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 the intrinsic Brownian motion of the entire condensate could interfere with diffusion measurements, confounding the interpretation of molecular mobility. Here we systematically assess and address this challenge with both experiments and simulations, using in vitro reconstituted condensates as simplified models of endogenous cellular assemblies. We show that tethering effectively suppresses the global translational and rotational Brownian motions of the entire condensate, eliminating inherent motion interference while preserving their spherical morphology. Quantitative analysis reveals that untethered condensates systematically overestimate 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, combining with simulations that sweep through the entire physiological parameter space, we provide a practical guideline for judging whether tethering is necessary in an experiment based on condensate size, diffusion type, and diffusion coefficient of the biomolecule of interest. Our findings establish surface tethering as a valuable and robust approach for accurate quantification of intra-condensate molecular dynamics, providing a methodological framework for future studies of membraneless organelles.