Extended Data Fig. 4: Single colour dSTORM imaging of the cell surface distribution of c-Met, S, and T, respectively, and dual-colour dSTORM imaging for nanodomain analysis of S and T on a same cell.

a, A549 cells were fixed and stained with different conditions: (i) with primary c-Met antibody followed by Alexa Fluor 647-labelled secondary antibody, (ii) with Alexa Fluor 532-labelled S, and (iii) with Alexa Fluor 647-labelled T. Scale bars represent 5 μm in the primary images and 1 μm in the zoomed-in images. b, The histogram showcases the frequency distribution of the different nanodomain sizes. Data is derived from 130 random nanodomains across 3 cells in three independent experiments. c, Schematic illustration of the dual-colour dSTORM imaging to visualize the nanodomains in the same cell labelled by S and T, both having comparable dissociation constants (shown in Supplementary Fig. 13 and 14). d, A549 cells underwent staining using Alexa Fluor 532-labelled S and Alexa Fluor 647-labelled T and subsequent fixation. The reconstructed dSTORM images for both Alexa Fluor 532-labelled S and Alexa Fluor 647-labelled T in a representative single cell were displayed. Scale bars indicate 5 μm in the original images and 1 μm in the magnified images. e, The graph depicts the average number of nanodomains per µm². f, The graph depicts the typical nanodomain areas. The data in e (n = 3) and f (n = 130) derive from 18 randomly chosen regions of interest (ROIs) with an area of 2 × 2 µm² from three cells in three independent experiments (a total of 130 nanodomains) and are presented as the mean ± S.D. There was no significant difference in the nanodomains of S and T captured in both channels, as determined by an unpaired two-tailed Student’s t-test (NS, not significant).

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