 格式保存风暴采集的原始数据。 注意:总是从较高的波长开始, 以避免其他颜色的漂白 (或活化)。由于 chamlide 室不密封, 因此有可能定期更改成像缓冲器, 例如测试不同的缓冲条件。使用488激光线耗尽 Alexa 555 染料是必要的, 只有当561激光功率是有限的 (与100兆瓦激光器)。</li>
</ol>7. 图像重建 (5 分钟)
<ol><li>在PAL 漂移工具中, 使用具有最大大小为8的自动段的基于模型的更正类型。在行中多次按应用, 直到更正漂移为止。这种基于模型的校正应用了一种基于交叉相关分析的算法来修正漂移。</li>
	<li>使用PAL 筛选器工具, 只需选择最佳本地化的分子, 即可改善风暴图像的外观。例如, 将本地化精度限制为 30 nm 和 PSF 到 120-180 nm。</li>
	<li>在PAL 渲染工具中使用像素分辨率 (5 或 10 nm), 以及高斯显示模式, 其扩展因子为 1. 选择 "使用95% 值呈现自动动态范围 HR", 并呈现具有90% 值的自动动态范围 SWF。选择 "呈现最佳质量"。</li>
	<li>在处理选项卡的palm菜单中选择palm 转换(后处理模式)。按选择, 然后应用。将创建的图像保存为格式。LSM (而不是. czi, 非常重要)。 注意:图像重建可以在采集后或以后使用采集软件的图像处理模式进行。在离线后处理模式下, 有可能重新分析不同参数值的栈 (峰值掩码大小, 峰值强度到噪声)。总是选择 "丢弃重叠分子" 而不是 "本地化前的平均值"。</li>
</ol>8. 估计风暴图像的定位精度 (10 分钟)
<ol><li>打开您的风暴原始数据并使用 "图像处理" 选项卡. 选择palm方法, 然后再次palm 。按选择。</li>
	<li>按应用以使用峰值掩码大小9和峰值强度到6的噪音 (或为图像选择的参数) 开始重建。</li>
	<li>选择矩形图形工具, 并在玻璃表面上的单个分子周围绘制原始图像上的矩形。那里通常有几个单一的分子。</li>
	<li>再次按应用, 仅分析矩形区域内存在的分子。</li>
	<li>在PAL 统计工具中, 选择绘图类型: 直方图和直方图源: X 位置或 Y 位置, 以可视化位置直方图。</li>
	<li>将表保存在图像下方左侧的定位列表中。</li>
	<li>使用数据分析软件, 创建 X 和 Y 位置的直方图 (图 1D)。</li>
	<li>用高斯匹配分布, 并保存σX和σY值。定位精度σSMLM估计由 (σX *σY) ^ 0.5。</li>
	<li>重复步骤 8.3-8.8 在尽可能多的分子和平均σSMLM
</li>
</ol>9. 渠道注册 (5 分钟)
<ol><li>使用斐济 (图像 J) 软件打开每种颜色的风暴图像。</li>
	<li>选择魔杖工具来测量每个 tetraspeck 珠子的位置。</li>
	<li>计算每个珠子和平均值的 X 和 Y 移位。</li>
	<li>使用 "平移" 命令, 用计算的 X 和 Y 移位转换第二个图像。</li>
	<li>使用 "合并通道" 命令合并两个图像。</li>
</ol>

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<li>Yang, Z., Wang, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glial+fibrillary+acidic+protein%3A+from+intermediate+filament+assembly+and+gliosis+to+neurobiomarker%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker.</a> Trends Neurosci. 38 (6), 364-374 (2015).</li>
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作者声明没有竞争的财政利益。

议定书中的关键步骤
我们提出了一个协议, 它最小化的 dSTORM 图像中的微管和 IFs 在胶质细胞中的伪影。可以在样品准备和成像的每一步创建工件: 固定、阻塞、immunolabeling、在采集过程中漂移、非最佳闪烁条件23。下面列出了最关键的步骤。
清洗盖玻片是限制显影在表面上的非特定约束, 从而限制 dSTORM 图像中的背景噪声的重...

