Fossil plants and modern microscopes

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How tomography helps peer into millions of years

The most abundant remains of fossil plants are spores and pollen. But they are very small, hundredths of a millimeter. To study them, you need to use not only a light microscope, but also high-resolution electron microscopes.

By the remains of fossil plants, paleobotanists judge what the plants of past eras were like. But in the fossil record, fossil plants are not preserved entirely, but in the form of separate pieces: leaves, branches, seeds, inflorescences, etc. Many millions of years ago, for example, due to a gust of wind or a suddenly crumbling shore of a reservoir, they broke away from the mother plant , got into the water, in an oxygen-free environment they were quickly covered with fine-grained sediment, which compacted and turned into a stone with scattered plant remains inside, which researchers have to find and study.

Partly we have to judge the whole plant. This part can be microscopic in size, such as pollen grains and spores (in tenths and hundredths of a millimeter). In the modern air, they annoy allergy sufferers, but in the same way they were full of the atmosphere of the distant past. Just as the botanists of modern plants can use pollen from the air to determine whose it is, so by pollen from a geological rock, one can judge which plants of the past produced it. Pollen and spores are the most abundant plant fossils; they are devoted to a special section of science – palynology. It was able to arise thanks to the invention of the light microscope. Here, the progress of palynology follows the advances in the invention of more and more advanced microscopes. The “workhorse” of palynology – the light microscope – allows you to distinguish details in a thousandth of a millimeter. And pollen and spores look incredibly beautiful under a light microscope. The dust particles can be decorated with thorns or mesh, cut with grooves and pores, provided with air sacs or fringes (photo 1). However, the capabilities of light microscopy are limited by the wavelength of light. With the help of visible light, objects can be distinguished, the size of which is not less than half the wavelength of the light wave, which is 0.2 microns (= 2×104 mm). Smaller details cannot be seen with a light beam. You need radiation with a shorter wavelength, and there is. This is a beam of electrons. Here, the wavelength is much shorter and allows you to distinguish objects of several angstroms – 107 mm (= one ten-millionth of a millimeter).

The use of electron microscopes, which began in the second half of the 20th century, increased the ability to distinguish the details of the structure of objects under study by a thousand times. This opened a new era for palynology. The shell of the pollen and spores under an electron microscope was full of very interesting details.

By the ultrafine structure of the shells observed in a transmission electron microscope (TEM), palynologists can judge which plants these pollen or spores belonged to. Although in a light microscope they all looked almost the same.

The design features of electron microscopes, which ensure their high resolution, impose many restrictions on the objects under study. For example, in TEM, only ultra-thin objects (50 nm thick = 5×10-5 mm) can be considered. This is because an electron beam can only pass through a fairly thin structure. This means that large objects (pollen and spores) should be cut into thin layers (photo 2). On ultrathin sections, the structure of the shell is clearly visible, but this is a two-dimensional projection, and looking at it, one can not quite correctly reconstruct the volumetric structures. It would be better to judge the whole by two projections, and there are special procedures for this.

A team of researchers from the Paleontological Institute and Moscow State University is studying two groups of higher plants of the Devonian age (which lived almost 400 million years ago). The descendants of one of them, heterosporous lycopods, have survived to this day. A small herb, Selaginella, can be seen in raised bogs or purchased from a flower shop. Representatives of the second group are Archeopteris, the first woody plants of our planet, unfortunately, completely died out. Although they were very different plants, their spores are remarkably similar to each other and come across in samples together. How can you tell them apart if all that remains of them is controversy?

Researchers believe this is an apparent similarity. It is reinforced by the fact that we are dealing with fossil material: with shells flattened and homogenized due to the millions of years of exposure to paleo temperatures and the pressure of geological rocks. Most likely, the shells of these spores were formed in different ways, from dissimilar structural elements. It is not possible to distinguish them from two-dimensional photographs of sections made using TEM. If it is possible to construct a three-dimensional model from a series of ultrathin sections, then it will probably be possible to find a difference in the structure of their shells, which means that these two groups of plants can be distinguished only by the remnants of their spores.

Traditional TEM can be helped by its new modification – analytical TEM with tomography function (photo 3). In recent years, it has been successfully used in the biology of modern organisms in the study of biological macromolecules and viral particles, and we are trying to adapt this method to fossil pollen grains and spores. For TEM tomography, semi-thin sections are used (for example, 250 nm thick = 2.5×10-4 mm), which are repeatedly photographed with a change in the angle of inclination from –70 to +70 degrees, in increments of one degree. From a series of such images, a summary file is generated that can be viewed as a video. On its basis, a special program builds a three-dimensional model. In the case of successful application of TEM tomography, we will be able to obtain more reliable three-dimensional reconstructions of the spore shells and convincingly distinguish between unrelated groups exhibiting convergent (unrelated) similarities.

Thus, the progress of microscopic technology allows us to look deeper and deeper into the ultrastructure of the pollen and spore envelope and to better understand the life of ancient plant communities.

Natalya Zavyalova (A. A. Borisyak Paleontological Institute, Russian Academy of Sciences), Svetlana Polevova (Faculty of Biology, Lomonosov Moscow State University)

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