<p>Embryonic stem cells expressing Cenp-A-mEos proteins were fixed and imaged. The corresponding movie (summed in <b>A</b>) was analysed with Peak Fit and the resulting list of localisations was separated between in vls (green) and out of vls (red) localisations using the vlsPALM thresholds defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125438#pone.0125438.g002" target="_blank">Fig 2C</a>. vlsPALM allows the identification of the in-focus localisations <b>(B)</b>. All localisations were plotted either as fitted <b>(C)</b> or in a super-resolved picture <b>(D)</b>, but coloured according to the vlsPALM filtering. Three categories of Cenp-A clusters were observed: some were almost entirely within the vls (<b>D-F</b>, 1); others were spanning one extremity of the vls, partly in the vls (<b>D-F</b>, 2); the last ones were entirely out of the vls (<b>D-F</b>, 3). <b>(E)</b> shows the diffraction-limited and super-resolved close-ups of the Cenp-A clusters defined in <b>(D)</b>. <b>(F)</b> displays the number of localisations in (green) and out of (red) the vls for each cluster. Such classification allows selecting in-focus clusters for further quantification and preventing under-counting due to undetectable out-of-focus emitters.</p
