我们非常感谢 EMBL (海德堡) 和海德堡大学电子显微镜核心设施 (EMCF) 的工作人员。我们还要感谢 Ulrike Herian、斯蒂芬妮 Kallis 和安德烈 Hellwig (海德堡大学), 以及艾伯施密特和 Renate Kunz (Ulm 大学) 的专家技术援助。右岸导流洞和他的小组 (U.H. 和红外 B) 的工作得到德意志 Forschungsgemeinschaft、SFB1129、TP11 和 TRR83、TP13 的支持。

1. 用于细胞培养的图案蓝宝石圆盘的制备
<ol><li>将图案的蓝宝石圆盘 (见材料表), 用一个字母数字图案刻在其表面上, 变成15毫升锥形离心管。 注: 或者, 传统的0.05 毫米厚蓝宝石光盘没有字母数字模式可以使用, 其中一个参考模式是由碳涂层他们与 finder 网格在顶部。为了达到这个目的, 在应用碳模式之前, 应该用乙醇清洗。</li>
	<li>用乙醇彻底清洗。用过滤纸把它们分散在培养皿里, 让它们干。把它们放在玻璃滑梯上, 用纤细的长镊子隔开。</li>
	<li>检查与倒置显微镜 (4X 放大) 是否在每个蓝宝石光盘上的坐标模式是可读的。 注: 如果情况并非如此, 请在镊子的帮助下翻转蓝宝石光盘。</li>
	<li>将蓝宝石光盘存储在培养皿中。 注意: 协议可以在这里暂停。</li>
</ol>2. 带图案蓝宝石圆盘的细胞培养
<ol><li>用紫外线 (UV) 交联剂对图案蓝宝石光盘进行消毒5分钟。</li>
	<li>把蓝宝石光盘带到细胞培养生物安全柜。在生物安全柜中用乙醇消毒长镊子。</li>
	<li>将细胞培养基的一半 (见材料表) 添加到细胞培养皿中。用长镊子小心地将蓝宝石盘转移到细胞培养皿中。</li>
	<li>种子 1 x 105 Huh7-Lunet 细胞, 稳定地表达 T7 聚合酶悬浮在细胞培养培养基的另一半, 上培养皿。 注: 计算要播种的细胞数, 在实验的终点, 细胞融合不应超过20–30%。高细胞密度会阻碍细胞在后期被 EM 迁移。此外, 过度融合可能导致细胞脱离蓝宝石圆盘。</li>
	<li>检查蓝宝石光盘是否留在培养基的底部, 字母数字坐标仍然可读与倒置显微镜。 注意: 如果光盘漂浮在细胞培养基中, 使用长镊子将光盘推到底部, 最终将其翻转回正确的方向。</li>
	<li>染第二天的单元格, 如先前在参照21,23中报告了转染代理制造商的指示。</li>
</ol>3. 在图案蓝宝石圆盘上生长细胞的 LM 成像
<ol><li>在实验结束点将蓝宝石圆盘转移到含有30–50µL/pH 无指示介质位置的金属成像板, 以减少非特异性背景荧光。 注: 此处描述的金属板 (图 2) 已在欧洲分子生物学实验室 (EMBL) 车间设计。如果没有这个板块或类似的, 玻璃底部细胞培养皿也可以使用, 而不是 LM。在无 pH 指标的培养基中改变正常细胞培养基是很重要的。还要注意, 长时间暴露时间应该避免, 因为它们可能导致漂白, 导致活性氧 (ROS) 积累和细胞的生理损伤24,25。</li>
	<li>在倒置的 widefield 荧光显微镜下对细胞进行成像。 注: 使用绿色荧光蛋白 (GFP) 标记的蛋白质时, 如代表结果所示, 应使用 450–490 nm 和500–550纳米激发和发射过滤器。</li>
	<li>首先获取单元格的低放大图像 (10–20X), 以分配表达 FPs 的单元格. 用差分干涉对比显微镜记录感兴趣细胞所在的图案蓝宝石圆盘的坐标。 注意: 缝合几个区域可能有助于更好地了解感兴趣的细胞/细胞的位置。还请注意, 相对比显微镜可以选择在这里使用。</li>
	<li>获取相同细胞/细胞的高放大率 (荧光和 DIC) 图像 (63–100X, 油浸泡目标), 以更好地辨别细胞内的蛋白质在胞内空间的定位。 注意: DIC 图像提供上下文信息。这些信息通常是至关重要的, 以关联 LM 图像与 EM 显微照片后, 因为最终的相关性更精确的 "解剖" 地标的细胞。由于一些蓝宝石光盘可能会打破在冷冻固定, 它是强烈建议准备 triplicates 每一个条件。此外, 有些细胞在 HPF 过程中与光盘分离, 而其他细胞的超微结构可能会由于冰冻损伤而包含工件。因此, 至少有两个区域的光盘应该通过 LM 映像, 以确保最终将有足够的细胞进行分析。重要的是, 这两个区域应该在光盘的反面。在这种方式下, 细胞嵌入的树脂块可以被切成两半, 这些区域的部分可以得到。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58154/58154fig2.jpg"> 图 2: 用于在图案蓝宝石光盘上生长的细胞的 LM 成像模式。(A)蓝宝石圆盘是用细镊子的帮助转移到成像板上的。(B)这个板块是专门设计在海德堡的 EMBL 车间, 以适应光显微镜, 其中蓝宝石光盘可以成像与细胞培养基没有 pH 值指示器。