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Lecturer(s)
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Ovečka Miroslav, prof. Mgr. Ph.D.
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Course content
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1. Light microscopy, imaging of fast processes in living plant cells using contrast modulation and video microscopy methods, wave nature of light and limitations caused by diffraction. Deconvolution, resolving power and wavelength of light, point spread function. 2. Fluorescence microscopy and its application in imaging of fluorescently labeled objects. Fluorescent labeling, genetically encoded fluorescent proteins, specifics of fluorescent labeling of proteins, lipids and carbohydrates. 3. Practical application of contrast modulation light microscopy methods in imaging of living plant cells. Practical use of epifluorescence microscope to detect autofluorescence and specific fluorescence in living cells. 4. Principle of confocal laser scanning microscopy, method of scanning optical sections, excitation and emission spectra, signal detection, stability of fluorescently labeled epitopes, separation and colocalization of fluorescent signals, implementation of superresolution using the Airyscan detector method. 5. Practical application of confocal laser scanning microscopy and Airyscan detector methods in scanning fixed and live plant cells, methods of multichannel scanning, time-lapse scanning and 3D reconstruction. 6. Laser microscopy with a rotating disk, principle of the method and characteristic components for live cell imaging and scanning of fast cellular processes. 7. Practical application of laser microscopy with a rotating disk, scanning of dynamic movement of fluorescently labeled organelles in plant cells, multichannel microscopy. 8. Spectral characteristics of emission in fluorescence, spectral characterization of fluorochromes and separation of emission spectra. Spectral scanning, spectral unmixing, online fingerprinting. 9. Practical application of confocal laser scanning microscopy using the spectral scanning method, separation of the overlap of emission spectra of multiple fluorochromes using the spectral unmixing method. 10. Special microscopic techniques. I Mesoscopic method Light-Sheet Fluorescence Microscopy (LSFM). Microscopy of developmental processes of living bulk samples in almost natural conditions. 11. Special microscopic techniques. II Super-resolution method Structured Illumination Microscopy (SIM). A method achieving super-resolution through structured illumination. 12. Special microscopic techniques. III Super-resolution methods Photo-Activated Localization Microscopy (PALM), Stochastic Optical Reconstruction Microscopy (STORM), Stimulated Emission Depletion (STED).
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Learning activities and teaching methods
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Demonstration, Laboratory Work
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Learning outcomes
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Obtaining extensive theoretical and practical experiences in the application of modern methods of light, fluorescence and laser microscopy of plant cells.
Acquainted with the principles of modern non-invasive microscopy techniques and their practical applications in plant cells. Students will gain knowledge about the practical application of advanced light microscopy methods, the use of DIC in the study of live unstained preparations, video microscopy and the capture of dynamic events. Also about fluorescence microscopy, documentation and software adjustment of image noise quality using deconvolution. For confocal laser scanning microscopy, its practical application in the study of plant cells. Also advanced methods of studying fluorescently labeled epitopes in live plant cells, colocalization, spectral analysis, FRAP, FLIP, FRET, physiological measurements. Real-time capture of fast cellular processes using spinning disk confocal microscopy, developmental light-sheet microscopy and super-resolution microscopy.
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Prerequisites
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unspecified
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Assessment methods and criteria
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Written exam
Participation and successful completion of a written exam in the form of a test with a minimum score of 75% of points obtained.
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Recommended literature
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Conn PM . (2010). Techniques in confocal microscopy.
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Cox G. (2012). Optical imaging techniques in cell biology. CRC Press.
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Goldman, R. D., Swedlow, J. R., & Spector, D. L. (2010). Live cell imaging : a laboratory manual . Cold Spring Harbor, N.Y.
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Haseloff J, Dormand E-L, Brand AH. (1999). Live imaging of green fluorescent protein. Methods in Molecular Biology, vol. 122: Confocal Microscopy Methods and Protocols .
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Hibbs A. R. (2004). Confocal Microscopy for Biologists.
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Murphy, D.B. (2001). Fundamentals of Light Microscopy and Electronic Imagine.
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Price RL, Jerome WG. (2010). Basic confocal microscopy. Heidelberg.
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Teubner, U., Bruckner, H.J. (2019). Optical imaging and photography.
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