Scientists have come a long way since Antonie van Leeuwenhoek discovered teeming colonies of previously invisible bacteria and protozoa by looking through her custom-made microscopes. The architecture of cells, organelles, proteins and even molecules has since been illuminated through the tree of life. Yet despite these advances, obstacles remain to comprehensively map the microscopic world. Before they can be observed under a microscope, tissues and cellular components must first be stained with dyes and fixatives and subjected to a lengthy preparation process.
In a new study published in the journal Plant science applications, scientists avoid the need for specimen staining by harnessing the natural autofluorescence of species tissue through the plant’s tree of life.
“Our work provides a generalized and cost-effective protocol for the preparation and visualization of plant samples that is also applicable to large research institutes and small plant science groups,” said Dr Timothy Pegg, Visiting Assistant Professor at Marietta College and senior author of the study.
When certain types of tissue in plants and animals absorb light, the electrons in their atoms receive a discharge of energy that propels them into an excited state. In the leaves of plants, these electrons become so unstable that they break free from their atoms and are used by the plant to fuel photosynthesis. In other tissues, excess energy is re-emitted as low-frequency light bright enough to be detected with specialized microscopes.
Autofluorescence has not always been considered a good thing. In cases where researchers must use dyes to visualize specific structures, the light-emitting properties of neighboring tissues can interfere by decreasing the contrast between different cell types.
But it can also be an indispensable resource for discovery. Autofluorescence has been used to detect early-onset cancers, as well as other diseases and conditions. It has been used to study how insects use their tongues and antennae to taste food, the mechanisms underlying lizard tail regeneration, and to analyze the diversity of microscopic plankton in marine environments.
Autofluorescence is also useful in plants, where it appears in everything from the hard tissues that give woody plants their stability, to the water-absorbing residues covering spores and pollen, to the diverse arsenal of toxic compounds. that plants produce to ward off potentials. predators.
Until now, however, researchers did not have a unique protocol for detecting autofluorescent light in plants. The lack of a unified standard approach is understandable, given that there are nearly half a million living species of terrestrial plants and algae, but Pegg and his colleagues are not discouraged. They selected 12 species from several key plant groups separated by more than 500 million years of evolutionary history, including pines, bryophytes, flowering plants and algae.
With the help of these representatives, they developed a cost-effective method of preserving fabrics without the need for stains or dyes.
While autofluorescence can often be visualized directly with confocal microscopes, it can also be induced or enhanced with various fixatives, including alcohols, ethanol, and compounds called aldehydes. Pegg and his colleagues chose five of the most effective from these for testing their plant specimens. After marinating in a fixative for 24 hours, the plants were rinsed, cut to the width of a human hair, and mounted on a clear slide for viewing.
When the researchers looked under the microscope, the miniature world of plant cells and organelles was brought to light. The rigid lines of the cell walls stood out in bas-relief from the tightly packed chlorophyll within. By focusing on particular wavelengths of light emitted by proteins, they were able to distinguish between the dense features of nuclei and the water and sugar-conducting tissues that meander between cells.
Most fixatives performed well in representative plants, with striking results, but algae turned out to be an exception. Most land plants have thick, reinforced cell walls that help prevent water loss while providing structural support, qualities that algae lack. Due to their more fragile cellular scaffolding, the ethanol and alcohol fixatives quickly penetrated the cell walls of the algae and sole liverwort (a plant closely related to mosses) used in the study, causing wrinkling. and deformation of organelles. For these samples, Pegg recommends sticking with aldehyde fixatives or reducing the time used in the sample preparation steps.
Most research labs also don’t have the high-powered confocal microscopes needed to view cellular structures at fine scales, but instead pay hourly rates to use equipment provided by their institution, a problem that Pegg and his colleagues have noted. hope their protocol can resolve.
“Our simple sample preparation technique can reduce the time researchers have to spend viewing samples on advanced microscopes,” said Dr. Robert Baker, assistant professor of biology at the University of Miami and senior author of the ‘study.
All of the chemicals and reagents used in the study are also inexpensive and readily available, meaning that just about anyone at a research institute can use this protocol to study subcellular interactions in plants.
Reference: “Algae to angiosperms: Autofluorescence for rapid visualization of plant anatomy between diverse taxa” by Timothy J. Pegg, Daniel K. Gladish and Robert L. Baker, July 2, 2021, Plant science applications.
DOI: 10.1002 / aps3.11437