作者感谢塔莫 Reisewitz 对手稿的批判性阅读, 以及对蓝鲨林和小百合萩的精心栽培和观察蓝藻, 吉孝城守的图像处理, 驰宏歌曲, Naoyuki 宫崎, 美代子Nagayoshi 帮助分割结构。这项工作得到了国家生理学科学研究所 (NIPS) 合作研究计划的支持, 张佑启

1. 蓝藻同步培养
<ol><li>养殖细长PCC 7942 在灭菌 BG 11 板 (在 9 cm 无菌塑料培养皿) 包含 1.5% (w/v) 琼脂和 0.3% (w/v) 硫代硫酸钠8。</li>
	<li>将板块放置在23摄氏度的生长室中, 亮度为50µE/米2/秒, 受 12 h light/12 h 暗循环的规限。</li>
	<li>每周将细胞转移到新鲜的 BG11 琼脂盘子上。 注: 在这种培养条件下, 琼脂上的培养基在一周后会出现活跃增殖细胞的绿色带。</li>
	<li>用火焰灭菌的铁丝环将细胞绿色的团块带到新鲜的 BG11 琼脂板上。在干净的长凳上这样做。</li>
</ol>2. 荧光显微镜监测
<ol><li>使用琼脂板上培养的细胞6天观察 DNA 压实。在光期结束时, 通过在细胞上浇注1毫升0.2 米蔗糖溶液, 从盘子中收集细胞。重复向单元格中注入解决方案, 以便收集大多数单元格。将悬浮细胞溶液转移到微管中进行 DNA 染色。</li>
	<li>添加 DNA 染色染料 (例如赫斯特 33342) 溶液到500µL 的悬浮细胞溶液中的一个微管, 最终浓度为1µg/毫升。然后, 把管子放在黑暗中10分钟。</li>
	<li>离心机1分钟, 在 2000 x克沉积的细胞。去掉上清, 加入10µL 0.2 米蔗糖溶液, 以获得致密的细胞悬浮液。</li>
	<li>将含有染色细胞的溶液的1µL 转移到滑动玻璃上, 用一个带有紫外滤镜的荧光显微镜, 用100X 和浸入式油放大的物镜, 将盖子滑动并观察。</li>
	<li>确认在大多数细胞的这一点上观察到 DNA 压实, 然后为下一个凝固步骤准备样品。</li>
</ol>3. 冷冻 HVET 的样品冻结法
<ol><li>设置一个冻结装置。用液态氮气填充油箱, 并在用聚四氟乙烯管连接油箱和燃烧室后开始冷却冷冻室。</li>
	<li>将液乙烷装入腔内的小铜锅内, 冷却到液氮温度。操作时戴眼镜, 因为液态乙烷是爆炸性的。</li>
	<li>辉光放电碳侧的孔碳涂层 EM 网格 (孔网格) 三十年代在 50 mA 使用等离子离子 bombarder。</li>
	<li>将1µL 的 BSA 金示踪剂 (15 nm) 应用到孔网格中作为基准标记。</li>
	<li>在 DNA 压实阶段将2.5 µL 整除细胞应用于孔网格。用过滤纸把多余的溶液涂抹掉。立即将栅格插入液乙烷中, 用一个柱塞在冻结装置中。</li>
	<li>将冻结的网格储存在液氮贮存中, 直至检查为止。</li>
</ol>4. 低温 HVET
<ol><li>在 1 MV 的高压下设置高压电能表。</li>
	<li>将冻结的网格安装在冷冻标本架中, 高压电能表预换热器到-150 摄氏度, 并将液氮放入冷冻工作站内, 并将其加载到高压电能表中。小心避免被冰污染。</li>
