1. Electron Microscopy Jim Atherton

2. Development of Light Microscope 1590 Hans Zacharias Janssen 1660 Robert Hook o Onserved cells (cork) 1 Klein, Aaron E. The Electron Microscope: A Tool of Discovery. New York,McGRaw-Hill 1974.

3. Development of Light Microscope 1670 Anton van Leeuwenhoek o Single lens o Observed bacteria (cavorting beasties) 1877 Ernst Abbe o Oil immersion maximized magnification 2

4. Development of Light Microscope Magnification Maximized by 1900 Resolution improvements continued Bacteria visible, viruses not 3

5. http://chemwiki.ucdavis.edu/Physical_Chemistry/Spectroscopy/Fundamentals/Electromagnetic_Radiation 4

6. 5

7. Development of Electron Microscope 1860’s o Electrodes in vacuum tube o Applied current o Radiation at cathode (green glow at opposite wall) 6

8. Development of Electron Microscope 1897 Joseph John Thompson o Deflected cathode rays with magnets o Negatively charged o Termed electron 7

9. Development of Electron Microscope 1923 Louis de Broglie o Electrons have wave and particle nature o Determined electron wavelengths 8

10. Development of Electron Microscope 1927 Germer H. Busch, Ernst Ruska o Lens: Magnetic and Electric fields o Lenses focus electrons 1931 – 1939 o Invention of electron microscope 9

11. Development of Electron Microscope First EMs resolution 100 Angstroms Now 1A Better resolution with: o Higher voltage = shorter electron wavelength 10

12. Development of Electron Microscope 1986 Nobel prize o Ernst Ruska o Gerd Binning and Heinrich Rohrer Scanning Tunneling Microscope 11

13. De Broglie’s Electron λ λ = wavelength h = Planck’s constant = 6.625 x 10 -34 12 p = electron momentum http://advanced-microscopy.utah.edu/education/electron-micro/

14. Electron λ related to voltage (1) 13 λ = wavelength e = electron charge = 1.6 x 10 -19 V = voltage m = mass = 9.1 x 10 -31 kg v = velocity

15. Electron λ related to voltage (2) 14

16. Electron λ related to voltage (3) 15

17. Electron λ related to voltage (4) 16 At: 100 keV 200 keV 300 keV 3.7 pm (picometer) 2.51 pm 1.96 pm

18. 17 Fluorescent Screen CCD Camera

19. Resolution 18 d = distance to resolve between two points n = lens refractive index α = semi angular aperture Lower wavelength, lower d, better resolution

20. EM versus Light Objective lens limits resolution to 1 angstrom 19 Light MicroscopeElectron Microscope Wavelength400-800nm0.0037nm Resolving Power200nm< 1nm

21. Transmission Electron Microscope 20 Illuminating System Specimen Stage Imaging System Image Recording System http://www.britannica.com/EBchecked/topic/602949/transmission-electron-microscope-TEM

22. 21 http://www.revisionworld.com/a2-level-level-revision/physics/fields-0/electric-fields

23. Illuminating System: Electron Gun 22 http://www.revisionworld.com/sites/revisionworld.com/files/imce/electron-gun.gif Types: o Tungston Hairpin o Lanthanum Hexaboride o Field Emission

24. Electron Gun Work Function: o Energy to remove electron from metal o Experimentally derived 23 Gun MaterialWork Function (eV) Tungsten4.55 Lanthanum Hexaboride2.66-2.91 Goebel, Dan M., Emily Chu. High Current Lanthanum Hexaboride Hollow Cathodes for High Power Hall Thrusters. International Electronic Propulsion Conference. Germany. 2011.