细胞质中间花丝 (IFs) 是10毫微米直径的人-或 heteropolymers 的细胞类型特定子集的 IF 蛋白。IFs 参与大量的细胞功能, 如细胞的运动, 增殖和压力反应。他们的关键作用是突出的事实, 90 以上的人类疾病直接造成的突变, 如果蛋白质;例如, 如果组合的变化伴随肿瘤生长和传播1,2,3。越来越多的证据表明, 三细胞骨架系统协同工作, 控制细胞极化, 分裂和迁移2,3的细胞功能。由于空间结构与功能之间存在着紧密的耦合, 因此, 要深入了解 IF 与其他细丝的结构组织, 更好地理解骨架的交叉谈话是至关重要的。本文提供了一种执行超分辨率技术双色 dSTORM (直接随机光学重建显微术)4的协议, 以及它如何被用于研究固定、培养的微管之间的相互作用2维 (2D) 中的单元格。
在过去的十年中, 已经开发出了一些超分辨率技术, 并在 2014年5中发现了诺贝尔化学奖的起源。在所有这些技术中都是基于单一分子定位的方法, 如棕榈6 (照片激活的本地化显微学), 使用 photoswitchable 荧光蛋白, 风暴7与对显影或 dSTORM4使用常规显影。所有这些方法都是基于相同的原理, 其中包括 (i) 大多数荧光记者切换成 "关闭" 状态 (非荧光), (ii) 将它们的子集随机激活为荧光状态, 以便将其空间位置与纳米精度, (iii) 重复这个过程, 以激活尽可能多的显影子集。最后的图像是重建使用所有的定位的活化分子, 提供一个横向分辨率下降到20-40 纳米。一些商业化的光学系统, 允许棕榈/风暴现在可以为生物学家进行例行实验。在这里, 一个这样的系统被用来研究微管和 IFs 的结构关联。在所有基于单分子定位的方法中, 选择了双色 dSTORM 技术, 因为它很适合观察粘附细胞的极薄层状区域 (< 1 µm), 并能为图像分辨率的提高提供显著的改善。最少的时间和金钱投资。事实上, dSTORM 是一种非常方便和多才多艺的技术, 是兼容的标准有机染料常规用于细胞染色和免疫荧光。
虽然一个颜色 dSTORM 图像相对简单, 使用荧光 Alexa6478, 但双色 dSTORM 需要优化实验条件, 以便两种染料在所选的缓冲区中能够正确闪烁, 特别是当有有限激光功率。在使用 dSTORM9、10、11、12、13和这些文件来设置多色成像的方法上已经有了优秀的论文, 这些论文详细解释了可能的来源工件和退化的图像分辨率以及如何克服它们。本文介绍了在细胞固定、染色、样品制备、影像采集和图像重建等方面的最佳实验条件, 以获取致密细胞质的图像, 如网络和微管在具有双色 dSTORM 的胶质细胞。简单地说, 在 Chazeau et al12中描述的提取/固定方法适用于胶质细胞, 并与固定后的步骤、最佳的抗体浓度、10毫米的风暴缓冲器在登入的9等中描述。发现是最佳的实验设置和样本类型。
胶质细胞主要表现为三种 IF 蛋白: 波形、巢和 GFAP (胶质酸性纤维蛋白)。这三种蛋白在星形胶质细胞14中被显示为共聚合。我们以前展示了使用超分辨率结构化光照显微镜 (SR SIM), 这三如果蛋白质可以发现在同一单一 IF 灯丝和他们显示相似的分布和动力学在胶质细胞15。由于三蛋白质之间的相似性, 波形染色被用作整个网络的记者。使用 dSTORM, 我们设法解决了如何, 如果表单捆绑沿微管, 这是不可能的衍射有限显微技术15。这些观察可能有助于了解波形 IFs 如何模板微管和调节其生长轨迹, 促进在细胞迁移过程中维护极性轴16。超分辨率图像提供了有关两个骨架子系统相互作用的关键信息, 并对空间关联和局部函数之间的联系进行了深入的了解, 该链接可以是单元类型特定的17。一般来说, 双色 dSTORM 可以用来研究骨架元素或其他类型的细胞器之间的串扰, 前提是在密度和特异性方面达到良好的染色条件。这项技术将有助于更好地描述在 astrogliosis 期间观察到的细胞骨架变化, 以及中枢神经系统最常见和最恶性肿瘤的胶质瘤, 如果蛋白质的表达被改变18 ,19,20,21。

