Zeiss Lightsheet Z.1, Operating Manual

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CHAPTER 4 - SYSTEM OPERATION
Carl Zeiss Left Tool Area and Hardware Control Tools Lightsheet Z.1
116 000000-1790-528 02/2013
If the
discard overlapping molecules
was
selected from the Settings drop down menu, all
identified peaks with overlapping circles will be
indicated in red and discarded (Fig. 168). If
ignore
overlap
or
account for overlap
was selected,
overlapping peaks will not be indicated and all
peaks will be circled in white.
−
If
discard overlapping molecules
is
selected, all overlapping peaks, indicated by
red circles wil be discarded. This will result in
the highest accuracy, which is defined as the
deviation of the fitted to the real peak
position and localization precision (the
standard deviation from the fit) (Fig. 169,
A ).
This method is therefore the method of
choice for single emiters and little overlap.
−
If ignore overlap is selected, all single and
overlapping peaks wil be taken into account.
However, fit accuracy will be decreased
when multi-emitter events are fitted as the
distribution of one emitter wil influence the
distribution of an overlapping one without
this fact taken into account. Hence accuracy
might suffer for multi-emitter events wheras
localization precision will be sustained.
(Fig. 169,
B
). This method is most suitable
for slightly overlapping molecules.
−
If
account for overlap
is selected all single
and multi-emitter events wil be taken into
account. For multi-emitters the overlap of
neighboring molecules is taken into account
so losses in accuracy are minimized. There
wil be a slight decrease in the localization
precision (Fig. 169,
C ). This method should
be used, when molecules heavily overlap.
Fig. 168 Preview window of the image
container for PALM processing with the
discard molecules (A), ignore overlap
(B) and account for overlap (C) selected
Fig. 169 Analyzed data set of Fig. 168 using discard overlapping molecules (A), ignore
overlap (B) and account for overlap (C).