Near-Freezing-Temperature Golgi Neuronal Staining for X-ray Imaging of Human Brain.

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)
Published Date: May 28, 2025

Abstract

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.

Authors

  • Feng Zhou
    Department of Urology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
  • Qiaowei Tang
    Institute of Materiobiology, College of Sciences, Shanghai University, Shanghai, 200444, China.
  • Xin Yan
    Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of the Ministry of Agriculture, Nanjing Agricultural University, 6 Tongwei Road, Nanjing, Jiangsu 210095, China.
  • Chao Ma
  • Yu Zhang
    College of Marine Electrical Engineering, Dalian Maritime University, Dalian, China.
  • Jichao Zhang
    Shanghai Synchrotron Radiation Facility, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, 201210, China.
  • Qian Li
    Emergency and Critical Care Center, Department of Emergency Medicine, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, China.
  • Lihua Wang
    Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China.
  • Jun Hu
    Jinling Clinical Medical College, Nanjing Medical University，Nanjing，Jiangsu 210002，China.
  • Xiaoqing Cai
    The National Center for Drug Screening and the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
  • Jiang Li
  • Ying Zhu
    China CDC Key Laboratory of Environment and Population Health, National Institute of Environmental Health, Chinese Center for Disease Control and Prevention, Beijing 100021, China.
  • Chunhai Fan
    Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China.

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