An engineered single-wavelength-excitable dual-color fluorescent probe for simultaneous imaging of polarity and...

Understanding complex biological processes like ferroptosis requires simultaneous monitoring of microenvironmental parameters, particularly polarity and viscosity. However, existing fluorescent probes lack multifunctionality for independent, real-time tracking. To address this, we developed CouPy⁺, the first single-molecule probe enabling dual-color imaging of polarity and viscosity dynamics during ferroptosis. Combining a twisted intramolecular charge transfer (TICT) mechanism with molecular rotors, CouPy ⁺ emits distinct green (polarity-sensitive) and deep-red (viscosity-sensitive) signals under a single excitation. This design allows concurrent yet independent parameter visualization in live cells, overcoming limitations of traditional tools. CouPy ⁺ exhibits dual-emission (green for polarity, deep-red for viscosity) under one excitation, enabling simultaneous, independent tracking in live cells. Mitochondria-localized, it discriminated cancer cells from normal cells via emission differences. During ferroptosis, CouPy ⁺ achieved the first real-time, two-color visualization, revealing concurrent nuclear polarity decreases and viscosity increases. In vivo studies confirmed its utility by detecting ferroptosis-associated changes in zebrafish. Control experiments validated the probe's specificity, with no cross-interference between polarity and viscosity responses. In living cells, polarity-dependent green emission decreased to 25 % of baseline levels, while viscosity-dependent red emission increased by 15 % during ferroptosis. Time-lapse imaging captured dynamic parameter shifts, highlighting CouPy⁺'s sensitivity to microenvironmental changes. Additionally, the probe's photostability and low cytotoxicity ensured reliable long-term imaging. Its dual-response capability provides unprecedented insights into the spatiotemporal coupling of polarity and viscosity during ferroptosis, a previously unattainable feat with single-parameter probes. CouPy⁺ is the first single-molecule probe for dual-color imaging of polarity and viscosity during ferroptosis, addressing a critical gap in multifunctional tools. Its TICT-rotor design establishes a blueprint for future multi-parameter probes. CouPy ⁺ provides a powerful tool for simultaneously tracking multiple microenvironmental parameters in live cells and zebrafish, facilitating a deeper mechanistic understanding of ferroptosis and related pathologies.