Ultrafast PALM is capable of imaging a view field as large as 640 × 640 pixels (35.3 × 35.3 µm ² ), often encompassing almost an entire live cell, at a data acquisition rate of 1 kHz using mEos3.2 (linked to paxillin; a total of 10,000 frames obtained for 10 s), while at the level of a single FA, it reveals an archipelago architecture of paxillin-enriched islands with a 33 nm mean diameter. (A and B) Typical reconstructed PALM (A) and diffraction-limited (B) images of mEos3.2-paxillin on ≈ 2/3 of the entire basal PM of a T24 cell (data acquisition for a view field of 640 × 640 pixels ≈35.3 × 35.3 µm²). PALM image: 3,526 × 3,526 pixels with a pixel size of 10 nm. Diffraction-limited image: 640 × 640 pixels with a pixel size of 55.1 nm. The yellow contours outline the cell peripheries determined in the diffraction-limited image on the right, using Sauvola’s local thresholding method with a local domain radius of 64 pixels, and k and r values of 0.5 and 128, respectively (Sauvola and Pietikäinen, 2000). Throughout this study, the reconstructions of PALM images with a pixel size of 10 nm were performed using the ThunderSTORM plugin for ImageJ (Ovesny et al., 2014) installed in the Fiji package (Schindelin et al., 2012) and Gaussian rendering with a localization precision of 29 nm (Fig. 2 A-d), whereas diffraction-limited images with a pixel size of 55.1 nm were generated by Gaussian rendering with a spot with an SD of 129 nm (Fig. 2 B). (C and D) Enlarged images of the domains enclosed in squares in A and B, respectively. The contours of the FAs in the PALM image shown in red (C) were determined by using the SR-Tesseler software based on Voronoï polygons (Levet et al., 2015), with a thresholding paxillin number density of 1.45. This contour is overlaid on the diffraction-limited image on the right (D). The contours of the FAs determined from the diffraction-limited image using the minimum cross entropy thresholding are shown in cyan (D). Comparison of the two FA contours indicates that the contour determined from the PALM image is slightly smaller than that determined from the diffraction-limited image and is more sensitive to much smaller FAs (or paxillin clusters outside larger FAs). (E) Further enlarged image from the squared domain in the PALM image in C. (F) The Voronoï polygon diagram of the PALM image in E, showing the contours of the FAs (red) and paxillin-enriched islands (dark green), using a thresholding paxillin number density of 1.45 for both contours. (G) Distribution of the paxillin-enriched island diameters obtained by the Voronoï tessellation analysis using the SR-Tesseler software. The mean diameter of the islands determined here was 59 nm, but after the correction for the effect of the 29-nm single mEos3.2-molecule localization precision on the SR-Tesseler segmentation (Fig. S2 C), the actual mean diameter of the islands was estimated to be 33 nm. (H) Distribution of the number of detections (localizations) of mEos3.2-paxillin molecules per detected paxillin island. The additional x-axis scale shows the estimated total (= mEos3.2-conjugated + endogenous) paxillin copy number per island. Although these values obtained in G and H (the mean paxillin island diameter of 33 nm containing a median of 36 copies of paxillin/island) would be quite informative, they need to be interpreted with caution. First, larger islands might be collections of smaller islands that could not be resolved. Second, the islands containing many other FA proteins, but only <6 paxillin copies, would be missed by definition. Third, paxillin-enriched islands with smaller diameters would be missed even though their diameters are ≥13 nm: This was evaluated by Monte Carlo simulations, which showed that ∼15, 29, and 82% of paxillin-enriched islands with diameters of 13, 20, and 30 nm, respectively, were detectable (when the protein density for the simulation was increased by a factor of 2 to 0.04 copies/nm², ≈30 and 57% of the 13- and 20-nm islands, respectively, were detectable). Approximately, 85–95% of the islands with ≥40 nm diameters were detectable (the localization precisions of 19 and 29 nm hardly affected the result). Taking these limitations into account, based on the data shown in G and H, we propose that the paxillin island diameters are mostly in the range of 13∼100 nm and that the paxillin nanoclusters containing <6 paxillin copies might either exist alone or be located in the islands enriched in other FA proteins. Furthermore, smaller FA-protein islands might contain a total of ≥6 copies of various FA proteins, but the copy number of each constituent protein species might be <6.