Imaging technologies have played a pivotal role in advancing biological research by enabling visualization of biological structures and processes. While traditional electron microscopy (EM) produces two-dimensional images, emerging techniques now allow high-resolution three-dimensional (3D) characterization of specimens in situ, meeting growing needs in molecular and cellular biology. Combining transmission electron microscopy (TEM) with serial sectioning inaugurated 3D imaging, attracting biologists seeking to explore cell ultrastructure and driving advancement of 3D EM reconstruction. By comprehensively and precisely rendering internal structure and distribution, 3D TEM reconstruction provides unparalleled ultrastructural insights into cells and molecules, holding tremendous value for elucidating structure–function relationships and broadly propelling structural biology. Here, we first introduce the principle of 3D reconstruction of cells and tissues by classical approaches in TEM and then discuss modern technologies utilizing TEM and on new SEM-based as well as cryo-electron microscope (cryo-EM) techniques. 3D reconstruction techniques from serial sections, electron tomography (ET), and the recent single-particle analysis (SPA) are examined; the focused ion beam scanning electron microscopy (FIB-SEM), the serial block-face scanning electron microscopy (SBF-SEM), and automatic tape-collecting lathe ultramicrotome (ATUM-SEM) for 3D reconstruction of large volumes are discussed. Finally, we review the challenges and development prospects of these technologies in life science. It aims to provide an informative reference for biological researchers.

成像技术通过实现生物结构和过程的可视化，在推进生物研究方面发挥了关键作用。传统电子显微镜 (EM) 产生二维图像，而新兴技术现在可以对样本进行原位高分辨率三维 (3D) 表征，满足分子和细胞生物学日益增长的需求。将透射电子显微镜 (TEM) 与连续切片相结合开创了 3D 成像，吸引了寻求探索细胞超微结构并推动 3D EM 重建进步的生物学家。通过全面、精确地呈现内部结构和分布，3D TEM 重建为细胞和分子提供了无与伦比的超微结构洞察，对于阐明结构-功能关系和广泛推动结构生物学具有巨大价值。在这里，我们首先介绍通过 TEM 中的经典方法进行细胞和组织 3D 重建的原理，然后讨论利用 TEM 和基于新 SEM 的现代技术以及冷冻电子显微镜 (cryo-EM) 技术。检查了连续切片、电​​子断层扫描 (ET) 和最近的单粒子分析 (SPA) 的 3D 重建技术；讨论了用于大体积 3D 重建的聚焦离子束扫描电子显微镜 (FIB-SEM)、串行块面扫描电子显微镜 (SBF-SEM) 和自动带收集车床超薄切片机 (ATUM-SEM)。最后，我们回顾了这些技术在生命科学领域面临的挑战和发展前景。旨在为生物学研究人员提供翔实的参考。