| Summary: | Biomolecules forming membraneless structures via liquid-liquid phase separation (LLPS) is a common event in living cells. Some liquid-like condensates can convert into solid-like aggregations, and such a phase transition process is related to some neurodegenerative diseases. Liquid-like condensates and solid-like aggregations usually exhibit distinctive fluidity and are commonly distinguished via their morphology and dynamic properties identified through ensemble methods. Emerging single-molecule techniques are a group of highly sensitive techniques, which can offer further mechanistic insights into LLPS and phase transition at the molecular level. Here, we summarize the working principles of several commonly used single-molecule techniques and demonstrate their unique power in manipulating LLPS, examining mechanical properties at the nanoscale, and monitoring dynamic and thermodynamic properties at the molecular level. Thus, single-molecule techniques are unique tools to characterize LLPS and liquid-to-solid phase transition under close-to-physiological conditions.
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