Zeiss LSM 880, Operating Manual

Page: 378 / 690

CHAPTER 1 - SYSTEM OPERATION
ZEISS Left Tool Area and Hardware Control Tools LSM 880
372 000000-2071-464 10/2014 V_01
5.4.19.2.2.2 Intensity Based Alignment
The
Intensity Based Alignment
option does not
need any fluorescent beads (fiducials) present in
the sample since registration is done based on
structural information within the image content. It
is therefore advisable to select the
Registration
Channel which contains clearly defined
fluorescent structures within the image. One can
chose more than one channel for registration. 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.
Registration can be done as a two-step process
with a preceding
Pre-Registration and afterwards
the Registration (Fig. 525).
The Pre-Registration performs the process in a
2D maximum intensity projection of the Z-Stacks;
while the
Registration
algorithm uses the
complete 3D information of the Z-Stacks.
It is advisable to perform first a
Pre-Registration
to move similar structures already into close
proximity, which might facilitate the Registration .
You can select the following
Pre-Registration Method
s from the drop down menu:
None
: No Pre-Registration is performed. Registration is then only done with the settings chosen in the
Registration Method menu.
Hardware Rotation : The Views are rotated solely based on the information from the rotation
positioning of the sample holder which are saved in the metadata.
Trans Hardware Rotation : The Views are rotated solely based on the information from the rotation
positioning of the sample holder which are saved in the metadata. Additionally the x, y, and z axis are
moved to match the Views.
Correlation Hardware Rotation : The Views are rotated solely based on the information from the
rotation positioning of the sample holder which are saved in the metadata. Additionally the x, y, and z
axis are moved to match the Views. Cross-correlation is used to determine the translation in x, y, and z.
To select the
Registration Method , use the drop-down menu.
Use Pre Register Only : No Registration process is performed; only the result of the Pre-Registration is
used.
Translation : The registration will correct along the x, y, and z axis only.
Trans Rotation : The registration will correct along the x, y, and z axis and also the rotation along x
and y.
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 from both illumination
sides.
Fig. 525 Processing – Lightsheet Processing.
Multiview Processing, Intensity
Based Alignment, Parameters