Abstract
Research on the comparative morphology of pollen grains depends crucially on the application of appropriate microscopy techniques. Information on the performance of microscopy techniques can be used to inform that choice. We compared the ability of several microscopy techniques to provide information on the shape and surface texture of three pollen types with differing morphologies. These techniques are: widefield, apotome, confocal and two-photon microscopy (reflected light techniques), and brightfield and differential interference contrast microscopy (DIC) (transmitted light techniques). We also provide a first view of pollen using super-resolution microscopy. The three pollen types used to contrast the performance of each technique are: Croton hirtus (Euphorbiaceae), Mabea occidentalis (Euphorbiaceae) and Agropyron repens (Poaceae). No single microscopy technique provided an adequate picture of both the shape and surface texture of any of the three pollen types investigated here. The wavelength of incident light, photon-collection ability of the optical technique, signal-to-noise ratio, and the thickness and light absorption characteristics of the exine profoundly affect the recovery of morphological information by a given optical microscopy technique. Reflected light techniques, particularly confocal and two-photon microscopy, best capture pollen shape but provide limited information on very fine surface texture. In contrast, transmitted light techniques, particularly differential interference contrast microscopy, can resolve very fine surface texture but provide limited information on shape. Texture comprising sculptural elements that are spaced near the diffraction limit of light (∼250 nm; NDL) presents an acute challenge to optical microscopy. Super-resolution structured illumination microscopy provides data on the NDL texture of A. repens that is more comparable to textural data from scanning electron microscopy than any other optical microscopy technique investigated here. Maximizing the recovery of morphological information from pollen grains should lead to more robust classifications, and an increase in the taxonomic precision with which ancient vegetation can be reconstructed.
Highlights
Pollen grains are an indispensable record of Earth’s vegetation and have multiple applications
The full width of a PSF measured at half its maximum (FWHM) is a useful measure of the resolution of a microscope system [30]
Concluding Remarks There appears to be no single optical microscopy technique that can satisfactorily capture both pollen shape and surface texture, and a combination of reflected and transmitted light techniques is required to maximize the recovery of morphological information from pollen grains
Summary
Pollen grains are an indispensable record of Earth’s vegetation and have multiple applications. The study of pollen is part of the discipline of palynology (the study of living and fossil spores, pollen and other palynomorphs), and in each of these examples of palynological investigation, assemblages of pollen grains are generally split into groups on the basis of their morphology. These groups often correspond to the taxonomic grouping of the parent plants and may represent species, genera or families. The pollen of angiosperms ranges from ,4.5 mm (the forget-me-not Myosotis sylvatica) to ,200 mm (the pumpkin Cucurbita pepo) [15], and as a result of this small size the acquisition of morphological information from pollen grains is intimately linked to microscopy
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