From Droplet to Destination: Compartment-Resolved Chemical Imaging of Agrochemical Translocation in Wheat Leaves by Broadband Coherent Raman Scattering

Abstract Chemical imaging of trace exogenous molecules within intact biological tissues remains a fundamental analytical challenge, demanding simultaneous molecular specificity, subcellular spatial resolution, and compatibility with complex endogenous backgrounds. In agrochemical research, spatially resolved mapping of active ingredient (AI) uptake and transport within plant tissues is central to understanding pesticide efficacy and environmental fate, yet current methods face fundamental trade-offs between sensitivity, molecular specificity, spatial resolution, and tissue preservation. Here, we introduce a label-free broadband coherent anti-Stokes Raman scattering (BCARS) imaging framework for in planta mapping of AI translocation in wheat (Triticum aestivum) leaves at submicrometer resolution. The approach couples hyperspectral BCARS microscopy with a linear discriminant analysis (LDA)-guided, region-wise spectral unmixing algorithm that operates on paired treated and untreated data sets to isolate AI-specific spectral signatures from the complex endogenous plant background, a regime in which conventional unmixing approaches such as MCR-ALS and direct reference fitting fail. Applied to two structurally and physicochemically diverse AIs, the triazole fungicide mefentrifluconazole (Revysol) and the pyridazine pyrazolecarboxamide insecticide dimpropyridaz (Axalion), the method resolves compartment-specific enrichment, apoplastic and symplastic partitioning, and xylem versus phloem long-distance translocation. Mefentrifluconazole exhibits limited mobility with preferential enrichment at secondary cell wall–symplast interfaces, consistent with its classification as a locally systemic fungicide whose residual efficacy depends on tissue-reservoir accumulation, and with its high hydrophobicity (log Kow = 3.4). Dimpropyridaz displays ambimobility across apoplast and symplast compartments with systemic transport through phloem and xylem, and spatial analysis distinguishes uptake, retention, and systemic transport zones within the leaf. The integrated platform delivers semiquantitative (relative, not absolute) AI abundance and spatial coverage metrics resolved by anatomical compartment, including apoplast, symplast, phloem, and xylem, enabling direct comparison of AI load across tissue compartments within a single experiment, without labeling, derivatization, or matrix deposition.