TU Dresden, DFG-Center for Regenerative Therapies Dresden, Tatzberg 47/49, 01307 Dresden, Germany.
Protist. 2012 Mar;163(2):296-305. doi: 10.1016/j.protis.2011.06.001. Epub 2011 Jul 20.
Fixation and preparation for electron microscopy of coccidian oocysts is a general problem. Especially in sporulated oocysts, proper fixation and resin infiltration are hindered by the robust oocyst wall. Conventional chemical fixation therefore leads to artefacts that obscure cellular details in the oocysts. In this study, sporulated oocysts of Eimeria nieschulzi were subjected to different fixation and embedding protocols: conventional chemical fixation and embedding in Spurr's resin, microwave-assisted fixation and processing followed by embedding in epon, and high pressure freezing followed by freeze substitution and epon embedding. The samples were finally studied by transmission electron microscopy. Many ultrastructural features of the oocyst wall and the sporozoites were already substantially improved after microwaved-assisted fixation and processing. However, the fine structural preservation still suffered from shrinkage and artificial extraction, which occured during dehydration and infiltration. High pressure freezing (HPF) and freeze substitution (FS) revealed much better preservation. Oocyst walls retained their ovoid shape, and the ultrastructure of sporozoites was well preserved with no signs of shrinkage or extraction. HPF and FS are therefore a suitable method for the ultrastructural analysis of coccidian oocysts.
球虫卵囊的电子显微镜固定和制备是一个普遍存在的问题。特别是在孢子化卵囊，适当的固定和树脂渗透受到坚固的卵囊壁的阻碍。因此，传统的化学固定会导致卵囊内细胞细节模糊的假象。在这项研究中，艾美耳属球虫的孢子化卵囊经历了不同的固定和包埋方案：常规的化学固定和 Spurr 树脂包埋、微波辅助固定和处理后包埋在环氧树脂中，以及高压冷冻后进行冷冻置换和环氧树脂包埋。最后通过透射电子显微镜进行研究。微波辅助固定和处理后，卵囊壁和裂殖子的许多超微结构特征已经得到了很大的改善。然而，在脱水和渗透过程中，收缩和人为提取仍然会对精细结构的保存造成影响。高压冷冻（HPF）和冷冻置换（FS）显示出更好的保存效果。卵囊壁保持椭圆形，裂殖子的超微结构保存良好，没有收缩或提取的迹象。因此，HPF 和 FS 是分析球虫卵囊超微结构的一种合适的方法。
