a, Temporal molecular brightness measurements, normalized to oligomeric state, for corresponding C-terminally tagged YFP constructs confirm that the β1-AR (normalized brightness= 0.965 ± 0.054, n = 47) and µOR (normalized brightness= 1.087 ± 0.051, n = 55) are largely monomeric. In contrast, CD28 results in an almost perfectly doubled brightness (brightness = 1.929 ± 0.111, n = 19). These experiments show that dimerization is not influenced by the N-terminal SNAP-tag used for single molecule studies. b, SpIDA brightness measurements with SNAP-tagged receptor constructs show that results also agree when SNAP-constructs are used in brightness measurements (normalized brightness values: SNAP-β1AR = 0.949 ± 0.118, n = 10; SNAP-µOR = 0.975 ± 0.081, n = 19; SNAP-µOR + 10 µM morphine = 0.987 ± 0.067, n = 16; SNAP-µOR + 10 µM DAMGO = 1.843 ± 0.121, n = 12; µOR (T279K) mutant 1.755 ± 0.076, n = 20; SNAP-CD28 = 2.1 ± 0.109, n = 19). Receptor expression levels used in these experiments are ≈ 40 receptors per µm2. Brightness values in are mean ± SEM and n = number of cells. The box shows IQR and whiskers are SD. P values were determined by one-way ANOVA and Tukey’s multiple comparison test. c, Acceptor photobleaching experiments on a confocal microscope with dual-color labeled SNAP-constructs also confirm the monomeric nature of β1AR and the µOR by linearly increasing FRET-efficiencies at high expression levels. In contrast, the dimer control SNAP-CD28 shows a steep increase and quick saturation of the measured FRET-efficiencies. d, The inactive T279D mutant of the µOR does not increase significantly the FRET- efficiency between receptor protomers. In contrast the T279K mutant shows significantly higher FRET-efficiencies over increasing expression. Due to the constitutive activity of this mutant, expression levels higher than depicted could not be achieved. Curves for SNAP-β1AR (blue) and SNAP-µOR-wt (black) are taken from panel c. Graphs show pooled experiments repeated independently n = 3 times on 3 different experimental days. Each datapoint represents a value from one individual cell.