	<li>在1,000X 的放大倍数下选择成像区域。调整 eucentric z 轴高度。</li>
	<li>将试样的舞台倾斜到--去, 并消除倾斜旋转的反弹。</li>
	<li>在10,000X 的放大倍数下调整目标位置附近的焦点。将6到10µm 的焦点设置为偏离聚焦图像。对于成像, 将剂量设置为 2 e/Å-2或更少的标本事先。</li>
	<li>在高压电能表的图像屏幕上用电流密度测量电子剂量。在电子胶片或数码相机上拍摄图像, 其放大倍数与聚焦过程相同。</li>
	<li>从2°到4°的倾斜角增量, 手动收集倾斜图像, 方法与 (4.5) 从。 注: 在许多现代电子显微镜中, 通过数码相机的组合自动获得倾斜系列。在这种情况下, 请按照说明书操作。对于底片, 用一台全力显影器在20°c 处开发出12分钟的胶片, 并在定影剂中固定10分钟. 用平板扫描仪将胶片以 4000 dpi (图像 0.635 nm/像素) 的分辨率数字化。在数码相机的情况下, 将采集到的图像直接放到下一个图像处理步骤中。如有必要, 请使用两到四 binning (尺寸递减因子) 和适当的软件 (例如ImageJ), 以中值过滤器减小图像大小。</li>
</ol>5. 层析重建
<ol><li>使用 IMOD 软件9中的命令 "tif2mrc" 或 "newstack", 从单个倾斜图像中制作堆栈图像文件。</li>
	<li>在 IMOD 中启动 eTomo GUI 软件并设置图像参数: 像素大小、基准直径、图像旋转等。然后创建脚本。</li>
	<li>根据材料表中列出的软件执行个别程序, 其中倾斜系列使用基准标记对齐 (平均残差小于 0.5)。最后, 利用 SIRT 算法在 IMOD 中重建3D 声像。</li>
	<li>从声像中提取感兴趣区域 (ROI), 并使用降噪过滤器: IMOD 中的各向异性扩散滤波器, EMAN10中的双侧滤波器, 或数学形态学滤波器 11等, 具有适当的参数来增强对比度。</li>
</ol>6. 利益特征的细分
注意: 下面描述的过程特定于所使用的软件 (参见材料表), 但是其他软件包可以使用。请参阅用户指南。
<ol><li>在3D 查看器窗口中, 打开阿米拉软件上的声像文件并生成 OrthoSlice。</li>
	<li>在 "细分编辑器" 窗口中, 通过选择一个新的 "标签字段" 来创建一个分割文件。</li>
	<li>手动跟踪感兴趣的特征 (FOI) 的边框。按照 FOI 通过所有声像切片。对于第二个 FOI, 创建一个新的 "材料" 并重复相同的操作。</li>
	<li>通过选择 "SurfaceGen" 菜单生成曲面呈现。要可视化分割的卷, 请选择 "SurfaceView" 菜单。要在3D 卷中移动、旋转和缩放, 请使用3D 查看器窗口中的工具。</li>
	<li>对于自动分割, 使用魔术棒工具。单击某个对象, 并调整显示和掩蔽中的滑块以覆盖值的范围, 以便对象由其功能完全选择。</li>
</ol>

