This chapter gives an overview how you can process your FIB-SEM stacks and align them. Consider the following workflow:

  1. Sorting of image files:
    With the function Sort SmartFIB Tiffs you can sort your .tiff image files created by SmartFIB according to channel name, number of pixels, image size, and spacing of the images, corresponding to slice thickness. Note that this function only works if the tiff files have their default names (e.g. channel0_slice_0001.tiff or slice_0001.tiff)! Do not rename your files before you use this function!
  2. Image import and conversion:
    With the special import functionality, you can import your FIB stack images and save them as a czi for further processing in ZEN. You can import the stack into ZEN (see Importing SmartFIB Tiffs) or into ZEN Connect (see Importing a SmartFIB Stack into ZEN Connect).
  3. Subset image creation:
    If you want to reduce the imported z-stack to a particular z-range and region before applying further processing steps, you can create a subset image of the imported FIB stack with the image processing function Create Image Subset.
  4. Replacing individual slices in the z-stack:
    If your stack contains image slices of bad quality which prevents further processing or segmentation, you can use the function Slices Replacement to replace those slices with the respective predecessor or successor. For more information, see also Replacing Z-Slices in a Z-Stack.
  5. Coarse alignment of the z-stack:
    To minimize shifts in x and y in the z-stack and correct a potential beam shift, you can use the image processing function Coarse Z-Stack Alignment to roughly align the z-stack. For more information, see also Aligning Z-Planes Manually.
  6. Image processing:
    Use the image processing functions to process your image and reduce artifacts. For general information about image processing, see also the chapter for the Image Processing Workflow.
  7. Automatic alignment of the z-stack:
    Make an automatic fine alignment of the planes in your z-stack with the processing function Z-Stack Alignment with ROI. For more information, see also Aligning Z-Planes Automatically (Based on a ROI).
  8. Equalization:
    Correct for variation of overall image intensity from image to image by equalizing the intensity value throughout the entire z-stack with the function Z-Stack Equalization.
  9. Cropping of a specific volume:
    Identify a particular region of interest and cut it out of your stack with the function Cut Out Regions. For more information, see also Cutting Out a Volume from a Z-Stack.
  10. Adding the processed z-stack to the ZEN Connect project:
    Add your processed z-stack into the correlative workspace of the ZEN Connect module. For this you can use the Add to Correlative Workspace button in the toolbar if you have an open project. In the correlative workspace you can then align several z-stacks and images (e.g. an overview image). For detailed information, see also Adding an Open Image to the ZEN Connect Project and Aligning Image Data.
    In order to import the data into a specific session in the ZEN connect project, right-click on the respective session and select the czi file. The transformation that was applied to the session will then also be applied to the newly imported image.