Serial Sectioning Tomography

Preparation for tomography in FIB-SEM

Compared to conventional TEM tomography, the 3D acquisition achieved in a Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) is completely different. Thanks to the gallium ion beam in addition to a conventional SEM with a large chamber, a much bigger sample can be put into the machine and tilted to 54 degrees (the angular difference between FIB and SEM on our system is 54°) with its top surface perpendicular to the FIB. Then the ion beam mill into the depth of a specific operator chosen area revealing structures in a new dimension other than the surface. In order to expose a cross-section with features at a certain depth, an appropriate volume of the material needs to be removed so that the electron beam can reach deep enough to image the target features. 

Tomographic acquisition

Once the area of interest with an adequate cross-section is finished preparing, towards a 3D acquisition process, a thin slice of material from the first cross-section under the chosen area will be milled away by the FIB and a proper electron image is then taken. This serial sectioning and imaging process is repeated until a sufficient amount of slice images have been obtained meaning a representative volume can be reconstructed. 

The image on the side shows an example of a raw stack of slice images obtained from a LED sample, with a constant slice thickness of 50 nm. 


Post-acquisition processing

The acquired raw image stack would need to be aligned to be free from any drift during the experimental process and then treated in dedicated software for visualisation and quantification, etc.

The video renders the reconstructed volume out of the aligned image stack as from the last section. As shown, once the processing is finished, the 3D volume can be manipulated freely for viewing, and with further treatment such as thresholding or segmentation, it's possible to only maintain certain parts of interest and remove others in order to have a better visualisation of target objects. 

3D to '4D'

The serial sectioning tomographic acquisition can be accompanied by other analytical imaging techniques, including EDS, EBSD and CL, to add more information about the explored volume. To gain insights other than a rendered volume can add another dimension of knowledge towards the material, thus analytical imaging combined 3D reconstruction is referred to as '4D'.

The video shows a volume reconstructed volume of a pure copper sample combining with EBSD maps. The colours represent copper crystal grains in the sample with different sizes and orientations. 

Notes


  • As described above, the material removed by FIB cannot be restored making this 3D technique destructive. 
  • Also, in the case of non-conductive materials, the sample needs to be coated before transferring into the machine, and the chosen area to undergo 3D acquisition needs to be covered by a much thicker protective layer to reduce influences from both beams. For this, please see GIS and FIB pages for more information. 

Available Equipment