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作者声明没有竞争的财政利益。

我们提出了一系列的协议, 以可视化的瞬态 DNA 压缩在蓝藻。基本概念类似于相关光和电镜 (克莱)12。此外, 在该方法中, 用荧光显微镜对活蓝藻进行监测, 在 EM 网格上快速冻结, 并直接通过高压低温电子层析成像进行可视化。作为第一个应用, DNA 压缩细菌细胞的详细结构在3D 成功地被形象化。目前, 这一程序是专门针对这个问题的, 但它将得到更广泛的应用, 并在某些情况下采用改进的方法。在此, 讨论了该方法的优点、局限性和未来的可能性。
该方法的优点之一是整个细胞的3D 可视化。1 MV 高压电能表成功地对 DNA 压实细胞中亚细胞细胞器的动态结构进行了可视化。然而, 由于图像对比度低, 正常细胞内的精细结构无法区分。在厚试样中增加非弹性和多重散射, 模糊了图像13。通过能量过滤器进行零损耗和最可能损耗图像滤波, 可以减少非弹性散射14、15, 从而提高图像对比度, 但对比 iMFP 厚的试样不起作用。随着试样厚度的增加, 零损耗和最可能损失峰值急剧减小。对于电子敏感的冰嵌入试样来说, 获得足够的信噪比是特别困难的。村田等已经表明, 1MV 扫描透射显微镜 (茎) 比一个明亮的场图像, 在5µm 厚度的塑料嵌入酵母细胞的图像对比, 其中图像对比主要由振幅对比13.然而, 预计撞击损伤对高加速电子的影响对损伤敏感的低温试样的辐照剂量产生了另一个限制16。应用 Zernike 相板17,18为高压电能表可能能够减少撞击伤害通过减少总剂量的未来。对厚样本使用高压电能表的另一个限制是, 提供高压电能表的用户设施在全球范围内是稀缺的。
使用另一种方法来观察厚的标本, 300 kV 的低温干细胞断层扫描显示了高对比度图像的冷冻水合试样的厚度超过了百纳米19。为了检索低温茎中的相对比度, 还介绍了电子 pthychographic 显微术, 其中在冷凝器透镜中的相板对调一个失真2D 探测器20的相位调制衍射。通过多个衍射的计算来检索图像。对于大型本机冷冻样品的直接快速3D 冷冻成像, 也可以使用低温-SEM-扫描电镜21, 用聚焦离子束和块面成像进行序列切片, 用于成像全水合冰冻标本。尽管这些技术扩大了生物标本的观赏范围, 但很难找到细菌的目标位置,例如标记细菌, 因为目标完全在冰下, 在修剪前无法辨认。
DNA 压实在蓝藻中产生明显的结构。DNA 压实细胞很容易分辨, 即使不被染色, 因为在细胞中不存在于正常细胞内的大密度偏倚。然而, 为了使细胞内更多的局部事件可视化, 有必要将荧光标记的 ROI 转移到电子显微镜中。对于相关的光和电子显微学 (克莱), 光镜图像和电子显微镜图像通常是相关的使用荧光乳胶珠或量子点在 EM 搜索网格12。除荧光外, 标签颗粒必须具有高的电子密度。它们可以准确和可靠地关联两个图像之间的位置。此外, 通过用低温显微镜确认标记区域, 两个显微镜之间可以实现 ROI 的完全重叠。在描述 DNA 压实的更详细的结构事件时, 这些粒子和低温光显微镜将成为未来更加强健和准确的相关性的不可缺少的工具。
本文通过同步培养、荧光显微镜和高压低温电子层析成像的结合, 说明了蓝藻 DNA 压实的瞬态结构特征。本协议的重点是对压实 DNA 的观察。将该方法与上述其他新技术相结合, 可以进一步深入研究 DNA 压实过程, 并对细菌中其他动态结构事件进行适当的改进。

