Two- and three-dimensional electron microscopy techniques: powerful tools for studying the brain under physiological and pathological conditions

We are in the midst of a revolution in the fields of neuroanatomy and electron microscopy. The monumental advancements in the neuroscience field during the last decade have led to unprecedented scientific discoveries about our brain and to the development of new technologies and applications that have significantly contributed to such advances. Conventional applications of transmission electron microscopy have revolutionized neurosciences and are critical for determining the fine morpho-functional characterization of brain cells and their connections. Electron microscopy has progressively evolved toward the development of both more sensitive approaches to unravel the bidimensional subcellular localization of proteins and tools that allow for the three-dimensional characterization of different nerve cells and their connections. The development of new technological advances in two- and three-dimensional electron microscopy to study and map the brain has led to the development of essential tools to decipher the complexity of the brain. For two-dimensional, the sodium dodecyl sulfate-digested freeze-fracture replica labeling technique is a technique with the main goal of chemically identifying the structural components viewed in freeze-fracture replicas and has significant advantages over conventional immunoelectron microscopic techniques for revealing the subcellular organization of proteins along the neuronal surface in the brain. For three-dimensional, volume electron microscopy methods can be applied to structural studies of cell components and organelles, just as conventional transmission electron microscopy has been traditionally applied, but with advantages derived from the possibility of three-dimensional visualization and analysis. The development of volume electron microscopy has greatly facilitated the study of brain structure and connectivity at the synaptic level. Dedicated software tools for the analysis of highly complex connectivity patterns in three dimension are evolving in parallel, allowing the extraction of relevant information from large datasets. Moreover, by applying these new methodologies, the field of pathology is expected to advance, potentially with the identification of the pathogenesis generating these diseases. This review aims to present the possibilities and fundamentals of two- and three-dimensional electron microscopy for high-resolution ultrastructural analyses of neurons and their connections. These technological tools have improved the ability to study the brain, thus providing new insights into brain structure and function.

摘要

我们正处于神经解剖学和电子显微镜领域的一场革命之中。过去十年间，神经科学领域取得了巨大进步，人们对大脑有了前所未有的科学发现，新技术和新应用的开发也为这些进步做出了重要贡献。透射电子显微镜的常规应用彻底改变了神经科学，对于确定脑细胞及其连接的精细形态和功能特征至关重要。电子显微镜逐步发展成为一种更灵敏的方法，既能揭示蛋白质的二维亚细胞定位，又能对不同神经细胞及其连接进行三维表征。二维和三维电子显微镜在研究和绘制大脑地图方面的新技术发展已成为解读大脑复杂性的重要工具。就二维而言，SDS消化冰冻断裂复型标记（SDS-FRL）技术是一种可对冻裂复制品中的结构成分进行化学鉴定的技术，在揭示大脑神经元表面蛋白质亚细胞组织方面具有显著优势。三维方面，体电子显微镜（VEM）方法可用于细胞成分和细胞器的结构研究，就像传统的透射电子显微镜一样，但其优势在于可进行三维可视化和分析。体电子显微镜的发展极大地促进了突触层面的大脑结构和连接研究。用于分析三维高度复杂连接模式的专用软件工具也在同步发展，从而可以从大型数据集中提取相关信息。此外，通过应用这些新技术，病理学领域研究有望取得进展，并有可能确定疾病的发病机制。此综述旨在介绍二维和三维电子显微镜在对神经元及其连接进行高分辨率超微结构分析方面的可能性和基本原理，这些技术工具提高了研究大脑的能力，从而使我们对大脑结构和功能有了新的认识。