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
A sense of scale
A light microscope
Interference and edge effects
Numerical aperture
Obtaining contrast
Four types of light microscopy
Bright-field microscopy
Phase-contrast microscopy
Differential-interference-contrast
microscopy
Dark-field microscopy
Image processing
Tissue sectioning
Fluorescence microscopy
Fluorescence dyes
Immunofluorescence
Image deconvulsion – removing the blur by computing
The confocal fluorescence microscope
Conventional vs. confocal fluorescence microscope
Confocal microscopy allows 3D recontruction of objects
Green fluorescent protein can be used to tag individual
proteins in living cells and organisms
GFP from jellyfish
Aqueoria victoria
Trichomes of Arabidopsis containing talin-GFP
Fluorescence resonance energy transfer (FRET)
Visualizing cell dynamics using caged
molecules
Determining microtubule flux in the mitotic
spindle with caged fluorescein linked to tubulin
Dynamic changes and photoactivation of GFP fluorescence
Fluorescence recovery photobleaching (FRAP)
Visualizing living cells: light-emitting indicators
Sperm entry into a fish egg
visualized with aequorin/Ca2+
Visualizing Ca2+ concentration by a fluorescent indicator
Neurone cell from the brain of a guinea pig – indicator fura-2
Introducing large molecules into cells
Laser tweezers
manipulating objects with higher refractive index within the cell
Total internal reflection fluorescence (TIRF) microscopy
can visualize single molecules
Single molecules can be manipulated
by atomic force microscopy (AFM)
Pulse-chase experiments: use of radioisotopes
Autoradiography: radioisotopically-labeled molecules
Transmission electron microscope
Electron microscopy
Limit of resolution 0.2 nm
(seen on a gold layer)
Chemical fixatives
A root tip cell visualized by electron microscope (Os stained)
Actin filaments by transmission EM
Localizing proteins by immunogold staining
3D reconstruction from serial sections
Electron microscope tomography
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)
Scanning electron microscope
Scanning electron microscopy
Stereocilia from a hair cell in the inner ear of a bullfrog
Scanning EM
Differential-interference
contrast LM
Transmission EM
Nuclear pore by scanning electrone microscopy
Freeze-fracture and freeze-etch electron microscopy
Thylakoid membranes of the chloroplast by freeze-fracture EM
Protein filaments in an insect muscle by freeze-etch EM
Single particle reconstruction
3D structure of 70S ribosome and RF2 from E. coli by cryo-EM
tomography (combined from 20,000 ribosomes)