Direct observation and quantification of single nanocondensates of the low complexity domain of TDP-43

Biomolecular condensates, formed by liquid–liquid phase separation, coordinate key cellular activities. Recent work has revealed the role of sub-micron assemblies, or nanocondensates, in the organisation of a significant portion of the proteome. Here, we introduce a single particle fluorescence spectroscopy framework to visualise and quantify individual nanocondensates in real time. Using the low-complexity domain of TAR DNA-binding protein 43 (TDP-43) as a model system, we show that this approach recapitulates the protein’s phase separation diagram across diverse conditions and reveals the rapid formation of TDP-43 nanoclusters at ten-fold lower concentrations than previously described. Fingerprinting of individual events provides quantitative measurements of size, density, and temporal evolution, while two-colours experiments capture dynamic exchange, coalescence and maturation into ThT-positive, amyloid-containing aggregates. Our results establish single particle detection as a quantitative tool for probing condensation formation, early liquid-liquid phase separation events and phase transition mechanisms in protein systems. Houx and colleagues introduce a single-particle fluorescence spectroscopy method that detects and quantifies nanoscale condensates, revealing their rapid formation, dynamic behaviour, coalescence and selective maturation into amyloid-like aggregates.