Z-Stack Alignment Wizard
This wizard is used to align the single images of a Z-Stack image. The wizard consists of 6 steps which are described in the following chapters:
Image Import
In this step you can load your acquired Z-Stack images which you want to align. Therefore simply click on the Load button and select the image file from the file system.
See also
Pre-Processing
In this step you can perform pre-processing functions on the loaded image, e.g. Stitching (only for tile images), Brightness and Contrast Correction (only for SEM images).
|
Parameter |
Description |
|---|---|
|
Apply Stitching button |
Only visible if a tile image is loaded. If you click on this button, stitching is performed automatically on the image. The stitching can be canceled (Undo) or repeated (Redo) by using the arrow buttons. |
|
Clip Limit |
Reduces noise in the image. The higher the Clip Limit , the lower the noise. The clip limit can be adjusted between 0 and 10%. |
|
Region Size |
Defines the region for histogram equalization. The smaller the area, the higher the contrast, but the noise will increase, too. The Region Size can be adjusted from 16 to 1024 px. |
|
Histogram Equalization |
If you click on this button, the SEM images are adapted to the selected values. The Histogram Equalization can be canceled (Undo) or repeated (Redo) by using the arrow buttons. |
See also
Image Review
This step is used for reviewing the single images of a Z-Stack. This is necessary because certain images might not be useful for 3D reconstruction due to problems during the image acquisition or sample preparation issues (wrinkles or ruptures within the section). These regions can be replaced either by the previous image or by the following image. To review the images, the images can be displayed as single 2D images in the 2D view or as images series in the Gallery view.
|
Parameter |
Description |
|---|---|
|
2D View |
If selected, you can review the single images of a Z-Stack image by using the 2D view. You can use the Z-Position slider to navigate through the single images. To replace an image select the image and click whether on the Replace with next or Replace with previous button. If you click on the Undo button, the last action performed will be undone. |
|
Gallery View |
If selected, you can review the single images by using the Gallery view. The single images of a Z-Stack image are displayed as an image gallery. If you found an image that does not meet your expectations, simply select the image and replace it by the next or previous image. |
See also
Alignment
In this step you perform the image alignment. Therefore simply click on the Start Alignment button. To cancel the alignment click on the Stop button.
Before you start the alignment, select one channel as reference channel (e.g. DAPI, because it stains the nucleus and the nucleus is a proper structure for performing alignment).
During alignment, a splitter view is visible. In the left container you can see the original images, in the right container you can see the aligned images.
See also
Manual Correction
In this step (optional) you can navigate through the aligned images and check the result of the alignment.
In case the results are unsatisfactory, you have the possibility to correct the alignment of the images manually. Misalignment can occur when no characteristic structures are visible within the images.
See also
Final Image Creation
Last but not least, in this step you create the final image.
|
Parameter |
Description |
|---|---|
|
Total |
If selected, the complete image will be used for the image creation. |
|
ROI |
If selected, only the ROI area will be used for the image creation. |
|
Create final image button |
Creates the final aligned Z-Stack image. |
Click Finish to exit the wizard.