DNA 压实是在一些蓝藻中发现的一种戏剧性的细胞质事件。当细长细长在每光/暗循环下培养12小时时, DNA 在光周期结束时就会凝结, 而在其他时间点1则明显不同于它的外观。有人建议, 这个过程是由一个昼夜节律时钟的基础上的凯蛋白2。Seki等人报告说, 赫斯特33342的 DNA 被压实在S. 细长细胞向光周期结束, 并在荧光显微镜下显示波浪状的杆形。被压缩的脱氧核糖核酸然后分离成二在杆的中心作为细胞划分, 并且最后回到正常均匀分布在每个女儿细胞3。然而, 它的瞬态性质和大尺寸的电子显微镜阻碍了结构分析。村田等结合多种方法, 包括同步培养、荧光显微、快速冷冻、高压电子低温层析 (HVET), 成功地确定了瞬态 DNA 压实的结构,包括聚磷酸盐体的动力学 (PPBs)4。该手稿通过结合实验程序, 对这种困难的材料进行了详细的视觉解释。
细长有一个蒴果形状, 长度为2到5µm, 宽度约为0.5 µm, 而完美的 DNA 压实在活细胞中只在很短的时间内出现。因此, 蓝藻 DNA 压实中发生的结构变化是未知的。为了通过电子显微镜对这些结构进行研究, 有必要克服两个主要的技术问题。一种是在接近当地条件下观察到整个细菌的厚标本, 另一种是动态结构的快速固定。对于第一个问题, 电子显微镜5的加速电压依赖于 iMFP 的非弹性平均自由路径。在透射电镜 (TEM) 的300伏, 它是小于 350 nm。例如, 当在 200kV TEM (iMFP ≈ 250 nm) 中观察到冰嵌入的蓝藻 (试样厚度≈ 600 nm) 时, 细胞内的结构很难观察。相比之下, 1 MV (iMFP ≈ 500 nm) 可以给和图像的细胞质结构在整个细胞 (图 1)。在本协议中, 作为解决方案的一部分, 采用了 1 MV 加速电压的高压电子显微镜 (高压电能表)。然而, 实施高压电能表的设施在世界范围内受到限制。讨论部分还讨论了可能的替代解决方案。第二个问题是通过低温电子显微镜 (冷冻 EM) 来解决的。这是一个强大的工具, 以可视化的动态结构在接近当地条件, 其中标本是迅速冻结在液态乙烷使用快速冻结装置, 和冻结的时刻是直接观察到最小的修改6。结合层析成像, 三维 (3D) 结构的快照可以从倾斜系列7重建。在本实验中, DNA 压实是在S. 细长中用同步培养法在每光/暗循环下复制的, 并通过荧光显微镜下的监测确定标本的凝固时间。
本文介绍的方法广泛适用于细菌细胞的动态结构研究,如细胞分裂、染色体分离和噬菌体感染, 并有可能开辟微生物研究的新途径。

<table><tbody><tr><td>Hoechst 33342 solution</td><td>Dojindo</td><td>346-07951</td><td>1mg/mL in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Agar Powder (for plant culture)</td><td>Wako</td><td>016-11875</td><td></td></tr><tr><td>Boric acid</td><td>Wako</td><td>027-02192</td><td>99.5%</td></tr><tr><td>Manganese chloride tetrahydrate</td><td>Wako</td><td>139-00722</td><td>99%</td></tr><tr><td>Zinc sulfate heptahydrate</td><td>Wako</td><td>264-00405</td><td>99.5%</td></tr><tr><td>Sodium molybdate</td><td>Wako</td><td>196-02472</td><td>99%</td></tr><tr><td>Copper sulfate pentahydrate</td><td>Wako</td><td>039-04412</td><td>99.5%</td></tr><tr><td>Cobalt nitrate hexahydrate</td><td>Wako</td><td>031-03752</td><td>99.5%</td></tr><tr><td>Sodium nitrate</td><td>Wako</td><td>191-02542</td><td>99%</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Wako</td><td>131-00405</td><td>99.5%</td></tr><tr><td>Calcium chloride dehydrate</td><td>Wako</td><td>031-00435</td><td>99.5%</td></tr><tr><td>Citric acid</td><td>Wako</td><td>036-05522</td><td>98%</td></tr><tr><td>EDTA-2Na</td><td>Dojindo</td><td>343-01861</td><td>99.5%</td></tr><tr><td>Sodium carbonate</td><td>Wako</td><td>197-01581</td><td>99.8%</td></tr><tr><td>Potassium phosphate dibasic</td><td>Wako</td><td>164-04295</td><td>99%</td></tr><tr><td>TES (Good&amp;rsquo;s buffer)</td><td>Dojindo</td><td>344-02653</td><td>99%</td></tr><tr><td>Ferric ammonium citrate</td><td>Wako</td><td>092-00802</td><td>1st Grade</td></tr><tr><td>Sodium thiosulfate pentahydrate</td><td>Wako</td><td>197-03585</td><td>99%</td></tr><tr><td>BSA gold tracer 15nm</td><td>Aurion</td><td>215.133</td><td></td></tr><tr><td>Quantifoil EM grid</td><td>Quantifoil MicroTools</td><td>R3.5/1 Copper grid</td><td></td></tr><tr><td>Electron films</td><td>Kodak</td><td>SO-163</td><td></td></tr><tr><td>Developer</td><td>Kodak</td><td>D19</td><td></td></tr><tr><td>Fixer</td><td>Kodak</td><td>Rapid fixer</td><td>Solution</td></tr><tr><td>Filter paper</td><td>Whatman</td><td>Grade 1</td><td></td></tr><tr><td>Growth chamber</td><td>NKsystem</td><td>LH-100SP</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Nikon</td><td>ECLIPSE 50i</td><td></td></tr><tr><td>High voltage TEM</td><td>HItachi</td><td>H1250M</td><td></td></tr><tr><td>Cryo-specimen holder for HVEM</td><td>Gatan</td><td></td><td></td></tr><tr><td>plunge-freezing device</td><td>Leica</td><td>EM CPC</td><td></td></tr><tr><td>Plasma Ion bombarder</td><td>Vacuum device</td><td>PIB-10</td><td></td></tr><tr><td>Liquid nitrogen storage</td><td>Taylor-Wharton</td><td>25LDB</td><td></td></tr><tr><td>Developing tank</td><td>Dosaka EM</td><td>TB-3-75</td><td></td></tr><tr><td>flatbed scanner</td><td>Nikon</td><td>Coolscan 9000ED</td><td></td></tr><tr><td>Segmentation software</td><td>FEI</td><td>Amira</td><td>https://www.fei.com/software/amira</td></tr><tr><td>Tomographic Reconstruction software</td><td>eTOMO</td><td></td><td>http://bio3d.colorado.edu/imod</td></tr></tbody></table>

