McEwen B F, Marko M
Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany 12201-0509, USA.
Methods Cell Biol. 1999;61:81-111. doi: 10.1016/s0091-679x(08)61976-7.
Transmission electron microscopy produces images that are projections of the original object, with the consequence that features from different depths of the specimen overlap and give a confusing image. This problem is overcome by reconstructing the object in 3D from a series of 2D views using either serial thin section reconstruction or electron tomography. In the serial section approach, the series of 2D views is generated from images of successive serial sections cut thin enough to be effectively 2D slices of the specimen. For electron tomography the series of 2D views is generated by tilting a single, usually thicker, section in the electron beam. Resolution in the depth dimension is limited to twice the section thickness for serial section reconstruction and is determined by the number of tilt views collected (i.e., by the fineness of the angular interval between successive tilt views) for electron tomography. Both methods produce distorted 3D reconstructions because of missing material and alignment difficulties in the case of serial sections and the limited angular tilt range in the case of electron tomography. However, techniques have evolved for minimizing and circumventing these distortions and, as long as the user is aware of the limitations, misinterpretations can be avoided. Since electron tomography provides better resolution (generally 5-20 nm), it is the method of choice for determining detailed structural interactions such as the depth of kinetochore MT penetration into the kinetochore outer plate. On the other hand, serial section reconstruction is more effective for projects that require tracking through a complete object in the specimen, such as counting the number of kinetochore MTs on each kinetochore. If the project requires finding a relatively small object in a large specimen (e.g., finding centrioles in an oocyte), then it is sometimes advantageous to cut thicker plastic sections and analyze them via stereo viewing. The mitotic spindle, however, is generally too complex to be analyzed via stereo viewing. Currently, collapse of plastic sections in the electron beam limits the utility of serial section electron tomography. Once a 3D reconstruction is completed it must be analyzed with the 2D medium of the screen on a computer monitor. The easiest approach is usually to walk through the 3D reconstruction volume slice by slice. However, in order to appreciate 3D interactions, and to communicate the results to others, it is generally necessary to segment key components from the rest of the volume and use modeling and rendering techniques. Rendered surface views can easily be color coded and provided with a number of depth cues to simulate the surface viewing encountered in everyday life. In some instances, it is useful to look through a smaller portion of the reconstruction volume with "X-ray vision." This can accomplished by using volume rendering to create a series of semitransparent views from different tilt angles.
透射电子显微镜所产生的图像是原始物体的投影，其结果是来自标本不同深度的特征相互重叠，形成令人困惑的图像。通过使用连续薄切片重建或电子断层扫描技术，从一系列二维视图中重建三维物体，可以克服这个问题。在连续切片方法中，一系列二维视图是由连续的薄切片图像生成的，这些切片薄到足以成为标本的有效二维切片。对于电子断层扫描，一系列二维视图是通过在电子束中倾斜单个（通常较厚）切片生成的。对于连续切片重建，深度维度的分辨率限制为切片厚度的两倍；对于电子断层扫描，分辨率则由收集的倾斜视图数量（即连续倾斜视图之间的角度间隔精细度）决定。由于连续切片存在材料缺失和对齐困难问题，以及电子断层扫描存在有限的角度倾斜范围，这两种方法都会产生失真的三维重建结果。然而，已经有技术来最小化和规避这些失真，只要用户了解其局限性，就可以避免错误解读。由于电子断层扫描提供了更好的分辨率（通常为5 - 20纳米），因此它是确定详细结构相互作用（如动粒微管穿透动粒外板的深度）的首选方法。另一方面，连续切片重建对于需要在标本中追踪完整物体的项目（如计算每个动粒上动粒微管的数量）更有效。如果项目需要在大标本中找到相对较小的物体（如在卵母细胞中找到中心粒），那么有时切更厚的塑料切片并通过立体观察进行分析会更有利。然而，有丝分裂纺锤体通常过于复杂，无法通过立体观察进行分析。目前，塑料切片在电子束中的塌陷限制了连续切片电子断层扫描的实用性。一旦完成三维重建，就必须在计算机显示器的二维屏幕介质上进行分析。最简单的方法通常是逐片浏览三维重建体积。然而，为了理解三维相互作用并将结果传达给他人，通常需要从体积的其余部分分割出关键组件，并使用建模和渲染技术。渲染的表面视图可以轻松地进行颜色编码，并提供许多深度线索，以模拟日常生活中遇到的表面观察。在某些情况下，用“X射线视觉”查看重建体积的较小部分会很有用。这可以通过使用体积渲染从不同倾斜角度创建一系列半透明视图来实现。