它由一个金属滑动, 75 毫米 x 25 毫米 x 1 毫米, 与4/5 孔铣入它与3毫米钻头适合蓝宝石盘的大小。另外, 小凹槽在孔的每边在90°角度被碾磨。这些孔使使用镊子在操作时更容易进入光盘。为使最佳成像条件, 玻璃盖玻片 0 (0.085 至0.13 毫米厚) 被粘在孔下面的 uv 胶水和放置在紫外线下硬化。(D)图案蓝宝石圆盘的高放大图像, 描绘了刻在其表面上的字母数字坐标。图案化的蓝宝石光盘可供商业使用 (见材料表)。<a href="https://www.jove.com/files/ftp_upload/58154/58154fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
4. 通过 HPF 在图案蓝宝石圆盘上生长的细胞的低温固定化
<ol><li>放置 "A" 和 "B" 铝载体为 HPF (参见材料表) 在培养皿与过滤纸浸泡与 1-hexadecene。浸入含有 1 hexadecene 的培养皿中包含细胞的图案蓝宝石圆盘。 注意: 移动光盘, 以洗涤细胞培养基远离。</li>
	<li>在 HPF 持有者的 "A" 和 "B" 铝载体之间组装蓝宝石光盘, 如下所示 (图 3B)。在底部, 放置一个 "B" 承运人, 其平面面朝上向上。将蓝宝石光盘放在这个平坦的一侧, 单元格朝上。添加一个 "A" 载体, 其0.1 毫米的深度面对细胞。 注: 由于图案蓝宝石光盘比传统蓝宝石光盘厚, 商用 "A" 载体 (设计用于与非图案0.05 毫米蓝宝石光盘) 必须削减到0.05 毫米在这0.2 毫米深度一侧在 EMBL车间, 以适应 HPF 持有人 (图 3A)。为此目的 , A 载体入到金属管的末端 , 以便它的 0 . 2 毫米边被暴露并且仍然保持 , 当削减它时。切割的一侧, 然后打磨, 使它是平坦的。另外, 一个特殊的铝载体可以使用在图案的蓝宝石光盘上 (商业上可用, 见材料表), 在这种情况下, HPF "三明治" 只组成蓝宝石光盘和这个载体。</li>
	<li>关闭 HPF 机的支架并冻结单元格。 注意: 此协议特定于特定的 HPF 计算机。其他 HPF 机器可以交替地使用6,26。在任何情况下, 请注意来自供应商的新一代机器的存在。 注意: 使用安全护目镜和听觉保护耳机。</li>
	<li>
将冷冻的 "三明治" 放在带有液氮的聚苯乙烯盒中, 用于临时贮存。 注意: 使用护目镜, 并与当地安全官员接触, 了解在处理液氮时的潜在危险。<ol>
<li>为了避免混淆, 将每个 "三明治" 放在一个单独的0.5 毫升离心管 (在聚苯乙烯盒内), 标有一个数字。 注意: 协议可以在这里暂停。如果不能立即执行冻结替代 (FS), 则可以将样品存储在箱内的低温管 (在顶部打孔) 内, 这些容器在低温液氮杜瓦瓶中的机架中 (见材料表)。这个液氮容器应该位于一个有氧气体传感器的房间里, 以防缺氧时窒息。 注意: 下面所述的所有程序 (步骤5和 6) 必须在生物安全柜中进行。此外, 大多数使用的试剂是危险的。在使用之前, 必须仔细阅读制造商提供的材料安全数据表, 并向安全官员询问当地的规则, 以确保安全处理。为正确处置这些二手材料, 以及正确使用以下所述设备, 还需要咨询当地研究所的卫生和安全程序。</li>
	</ol>
</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58154/58154fig3.jpg"> 图 3: HPF 两个铝载体之间的图案蓝宝石圆盘组装模式。(a)传统的 "A" 载体的切割视图, 必须从0.2 毫米到0.05 毫米的更深一侧切割. (B) 0.16 毫米厚蓝宝石光盘, 其表面蚀刻的字母数字图案被组装成两个铝载体 HPF, 如下所示: 蓝宝石圆盘放置在 "B" 载体的平边上, 单元格朝向向上。一个切碎的 "A" 载体, 其0.1 毫米的深度面对细胞被放置在顶部关闭 "HPF 三明治"。铝载体和被图案的蓝宝石盘是商业上可利用的 (参见材料表)。<a href="https://www.jove.com/files/ftp_upload/58154/58154fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
5. 冷冻固定细胞的 FS
<ol><li>用液氮填满 FS 机 (见材料表)。将温度设置为-90 摄氏度, 等待机器达到此温度。</li>
	<li>用玻璃蒸馏丙酮制备含0.2% 锇毒气 (OsO4) (v/v)、0.1% 醋酸铀 (UA) 的 fs 介质, 而 fs 机则冷却。 注: 准备, (例如) 12 毫升的 FS 介质, 混合600µL 4% OsO4与0.012 克的 UA 在一个小的玻璃容器。将这种混合物在超声波浴装置中溶解5分钟. 用吸管加入11.4 毫升的玻璃蒸馏丙酮, 混合均匀。</li>
