I' Basics of Electron Microscopy

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I. Basics of Electron Microscopy

• Electro-magnetic lenses
• Scanning Transmission EM (STEM)
• Mass determination
• Elemental mapping
• Scanning EM (SEM)
• Surface relief
• Transmission EM (TEM)
• Structures
• Imaging or Diffraction
• 0.03 Å theoretical resolution1.0 Å practical
  resolution

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II. Electron Source

• Tungsten filament (thermionic)
• Tungsten crystal (field emission) 100x brighter
• Electrons Accelerated to defined energy behave as
  a wave.
• ??kV (100 1000 kV with 200kV-gt 0.025 Å)

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III. Lenses and deflection coils

• Lenses focus beam and form images
• Coils control beam direction

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IV. Column Design

• Inverted light microscope
• Gun
• Condenser lens(es)
• Objective lens
• Projection lens (ocular)
• Apertures

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V. Detectors

• Film 3.5 x 4 cm, small grain size
• CCD fast readout, good dynamic range, poor
  resolution
• Imaging plates excellent dynamic range, high cost

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VI. Specimen Preparation

• Vacuum Conductivity Contrast Radiation
  Damage
• SEM gold coat
• STEM freeze-dry, carbon coat
• TEM
• plastic section positive stain
• Protein suspension/crystal
• Negative stain
• Frozen-hydrated

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VII. Structure Determination

• Tissue
• tomography
• Crystals
• crystallography
• Isolated complexes
• single particle analysis

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VIII. 3D reconstruction

• Projection theorem

• Tilt Reconstruction

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IX. 2D crystallography

• Fourier Transform composed of 2D lattice lines

• Individual Projections sample lattice lines

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IX. 2D Crystallography (cont)

• Electron Diffraction amplitudes
• Images phases

• Max tilt /- 60º missing cone

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X. Helical Reconstruction
planar 2D lattice
Cylindrical (helical) 2D lattice
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X. Helical reconstruction (cont)
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X. Helical reconstruction (cont)
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X1. Electron tomography
EM control
Data collection
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coordination
-60
CCD control
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3D-reconstruction by back projection
medical imaging - CAT scan, MRI
mathematical foundation
Horiz proj
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Reconstructed Image Segmentation
Plakoglobin plakophilin
cadherin
membrane
desmoplakin
Sample Thickness 50 nm CCD DATA
0.931 nm/pixel Theoretical resolution
2.1 nm Practical Resolution 4-5nm or better
Intermediate Filament
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