Spirocyclization-Switchable Dual-Channel Fluorescent Probe for Synchronous Mitochondria and Lipid Droplet Visualization in Atherosclerotic Lesions

Atherosclerosis (AS) develops from aberrant crosstalk between impaired mitochondria and excessive lipid droplet accumulation within macrophage-originated foam cells, yet conventional fluorescent probes cannot simultaneously track these two organelles or produce distinct dual-channel signals responding to diverse intracellular microenvironmental conditions. In this work, four cyclization-activated D−π−A fluorophores were rationally designed and synthesized, from which the dual-emission probe PODB was screened as the optimal candidate. PODB displays characteristic dual-channel fluorescence shifts when exposed to altered intracellular pH, viscosity, and polarity: it produces green fluorescence localized at mitochondria (Pearson’s r = 0.93) and near-infrared (NIR) emission accumulated in LDs (Pearson’s r = 0.95). Owing to its superior photophysical properties, PODB enables static super-resolution imaging to visualize mitochondrial structural fragmentation and altered intracellular microenvironment after fixed oxidized low-density lipoprotein stimulation during foam cell maturation. In vivo assays using ApoE−/− atherosclerotic mice verify that the probe’s NIR fluorescence intensity correlates with the plaque lipid load. Furthermore, PODB allows qualitative inter group comparison of plaque-associated NIR fluorescence signals among atherosclerotic mice receiving single-dose drug intervention. This dual-channel fluorescent probe provides a practical molecular tool to visualize correlated morphological changes of mitochondria and lipid droplets under pathological microenvironments for AS research.