	<li>填充1.5 毫升离心管与200-500 µL 的 fs 介质, 关闭盖子, 并将其转移到 FS 机器。等待10分钟的 FS 介质冷却。</li>
	<li>将含有蓝宝石圆盘的冰冻 "三明治" 转移到含有 FS 介质的管中, 用冷却的长镊子将其与液态氮气中的细胞结合使用。 注意: 使用低温防护手套和护目镜。 注: HPF "三明治" 可以在处理过程中自发分解。在这种情况下, 确保冻结蓝宝石光盘转移到离心管。铝载体可以被丢弃。</li>
	<li>运行 FS 如下, 直到第二天27: 从-90 °c 到-80 °c 为 8 h;从-80 °c 到-50 °c 为 8 h;从-50 °c 到-20 °c 为 2 h;从-20 °c 到0°c 为 2 h。</li>
</ol>6. 冷冻替代样品的树脂嵌入
注意: 下面描述的所有程序必须在生物安全柜中进行。此外, 大多数使用的试剂是危险的。在使用之前, 必须仔细阅读制造商提供的材料安全数据表, 并向安全官员询问当地的规则, 以确保安全处理。为正确处置这些二手材料, 以及正确使用以下所述设备, 还需要咨询当地研究所的卫生和安全程序。
<ol><li>取出 FS 机器上的替代样品, 放在冰上20分钟。</li>
	<li>将样品常温保存20分钟, 用玻璃蒸馏丙酮彻底清洗 (3 次, 每10分钟)。如果在 FS 后仍在离心管中, 用长镊子丢弃铝载体。</li>
	<li>用 1 h 孵化在25%、50% 和75% 树脂中的四步环氧树脂系列中渗透细胞 (在剩余的蓝宝石圆盘上), 然后用100% 树脂进行隔夜孵化。第二天将100% 树脂换成1h。</li>
	<li>小心地将蓝宝石圆盘放入装有100% 树脂的试剂浴中的流动环中。这些环被用作10蓝宝石圆盘的聚合模。 注: 蓝宝石光盘必须完全推到底部, 坐标朝上以可读的方式向上。这可以用一双双目连接到抽油烟机的帮助下进行检查。另外, 可以使用生物安全柜内的双目。重要的是, 有数字 (1-10) 刻在试剂的底部, 有助于识别样品。 另外, 为样品的证明一个纸标记可以包括在树脂块。</li>
	<li>聚合在烤箱中的样品在60°c 为48小时。</li>
</ol>7. 从聚合树脂块中去除图案蓝宝石圆盘
<ol><li>从烤箱中取出含有电池的硬树脂块。 注意: 协议可以在这里暂停。如果不这样做, 在切割之前, 让这些块冷却到室温。</li>
	<li>用剃刀刀片切割嵌入模具, 以获得树脂块。使用镊子拆卸圆柱形树脂块。 注: 如果在聚合前未将纸标签包括在树脂块中, 则用永久性标记将这些块编号, 并将其存储在1.5 毫升离心管中。协议可以在这里暂停。</li>
	<li>将含有蓝宝石圆盘的树脂块的尖端浸入含有液氮的聚苯乙烯盒中, 直到它停止 "冒泡"。然后, 将树脂块的尖端浸入沸腾的 H2O。</li>
	<li>重复上述两个步骤, 尽可能多次, 直到蓝宝石光盘脱落的树脂块。 注意: 使用护目镜和低温防护手套。 注: 如果此操作不起作用, 请使用剃刀刀片在光盘周围移去一点聚合树脂, 然后再试一次。协议可以在这里暂停。</li>
</ol>8. 透射电镜靶向修整和超薄切片
<ol><li>用 ultramicrotome 的双目检查块面, 在树脂块面上识别出感兴趣的细胞所在的区域。 注: 图案蓝宝石圆盘上的坐标图案的负压印保留在块面上。这允许标识感兴趣的单元格所在的区域以进行目标修剪。LM 图像必须垂直翻转, 以方便查找同一位置的块面。</li>
	<li>将含有所感兴趣的细胞/细胞的嵌入细胞单层修剪成一个具有梯形 (约200µm x 250 µm 平均尺寸) 的小型扁平金字塔, 并配以干净的刀片。</li>
	<li>用35°金刚石刀获得嵌入细胞的70纳米超薄截面。</li>
	<li>使用超细镊子将部分放在 EM 插槽网格上。 注:应避免使用网状网格, 因为网格栏可以屏蔽部分。</li>
	<li>将网格存储在网格框中。 注意: 协议可以在这里暂停。</li>
</ol>9. 超薄断面的 TEM 分析
<ol><li>将插槽网格放入透射电镜的持有者。获取低放大率 (概述) 图像, 其中所感兴趣单元格的完整单元格配置文件可见。 注::通过使用单元格配置文件和单元格的位置作为参考, 与 LM 之前获取的 DIC 视图进行比较, 可以找到感兴趣的单元格/单元格。</li>
	<li>获取高放大图像的细胞/细胞的兴趣, 尝试和发现, 在超微结构水平上的特点, 对应的荧光信号, 以前观察到 LM。 注: 蒙太奇图像可以获得在高放大率有一个全面的分辨率细胞/细胞的兴趣。此外, 在与荧光图像进行比较之前, 通常需要在连续的序列切片中获取同一个细胞的图像, 以便能够在3D 重建细胞。</li>
</ol>

