High-resolution Brillouin imaging of protein condensates in living cells

Mechanical properties of biomolecular condensates play a central role in cellular function and disease, yet their quantitative characterization in living cells remains technically challenging. Brillouin microscopy offers a label-free and non-contact approach to probe viscoelastic properties at subcellular resolution, but its broader application is limited by spectral instabilities and calibration issues. Here, we report an enhanced Brillouin microscope incorporating an electrooptic modulator (EOM) that acts as an internal frequency reference, spectrometer calibrator, and stabilizing element. This approach enables automated, long-term measurements with improved spectral precision and robustness. We demonstrate the performance of the system through stability tests on water reference samples and apply it to the investigation of protein condensates in living cells. Representative measurements reveal distinct Brillouin signatures for different condensate states, which are compared with fluorescence recovery after photobleaching (FRAP) experiments. These results illustrate the potential of the EOM-based Brillouin microscope as a reliable platform for long-term, user-independent biomechanical imaging in biological systems.