visualization of dna compaction in cyanobacteria by high-voltage cryo-electron tomography

该协议描述了如何可视化蓝藻中的瞬态 DNA 压缩。DNA 压实是最近发现的一个戏剧性的细胞质事件发生在一些蓝藻细胞分裂之前。然而, 由于细胞大小和瞬态特性的存在, 很难对结构进行详细的研究。为了克服这些困难, 首先, 蓝藻细长细长PCC 7942 中的 DNA 压实是通过同步培养, 每光/暗循环的12小时重现性产生的。其次, 用荧光显微镜对 DNA 压实进行监测, 快速冷冻捕获。第三, 采用高压低温电子层析成像技术, 在三维 (3D) 中可视化了 DNA 压缩细胞的详细结构。这套方法广泛适用于研究细菌中的瞬态结构,如细胞分裂、染色体分离、噬菌体感染等, 通过荧光显微镜观察, 直接可视化低温电子层析成像在适当的时间点。

在一个精确的同步文化下每光/暗周期12小时, 标记与赫斯特的 DNA 显示在黑暗条件下正常均匀分布 (图 2a)。但是, 它在光周期内逐渐压缩单元格, 并在光周期结束时显示为一个波浪状杆样结构 (图 2b)。最后, 杆分在中心 (图 2c中的箭头) 和它的两个部分分布到子细胞 (图 2d)。细胞分裂后, 压实 dna 立即消失, dna 返回正常均匀分布。
当 DNA 压实最后阶段的细胞整除被立即转移到孔网格中, 在液态乙烷中快速冻结时, 1 MV 低温高压电能表观察到冰冻的网格, 蓝藻的内部结构包括DNA, 囊状体膜层, 细胞壁和 PPBs, 出现在一个快照中, 在冰冻的时刻 (图 3a)。许多细胞在细胞中显示出明显的 DNA 压实 (图 3a中的白色箭头), 很容易与正常细胞 (图 3b中的白色箭头) 区分开来。有些在细胞分裂之前显示了收缩在单元的中心 (图 3a的黄色箭头)。
在3D 断层中, 细胞的主要器可以被分割;细胞壁, 囊体膜层, DNA 和 PPBs 可以区分 (图 4)。特别地, 压缩 dna 被分离了在细胞质的一个明显的空白在那里 dna 被低密度材料围拢, 并且类囊体膜层被扭曲沿波浪杆的被压缩的脱氧核糖核酸。新发现 PPBs 的动态行为: 在 dna 压实细胞中, 许多小 PPBs 被认为是附着在 dna 上的, 而在正常细胞中却是大而少。此外, 大多数 PPBs 出现成对, 一些 DNA 似乎是在分离过程中的 PPBs。这表明, PPBs 本身被分为两个 dna 复制和功能作为磷酸盐供应商的 dna 合成。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57197/57197fig1.jpg"> 图1。冰嵌入正常蓝藻的低温 EM 图像,细长PCC 7942 在不同的加速电压。a) 200kV 和b) 1000kV。刻度条 = 500 毫微米。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57197/57197fig1large.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/57197/57197fig2.jpg"> 图 2: 蓝藻细长细胞的荧光显微镜.在同步培养后12小时每光/暗循环, 细胞被染色的赫斯特33342。(a)在2小时后的黑暗状态的细胞显示统一的 DNA 标记。许多细胞的 DNA 在光周期中逐渐凝结。它最终形成了一个粗波浪棒(b)典型的 DNA 压实。在这个阶段以后, 被压缩的脱氧核糖核酸结构快速地划分在他们的中心 (箭头) 在细胞分裂(c)期间, 并且二个片断分离入子细胞(d).在黑暗时期, 女儿细胞又回到了统一的 DNA 标记上。刻度条 = 2 µm.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57197/57197fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57197/57197fig3.jpg"> 图 3: 在非晶态冰中嵌入的蓝藻的低温高压电能表图像.(a)显示在0°倾斜处的原始图像。图像是在光期结束时拍摄的。有些细胞显示波浪状的杆形 DNA 体, 类似于真核凝聚染色体 (白色箭头)。在正常细胞中, 蓝藻在细胞质中具有明显的 DNA 结构 (白色箭头)。有些陈列收缩在细胞的中心象期望在细胞分裂之前 (黄色箭头)。(b) xy、xz、yz 3D 声像片。黄色虚线显示切片的交集。刻度条 = 2 µm.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57197/57197fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57197/57197fig4.jpg"> 图 4: 含有压缩 DNA 的细胞的超微结构.主要成分是分段: 细胞壁 (亮黄色), 囊体膜 (红色) 和 DNA (蓝色)。(a)显示3D 声像的 z 切片。(b)所有部分。表示单元分离的收缩出现在单元格的中心 (箭头)。PPBs 被建模为黄色或橙色球体;每个橙色球体代表最接近的黄色球体的对应。刻度条 = 500 毫微米。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57197/57197fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography at JoVE.com

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

该协议描述了如何可视化蓝藻中的瞬态 DNA 压缩。采用同步培养、荧光显微镜、快速冷冻、高压低温电子层析成像等技术进行监测。提出了这些方法的一个协议, 并讨论了未来的应用和发展。

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

visualization-dna-compaction-cyanobacteria-high-voltage-cryo-electron

