Probing Diffusion and Viscosity in Multiphase Coacervates and Cellular Condensates with Raster Image Correlation Spectroscopy

Raster image correlation spectroscopy (RICS) is a nondestructive, fluorescence-based method to probe molecular diffusion and effective viscosity in biomolecular condensates. By measuring fluorescence intensity autocorrelations of fluorescent probes in a scanning confocal microscope image, RICS enables the calculation of effective, local condensate viscosities both in reconstituted condensates and in living cells. Here we demonstrate the use of RICS as a powerful tool to probe local diffusion and length-scale-dependent viscosities in multiphase coacervates and stress granules without disrupting them. We quantify the local viscosity in core and shell regions of multiphase coacervates, and inside stress granules and the surrounding cytoplasm in HEK293T cells without relying on labeled proteins. By comparing different probe types, we further show that globular probes such as folded proteins and quantum dots experience a length-scale-dependent viscosity in condensates, whereas dextran-based polymeric probes exhibit an apparent size-independent diffusivity over three orders of magnitude in molecular weight. We attribute these findings to a progressive collapse with increasing size of the polymer chains inside condensates, combined with a weakening of sticky probe-condensate interactions. These findings emphasize the need for due care when using probes to characterize condensate properties. Altogether, our results show that RICS is a useful tool for studying condensate properties that can be accessed on conventional confocal microscopes.