A head-to-tail dimeric J-aggregate probe for rapid and ratiometric imaging of mitochondrial 5-HT via dual-anchoring.

The precise role of 5-hydroxytryptamine (5-HT) in depression remains mechanistically unresolved. While synaptic deficits dominate the monoamine hypothesis, emerging evidence points to mitochondrial dysfunction as a key factor. However, progress has been hindered by the lack of tools capable of directly, specifically, and quantitatively tracking real-time 5-HT dynamics within mitochondria. Here, we present FPY, a ratiometric fluorescent probe featuring unique head-to-tail dimeric J-aggregation via F-π interactions. FPY operates via a synergistic covalent-noncovalent dual-mode-anchoring mechanism, producing a near-infrared-to-visible ratiometric fluorescence response toward 5-HT with superior selectivity, a low detection limit (7.68 nM), and rapid kinetics (1.54 s). Leveraging its intrinsic mitochondrial localization, we achieve the first real-time visualization and ratiometric quantification of mitochondrial 5-HT dynamics in neurons. Critically, we reveal significant 5-HT depletion across key brain regions in a murine depression model and provide direct molecular evidence that the therapeutic action of monoamine oxidase inhibitors involves restoration of mitochondrial 5-HT pools, revealing a strong correlation between mitochondrial 5-HT dysregulation and depressive pathophysiology. This study provides fundamental insights into depression and introduces a novel covalent-noncovalent dual-anchoring strategy for advanced molecular probe design.