Single-molecule FRET (smFRET) measurements of 80 nM (ATTO532)-hp-RNA35-(ATTO647N) RNA hairpin in G3BP1:polyA RNA. Top: Representative total fluorescence intensity (right) and corresponding fluorescence lifetime image (FLIM) (left) image color coded with the donor intensity averaged fluorescence lifetime. Bottom: Contour plots of the relative donor lifetime τD(A)/τD(0)versus FRET efficiencyEof single-RNA-hairpin molecules outside (left, position ×, blue, in top image) and inside the condensates (right, position +, orange, in top image). ProjectedE-histograms shown on top. For contour plots and histograms, 10 confocal scanning images with 10 positions inside and outside condensates were recorded and pooled, respectively. Scale bar, 10 μm.