作者没有什么可透露的。

这里介绍的克莱克方法研究了病毒蛋白表达对细胞膜的影响, 在阐明 HCV 复制相关结构, 主要是双膜泡 (DMVs)21, 以及确定构成这些 HCV 诱发结构所需的关键构件23。请注意, 在我们的第一个工作使用克莱研究 HCV 复制21, 有一个稍微修改版本的协议, 这里描述的应用。在这项研究中, 传统的0.05 毫米厚蓝宝石圆盘, 没有字母数字模式, 被用来在其中一个参考模式是由碳涂层他们与一个 finder 网格在顶部 (见材料表)。此版本的当前协议最终可以应用, 其优点是可以直接使用 HPF 的 "A" 载体, 而无需将其剪切下来, 如图 3A所示。或者, 正如协议中所提到的, 一个较厚的 "a" 载波可以使用图案蓝宝石光盘 (见材料表), 而不需要 "B" 载体。
有趣的是, 这个协议不仅可以应用于研究 BSL-1 样本, 如在这里和其他地方19,23中所描述的病毒蛋白转染的细胞, 而且还能研究病毒感染的细胞。虽然与人类病原体的工作通常限于 BSL-2 和 BSL-3 实验室, 但在一些国家, 在这些生物安全条件下仍有可能进行冷冻固定。在那些无法玻璃化的 BSL-2 和 BSL-3 实验室里, 由于当地的规定或缺乏 HPF 机器, 如果事先进行化学固定, 可以使用这种方法制备病毒感染细胞,即在离开 BSL-2 或 BSL-3 设施之前。此外, 醛需要立即淬火后, 固定保持荧光, 而其余的协议是相同的, 这里描述。这种技术可以被认为是多余的, 因为细胞是固定的两次, 化学和通过玻璃化。然而, 这种双固定协议确实导致更好地保存 HCV 诱导的 DMVs 与 DMVs 发现的细胞仅在化学固定21。
要进一步修改和排除故障, 读者可以在本手稿的整个协议部分中引用注释。这些注释描述了避免的陷阱, 以及克服在执行此方法时可能出现的困难的替代方案。
应用这种技术的主要必要条件是 HPF 机。当冷冻固定的细胞通过 HPF 是不可行的 (由于缺乏 HPF 机) 或不需要 (当膜保存不需要最佳), 细胞可以化学修复, 随后准备和分析的 EM8, 9,10,11,12,13,14,15,16,17,18 ,19。此选项不需要使用蓝宝石光盘, 而是用网格化模式的细胞培养皿来重新定位细胞或细胞簇。使用这些菜肴的主要优点是它们的直径比蓝宝石圆盘大, 允许对较大的表面积进行筛选。因此, 此克莱协议的应用已成功地应用于研究抗病毒化合物对 HCV15的作用, 或可视化 noroviruses19结构性蛋白诱导的膜重排。另一个可以限制此方法性能的问题是缺少商用 FS 设备。在这种情况下, 可以使用基本的自制 FS 系统。虽然自动 FS 设备可能减少处理事故, 自制的设备是成功地使用, 例如在肯特麦当劳30和保罗瓦尔特的实验室。
关于化学固定, 这里描述的协议确保了细胞内结构的最佳保存20。因此, 如果上面提到的 HPF 和 FS 设备可用, 玻璃化转变感兴趣的细胞将是首选。
这种克莱方法的未来选择包括使用这种方法不仅可以在超微结构水平上获取2D 信息, 而且还可以获取关于病毒引起的膜和细胞器改变的结构的3D 信息。3维 EM 方法, 包括电子层析成像 (ET) 和聚焦离子束扫描电子显微镜 (谎言-SEM) (广泛描述在29), 也可以适用于已编写的细胞, 这是目前的协议21后, 31。另外, 当使用共焦显微镜时, 也可以在 LM 水平上获得3D 信息, 这样可以获得 z 栈。事实上, 当需要 LM 和 EM 数据集之间的精确关联时, 强烈建议使用此选项 (如17所示)。3D z 栈中包含的信息有助于改善与 2D TEM 图像的相关性。因此, 在这种情况下, 最好的拟合 LM 和 EM 图像可以被选择, 然后接受一个相关的软件可用, 如欧共体-克莱克冰插件 (http://icy.bioimageanalysis.org/)32或标志性的对应插件图像 J (http://imagej.net/Landmark_Correspondences), 导致生成重叠的 LM EM 图像。
