Electron Microscopy
Full course description
Electron microscopy (EM) is currently the imaging technique providing the highest resolution in objects. The resolution that can be reached today is sub-atomic for hard materials and atomic for biological specimens. Different types of electron microscopes are adapted in their configuration to different type of samples and different type of applications. The applications of the EM in situ atomic resolution go from deciphering the structure of proteins key for all genetic diseases and infections, to characterizing the composition of nanomolecular machines in its normal and aberrant form in order to make new drug and or vaccines based on 3D structural information.
In this course, the EM applications targeted are mainly in the fields of life sciences and medicine. The possibility of studying the fine three-dimensional structure of proteins in their natural environment, the cells, where they are working in a dynamic manner with many other biomacromolecules, makes the electron microscope an essential and very powerful tool to aid in the search for new therapeutic targets and new treatments like drugs or vaccines against the molecules responsible for a disease. The cell functions are the result of the combination of the work of thousands of different proteins with each other and with other molecules and this can only be analysed using an in situ high-resolution technique preserving the small crowded cellular environment where all these molecules work together. Cryo-electron microscopy makes this possible. At the same time, applying different EM techniques, it is possible to describe the cellular characteristics in tissues where the proteins are playing their role.
However, not only medicine can benefit from EM but also animal, plant cells, bacteria and viruses are being characterized by EM. The role of certain molecules involved in many cellular events can be addressed by high resolution imaging techniques providing crucial information to agricultural and environmental studies. The electron microscope is a very powerful and necessary tool for the investigations of new biological models replacing the use of animal experiments. Mini-organs (organoids), cellular aggregates, artificial tissues etc. are now under development and will be the future support of the biological and medical research.
EM is a field in continuous and very fast expansion. Other professional areas closely connected to EM developments are engineering, informatics, image processing, data management, machine learning and artificial intelligence. These professional fields will benefit from the knowledge acquired in this course.
Course objectives
After completing this course, you are able to:
- Explain basic principles of EM instrumentation:
- Transmission electron microscopy (TEM), scanning electron microscopy (SEM), STEM, focused ion beam SEM microscopy (FIB-SEM), use of different electrons acceleration voltage in TEM and SEM, use of contrast strategies, use of low irradiation techniques, use of electron diffraction, use of CCD detectors and electrons direct detector devices (DDD) and coating techniques for the FIB work.
- Utilise specific techniques of recording:
- Automation of 2D projection, single particle data acquisition, tomography, single tilt, block face SEM and Slicing and imaging. All at room temperature and at cryogenic conditions.
- Prepare samples and apply molecular labelling techniques to reach all information from samples:
- Chemical fixation and room temperature dehydration and embedding, ultramicrotomy, Tokuyasu cryo-sectioning, vitrification by plunge freezing, jet freezing and high pressure freezing, cryo-lamella fabrication, freeze-substitution, immunolabelling).
- Analyse and process acquired data
- Single particle analysis, helical reconstruction, tomographic reconstruction, subtomogram averaging and 3D volume imaging techniques.
- Work in teams to select the right type of EM instrument and demonstrate an innovative attitude by defining a (novel) technique for specific problems.
Recommended reading
Recommended
Recent methodological literature regarding electron microscopy will be provided in lectures and tutorials. The following books are recommended if you are interested in specific topics and/or desire to have more information:
- Hawkes, P. W., & Spence, J. C. (Eds.). (2019). Springer Handbook of Microscopy. Springer. https://doi.org/10.1007/978-3-030-00069-1
- Egerton, R. F. (2005). Physical Principles of Electron Microscopy (2nd. ed). Springer. https://doi.org/10.1007/978-3-319-39877-8
- Goodhew, P.J., Humphreys, J., & Beanland, R. (2000). Electron Microscopy and Analysis (3rd ed). Taylor & Francis.
Software
- RELION
- MATLAB
- Coot