Label-free composition determination for biomolecular condensates with an arbitrary number of components

Many cellular compartments are protein-rich biomolecular condensates demixed from the cyto- or nucleoplasm. Although condensates are defined by their molecular composition, traditional approaches to measure composition are inefficient or require confounding labels, and are typically limited to few components. Here, we describe a label-free method to measure the shape and composition of micron-sized condensates based on quantitative phase microscopy and the physics of sessile droplets. This method has a precision better than 2%, requires 1000-fold less material than bulk techniques, and exposes systematic errors as large as 50-fold in common approaches based on fluorescence intensity ratios. Further, we show that this method can be used in combination with standard dilute-solution detection methods to determine the composition of condensates containing an arbitrary number of molecular components. We demonstrate this explicitly by measuring the tie-lines and binodals for a ternary mixture containing RNA and the full-length RNA-binding protein FUS. In addition to recovering the expected re-entrant behavior in the dilute binodal branch with increasing total RNA concentration, our measurements reveal an unexpected kink in the condensed binodal branch above which the condensed phase maintains a constant polymer volume fraction over an extended range. Interestingly, this density is comparable to that of many condensates in vivo and significantly lower than that typical of protein condensates in binary systems, underscoring the ability of multi-component reconstitutions to more faithfully reflect in vivo conditions. Finally, we discuss the fundamental role of composition in controlling condensate mechanical, dielectric and surface properties, which are expected to collectively underly biological functionality.