Mitochondria and Neuromast Tagging With Fluorescent Gallium‐Triapine Analogues: In Cellulo MP FLIM and Zebrafish Live Imaging

ABSTRACT We report on the synthesis and imaging‐based investigations of fluorescent thiosemicarbazone–BODIPY conjugates and their gallium(III), indium(III), and iron(III) complexes, and their cellular and in vivo distribution using multiphoton fluorescence lifetime imaging microscopy (MP‐FLIM) in living cancer cells and live zebrafish. A panel of novel tridentate thiosemicarbazone ligands and their corresponding lipophilic and monocationic complexes with 1:2 metal‐to‐ligand stoichiometry was synthesized. These compounds are structurally related to clinically relevant thiosemicarbazone‐based systems with imaging and diagnostic applications. Comprehensive analytical characterization confirmed these probes’ identity, while their complex solution behavior was evaluated under biologically relevant conditions. Cellular uptake and subcellular distribution were investigated in HeLa and PC‐3 cells using correlated confocal microscopy and MP‐FLIM, revealing preferential mitochondrial localization for selected gallium complexes. In vivo imaging in zebrafish further unraveled the biodistribution of these fluorescent metal complexes, including selective labelling of neuromast structures. The combined in vitro and in vivo imaging data provide direct insight into the speciation, stability, and spatial distribution of thiosemicarbazone metal complexes in complex biological systems. This work establishes new fluorescent thiosemicarbazones as versatile platforms for studying metal complex behavior and biodistribution by advanced optical imaging techniques, opening new opportunities for advanced development of new theranostic applications.