THE MORPHOLOGY OF POLLEN AND SPORES: WHICH MICROSCOPY TECHNIQUE?

Research on the comparative morphology of pollen and spores depends crucially on the application of appropriate microscopy techniques. However, a bewildering array of techniques is available and selecting the correct technique is not always straightforward. We compared the ability of several microscopy techniques to provide information on the 3-dimensional shape and surface texture of three pollen types with differing morphologies. The microscopy techniques that were compared are: widefield, apotome, confocal and two-photon microscopy (reflected light techniques), and brightfield and differential interference contrast microscopy (transmitted light techniques). The three pollen types that were used to contrast the performance of each technique are: Croton hirtus (Euphorbiaceae), Mabea occidentalis (Euphorbiaceae) and Agropyron repens (Poaceae). We find that no single microscopy technique provides an adequate picture of both the 3-dimensional shape and surface texture of any of the three pollen types investigated in this study. Reflected light techniques, particularly confocal and two-photon microscopy, best capture 3-dimensional shape but provide little 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 3-dimensional shape. The wavelength of incident light has a noticeable effect on the recognition of surface texture; the diffraction-limited texture of Agropyron repens is well resolved using a 561nm laser, but is poorly resolved using a 780nm two-photon laser. This study demonstrates that several microscopy techniques are required to capture the full range of morphological features of a single pollen grain or spore, and that the use of a single technique will result in a substantial loss of morphological information.