Imaging-based quantitative assessment of biomolecular condensates in vitro and in cells.

The formation of biomolecular condensates contributes to intracellular compartmentalization and plays an important role in many cellular processes. The characterization of condensates is however challenging, requiring advanced biophysical or biochemical methods that are often less suitable for in vivo studies. A particular need for easily accessible yet thorough methods that enable the characterization of condensates across different experimental systems thus remains. To address this, we present PhaseMetrics, a semi-automated FIJI-based image analysis pipeline tailored for quantifying particle properties from microscopy data. Tested using the FG-domain of yeast nucleoporin Nup100, PhaseMetrics accurately assesses particle properties across diverse experimental setups, including particles formed in vitro in chemically defined buffers or Xenopus egg extracts and cellular systems. Comparing the results with biochemical assays, we conclude that PhaseMetrics reliably detects changes induced by various conditions, including the presence of polyethylene glycol, 1,6-hexanediol, or a salt gradient, as well as the activity of the molecular chaperone DNAJB6b and the protein disaggregase Hsp104. Given the flexibility in its analysis parameters, the pipeline should also apply to other condensate-forming systems, and we show its application in detecting TDP-43 particles. By enabling the accurate representation of the variability within the population and the detection of subtle changes at the single-condensate level, the method complements conventional biochemical assays. Combined, PhaseMetrics is an easily accessible, customizable pipeline that enables imaging-based quantitative assessment of biomolecular condensates in vitro and in cells, providing a valuable addition to the current toolbox.