Feeding a population of 9 billion in 2050 coupled with the stressors of a changing climate has become a key driver in the progress of plant science and technology. A method increasingly used to address such effects is the technique of Raman microscopy, which provides a real-time, non-invasive, technique to exploring cellular changes in biological samples.
Plant. Image Credit: Romolo Tavani/Shutterstock.com
The development and recent advances of Raman microscopy
Microscopic methods have been increasingly applied to improve our understanding of the structure, chemical composition, and properties of biological cells, tissues, and organs. Named after the Indian physicist, Sir Chandrasekhara Venkata Raman, whose pioneering work on the light-scattering properties in liquids earned him a Nobel Prize in 1930, Raman microscopy was first developed in the 1980s and now provides a wide range of biological information.
Raman microscopy is used to measure the Raman spectrum (i.e., vibrational modes of target molecules) of a point on a sample and is typically composed of a standard optical microscope, an excitation laser, laser rejection filters, a spectrometer or monochromator, and an optical detector.
This method of direct imaging examines the field of view to determine the Raman shift (wavenumber derived from light scattering) of molecules within a cell culture, capable of narrowing to fields less than 1 μm in range. More recently, the technique has been extended to measure direct chemical imaging over the whole field of view of a 3-dimensional sample, improving the technique from a 2D sample.
Furthermore, progressing from a 3D view, the development of the global Raman imaging has now been popularized. Contrasting to the direct imaging method, this technique is used to characterize large-scale devices and can map different compounds and dynamics together within samples. Consequently, Raman microscopy has become a commonly used and reliable method conferring the ability to track key changes in biological samples, from protein and cellular dynamics to organ functioning, using a non-destructive method.
Raman microscopy in biological research
In research, Raman microscopy provides an accurate and reliable tool to examine the chemical and structural information of samples. The main advantage this method confers is that it does not rely on staining or any intricate sample preparation that may be invasive to the sample.
These benefits were further discussed in a 2007 review from scientists at the Max Planck Institute highlighting the different Raman techniques in relation to plant science. The review focused on how this method is central for the investigation of structural cell wall components, valuable plant substances, metabolites, and inorganic substances.
By considering the molecular vibration, rotation, and other low‐frequency modes of key molecules, the Raman scattering effect provides a considerable range of applicable information in plant science. Such characteristics can provide extensive information in plant science, particularly when addressing emerging issues of environmental change and the growing need for food research.
Applications of Raman microscopy in plant science
Raman microscopy can be applied to understand a range of impacts in plant research. In recent studies, this method has been used to improve the detection and movement of pesticides, the mapping of functional components, and the structural elements of cell walls.
First, the Raman microscopy method allows real-time monitoring of pesticide translocation. The study published by American food scientists in 2019 used Raman microscopy to track pesticide concentrations through tomato plants over time. The researchers found that pesticide signals first appeared along the midrib in the lowest leaves and moved distally to the edge of the leaves in tomato plants. The scientists also combined with imaging method with the surface-enhanced Raman spectroscopy technique, which was able to detect thiabendazole in the trichomes of the leaves.
Such findings are strategic when considering the mechanisms of pesticide resilience in pest species but can also prove of significant value when designing more effective pesticides in the future. This is of particular importance to stabilize elements of food security, which often relies on the use of effective pesticides.
Second, Raman microscopy was used to explore the in vivo diagnostics of early abiotic plant stress response. In this 2017 study, a team of American researchers was able to detect various stress levels in Coleus (Plectranthus scutellarioides) plants in response to high soil salinity, drought, chilling exposure, and light saturation. Raman microscopy was used to determine subtle changes in reactive oxygen-scavenging pigments, finding a unique negative correlation in concentration levels of anthocyanins and carotenoids, indicating that plants have developed key resistances to a range of abiotic stressors.
As such, Raman microscopy can also be used to explore the effects of changing environmental conditions, a key component when considering global climate change and its implications on future food security.
The final example of employing Raman microscopy is its use in the development of mapping systems. This was particularly well demonstrated by German scientists in 2018, who assembled 55 maps of root, stem, and leaf tissues in the Cucumber, Cucumis sativus, to design comprehensive imaging of plant cell walls. By combining such many maps across tissues, the team was able to improve the understanding of cellular dynamics, providing additional comparisons on the effectiveness of various methods as well.
Altogether, Raman microscopy has gained in popularity thanks to its widespread potential of applications. This technique is also improving rapidly, with recent studies coupling Raman microscopy with confocal and other types of microscopy and mapping. This has extended the method to collect semi-automatic data, integrating it into color images based on the received data.
Ultimately, the applications and refinement of Raman microscopy will contribute towards scientific progress as well as the knowledge of food-related research and improvements in terms of food security, establishing it as a promising technique for future advances.
- Altangerel, N., Ariunbold, G. O., Gorman, C., Alkahtani, M. H., Borrego, E. J., Bohlmeyer, D., Hemmer, P., Kolomiets, M. V., Yuan, J. S., & Scully, M. O. (2017). In vivo diagnostics of early abiotic plant stress response via Raman spectroscopy. Proceedings of the National Academy of Sciences, 114(13), 3393–3396. doi:10.1073/pnas.1701328114
- Gierlinger, N., & Schwanninger, M. (2007). The potential of Raman microscopy and Raman imaging in plant research. Spectroscopy, 21(2), 69–89. doi:10.1155/2007/498206
- Yang, T., Doherty, J., Guo, H., Zhao, B., Clark, J. M., Xing, B., Hou, R., & He, L. (2019). Real-Time Monitoring of Pesticide Translocation in Tomato Plants by Surface-Enhanced Raman Spectroscopy. Analytical Chemistry, 91(3), 2093–2099. doi:10.1021/acs.analchem.8b04522
- Yang, Y., Wang, X., Zhao, C., Tian, G., Zhang, H., Xiao, H., He, L., & Zheng, J. (2017). Chemical Mapping of Essential Oils, Flavonoids and Carotenoids in Citrus Peels by Raman Microscopy. Journal of Food Science, 82(12), 2840–2846. doi:10.1111/1750-3841.13952
- Zeise, I., Heiner, Z., Holz, S., Joester, M., Büttner, C., & Kneipp, J. (2018). Raman Imaging of Plant Cell Walls in Sections of Cucumis sativus. Plants, 7(1), 7. doi:10.3390/plants7010007