Zeiss LSM 880, Operating Manual, Page: 339 / 690

CHAPTER 1 - SYSTEM OPERATION
LSM 880 Left Tool Area and Hardware Control Tools ZEISS
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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. 449). 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 will 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. 450,
A
).
This method is therefore the method of
choice for single emitters and little overlap.
−
If ignore overlap is selected, all single and
overlapping peaks will be taken into
account. However, fit accuracy will be
decreased when multi-emitter events are
fitted as the distribution of one emitter will
influence the distribution of an overlapping
one without this fact taken into account.
Hence accuracy might suffer for multi-
emitter events whereas localization precision
will be sustained. (Fig. 450,
B ). This method
is most suitable for slightly overlapping
molecules.
−
If account for overlap is selected all single
and multi-emitter events will be taken into
account. For multi-emitters the overlap of
neighboring molecules is taken into account
so losses in accuracy are minimized. There
will be a slight decrease in the localization
precision (Fig. 450,
C
). This method should
be used, when molecules heavily overlap.
Fig. 449 Preview window of the image
container for PALM processing with
the discard molecules (A), ignore
overlap (B) and account for overlap
(C) selected
Fig. 450 Analyzed data set of Fig. 449 using discard overlapping molecules (A),
ignore overlap (B) and account for overlap (C).