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
Summary
以保留神经流程超微结构分析，我们描述了一种协议，用于在电子显微镜网格后用快速冷冻原代神经元的电镀，得到的样品悬浮在一层玻璃的冰。这些样本可以检查与一个低温电子显微镜可视化结构在纳米尺度。
Abstract
突起，无论是树突和轴突，是神经元细胞过程，使电脉冲的神经元之间的传导。定义突起的结构是如何理解这些过程动议，支持突触宣传材料和信号的关键。电子显微镜（EM）传统上被用来评估突起内的超微结构特征，但是，接触有机溶剂的脱水和树脂的嵌入过程中，可能会扭曲结构。一个重要的未满足的目标是有关程序，以便不被这种假象影响结构性评估的制定。在这里，我们已经建立了种植和速冻对整个电子显微镜网格其次是他们检查与低温电子断层扫描（冷冻ET）不同的主神经元的轴突详细的和可重复的协议。这种技术允许冷冻，水合突起在纳米级分辨率的三维可视化，便于ASSEssment他们的形态差异。我们的协议产生背根神经节（DRG）突起，和海马神经突在他们接近天然状态的可视化前所未有的看法。因此，这些方法为未来在正常的神经元和那些由神经系统疾病影响的突起研究奠定了基础。
Introduction
神经元建立复杂的电路必需的中枢和周围神经系统的通过阐述树突接收信息和轴突，往往是相当冗长的，与下游神经元进行通信的功能。神经突增生起到胚胎发育和神经细胞的分化和维持轴突的过程中的基础性作用支持神经系统的审慎的功能。神经炎过程还具有批判性的神经损伤和再生，以及神经系统疾患。神经元结构的研究是至关重要的了解双方的正常和病变的大脑。幸运的是，生理有关的神经元细胞培养系统存在，可以概括复杂和异构的细胞结构。基于坚实的实验平台，有效的可视化策略，使神经元形态的定性和定量分析的澄清是必要的。特别有用会是一个详细的方法，提供了用于可视化的神经突，既轴突和树突在纳米尺度一致的平台。
传统的电子显微镜需要脱水和树脂包埋过程中使用有机溶剂，可以从自己的真实状态引起扭曲的标本。迄今为止，在纳米尺度结构最大表征是基于较大的细胞或组织所承受这样苛刻的化学物质-从而限制的结果4,9,25解释。此外，对于电子束的穿透，切片所需的生物体或细胞的突起呈现的厚度大于1微米的12。最后，切片或磨碎的组织结果集合中的离散量特定的数据集，使突起的细长的特点繁琐的定义。即使冷冻电镜，其中切片冷冻水合的样品是可能的，该方法引入了压缩ARTIF使徒行传1。
近年来，研究人员已经学会了如何直接在EM电网和速冻它们在液态乙烷采用低温ET 8,10,18,23随后可视化轴突生长的海马神经元。然而，这些研究要么使用一个特制的装置10,23，或对产生足够薄玻璃冰常规可视化8,18的印迹步缺乏细节。例如，一项研究建议使用30-40秒，吸干了EM网格10，但是，这个值进行了优化不能用于一般用途，但具体是对于定做暴跌冷冻设备。使用一个特制的设备，而不是市售17保持湿度暴跌冻结的样品前可能构成障碍的广泛的可重复性。
虽然这些研究已经破土动工了冷冻ET可视化突起，我们已经迈出了一步，探讨AP冷冻的ET plicability各种神经元标本（海马及背根神经节神经元）的。此外，我们将讨论这两种最优和次优的结果，以及人们可以利用冷冻ET此类标本遇到潜在的文物。
定义一个详细的技术维护和在纳米尺度可视化整个轴突在接近本机的状态将加强开展超微结构研究的能力，更多的研究者。为此，我们使用市售设备来制备未定影的，未染色的神经元来可视化突起描述一种有效和详细的协议。这是迈向健康的详细轴突的超微结构，并奠定基础，了解什么是结构上的差异存在于神经系统疾病模型的重要的第一步。因为冷冻的ET能在纳米尺度解决在3-D未定影的，未染色的神经突的特点，该方法将使得能够作为永远脱颖而出定义神经炎架构12。
Protocol
Representative Results
Discussion
我们证明了大鼠胚胎神经元（背根神经节和海马）可以生长在金电子显微镜（EM）的网格，并冻结在玻璃冰足够薄，其轴突使用2-D冷冻电镜和3-D冷冻待成像ET。虽然海马神经元突起先前已使用冷冻ET 8,10,18,23成像，协议使用市售的设备一直缺乏足够详细的成功复制。此外，虽然他们的研究已经率先使用冷冻ET的可视化海马突起，我们的研究探讨低温ET的适用性，以不止一种类型的神经元标本?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究已经由美国国立卫生研究院资助（PN2EY016525和P41GM103832）的支持。二手烟是由来自WM凯克中心对墨西哥湾沿岸财团（美国国立卫生研究院批准号：T32EB009379）的跨学科生物科学训练的纳米生物学跨学科研究生培养方案的研究奖金支持。
SHS解剖，成长和陶瓷DRG和海马细胞;收集的冷冻电镜和背根神经节和海马轴突冷冻 – 经济数据，重建和颜色标注的倾斜系列。 MRG解剖和MNR的实验室提供的海马细胞。资深大律师协助的倾斜系列注释。二手烟是如何解剖和神经生长训练的CW。 SHS，WCM和厕所构思的实验。自蔓延高温合成制备的手稿与其他作者的输入。
这部影片的拍摄地在该中心为t的Biozentrum的细胞成像和NanoAnalytics（C-CINA）他巴塞尔大学。 C-CINA集成部的苏黎世联邦理工学院，位于巴塞尔，瑞士的生物系统科学与工程（D-BSSE）。
