Single-molecule microscopy of biomolecular condensates: From liquid droplets to heterogeneous networks

Single-molecule microscopy has transformed our view of biomolecular condensates―membraneless organelles that organize cellular biochemistry and are frequently dysregulated in disease―revealing them not as simple liquid droplets, but as spatially heterogeneous and percolated networks that can undergo time-dependent physical aging and gelation. Here, we summarize how single-particle tracking, single-molecule-fluorescence resonance energy transfer and super-resolution microscopy resolve molecular motion, confinement, and conformational dynamics to link nanoscale behaviors to mesoscale condensate material properties and biological function. In vitro reconstitution affords mechanistic control, whereas emerging live-cell imaging probes physiological context. Photobleaching, phototoxicity, and autofluorescence remain challenges that are increasingly mitigated by optimized fluorophore and label-free approaches. Concurrently, deep-learning pipelines automate analysis and expose hidden heterogeneities. Further integrating artificial intelligence and imaging advances will be essential for decoding condensate structure–function relationships in health and disease.