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Molecular imaging tools that can image plant metabolism and effects of external agricultural treatments in the micro-environment of plant tissues are significant for further understanding plant biology and optimizing the formulation of new agricultural products. Mass spectrometry, a common tool used by plant biologists, is unable to resolve nano-crystalline active ingredients (AIs) on the leaf surface nor achieve 3D molecular imaging of living plants. To address that, multiphoton microscopy (MPM) and fluorescence lifetime imaging microscopy (FLIM) are combined to achieve sub-cellular, depth-resolved fluorescence lifetime of both AIs and intrinsic proteins/pigments (e.g., chlorophyll and/or cytosolic NADH) after the herbicide treatment application. Here we present a method using a custom-designed, high-speed MPM-FLIM system, “Instant FLIM”, to achieve real-time, unlabeled 3D functional molecular imaging of intrinsic proteins and pigments in optically thick and highly scattering plant samples with the application of external treatments. To validate the capability of MPM-FLIM to measure intrinsic proteins and pigments within plant tissues, we present the results of unlabeled bluegrass blades samples. To demonstrate simultaneous imaging of 3D molecular plant tissue and the agricultural AI nano-crystals deposition and formation, we evaluate the performance of the MPM-FLIM by applying commercial herbicide product to gamagrass blade sample. Additionally, to measure the herbicide-induced cellular-level functional responses within living plant tissues, 3D time-resolved molecular MPM-FLIM imaging of hemp dogbane leaf with herbicide is performed. Results demonstrate MPM-FLIM is capable of 3D simultaneous functional imaging of label-free living plant tissues and the quantitative measurements of the location and formation of AI nanocrystals within the plant tissues.
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