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.

Establishment of a Method for the Introduction of Poorly Water-Soluble Drugs in Cells and Evaluation of Intracellular Concentration Distribution Using Resonance Raman Imaging

“…As mentioned in , we have reported the determination of the protein concentration in an LLPS droplet in buffer solution using the Raman band of water outside the droplets as an intensity standard. − Quantification using the water Raman band is based on the fact that the concentration of water in liquid water is extremely high and constant. In this study, we applied this method to quantify the intracellular components.…”

“…We also applied this method to cells and succeeded in quantifying the concentration of nucleic acids in a living cell by normalizing their Raman intensities with the O−H stretching band of water outside the cells as an intensity standard, utilizing the constant concentration of extracellular water. 14 In this study, we applied the concentration quantification method to quantify intracellular drug molecules and obtain the concentration distribution in a living cell. Many drug molecules have low water solubility, making drug uptake into cells problematic for drug delivery.…”

“…In previous studies, we propose that the Raman band of water can be used as an intensity standard for determining the concentration of the target molecule using Raman microscopy. − We demonstrated the concentration quantification using liquid droplets of the protein formed by liquid–liquid phase separation (LLPS) in a buffer solution. − Taking advantage of the fact that the concentration of water molecules in water outside the droplet is constant regardless of the sample, we normalized the Raman band of the protein in a droplet by the O–H stretching Raman band of water outside the droplet and determined the protein concentration within a single droplet in a label-free manner. We also applied this method to cells and succeeded in quantifying the concentration of nucleic acids in a living cell by normalizing their Raman intensities with the O–H stretching band of water outside the cells as an intensity standard, utilizing the constant concentration of extracellular water …”