Innovative Method for Evaluating the Optical Properties of Liquid–Liquid Phase Separations Using Laser Scanning Microscopic Spectroscopy

Abstract

ABSTRACT Liquid–liquid phase separation (LLPS) plays a central role in intracellular compartmentalization and gene regulation. Although optogenetic optoDroplet systems are widely used to study LLPS in living cells, the mechanisms underlying the transition from dynamic liquid‐like condensates to operationally defined gel‐like assemblies remain poorly understood because intermediate assembly states are difficult to monitor in situ. Here, we developed a confocal laser microspectroscopy‐based approach to characterize the assembly dynamics of the fused sarcomas (FUS)n‐mCherry‐CRY2 optoDroplet system in living NIH3T3 cells. Quantitative fluorescence analysis enabled the identification and discrimination of three distinct assembly states: monomeric, dimer/oligomer intermediate, and gel‐like. Passive micro‐rheology, fluorescence recovery after photobleaching (FRAP), and automated morphological analyses further demonstrated progressive reductions in molecular mobility and increasing structural rigidity during condensate maturation. Three‐dimensional spatial analysis revealed that mature condensates exhibit a distinct core–shell organization consisting of an operationally defined gel‐like core, a dimer/oligomer‐rich intermediate layer, and an outer monomer‐rich boundary layer with liquid‐like properties. Upon cessation of blue‐light stimulation, the outer layer rapidly dissolved, whereas the central gel‐like core remained intact, indicating the acquisition of physical irreversibility. These findings establish fluorescence microspectroscopy as a quantitative approach for resolving molecular assembly states during condensate maturation and demonstrate that gelation proceeds from the condensate center toward the periphery. This framework provides new insights into pathological liquid‐to‐gel phase transitions associated with neurodegenerative diseases.