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Probing Diffusion and Viscosity in Multiphase Coacervates and Cellular Condensates with Raster Image Correlation Spectroscopy

Brent S. Visser, Iris B. A. Smokers, Wojciech P. Lipiński, and Evan Spruijt*

  • Radboud University, Institute of Molecules and Materials (IMM), Heyendaalseweg 135, 6525 AJ, Nijmegen, The Netherlands

  • *Contact author: evan.spruijt@ru.nl
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PRX Life 4, 033007 – Published 27 July, 2026

DOI: https://doi.org/10.1103/msq9-61tz

Abstract

Raster image correlation spectroscopy (RICS) is a nondestructive, fluorescence-based method to probe molecular diffusion and effective viscosity in biomolecular condensates. By measuring fluorescence intensity autocorrelations of fluorescent probes in a scanning confocal microscope image, RICS enables the calculation of effective, local condensate viscosities both in reconstituted condensates and in living cells. Here we demonstrate the use of RICS as a powerful tool to probe local diffusion and length-scale-dependent viscosities in multiphase coacervates and stress granules without disrupting them. We quantify the local viscosity in core and shell regions of multiphase coacervates, and inside stress granules and the surrounding cytoplasm in HEK293T cells without relying on labeled proteins. By comparing different probe types, we further show that globular probes such as folded proteins and quantum dots experience a length-scale-dependent viscosity in condensates, whereas dextran-based polymeric probes exhibit an apparent size-independent diffusivity over three orders of magnitude in molecular weight. We attribute these findings to a progressive collapse with increasing size of the polymer chains inside condensates, combined with a weakening of sticky probe-condensate interactions. These findings emphasize the need for due care when using probes to characterize condensate properties. Altogether, our results show that RICS is a useful tool for studying condensate properties in situ that can be accessed on conventional confocal microscopes.

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This article appears in the following collection:

Collection on Physics of Biomolecular Condensates

From the fundamentals of phase separation, dynamics and structure of condensates in biological settings, to advanced imaging methods, this special collection presented by PRX Life showcases exciting and timely work on the physics of biomolecular condensates.

This Invited Collection is coordinated by PRX Life Associate Editor Christoph Weber, in consultation with Jerelle Joseph, Simon Alberti, and Tanja Mittag. Every article published in this collection underwent a rigorous peer review process, adhering to the same high standards applied to all papers handled by the journal. The PRX Life editorial team managed the peer review and made all editorial decisions.

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PRX Life 4, 033007– Published 27 July, 2026
  • Received 10 November 2025
  • Accepted 16 June 2026
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Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article's title, journal citation, and DOI.

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