Single-particle light scattering reveals structural heterogeneity of biomolecular condensates

Biomolecular condensates can be heterogeneous in composition, morphology, and material properties, but resolving this heterogeneity at the level of individual submicrometer condensates remains challenging. Here, we combine off-axis holographic imaging with quantitative light-scattering analysis and particle tracking to simultaneously measure the size, optical properties, interfacial structure, and hydrodynamic mobility of hundreds of individual condensates per minute. Applied to condensates formed by the N-terminal domain of Ddx4 (Ddx4N1), the method reveals two populations with distinct scattering signatures: compact particles with well-defined interfaces and structurally heterogeneous, non-compact assemblies. Their relative abundance varies systematically with ionic strength. Synthetic polymer controls show that pronounced structural heterogeneity is not a generic consequence of phase separation and suggest a role for interaction heterogeneity in promoting competing mesoscale structures. Mass-size scaling and Brownian aggregation simulations further show that aggregation with incomplete fusion of smaller protein-rich units provides a physical model for the non-compact population. Simultaneous optical and diffusion measurements further show that the two populations have distinct hydrodynamic properties. These results demonstrate that chemically homogeneous condensate-forming systems can populate distinct mesoscale structures that are obscured by ensemble-averaged measurements.