一种在透射电镜中获取微生物连续超薄切片的方法
Summary
本研究为在透射电子显微镜下获取无昂贵设备的微生物的连续超薄切片提供了可靠、简便的程序。
Abstract
在透射电镜高倍率下观察三维细胞和细胞成分, 需要制备试样的超薄切片。虽然准备连续超薄的部分被认为是非常困难的, 它是相当容易的, 如果正确的方法是使用。本文介绍了一种安全获取微生物连续超薄切片的 step-by 步骤。该方法的要点是: 1) 使用试样的大部分, 调整试样的表面和刀口, 使其相互平行;2) 在狭缝网格上检索一组连续剖面时, 要在组中切割连续节, 避免在分离时使用一对头发束的困难;3) 使用 “节-保持回路”, 避免混淆节群的顺序;4) 使用 “水表面提升回路”, 并确保剖面位于水的顶端, 并首先接触网格, 以便将它们放置在栅格上所需的位置;5) 使用铝架上的支撑膜, 使其更容易恢复网格上的剖面, 避免支撑膜起皱;6) 使用染色管, 避免用镊子意外折断支撑膜。这种新的方法可以毫不费力地获得连续的超薄切片。该方法可以对3D 高分辨率的微生物细胞结构进行分析, 使用自动胶带收集 ultramicrotome 法和连续块面或聚焦离子束扫描电子显微镜是无法实现的。
Introduction
适当的连续超薄切片技术是必不可少的研究细胞和细胞成分三维在电子显微镜水平。研究了酵母细胞周期中纺锤极体的动态变化, 并揭示了其在细胞周期和重复时间上的形态学改变1,2,3, 4,5。在 2006年, 我们创造了一个新词 ‘ structome ‘ 通过结合 “结构” 和 “-“, 并定义为 ‘ 定量和三维结构信息的整个细胞在电子显微镜水平 ‘ 6,7。
通过 structome 分析, 需要连续超薄切片技术, 发现酵母细胞的酿酒酵母和外 dermatitidis有大约20万核糖体7,8, 一个大肠杆菌细胞有2.6万核糖体9, a 结核分枝杆菌细胞有1700核糖体10 , Myojin 螺旋菌只有300核糖体11。这些信息不仅可以用来估计每个生物体的生长速率, 也有助于识别物种的9。
此外, structome 分析导致发现一个新的有机体;Parakaryon myojinensis在日本海岸的深海中发现, 其细胞结构介于原和真核之间的中间,121314,15。目前, 连续超薄切片技术被认为是非常困难的, 需要很长时间才能掌握。在这项研究中, 我们开发了一个可靠的方法, 任何人都可以进行连续超薄切片无困难。
Protocol
注: 本研究中使用的标本为微生物, 在液氮中迅速冷冻, 用丙烷取代含有2% 锇氧化的丙酮, 并嵌入环氧树脂1,2,3, 4,5,6,7,8,9,10,<sup class="xref"…
Representative Results
在该协议中, 三-狭缝网格用于拾取串行段。网格是由镍或铜制成的。串行部分放置在中间狭缝上。双方的狭缝是必要的, 以查看的部分时, 他们拿起网格。在用镊子 (图 11d) 拾取时, 为了使网格与串行剖面保持平行, 手柄弯曲 (图 6c, 右)。一个小手柄有利于防止网格主体的弯曲, 在拾取时会引起严重的问题, 并防止在染色过程中掉节…
Discussion
这里提出的方法不需要昂贵的设备。它只需要一个铝架 (图 3)、三狭缝网格 (图 6c)、节保持环路 (图 10a)、water-surface 提升环路 (图11a) 和染色管 (图 13)。目前的方法有许多特点。试样的大部分用于调整试样的表面和刀口, 使其彼此平行。在将一组串行剖面检索到狭缝网格时, 将序列切片分…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们衷心感谢茂雄的宝贵建议和讨论。我们还感谢约翰和 Sumire Eckstein 对手稿的批判性阅读。
Materials
| Formvar making apparatus | Nisshin EM Co. Ltd., Tokyo | 652 | W 180 x D 180 x H 300 mm |
| Glass slide | Matsunami Co. Ltd., Osaka | – | 76 x 26 x 1.3 mm |
| Aluminum rack with 4-mm holes | Nisshin EM Co. Ltd., Tokyo | 658 | W 30 x D 25 x H 3 mm, Refer to this paper |
| Stereomicroscope | Nikon Co. Ltd., Tokyo | – | SMZ 645 |
| LED illumination for stereomicroscope | Nikon Co. Ltd., Tokyo | – | SM-LW 61 Ji |
| Trimming stage | Sunmag Co.Ltd., Tokyo | – | Tilting mechanism equipped, Refer to this paper |
| LED illumination for trimming stage | Sunmag Co.Ltd., Tokyo | – | Refer to this paper |
| Ultrasonic trimming blade | Nisshin EM Co. Ltd., Tokyo | 5240 | EM-240, Refer to this paper |
| Diamond knife for trimming | Diatome Co. Ltd., Switzerland | – | 45° |
| Diamond knife for ultrathin sectioning | Diatome Co. Ltd., Switzerland | – | 45° |
| Ultramicrotome | Leica Microsystems, Vienna | – | Ultracut S |
| Mesa cut | Leica Microsystems, Vienna | – | Mirror |
| 0.5% Neoprene W solution | Nisshin EM Co. Ltd., Tokyo | 605 | |
| Special 3-slit nickel grid | Nisshin EM Co. Ltd., Tokyo | 2458 | Refer to this paper |
| Special 3-slit copper grid | Nisshin EM Co. Ltd., Tokyo | 2459 | Refer to this paper |
| Section-holding loop | Nisshin EM Co. Ltd., Tokyo | 526 | Refer to this paper |
| Water-surface-raising loop | Nisshin EM Co. Ltd., Tokyo | 527 | Refer to this paper |
| Staining tube | Nisshin EM Co. Ltd., Tokyo | 463 | Refer to this paper |
| Multi-specimen holder | JEOL Co. Ltd., Tokyo | – | EM-11170 |
| JEM-1400 | JEOL Co. Ltd., Tokyo | – | Transmission electron microscope |
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Citar este artigo
Yamaguchi, M., Chibana, H. A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy. J. Vis. Exp. (131), e56235, doi:10.3791/56235 (2018).