Single FM imaging of WT-mGFP and D71A-mGFP before and after the addition of the nonfluorescent ligand FP, for comparing liganded D71A with unliganded WT. (A) A schematic drawing of WT-mGFP and D71A-mGFP. The ligand was not labeled in the experiments shown here. (B) A representative TIRF single-molecule image of WT-mGFP expressed on the bottom cell membrane of a live CHO cell (no ligand). Yellow arrowheads and red arrows indicate spots with intensities < and >18 AU, respectively (see C and the legend for Fig. 2 A). (C) The distributions of the fluorescence signal intensities of individual spots. They were fitted with the sum of two Gaussian functions: the best-fit functions for the top two boxes became single Gaussian functions, whereas those for the bottom four boxes were the sum of two Gaussian functions. First and second panels, Monomer-reference molecules of mGFP. First box, mGFP expressed in and purified from E. coli, and nonspecifically adsorbed on coverslips. Second panel, mGFP-Gβ1, a subunit of trimeric G-protein, expressed in the plasma membrane of CHO cells. Note that the distributions of these two specimens are very similar to each other. Third and fourth panels, WT-mGFP expressed on the cell surface before and after the addition of 100 nM FP ligand (nonlabeled), respectively. The spots with signal intensities >40 AU probably represent molecules assembled in the internalization apparatuses. Fifth and sixth panels, D71A-mGFP expressed on the cell surface before and after the addition of 100 nM FP ligand, respectively.