透射电子显微镜(TEM)具有优异的纳米尺度显微成像性能, 因此被广泛应用于材料科学、物理学和生物学等学科的超微结构研究领域。文中根据应用TEM技术研究大孢子化石壁超微结构的实践体会, 系统归纳和总结了大孢子化石TEM样品制备的前处理过程, 主要步骤包括: 材料选取、梯度脱水、包埋剂配比、梯度渗透、包埋、聚合、超薄切片和染色。运用上述TEM实验技术, 文中以云南省曲靖市沾益区龙华山剖面中泥盆统吉维特阶上双河组分散拟网龙华山大孢(Longhuashanispora reticuloides Lu and Ouyang, 1978)的壁层超微结构为研究案例, 揭示了这类孢子壁主要由三层薄壁组成, 包括内部基底层、中部疏松层和外部致密层, 其射线唇基底层下部具有近平行排列的多细纹带结构。此类大孢子的多细纹带结构与从加拿大新不伦瑞克省下泥盆统埃姆斯阶Campbellton组同孢植物化石Leclercqia complexa Banks et al., 1972中发现的原位小孢子射线唇基底层下部的多细纹带结构特征非常相似。此外, 两者都具有相似的完全弓形脊和远极表面刺瘤状二型纹饰。因此综合外壁超微结构和纹饰形态特征, 文中认为Longhuashanispora reticuloides的母体植物可能属于同孢植物向异孢植物演化过程中的过渡类型植物, 并与Leclercqia complexa具有较近的亲缘关系。当前研究表明TEM技术在孢粉化石壁超微结构研究领域中具有独特优势, 可为深入研究大孢子形态分类和揭示其与母体植物的亲缘关系提供新的线索, 也可以被推广和应用到其它微体有机壁类化石的超微结构研究。

The transmission electron microscope (TEM) has an excellent performance on revealing the nanoscale structure of the most solid body. It thus has been widely used in various studies of Materials Science, Physics, Biology as well as some associated areas. Here we summarize the detailed laboratory procedures of the sample preparation for TEM, including the pretreatment process, material selection, gradient dehydration, resin preparation, embedding, polymerization, ultrathin section preparation, and staining based on our recent study of the fossil megaspore ultrastructure. By using the TEM techniques described here, the exine ultra-structure of Longhuashanispora reticuloides Lu and Ouyang, 1978, which was recovered from the Middle Devonian Givetian Shangshuanghe Formation of the Longhuashan section in Zhanyi County, Yunnan Province was investigated in detail. Its exine is composed outwardly by the innermost multilamellate zones, the basal lamina, the spongy region, and the solid region. Long-huashanispora reticuloides from the Longhuashan section shows the closest affinity with the fossil plant Leclercqia complexa by the similar spinous-verrucate biform processes and the multilamellate zones at the base of the labrum of in situ microspores of L. complexa. Longhuashanispora reticuloides shares ultrastructural and morphological characteristics of in situ spores yielded by both the heterosporous and homosporous ligulate lycopsids. Its parent plant probably represents a transitional form from the ho-mosporous ligulate lycopsids to the heterosporous ligulate lycopsids. The present study demonstrates that scientists can obtain a clear and intact TEM image of fossil megaspore through experimental procedures described here. Thus, TEM deserves to be ap-plied in the studies of other fossils with an organic wall.