Zeiss LSM 880, Operating Manual

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CHAPTER 1 - SYSTEM OPERATION
LSM 880 Left Tool Area and Hardware Control Tools ZEISS
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−
Account for overlap
(multi-emitter): multi-
emitter events will be fitted taking into
account the overlap. If selected, the
Maximum cluster size slider and input box
become available (Fig. 443). Here you can
select the maximum number of molecules in
the cluster that are used for computation. So
a number of 10 means, for example, that
clusters with 10 or less peaks will be taken,
and thoise exceeding 10 will be discarded.
We recommend to limit custer size to 10 as
otherwise computation can become time
consuming.
White circles indicate that the particular emitters
are used (if channel color is grey; otherwise the
channel color is used), red circles that the emitters
are discarded for the fitting process. For account of
overlap the color stays white or in the channel
color regardless if a cluster may be discarded due
to the
Maximum cluster size setting.
The Dimension drop down menu will display the
dimensionality (2D or 3D) of the selected PALM
image, which is automatically recognized if a .czi
format is loaded (Fig. 444). It is possible to overrule
the automatically chosen value.
In the
Channel drop down menu you can select
the channel for which the Processing should be
conducted for a multi-channel image.
Expand the
Peak Finder field to have access to the
peak selection filters (Fig. 445). Two filters are
available:
−
Peak mask size
−
Peak intensity to noise
Use the sliders or input spin boxes to set the
corresponding values.
We recommend to start with the default
values for the peak mask size (9 for 2D; 19
for 3D data) and then optimize on behalf of
the results. Note that in 3D-PALM using
PRLIM (phase ramp localization imaging
microscopy) with a double phase ramp, the
PSF is split into two lobes (
Fig. 446
) that have
a larger expansion than the classical PSF.
F
ig. 443 Maximum cluster size field
Fig. 444 Dimension drop down menu
Fig. 445 Peak Finder Expansion field
Fig. 446 Double lobed PSF created
by a double phase-ramp in
the pupil plane