Johkura Kohei, Usuda Nobuteru, Tanaka Yoshihiro, Fukasawa Motoaki, Murata Kazuyoshi, Noda Toru, Ohno Nobuhiko
Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
Department of Cell Biology and Anatomy, Fujita Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan.
Microscopy (Oxf). 2022 Oct 6;71(5):262-270. doi: 10.1093/jmicro/dfac024.
The Golgi apparatus, which plays a role in various biosynthetic pathways, is usually identified in electron microscopy by the morphological criteria of lamellae. A 3-dimensional analyses with serial block-face scanning electron microscope (SBF-SEM), a volume-SEM proficient in obtaining large volumes of data at the whole-cell level, could be a promising technique for understanding the precise distribution and complex ultrastructure of Golgi apparatus, although optimal methods for such analyses remain unclear since the observation can be hampered with sample charging and low image contrast, and manual segmentation often requires significant manpower. The present study attempted the whole-cell observation and semi-automatic classification and segmentation of the Golgi apparatus in rat hepatocytes for the first time by SBF-SEM via ZIO staining, a classical osmium impregnation. The staining electron-densely visualized individual Golgi lamellae, and their ultrastructure could stably be observed without any noticeable charging. The simple thresholding of the serial images enabled the efficient reconstruction of the labeled Golgi apparatus, which revealed plural Golgi apparatus in one hepatocyte. The combination of the heavy metal-based histochemistry of zinc, iodine and osmium (ZIO) staining and SBF-SEM was useful in the 3-dimensional observation of the Golgi apparatus at the whole-cell level because of two technical advantages: (i) visualization of the Golgi apparatus without any heavy metal staining and efficient acquisition of the block-face images without additional conductive staining or any devices for eliminating charging; (ii) easy identification of the staining and hassle-free, semi-automatic classification and segmentation by simple thresholding of the images. This novel approach could elucidate the topographic characteristics of the Golgi apparatus in hepatocytes.
高尔基体在各种生物合成途径中发挥作用，在电子显微镜下通常根据薄片的形态学标准来识别。连续块面扫描电子显微镜（SBF-SEM）是一种能够在全细胞水平获取大量数据的体视扫描电子显微镜，三维分析有望成为了解高尔基体精确分布和复杂超微结构的技术，尽管由于样品充电和低图像对比度可能会妨碍观察，且手动分割通常需要大量人力，此类分析的最佳方法仍不明确。本研究首次通过SBF-SEM和ZIO染色（一种经典的锇浸渍法）对大鼠肝细胞中的高尔基体进行全细胞观察以及半自动分类和分割。这种染色使高尔基体的各个薄片在电子密度上清晰可见，并且可以稳定地观察到它们的超微结构，而不会出现明显的充电现象。对连续图像进行简单的阈值处理就能高效重建标记的高尔基体，结果显示一个肝细胞中有多个高尔基体。锌、碘和锇（ZIO）的重金属基组织化学染色与SBF-SEM相结合，在全细胞水平对高尔基体进行三维观察时很有用，这得益于两个技术优势：（i）无需任何重金属染色就能使高尔基体可视化，并且无需额外的导电染色或任何消除充电的装置就能高效获取块面图像；（ii）染色易于识别，通过对图像进行简单的阈值处理就能轻松实现半自动分类和分割。这种新方法可以阐明肝细胞中高尔基体的拓扑特征。