25. Electron Gun Thermoionic emission Current enhanced with Schottky emission 24 J = current density A = constant r = electron reflection coefficient T = temperature k = Boltzman’s constant f = work function

26. Beam Characteristics Wavelength o Affects resolution Temporal Coherency o wavelength (or energy) range of a beam o Affects resolution 25

27. Beam Characteristics Brightness: affects magnification 26 i = beam current d = source diameter α = semi-angle of beam divergence

28. Different Gun Types 27 TungstenLaB 6 FEG Brightness (A/cm 2 str)10 5 10 6 10 8 Lifetime (h)4-100200-1000>1000 Source Size (um)30-1005-10< 5 x 10 -3 Energy Spread (eV)1-31-21 Vacuum (Torr)10 -5 10 -7 10 -11

29. Vacuum 28 Vacuum Torr Atoms/cm 3 Distance between atoms (m) Mean Free Path 76010 19 5 x 10 -9 10 -7 10 -2 10 14 2 x 10 -7 10 -2 10 -7 10 9 1 x 10 -5 10 3 10 -10 10 6 1 x 10 -4 10 6

30. Illuminating System: Lens http://web.eng.fiu.edu/wangc/Lenses%20and%20Apertures%20of%20A%20TEM.pdf Condensor Lens (4)

31. Electromagnetic Lens Iron pole houses coil of copper wires Current through copper wires creates magnetic field 30 http://web.eng.fiu.edu/wangc/Lenses%20and%20Apertures%20of%20A%20TEM.pdf

32. Condensor Lens Magnetic field of lens: o Focal length changed by varying current 31 μ o = permeability of free space I = current B = magnetic field strength

33. Question How is focal length of electromagnetic lenses controlled? Change magnetic field current 32

34. Condensor Lens Effectively, it decreases source diameter Focuses electron beam Limits current in beam (with aperture) Controls beam diameter 33

35. Condensor Lens 34 Double Condensor Lens with Aperture http://www.matter.org.uk/tem/lenses/first_condenser_lens.htm C-1 1.smaller gun crossover image (smaller diameter of source brightness equation) 2.Control minimal spot size

36. Condensor Lens 35 Double Condensor Lens with Aperture C-2 controls: 1.Convergence of beam at specimen 2.Diameter of specimen illuminated area

37. Condensor Lens Aperture 36 Aperture controls Fraction of beam hitting specimen (illumination)

38. Electron Beam & Specimen Electron as particle o Electrostatic interaction with sample electrons o Also interact with sample nucleus 37

39. Electron Beam & Specimen Electron as wave o Interaction with atoms results in diffraction 38 http://physics.bu.edu/ulab/modern/Electron_Diffraction.pdf

40. Electron Beam and Specimen Waves 1 and 2 will constructively interfere if o Angle of incidence = angle of reflection o Difference in path length = integer 39 http://physics.bu.edu/ulab/modern/Electron_Diffraction.pdf

41. Electron Beam & Specimen 40 Brag’s Law n = integer d = distance between atomic planes θ = beam angle to plane http://physics.bu.edu/ulab/modern/Electron_Diffraction.pdf 1 2

42. 41 http://www.st-andrews.ac.uk/seeinglife/science/imaging/phase-cont/phase-cont.html

43. 42 http://philschatz.com/physics-book/contents/m42576.html

44. Imaging System 43 Illuminating System Specimen Stage Imaging System http://www.britannica.com/EBchecked/topic/602949/transmission-electron-microscope-TEM

45. Imaging System Objective Lens o Image inverted and magnified Objective aperture o Allows for selection of electrons o Improves contrast 44

46. Imaging System Intermediate lens o Magnifies image o Allows to focus either on image or diffraction pattern 45

47. Imaging System Projector lens o Further magnification 46

48. Transmission Electron Microscope 47 http://www.britannica.com/EBchecked/topic/602949/transmission-electron-microscope-TEM

49. Specimen Preparation o Similar for SEM and TEM Dehydrate (Vacuum) o Fix, cross-link specimen (glutaraldehyde) o Dehydrate with alcohol or acetone Coat with metal (gold/palladium) For TEM, cut in slices 50-100nm 48

50. Specimen Preparation TEM o For solution: Mix solution with metal salt which provides shadow around microbes 49

51. Sample Preparation SEM o Remove water Use fixative to cross link tissue together dehydrating with alcohol o Stain with heavy metals 50

52. Cost SEM o $50-100 per hour o New Tabletop SEM, COXEM, EM-30 $74,000 TEM o $50-135 per hour o $95,000: Jeol 1200EXII o $95,000: Philips EM10 o $100,000: Hitachi 7000 51 http://www.microscopemaster.com/transmission-electron-microscope.html http://www.labx.com/electron-microscope