Russian Academy of Sciences, V.L. Komarov Botanical Institute, St. Petersburg, Russia.
Russian Academy of Sciences, A.A. Borissiak Paleontological Institute, Moscow, Russia.
J Microsc. 2018 Mar;269(3):291-309. doi: 10.1111/jmi.12639. Epub 2017 Sep 21.
We have tested possibilities and limitations of confocal laser scanning microscopy to study the morphology of pollen and spores and inner structure of sporoderms. As test objects, we used pollen grains of the modern angiosperm Ribes niveum (Grossulariaceae) and Datura metel (Solanaceae), fossil angiosperm pollen grains of Pseudointegricorpus clarireticulatum and Wodehouseia spinata dated to the Late Cretaceous, fossil gymnosperm pollen grains of Cycadopites-type dated to the Middle Jurassic, and fossil megaspores Maexisporites rugulaeferus, M. grosstriletus, and Trileites sp. dated to the Early Triassic. For comparative purpose, we studied the same objects with application of conventional light, scanning electron (to entire pollen grains and spores or to semithin sections of their walls), or transmission electron microscopy. The resolution of confocal microscope is much lower than that of electron microscopes, as are its abilities to reconstruct the surface patterns and inner structure. On the other hand, it can provide information that is unreachable by other microscopical methods. Thus, the structure of endoapertures in angiosperm pollen grains can be directly observed. It is also helpful in studies of asymmetrical pollen and pollen grains bearing various appendages and having complicated exine structure, because rotation of 3-D reconstructions allows one to examine all sides and structures of the pollen grain. The exact location of all visible and concealed structures in the sporoderm can be detected; this information helps to describe the morphology and inner structure of pollen grains and to choose necessary directions of further ultrathin sectioning for a transmission electron microscopical study. In studies of fossil pollen grains that are preserved in clumps and stuck to cuticles, confocal microscope is useful in determining the number of apertures in individual pollen grains. This can be done by means of virtual sections through 3-D reconstructions of pollen grains. Fossil megaspores are too large and too thick-walled objects for a confocal study; however, confocal microscope was able to reveal a degree of compression of fossil megaspores, the presence of a cavity between the outer and inner sporoderm layers, and to get some information about sporoderm inner structure.
我们已经测试了共聚焦激光扫描显微镜在研究花粉和孢子的形态以及孢子壁内层结构方面的可能性和局限性。作为测试对象，我们使用了现代被子植物白鲜 Ribes niveum（卫矛科）和曼陀罗 Datura metel（茄科）的花粉粒、晚白垩世的化石被子植物花粉粒 Pseudointegricorpus clarireticulatum 和 Wodehouseia spinata、中侏罗世的化石苏铁类花粉粒 Cycadopites-type 以及早三叠世的化石大孢子 Maexisporites rugulaeferus、M. grosstriletus 和 Trileites sp.。为了进行比较，我们还应用常规光、扫描电子显微镜（用于整个花粉粒和孢子，或其壁的半薄切片）或透射电子显微镜研究了相同的物体。共聚焦显微镜的分辨率远低于电子显微镜，其重建表面图案和内层结构的能力也较低。另一方面，它可以提供其他显微镜方法无法获得的信息。因此，可以直接观察被子植物花粉粒中的内孔结构。它在研究不对称花粉以及具有各种附属物和复杂外壁结构的花粉粒方面也很有帮助，因为 3D 重建的旋转可以使人们检查花粉粒的所有侧面和结构。可以检测到孢子壁中所有可见和隐藏结构的确切位置；这些信息有助于描述花粉粒的形态和内层结构，并选择进行透射电子显微镜研究所需的进一步超薄切片方向。在研究保存在团块中并粘在角质层上的化石花粉粒时，共聚焦显微镜有助于确定单个花粉粒中的孔数。这可以通过穿过花粉粒的 3D 重建进行虚拟切片来完成。化石大孢子是太大且外壁太厚的物体，无法进行共聚焦研究；然而，共聚焦显微镜能够揭示化石大孢子的压缩程度、外孢子壁层和内孢子壁层之间存在腔隙，并获得有关孢子壁内层结构的一些信息。