当需要时间信息来理解某一事件的动力学时, 时移成像可以用来监测活细胞的动态与 EM 的结合。在有兴趣的情况下, 细胞立即修复, 产生一个 "冰冻的快照", 随后可以通过 EM 进行分析, 提供关于固定化时该特定时刻的详细超微结构信息。为了获得 "冰冻快照", 观察后实时, 细胞可以要么化学固定33或冷冻固定6。由于许多细胞过程的发生速度比化学固定的扩散过程快, 如果可能的话, 应进行超快速冷冻。然而, 重要的是要考虑到 HPF 机在其有效时间分辨率34不同。
此外, 尽管本协议设计用于在环氧树脂中嵌入细胞, 细胞也可以嵌入低粘度树脂, 如 Lowicryls、lr 白色或 lr 金。使用这些嵌入介质可以保留抗原性35、36以及荧光37、38 , 因此主要用于嵌入后的部分 immunolabeling39,40和在部分克莱姆 41,42,43,44, 其中 LM 是在嵌入后完成。这两种方法 (免疫 EM 和截面克莱克) 必须是关键的那些非特征结构可以很容易地找到通过 TEM 和/或作为控制 miscorrelation 之间的 LM 和 EM 信号。同样, 可以通过两种成像方式 (LM 和 EM) 可视化的抗体进行标记, 以便在 lm (预嵌入阶段) 识别转染的细胞, 从而实现更精确的定位。GFP 信号通过其特定标记的免疫 EM (后嵌入阶段)。然而, 必须考虑到, 通透是在 LM 允许进入细胞内空间的抗体之前进行的, 这可能导致在 EM 水平上对细胞体系结构进行次优保存。有趣的是, 这个协议也非常适合于多颜色的实验, 可以实现使用其他荧光标签, 除了 GFP (如图所示)。最后, 根据正在处理的问题, 有许多假定的可能性, 在 LM 和/或 EM 两侧修改此协议。有关其他替代协议的全面说明, 读者可参考5、46。不管显微镜的方法如何组合, 结果是信息的增益, 使我们能够更好地理解病毒及其蛋白质在现实生活中与宿主的相互作用。
此方法中最关键的步骤是从感兴趣的单元格中收集序列部分。正如代表成果科所强调的那样, 这需要专家工作人员, 以及大量的耐心。重要的是, 这一步对于在 EM 级别找到单元格是非常重要的, 原因有两个。首先, 在这种使用预嵌入 LM 的克莱姆协议中, 该坐标仅在 lm 级和嵌入后的树脂块面上可见。然而, 他们将不可见的部分, 通过 TEM。因此, 必须用剃刀刀片仔细地完成块面上的目标修剪 (ROIs), 以确保随后获得的部分包含表示给定 FP 的单元格。其次, "扫描" 几个部分是必要的, 以找到最佳覆盖之间的 LM 和 EM 收购。与在嵌入后阶段执行 lm 的方法相反, 在这种情况下, lm EM 覆盖不那么精确。低叠加精度是由于 LM 与 em 轴向分辨率的差异、em 样品加工过程中的收缩和切片42中的压缩。然而, 有效的追踪方法, 如使用地标, 有助于找回细胞。这包括一个细胞的位置到另一个, 以及细胞的形状及其细胞核。在这方面, 正如《议定书》所解释的, DIC 图像提供了对改善相关性至关重要的细胞的 "解剖" 信息。或者, 细胞核或其他可识别的细胞器 (如线粒体或脂滴) 可以在 LM 之前被染色, 用作地标。
最后, 值得一提的是, 虽然本手稿的重点是利用这一技术进行病毒学研究, 但这一实验设计的范围可以扩大, 以解决更多的一般生物学问题。

