Abstract To unravel the biological functions of membraneless liquid condensates it is crucial to develop a quantitative understanding of the physics underlying their dynamics. Key processes within such condensates are diffusion and material exchange with their environment. Experimentally, diffusive dynamics are typically probed via fluorescent labels. However, to date we lack a physics-based quantitative framework for the dynamics of labeled condensate components. Here, we derive the corresponding theory, building on the physics of phase separation, and quantitatively validate this framework via experiments. We show that using our theory we can precisely determine diffusion coefficients inside liquid condensates via a spatio-temporal analysis of fluorescence recovery after photobleaching (FRAP) experiments. We showcase the accuracy and precision of our approach by considering space- and time-resolved data of protein condensates and two different polyelectrolyte-coacervate systems. Strikingly, our theory can also be used to determine the diffusion coefficient in the dilute phase and the partition coefficient, without relying on fluorescence measurements in the dilute phase. This bypasses recently described quenching artefacts in the dense phase, which can underestimate partition coefficients by orders of magnitude. Our experimentally verified theory opens new avenues for theoretically describing molecule dynamics in condensates, measuring concentrations based on the dynamics of fluorescence intensities and quantifying rates of biochemical reactions in liquid condensates.

Quantitative Theory for the Diffusive Dynamics of Liquid Condensates