Fluorescence-based mapping of dielectric permittivity of condensates and their environment

Membraneless organelles, formed through liquid-liquid phase separation, are essential for cellular organization and function. One important property of these biomolecular condensates is their dielectric permittivity (or micropolarity), which influences their viscosity, structural integrity, protein conformations, and hydration levels. Since permittivity reflects a medium’s polarity, condensates with large permittivity contrasts also tend to have high surface tensions, affecting their behavior in the cellular environment. While environment-sensitive fluorophores are commonly used to probe condensate permittivity, existing methods have limited measurement ranges and often cannot simultaneously resolve both dense and dilute phases, especially when there are large permittivity contrasts. Furthermore, calibration curves based on ratiometric standards can be sensitive to the choice of solvent, complicating data interpretation and comparison. Here, we introduce a broadband fluorescence-based technique using the water-soluble dye 2-acetyl-6-(dimethylamino)naphthalene (ACDAN), which allows for precise measurement of condensate permittivity. Our findings reveal a surprisingly broad permittivity range, spanning from 5ε ₀ to 70ε ₀ . This large range provides new insights into the interactions of condensates with cellular membranes and other cellular structures. Importantly, this method can efficiently probe both protein-rich and protein-depleted phases, enabling a sensitive and direct readout of a mixture’s position on its phase diagram. Because it relies on fluorescence imaging, this technique is highly versatile for studying condensates in both live-cell and in vitro systems. Our results demonstrate that this approach is robust across different analytical platforms and detection equipment, offering a convenient and reliable tool for measuring the dielectric properties of condensates and other microheterogeneous systems.