将两种显微方法结合起来, 以获得更好的特定生物学过程的图片的想法是相当古老的。因此, 第一次关于使用 "相关显微学" 的病毒的研究发表于 1960年, 分别为两份出版物1、2。在该研究中, 作者通过两种显微技术分析了腺病毒诱发细胞核形态学的变化。在第一个出版物中, 报告了描述与腺病毒感染有关的形态学细节的电子显微术 (EM) 观察1。在第二个出版物中, em 观察到的不同结构与光镜 (LM) 的组织化学染色模式的图像相关, 以确定2以前观察到的结构的性质。
然而, 在这些早期研究中, 他们的观察是使用不同的受感染细胞作为独立实验进行的。事实上, "相关性" 是指从两种成像方式中获取信息的组合, 以了解某种现象, 比较通过不同化验得出的所有发现, 以了解特定生物过程。
目前, 术语相关显微学, 也称为相关的光和电子显微镜 (克莱), 被应用到越来越多的方法 (参考文献3,4,5), 与共同性两种成像技术 (LM 和 EM) 都是在相同的样本上进行的。这两种方法的结合结果, 从而, 在多模态, 多尺度和多维分析的样本3。其优点是 LM 可以提供许多不同细胞的广泛概述, 从而能够识别出在异质细胞群体中表达感兴趣的蛋白质或蛋白质的细胞亚群。EM 克服了 LM 的分辨率限制, 为特定胞内事件提供了更高分辨率的图像。此外, EM 使非荧光亚细胞环境的可视化, 包括所有的膜束缚细胞器, 大型大分子络合物 (如核糖体, 中心粒等) 和骨架元素, 从而提供额外的空间信息, 所谓的 "参考空间"6, 并给出了由 LM 检测到的荧光点的上下文。
在过去的几年中, 克莱已成为一个强大的工具, 不仅为细胞生物学家5, 但也为病毒学家 (参考7) 愿意了解复杂的病毒细胞相互作用, 导致成功的病毒传播。因此, 了解病毒如何修改细胞膜和细胞器以自身的利益是发展抗病毒药物根除致病性病毒的必要条件。
在这里, 一个克莱姆方法被描述, 允许检测由 LM 的细胞表达病毒蛋白融合到荧光蛋白 (FP)。这些细胞随后被冷冻固定, 并进一步准备通过透射电镜 (TEM) 进行超微结构分析, 以获得新的洞察力, 这些蛋白质的表达如何重新排列细胞内膜 (图 1)。在迄今发表的8、9、10、11、12、13、14 的病毒学研究中, 克莱已被化学固定细胞所执行. ,15,16,17,18,19。这主要是因为在生物安全 level-2 和-3 (BSL-2 和 BSL-3) 实验室中, 需要为生物安全原因而灭活感染性物质, 在那里, 通常不可能对细胞进行冷冻固定。然而, 对于那些需要最佳保存细胞膜的问题, 通过高压冷冻 (HPF) 玻璃化是非常推荐的20。在这些情况下, 可以应用此处描述的克莱协议。有趣的是, 特别是在使用传染性标本时, HPF 可以在以前化学灭活的样品上进行, 例如在 BSL-2 和 BSL-3 实验室。化学固定后 HPF 的结合是一种可能的利润至少部分从冷冻保存方法21,22的优势。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58154/58154fig1.jpg"> 图 1: 通过对单元格进行分析的工作流的示意图表示形式克莱.首先, 通过 LM 对有图案的蓝宝石圆盘上生长的细胞进行分析, 在它们处理前对其进行局部化表达。一旦定位, 细胞立即由 HPF 和 FS 固定, 随后嵌入树脂。树脂聚合后, 细胞生长的支持 (= 蓝宝石圆盘) 必须从树脂块中除去。包含嵌入单元格的块被修剪成一个小的梯形, 其余的单元表示 FPs, 用菱形刀切片。在缝隙网格上采集超薄切片, 经 TEM 进一步检查, 获得这些细胞的微结构信息。此图经参考29的许可而改编和修改。<a href="https://www.jove.com/files/ftp_upload/58154/58154fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table border="0" cellpadding="0" cellspacing="0" style="width:2451px;" width="2450">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">UV Crosslinker</td>
			<td style="width:392px;">Stratagene</td>
			<td style="width:149px;">400072</td>
			<td style="width:1539px;">Stratalinker 1800, 230Vac, equipped with 254-nm UV light bulbs, 8-watts/each.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Patterned sapphire discs</td>
			<td>Engineering Office M. Wohlwend, Sennwald, Switzerland</td>
			<td>627</td>
			<td>They have a 0.3-mm diameter and are 0.16-mm thick, as well as an an etched alphanumeric pattern to allow for re-location of the cells. They can be only ordered per e-mail: martin-wohlwend@bluewin.ch.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Slim and long tweezers</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>72919-SS</td>
			<td>&#34;Style SS&#34;, for handling sapphire discs.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Cell culture medium</td>
			<td style="width:392px;">Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:149px;">41965-062</td>
			<td>Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 2 mM L-glutamine, nonessential amino acids, 100 units penicillin per ml, 100 &#181;g streptomycin per ml and 10% fetal calf serum (DMEM complete, see below). Package containing 10 bottles, 500 ml/each.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">L-glutamine</td>
			<td style="width:392px;">Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:149px;">25030-123</td>
			<td style="width:1539px;">Package containing 20 bottles of 100 ml/each.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Nonessential amino acids</td>
			<td style="width:392px;">Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:149px;">11140-068</td>
			<td style="width:1539px;">Package containing 20 bottles of 100 ml/each.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Penicillin/Streptomycin</td>
			<td style="width:392px;">Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:149px;">15140-163</td>
			<td style="width:1539px;">Package containing 20 bottles of 100 ml/each.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Fetal calf serum</td>
			<td style="width:392px;">Sigma Aldrich, St. Louis, MI, USA</td>
			<td>F7524</td>
			<td style="width:1539px;">Bottle of 50 ml.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Inverted microscope</td>
			<td style="width:392px;">Olympus Deutschland, Hamburg, Germany</td>
			<td style="width:149px;">CKX41</td>
			<td>It is an inverted microscope with trinocular head options and fluorescence upgrade capability.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Huh7-Lunet cells stably expressing the T7 RNA-polymerase</td>
			<td style="width:392px;">Avaliable at Prof. Dr. Ralf Bartenschlager laboratory</td>
			<td style="width:149px;">Not available</td>
			<td style="width:1539px;">Contact Prof. Bartenschlager at: ralf.bartenschlager@med.uni-heidelberg.de.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Mirus TransIT-LT1 Transfection Reagent</td>
			<td>Mirus Bio LLC, Madison, USA</td>
			<td style="width:149px;">MIR 2304</td>
			<td style="width:1539px;">Broad spectrum, low toxicity, DNA transfection reagent. Vial of 0.4 ml.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Inverted widefield fluorescence microscope</td>
			<td style="width:392px;">Carl Zeiss Microscopy GmbH, Germany</td>
			<td style="width:149px;">Zeiss Observer.Z1</td>
			<td style="width:1539px;">Inverted fluorescence microscope for experiments involving living cell cultures.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-hexadecene</td>
			<td style="width:392px;">Merck, Darmstadt, Germany</td>
			<td>8220640100</td>
			<td style="width:1539px;">Bottle of 100 ml.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#34;A&#34; aluminium holders for high pressure frezing (HPF)</td>
			<td>Engineering Office M. Wohlwend, Sennwald, Switzerland</td>
			<td>241</td>
			<td>This is the holder that has been used for this protocol and has 2 different depths of 0.1 and 0.2-mm. It can be only ordered per e-mail: martin-wohlwend@bluewin.ch.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#34;B&#34; aluminium holders for HPF</td>
			<td>Engineering Office M. Wohlwend, Sennwald, Switzerland</td>
			<td>242</td>
