In situ Quantification of Biomolecular Concentration of Cytoplasmic Membraneless Organelles in a Living Cell

ABSTRACT Liquid droplets formed via intracellular Liquid–liquid phase separation (LLPS) are called membraneless organelles and provide enzymatic reaction fields for maintaining cellular homeostasis, while they can be sources of protein aggregates and fibrils, causing neurodegenerative diseases. To understand the nature of intracellular liquid droplets, it is essential to quantify liquid droplets inside a living cell. Here, we performed near-IR fluorescence and Raman imaging to quantify chemical components inside stress granules (SGs) formed via LLPS in living cells under oxidative stress. The Raman images of stressed cells indicate the concentration of nucleic acids in the SGs was 20% higher than surrounding cytoplasm, while the lipid concentration was lower. The intensity of biomolecular C–H bands relative to the water band shows the net concentration of biomolecules was almost the same inside and outside the SGs, indicating intracellular droplets are not highly condensed, but the crowding environments are similar to the surroundings. TOC GRAPHICS