Single fluorogen imaging reveals spatial inhomogeneities within biomolecular condensates.

Recent investigations have suggested that biomolecular condensates are viscoelastic materials. This implies that material properties of condensates are governed by internal microstructures. Furthermore, computations show that the internal organization in protein condensates is spatially inhomogeneous, featuring hub-and-spoke-like percolated networks of molecules. Here, we test these predictions using imaging of single fluorogenic dyes that are turned-on in response to specific chemical microenvironments. We deployed Nile blue (NB), Nile red (NR), and merocyanine 540 (MC540) for epifluorescence and single-molecule localization microscopy imaging of condensates formed by intrinsically disordered, low-complexity domains of proteins. Imaging with NB reveals internal environments that are uniformly hydrophobic, whereas NR shows preferential binding to hubs that are more hydrophobic than the surrounding background within condensates. Finally, imaging with MC540 suggests that interfaces of condensates are unique chemical environments. Overall, the high spatiotemporal resolution and environmental sensitivity of single-fluorogen imaging reveals spatially inhomogeneous organization of molecules within condensates.