<p>(a) SpoIIIE is observed during all stages of the cell cycle. Individual cells were recognized and classified as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001557#pbio-1001557-g001" target="_blank">Figure 1f</a>. Pixel size was 110 nm. From each 55 ms image, we automatically determined the localization of each single molecule in the image by using MTT <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001557#pbio.1001557-Serge1" target="_blank">[57]</a>. Each of these localizations is called a single-molecule event. In our pointilist representation, each single-molecule event is represented by a single green dot, whereas membrane stain is shown in white (see SM10 in Methods S1 for more details). Cells without septum were classified as stage 1 (vegetative/pre-divisional, left panel). Cells having a symmetric division septum were classified as stage 2 (division, middle panel), whereas those showing an asymmetric septum (at 1/5<sup>th</sup> or 4/5<sup>th</sup> of the total cell length) were classified as stage 3 (sporulating, right panel). (b) SpoIIIE clusters were automatically detected and classified depending on their size and composition. FWHM, full width at half maximum. (c) Analysis of the cluster size distribution versus the number of single-molecule events shows two distinct cluster types: PALM-limited clusters (red dots) have a size equal or smaller (∼45 nm FWHM) than the resolution of PALM in our conditions and contain a large number of events (>1,000), whilst dynamic clusters (orange dots) are large (>100 nm FWHM) and contain fewer events (<1,000). (d) The size of PALM-limited clusters is independent of cell cycle stage and the most typical size is ∼45 nm FWHM. (e) PALM-limited cluster sizes as a function of imaging time (total time used to image each single cluster) show that these clusters are extremely stable.</p
