1 / 46

12. Methods of molecular biology I I : Visualizing cells

Molekulární biologie (KBC/MBIOG) Ivo Frébort Alberts et al. (2008) Molecular Bi o logy of the Cell, 5th ed. Garland Science, New York. 12. Methods of molecular biology I I : Visualizing cells. A sense of scale. A light microscope. Interference and edge effects. Numerical aperture.

star
Download Presentation

12. Methods of molecular biology I I : Visualizing cells

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Molekulární biologie(KBC/MBIOG)Ivo FrébortAlberts et al. (2008) Molecular Biology of the Cell, 5th ed.Garland Science, New York 12. Methods of molecular biology II: Visualizing cells

  2. A sense of scale

  3. A light microscope

  4. Interference and edge effects

  5. Numerical aperture

  6. Obtaining contrast

  7. Four types of light microscopy Differential-interference-contrast microscopy Bright-field microscopy Phase-contrast microscopy Dark-field microscopy

  8. Image processing

  9. Tissue sectioning

  10. Fluorescence microscopy

  11. Fluorescence dyes

  12. Immunofluorescence

  13. Image deconvulsion – removing the blur by computing

  14. The confocal fluorescence microscope

  15. Conventional vs. confocal fluorescence microscope

  16. Confocal microscopy allows 3D recontruction of objects

  17. 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

  18. Fluorescence resonance energy transfer (FRET)

  19. Visualizing cell dynamics using caged molecules Determining microtubule flux in the mitotic spindle with caged fluorescein linked to tubulin

  20. Dynamic changes and photoactivation of GFP fluorescence

  21. Fluorescence recovery photobleaching (FRAP)

  22. Visualizing living cells: light-emitting indicators Sperm entry into a fish egg visualized with aequorin/Ca2+

  23. Visualizing Ca2+ concentration by a fluorescent indicator Neurone cell from the brain of a guinea pig – indicator fura-2

  24. Introducing large molecules into cells

  25. Laser tweezers manipulating objects with higher refractive index within the cell

  26. Total internal reflection fluorescence (TIRF) microscopy can visualize single molecules

  27. Single molecules can be manipulated by atomic force microscopy (AFM)

  28. Pulse-chase experiments: use of radioisotopes

  29. Autoradiography: radioisotopically-labeled molecules

  30. Transmission electron microscope

  31. Electron microscopy Limit of resolution 0.2 nm (seen on a gold layer) Chemical fixatives

  32. A root tip cell visualized by electron microscope (Os stained)

  33. Actin filaments by transmission EM

  34. Localizing proteins by immunogold staining

  35. 3D reconstruction from serial sections Electron microscope tomography

  36. 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)

  37. Scanning electron microscope

  38. Scanning electron microscopy Stereocilia from a hair cell in the inner ear of a bullfrog Differential-interference contrast LM Transmission EM Scanning EM

  39. Nuclear pore by scanning electrone microscopy

  40. Freeze-fracture and freeze-etch electron microscopy

  41. Thylakoid membranes of the chloroplast by freeze-fracture EM

  42. Protein filaments in an insect muscle by freeze-etch EM

  43. Single particle reconstruction

  44. 3D structure of 70S ribosome and RF2 from E. coli by cryo-EM tomography (combined from 20,000 ribosomes)

More Related