EM Processing Toolbox

This module offers functionality for the processing of FIB-SEM stacks. This chapter describes how the different functions of the EM Processing Toolbox can be used to process a FIB-SEM-stack acquired with SmartFIB in the ZEN software. Note that parts of this special workflow also require functionalities of the ZEN Connect module. To make yourself familiar with this module, see also the documentation for ZEN Connect.

Workflow Overview

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

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!
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).
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.
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.
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.
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.
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).
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.
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.
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.

Aligning Z-Planes Manually

If you have an imported FIB-SEM stack which requires manual pre-alignment before using the automatic z-alignment, you can use the image processing function Coarse Z-Stack Alignment for a manual alignment of your stack. See the following instruction.

You have opened your (imported) z-stack that needs alignment in ZEN.

On the Processing tab, select the image processing function Coarse Z-Stack Alignment.
Click on the Setup button.
The Coarse Z-Stack Alignment Setup opens.
The current z-plane (selected by the Z-Position slider in the Dimensions tab) is displayed in cyan and the following z-plane is displayed in red.
If you have an image with multiple channels, select the channel which should be displayed in the Image View with the Channels tool.
With the Z-Position control in the Dimensions tab, go to the z-plane where the image needs an alignment.
Select the Speed with which your alignment should be performed.
Shift the following z-planes with the arrow buttons in the top left of the setup until the x/ y shift in the z-stack seems to be eliminated. Alternatively, you can also use the arrow keys on your keyboard.
If necessary, adjust the speed step size during the alignment.
Repeat the steps 4 to 6 until all shifts in the z-stack are corrected.
Every shift is displayed in the Shift List on the left side of the setup.
Click on Finish to save the changes and close the setup.
The Shift List with all the shifts is displayed in the Parameters tool.
On the top of the Processing tab, click on Apply.

You have now aligned the planes in your z-stack manually to correct a shift in x and/or y within the z-stack.

Aligning Z-Planes Automatically (Based on a ROI)

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.

You have opened your z-stack in ZEN.

On the Processing tab, select the image processing function Z-Stack Alignment with ROI.
The method's options are displayed in the Parameters tool.
For Quality, select the quality for the image alignment.
For Registration Method, select whether the alignment to optimize the z-stack should perform a translation, a rotation, or both.
For Interpolation, select the interpolation method that should be used for the alignment.
Under Region, select Rectangle Region in the drop-down menu.
In the 2D view, draw a region of interest into your image to mark the structure(s)/area of particular interest.
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.
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.
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.
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.
At the top of the Processing tab, click on Apply.

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.

Replacing Z-Slices in a Z-Stack

With the processing function Slices Replacement you can replace slices of a z-stack with the previous or next slice in the stack.

On the Processing tab, select the method Slices Replacement.
In the Dimensions tab, use the Z-Position slider or input field to select the slice you want to replace.
Click on Replace with Next to replace the selected slice with the next one, or click on Replace with previous if you want to replace the slice with the previous one.
If you want to replace other slices as well, repeat the steps 2 and 3 for each slice.
Each slice is listed in the Replacement Table on the left.
Click on Apply.

You have now replaced the selected slice(s) with its previous and/or next one(s).

Replacing multiple slices

If you have several slices in your z-stack which you want to replace with the following or preceding slice, you can also take the following workflow:

On the Processing tab, select the method Slices Replacement.
Open the Gallery view of your z-stack.
In the Gallery view, press the Ctrl button and select all the slices you want to replace. As an example, you select the slices 34, 37, and 38.
Click on Replace with Next to replace the selected slices with the next ones, or click on Replace with previous if you want to replace the slices with the previous ones.
Each slice is listed in the Replacement Table on the left. As an example, clicking Replace with previous would replace slice 34 with 33, and 37 and 38 with the slice 36.
Click on Apply.

You have now replaced the selected slices with its previous and/or next ones.

Cutting Out a Volume from a Z-Stack

With the processing function Cut Out Regions you can define a region in your z-stack and cut it out as a volume.

