Measuring concentration and diffusivity within biomolecular condensates using calibration-free scanning fluorescence correlation spectroscopy.

There is a growing consensus that cells can regulate biochemical activity through membrane-less organelles, also known as biomolecular condensates. Unfortunately, the mechanisms underlying the interplay between phase separation and biochemical reactions are still unclear. Since biochemical reactions depend strongly on the local concentrations and diffusivities of molecules in the dense phase, accurately characterizing these parameters is essential for understanding biochemical regulation within phase-separated condensates. Fluorescence correlation spectroscopies can measure these properties but are limited by their need for calibration standards. Here, we present a calibration-free method based on temporal line scan fluorescence correlation spectroscopy and sinusoidal scan fluorescence correlation spectroscopy to quantify concentrations and diffusivities of molecules in the dilute and dense phases. We showcase the potential of the approach by measuring the full phase diagram of the intrinsically disordered region of the DEAD-box protein Ddx4, as well as the diffusivities of recruited client molecules in the dense phase. We show that the diffusivity of different client molecules decreases as their concentration in the dense phase increases. Such a drastic decrease in diffusivities may explain the stability of certain aggregation-prone proteins in the dense phase despite their high local concentrations.