Seeing the Chemistry of Biomolecular Condensates: In Situ Mapping of Composition and Water Content.

Biomolecular condensates are membraneless cellular organelles that form via liquid-liquid phase separation of proteins and nucleic acids. Their functional roles are tightly coupled to material properties like viscosity and hydrophobicity, which serve as key markers of cellular state. However, conventional determination of condensate composition and water content relies on invasive procedures that alter their thermodynamic state or can damage samples. Here, we introduce Raman spectroscopy coupled with spectral phasor analysis as an in situ, label-free approach to estimate the chemical profiles and relative molecular contributions within both the dense and dilute phases of biomolecular condensates. This method outperforms traditional regression and deconvolution approaches, enabling estimation of client molecule partitioning. By accounting for contributions of the protein backbone to the Raman spectra of condensates, we assess the signature of "solid-like" hydrogen-bonded water from protein hydration, revealing that most water molecules within condensates retain bulk, "liquid-like" properties. Finally, using environment-sensitive fluorescent probes, we demonstrate that macromolecular structure and water content-rather than hydrogen bonding alone-drive condensate hydrophobicity; notably, the dense phase remains predominantly water-rich even at low apparent dielectric constants.