Zhou Feng, Tang Qiaowei, Yan Xin, Ma Chao, Zhang Yu, Zhang Jichao, Li Qian, Wang Lihua, Hu Jun, Cai Xiaoqing, Li Jiang, Zhu Ying, Fan Chunhai
CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, 201800, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Adv Sci (Weinh). 2025 Aug;12(30):e04468. doi: 10.1002/advs.202504468. Epub 2025 May 28.
Achieving detailed neuronal structural information in large-volume brain tissue has been a longstanding challenge in human brain imaging. A key obstacle arises from the trade-off between staining efficiency and tissue autolysis. Traditional Golgi staining, typically conducted at room temperature or 37 °C to optimize staining efficiency, leads to rapid autolysis of brain tissue, resulting in the loss of fine structural details. Here, a near-freezing temperature (NFT) staining strategy in post-mortem frozen (PMF) human brain samples are presented, using a mercury chloride-based method under ice-water bath conditions. In contrast to the 37 °C Golgi staining, this NFT-based method significantly reduces tissue autolysis, preserving fine neuronal structures. Notably, neuronal counts in the same field of view increased by 5.5-fold, and dendritic spine density increases by 22-fold. Using this approach, uniform staining of millimeter-thick is achieved, centimeter-scale human brain slices and integrated it with synchrotron-based X-ray microscopy to perform micrometer resolution 3D reconstructions of the cerebellum and frontal lobe. This novel technique offers a powerful tool for the fine-structural imaging of large-volume brain tissue, providing new insights into the intricate organization of neural networks.
在大体积脑组织中获取详细的神经元结构信息一直是人类脑成像领域长期面临的挑战。一个关键障碍源于染色效率与组织自溶之间的权衡。传统的高尔基染色通常在室温或37°C下进行以优化染色效率，但会导致脑组织快速自溶，从而导致精细结构细节的丢失。在此，本文介绍了一种在死后冷冻（PMF）的人脑样本中采用基于氯化汞的方法在冰水浴条件下进行的近冰点温度（NFT）染色策略。与37°C的高尔基染色相比，这种基于NFT的方法显著减少了组织自溶，保留了精细的神经元结构。值得注意的是，同一视野中的神经元计数增加了5.5倍，树突棘密度增加了22倍。使用这种方法，可以实现毫米厚、厘米级人脑切片的均匀染色，并将其与基于同步加速器的X射线显微镜相结合，对小脑和额叶进行微米分辨率的三维重建。这项新技术为大体积脑组织的精细结构成像提供了一个强大的工具，为神经网络的复杂组织提供了新的见解。
