Abstract
Liquid-liquid phase separation (LLPS) of biomolecules is a fundamental cellular process that is essential for maintaining homeostasis and facilitating biochemical activities. On the other hand, aberrant phase separation alters condensate fluidity and causes a transition from liquid-like condensates to solid-like condensates, which may lead to the formation of the pathological aggregations often observed in neurodegenerative diseases. Condensate fluidity is usually assessed by the fluorescence recovery after photobleaching. Here, we reveal that the fluorescence lifetimes of several fluorescent proteins are sensitive to LLPS and the liquid-to-solid transition. Furthermore, we identify several key residues that regulate the sensitivity of fluorescence lifetimes toward phase separation. Thus, we apply fluorescence lifetime imaging microscopy (FLIM) to visualize LLPS and the liquid-to-solid transition in living cells, demonstrating that FLIM is a nondestructive method for tracking changes in condensate fluidity in real time.