Stabilized real-time Brillouin microscopy reveals fractal organization of protein condensates in living cells

Mechanical alterations of protein condensates are increasingly recognized in the etiology of several neurodegenerative diseases, yet their characterization remains technically challenging. Although Brillouin microscopy could offer a promising solution, its use is hindered by instrumental instabilities demanding frequent adjustments and manual calibrations with reference materials. Here, we present an enhanced Brillouin Microscope that incorporates an electro-optic modulator, serving simultaneously as frequency reference, spectrometer calibrator, and temporal stabilizer. This integration enables robust, real-time spectral stability over multiple days in a fully automated workflow. Using this system, we quantified the mechanical properties of several protein condensates in living cells and validated our findings with FRAP measurements. The correlation between techniques reveals a fractal internal architecture of the protein condensates, providing important insights into their physical nature. Our innovative method thus offers a unique framework for distinguishing physiological from pathological condensates, paving the way for long-term, user-independent and high-precision mechanical measurements in living cells.