Module 5
Volume Scanning Electron Microscopy (ScopeM, ETHZ, campus Hönggerberg)
The third dimension is of utmost importance for understanding the complex structural context of specimen, in biology as well as in material sciences. This module provides an overview and compares scanning electron microscopy (SEM)-based techniques for volume imaging: (1) focused ion beam-SEM (FIB-SEM), (2) serial blockface SEM (SBF-SEM) and (3) array tomography. For the first two methods, a fresh blockface is created in situ in the SEM employing either a focused ion beam, or a diamond knife respectively, and volume data is acquired by alternating cutting and imaging of the fresh blockface. For array tomography on the other hand, ribbons of sections (i.e. serial sections) are imaged. These are prepared using conventional ultramicrotomy and mounted onto a conductive support for SEM. We will acquire 3D data of the same sample type using all three techniques, and cover the methodology and sample preparation techniques for the respective methods. The module will be rounded off by image processing sessions covering the basics of post-processing of volume data, visualization and 3D modelling of structures of interest. Samples will be provided.
Contact:
Dr. Miriam Lucas
ScopeM, ETHZ
miriam.lucas@scopem.ethz.ch
+41 44 633 4424
Learning outcomes:
- Become familiar with 3D electron microscopy imaging
- Understand the advantages and disadvantages of the presented 3D imaging techniques and be able to select the optimum approach for your research
- Understand sample requirements and respective preparation techniques for volume SEM
- Visualize, process, evaluate and interpret different 3D microscopic data types
Practical activities:
- Tips and tricks for serial sectioning
- Choosing a relevant region of interest for image stack acquisition
- Acquisition of 3D image stacks assisted by ScopeM-staff
- Inspection and evaluation of the resulting 3D data, and comparison of the presented imaging techniques
- Processing and visualization of the resulting 3D data using Fiji Amira 3D software
- Discussion of relevant problems concerning preparation acquisition and data interpretation
Prerequisites:
- Knowledge of electron microscopy theory
- Ideally practical experience with basic SEM operation