Figure 1: FLImP measurement of pairwise EGF separations.

(a) Cartoon of an EGFR monomer, a two-ligand active dimer, and an EGFR sequence diagram. (b) Steps to determine EGF separations using FLImP¹⁵: (1) TIRF images are collected from intact cells; (2) spots from individual complexes are tracked to derive intensity time courses; and (3) a spot image of a complex containing two fluorophore-conjugated EGF ligands (red dots) features two intensity levels and decays to zero in two bleaching steps; when one fluorophore bleaches, the centroid position shifts. If more than two steps occur, the lowest two are analysed. (4) A global least-squares seven-parameter-fit is used to identify the best intensity, x-y positions and the full-width at half-maximum of the point spread function for each fluorophore, from which their separation is calculated with a precision determined by the localization error; (5) Example systems of a two-ligand dimer and tetramer, a three-ligand tetramer, and a mixture of a dimer and a tetramer. (6) The empirical posterior distributions (or FLImP measurement) of pairwise ligand separations obtained for each example system with their 69% confidence intervals highlighted. The size of the latter depends on the combined localization errors of the two molecules¹⁵. FLImP measurements with confidence intervals smaller than the required resolution are retained in a histogram, generating a so-called FLImP distribution that is fitted by the sum of a discrete number of Rician peaks (Supplementary Fig. 3a). (c) FLImP distribution (grey) of CF640R fluorophore-conjugated EGF on CHO cells (∼10⁵ copies of wild-type EGFR per cell) treated with 4 nM EGF at 4 °C with chemical fixation, compiled from 30 FLImP measurements with confidence intervals <4.8 nm. The distribution is decomposed into a sum of six Rician peaks. Positions and error estimates are shown in the inset. (Details in Supplementary Methods.) The peak positions (and error bars) reflecting the expected dimers and tetramers are marked above the plot. The optimal number of peak components (colour lines) and the best-fit (black line) were determined using a Bayesian information criterion and Bayesian parameter estimation (Supplementary Figs 3b and 4a, and Supplementary Methods).