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Molekulární biologie(KBC/MBIOG)
Ivo Frébort
Alberts et al. (2008) Molecular Biology of the Cell, 5th ed.Garland Science, New York
12. Methods of molecular biology II:Visualizing cells
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A light microscope
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Interference and edge effects
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Numerical aperture
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Obtaining contrast
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Four types of light microscopy
Bright-field microscopy
Phase-contrast microscopy
Differential-interference-contrast microscopy
Dark-field microscopy
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Tissue sectioning
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Fluorescence microscopy
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Fluorescence dyes
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Immunofluorescence
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Image deconvulsion – removing the blur by computing
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The confocal fluorescence microscope
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Conventional vs. confocal fluorescence microscope
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Confocal microscopy allows 3D recontruction of objects
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Trichomes of Arabidopsis containing talin-GFP
Green fluorescent protein can be used to tag individual proteins in living cells and organisms
GFP from jellyfish Aqueoria victoria
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Fluorescence resonance energy transfer (FRET)
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Visualizing cell dynamics using caged molecules
Determining microtubule flux in the mitotic spindle with caged fluorescein linked to tubulin
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Dynamic changes and photoactivation of GFP fluorescence
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Fluorescence recovery photobleaching (FRAP)
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Visualizing living cells: light-emitting indicators
Sperm entry into a fish egg visualized with aequorin/Ca2+
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Neurone cell from the brain of a guinea pig – indicator fura-2
Visualizing Ca2+ concentration by a fluorescent indicator
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Introducing large molecules into cells
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Laser tweezers
manipulating objects with higher refractive index within the cell
09_1.mov
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Total internal reflection fluorescence (TIRF) microscopy can visualize single molecules
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Single molecules can be manipulated by atomic force microscopy (AFM)
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Pulse-chase experiments: use of radioisotopes
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Autoradiography: radioisotopically-labeled molecules
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Transmission electron microscope
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Electron microscopy
Limit of resolution 0.2 nm (seen on a gold layer)
Chemical fixatives
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A root tip cell visualized by electron microscope (Os stained)
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Actin filaments by transmission EM
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Localizing proteins by immunogold staining
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3D reconstruction from serial sectionsElectron microscope tomography
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Electron-microscopic autoradiography
Moving of insulin (labeled by 3H-leucine feeding) from ER to Golgi for secretion (45 min)
Staining with photographic emulsion (silver grains)
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Scanning electron microscope
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Scanning electron microscopy
Stereocilia from a hair cell in the inner ear of a bullfrog
Scanning EMTransmission EMDifferential-interference
contrast LM
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Nuclear pore by scanning electrone microscopy
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Freeze-fracture and freeze-etch electron microscopy
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Thylakoid membranes of the chloroplast by freeze-fracture EM
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Protein filaments in an insect muscle by freeze-etch EM
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Single particle reconstruction
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3D structure of 70S ribosome and RF2 from E. coli by cryo-EM tomography (combined from 20,000 ribosomes)