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The course is focused on explaining how electron microscopes work and how can be used for the observation of biological objects. The course can be divided into three parts:
1/ physical principles of electron microscopy and description of instrumentation: Properties of accelerated electrons, basic components of electron microscopes – sources of primary electrons, lenses and their aberrations, the interaction of primary electrons with specimen atoms, formation of images in TEM and SEM, construction of TEM and SEM. 2/ specimen preparation of biological objects for TEM and SEM: Chemical methods (fixation, dehydration, embedding), cutting of ultrathin sections, cryo-methods (high-pressure freezing, plunge freezing, cryo-cutting, freeze substitution), special methods for SEM (critical point drying, freeze fracturing, freeze-drying, methods of specimen coating by conductive layer), cellular ultrastructure and interpretation of electron-microscopic images. 3/ advanced methods of current biological electron microscopy: high-resolution electron microscopy in biology (SPA), immunolocalization of molecules of interest in cell ultrastructure, volume electron microscopy (electron tomography, dual-beam SEM, serial block-face SEM, micro-array tomography), data recording and processing. The course consists of a theoretical part (lectures) and several practical tasks. Recommended literature: https://www.thermofisher.com/cz/en/home/electron-microscopy/life-sciences.html https://en.wikipedia.org/wiki/Electron_microscope https://en.wikipedia.org/wiki/Transmission_electron_microscopy https://en.wikipedia.org/wiki/Scanning_electron_microscope https://myscope.training MJ Dykstra, LE Reuss: Biological Electron Microscopy – Theory, Techniques, and Troubleshooting. 2nd Edition. Kluwer Academic Plenum Publisher. ISBN 0-306-47749-1. 2003 Last update: Hyliš Miroslav, RNDr., Ph.D. (23.02.2026)
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1. Why electrons? 2. How does a transmission electron microscope work? 3. How does a scanning electron microscope work? 4. Methods of biological specimen preparation at room temperature. 5. How to cut section with the thickness below 100 nm? 6. How to vitrify water in biological samples? 7. Methods of biological sample preparation for SEM. 8. Cellular ultrastructure and interpretation of electron-microscopic images. 9. Immunolabeling methods – the tool how to localize and identify molecules in cell structure. 10. Volume electron microscopy – from 2D to 3D imaging. 11. How to visualise macromolecules in TEM? 12. How to record electron microscopic images and how to process them. Last update: Hyliš Miroslav, RNDr., Ph.D. (12.01.2022)
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After successful completion of the course, the student: - demonstrates advanced understanding of the physical principles of electron microscopy, including electron–specimen interactions, image formation, and instrument design, and critically compares the capabilities, limitations, and appropriate areas of application of transmission (TEM) and scanning (SEM) electron microscopy, - acquires the ability to operate TEM and SEM with a high degree of independence, optimizes imaging conditions and image acquisition parameters, and adapts microscope settings to different specimen types, preparation strategies, and experimental objectives, - independently designs, selects, and performs complex preparation workflows for biological samples for TEM and SEM, ranging from chemical fixation through semi-thin and ultrathin sectioning to various cryogenic methodologies and specialized techniques for 3D SEM and TEM, - critically evaluates specimen quality and preservation, identifies and explains artifacts arising during sample preparation and imaging, and assesses the integrity of cellular ultrastructure, macromolecular organization, and three-dimensional tissue architecture, - analyzes, interprets, and places electron microscopy data in a biological context, including data obtained using high-resolution and volume imaging approaches, and integrates image analysis with biological interpretation, - selects, applies, and critically evaluates advanced electron microscopy methods (e.g. immunolabeling, single-particle analysis, volume electron microscopy, microanalysis) to address specific biological questions and research problems, including data acquisition, processing, and analysis. Last update: Hyliš Miroslav, RNDr., Ph.D. (02.02.2026)
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