Miranda Kildare, Girard-Dias Wendell, Attias Marcia, de Souza Wanderley, Ramos Isabela
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica, Carlos Chagas Filho and Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens-Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Diretoria de Metrologia Aplicada a Ciências da Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMETRO), Xer, é, m, Rio de Janeiro, Brazil.
Mol Reprod Dev. 2015 Jul-Aug;82(7-8):530-47. doi: 10.1002/mrd.22455. Epub 2015 Feb 4.
Early applications of transmission electron microscopy (TEM) in the life sciences have contributed tremendously to our current understanding at the subcellular level. Initially limited to two-dimensional representations of three-dimensional (3D) objects, this approach has revolutionized the fields of cellular and structural biology-being instrumental for determining the fine morpho-functional characterization of most cellular structures. Electron microscopy has progressively evolved towards the development of tools that allow for the 3D characterization of different structures. This was done with the aid of a wide variety of techniques, which have become increasingly diverse and highly sophisticated. We start this review by examining the principles of 3D reconstruction of cells and tissues using classical approaches in TEM, and follow with a discussion of the modern approaches utilizing TEM as well as on new scanning electron microscopy-based techniques. 3D reconstruction techniques from serial sections and (cryo) electron-tomography are examined, and the recent applications of focused ion beam-scanning microscopes and serial-block-face techniques for the 3D reconstruction of large volumes are discussed. Alternative low-cost techniques and more accessible approaches using basic transmission or field emission scanning electron microscopes are also examined.
透射电子显微镜（TEM）在生命科学中的早期应用极大地促进了我们目前在亚细胞水平上的理解。这种方法最初仅限于三维（3D）物体的二维呈现，却彻底改变了细胞生物学和结构生物学领域——对于确定大多数细胞结构的精细形态功能特征起到了重要作用。电子显微镜已逐渐朝着开发能够对不同结构进行3D表征的工具发展。这借助了各种各样的技术得以实现，这些技术日益多样化且高度复杂。我们在这篇综述中首先探讨使用TEM经典方法对细胞和组织进行3D重建的原理，接着讨论利用TEM的现代方法以及基于扫描电子显微镜的新技术。研究了来自连续切片和（冷冻）电子断层扫描的3D重建技术，并讨论了聚焦离子束扫描显微镜和连续块面技术在大体积3D重建中的最新应用。还研究了使用基本透射或场发射扫描电子显微镜的替代低成本技术和更易获取的方法。
