Abstract 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 quantify Brillouin shifts of several protein condensates in living cells and validate our findings with FRAP. The correlation between techniques reveals a fractal internal architecture of the condensates, providing important insights into their physical nature while probing the mechanical behavior of entire compartments containing multiple protein species. Our method offers a unique framework for distinguishing physiological from pathological condensates, paving the way for long-term, user-independent, high-precision mechanical measurements in living cells.
Testi et al. (Thu,) studied this question.