Skip to content

iSBEM User Guide

Created: 20.05.2025, Yanneck Klenz

Edited: 20.04.2026, Fiona Young

This is a step-by-step user guide for a typical workflow using iSBEM. It describes the steps depicted in the accompanying PDF, which is referenced throughout ("slide x").

Before "Targeting"

  • Approach block, when cutting, adjust the focus stigmators and wobbler
  • Acquire Overview stack (Z = 1 µm) with a cutting thickness matching the settings for the actual acquisition (e.g. 100 µm)
  • When importing X-ray as TIFF, check if the correct pixel size is saved in the image properties

Import Datasets and preprocess ROIs > "Targeting"

  • Go to "Targeting" (top right menu)
  • Import X-ray tomogram (OME-zarr or TIFF format) (slide 2) and adjust contrast, if needed (slide 3)
  • Import labels (TIFF) (slide 3)

Import menu

  • Dilate ROIs, create a buffer in all dimensions (slide 4)
  • Run connected components and merge nearby labels to avoid double scanning (adjust merge tolerance based on tile size) => create individual ROIs
  • Save adjusted ROIs by selecting "Export labels" (slide 7)

Label settings

  • Go to "acquisition" (top right menu) > set EM overview directory > move slider to the left until EM overview is visible (slide 8 -10)

Load overviews

Rotation Transformation > "Registration"

  • Go to "Registration" > show rotation => second napari window pops up with X-ray volume in cyan
  • Drag and rotate the image (in the pop-up window) to match EM overview (slide 12)
  • Adjust the position to match X-ray to EM overview (slide 12)
  • Adjust the rotation in Z-X and Z-Y to match both images (slide 13)

Rotation controls

  • Close second window > "apply rotation" > message "Image rotated successfully!"

2D alignment > "Registration"

  • Select X-ray image (left menu) > click "transform" bottom (top left) > rotate and move X-ray image next to EM overview (slide 16)

Transform

  • Tick reverse Z direction, if both datasets do not move in the same direction when changing the bottom slider (slide 17)
  • Click "move up" or "move down" to match the display of the X-ray to the EM overview (slide 18) as accurately as possible
  • can adjust "Move amount (µm)" to be more accurate
  • If the overview is no longer depicted in the viewer, go to "Acquisition" > "Reset view"

Z alignement

  • Press "Start" under 2D alignment > "point" layer for each modality should be added to the layer list on the left (slide 19)
  • Select landmarks on EM and X-ray sequentially for 2D registration (images should switch automatically)
  • Alignment is done in real-time > after each point is added, evaluate the output

Landmark registration

  • Using the drop-down menu, select the desired transformation method (Euclidean, Similarity, Affine ) -> output is updated immediately > can compare results of different methods (slide 21)
  • Satisfied with the result of 2D alignment > press "stop" (slide 22)
  • Save rotation and 2D alignment as ".txt" by clicking "Save transform"

ROI selection > "Acquisition"

  • Go to the "Acquisition" tab (top right menu)
  • In the drop-down menu under "ROI settings," select "ROIs" (slide 24)
  • By double-clicking on "ROI ID" in the depicted table, the respective ROI is shown in the center of the napari viewer -> can be used to check each ROI
  • Set the acquisition settings, including cutting thickness and pause options, as required (slides 25-27)
  • Press "Start server" (slide 24)

TCP server

  • Go to SBEMimage > tick "Use TCP" > click on the "..." symbol next to it
  • In the popup window, select "Sync data" > click "Ok" (slide 28)
  • ROIs should appear in the SBEMimage Viewport > check if positions are correct

SBEMimage TCP server

  • Adjust the focus and the stigmators
  • In acquisition settings, define target depth (e.g. 10. µm) and cutting thickness (e.g. 100 nm) and start acquisition
  • After the defined target depth is reached, re-register the two volumes in napari
  • Continue with the acquisition

Resuming from saved transformation (slides 30-38)

  • Import X-ray and saved labels as usual
  • Saved labels should be dilated and merged
  • Load EM overview as described
  • Go to "Registration" > click "Upload transform" > select the last saved transformation

Upload transform

  • Should apply rotation and 2D transform on the X-ray
  • Resume with "ROI selection" as described above (slide 24, 25)