Biological condensates form percolated networks with molecular motion properties distinctly different from dilute solutions.

Formation of membraneless organelles or biological condensates via phase separation and related processes hugely expands the cellular organelle repertoire. Biological condensates are dense and viscoelastic soft matters instead of canonical dilute solutions. To date, numerous different biological condensates have been discovered, but mechanistic understanding of biological condensates remains scarce. In this study, we developed an adaptive single-molecule imaging method that allows simultaneous tracking of individual molecules and their motion trajectories in both condensed and dilute phases of various biological condensates. The method enables quantitative measurements of concentrations, phase boundary, motion behavior, and speed of molecules in both condensed and dilute phases, as well as the scale and speed of molecular exchanges between the two phases. Notably, molecules in the condensed phase do not undergo uniform Brownian motion, but instead constantly switch between a (class of) confined state(s) and a random diffusion-like motion state. Transient confinement is consistent with strong interactions associated with large molecular networks (i.e., percolation) in the condensed phase. In this way, molecules in biological condensates behave distinctly different from those in dilute solutions. The methods and findings described herein should be generally applicable for deciphering the molecular mechanisms underlying the assembly, dynamics, and consequently functional implications of biological condensates.