LLPS droplet size estimation via UV–Vis spectroscopy using a microplate reader

Liquid-liquid phase separation (LLPS) is critical to the formation of membraneless organelles within cells. Monitoring the size and dynamics of LLPS droplets is essential to understanding their function. However, conventional methods, such as microscopy and dynamic light scattering (DLS), have limitations, including low throughput and complex setups. Here, we present a simple, label-free, high-throughput method for estimating droplet size using ultraviolet-visible (UV-Vis) spectra obtained using a standard microplate reader. This method is theoretically supported by Mie scattering theory. By analyzing the spectral shape and comparing it to that of glass beads of known sizes, our method enables the quantitative estimation of LLPS droplet size in a minimally invasive manner. We validated our approach using peptide droplets of known sizes, then applied it to monitor the growth of protein condensates formed by vesicle-associated membrane protein-associated protein B (VAPB). This technique offers a convenient, scalable alternative for screening LLPS dynamics in various biomolecular systems.