Life’s tiny droplets: A novel method revealing biological condensate composition

Our cells are bustling with tens of thousands of types of molecules at any given time. Within this complex environment, proteins often come together with RNA or DNA and, through a process called phase separation, form ‘droplets’ known as biomolecular condensates. Numerous cellular processes are governed by condensates, yet understanding their makeup is still an active area of research. “When you want to build something, it’s not just a question of which ingredients to use, but also of how much. The ratios are essential,” says Dr. Patrick McCall, an independent research associate at the Leibniz Institute of Polymer Research Dresden (IPF) and the lead author of the study. “With condensates, the very same principle applies. We often know which components are present, but not the specific proportions inside a condensate that ultimately define its composition”. The mix of components inside a condensate are key in determining how it behaves and what it does. An analogy can be made to baking; both cake and cookies can be made with similar ingredients, but the specific ratios in the mixture are key in determining which comes out of the oven.

But how can these ratios be studied? Scientists have traditionally ‘tagged’ the individual components of a condensate, using fluorescent labels to measure their concentration. However, recent research has revealed that tagging and quantification can introduce significant errors. Commonly used tags are large enough to substantially alter the target molecule’s properties, behaviour, and even its tendency to phase separate. To complicate things further, their apparent brightness in the condensate can be unreliable. While existing “label-free” methods avoid the problems with fluorescent tags entirely, they typically struggle to distinguish between different components in a condensate. As a result, reliable measurements have been limited to simplified condensates reconstituted in a test tube from only one or two components. To understand the complex makeup of more realistic condensates, a new label-free approach to measure the components was needed. Recognising this gap, Dr. McCall initially began exploring new ways to measure condensate composition during his postdoctoral work, carried out jointly in the labs of Jan Brugués (now at the Cluster of Excellence Physics of Life) and Anthony Hyman, at the Max Planck Institute for Cell Biology and Genetics (MPI-CBG). Building on this lengthy collaboration with colleagues at both PoL and MPI-CBG, seminal findings have now been published in Nature Chemistry, highlighting a novel, label-free method to measure the composition of multi-component condensates. This is an essential step towards understanding the physical properties and function of these droplets. 

Their new method, Analysis of Tie-lines and Refractive Index (ATRI), combines two physical concepts – the refractive index and tie-line – to determine the composition of a condensate. The refractive index of a medium describes how much light bends when passing through it. Using quantitative phase imaging (QPI), a powerful label-free microscopy tool, the authors could measure the refractive index difference between micron-sized condensates and the surrounding medium, the “dilute phase”, to determine the concentration. When a condensate is made of just a single protein, the QPI measurement immediately provides an estimate of concentration inside the condensate. However, certainty turns to ambiguity when more than one component is present, as different combinations of molecules can produce the same refractive index. This makes it difficult to determine the exact ratios of each component inside multi-component condensates. To resolve this confusion, ATRI uses a tie-line. The tie-line is a fundamental concept in physical chemistry that relates the compositions of two phases following phase separation – the condensate and the surrounding dilute phase – to the composition of the overall system. The key realization behind ATRI is that combining refractive index measurements with the tie-line graphically results in two lines that meet at one specific point: the point representing condensate composition. Using the refractive index and tie-line as inputs, ATRI generates a set of equations to pinpoint the concentration of multiple different components in a condensate, thereby determining its composition. This technique can be applied even in mixtures with many different molecules, and even when only tiny quantities are available.

Utilising their new approach, the authors successfully revealed the composition of complex condensates in unprecedented detail, resolving the concentrations of five molecules. At the time of publication, experiments capable of resolving more than two proteins in a condensate without using fluorescence have not been reported previously, highlighting the significance of this new method. By revealing the composition of condensates quantitatively, their behaviour and properties may be predicted with greater accuracy than ever before, paving the way for new insights. In addition, researchers can now use ATRI to study how condensates react to variation in the abundance of a particular component, mimicking changes in gene expression that normally happen in a cellular environment. Such experiments will help uncover the contribution of individual components to overall condensate behaviour and function. Beyond basic science, the new method has the potential to drive biomedical advances as well. Condensates play a role in numerous disorders and, by exposing how a condensate’s molecular makeup responds to promising compounds, ATRI may aid the development of potent therapeutics and treatments in future.

Information about the publication:

Investigators: Patrick M. McCall, Kyoohyun Kim, Anna Shevchenko, Martine Ruer-Gruß, Jan Peychl, Jochen Guck, Andrej Shevchenko, Anthony A. Hyman, and Jan Brugués. 

Funding: This study was supported by Volkswagen ‘Life’ grant number 96827 and the Deutsche Forschungsgemeinschaft (DFG) under Germany's Excellence Strategy, Cluster of Excellence Physics of Life of TU Dresden (EXC-2068–390729961). Researchers were additionally supported by an ELBE Postdoctoral Fellowship from the Center for Systems Biology Dresden and the Biocondensate Emerging Topic at the IPF. 

Study: Patrick M. McCall, Kyoohyun Kim, Anna Shevchenko, Martine Ruer-Gruß, Jan Peychl, Jochen Guck, Andrej Shevchenko, Anthony A. Hyman, Jan Brugués. (2025): A label-free method for measuring the composition of multi-component biomolecular condensates. Nature Chemistry. DOI: 10.1038/s41557-025-01928-3

About the Cluster of Excellence Physics of Life

Physics of Life (PoL) is one of five Clusters of Excellence at TU Dresden. PoL’s aim is to identify the physical laws underlying the organization of life in molecules, cells, and tissues. In the cluster, scientists from physics, biology, and computer science investigate how active matter in cells and tissues organizes itself into given structures and gives rise to life. PoL is funded by the DFG within the framework of the Excellence Strategy. It is a cooperation between scientists of the TU Dresden and research institutions of the DRESDEN-concept network, such as the Max Planck Institute for Molecular Cell Biology and Genetics (MPI-CBG), the Max Planck Institute for the Physics of Complex Systems (MPI-PKS), the Leibniz Institute of Polymer Research (IPF) and the Helmholtz-Zentrum Dresden-Rossendorf (HZDR).

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