The wizard contains the following 6 steps:

  • Image Import
  • Pre-Processing
  • Image Review
  • Alignment
  • Manual Correction
  • Final Image Creation
  1. In the CAT tool click on Start Z-Stack Alignment Wizard.
  2. You will see the first step Image Import of the wizard.
  3. Click on Load Image and select the acquired Z-Stack image from the file system. In our example we choose the LM image. The same process has to be performed for the SEM image afterwards.
  4. You will see the Z-Stack image in the center screen area.
  5. Click on Next.
  6. You will see step 2/6 Pre-Processing.
  7. If your image is a tile image, click on Apply Stitching to correct the offset between the individual tiles.
  8. If your image is a SEM image, click on Histogram Equalization to adjust the image display.
  9. Click on Next.
  10. You will see step 3/6 Image Review. Note that in this step no image acquisition is possible. You can just replace an image by the next or previous image of the Z-Stack.
  11. Select the image to be replaced by clicking on it with the mouse and click on Replace with next or Replace with previous. Alternatively, you can press the N or the P key. Note that the table of the replaced image will not be saved.
  12. Click on Next.
  13. You will see step 4/6 Alignment.
  14. If you have acquired a multi-channel image, select the reference channel from the Channel list.
  15. Click on Start Alignment.
  16. The alignment of the Z-Stack image will be performed automatically. After the alignment you will see the original Z-Stack image in the left image and in the right image you will see the aligned Z-Stack image.
  17. Click on Next.
  18. You will see step 5/6 Manual Correction.
  19. If you browse through the Z-Stack by using the Z-Position slider and you still realize a shift between the single Z-Stack images, you can perform a manual correction of the single Z-Stack images. Therefore continue as follows:
  20. Click on Define direction.
  21. A red arrow will appear in the right and in the left image.
  22. Place the arrow in the left image at a prominent structure in the image which is easy to recognize through the full Z-Stack.
  23. Select the right image and browse through the Z-Stack by using the Z-Slider on the Dimensions tab.
  24. When you realize a shift in an image, adjust the arrow in the right image so that it matches with the prominent structure marked with the arrow in the left image. Note that you have to check and adjust the arrow for each image of the Z-Stack which does not match the position.
  25. Click on Apply.
  26. The correction will be applied. If you want to correct the Z-Stack at another position, simply repeat the procedure with another prominent structure. Of course you can undo and redo actions by using the Undo/Redo buttons.
  27. Click on Next.
  28. You are in step 6/6 Create final Z-Stack.
  29. If you want to create the Z-Stack image from the complete image, select Total and click on Create final image.
  30. If you want to create the Z-Stack image from the marked ROIs, select ROI. Note that you can adjust the marked ROI in its size and position here. If you have marked more than one ROI, you can switch between the ROIs by using the Scene slider on Dimension tab.
  31. Click on Create final image.
  1. You have successfully aligned and created the Z-Stack image. Of course you have to repeat the process for the SEM image that was acquired.