基于高压冷冻固定的脑组织体电镜样品制备方法

基于高压冷冻固定的脑组织体电镜样品制备方法

王方方¹，梁雅坤¹，朱  宏²，华云峰^1*，常海双^1*

（1. 上海交通大学医学院，附属第九人民医院，上海精准医学研究院，上海200125；2. 临港实验室，上海 201602）

摘　要　　高压冷冻（high pressure freezing, HPF）是生物样品固定的前沿方法，制备的样品主要用于电子显微镜观察。相比于传统化学固定，高压冷冻固定的样品被认为更接近其生理状态，这对于细胞超微结构的研究至关重要。本研究尝试对快速分离的小鼠大脑皮层组织进行高压冷冻固定，并进行冷冻替代、重金属染色和树脂包埋。通过连续超薄切片以及电子断层扫描成像，首次获得了接近在体状态的完整神经突触重构。实验结果表明，该样品制备方案获取脑组织大尺度体电镜数据具有可行性，为后续的神经连接组学以及突触形态组学研究提供了坚实、可靠的技术支撑。

关键词　　高压冷冻固定；脑组织样品制备；连续超薄切片；电子断层扫描；神经突触

中图分类号：Q24; Q-336; Q952　　文献标识码：B Doi:10.3969/j.issn.1000-6281.2025.05.010

Preparation of high-pressure Cryo-fixed cortical tissue for volume electron microscopy

WANG Fangfang¹, LIANG Yakun¹, ZHU Hong², HUA Yunfeng^1*, CHANG Haishuang^1*

(1. Shanghai Institute of Precision Medicine, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125; 2. Lingang Laboratory, Shanghai 201306, China)

Abstract　　High-pressure freezing (HPF) is a cutting-edge method for biological sample fixation that preserves ultrastructures for electron microscopy. Compared to conventional chemical fixation, HPF maintains the physiological state of samples, which is essential for cellular ultrastructure analysis. In this study, we applied HPF to rapidly dissected mouse cerebral cortical tissue, followed by freezing substitution, heavy metal staining, and resin embedding. Using serial ultrathin sectioning and electron tomography imaging, we successfully reconstructed intact synapse in a near-native state for the first time. Experimental results demonstrate the feasibility of this sample preparation pipeline for acquiring large-scale volume electron microscopy data of brain tissues, providing a reliable technical foundation for future research in connectomics and synaptic morphomics.

Keywords　　High-pressure freezing fixation; Preparation of brain tissue; Serial sectioning; Electron tomography; Synapse

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