<table><tbody><tr><td>Minimum Essential Media (MEM)</td><td>Gibco</td><td>41090-028</td><td>cell culture</td></tr><tr><td>non essential amino acid</td><td>Gibco</td><td>1140-050</td><td>cell culture 1/100e</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td>cell culture 1/100e</td></tr><tr><td>F&amp;oelig;tal Bovine Serum</td><td>Gibco</td><td>10270-106</td><td>cell culture 1/10e</td></tr><tr><td>0,05 % Trypsin-EDTA (1X)</td><td>Gibco</td><td>25300-054</td><td>cell culture</td></tr><tr><td>PBS 10x</td><td>fischer scientific</td><td>11540486</td><td>cell culture</td></tr><tr><td>coverslip 18 mm n&amp;deg;1,5H</td><td>Marienfield/Dominic dutsher</td><td>900556</td><td>coverslips</td></tr><tr><td>paraformaldehyde (PFA) solution</td><td>Sigma</td><td>F8775</td><td>cell fixation</td></tr><tr><td>glutaraldehyde</td><td>Sigma</td><td>G5882</td><td>cell fixation</td></tr><tr><td>triton X100</td><td>Sigma</td><td>T9284</td><td>non ionic surfactant. Used for cell fixation</td></tr><tr><td>sodium borohydride (NaBH4)</td><td>Sigma</td><td>452904-25ML</td><td>cell fixation</td></tr><tr><td>BSA</td><td>Sigma</td><td>A7906</td><td>sample passivation 3% in PBS</td></tr><tr><td>Monoclonal Anti-Vimentin (V9) antibody produced in mouse</td><td>Sigma</td><td>V6630</td><td>primary antibodies</td></tr><tr><td>Rat anti alpha tubulin</td><td>Biorad</td><td>MCA77G</td><td>primary antibodies</td></tr><tr><td>Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 555</td><td>Fisher scientific</td><td>10635923</td><td>secondary antibodies</td></tr><tr><td>Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647</td><td>Fisher scientific</td><td>10226162</td><td>secondary antibodies</td></tr><tr><td>TetraSpeck Microspheres, 0.1 &amp;micro;m, fluorescent blue/green/orange/dark red</td><td>Fisher scientific</td><td>T7279</td><td>fluorescent beads</td></tr><tr><td>Chamlide magnetic chamber</td><td>Live Cell Instrument</td><td>CM-B18-1</td><td>magnetic sample holder, maximum volume 1,2 mL</td></tr><tr><td>Cysteamine (MEA)</td><td>Sigma</td><td>30070</td><td>for the blinking buffer</td></tr><tr><td>glucose oxidase from Aspergillus niger</td><td>Sigma</td><td>G0543</td><td>for the blinking buffer</td></tr><tr><td>glucose</td><td>sigma</td><td></td><td>for the blinking buffer</td></tr><tr><td>catalase from bovine liver</td><td>Sigma</td><td>C9322</td><td>for the blinking buffer</td></tr><tr><td>Tris (trizma)</td><td>Sigma</td><td>T1503</td><td>for the blinking buffer</td></tr><tr><td>Wash-N-Dry coverslip rack</td><td>Sigma</td><td>Z688568</td><td></td></tr><tr><td>Parafilm</td><td>Sigma</td><td>P7793-1EA</td><td>paraffin film</td></tr><tr><td>ultrasonic cleaner</td><td>Fisher scientific</td><td>15-335-6</td><td></td></tr><tr><td>plasma cleaner</td><td>Harrick plasma</td><td>PDC-32G-2</td><td></td></tr></tbody></table>

imaging intermediate filaments and microtubules with 2-dimensional direct stochastic optical reconstruction microscopy

细胞骨架由肌动蛋白丝、微管和中间细丝 (IF) 组成, 在控制细胞膜的形状、极性和运动方面起着关键作用。肌动蛋白和微管网络的组织已被广泛研究, 但 IFs 尚未充分的特点。IFs 的平均直径为 10 nm, 形成了整个细胞细胞质的网络。它们与肌动蛋白和微管通过分子马达和骨架连接器物理相关。这种紧密的关联是监管机制的核心, 它确保了对大多数细胞功能所需的三骨架网络的协调调节。因此, 必须单独和其他骨架网络一起可视化 IFs。然而, 如果网络是非常密集的大多数细胞类型, 特别是在胶质细胞, 这使得它的分辨率很难实现与标准荧光显微镜 (横向分辨率的 250 nm)。直接随机光学重建显微镜 (dSTORM) 是一种技术, 允许增益的横向分辨率的一个数量级。在这里, 我们表明, 侧向 dSTORM 分辨率足以解决 if 网络的稠密组织, 特别是如果管束周围的微管。这种紧密的关联很可能参与这两个网络的协调调节, 并可以解释波形 IFs 模板和稳定微管组织以及可能影响微管依赖性水泡的贩运。更一般地, 我们展示了如何观察两个骨架成分的双色 dSTORM 技术, 为他们的相互互动带来了新的洞察力。

显微镜配备50兆瓦 405 nm 和100兆瓦 488, 561 和 642 nm 固态激光器, 一个 EMCCD 512x512 相机, 阿尔法计划, 一个人的 100 x/1.46 目标和波段通行证 570-650/长通行证655发射过滤器被用于代表性的结果如下。
图 1A给出了在原始图像获取过程中应使用的分子密度和信噪比的示例。在相邻的分子间至少有1µm 的质量图像。在?...

