Zeiss LSM 880, Руководство по эксплуатации

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CHAPTER 1 - SYSTEM OPERATION
LSM 880 Left Tool Area and Hardware Control Tools ZEISS
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Expand the
Localizer
field to have access to the fit
model used to localize peaks (Fig. 452). Three fit
models are available that can be selected by the
Fit
Model
drop down menu (Fig. 453) if 2D is
selected from the Dimension drop down menu:
−
x,y 2D Gauss Fit
−
Mask fit
−
File PSF
If 3D is selected from the Dimension drop down
menu, only
File PSF
is available. You should load
an Experimenatl PSF with linked look up table for
localization precision in order to obtain localization
precisions in z in addition to x and y.
If
x,y 2D Gauss Fit
is selected, the intensity
distributions of the identified peaks are fitted to a
full 2D Gaussian function. The fit in each direction
takes into account the influence of the other
direction (x,y 2D Gauss).
If
Mask Fit is selected, a Gaussian mask with a
defined PSF width is used to match as best as
possible the intensity distribution of a peak in x
and y (Fig. 454). The PSF width can be set via the
PSF Width slider or input spin box. Default value is
the theoretical calculated PSF based on the
objective lens and laser line used.
Note that with the Mask Fit only one
predefined PSF width will be used.
Computation of the Fit will be faster for the
Mask Fit , but less precise.
If
File PSF
is selected, you can select a PSF that will be used (Fig. 455). To this end press the
PSF file
button. This will open Windows Explorer, where you can select an approriate file, the name of which will
be displayed in the
PSF file
display box.
The fit model in 2D defines the PSF width, which will be used to fit to the experimental results. It
does not correspond to the size of the peak that is defined by the Peak Mask Size and that in the
end will define the overlapping degree of neighboring peaks. In 3D the PSF with the attached look
up table is a simulation where the PSF was modulated.
The settings of the
Localizer
will not influence the peak finding process and hence the preview
image.
In the Processing
PALM
function, peak finding and localization are always linked. If you want to
just view localized peaks (molecules), you might select in the PAL-Render tab of the PALM image
in the
Display
drop down menu
Centroids
.
Fig. 452 Localizer Expansion field with x,y
2D Gauss Fit selected
Fig. 453 Fit Model drop down menu
Fig. 454 Localizer Expansion field with Mask
Fit selected
Fig. 455 File PSF menu