.(a) Left column (a, d, g, j): schematic of different defined mixtures of two labeled proteins, in which one protein is labeled with JFX650 only and the other is labeled with both JFX650 and JF549. In (g) and (j), each subunit of the 60-mer is fused to SNAPf or Halo-SNAPf, though only one label is displayed for clarity. (b) Center column (b, e, h, k): inferred diffusion spectra of PAPA (green-reactivated) and direct reactivation (DR) (violet-reactivated) trajectories pooled from 20 cells (b, e, k) or 10 cells (h). (c) Right column: fraction bound (c, f) or fraction slow-diffusing (i, l) of PAPA and DR trajectories from individual cells, obtained from fits to a two-state model (c, f) or three-state model (i, l). Paired, two-tailed t-tests of the comparisons in (c), (f), (i), and (l) showed all differences to be statistically significant with p=9 × 10–9, 8 × 10–8, 1 × 10–5, and 4 × 10–11, respectively.