Liquid-Liquid Phase Separation from the Viewpoint of Molecular Crowding Environment: A Raman Imaging Study

We present the results of Raman imaging of molecular crowding environments in a living cell and a liquid droplet formed by liquid-liquid phase separation. Using the Raman band of water as an internal intensity standard, we propose an in situ quantification method for evaluating biomolecular concentrations. Based on these concentration measurements, intracellular crowding environments can be quantitatively assessed. A single liquid droplet in a buffer solution is found to have a very high biomolecular concentration, exceeding a few millimolar concentrations, and the concentration within a droplet varies depending on the surrounding environment. The biomolecular concentration in a droplet increases as conditions facilitate droplet formation, and we propose a model in which changes in the surrounding environment lead to the formation of dense droplets, which subsequently transform into aggregates. Concentration quantification in a single droplet was also performed in a living cell, demonstrating that the biomolecular concentration in a droplet is not much different from that in the surrounding intracellular environment. This result indicates that within a droplet in a cell, certain molecules are highly concentrated, while other molecules are excluded, and the overall concentration is comparable to that of the surrounding intracellular environment. The droplet formation within a cell can be regarded as a redistribution of biomolecules constituting a molecular crowding environment.