Abstract：
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.
摘要：
植物细胞中的液泡是具有独特特征的最突出的细胞器，包括裂解功能，蛋白质和糖的储存，细胞体积平衡，和防御反应。尽管它们的主要尺寸和功能多功能性，植物中液泡的性质和生物发生本身仍然难以捉摸，已经提出了几种模型。最近,我们使用全细胞3D电子断层扫描（ET）技术以纳米分辨率研究了液泡的形成和分布，并证明了小液泡来自多囊体成熟和融合。良好的样品制备是获得高质量电子层析成像图像的关键步骤。在这一章中,我们提供了拟南芥根细胞中高分辨率ET的详细样品制备方法，包括高压冷冻,随后的冷冻替代固定，嵌入,和连续切片。
