Name: imaging intermediate filaments and microtubules with 2-dimensional direct stochastic optical reconstruction microscopy
Uploaded: 2018-03-06T13:30:00.000+00:00
Duration: 14 min 23 s
Description: Watch this Scientific Journal Video about Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy at JoVE.com

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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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要步骤。
我们建议用戊二醛和海卫一混合的萃取和固定溶液的步骤来固定样品, 然后用粉煤灰 (多聚甲醛) 固定 (步骤 n°3)。该固定协议是从 Chazeau et al12: 我们减少了提取/固定步骤孵化时间到六十年代 (步骤 3.4), 删除葡萄糖从细胞骨架缓冲, 并跳过的额外步骤的通透。这种方法的优点是自由移动的骨架子单元被耗尽, 允许超分辨率成像, 背景12更少。更一般的固定步骤是关键的, 因为不好的固定 (使用旧的粉煤灰, 冷的解决方案, 太长/短孵化步骤) 导致部分破坏的灯丝网络 (破碎微管, 微管, 不达到细胞前面和/或在低密度)。请注意, 也可以使用甲醇固定, 特别是如果只有波形网络映像如勒迪克 et al15中所述。但是, 可以在单元格内观察到背景噪音 (图 5)。虽然用甲醇固定电池获得的 dSTORM 图像质量较差, 但它们仍然提供了有关 IF 和微管网络应该是什么样子的信息, 例如长丝密度。只有戊二醛的固定效果非常差, 尽管它是微管固定的最佳协议23。
在染色部分, 使用抗小鼠抗体, 最大限度地减少交叉反应与抗体开发的大鼠是很重要的。由于这一原因, 我们不能使用晶圆碎片 (抗鼠), 因为它们与老鼠抗蛋白除了鼠标反波形外, 相互反应不特别。
由于激光功率有限, 有必要使用488和561纳米激光线将 Alexa555 染料泵入暗态。高488激光功率 (~ 50%) 在采集过程中也需要观察良好的闪烁和低工作周期 (荧光在状态下花费的时间的百分比), 尽管它也增加了背景噪声。TIRF 光照模式限制了显影在薄层 (200 nm 以上的玻璃盖玻片) 的激发。通过减小背景光, 提高信噪比, 同时降低励磁功率。希洛模式允许轴向切片, 并可以有效地优化信噪比。因此, 它是一个良好的中间 TIRF 和免疫。使用希洛模式是非常重要的, 以激发有效的 Alexa555 显影, 这需要高激光功率闪烁正确。
另一个关键步骤是获取原始数据 (步骤 6)。在获取影片时, 有必要调整采集参数 (曝光、增益、TIRF, 甚至聚焦)。保持明亮显影的最佳密度非常重要 (图 1A): 它必须足够低, 可以在空间上分离每个染料, 但足够高, 可以在 40 000 帧后可视化整个结构。如果活化分子的数量太低, 应获取更长的栈。我们获得了良好的图像与5000定位每µm²为 IFs, 2 500 定位每 cm²为微管。
方法的修改和疑难解答
使用新鲜的样品和优化的成像缓冲器是很重要的。pH 值应精确调整, 特别是对于多边环境协定。
波形和微管在用标准的广域显微技术 (WF) 成像时应该看起来均匀地贴上标签。如果长丝看起来与经典显微术有间断/虚线, 这将被放大时使用超分辨率。在这种情况下, 最好准备新的样品。未优化的固定条件 (老煤灰等) 可能导致微管看起来支离破碎。玻璃表面的高背景可能是由于低效率的堵塞引起的。在这种情况下, BSA (牛血清白蛋白) 可以由血清 (胎牛血清) 取代, 并用于每一个孵化步骤。
