Mesoscale properties of phase-separated polymers under confinement and their size scaling

Sensitive visualization and conformational control of biopolymer interactions at super-molecular dimensions is important because it is at these scales that biopolymers undergo liquid-liquid phase separation inside cells. For example, the phase separation of proteins and nucleic acids from other material inside the cell results in the formation of non-membranous organelles (NMOs). These structures have important functions, but it is unclear how the nanoscale confinement inside the cell may regulate their physical properties. We address this question by using confinement microscopy to gently load polymers into a nanofabricated array of pits. We have tested this approach using solutions of two water-soluble polymers, polyethylene glycol and dextran, as a model system, as well as a reconstituted model of NMOs phase separated from a solution mixture of protein and nucleic acid. Droplets of various sizes (from less than 1 to 20 μm in diameters) were formed under confinement. Particle tracking microrheology was performed to study the mesoscale properties of phase separated droplets in order to explore the relationship between droplet sizes and their internal diffusion properties. In both systems, crowding-induced sub-diffusion as well as spatial heterogeneity are observed.