Department of Neurology, University Hospital Würzburg, 97080 Würzburg, Germany.
Institute of Physiology, Department of Neurophysiology, University of Würzburg, 97070 Würzburg, Germany.
Int J Mol Sci. 2020 Mar 2;21(5):1708. doi: 10.3390/ijms21051708.
The size of the synaptic subcomponents falls below the limits of visible light microscopy. Despite new developments in advanced microscopy techniques, the resolution of transmission electron microscopy (TEM) remains unsurpassed. The requirements of tissue preservation are very high, and human post mortem material often does not offer adequate quality. However, new reprogramming techniques that generate human neurons in vitro provide samples that can easily fulfill these requirements. The objective of this study was to identify the culture technique with the best ultrastructural preservation in combination with the best embedding and contrasting technique for visualizing neuronal elements. Two induced neural stem cell lines derived from healthy control subjects underwent differentiation either adherent on glass coverslips, embedded in a droplet of highly concentrated Matrigel, or as a compact neurosphere. Afterward, they were fixed using a combination of glutaraldehyde (GA) and paraformaldehyde (PFA) followed by three approaches (standard stain, Ruthenium red stain, high contrast en-bloc stain) using different combinations of membrane enhancing and contrasting steps before ultrathin sectioning and imaging by TEM. The compact free-floating neurospheres exhibited the best ultrastructural preservation. High-contrast en-bloc stain offered particularly sharp staining of membrane structures and the highest quality visualization of neuronal structures. In conclusion, compact neurospheres growing under free-floating conditions in combination with a high contrast en-bloc staining protocol, offer the optimal preservation and contrast with a particular focus on visualizing membrane structures as required for analyzing synaptic structures.
突触亚组件的大小低于可见光显微镜的分辨率。尽管先进显微镜技术有了新的发展，但透射电子显微镜（TEM）的分辨率仍然是无与伦比的。组织保存的要求非常高，而人类死后的材料通常质量不够好。然而，新的重编程技术可以在体外生成人类神经元，从而提供了容易满足这些要求的样本。本研究的目的是确定具有最佳超微结构保存的培养技术，结合最佳的包埋和对比技术，以可视化神经元元素。从健康对照受试者中分离得到的两种诱导神经干细胞系在玻璃载玻片上贴壁培养、嵌入高度浓缩的基质胶液滴中或作为紧密的神经球进行分化。然后，使用戊二醛（GA）和多聚甲醛（PFA）的组合固定，随后使用三种方法（标准染色、钌红染色、高对比度整体染色），在超薄片和 TEM 成像之前使用不同的膜增强和对比步骤组合。紧密的自由浮动神经球显示出最佳的超微结构保存。高对比度整体染色特别能清晰地染色膜结构，并能高质量地可视化神经元结构。总之，在自由浮动条件下生长的紧密神经球与高对比度整体染色方案相结合，提供了最佳的保存和对比度，特别注重观察膜结构，以分析突触结构。
