Investigating the Fundamental Limitations of Real-Time, In Vivo Multiphoton Fluorescence Lifetime Imaging Microscopy and Its Application to Agricultural Herbicide Treatments in Plants

Multiphoton microscopy (MPM) and frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) are both powerful tools in biological studies and the combination of these two techniques have enabled three-dimensional, molecular and functional imaging in vivo. The integration of MPM-FD-FLIM combines the benefits of MPM, such as reduced photodamage and increased penetration depth within tissues, and advantages of FLIM, including the ability to differentiate fluorophores with overlapping emission spectra and the sensitivity to the changes in the microenvironment. In this work, both theoretical analysis and experimental applications of a custom-built, high-speed MPM-FD-FLIM system, “Instant FLIM”, are investigated and demonstrated in order to more thoroughly study and explore the capability of the technology. In the first part of the work, theoretical research on the spatial resolution of FD-FLIM is discussed and investigated. The resolution limit of FLIM system and how it can be different with that of the conventional intensity-based microscopy system has always been a gap in the research areas. We aim to fill that gap by presenting an analytical model of adjacent fluorophores to look into the underlying properties of the spatial resolution of FLIM and how it can affect and distort the measurements. We then propose a super-resolution FLIM method that utilizes localization super-resolution techniques to achieve resolution enhancement and the FLIM results are demonstrated both numerically and experimentally. The limitations of the method are then discussed; followed by the proposed important future direction of this work which is Richardson-Lucy deconvolution method as the distortion correction for FLIM. Second part of the work focuses on the experimental applications of the MPM-FD-FLIM system to evaluate its performance and capability. The development of effective and safe agricultural treatments requires sub-cellular insight of the biochemical effects of treatments in living tissue in real-time. Industry-standard mass spectroscopic imaging lacks real-time in vivo capability. As an alternative, MPM-FLIM system allows for 3D sub-cellular quantitative metabolic imaging but is often limited to low frame rates. To resolve relatively fast effects and dynamics (e.g., photosynthesis inhibiting treatments), high-frame-rate MPM-FLIM is necessary. In this work, we experimentally demonstrate and evaluate the MPM-FLIM system as a time-resolved 3D sub-cellular molecular imaging system in highly scattering, living plant tissues. We demonstrate simultaneous imaging of cellular autofluorescence and crystalline agrochemical crystals deposition within plant tissues. We further quantitatively investigate the herbicidal effects of two classes of agricultural herbicide treatments, photosystem II inhibiting herbicide (Basagran) and auxin-based herbicide (Arylex), and successfully demonstrate the capability of our MPM-FD-FLIM system to measure biological changes over a short time with enhanced imaging speed. Results indicate that high-frame-rate 3D MPM-FD-FLIM achieves the required fluorescence lifetime resolution, temporal resolution, and spatial resolution to be a useful tool in basic plant cellular biology research and agricultural treatment development.