Diameter distributions of the islands of various FA-proteins in the MEFs, obtained by the tessellation analysis of the dSTORM images, the effect of elongating the spot merging distance in the dSTORM and PALM image processing on the paxillin island diameter distribution (MEF cells), and representative dSTORM image sequences of HMSiR-labeled Halo-paxillin on the basal PM of T24 cells (similar representative image sequences in MEFs are shown in Fig. 7 ). (A) Diameter distributions of various FA-protein islands. All FA-proteins were Halo-tagged at their N-termini and labeled with HMSiR. The arrowheads indicate the mean values. After the correction for single-molecule localization precisions of 19 nm for dSTORM images of HMSiR (Fig. S3), the true mean diameters of the islands were estimated to be 24–31 nm. (B) The effects of the merging distances (the threshold distances to identify the detected spots as those representing the same molecule) on the characteristics of the paxillin islands. We generally use $2∙3$ σ and $2∙2$ σ (σ = single-molecule localization error) for dSTORM (81 nm) and PALM images (82 nm), respectively (bottom row, which are reproduced from Fig. 5 D). In the bottom row, the results using the extended merging distances of $2∙5$ σ = 134 nm for dSTORM and 205 nm for PALM are shown. (C) Typical dSTORM image sequences of HMSiR-labeled Halo-paxillin on the basal PM of live (top panel) and fixed (bottom panel) T24 cells, shown every 10 s (data acquisition of 2,500 frames) (top rows) and those of the expanded square regions superimposed by the contours of FAs (white) and paxillin islands (green) determined by the Voronoï segmentation analysis (bottom rows). The results obtained in T24 cells are similar to those obtained by using MEFs (Fig. 7 A and B).