Zeiss LSM 880, Operating Manual

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CHAPTER 1 - SYSTEM OPERATION
ZEISS Left Tool Area and Hardware Control Tools LSM 880
336 000000-2071-464 10/2014 V_01
If the
Average before Localization
check box
remains inactive, every peak (event) will be
localized independently and is regarded as being
derived from one molecule. Later on, peaks can be
grouped belonging to one molecule in the image
container in the
PAL-Grouping
tab of the image
( localization before grouping ). If the check box
is ticked, however, the system will group peaks
(events) based on the filter settings that become
available (Fig. 456) before localizing the grouped
peaks (
grouping before localization ).
When localization before grouping is performed, the single peaks are first localized and then the
localizations averaged. When
grouping before localization
is performed, than the peaks are first
averaged and then localized.
There will be two filter settings when one performs
grouping before localization:
−
Filters for
Drift Correction
based on fiducial
markers (like gold particles).
−
Filters for Grouping the peaks (events).
Expand the Drift Correction field to have access
to the Drift Correction tool (Fig. 457). If you want
to perform a drift correction the Drift Correction
check box must be ticked.
Note that for lateral drift correction the system needs to identify at least one fiducial marker based
on the
Drift Correction
filter settings. Fiducial markers could be gold particles deposited on the
glass surface of the dish. If the system cannot find a fiducial, no drift correction will be performed
and in the
PAL-Drift
tab of the image no fiducials will be listed in the
Fiducial
table.
You can set two parameters to be associated with a fiducial by using the sliders or the spin input boxes:
−
Min on time
[
%]. This defines the minimal number of frames out of the total frames acquired in
percentage the molecule has to be on in order to be regarded as a fiducial. E. g. 75 % means that
the molecule has to be on at least ¾ of all the frames.
−
Capture radius [pixel]. This defines the radius around a fiducial in which no other fiducial shoud
be localized in order to be taken as a fiducial. E. g. if 2 pixels was set and within two pixels another
fiducial is present, than both fiducials will be discarded.
Note that the settings of the Drift Correction will not influence the Preview image.
Note that using
Drift Correction
in the Processing tool is irreversible. Therefore, in the created
image all drift related functions in the PAL-Drift tab will be inaccessible and greyed out.
Fig. 456 Localizer Expansion field with
Average before localization
selected
Fig. 457 Drift correction panel