准备厚生物样品的观察扫描透射电子断层扫描
Summary
This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.
Abstract
This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.
Introduction
自从70年代初，断层扫描透射电子显微镜（TEM）已被广泛地用于生物标本1，2，3的结构表征方法。透射电子断层扫描的吸引力是，它可用于在纳米尺度，研究了广泛的生物结构的，从细胞的结构和细胞器的大分子复合物和蛋白质的结构的超微结构。然而，透射电子断层扫描不能用来研究非常厚的样品（大于0.5微米）。的确，厚的样品产生太多的散射电子，产生低信噪比（SNR）的图像。另外，断层摄影涉及倾斜标本的图像的集合，将样品与倾斜角度增加的表观厚度。即使非弹性散射可以被过滤出用能过滤器，需要的高信噪比图像电子的临界量在TEM是勉强达到。因此，厚的生物标本只用了4切片研究。
有些样品不能被切片：它们被切割时一些可能会降低，和其他需要的全部进行研究，以了解它们的复杂性。另一种方法是在扫描模式5，6，7，8使用的TEM。在扫描透射电子显微镜（STEM），电子的光路是与在常规的TEM不同。流过试样而不散射电子可以与亮场（BF）检测器9的光轴被收集，而那些弹性散射可以在从与暗场（DF）检测器的光轴以一定角度被收集。干的另一优点是，聚焦的电子束在所述样品的表面扫描，从而使该像素由像素集合的图像。即使通过样品10，而电子束变宽，这种特殊的收集方案是非弹性散射电子的比常规的TEM不太敏感。此外，不存在后透镜试样在STEM，从而避免可能发生在TEM色差。相机长度可以调整，以使高炉检测器主要检测未散射电子。使用DF探测器研究厚的样品不建议，因为多次散射，从而产生不准确的图像。取而代之的是，高炉检测器可以用来11。而干可以产生高信噪比的图像，其具有因为相比TEM相对高的电子束会聚的相对低的深度的领域中，降低，可以从厚要回收的的深入信息量标本。在像差校正STEM显微镜，其中该会聚角可高达30兆拉德的情况下，使得即在焦点中的信息，从只有几纳米的焦平面起源的深度的领域可以是足够低。设置在并行模式下的电子束增强了深度的领域的电子束的决议12的损害。然而，这种设置并不总是可能的。
每当有必要使用一个会聚电子束，人们必须使用学习厚样品时，可增强深度的领域的电子束的技术。最近的研究已经报道了采集在不同焦平面多个图像的整个样品回收的在焦信息13，14的最大量。两项研究描述不同的方式来处理来自不同的焦平面的信息。 Hovden 等。结合在傅立叶空间中被收集在不同的焦平面的图像，并且直接从3D逆傅立叶获得的最终重建变换13。与此相反，达门等。开发出会聚光束重建引擎在现实空间重构来自各种焦平面14中的3D体积。我们的实验室还开发了厚成像生物标本的方法。我们的策略是从在上述两种方法的不同，我们合并是在聚焦在各焦平面中的信息，并使用平行光束投影15重构了最终的3D体积在现实空间。我们的目的是开发，可以很容易在任何电子显微镜实验室中进行的方法。为此，我们的目的是收集焦点图像的时间也是有限的，与常规断层实验的时间框架。此外，我们提出的方法C乌尔德适于与不同类型的对齐和重建软件的使用。
在本刊从2015年15的背景下，我们想可视化和表征景深-的恢复，所以我们用25毫弧度大收敛半的角度。在这里，我们提出了一个一步一步的协议根据我们的实验室在2015年15开发的方法在STEM通过焦成像表现，以及我们提出从2015年数据是如何处理。这种方法恢复整个厚（750 nm）的生物样品中，重点从几个焦点平面信息，使高品质的3D重建。如果相关的，在这种方法与其他团体使用的方法的差异还提出。
Protocol
Representative Results
Discussion
在这篇文章中，我们提出了一个常规的样品制备协议与一步一步的指导使用STEM通过焦点断层扫描厚的生物样品进行三维分析一起。生物标本的树脂嵌入已使用了几十年26，和适于不同种类的样品替代协议可以在整个文献27中找到。与此相反，成像厚的样品在STEM使用通过焦方法是一种新的任务，并当它变得更普遍的方法将最有可能得到改善。
<p class="jove_content"…Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was funded by two ANR grants (ANR-11-BSV8-016 and ANR-10-IDEX-0001-02). We also acknowledge the PICT-IBiSA for providing access to chemical imaging equipment.
Materials
| Phosphate Buffered Saline | Sigma-Aldrich | P4417 | |
| Ethanol | Sigma-Aldrich | 2860 | |
| Epoxy resin | EMS | 14120 | |
| Paraformaldehyde | Sigma-Aldrich | P6148 | Add paraformaldehyde powder to PBS heated at approximately 60 °C. Increase pH by adding 1 N NaOH until no PFA powder is visible. |
| Glutaraldehyde | Sigma-Aldrich | G5882 | |
| Osmium Tetroxyde | EMS | 19150 | |
| Uranyl Acetate | Sigma-Aldrich | 73943 | |
| Gelatin Capsule | EMS | 70110 | |
| Triming and Histo knives | LFG Distribution | Diatome diamond knives | |
| Electron Microscopy copper grid | LFG Distribution | G200-Cu | |
| Grid Coating Pen | LFG Distribution | 70624 | |
| Specimen Holder | JEOL | EM-21311 HTR | |
| Electron Microscope | JEOL | JEM-2200FS |
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