LEARNING OUTCOMES:
1. Overmaster the basic skills necessary for independent elaboration and interpretation of light microscopy samples.
2. Choose the appropriate method for the preparation of biological samples for transmission electron microscopy.
3. Choose the appropriate method for the preparation of biological samples for scanning electron microscopy.
4. Apply and use methods for the preparation of biological samples for research of cells and tissues ultrastructure.
5. Analyze and interpret biological ultrastructures obtained by the above mentioned techniques and assess their quality.
COURSE CONTENT:
This is a course in which students will gain a detailed insight into the basic principles and application of special light microscopy as well as transmission and scanning electron microscopy. Students will acquire basic knowledge on preparation of biological samples for transmission and scanning electron microscopy.
LECTURES
1. Theoretical basis of light microscopy and working principle and application of specific types of microscopes (dark field, phase contrast, differential interference contrast, polarization microscopy, fluorescence microscopy, confocal laser scanning microscopy);
2. The basic principles of electron microscopy (EM);
3. Components of transmission electron microscope (TEM);
4. Electron gun, types of cathodes, electromagnetic lenses and system for vacuum achievement in TEM;
5. Types of electron interaction with the sample;
6. Preparation of biological samples for TEM (chemical fixation and fixation by freezing, dehydration, embedding, sectioning);
7. Contrast methods in TEM (positive and negative staining, metal shadowing);
8. Production of replicas and method of fracturing / etching of frozen material;
9. High voltage electron microscopy;
10. Components of scanning electron microscope (SEM);
11. Types of signals in the SEM;
12. Preparation of biological samples for SEM (fixation, dehydration, drying, applying conductive layer);
13. Special types of SEM (Low vacuum SEM, Environmental SEM);
14. Application of electron microscopy in biological research (immunocytochemistry, enzymatic cytochemistry, autoradiography) and image analysis in electron microscopy;
15. Analytical methods in EM (X-ray analysis, electron diffraction, EELS) and Scanning Probe Microscopes (atomic force microscopy, scanning tunneling microscopy).
PRACTICAL WORK:
1. Components of the light microscope, resolving power of the microscope and the microscope as a measuring instrument;
2. Application of phase contrast microscope and differential interference contrast microscope;
3. Application of polarizing microscope and dark filed microscope;
4. Application of fluorescence microscope and confocal laser scanning microscope;
5. Fixation, postfixation and dehydration of biological tissues /cells for transmission electron microscope;
6. Use of transitional solvents and embedding biological samples in epoxy resin;
7. Crafting the glass knives;
8. Making support films and sputter coating the grids for electron microscopy;
9. Negative staining and metal shadowing using vacuum: viruses, macromolecules and particles;
10. Sectioning the material with the ultramicrotome. Making and contrasting the semi thick and ultrathin sections;
11. Work with the transmission electron microscope;
12. Work with the scanning electron microscope;
13. Recording and production of the micrographs of various biological samples;
14. Analysis and interpretation of biological ultrastructures;
15. Measurements, morphometry and computerized image analysis.
|
Pristupačnost
