Label‐Free Quantitative Analysis of Coacervates via 3D Phase Imaging

Abstract Coacervation is considered as a ubiquitous mechanism for assembling biomolecular materials outside cells and organizing membraneless organelles inside cells. Despite the importance of mapping binodals to understand the driving forces and thermodynamics of coacervate, quantifying protein concentration within a droplet is significantly challenging owing to its dynamic and viscous nature. A direct imaging‐based method is presented to quantify coacervate using real‐time 3D quantitative phase imaging. The proposed method utilizes the measurements of the refractive index tomograms of individual coacervates and retrieves the protein concentration and volume of individual protein droplets exploiting light‐scattering analysis. The retrievals of accurate protein concentrations are demonstrated in droplets, whereas conventional fluorescence‐based techniques present underestimations. With its simple, direct, real‐time, and quantitative analysis capability, the present method can be utilized in various protein analyses and quantifications for the study of coacervation both in vitro and in vivo.