Fluorescence-Based Microviscosity Mapping in Membraneless Organelles

High Resolution Image Download MS PowerPoint Slide Membraneless organelles (MLOs) are cellular biomolecular condensates formed by liquid–liquid phase separation. Their biological functions are intimately linked to their material properties, including viscosity. Condensate viscosity is determined by the size, shape, concentration, and molecular interactions between MLO components. It impacts the diffusion of MLO constituents and the selective permeability of the condensate, thereby regulating the rate of biochemical reactions. Viscosity modifications associated with the liquid-to-gel transition of the condensates are related to pathologies. Current experimental approaches for characterizing the material properties of cellular condensates remain limited. In this study, we report the use of BODIPY-based molecular rotors, in combination with fluorescence lifetime imaging microscopy (FLIM), to monitor the microviscosity of cellular MLOs directly in living cells. The fluorescence lifetime of BODIPY derivatives increases with the viscosity of their microenvironment, enabling a quantitative assessment of microviscosity within condensates. HaloTag technology was employed to specifically label MLO components. Our findings reveal that the nucleolus exhibits higher apparent viscosity than the surrounding nucleoplasm and that microviscosity varies across nucleolar subcompartments. Furthermore, nucleolar reorganization induced by the inhibition of rRNA synthesis results in a measurable increase in microviscosity. Finally, we demonstrate that the apparent microviscosity of stress granules is lower than that of the nucleoli. Overall, the presented results demonstrate the strong potential of BODIPY-based molecular rotors as versatile and powerful tools for probing local microviscosity in cellular membraneless organelles.