Interface Dynamics in Biomolecular Condensates by High‐Speed AFM

ABSTRACT Biomolecular condensates are adaptive soft materials whose interfacial properties play essential roles in cellular organization and numerous biological processes. While their bulk properties are routinely characterized using optical techniques, the structure and dynamics of their interfaces, critical for molecular recruitment and interactions with cellular components, remain largely inaccessible due to the interface thickness. Here we demonstrate that high‐speed atomic force microscopy (HS‐AFM) can investigate the interface dynamics of biomolecular condensates with nanometer spatial and sub‐second temporal resolution, which is not accessible to optical techniques. We identify and overcome key experimental challenges, including cantilever instabilities induced by condensates and the need for precise force control under low surface tension (10 −5 to 10 −3 N m −1 ) conditions. We show that optimized sample preparation and environmental control ensure stable imaging. We demonstrate the capabilities of this method using 1) the prion‐like domain of the plant protein EARLY FLOWERING 3 (ELF3), revealing confined dynamics of protein multimers, and 2) a minimal peptide system (WGR‐4), exhibiting time‐dependent progressive interfacial aggregation. By extending the application of HS‐AFM from single biomolecules to soft biological and liquid interfaces, this approach provides direct access to the nanoscale organization and dynamic evolution of biomolecular condensate interfaces.