Quantification of nanocondensates formation at the single molecule level

Understanding the molecular mechanisms of biomolecular condensate formation through liquid-liquid phase separation is crucial for deciphering cellular cues in normal and pathological contexts. Recent studies have highlighted the existence of sub-micron assemblies, known as nanocondensates or mesoscopic clusters, in the organization of a significant portion of the proteome. However, as smaller condensates are invisible to classical microscopy, new tools must be developed to quantify their numbers and properties. Here, we establish a simple analysis framework using single molecule fluorescence spectroscopy to quantify the formation of nanocondensates diffusing in solution. We used the low-complexity domain of TAR DNA-binding protein 43 (TDP-43) as a model system to show that we can recapitulate the phase separation diagram of the protein in various conditions. Single molecule spectroscopy reveals rapid formation of TDP-43 nanoclusters at ten-fold lower concentrations than described previously by microscopy.