Real-Time Brillouin Microscopy for Linewidth Imaging of Protein Condensates in Living Cells

We propose a label-free, high-resolution approach to investigate pathological protein aggregation and aberrant phase behavior in living cells. Intracellular protein aggregates associated with neurodegenerative diseases are increasingly recognized for their impact on cellular mechanics and pathophysiology. Proteins involved in these disorders tend to misfold and form insoluble inclusions, via aberrant liquid–to solid phase transitions within stress granules and other biomolecular condensates. Despite their importance, probing the viscoelastic properties of these heterogeneous assemblies in living cells remains challenging due to their small size and rapid molecular turnover. Building on our previous study on Brillouin frequency shifts, here we employ a stabilized Brillouin microscope to quantify the full width at half maximum of the Brillouin peak, revealing viscosity-related mechanical signatures of pathological condensates. Combined with FRAP, our analysis shows that ALS-related proteins form condensates with broader linewidths than physiological stress granules, indicating increased viscosity and a more solid-like state. These findings demonstrate that Brillouin linewidth imaging can distinguish liquid-like from solid-like condensates in situ and uncover dissipative mechanical alterations relevant to neurodegenerative disease mechanisms.