如果标签密度太高 (图 1B), 并且不能在黑暗状态下充分推送, 即使具有最高的激光功率, 次级抗体的数量也应该降低 (比减少主要抗体的数量要好)。但是, 这需要准备新的样品。一般而言, 有必要对使用的二次抗体的数量进行经验主义的评估。
如果显影不正常闪烁 (在仅一帧中它们应该处于亮状态), 请检查成像缓冲区的 ph 值是否正确 (ph 值 8.3)。如果问题仍然存在, 请准备新的解决方案 (特别是多边环境协定)。始终检查 TIRF 准直器是否在 (TIRF u_HP) 上。与有限的激光功率, 获得正确的闪烁可能是一个挑战 (上和关闭率, 以及染料亮度取决于激光功率, 正如在范 de 林德 et 和 al11) 中所述。如果激发激光器的更多功率可用14, 则可以探索其他类似 Alexa488 或 Atto488 的染料。
如果显影在 40 000 帧结束前停止闪烁, 请更改可能已被氧化的图像缓冲区。验证缓冲区更改后缓冲区和盖子之间没有气泡。密封样品可以在一个原始的几个小时成像。
如果风暴电影是在常规的 TIRF 显微镜下获得的, 配备了 EMCCD, 可以使用雷暴24重建图像。对于漂移校正、图像滤波和图像渲染, 也有类似的选择。雷暴是一个插件使用与斐济/imageJ 软件。与制造商软件和雷暴 (图 6) 获得了类似的结果。
小床 (cyclooctatetraene) 可以添加到成像缓冲器中, 以提高单分子的定位精度。它增加了每个周期为 Alexa64725发出的光子数。当将 2 mm 的小床添加到步骤5中描述的风暴缓冲区或100毫米的环境协定 (如参考25中所述) 时, 确实可以看到更好的定位精度 (下至 6 nm)。然而, 在这些条件下, 在采集过程中明亮分子的数量比没有小床的速度要快得多, 限制了在采集结束时分子定位的总数, 并可能导致 IF 网络的取样不足。据报道, 其他成像缓冲器与 Alexa 染料一起工作良好, 例如使用乳糖酶和 oxyrase 作为氧气清除系统26。在本协议中, 我们报告了缓冲成分, 为我们的样本类型和成像设置提供了最佳的分辨率, 但每个样本类型都需要缓冲区优化, 除了标签和固定条件优化。
方法的限制和可能的改进
这里提出的方法仅限于固定单元和2D 图像。使用一个 TIRF 显微镜和 EMCCD 的标准成像, 最限制因素是激光功率 (100 兆瓦为 561 nm 激光是不够的, 使 Alexa555 染料闪烁)。532纳米激光线可能更有效地激发这些染料。该方法可能的改进是使用光学散光27执行 3D dSTORM。这将需要对协议进行最少的修改, 除了较低的标签密度和增加的帧数。3D 双色风暴图像可以更好地查看环绕微管的 IFs, 尤其是束丛。注意, 肌动蛋白花丝也可以观察使用相同的固定方法和优化量的荧光罗丹明8。3种颜色风暴可以用来观察三骨架子系统的组织。
使用主和二级抗体会增加感兴趣对象的大小, 因为次级抗体从目标到 20 nm。通常情况下, 25 nm 直径微管将有一个宽度约50毫微米后, 标签。提高目标和染料之间距离的一种可能性是直接用适当的染料标记主要抗体。理想的 nanobodies 标记有机染料应使用28。
我们提供了一个简单的方法来估计一个单一分子的平均定位精度后, Endesfelder et al22。同时考虑定位精度和标记密度的图像的整体分辨率也可以使用傅立叶环相关方法29来测量。
对于频道的注册, 很难完全覆盖在视野中的所有珠子。在 Chazeau et 12中可以找到更复杂的更正。
显影的闪烁和探针的漂白所产生的限制可以通过使用 dna-颜料 (在纳米尺度地形中成像的 dna 点积累) 来克服30。该方法通过对短染料标记寡核苷酸的瞬态结合, 对其互补靶点进行单分子定位所需的闪烁。
结论
本文提出了一个健壮的协议, 执行双色 dSTORM 成像在2维度的骨架花丝在固定的细胞。详细描述了在我们的样本类型中, 在固定、immunolabeling 和成像缓冲器方面找到的最佳实验条件. 然后, 提出了原始数据采集的过程和必须记录的闪烁图像类型 (分子密度、信噪比、荧光任务周期等), 以及如何重建 dSTORM 图像、纠正漂移和过滤数据。这些步骤对于双色 dSTORM 成像来说是通用的, 而不是特定于样本类型的。最后, 给出了该技术如何帮助解决两个耦合骨架网络的稠密组织。

