Capturing the Surface Texture and Shape of Pollen: A Comparison of Microscopy Techniques

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.

On the Taxonomic Resolution of Pollen and Spore Records of Earth’s Vegetation

“…This comment alludes to the idea that an increase in the resolution of a microscope (not to be confused with magnification) can increase the amount of morphological information that is recovered from individual specimens, which can in turn improve the classification of pollen and spores (Sivaguru et al 2012). This idea is highlighted by the example of grass pollen, which is a classic case of low taxonomic resolution (Mander et al 2013).…”

“…The SEM has been used extensively in palynology because of its ability to image structures that are just a few nanometers in size, and although specimen preparation and image acquisition time have been barriers to the use of the SEM in routine palynological studies (e.g., Faegri et al 1992), recent work has shown that SEM images of surface ornamentation can be used by human analysts to classify species of grass pollen (Mander et al 2013(Mander et al , 2014. Von Post's comment also foreshadows the idea that different microscopy techniques are suited to the analysis of different morphological features of sporomorphs (Sivaguru et al 2012). Continuing the example of grass pollen, phase-contrast microscopy has been employed to acquire information on the organization of the exine (Faegri et al 1992;Beug 2004), and this has been used to generate broad groupings of grass pollen morphotypes (Beug 2004) and to distinguish Tripsacum pilosum, which has clumped tectal columellae, and Zea mays ssp.…”

“…parviglumis (Balsas teosinte), which has uniformly distributed tectal columellae (Holst et al 2007). Such features of the grass pollen exine cannot be viewed with an SEM because this instrument provides information only from the surface of a specimen (Sivaguru et al 2012). TEM also provides data on exine organization, and this technique has been useful in preQuaternary time for clarifying the systematic position of sporomorphs that have not been found in situ in a fossilized reproductive structure (e.g., Doyle et al 1990;Taylor 1995;Mander et al 2012).…”

“…Reflected-light (fluorescence) techniques, such as confocal and apotome microscopy, have been used to generate taxonomically significant data on the three-dimensional shape of entire pollen grains (Ronneberger et al 2002a(Ronneberger et al , 2002b(Ronneberger et al , 2007Punyasena et al 2012; figs. 3B, 4L) and structures within pollen grains (Hochuli and Feist-Burkhardt 2013), which can be difficult to acquire with transmitted-light techniques because of their relatively poor optical-sectioning capabilities (Sivaguru et al 2012).…”

How to reveal subsurface defects in Kevlar® composite materials after an impact loading using infrared vision and optical NDT techniques?

“…The same considerations can be made for other fields (i.e., biological [5], geological [6], and art [7] fields), where the use of a microscope instead of a camera is needed for the experiments. In the latter case [7], the combination of reflectographic and transmittographic images is a well-known method.…”