Körtje K H, Paulus U, Ibsch M, Rahmann H
Institute for Zoology, University Hohenheim, Stuttgart, Germany.
J Microsc. 1996 Jul;183(Pt 1):89-101.
Electron microscopy of nervous tissue requires on the one hand nanometre resolution for the analysis of fine structures of nerve cell contacts, for instance synaptic vesicles, synaptic membranes and associated organelles. On the other hand, the visualization of the three-dimensional organization of nervous tissue on the level of dendrites and neurites is essential for the understanding of neuronal integration and also for a stereological evaluation of quantitative parameters such as size and shape of synaptic contact zones, number and distribution of synaptic vesicles, organization of cytoskeleton and distribution of organelles like mitochondria and endoplasmic reticulum. Therefore, it is necessary to have access to the fine structure and to the spatial organization within one sample. Energy-filtering transmission electron microscopy (EFTEM) enables the imaging of sections up to 1 micron thickness with a high resolution because the chromatic error due to inelastic scattering is avoided by selecting electrons within a restricted energy-loss range for imaging. The contrast depends strongly upon the section thickness, the selected energy range and the composition of the sample, so that optimum imaging conditions can be found for each object. Different staining protocols enable either a high global contrast or a selective staining of peculiar tissue properties. The three-dimensional organization can be visualized with stereopairs or with extended tilt series, which shows that characteristic structures as the synaptic junctions are detectable only within a narrow range of orientations to the electron beam. This is especially important for quantitative approaches with stereological tools which profit generally from the fact that a wide range of section thickness is available with EFTEM. EFTEM is therefore a powerful tool for the imaging of thick sections of biological materials with attractive possibilities of contrast tuning and advantages for stereological quantifications. The main benefit is the rapid and effective visualization of the three-dimensional organization of cells and tissues.
神经组织的电子显微镜检查一方面需要纳米级分辨率来分析神经细胞接触的精细结构，例如突触小泡、突触膜和相关细胞器。另一方面，在树突和神经突水平上对神经组织三维结构的可视化对于理解神经元整合以及对诸如突触接触区的大小和形状、突触小泡的数量和分布、细胞骨架的组织以及线粒体和内质网等细胞器的分布等定量参数进行体视学评估至关重要。因此，有必要在一个样本中同时观察到精细结构和空间组织。能量过滤透射电子显微镜（EFTEM）能够对厚度达1微米的切片进行高分辨率成像，因为通过在有限的能量损失范围内选择电子进行成像，避免了非弹性散射引起的色差。对比度在很大程度上取决于切片厚度、所选能量范围和样品组成，因此可以为每个对象找到最佳成像条件。不同的染色方案可以实现高整体对比度或对特殊组织特性进行选择性染色。三维结构可以通过立体对或扩展倾斜系列来可视化，这表明像突触连接这样的特征结构只有在与电子束的狭窄取向范围内才能被检测到。这对于使用体视学工具的定量方法尤为重要，这些方法通常受益于EFTEM能够提供多种切片厚度这一事实。因此，EFTEM是一种强大的工具，可用于对生物材料厚切片进行成像，具有吸引人的对比度调节可能性以及体视学定量优势。主要优点是能够快速有效地可视化细胞和组织的三维结构。