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。在良好的闪烁条件下, 显影应该只存在于一个框架中。良好的闪烁要求调整成像缓冲条件 (pH, 还原剂, 氧气清除系统) 和激光功率, 影响显影的上和外速率, 因此工作周期 (荧光花费的时间的百分比on 状态)9,11。图 1B相反, 给出了一个原始图像的示例, 其中分子密度过高, 无法解析单个分子。
图 1C显示了由 20 000 帧组成的制造商软件检测和分析的所有分子的本地化精度的典型直方图。此直方图对应于图 3B中显示的微管网络的 dSTORM 图像。制造商软件提供的本地化精度值与步骤8中估计的值有很好的一致性, 使用在不同时间点22 (图 1D) 上本地化的单个分子的指向精度。
图 2A显示了波形花丝 immunolabeled 与 Alexa647 dSTORM 图像的典型示例。与标准宽场显微镜 (图 2B-c) 获得的图像相比, 分辨率的增加可以清楚地观察到。图 2D中表示的荧光强度剖面显示超分辨率显微成像允许解析波形束。dSTORM 分辨率足以计算束中存在的花丝数。
图 3显示了波形和蛋白网络的双色 dSTORM 图像的典型示例, 分别 immunolabeled Alexa647 和 Alexa555。两个网络的叠加表明, 波形束通常沿微管进行局部定位, 为两个骨架子系统之间的耦合提供关键的结构信息。
图 4说明了在非最佳条件下获得的微管 dSTORM 图像的各种示例。首先, 使用 Alexa488 和带有143毫米 beta 巯基乙醇的缓冲区 (而不是使用9、10、11) 和一个氧气清除系统 (由0.5 毫克/毫升葡萄糖氧化酶和40µg/毫升组成的另一种还原代理).过氧化氢酶), 显影闪烁, 但不够明亮, 精确本地化 (太低的光子数) (图 4A)。第二, 使用 Alexa555 与143毫米β巯基乙醇, 50 毫米和氧气清除系统, 显影不能被抽到他们的黑暗状态, 因此没有很好的空间分离 (太高的责任周期, 即在 "上" 状态的时间的一部分太长) (图 4B)。最后, 使用 Alexa568, 与143毫米 beta 巯基乙醇, 50 毫米和氧气清除系统, 显影是不明亮的, 并缓慢地闪烁着非常长的 "on" 和 "关闭" 状态 (低光子数和太高的工作周期)(图 4C)。在所有这些非最佳条件下, 超分辨率图像中的微管网络呈现出很差的表现。总之, 在相应的成像缓冲区中, 最佳条件要求显影具有高光子数和低工作周期9,10 。请注意, 优化标记密度和成像缓冲区条件以获得良好的闪烁条件是双色 dSTORM 的标准程序, 不特定于 IF 微管成像。
根据显影定理, 有必要至少每 10 nm 有一个分辨率为 20 nm。扩展到2D 结构, 显影估计每个µm2上的 104标记是必需的。由于 if 网络密度较大, 其花丝较薄 (10 毫微米 vs 25 毫微米直径没有主、二级抗体) 与微管网络相比, 我们观察到, 要描述 if 网络, 需要加倍定位。我们获得了良好的图像与 5 000 定位/µm2为 IF 和2500定位/µm²的微管, 获得 40 000 帧和 20 000 帧分别。获得最高分辨率的图像, 其定位精度为σSMLM ~ 8-12 nm, 估计了协议的步骤 8 (在不同时间点22处定位的单个分子的指向精度)。这种定位精度的估计与制造商软件提供的值有很好的一致, 它显示在原始数据中从所有检测到的分子中提取的定位精度直方图中 (如图 1C所示)。如果标签密度足够高, 这种定位精度可以提供一个分辨率为 30 nm (2.35 * 精度) 的图像。我们经常观察到波形灯丝的全宽在半最大 (FWMH) 的 ~ 40 毫微米和微管与宽度 FWMH 55 nm。由于图像分辨率取决于定位精度和定位密度, 我们提供了实验条件, 使最佳结果考虑到两个标准。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57087/57087fig1.jpg"> 图 1: 在采集和定位精度估计过程中, 单个分子的最佳密度。 (A-B)左:代表性原始图像从一个单一的框架与最佳密度的单一分子在明亮的状态(a)和具有太高密度(B)在风暴采集期间 (步骤 6.12.) 用抗波形和抗小鼠染色的固定 U373 细胞Alexa647。右:原始图像, 其中的荧光水平高于峰值强度到噪声阈值 (设置为 6) 周围的圆圈 (白色或红色)。圆直径由峰值掩码大小 (设置为9像素) 设置, 颜色确定分子是否重叠 (红色) (白色)。请注意, 单个分子的高密度 (B) 导致了大量的重叠分子, 这是没有考虑到风暴图像重建。一个嵌入:图, 显示红色中单个分子的典型荧光强度剖面, 其高斯拟合为黑色。在此示例中, 定位精度为σx = 6.7 nm。缩放条, 1 µm. (C)制造商软件检测和分析的所有分子的定位精度的典型直方图。它可以可视化, 并在获取原始数据期间定期更新, 因为在线处理。此直方图对应于图 3B中显示的微管网络的 dSTORM 图像。(D)左:重建后, 单分子的风暴图像呈现在玻璃表面。右:高斯拟合后得到的 X 和 Y 位置的直方图。高斯拟合 X 和 Y 分布给出了定位精度的估计。<a href="/files/ftp_upload/57087/57087fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57087/57087fig2.jpg"> 图 2: 波形网络的代表 dSTORM 映像.dSTORM 图像(A),对应的标准宽域图像(B)和叠加(C)显示 U373 胶质细胞的前缘, 固定如协议所述, 并为波形与 Alexa647 染色。底部: 高亮区域的缩放。(D)沿黄色线的横向强度剖面在 A 和 b 刻度线的缩放图像中, 2 µm 和 500 nm 缩放。<a href="/files/ftp_upload/57087/57087fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57087/57087fig3.jpg"> 图 3: 代表波形和微管网络的双色 dSTORM 图像。(A)波形网络上沾有 Alexa647 (与图 2A相同)。使用 Alexa555 标记的(B)微管网络。(C)覆盖波形 (红色) 和微管 (青色) 网络, 右侧有一个缩放区域。波形的 dSTORM 图像得到了150万定位的 40 000 帧。用 850 000 定位 20 000 帧, 得到了微管的 dSTORM 图像。缩放栏, 2 µm 和500毫微米在变焦。<a href="/files/ftp_upload/57087/57087fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57087/57087fig4.jpg"> 图 4: 微管的非最佳成像条件示例.当显影眨眼, 但不够明亮, Alexa488 标签蛋白在 U373 胶质细胞和143毫米 BetaMercaptoethanol 和氧气清除器在成像缓冲区(A),当显影是足够明亮, 但不能正确耗尽在黑暗状态 (在最大激光电源可利用) 和慢慢地眨眼与 Alexa555 标记蛋白在143毫米 BetaMercaptoethanol, 50 毫米多边环境协定和氧气清道夫缓冲(B)和当显影慢慢地眨眼并且不明亮使用 Alexa568在包含143毫米 BetaMercaptoethanol、50毫米和氧气清除器(C)的风暴缓冲器中标记蛋白。在所有这些情况下, dSTORM 图像都有退化的分辨率。缩放条, 2 µm.<a href="/files/ftp_upload/57087/57087fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57087/57087fig5.jpg"> 图 5: 具有代表性的双色 dSTORM 图像波形和微管网络的 U373 电池固定的冷甲醇5分钟.(A)波形和(B)微管网络, 分别标有 Alexa647 和 Alexa555, 并带有突出显示的缩放。注意在细胞质中的细胞前面有单分子存在, 从而降低了灯丝网络的全球分辨率。缩放栏, 2 µm 和500毫微米在变焦。<a href="/files/ftp_upload/57087/57087fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57087/57087fig6.jpg"> 图 6:dSTORM 重建图像的比较 重建 dSTORM 图像由制造商软件 (A) 和雷暴 (斐济插件) (B)。(C) 在洋红和 (B) 中用绿色覆盖 (A)。缩放栏, 2 µm 和500毫微米在变焦。<a href="/files/ftp_upload/57087/57087fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Universidad San Sebastian

