Investigating the Fundamental Limitations of Real-Time, <em>In Vivo</em> 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.多光子显微镜（Multiphoton Microscopy, MPM）与频域荧光寿命成像显微镜（Frequency-Domain Fluorescence Lifetime Imaging Microscopy, FD-FLIM）均为生物学研究中的强力工具，二者结合可实现活体三维、分子与功能成像。MPM-FD-FLIM融合技术整合了MPM的优势：如降低光损伤、提升组织内穿透深度，同时兼具荧光寿命成像显微镜（Fluorescence Lifetime Imaging Microscopy, FLIM）的长处——可区分发射光谱重叠的荧光团，且对微环境变化具有敏感性。本研究针对一套定制化高速MPM-FD-FLIM系统“Instant FLIM”开展理论分析与实验应用研究，以全面探究该技术的性能潜力。 研究的第一部分围绕FD-FLIM的空间分辨率展开理论研究。FLIM系统的分辨率极限，以及其与传统强度型显微镜系统的分辨率差异，一直是该研究领域的空白。我们通过构建相邻荧光团的分析模型，深入解析FLIM空间分辨率的内在特性，以及其对测量结果的影响与畸变，以期填补这一研究空白。随后，我们提出一种利用定位超分辨率技术实现分辨率提升的超分辨FLIM方法，并通过数值模拟与实验验证了该FLIM成像效果。最后讨论了该方法的局限性，并提出本研究未来的重要研究方向：采用Richardson-Lucy去卷积方法作为FLIM的畸变校正手段。 研究的第二部分聚焦于MPM-FD-FLIM系统的实验应用，以评估其性能与能力。开发高效安全的农业处理技术，需要实时获取活体组织中处理剂生化效应的亚细胞层面信息。行业标准的质谱成像技术无法实现实时活体成像。作为替代方案，MPM-FLIM系统可实现三维亚细胞定量代谢成像，但通常受限于较低的帧率。为捕捉相对快速的生理过程与动态变化（如光合作用抑制处理），亟需高帧率MPM-FLIM技术。本研究通过实验验证并评估了该MPM-FLIM系统作为时间分辨三维亚细胞分子成像系统，在高散射性活体植物组织中的应用效果。我们实现了植物组织内细胞自发荧光与结晶性农用化学品沉积的同步成像。进一步定量研究了两类农用除草剂处理的除草效应：光系统II抑制剂类除草剂（Basagran）与生长素类除草剂（Arylex），并成功证明该MPM-FD-FLIM系统可在提升成像帧率的前提下，短时间内检测生物变化。实验结果表明，高帧率三维MPM-FD-FLIM系统可实现所需的荧光寿命分辨率、时间分辨率与空间分辨率，有望成为植物细胞基础生物学研究与农业处理技术开发的实用工具。