Fig. 4: Live-cell topological mapping and 3D single-molecule tracking by MAxSIM.

a, b Live-cell 3D basal surface morphology mapping of SKBR3 cells at 1.9 s per topology map, equivalent to 0.52 Hz. The incident angle range was (19°, 35°) with 0.5° step size, and a 50-ms exposure time was used (Supplementary Video 1). The images show 3D topology, zoomed-in views of specific regions marked by white boxes, and 2D NELD images at time 0 a and 20.9 s b. c A 3D topology snapshot of single-molecule tracking movie of αHER2 Fab’:QD605 conjugate-labeled HER2 and WGA-555 in the basal cell surface of live SKBR3 cells, while both chromophores were simultaneously excited at 560 nm. Single-molecule tracking was obtained from all 68 frames used for angle scanning, which was required to reconstruct one height map. The incident angle range was (19°, 53°) with 0.5° step size, and 50-ms exposure time was used (Supplementary Video 2). Summed trajectories of HER2 single molecules are shown in black lines. d A snapshot 3D topology PM map (at t = 0 s; Supplementary Video 3) using WGA-555 staining overlaid with summed trajectories of HER2 single molecules (black lines). A median filter with kernel size of 2 was applied to reduce noise a–d. Scale bars = 10 µm a–d.