Azo-Enhanced Raman Rotors: Bridging Raman Scattering and Fluorescence for Hyperspectral Imaging of Live-Cell Microviscosity Dynamics

Azobenzene derivatives are well-known molecular photoswitches that undergo tunable changes in the geometry, dipole moment, and electronic structure upon light irradiation. While their tunable absorption and fluorescence have inspired diverse applications in sensing various physical and chemical environments, recent advances have shown that azobenzene conjugates also serve as powerful Raman reporters through azo-enhanced Raman scattering. Although Raman scattering, a narrow-band vibrational signature, and fluorescence, a broadband electronic emission, are both valuable photophysical processes, their distinct energy-level transitions pose challenges for simultaneously interpreting both signals in a single measurement, especially for measuring heterogeneous biological systems. In this study, we fine-tuned a class of azo-enhanced Raman rotors for hyperspectral imaging and leveraged their fluorescence-versus-Raman intensity ratio to report microviscosity in live cells. This approach provides self-calibrated readouts, enabling accurate visualization of microviscosity changes within organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum. Our findings demonstrate an effective strategy to bridge the spectral gap between Raman scattering and fluorescence, offering a robust method for probing physicochemical heterogeneity in biological systems. The ability to simultaneously harness both photophysical processes in a single molecular platform opens new avenues for advanced hyperspectral bioimaging, cellular diagnostics, and mechanistic studies of heterogeneous nonbiological interfaces.