Real-time, depth-resolved, in vivo multiphoton fluorescence lifetime imaging microscopy of agricultural herbicide treatments in plants.

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, multiphoton fluorescence lifetime imaging microscopy (MPM-FLIM) allows for 3D sub-cellular quantitative metabolic imaging but is often limited to low frame rates. To resolve relatively fast effects (e.g., photosynthesis inhibiting treatments), high-frame-rate MPM-FLIM is needed. In this paper, we demonstrate and evaluate a high-speed MPM-FLIM system, "Instant FLIM", 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 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 the MPM-FLIM system to measure biological changes over a short time with enhanced imaging speed. Results indicate that high-frame-rate 3D MPM-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.