Fig. 1: Large-scale single-molecule imaging analysis of fluorescently labeled EGFR for drug screening.
a Schematic flow of single-molecule imaging using AiSIS for drug screening. b Optics of the autofocus device. Slit images, which reflect deviation from the in-focus position of the objective lens, are generated with switching illumination and used for feedback control. c Automated single-molecule tracking. Left, representative images of EGFR-mEGFP observed with AiSIS. Scale bar, 5 μm. Middle left, time series images of the squared region in the left panel. Scale bar, 2 μm. Representative images obtained by single-molecule imaging are displayed. Middle right, trajectories of EGFR (>8 frames). Right, time series of step length (top) and brightness (bottom) of a spot in the left panels. d Heatmaps of diffusion coefficients and brightness measured before and 5 min after the EGF addition. The average distributions over the cells are shown at the bottom. Black and red lines with shaded areas indicate the average with SD before and after EGF addition, respectively. e Time development of MSDs (mean square displacements) for either untreated or gefitinib-treated (10 μM for 1 hour) cells without (black) and with EGF (300 nM, red). The acquisition of single-molecule images began 1 min after the EGF addition. Data are from 60 cells for each condition (Supplementary Table 3). f Relative changes in MSD500ms and fold changes of phosphorylation obtained from western blotting (bottom) against different EGF concentrations. Single-molecule imaging and the phosphorylation assay began 1 and 2 min after the EGF addition, respectively. Data of MSD and phosphorylation are from 140 cells and 7 independent experiments, respectively, for each EGF concentration. M indicates molecular weight marker. g Dose-dependency of gefitinib on MSD. Top, MSD versus gefitinib concentration. Bottom, ratios between MSD with and without EGF. Colored curves correspond to the indicated EGF concentrations. Single-molecule imaging began 1 min after the EGF addition. Data are from 33 cells for each concentration. Shaded areas (d, e, and g) and error bars (f) indicate the SD and SE around the mean, respectively. Source data are provided as a Source Data file.
