Extended Data Fig. 9 |. SmFRET-RAP data for S_f-mGluR2.

a, Relationship between the number of acceptor and donor particles (nParticles) recovered 2 – 3 minutes after photobleaching and the total-background corrected acceptor and donor fluorescence per cell area. The number of cells for each point is 8. b, Surface densities of donor and acceptor labeled receptors before bleaching for the smFRET-RAP experiments (the median density of total labeled (acceptor + donor) receptors was ~ 4.0 molecules/μm² (donor-to-acceptor ratio ~1:1)) compared to (c) those used for smFRET at lower expression levels reproduced from Extended Data Fig. 2b for comparison. Dots represent the number of acceptors (nAcc) and donors (nDon) per area for individual cells. Box plot details are described in the legend of Extended Data Fig. 2b. d, Representative acceptor and donor image sequence during smFRET with corresponding smFRET trajectory (red and green lines). Scale bar, 5 μm. Purple arrow at 10.64 s indicates a second donor particle that overlaps briefly with the particle showing smFRET. These occurrences do not influence the number of FRET events or their lifetime. e, Representative smFRET trajectory and fluorescence- and FRET-time traces derived from the image sequence in (d) where the donor and acceptor emission are anticorrelated upon acceptor photobleaching. f, Distribution of the duration of smFRET-RAP events comprised of 7,529 smFRET-RAP trajectories from 8 cells with single-exponential decay constant τ.