Universidad San Sebastian

Research

JoVE Journal

Biology

9.0K 次观看.  National Institute for Physiological Sciences. 该协议描述了如何可视化蓝藻中的瞬态 DNA 压缩。采用同步培养、荧光显微镜、快速冷冻、高压低温电子层析成像等技术进行监测。提出了这些方法的一个协议, 并讨论了未来的应用和发展。

视频: Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts.
Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.

To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.

用于可视化神经突在冷冻水合状态下使用低温电子断层扫描准备初级神经元

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the ...

科研

神经科学

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

生物工程

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

冷冻电子断层扫描 的糖核 酸的准备和冷冻-FIB微加工

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

生物化学

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.
Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2
In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

细菌细胞的电子Cryotomography

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

生物学

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

应用活细胞成像和低温电子断层扫描解决军团菌肺瘤点/Icm分泌系统的时空特征

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

免疫与感染

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms

Efficient methods for the extraction of features of interest remain one of the biggest challenges for the interpretation of cryo-electron tomograms. Various automated approaches have been proposed, many of which work well for high-contrast datasets where the features of interest can be easily detected and are clearly separated from one another. Our inner ear stereocilia cryo-electron tomographic datasets are characterized by a dense array of hexagonally packed actin filaments that are frequently cross-connected. These features make automated segmentation very challenging, further aggravated by the high-noise environment of cryo-electron tomograms and the high complexity of the densely packed features. Using prior knowledge about the actin bundle organization, we have placed layers of a highly simplified ball-and-stick actin model to first obtain a global fit to the density map, followed by regional and local adjustments of the model. We show that volumetric model building not only allows us to deal with the high complexity, but also provides precise measurements and statistics about the actin bundle. Volumetric models also serve as anchoring points for local segmentation, such as in the case of the actin-actin cross connectors. Volumetric model building, particularly when further augmented by computer-based automated fitting approaches, can be a powerful alternative when conventional automated segmentation approaches are not successful.

