Zeiss Lightsheet Z.1 Operating Manual

Contents

CHAPTER 4 - SYSTEM OPERATIONCarl Zeiss Left Tool Area and Hardware Control Tools Lightsheet Z.1116 000000-1790-528 02/2013If the discard overlapping molecules wasselected from the Settings drop down menu, allidentified peaks with overlapping circles will beindicated in red and discarded (Fig. 168). If ignoreoverlap or account for overlap was selected,overlapping peaks will not be indicated and allpeaks will be circled in white.− If discard overlapping molecules isselected, all overlapping peaks, indicated byred circles wil be discarded. This will result inthe highest accuracy, which is defined as thedeviation of the fitted to the real peakposition and localization precision (thestandard deviation from the fit) (Fig. 169, A).This method is therefore the method ofchoice for single emiters and little overlap.− If ignore overlap is selected, all single andoverlapping peaks wil be taken into account.However, fit accuracy will be decreasedwhen multi-emitter events are fitted as thedistribution of one emitter wil influence thedistribution of an overlapping one withoutthis fact taken into account. Hence accuracymight suffer for multi-emitter events wheraslocalization precision will be sustained.(Fig. 169, B). This method is most suitablefor slightly overlapping molecules.− If account for overlap is selected all singleand multi-emitter events wil be taken intoaccount. For multi-emitters the overlap ofneighboring molecules is taken into accountso losses in accuracy are minimized. Therewil be a slight decrease in the localizationprecision (Fig. 169, C). This method shouldbe used, when molecules heavily overlap.Fig. 168 Preview window of the imagecontainer for PALM processing with thediscard molecules (A), ignore overlap(B) and account for overlap (C) selectedFig. 169 Analyzed data set of Fig. 168 using discard overlapping molecules (A), ignoreoverlap (B) and account for overlap (C).