Module 4
3D Correlative Light and Electron Microscopy (ScopeM, ETHZ, campus Hönggerberg)
Correlative Light and Electron Microscopy (CLEM) combines the best of two worlds: large-scale imaging using light microscopy and high-resolution electron microscopy. This module covers the basics of methodology and preparation techniques needed to perform light and electron microscopic investigations on identical sample areas. Starting with imaging of living cells, we will in the following prepare these samples for EM, and acquire focused-ion beam SEM (FIB-SEM) and serial blockface SEM (SBF-SEM) data of the exact same cells. You will learn to relocate a previously imaged region of interest in the EM, collect correlative 3D data sets and get an understanding of the different image characteristics. The practical work will be rounded off by image processing sessions, covering the basics of post-processing of volume data, aligning the acquired LM and EM data in 3D, visualizing the correlation, and creation of 3D models. Samples will be provided.
Contact:
Dr. Miriam Lucas
ScopeM, ETHZ
miriam.lucas@scopem.ethz.ch
+41 44 633 4424
Learning outcomes:
- Get familiar with the workflow for different CLEM-approaches
- Understand the requirements for CLEM experiments with respect to sample preparation and choice of microscopy techniques, and be able to design own experiments
- Visualize, process evaluate, and interpret different 3D microscopic data types
Practical activities:
- Acquisition of light microscopic data of living cells with light microscopy, and selection of regions of interest for optimal correlation with electron microscopy data
- Preparation of the previously imaged cell culture samples for EM
- Relocation of the region of interest in the electron microscope and stack acquisition, assisted by ScopeM staff
- Inspection, evaluation and processing of the resulting 3D data and visualization of the correlation using Fiji and Amira 3D software
- Discussion of relevant problems concerning preparation acquisition and data interpretation
Prerequisites:
- Knowledge of theory for light and electron microscopy
- Practical experience in LM, and ideally with basic SEM operation