体积电子显微镜技术在植物研究中发挥着重要作用,从了解细胞器和单细胞形式到发育研究,环境效应和微生物与大型植物结构的相互作用,仅举几例。由于中央空泡的空隙很大,细胞壁和蜡质角质层,许多植物组织在试图获得高质量的形态时面临挑战,用于高分辨率体积EM研究的金属染色和足够的电导率。这里,我们应用了强大的常规化学固定策略来解决植物样品的特殊挑战,但不限于,串行块面和聚焦离子束扫描电子显微镜。该方案的化学是从为改善和均匀染色大脑体积而开发的方法进行修改的。简而言之,主要固定是在多聚甲醛和戊二醛中用孔雀石绿,然后用四氧化锇进行次要固定,亚铁氰化钾,硫代碳酰肼,四氧化锇,最后是乙酸铀酰和天冬氨酸铅染色。然后将样品在具有环氧丙烷转变的丙酮中脱水并包埋在硬配方Quetol651树脂中。将样品修剪并用银环氧树脂安装,金属涂层和成像通过连续块面扫描电子显微镜和聚焦电荷补偿电荷抑制。高对比度的植物烟草和浮萍叶细胞结构很容易看到,包括线粒体,高尔基,内质网和核包膜,以及突出的叶绿体类囊体膜和花岗岩堆中的单个薄片。该样品制备方案作为常规植物体积电子显微镜的可靠起点。
Volume electron microscopy techniques play an important role in plant research from understanding organelles and unicellular forms to developmental studies, environmental effects and microbial interactions with large plant structures, to name a few. Due to large air voids central vacuole, cell wall and waxy cuticle, many plant tissues pose challenges when trying to achieve high quality morphology, metal staining and adequate conductivity for high-resolution volume EM studies. Here, we applied a robust conventional chemical fixation strategy to address the special challenges of plant samples and suitable for, but not limited to, serial block-face and focused ion beam scanning electron microscopy. The chemistry of this protocol was modified from an approach developed for improved and uniform staining of large brain volumes. Briefly, primary fixation was in paraformaldehyde and glutaraldehyde with malachite green followed by secondary fixation with osmium tetroxide, potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide and finally uranyl acetate and lead aspartate staining. Samples were then dehydrated in acetone with a propylene oxide transition and embedded in a hard formulation Quetol 651 resin. The samples were trimmed and mounted with silver epoxy, metal coated and imaged via serial block-face scanning electron microscopy and focal charge compensation for charge suppression. High-contrast plant tobacco and duckweed leaf cellular structures were readily visible including mitochondria, Golgi, endoplasmic reticulum and nuclear envelope membranes, as well as prominent chloroplast thylakoid membranes and individual lamella in grana stacks. This sample preparation protocol serves as a reliable starting point for routine plant volume electron microscopy.