Study liquid–liquid phase separation with optical microscopy: A methodology review

Intracellular liquid–liquid phase separation (LLPS) is a critical process involving the dynamic association of biomolecules and the formation of non-membrane compartments, playing a vital role in regulating biomolecular interactions and organelle functions. A comprehensive understanding of cellular LLPS mechanisms at the molecular level is crucial, as many diseases are linked to LLPS, and insights gained can inform drug/gene delivery processes and aid in the diagnosis and treatment of associated diseases. Over the past few decades, numerous techniques have been employed to investigate the LLPS process. In this review, we concentrate on optical imaging methods applied to LLPS studies. We begin by introducing LLPS and its molecular mechanism, followed by a review of the optical imaging methods and fluorescent probes employed in LLPS research. Furthermore, we discuss potential future imaging tools applicable to the LLPS studies. This review aims to provide a reference for selecting appropriate optical imaging methods for LLPS investigations.