Condensate Growth Analysis Platform for Proteins Using Ultra-Widefield Dark-Field Microscopy and Image Analysis

Biomolecular condensates, which are membraneless organelles formed through liquid-liquid phase separation, serve as fields that regulate chemical reactions and functions by linking functionally related molecules. Accumulated data from the field of phase separation chemistry suggests that biomolecular condensates form and grow via the widely involved mechanisms, including diffusion-limited growth, fusion, and Ostwald ripening. However, tracking individual emerging or growing condensates is required to differentiate and quantify these mechanisms. In this study, we developed a label-free condensate growth analysis platform based on ultra-widefield dark-field microscopy and image analysis. Our system enables long-term detection for at least 30 min, high-time-resolution imaging at 0.1 s, and an ultra-wide imaging area of 5.8 mm². Using this platform, we characterized the growth processes of approximately 10,000 condensates in two model proteins, HP1α and FUS, by counting the occurrences of the three growth mechanisms. For these two proteins, condensates form through diffusion-limited growth and unexpectedly primarily grow via fusion rather than Ostwald ripening, either simultaneously or with a short lag time. Our results demonstrate that the platform can analyze the formation and growth of various biomolecular condensates in vitro without labeling the sample.