Watch this Scientific Journal Video about Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy at JoVE.com

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

2维直接随机光学重建显微成像中间丝和微管

该方法的总体目标是给出从样品制备到图像采集和重建的最佳实验条件, 以执行固定细胞中微管和中间丝的2D 双色 dSTORM 图像。

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

imaging-intermediate-filaments-microtubules-with-2-dimensional-direct

Research

JoVE Journal

Biology

10.8K 次观看.  Institut Pasteur. 该方法的总体目标是给出从样品制备到图像采集和重建的最佳实验条件, 以执行固定细胞中微管和中间丝的2D 双色 dSTORM 图像。

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

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

科研

生物工程

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

生物化学

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

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

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

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

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

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

免疫与感染

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

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

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

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

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

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and&#160;wide-field&#160;illumination&#160;microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.&#160;

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

实现干扰反射显微镜,实现无标签、高速微管成像

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...

Quantifying Cytoskeleton Dynamics Using Differential Dynamic Microscopy

Cells can crawl, self-heal, and tune their stiffness due to their remarkably dynamic cytoskeleton. As such, reconstituting networks of cytoskeletal biopolymers may lead to a host of active and adaptable materials. However, engineering such materials with precisely tuned properties requires measuring how the dynamics depend on the network composition and synthesis methods. Quantifying such dynamics is challenged by variations across the time, space, and formulation space of composite networks. The protocol here describes how the Fourier analysis technique, differential dynamic microscopy (DDM), can quantify the dynamics of biopolymer networks and is particularly well suited for studies of cytoskeleton networks. DDM works on time sequences of images acquired using a range of microscopy modalities, including laser-scanning confocal, widefield fluorescence, and brightfield imaging. From such image sequences, one can extract characteristic decorrelation times of density fluctuations across a span of wave vectors. A user-friendly, open-source Python package to perform DDM analysis is also developed. With this package, one can measure the dynamics of labeled cytoskeleton components or of embedded tracer particles, as demonstrated here with data of intermediate filament (vimentin) networks and active actin-microtubule networks. Users with no prior programming or image processing experience will be able to perform DDM using this software package and associated documentation.

Differential dynamic microscopy (DDM) combines features of dynamic light scattering and microscopy. Here, the process of using DDM to characterize reconstituted cytoskeleton networks by quantifying the subdiffusive and caged dynamics of particles in vimentin networks and the ballistic motion of active myosin-driven actin-microtubule composites is presented.

使用差分动力学显微镜定量细胞骨架动力学

Cells can crawl, self-heal, and tune their stiffness due to their remarkably dynamic cytoskeleton. As such, reconstituting networks of cytoskeletal ...

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 ...

生物学

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy 3D-SIM

The present protocol describes a method to visualize and measure actin rings and other components of the membrane periodic skeleton of the axon initial segment using cultured rat hippocampal neurons and 3D-structured illumination microscopy (3D-SIM).

使用3D结构照明显微镜（3D-SIM）测量轴突初始段的膜周期性骨架的性质

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to ...

神经科学

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

三维细胞外囊泡的直接随机光学重建显微镜

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...

2维直接随机光学重建显微成像中间丝和微管

摘要

探索更多视频

相关显微镜的动力相互作用的三维结构分析

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

通过TIRF显微镜在体外同时可视化交联和单微管的动力学

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

实现干扰反射显微镜,实现无标签、高速微管成像

使用差分动力学显微镜定量细胞骨架动力学

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）

使用3D结构照明显微镜（3D-SIM）测量轴突初始段的膜周期性骨架的性质

三维细胞外囊泡的直接随机光学重建显微镜

2维直接随机光学重建显微成像中间丝和微管

摘要

探索更多视频

相关显微镜的动力相互作用的三维结构分析

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

实现干扰反射显微镜,实现无标签、高速微管成像

使用差分动力学显微镜定量细胞骨架动力学

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）

使用3D结构照明显微镜（3D-SIM）测量轴突初始段的膜周期性骨架的性质

三维细胞外囊泡的直接随机光学重建显微镜

通过TIRF显微镜在体外同时可视化交联和单微管的动力学