Lipid rafts are small biomembrane functional units, formed as the result of the lateral phase separation of phospholipids. Phospholipid phase separation plays a crucial role in the spatial organization of biomolecules in life activities. Here, we study the kinetics of multi-component phospholipid phase separation quantitatively using the single domain characterization methods including the movement tracking and radial fluctuation analyses, which provide valuable information about the physical and mechanical properties of the bulks and domains. The study is carried out in a low line tension condition similar to that in cells. The order of magnitude of line tension is~0.1 pN as estimated from the radial fluctuation analysis. Fluorescence microscopy characterization shows that domains mainly coarsen through the coalescence pathways, while the evaporation-condensation is negligible. Through the tracking of domains, it is found that the bulk viscosity dominates the dynamics of domain coalescence. The coalescence of domains produces strong hydrodynamic flows in low viscosity bulk, which promotes the non-Brownian motion of surrounding domains, accelerating the lateral diffusion and coalescence of the domains. However, these hydrodynamic flows reduce significantly in high viscosity bulk. The domains rely mainly on Brownian motion to diffuse in this highly viscous medium, resulting in the slow lateral diffusion and low coalescence. Picking the domains following Brownian motion, the viscosities of liquid ordered bulk and liquid disordered bulk are determined to be in the range of 10^-8-10^-7 Pa·s·m and 10^-9 Pa·s·m from the Hughes-Pailthorpe-White empirical relation. Furthermore, we observe a bulk-viscosity-dependent scaling relation between the domain size and coarsening time in experiments. A theoretical model of domain diffusion and coalescence is established to understand the scaling relation. If the bulk viscosity is low, the hydrodynamic flow produces a high power exponent of 1.0. And if the bulk viscosity is high, the Brownian diffusion produces a low power exponent of 0.5. In addition, we demonstrate that the bulk viscosity can be regulated through the relative content of cholesterol. 1,6-Diphenyl-1,3,5-hexatriene fluorescence anisotropy characterization exhibits that the increase of cholesterol in liquid ordered and liquid disordered bulks disorders and orders the phospholipid packing, thus decreasing and increasing the bulk viscosity, respectively. It is expected that this viscosity regulation strategy can be used to control the multicomponent phospholipid phase separation. In all, our study deepens the understanding of the physical mechanism underlying the formation of lipid rafts. It also provides a reference for regulation of the biomolecule distribution in cell membranes.

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