Materials
| Dumont #7 Tweezers | Electron Microscopy Sciences | 72803-01 | For handling EM grids |
| Glass bottom dishes | MatTek Corp. | P35G-1.5-10C | For growing sample |
| Electron microscopy grids | Quantifoil | Holey Carbon, Au 200, R 2/2 | For growing sample |
| Calcium-free filter paper | Whatman | 1541-055 | For blotting sample |
| Large flat point long tweezers | Excelta Corporation | E003-000590, 25-SA | For blotting sample |
| Vitrification device and tweezers | FEI | Vitrobot Mark III | For freezing sample |
| Mini grid storage boxes | Ted Pella, Inc. | 160-40 | For storing EM grids |
| Cryo transfer holder | Gatan | 626 Single Tilt Liquid Nitrogen Cryo Transfer Holder | For imaging samples |
| Semi-automated tilt series acquisition software | SerialEM | http://bio3d.colorado.edu/SerialEM/ | For imaging samples |
| Image processing software | IMOD eTomo | http://bio3d.colorado.edu/imod/ | For image processing |
| Transmission electron microscope for cryoEM | JEOL, Tokyo | 200-kV JEM2100 LaB6 electron microscope | For imaging samples |
| 4k x 4k CCD camera | Gatan | N/A | For imaging samples |
| 3-D annotation software | Visage Imaging GmbH | Amira/Avizo | For processing 3-D data |
| Software for digitally stitching 2-D images | Adobe | Adobe Photoshop | For processing 2-D data |
| DMEM, High Glucose | Invitrogen | 11965-118 | For hippocampal culture |
| Boric acid | Sigma Aldrich | B-0252 | For hippocampal culture |
| Sodium tetraborate | Sigma Aldrich | B-9876 | For hippocampal culture |
| Poly-L-lysine | Sigma Aldrich | P2636-500MG | For hippocampal culture |
| Filter system | Corning | 430758 | For hippocampal culture |
| Neurobasal medium | Invitrogen | 21103-049 | For DRG culture |
| B-27 supplement | Invitrogen | 17504-044 | For DRG culture |
| Penicillin/Streptomycin | Invitrogen | 15140-122 | For DRG culture |
| Glutamax | Invitrogen | 35050-061 | For DRG culture |
| Recombinant rat b-NGF | R&D Systems | 556-NG | For DRG culture |
| Uridine | Sigma | U3003-5G | For DRG culture |
| 5'-Fluoro-2'-deoxyuridine | Sigma | F0503-100MG | For DRG culture |
| Matrigel | BD Biosciences | 356234 | For DRG culture |
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