Molecular Machine‐Driven Fluorogenic Probes for Motion–Signal Conversion and Hydrophilicity Retention in Live‐Cell Nanoscopy

ABSTRACT Traditional fluorogenic and cell‐permeable molecular probes for live‐cell nanoscopy mainly rely on structural or hydrophilicity changes to generate intracellular fluorescence signals. Here, we introduce molecular machines into fluorogenic probe design. As a proof of concept, malachite green (MG) was coupled to spirocyclization‐free rhodamine to construct the water‐soluble fluorogenic probe MG‐Rho. We define “motion–signal conversion” as the transduction of intramolecular motion of a molecular rotor into corresponding changes in fluorescence signal output. Mechanistic studies support an electron‐transfer‐mediated quenching model in which photoexcitation of the rhodamine signal module is followed by rapid electron transfer to the MG switch module. Free motion of the MG rotor favors this electron‐transfer‐coupled non‐radiative deactivation and maintains a weakly emissive state, whereas restriction of MG motion suppresses the quenching pathway and restores rhodamine fluorescence. MG‐Rho exhibits excellent water solubility and photostability and enables mitochondrial cristae staining and long‐term imaging of mitochondrial dynamics in living cells. This work establishes a modular strategy that couples molecular motion to electron‐transfer‐regulated fluorescence output while retaining probe hydrophilicity for dynamic imaging in aqueous biological environments.