Intracellular biomolecular condensates viewed by Raman microscopy: Composition, crowding, and hydration

Liquid-liquid phase separation (LLPS) generates high-concentration biomolecular liquid droplets and contributes to diverse intracellular processes. Intracellular droplets contain not only proteins that drive LLPS but also various coexisting components and abundant hydration water, and their properties depend strongly on both the constituent components and the surrounding intracellular environments. However, the quantitative understanding of these factors within living cells remains limited. Raman microscopy is well suited for this challenge, enabling label-free, direct analysis of structures and concentrations of constituent molecules and hydration water in droplets, while also providing information on intracellular molecular crowding environments. In this review, we summarize recent Raman microscopic studies on intracellular droplets, focusing on coexisting components, structural characterization, hydration water, and their relationships with molecular crowding.