Here, we present a protocol to place simplified volumetric models into noisy, complex, tomographic 3D volumes. This allows the fast segmentation of actin filament densities, detection of systematic filament bending and of gaps in hair bundle filaments, as well as convenient quantification of volumetric model properties, such as distances.

Efficient methods for the extraction of features of interest remain one of the biggest challenges for the interpretation of cryo-electron tomograms. ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

通过冷冻软X射线断层扫描对细胞进行3D制图描述

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

冷冻电子断层扫描远程数据收集和断层扫描平均

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryogenic electron microscopy (cryo-EM) relies on the imaging of biological or organic specimens embedded in their native aqueous medium; water is solidified into a glass (i.e., vitrified) without crystallization. The cryo-EM method is widely used to determine the structure of biological macromolecules recently at a near-atomic resolution. The approach has been extended to the study of organelles and cells using tomography, but the conventional mode of wide-field transmission EM imaging suffers a severe limitation in the specimen thickness. This has led to a practice of milling thin lamellae using a focused ion beam; the high resolution is obtained by subtomogram averaging from the reconstructions, but three-dimensional relations outside the remaining layer are lost. The thickness limitation can be circumvented by scanned probe imaging, similar to the scanning EM or the confocal laser scanning microscope. While scanning transmission electron microscopy (STEM) in materials science provides atomic resolution in single images, the sensitivity of cryogenic biological specimens to electron irradiation requires special considerations. This protocol presents a setup for cryo-tomography using STEM. The basic topical configuration of the microscope is described for both two- and three-condenser systems, while automation is provided by the non-commercial SerialEM software. Enhancements for batch acquisition and correlative alignment to previously-acquired fluorescence maps are also described. As an example, we show the reconstruction of a mitochondrion, pointing out the inner and outer membrane and calcium phosphate granules, as well as surrounding microtubules, actin filaments, and ribosomes. Cryo-STEM tomography excels in revealing the theater of organelles in the cytoplasm and, in some cases, even the nuclear periphery of adherent cells in culture.

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryo-STEM tomography provides a means to visualize organelles of intact cells without embedding, sectioning, or other invasive preparations. The 3D resolution obtained is currently in the range of a few nanometers, with a field of view of several micrometers and an accessible thickness in the order of 1 &#181;m.

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

摘要

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此视频中的章节

用于可视化神经突在冷冻水合状态下使用低温电子断层扫描准备初级神经元

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

冷冻电子断层扫描的糖核酸的准备和冷冻-FIB微加工

细菌细胞的电子Cryotomography

应用活细胞成像和低温电子断层扫描解决军团菌肺瘤点/Icm分泌系统的时空特征

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms

通过冷冻软X射线断层扫描对细胞进行3D制图描述

冷冻电子断层扫描远程数据收集和断层扫描平均

通过冷冻STEM断层扫描原位可视化细胞器

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

摘要

探索更多视频

此视频中的章节

用于可视化神经突在冷冻水合状态下使用低温电子断层扫描准备初级神经元

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

冷冻电子断层扫描 的糖核 酸的准备和冷冻-FIB微加工

细菌细胞的电子Cryotomography

应用活细胞成像和低温电子断层扫描解决军团菌肺瘤点/Icm分泌系统的时空特征

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms

通过冷冻软X射线断层扫描对细胞进行3D制图描述

冷冻电子断层扫描远程数据收集和断层扫描平均

通过冷冻STEM断层扫描原 位 可视化细胞器

冷冻电子断层扫描的糖核酸的准备和冷冻-FIB微加工

通过冷冻STEM断层扫描原位可视化细胞器