1. The electron microscope: the contrast transfer function (CTF) Javier Vargas Centro Nacional de Biotecnología-CSIC jvargas@cnb.csic.es

2. What is an electron microscope? Why electron microscopes?

3. What is an electron microscope? An electron microscope is a tool for obtaining projection images of very small biological objects Evolution

5. Important characteristics of cryoEM images 1) Projection images 2) Phase contrast and WPA

6. Projection images

7. Images are formed by phase contrast

8. projection images

9. 3DEM as an inverse problem

10. Limitations of cryo-electron microscopy 1) Radiation damage.

11. Limitations of cryo-electron microscopy 1) Radiation damage. 2) Electron lenses.

12. Limitations of cryo-electron microscopy 1) Radiation damage. 2) Electron lenses. 3) The samples are very small.

13. Limitations of cryo-electron microscopy 1) Radiation damage. 2) Electron lenses. 3) The samples are very small. 4) Presence of ice.

14. Limitations of cryo-electron microscopy 1) Radiation damage. 2) Electron lenses. 3) The samples are very small. 4) Presence of ice. 5) Charging: non conductive samples charge up and act like lenses.

15. Limitations of cryo-electron microscopy 1) Radiation damage. 2) Electron lenses. 3) The samples are very small. 4) Presence of ice. 5) Charging: non conductive samples charge up and act like lenses. 6) Expensive. Titan Krios around 1000€/day (NeCEN)

16. Limitations of cryo-electron microscopy 1) Radiation damage. 2) Electron lenses. 3) The samples are very small. 4) Presence of ice. 5) Charging: non conductive samples charge up and act like lenses. 6) Expensive. Titan Krios around 1000€/day (NeCEN) We obtain very noisy images

17. Limitations of cryo-electron microscopy 1) Radiation damage. 2) Electron lenses. 3) The samples are very small. 4) Presence of ice. 5) Charging: non conductive samples charge up and act like lenses. 6) Expensive. Titan Krios around 1000€/day (NeCEN) We require a lot of images

18. Why use electrons?: AdvantagesDisadvantages Visible light

19. Why use electrons?: AdvantagesDisadvantages Visible light Not very damaging Easily focused

20. Why use electrons?: AdvantagesDisadvantages Visible light Not very damaging Easily focused Long wavelengths (~400 nm) Poor Penetration X rays

21. Why use electrons?: AdvantagesDisadvantages Visible light Not very damaging Easily focused Long wavelengths (~400 nm) Poor Penetration X raysSmall wavelength (Angstromgs) Good penetration

22. Why use electrons?: AdvantagesDisadvantages Visible light Not very damaging Easily focused Long wavelengths (~400 nm) Poor Penetration X raysSmall wavelength (Angstromgs) Good penetration Hard to focus Damage Samples

23. Why use electrons?: AdvantagesDisadvantages Visible light Not very damaging Easily focused Long wavelengths (~400 nm) Poor Penetration X raysSmall wavelength (Angstromgs) Good penetration Hard to focus Damage Samples ElectronsSmall wavelength (pm) Can be focused

24. Why use electrons?: AdvantagesDisadvantages Visible light Not very damaging Easily focused Long wavelengths (~400 nm) Poor Penetration X raysSmall wavelength (Angstromgs) Good penetration Hard to focus Damage Samples ElectronsSmall wavelength (pm) Can be focused Damage Samples Poor Penetration

28. Electrons energy Electrons wavelength

29. Why electron microscopes?

30. electron microscopy light microscopy Why electron microscopes?

31. electron microscopes Electrons energy Electrons wavelength

32. The contrast transfer function (CTF)

33. Main idea

34. Perfect system: the image of a point is a point Real system: the image of a point is a spot Hubble telescope was myopic !!!

35. Perfect system: the image of a point is a point Real system: the image of a point is a spot Hubble telescope was myopic !!!

36. Perfect system: the image of a point is a point Real system: the image of a point is a spot

37. Introduction

38. There is no any perfect real system!!

39. Transfer functions Frequency increase

40. Transfer functions basstreble

41. Transfer functions

42. CTF

43. Transfer functions

44. 8 Ǻ With phase-plate

49. Matlab Script to simulate the CTF

50. How a real microscope distort the ideal projections? Assuming a LTI system

52. Defocus = 0 A. Astigmatism = 0 A.

53. Defocus = 0 A. Astigmatism = 0 A.

54. Defocus = 0 A. Astigmatism = 0 A.

55. Defocus = 1000 A (0.1 um). Astigmatism = 0

56. Defocus = 10000 A (1um) Astigmatism = 0

57. Defocus = 5500 A (0.55 um) Astigmatism = 4500 A (0.45 um)

58. How a real microscope distort the ideal projections? Assuming a LTI system

59. A little bit of theory… CTF is important because: 1. Image restoration (deconvolution) 2. Micrograph screening

60. 1. Image restoration (deconvolution) Problem: CTF have zeros Is not well defined at some points

61. Wiener filter No problems in frequencies 1. Image restoration (deconvolution)

62. CTF is important because: 2. Micrograph screening

63. CTF is important because: 2. Micrograph screening

64. Questions ?