With the image processing function Z-Stack Alignment with ROI you can perform an automatic alignment of the z-planes in a stack. This alignment can be based on a particular region of interest that you can draw into your image.

  1. You have opened your z-stack in ZEN.
  1. On the Processing tab, select the image processing function Z-Stack Alignment with ROI.
  2. The method's options are displayed in the Parameters tool.
  3. For Quality, select the quality for the image alignment.
  4. For Registration Method, select whether the alignment to optimize the z-stack should perform a translation, a rotation, or both.
  5. For Interpolation, select the interpolation method that should be used for the alignment.
  6. Under Region, select Rectangle Region in the drop-down menu.
  7. In the 2D view, draw a region of interest into your image to mark the structure(s)/area of particular interest.
  8. In the Dimensions tab, use the Z-Position slider to check if the region of interest needs to be adjusted. Note: The region of interest cannot be set for each plane individually. Make sure that the drawn region is big enough to contain the structure(s)/area of interest in every z-plane.
  9. Adjust the region of interest if necessary. You can adjust and move it directly in the 2D view or with the input fields in the Parameters tool.
  10. If you want your output image to have the same size as your input image, activate the checkbox Keep Size. The image will be cropped in the process, in the case that pixels are shifted out of the area defined by the input image.
  11. Under Channel Component, select one of the image channels whose alignment transformation matrix is then also applied to the other channel(s). If an alignment should be calculated for each channel individually, deactivate the checkbox Single Component. This step is only applicable for images with multiple channels.
  12. At the top of the Processing tab, click on Apply.
  1. The z-planes of your image are now aligned automatically. The progress of the alignment process is displayed in the progress bar on the bottom of the ZEN software.