Liquid-liquid phase separation (LLPS) plays an important role in a variety of biological processes and is also associated with protein aggregation in neurodegenerative diseases. Quantification of LLPS is necessary to elucidate the mechanism of LLPS and the subsequent aggregation process. In this study, we showed that ataxin-3, which is associated with Machado-Joseph disease, exhibits LLPS in an intracellular crowding environment mimicked by biopolymers, and proposed that a single droplet formed in LLPS can be quantified using Raman microscopy in a label-free manner. We succeeded in evaluating the protein concentration and identifying the components present inside and outside a droplet using the O-H stretching band of water as an internal intensity standard. Only water and protein were detected to be present inside droplets with crowding agents remaining outside. The protein concentration in a droplet was dependent on the crowding environment, indicating that the protein concentration and intracellular environment should be considered when investigating LLPS. Raman microscopy has the potential to become a powerful technique for clarifying the chemical nature of LLPS and its relationship with protein aggregation.
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