Tracking Galvoxite on Leaf Surface with Fluorescence Lifetime Imaging: Film Forming and Delivery Capabilities Towards Citrus Crop Protection

Huanglongbing (HLB, citrus greening disease) continues to severely impact global citrus production, motivating the development of foliar treatments capable of protecting leaf surfaces and enabling targeted agrochemical delivery. Using optical and electron spectroscopy and imaging, a novel crop protection formulation, Galvoxite, was investigated. We tracked the location of Galvoxite on the leaf surface with advanced imaging and spectroscopic methods, including time-correlated single photon counting (TCSPC), fluorescence lifetime imaging (FLIM), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS). A fluorescently labeled Galvoxite derivative containing tris(2,2′-bipyridyl)ruthenium(II) chloride (RuGalvoxite) was developed for this purpose. Steady-state fluorescence measurements revealed substantial spectral overlap between citrus leaf autofluorescence dominated by chlorophyll and the Ru-based fluorescent probe over the 550–750 nm spectral region, as is typical for organic dyes. However, the Ru-based dye also allows for temporal separation from a fluorescent background due to its long excited state lifetime. TCSPC data show that the fluorescence lifetime contrast between RuGalvoxite and the leaf fluorescence background provides excellent selectivity. Untreated citrus leaves displayed short excited-state lifetimes near 2 ns, whereas RuGalvoxite exhibited long-lived emission with average lifetimes ranging from several hundred nanoseconds up to localized microsecond-scale behavior in FLIM measurements. The RuGalvoxite decay behavior was characterized by predominantly monoexponential dynamics, attributed to dilution-induced reduction of Ru-based dye aggregation and suppression of non-radiative decay pathways. FLIM images reveal spatially localized long-lifetime domains on treated leaves that are clearly distinguishable from the short-lived autofluorescence background of untreated tissue. The bottom leaf surface exhibited more uniform film formation and preferential localization of RuGalvoxite near and within stomatal pores, whereas the waxy top surface promoted accumulation near glandular trichome-like structures. SEM imaging confirmed the formation of deposited film domains on the leaf surface, while EDS mapping identified Zn-rich regions corresponding to Galvoxite. These results demonstrate that fluorescence lifetime spectroscopy and imaging provide sensitive approaches for tracking foliar delivery systems in highly autofluorescent plant environments. The observed localization behavior and film-forming properties support the potential use of Galvoxite as a platform for targeted micronutrient or agrochemical delivery to stomatal regions of citrus leaves.