Surface-Tethering Enhances Precision in Measuring Diffusion Within 3D Protein Condensates

Biomolecular condensates, or membraneless organelles, play pivotal roles in cellular organization by compartmentalizing biochemical reactions and regulating diverse processes such as RNA metabolism, signal transduction, and stress response. Super-resolved imaging and single-molecule tracking are essential for probing the internal dynamics of these condensates, yet intrinsic Brownian (thermal capillary wave) motion of the entire condensate in vitro could introduce artifacts into diffusion measurements, confounding the interpretation of molecular mobility. Here, we systematically assess and address this question using both experiments and simulations. We deploy three surface-tethering strategies-using biotinylated DNA, protein, or antibody tethers-to immobilize FUS protein condensates on passivated glass surfaces. We show that tethering effectively suppresses the global translational and rotational Brownian motion of the entire condensate, eliminating inherent measurement artifacts while preserving their spherical appearance and native liquid-like properties. Quantitative analysis reveals that untethered condensates systematically overestimate or underestimate molecular diffusion coefficients and step sizes, particularly for slowly diffusing structured mRNAs, while rapidly diffusing unstructured RNAs are unaffected due to temporal scale separation. Comparative evaluation of tethering strategies demonstrates tunable control over condensate stability and internal dynamics, with implications for optimizing experimental design. Finally, simulations spanning the full physiological parameter space enable us to provide practical guidelines for assessing whether, and to what extent, tethering is beneficial, based on condensate size and the diffusion properties of the biomolecule of interest. Our findings establish surface tethering as a necessary and robust approach for accurate quantification of intra-condensate molecular dynamics, providing a methodological framework for future studies of membraneless organelles.