			<td>This is the holder that has been used for this protocol.It is 0.3-mm thick.They can be only ordered per e-mail: martin-wohlwend@bluewin.ch.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">&#34;A&#34; aluminium holder for HPF (special for working with patterned sapphire discs)</td>
			<td>Engineering Office M. Wohlwend, Sennwald, Switzerland</td>
			<td style="width:149px;">737</td>
			<td style="width:1539px;">This is the holder than can be used alone with the patterned sapphire disc (as an alternative to the current protocol), without the need of a &#34;B&#34; holder or the edition of the &#34;A&#34; holder. It is 0.84-mm thick. It can be only ordered per e-mail: martin-wohlwend@bluewin.ch.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Sapphire discs</td>
			<td>Engineering Office M. Wohlwend, Sennwald, Switzerland</td>
			<td style="width:149px;">405</td>
			<td style="width:1539px;">These sapphires discs are the &#34;conventional&#34; one, with a 0.3-mm diameter and a thickness of 0.05 mm . They can be also used for CLEM as an alternative instead of patterned sapphire discs.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Finder grids</td>
			<td>Electron Microscopy Sciences, Hatfield, Philadelphia, USA</td>
			<td>LF135-Cu</td>
			<td style="width:1539px;">These 135 mesh grids are used to create an alphanumeric pattern on &#34;conventional&#34; sapphire discs (described above) via carbon coating, so that they can be used for CLEM. 100 grids/vial.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HPF machine</td>
			<td style="width:392px;">ABRA Fluid AG, Widnau, Switzerland</td>
			<td style="width:149px;">HPM 01</td>
			<td style="width:1539px;">This HPF machine has been used for this protocol.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">HPF machine</td>
			<td style="width:392px;">Leica Microsystems, Vienna, Austria</td>
			<td style="width:149px;">EMPACT 2</td>
			<td style="width:1539px;">&#34;EMPACT 2&#34;, as an alternative to the use of the HPM machine that it has been used in this protocol (described above).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Cryo-tubes</td>
			<td style="width:392px;">Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:149px;">Z763667-500EA</td>
			<td style="width:1539px;">For long term-storage of cryo-immobilized samples in liquid nitrogen. Package containing 500 cryovials.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Liquid nitrogen dewar</td>
			<td>Cole-Parmer GmbH, Wertheim, Germany</td>
			<td style="width:149px;">GZ-05094-60</td>
			<td style="width:1539px;">For long term-storage of cryo-immobilized samples in liquid nitrogen, equipped with cryo-racks with a capacity of 1600 cryo-tubes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Automatic freeze substitution (AFS) machine</td>
			<td style="width:392px;">Leica Microsystems, Vienna, Austria</td>
			<td style="width:149px;">EM AFS2</td>
			<td>This machine performs freeze substitution and progressive lowering of temperature (PLT) techniques and allows low temperature embedding and polymerization of resins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Osmium tetroxide (OsO4)</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>19150</td>
			<td>4% aqueous solution, one box containing 10 x 2 ml ampules.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uranyl-Acetate (UA)</td>
			<td>Serva, Heidelberg, Germany</td>
			<td>77879.01</td>
			<td>Bottle containing 25 grs of (UA)-2 H2O.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Sonicator</td>
			<td style="width:392px;">Bandelin Electronic, Berlin, Germany</td>
			<td style="width:149px;">329</td>
			<td style="width:1539px;">&#34;Sonorex Super RK 31&#34; is a high-power ultrasonic cleaning bath for aqueous cleaning solution that is used in this protocol to mix OsO4 and Ua when preparing the FS medium.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass-distilled acetone</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>10015</td>
			<td>Bottle of 100 ml.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Stereomicroscope</td>
			<td style="width:392px;">Leica Microsystems, Vienna, Austria</td>
			<td style="width:149px;">Leica M80</td>
			<td style="width:1539px;">Routine stereomicroscope for daily inspections, equipped with a camera for capturing images.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Epoxy resin</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td style="width:149px;">14120</td>
			<td style="width:1539px;">Embed 812 is a kit containing several components that must be mix together in the proportions given by the manufacturers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Flow through rings</td>
			<td>Leica Microsystems, Vienna, Austria</td>
			<td>16707157</td>
			<td>Package containing 100 pieces.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Reagent baths</td>
			<td>Leica Microsystems, Vienna, Austria</td>
			<td>16707154</td>
			<td>Package containing 100 pieces.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Ultramicrotome</td>
			<td>Leica Microsystems, Vienna, Austria</td>
			<td>Leica EM UC7</td>
			<td>Ultramicrotome for preparation of semi- and ultrathin sections at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra 35&#176; diamond knife</td>
			<td>Diatome Ltd., Nidau 
			Switzerland</td>
			<td>AGG339-735</td>
			<td>This knife have an edge length of 3 mm.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra fine tweezers</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>E78318</td>
			<td style="width:1539px;">&#34;Style 3X&#34;, for handling EM grids.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EM slot grids</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>G2010-Cu</td>
			<td>100 grids/vial.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EM grid storage box</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>71155</td>
			<td>It has capacity for 100 grids.</td>
		</tr>
	</tbody>
</table>

