1. 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

2. A sense of scale

4. A light microscope

5. Interference and edge effects

6. Numerical aperture

7. Obtaining contrast

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

9. Image processing

10. Tissue sectioning

11. Fluorescence microscopy

12. Fluorescence dyes

13. Immunofluorescence

14. Image deconvulsion – removing the blur by computing

15. The confocal fluorescence microscope

16. Conventional vs. confocal fluorescence microscope

17. Confocal microscopy allows 3D recontruction of objects

18. 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

19. Fluorescence resonance energy transfer (FRET)

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

21. Dynamic changes and photoactivation of GFP fluorescence

22. Fluorescence recovery photobleaching (FRAP)

23. Visualizing living cells: light-emitting indicators Sperm entry into a fish egg visualized with aequorin/Ca 2+

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

25. Introducing large molecules into cells

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

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

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

29. Pulse-chase experiments: use of radioisotopes

30. Autoradiography: radioisotopically-labeled molecules

32. Transmission electron microscope

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

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

35. Actin filaments by transmission EM

36. Localizing proteins by immunogold staining

37. 3D reconstruction from serial sections Electron microscope tomography

38. Electron-microscopic autoradiography Moving of insulin (labeled by 3 H-leucine feeding) from ER to Golgi for secretion (45 min) Staining with photographic emulsion (silver grains)

39. Scanning electron microscope

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

41. Nuclear pore by scanning electrone microscopy

42. Freeze-fracture and freeze-etch electron microscopy

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

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

45. Single particle reconstruction

46. 3D structure of 70S ribosome and RF2 from E. coli by cryo-EM tomography (combined from 20,000 ribosomes)