Two-Photon Probe Executes the Restrained-Vibration Mechanism for Pathological Viscosity Detection In Vivo

The fluorescence efficiency is closely linked to the vibration relaxation process. In high-viscosity conditions, the vibration relaxation process is restricted, significantly enhancing fluorescence intensity. Based on this principle, we successfully designed and synthesized a viscosity-sensitive two-photon fluorescent probe, DCM1. Through density functional theory calculations, we elucidated the molecular conformational changes and the regulation of energy dissipation mechanisms under high-viscosity conditions. In glycerol solution, the fluorescence intensity of DCM1 is 36 times higher than that in aqueous solution, demonstrating its high sensitivity to microenvironmental viscosity changes. Furthermore, DCM1 exhibits excellent optical properties under two-photon excitation. At an excitation wavelength of 1000 nm, the two-photon absorption cross section reaches 181 GM. Leveraging these characteristics, DCM1 was successfully applied in two-photon fluorescence imaging experiments with live zebrafish, achieving high-resolution deep-tissue fluorescence imaging with a maximum penetration depth of 154 μm. This result demonstrates that the DCM1 probe can not only sensitively respond to viscosity changes in biological systems but also achieve deep-tissue fluorescence imaging, providing a novel tool for the diagnosis and mechanistic study of high-viscosity-related disease.