A pollination biologist from Stellenbosch University is breaking new ground in his field by using quantum dots to track and label individual pollen grains. His novel, low-cost method could enable biologists to track the whole pollination process, from the first visit by a pollinator to the endpoint — either the pollen grain’s successful transfer to another flower’s stigma or its loss along the way. Corneile Minnaar, a pollination biologist in the Department of Botany and Zoology at Stellenbosch University, developed a technique that uses quantum dots to track individual pollen grains. This field of research that has been hampered by lack of a universal method to track pollen. Courtesy of Ingrid Minnaar. Despite over 200 years of detailed research on pollination, scientist Corneile Minnaar says, researchers do not know for sure where most of the microscopically tiny pollen grains actually land once they leave flowers. Minnaar came upon the idea for his pollen-tracking method after reading an article on the use of quantum dots to track cancer cells in rats (https://doi.org/10.1038/nbt994). When exposed to UV light, quantum dots emit extremely bright light in both visible and IR wavelengths. Minnaar surmised that quantum dots with “fat-loving” (lipophilic) ligands would theoretically stick to the fatty outer layer of pollen grains, called pollenkitt. The glowing colors of the quantum dots could then be used to uniquely “label” pollen grains. Minnaar and his team tested the suitability of nontoxic CuInSexS2−x/ZnS (core/shell) quantum dots with oleic-acid ligands as pollen-grain labels. Using a micropipette, they dispensed quantum dots dissolved in hexane in minute volumes (0.15 to 0.5 μl) directly onto dehisced anthers of four different plant species from four different families. After application, the hexane solvent evaporated immediately, leaving behind quantum dots that remained attached to pollen grains of the four plant species even after agitation in a polar solvent, suggesting a lipophilic interaction between oleic-acid ligands on quantum dots and pollenkitt surrounding pollen grains. This bee was caught after it visited a flower where the pollen grains were labeled with quantum dots. Under the microscope, one can see where the pollen was placed and actually determine which insects carry the most pollen from which flower. Courtesy of Corneile Minnaar. The team also showed that most pollen grains within anthers of the same four plant species were labeled with quantum dots after applying a volume of quantum-dot solution sufficient to cover an individual anther. To test whether quantum-dot pollen labels influenced pollen transport, they conducted pollen transfer trials using captively reared honeybees to ensure the bees were free of external pollen prior to the experiments. They found no difference in pollen transport to recipients from donor flowers with labeled versus unlabeled pollen grains. The researchers’ next step was to find a cost-effective way to view the fluorescing pollen grains under a field dissection microscope. “I decided to design a fluorescence box that can fit under a dissection microscope,” Minnaar said. “And, because I wanted people to use this method, I designed a box that can easily be 3D-printed at a cost of about R5,000 (about $355 US), including the required electronic components.” Files for 3D-printing the box can be found online at https://doi.org/10.1111/2041-210X.13155. So far, the use of quantum dots with the excitation box has proven to be an easy and relatively inexpensive method to track individual pollen grains. “I’ve done studies where I caught the insects after they have visited the plant with quantum-dot labeled anthers, and you can see where the pollen is placed, and which insects actually carry more or less pollen,” Minnaar said. But the post-labeling part of the work still requires hours and hours of counting and checking. “I think I’ve probably counted more than a 100,000 pollen grains these last three years,” Minnaar said. Quantum dot nanotechnology could allow direct assessments of pollen movement in most angiosperms, the researchers believe, and could help to quantify, among other things, the magnitude and frequency of pollen loss during various stages of the pollen export process. “Most plant species on Earth are reliant on insects for pollination, including more than 30 percent of the food crops we eat,” Minnaar said. “With insects facing rapid global decline, it is crucial that we understand which insects are important pollinators of different plants — this starts with tracking pollen.” The research was published in Methods in Ecology and Evolution (https://doi.org/10.1111/2041-210X.13155). Corneile Minnaar, a pollination biologist from Stellenbosch University in South Africa, is using quantum dots to track the fate of individual pollen grains, and then viewing them with a custom-made excitation box that can fit under a field microscope. This is breaking new ground in a field of research that has been hampered by the lack of a universal method to track pollen for over a century. Courtesy of Stefan Els, Corneile Minnaar, and Ingrid Minnaar.