correlative light electron microscopy (clem) for tracking and imaging viral protein associated structures in cryo-immobilized cells

由于其高分辨率, 电子显微镜 (EM) 是病毒学家不可缺少的工具。然而, 通过 EM 对病毒感染或转染细胞进行分析的主要困难之一是感染或染毒效率低下, 阻碍了对这些细胞的检查。为了克服这一困难, 光镜 (LM) 可以首先执行分配的亚群感染或转染细胞。因此, 利用荧光蛋白 (FPs) 融合到病毒蛋白, LM 用于记录 "正转染" 细胞的位置, 表达 FP 和增长的支持与字母数字模式。随后, 通过高压冷冻 (HPF)、冷冻替代 (FS) 和树脂嵌入进一步处理 EM 细胞。超快速冷冻步骤确保了对所选细胞的优良膜保存, 然后通过透射电镜 (TEM) 在超微结构水平上进行分析。本文提供了一步一步的相关光电子显微镜 (克莱) 工作流, 详细描述了样品的制备、成像和相关性。实验设计也可用于解决许多细胞生物学问题。

使用这里介绍的过程 (图 1), 稳定表达 T7-RNA 聚合酶的 Huh7-Lunet 细胞被播种在有图案的蓝宝石圆盘上, 随后转染为表达丙型肝炎病毒 (HCV) 两个领域的质粒。结构性蛋白 NS5A, 用 GFP (pTM_AH-D1-GFP) 标记 (图 4)。第二天, 这些细胞生长的图案蓝宝石光盘被从细胞培养皿中取出, 并通过 LM 成像 (图 4,图 2)。那些表达 AH-D1-GFP 的细胞是由细胞质中存在的绿色荧光识别出来的, 记录下来保存它们在蓝宝石圆盘的坐标模式中的位置 (图 5A, 5B)。立即在 LM 检查之后, 含有感兴趣细胞的蓝宝石圆盘组装在两个铝载体之间 (图 3), 并受 HPF 的冷冻固定。细胞随后被冷冻取代, 随后嵌入环氧树脂中。
从聚合树脂块中取出蓝宝石光盘后, 在块面上可以看到字母数字模式的印记, 从而可以检索感兴趣的单元格所在的区域。其余的树脂块被修剪, 以产生一个小的梯形, 窝藏细胞的利益。从该梯形中获得了一系列超薄剖面, 并在 EM 槽网格上采集, 并通过 TEM 进一步分析 (图 5C)。请注意, 虽然可行的28是劳动密集型的, 但需要训练有素和熟练的人员, 但要手动连续的部分获得。同样重要的是, 当受到这个克莱协议 (图 5a) 时, 细胞有一个低的融合, 以保证快速识别以前由 LM 识别的同一单元格。否则, 有更高的细胞融合可能会大大减慢细胞在 EM 水平上的成功定位。
透射电镜分析感兴趣的细胞的几个超薄的部分 (图 5D, 5E), 表达 AH-D1-GFP, 揭示了在其细胞内空间的大量堆积的可变形态的囊泡从周围的细胞质由一个单一的脂质双层 (图 5E)。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58154/58154fig4.jpg"> 图 4: 工作流的示意图表示形式, 执行在中描述的克莱姆示例图 5.Huh7-Lunet 细胞稳定表达 T7-RNA 聚合酶被转染的 DNA 质粒编码的双亲螺旋 (AH) 和领域 1 (D1) HCV NS5A 蛋白, 标签在其 C 终端与 GFP (pTM_AH-D1-GFP)。该部分的 NS5A 蛋白的表达控制转录由 T7 启动子 (T7 Pm) 和 translationally 的脑心肌炎病毒 (EMCV) IRES (内部核糖体入口点) 元素, 由二级结构表示。采用 LM AH-D1-GFP 检测二十四小时后转染 (24 hpt) 细胞, 并对其进行表达, 并立即 HPF。<a href="https://www.jove.com/files/ftp_upload/58154/58154fig4large.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/58154/58154fig5.jpg"> 图 5: 一个克莱过程的例子。(A) Huh7-Lunet 细胞通过荧光显微镜对 T7 聚合酶进行稳定表达, 以在24小时后, 用质粒 pTM_AH-D1-GFP 转染后, 将 GFP 阳性细胞分配给生长在图案蓝宝石圆盘上。(B)在白色虚线矩形内选定的单个单元格的放大图像更高, 由克莱克分析。(C) HPF、FS 和树脂嵌入后, 同一细胞超薄截面的 TEM 图像。(D)在包含感兴趣单元格的插槽 EM 网格上串行 70 nm 超薄截面的示意图表示。(E)左: 透射电镜对感兴趣细胞的两个部分的概述。右侧: 在左边的黄色矩形中选择的胞内区域的高放大率透射电镜图像, 揭示了由细胞质通过单一膜分隔出来的大量囊泡。<a href="https://www.jove.com/files/ftp_upload/58154/58154fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells at JoVE.com

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

低温固定化细胞跟踪和成像病毒蛋白相关结构的相关光电子显微镜 (克莱)

采用相关光电子显微镜 (克莱) 方法, 通过光学显微术 (LM) 在细胞内的电子显微术 (EM) 对病毒诱导的胞内结构进行图像分析。LM 和 EM 作为一种混合成像方法, 实现了病毒宿主交互的集成视图。

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

correlative-light-electron-microscopy-clem-for-tracking-imaging-viral

Research

JoVE Journal

Bioengineering

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

80.9K 次观看.  European Molecular Biology Laboratory. 采用相关光电子显微镜 (克莱) 方法, 通过光学显微术 (LM) 在细胞内的电子显微术 (EM) 对病毒诱导的胞内结构进行图像分析。LM 和 EM 作为一种混合成像方法, 实现了病毒宿主交互的集成视图。

视频: Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

低成本的低温光学显微镜舞台制作相关的光/电子显微镜

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

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, ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

近同步激光扫描在活细胞上的共和和原子力显微镜（康波卡尔）

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

低温固定化细胞跟踪和成像病毒蛋白相关结构的相关光电子显微镜 (克莱)

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三维光学分辨率的光声显微镜

低成本的低温光学显微镜舞台制作相关的光/电子显微镜

病毒粒子为

相关显微镜的动力相互作用的三维结构分析

荧光淬一个脂质体包封的近红外荧光团的作为一个工具

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

相关轻型和电子显微镜使用量子点纳米粒子

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

近同步激光扫描在活细胞上的共和和原子力显微镜（康波卡尔）

用于微生物化学和蛋白质生产的光控发酵