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.