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 ...

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

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 ...

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

直接测量重组活性微管束内的力

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as ...

可定制的荧光显微镜，用于可视化细胞骨架网络

Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the dynamics of these 3D, dense networks calls for optical sectioning, which is often associated with fluorescence confocal microscopes. However, recent developments in light-sheet fluorescence microscopy (LSFM) have established it as a cost-effective and, at times, superior alternative. To make LSFM accessible to cytoskeleton researchers less familiar with optics, we present a step-by-step beginner's guide to building a versatile light-sheet fluorescence microscope from off-the-shelf components. To enable sample mounting with traditional slide samples, this LSFM follows the single-objective light-sheet (SOLS) design, which utilizes a single objective for both the excitation and emission collection. We describe the function of each component of the SOLS in sufficient detail to allow readers to modify the instrumentation and design it to fit their specific needs. Finally, we demonstrate the use of this custom SOLS instrument by visualizing asters in kinesin-driven microtubule networks.

作者聚焦:通过光片显微镜推进远非平衡材料知识

This protocol describes in detail how to build a single-objective, light-sheet fluorescence microscope and its usage for visualizing cytoskeleton networks.

设计和构建用于细胞骨架网络可视化的可定制、单物镜、光片荧光显微镜

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the ...

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

此视频中的章节

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

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）

三维细胞外囊泡的直接随机光学重建显微镜

直接测量重组活性微管束内的力

设计和构建用于细胞骨架网络可视化的可定制、单物镜、光片荧光显微镜

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

此视频中的章节

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

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）

三维细胞外囊泡的直接随机光学重建显微镜

直接测量重组活性微管束内的力

设计和构建用于细胞骨架网络可视化的可定制、单物镜、光片荧光显微镜

通过TIRF显微镜在体外同时可视化交联和单微管的动力学