State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, China.
Centre for Cell and Developmental Biology, State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China.
Methods Mol Biol. 2024;2841:179-188. doi: 10.1007/978-1-0716-4059-3_17.
Vacuoles in plant cells are the most prominent organelles that harbor distinctive features, including lytic function, storage of proteins and sugars, balance of cell volume, and defense responses. Despite their dominant size and functional versatility, the nature and biogenesis of vacuoles in plants per se remain elusive and several models have been proposed. Recently, we used the whole-cell 3D electron tomography (ET) technique to study vacuole formation and distribution at nanometer resolution and demonstrated that small vacuoles are derived from multivesicular body maturation and fusion. Good sample preparation is a critical step to get high-quality electron tomography images. In this chapter, we provide detailed sample preparation methods for high-resolution ET in Arabidopsis thaliana root cells, including high-pressure freezing, subsequent freeze-substitution fixation, embedding, and serial sectioning.
植物细胞中的液泡是最显著的细胞器，具有独特的功能，包括溶酶体功能、蛋白质和糖的储存、细胞体积平衡和防御反应。尽管它们的体积较大且功能多样，但植物液泡本身的性质和生物发生仍然难以捉摸，已经提出了几种模型。最近，我们使用全细胞三维电子断层扫描（ET）技术在纳米分辨率水平上研究液泡的形成和分布，并证明小液泡是由多泡体成熟和融合而来的。良好的样品制备是获得高质量电子断层扫描图像的关键步骤。在本章中，我们提供了用于拟南芥根细胞高分辨率 ET 的详细样品制备方法，包括高压冷冻、随后的冷冻置换固定、包埋和连续切片。
