Advanced applications of Raman spectroscopy and super-resolution imaging of biological and plant materials
Plants are an essential resource for the Earth, yet there is still a significant lack of knowledge about the cells, tissues, and organs of plants compared to the human body. There is a great need for sensitive methods to analyze plants and biological materials to gain an in situ understanding of processes at the molecular and cellular level. Raman spectroscopy has many advantages as an analytical method to help fill this knowledge gap: it can differentiate chemical structures with high sensitivity, it is non-destructive, it requires minimal sample preparation compared to many other techniques, and it can reveal quantitative chemical abundances. For these reasons, Raman spectroscopy can non-destructively identify and semi-quantify valuable and essential plant metabolites. In combination with a microscope and mechanized stage, Raman spectroscopy can also reveal the spatial dependence of plant metabolites. In this dissertation, the following three topics are discussed: a developed multimodal imaging method to study gene function in plants, the potential for Raman spectroscopy to infer relative plant metabolite content for optimal harvest, and the prospects of using inorganic semiconductor quantum dots with saturated excitation microscopy for subdiffraction imaging in biological materials. Also four collaborative research projects are discussed that use fluorescence microscopy and Raman spectroscopy to characterize inorganic materials. The first topic introduces a multimodal imaging method that harnesses Raman spectroscopy and mass spectrometry to reveal the cellular-level biochemical changes from virus-induced gene silencing (VIGS). VIGS is a powerful technique to study gene function, but few tools can study the spatial dependence of its effects at the cellular level. The combined imaging methods uncovered the spatial distribution of the effects from silencing the phytoene desaturase gene within maize via the Foxtail mosaic virus vector. Raman spectroscopy revealed that the localized downstream carotenoid expression was reduced in the silenced locations but not eliminated. The complementary mass spectrometry signal for phytoene showed an abundance within the same locations. This is the first instance in which biochemical changes resulting from VIGS at the cellular level were spatially characterized by traditional analytical instrumentation. The imaging method is advantageous for studying plant tissues, including for the biochemical changes that result from gene silencing at the cellular level of expression in stems, leaves, and roots. The second topic discusses how Raman spectroscopy was developed as a fast and simple alternative method for determining relative plant metabolite abundance in situ. The Raman peak area ratio analysis was performed on KI110 and Native spearmint to infer the optimal harvest time for rosmarinic acid extraction. The rosmarinic acid abundance revealed a cellular structure dependence within epidermal cells and trichomes on the adaxial leaf surface. The relative abundance of rosmarinic acid with reference to leaf age was also investigated and found to be statistically different in some instances. A chemometric model was developed to establish a quantitative relationship between Raman and high-performance liquid chromatography (HPLC) analyses of plant leaves and subsequently extracted rosmarinic acid, respectively. This calibration model was built with partial least-squares regression and can relay a quantitative rosmarinic concentration for new plant leaf samples in milligram per gram values. Raman spectroscopy can be a faster alternative to HPLC and has the potential to be used in the field for quick analysis of the relative abundance of rosmarinic acid to harvest with the highest yield. The third topic introduces the feasibility of using inorganic semiconductor CdSe/ZnS quantum dots (QDs) as a luminescent probe in biological cells with a lab-built saturated excitation (SAX) microscope for subdiffraction imaging. This is the first instance in which these QDs were tested as a luminescent probe for subdiffraction biological imaging with SAX microscopy. The excitation modulation frequency (ω) was demodulated for signal detection at higher harmonics (nω,n = 2, 3, etc.). To test the practicality of the QDs, control experiments were completed to mimic the same conditions that were subsequently used for cellular studies that included dried on a glass substrate and an aqueous environment. The QDs revealed different saturation characteristics based upon the sample environment (dried versus aqueous). The dried environment achieved a high harmonic signal with relatively low saturation intensities, but the cellular preparation in a dried environment was not tested. The aqueous environment required the saturation intensity to be very high, which is unfavorable for cellular experiments. The aqueous cells labeled with the QDs was measured and the image constructed using the fundamental frequency; imaging with higher harmonics was not possible without sample damage. This work has laid the foundation for understanding the photoluminescence saturation of these QDs and their compatibility for use within targeted cellular studies to obtain subdiffraction imaging with SAX microscopy. In summary, this dissertation emphasizes Raman spectroscopy and SAX microscopy for novel application analyses of plants and targeted cellular studies. Two collaborative research works are also reviewed within this dissertation in which fluorescence microscopy and Raman spectroscopy aid in the characterization of inorganic materials and catalysts.