Islands of various FA proteins and their loose clusters of ≈320 nm in diameter. (A) Typical overlaid PALM and dSTORM images simultaneously obtained in a live MEF for mEos3.2-paxillin and five other HMSiR-labeled Halo-linked FA molecules (data acquisition at 1 kHz for 10 s). (B) The contours of the FA and the islands of paxillin and other FA proteins were identified by the Voronoï segmentation analysis of the images in the regions enclosed within squares shown in A (expanded and overlaid on the images). (C) Autocorrelation functions obtained from the dSTORM data for the FA proteins (calculated for all of the fluorescent spots localized in the detected islands; see the legend to Fig. 5 F) with their best-fit functions. Integrins β3 and β1 did not exhibit the longer autocorrelation component, whereas they exhibited short-range auto-correlation lengths (2×ξ1 values of 70 and 66 nm, respectively) slightly greater than those for talin, FAK, vinculin, and paxillin (2×ξ1 values of 40∼46 nm). However, these slightly greater 2×ξ1 values are likely spurious, as the mean diameters of the islands enriched in integrins β3 and β1, after localization error correction, are 57 nm in the island diameter distributions (Fig. S4 A), consistent with those of paxillin, talin, FAK, and vinculin (48–62 nm; Fig. S4 A). Therefore, we conclude that the longer short-range autocorrelation lengths of integrins β3 and β1 (2×ξ1 values of 70 and 66 nm, respectively) are probably due to the slight mixing of the longer correlation component, which could not be resolved. (D) Crosscorrelation functions between the dSTORM spots of Halo-linked FA proteins and the PALM spots of mEos3.2-paxillin in the detected islands (see the legend to Fig. 5 G).
