Fig. 6: A schematic illustration of homoFRET imaging to study modulation in internal organization and molecular packing within phase-separated assemblies formed in vitro and in situ.

Phase separation into biomolecular condensates results in energy migration via homoFRET as evident from the loss in steady-state anisotropy within droplets. The effect of phase separation modulators such as RNA, ATP, and post-translation methylation is readily captured by our homoFRET measurements which reveal the formation of a denser protein network within heterotypic protein-RNA condensates leading to further reduction in anisotropy values. ATP and methylation result in a less dense droplet interior and hence decreased energy migration as indicated by the rise in the steady-state anisotropy. Anisotropy imaging within cells detects the differential molecular packing and intermolecular proximities within various nuclear and cytoplasmic assemblies.