Determining f and 2D-K_D. (A) P_Dspot^T+A plotted as a function of NDDS (green; for each point, n_cell = 3, examining a total of ∼500 spots). Black keys and the line, P_Dspot^A (simulation). (B) P_Dspot^T (equal to P_Dspot^T+A − P_Dspot^A) plotted as a function of NDDS. (C) 2[D], the number density of molecules in true dimers, plotted as a function of the number density of expressed molecules (ND_mol), and fitted with Eq. 5 (red curve). The error bars are greater for samples at higher number densities (particularly the points around 2.3–2.5 copies/µm²) because of the crowding of the fluorescent spots in the image. To address this point, we fitted the data, assuming that the largest or the smallest values given by the error bar for the highest number density point are correct. The obtained 2D-K_D was 5.6 and 2.8 copies/µm², respectively, whereas the present estimate is 3.6 ± 0.58 copies/µm².