Abstract
Biomolecular condensates are essential for cellular organization and function, yet understanding how chemical and physical factors govern their formation and dynamics has been limited by a lack of noninvasive measurement techniques. Conventional microscopy methods often rely on fluorescent labeling and substrate immobilization, which can perturb the intrinsic properties of condensates. To overcome these challenges, we apply label-free, contact-free holographic video microscopy to study the behavior of a condensate-forming protein in vitro. This technique enables rapid, high-throughput, and precise measurements of individual condensate diameters and refractive indexes, providing unprecedented insight into size distributions and dense-phase macromolecular concentrations over time. Using this method, we investigate the kinetics of droplet growth, aging, and equilibrium dynamics in the model condensate-forming protein PopZ. By systematically varying the concentration and valence of cations, we uncover how multivalent ions influence condensate organization and dynamics, a hypothesis we further test using super-resolution microscopy. Our findings reveal that PopZ droplet growth deviates from classical models such as Smoluchowski coalescence and Ostwald ripening. Instead, we show that condensate growth is consistent with gelation at the critical overlap concentration. Holographic microscopy offers significant advantages over traditional techniques, such as differential interference contrast microscopy, delivering reproducible measurements and capturing condensate dynamics with unparalleled precision. This work highlights the power of holographic microscopy to probe the material properties and mechanistic underpinnings of biomolecular condensates, paving the way for deeper insights into their roles in synthetic systems.