You have opened your z-stack in ZEN.

On the Processing tab, select the Method Cut Out Regions.
Under Parameters, click on Define Regions.
The Define Regions setup opens.
In the Dimensions tab, use the Z-Position slider to go to the first z-slice where you want to mark your region of interest.
In the Region Edit section on the left, click on Draw and select the type of region you want to use (rectangular, circular, polygonal,...).
In the image, draw your so-called support region to mark the structure of interest.
Repeat the previous steps for the last slice where your structure of interest appears as well as for those slices in between where significant changes (in shape and/or position) of this structure take place.
Click on Interpolate.
Interpolated regions are created for all slices between the support regions. The interpolated regions are displayed with a slightly darker color.
Use the Z-Position slider to move through your stack and examine whether the interpolated regions satisfyingly cover the structure of interest in your stack.
If you find a slice where the interpolated region does not cover your structure of interest, draw a new support region on this slice and click on Interpolate again.
When the entire structure is marked satisfyingly by the interpolated regions, click on Finish.
The Define Region setup closes and your image with the newly created regions is displayed in the Analysis view.
On the Processing tab, click on Apply.

Your marked structure is now cut out of the z-stack and opened as a new image.

Importing SmartFIB Tiffs

In ZEN you can import SmartFIB stacks of Crossbeam microscopes. The orientation of these stacks differs from standard z-stack acquisition, as the acquired images are tilted by a certain angle compared to a z-stack acquired on a light microscope. The import function calculates this tilt from the metadata of the image. If the import finds no metadata concerning the tilt angle and the user does not enter a value for the sample angle, it uses a default angle of 54 degrees (default angle between FIB and SEM column at the Crossbeam) and the image is rendered with a 90 degree tilt when displayed in a ZEN connect project. Alternatively, you can enter the angle of your sample during import, e.g. as set during acquisition of the stack with SmartFIB, and the import then calculates the tilt angle based on this sample angle.
During import, the XY offset metadata of the individual slices is ignored by default and only the offset of the first tiff file is considered. This default avoids the creation of a slanted z-stack, however in certain cases, such as on-grid-thinning configuration, the XY offset of the individual slices needs to be taken into account.

On the Processing tab, select the image processing function Import SmartFIB TIFFs.
The function settings are displayed in the Parameters tool.
Click on Select Files.
A file browser opens.
Select the images you want to import as FIB stack.
Note: Select only images with consistent metadata with respect to number of pixels, image size, and spacing of the images (i.e. use the Sort SmartFIB tiffs function before importing the data).
Note: To make sure the stack is composed/ ordered correctly, watch out how the images are sorted in the explorer and in which order you choose them.
Enter a File name for the FIB stack.
If you import images without scaling information, deactivate the Auto checkbox for XY-Scaling and manually enter the information.
Note: ZEN currently cannot determine automatically if scaling information is present.
You can set the slice distance manually if you deactivate the Auto checkbox for Z-Spacing. This step is optional and should only be done if you have reason to believe the information calculated with the metadata is incorrect. Leave the Auto checkbox activated and the slice distance is automatically calculated with information saved in the metadata of the images.
Note: When you set the slice distance manually, the information in the metadata is ignored.
If you know the angle of your sample, deactivate the Auto checkbox for Sample Angle and enter it. Otherwise the tilt for the image is calculated using the metadata, or the sample angle is set to the default of 54 degrees (default angle between FIB and SEM column at the Crossbeam) and the image is rendered with a 90 degree tilt (if no information is available in the metadata).
If you want to consider the xy offset metadata of the individual slices for the import, activate the checkbox Read XY Offsets. Note that this can lead to a slanted z-stack depending on the sample and the metadata, assuming tilt correction was used during acquisition with SmartFIB (e.g. if the metadata contain incorrect offset information).
Click on Apply.

The FIB stack is now imported into ZEN and a czi-file is created.
Note: When importing larger image files, it may take a while until the entire stack is visible in the viewer.