Potential of CLSM in studying some modern and fossil palynological objects

Abstract

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.

Improving the taxonomy of fossil pollen using convolutional neural networks and superresolution microscopy

“…S2), allowing us to separate morphotypes that appear indistinguishable under the lower resolution of traditional brightfield microscopy (9). The morphological detail visible in Airyscan has been shown to be comparable to EM (8,9); and as a nondestructive optical technique, Airyscan opens up the possibility of imaging and analyzing far greater numbers of fossil specimens than would be possible with EM (8,11,12). We were able to capture the morphological diversity in a low-abundance morphospecies like Striatopollis because Airyscan works with specimens directly on a microscope slide.…”

“…Electron microscopy (EM) methods like scanning electron microscopy (SEM, <5-nm resolution) and transmitted electron microscopy (TEM, 0.2-nm resolution) have traditionally been used to increase visual resolution and morphological detail (2,10). However, EM requires destructive preparation methods that are often too time intensive to scale (9,11,12). An alternative high-resolution optical imaging technique-Airyscan confocal superresolution microscopyproduces images comparable to EM, but is nondestructive and uses material mounted on slides (9).…”

Maiaspora: a new miospore genus with enigmatic sculpture from the late Visean of European Russia

“…Confocal laser scanning microscopy was carried out with a Zeiss LSM 780 (Carl Zeiss AG) CLSM at the Core Centrum ‘Cell and Molecular Technologies in Plant Science’ in Komarov Botanical Institute RAS (BIN) following the techniques of Gavrilova (2014) and Gavrilova et al . (2018). Unstained specimens were mounted on slides in liquid glycerin.…”

“…‘Transparent’, ‘mixed’ and ‘surface’ 3D visualization techniques were applied (Gavrilova et al . 2018). Optical sections and virtual, arbitrarily directed sections through the reconstructions were applied in order to study and describe morphological features of spore walls.…”

Pollen morphological study in subfamily Papilionoideae using Confocal Laser Scanning Microscopy

“…The pollen morphological characters of these are not very distinguishable under Light Microscope and are in general grouped either under subfamily or generic level which fails to provide any clue related to the climate-culture relationship. The CLSM study in permanent palynological slides, on the other hand, makes it easier to see high-resolution images of species that are different within the same genus (Gavrilova et al, 2018). We, therefore, attempted to describe the pollen morphology and morphometry of the 15 species under 14 genera (Table 1) of subfamily Papilionoideae using LM and CLSM contributing significant pollen characters of taxonomic importance.…”