Nucleic Acid-Rich\nStress Granules Are Not Merely Crowded\nCondensates: A Quantitative Raman Imaging Study

Liquid droplets,\nformed by intracellular liquid–liquid phase\nseparation (LLPS), are called membraneless organelles. They provide\ntransient enzymatic reaction fields for maintaining cellular homeostasis,\nalthough they might transform into aggregates, leading to neurodegenerative\ndiseases. To understand the nature of intracellular droplets, it is\ncrucial to quantify the liquid droplets inside a living cell as well\nas to elucidate the underlying biological mechanism. In this study,\nwe performed near-infrared fluorescence and Raman imaging to quantify\nchemical components inside stress granules (SGs) formed by LLPS in\nliving cells. The Raman images reveal that the nucleic acid concentration\ninside the SGs was more than 20% higher than the surrounding cytoplasm,\nwhereas the lipid concentration was lower. Quantitative Raman intensity\nanalysis using a water Raman band as an internal standard enables <i>in situ</i> concentration determination of nucleic acids in\nthe SGs and other organelles. The intensity of the biomolecular C–H\nbands relative to the water band indicates that the crowding environment\ninside the SGs depends on the stress type; under oxidative stress,\nthe inside of the SGs was nearly identical to the outside, whereas\nit was sparser in hyperosmotic stressed cells, suggesting that the\nhigh concentrations of nucleic acids play a pivotal role in maintaining\nthe environments inside the SGs. These results demonstrate that intracellular\ndroplets are not always highly condensed.