Zeiss LSM 880, Operating Manual

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CHAPTER 1 - SYSTEM OPERATION
LSM 880 Left Tool Area and Hardware Control Tools ZEISS
10/2014 V_01 000000-2071-464 369
5.4.19.2.2.1 Landmark Alignment
For the
Landmark Alignment
(Fig. 522), the
sample must have been embedded with
appropriate fluorescent beads (fiducials) which can
be recognized by the software. The beads can be
present in the same channel as the image
information. Another option is to image the beads
alone in a second channel.
Select the Channel in which the beads are present
by clicking the according
Registration Channel
button. One can select more than one channel for
the registration process. For each chosen channel
and individual registration will be performed; it is
therefore advisable to use one channel for the final
registration. To test which channel will lead to the
best results, all channels can be chosen for later
comparison of the results.
To define which structures within the images are
the beads to be used for the Landmark Alignment,
use the sliders and input boxes with arrows of
Threshold: brightness of beads,
Size: size of the beads, and
Size Variation:
how much do the beads differ in
size.
All values with these parameters are arbitrary units.
To check if the settings recognize the beads of the image, press the
Preview button. This will circle all
elements recognized as beads in the chosen and displayed image frame. Use the sliders in the
Dimensions View control to change to a different view, z-slice, and time point and press again the
Preview
button. Control by using several different frames to see if the beads are recognized correctly
before proceeding. Adjust the settings for Threshold , Size , and Size Variation until this is
accomplished.
If your data was acquired with dual side illumination which has not yet been fused, the method to fuse
both illumination sides is available in the
Dual Side Fusion drop-down menu ( Mean Fusion , DFT
Fusion
,
Maximum Fusion
).
Mean Fusion : The average intensity for each pixel is calculated using the values of both illumination
sides.
DFT Fusion
(Discrete Fourier Transform Fusion): The Fusion is performed based upon the published
method ("Multiple imaging axis microscopy improves resolution for thick-sample applications", Jim
Swoger, Jan Huisken, and Ernst H. K. Stelzer, OPTICS LETTERS / Vol. 28, No. 18 / September 15, 2003).
Fine structures and details are more visible after the fusion, which might be worsened when using Mean
Fusion. However, this process can create noticeable artifacts in the resulting image.
Maximum Fusion : The intensity of each pixel of both illumination images is compared and the value
with the higher intensity is used for the resulting image.
Fig. 522 Processing – Lightsheet Processing.
Multiview Processing, Landmark
Alignment Parameters