Department of Anatomy, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan.
Department of Anatomy, Division of Histology and Cell Biology, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan.
Microscopy (Oxf). 2023 Aug 4;72(4):279-286. doi: 10.1093/jmicro/dfad002.
Recent advances in volume electron microscopy (EM) have been driving our thorough understanding of the brain architecture. Volume EM becomes increasingly powerful when cells and their subcellular structures that are imaged in light microscopy are correlated to those in ultramicrographs obtained with EM. This correlative approach, called correlative light and volume electron microscopy (vCLEM), is used to link three-dimensional ultrastructural information with physiological data such as intracellular Ca2+ dynamics. Genetic tools to express fluorescent proteins and/or an engineered form of a soybean ascorbate peroxidase allow us to perform vCLEM using natural landmarks including blood vessels without immunohistochemical staining. This immunostaining-free vCLEM has been successfully employed in two-photon Ca2+ imaging in vivo as well as in studying complex synaptic connections in thalamic neurons that receive a variety of specialized inputs from the cerebral cortex. In this mini-review, we overview how volume EM and vCLEM have contributed to studying the developmental processes of the brain. We also discuss potential applications of genetic manipulation of target cells using clustered regularly interspaced short palindromic repeats-associated protein 9 and subsequent volume EM to the analysis of protein localization as well as to loss-of-function studies of genes regulating brain development. We give examples for the combinatorial usage of genetic tools with vCLEM that will further enhance our understanding of regulatory mechanisms underlying brain development.
近年来，体式电子显微镜（EM）的发展推动了我们对大脑结构的深入理解。当在光镜下成像的细胞及其亚细胞结构与 EM 获得的超微结构相关联时，体式 EM 变得越来越强大。这种关联方法称为相关光和体式电子显微镜（vCLEM），用于将三维超微结构信息与生理数据（如细胞内 Ca2+动力学）相关联。表达荧光蛋白和/或大豆抗坏血酸过氧化物酶的工程形式的遗传工具允许我们使用包括血管在内的自然标志物进行 vCLEM，而无需免疫组织化学染色。这种无免疫染色的 vCLEM 已成功应用于体内双光子 Ca2+成像以及研究从大脑皮层接收各种特化输入的丘脑神经元的复杂突触连接。在这篇综述中，我们概述了体式 EM 和 vCLEM 如何促进对大脑发育过程的研究。我们还讨论了使用簇状规则间隔短回文重复相关蛋白 9 对靶细胞进行遗传操作，然后进行体式 EM 分析蛋白质定位以及研究调节大脑发育的基因的功能丧失研究的潜在应用。我们给出了遗传工具与 vCLEM 组合使用的示例，这将进一步增强我们对大脑发育背后的调节机制的理解。
