During FIB-SEM image acquisition, the alignment of the image slices is not maintained due to specimen drift or image distortion, for example. This typically occurs along the Z-axis. Therefore, Slice Alignment is necessary.

Two options are available for the Alignment Method:

• The Correlation algorithm shifts the slices in the X- and Y-directions until the regions within the defined region of interest (ROI) are as similar as possible.
• The SIFT (Scale-Invariant Feature Transform) algorithm recognizes features and attempts to map them to each other by shifting and rotating the slices. This method works well when the features are similar across the different slices. More detailed information on this method can be found here.

The choices for ROI First Slice and ROI Last Slice for X and Y define the region, in which the alignment is examined. Based on the findings for that region, the slice alignment is performed on the entire image. Please note that this may result in unavoidable boundary effects, which may necessitate cropping the image at its boundaries. The rectangle resulting from the First Slice and Last Slice values is visualized in red in the 2D Slice Visualization section in the X-Y-plane if Slice Alignment is chosen as Overlay.

In the Correlation method, the Radius defines the maximum shift between two slices that will be searched. For many FIB-SEM images, the default radius of 10 voxels is sufficient. However, if the slices are shifted by more than 10 voxels, the radius setting needs to be increased.

For the SIFT method, the SIFT Threshold for the image features can be defined as Low, Medium, or High. A higher threshold uses only the most significant image features, while a lower threshold uses more image features. If the alignment does not modify the image, choose a lower threshold. If, on the other hand, the alignment is incorrect, a higher threshold may be better. When the threshold is set to Automatic, it is adjusted automatically.

The example below shows a 2D slice from the X direction of an artificially misaligned gray value image (left). After applying the Slice Alignment tool with the default settings, the misalignment is completely corrected. Note the offsets on the left and right boundaries of the aligned image. The image size in the X and Y directions has increased to include all aligned slices in the 3D image without losing information.