C9940 3-Dimensional
Transmission Electron Microscopy
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Sylabus
 Lecture 1: Anatomy of the electron microscope
 Lecture 2: Electron microscopy in biological applications,
sample preparation for electron microscopy
 Lecture 3: Principles of image formation, contrast transfer
function, image alignment in 2D
 Lecture 4: 3D reconstruction methods in TEM
 Lecture 5: Interpretation and optimization of cryo-EM maps
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Resources
Literature:
– J. Frank, Three-dimensional electron microscopy of macromolecular assembliesvisualization
of biological molecules in their native state.
– J. Frank, Electron Tomography: Methods for Three-Dimensional Visualization of Structures in
the Cell
– Williams et al., Transmission electron microscopy
Video courses (youtube):
– Grant Jensen
– NRAMM SEMC
– Cryo-EM14 (LMB)
– Cryo-EM17 (LMB)
C9940 3-Dimensional
Transmission Electron Microscopy
Lecture 1: Electron Microscope
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Electron
rest mass: 9.109 e-31 kg
charge: -1.61 e-19 C
spin: 1/2
Electron in electric and magnetic field
(Lorentz force)
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Electron
Dual character of electron
Acceleration Voltage [kV] Non-relativistic wavelength [pm] Relativistic wavelength [pm]
2 27.35 27.32
20 8.65 8.57
100 3.87 3.69
200 2.73 2.50
300 2.23 1.96
Abbe diffraction limit
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Scales in electron microscopy
1 mm 1 um 1 nm
Tick (ESEM) Plant cell (TEM)
Plant (SEM)
Bacteria (SEM)
Virus (TEM)
Bacteriophage (TEM)
RNA polymerase (TEM)
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Electron microscopes
TEM SEM
STEM
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Electron microscopes
TEM SEM
STEM
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Electron microscopes
TEM FIB/SEM
STEM
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Interaction of electrons with matter
Mean free path
Williams et al. 2009
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Interaction of electrons with matter
SEM
TEM Williams et al. 2009
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Optical vs. TEM microscope
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Optical vs. TEM microscope
source
condensor
objective
detector
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Electron source
- thungsten filament
- LaB6 crystal
- Field Emission
Gun
Probe
size
Work
function
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Electron source
- thungsten filament
- LaB6 crystal
- Field Emission
Gun
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Electron source - LaB6
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Electron source - FEG
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Electron source - accelerator
E=U.
e
U=300kV =>
λ=1.97pm
U=200kV ??
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Lenses – ray diagram
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Electromagnetic lenses
Lorentz
force:
Magnetic lenses rotate
image
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Electromagnetic lenses
Power of the magnetic lens can be
changed
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Electromagnetic lenses
Illumination on the detector changes with change of lens
power
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Lens assembly - appertures
Apperture size: ~100um
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Lens assembly - appertures
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Lens assembly - stigmators
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Lens aberrations - chromatic
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Lens aberrations - spherical
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Detectors
CCD – charge coupled device
CMOS – complementary metal oxide semiconductor
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Detectors
DQE – detective quantum efficiency
- probability to detect an
electron
- DQE ~ sin(x)/x
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Vacuum system
- roughing pump (105
– 10-4
Pa)
- turbo molecular pump (10-2
– 10-
8
Pa)
- ion getter pump ( up to 10-9
Pa)
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Vacuum system
- roughing pump (105
– 10-4
Pa)
- turbo molecular pump (10-2
– 10-
8
Pa)
- ion getter pump ( up to 10-9
Pa)
90.000
rpm
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Vacuum system
- roughing pump (105
– 10-4
Pa)
- turbo molecular pump (10-2
– 10-
8
Pa)
- ion getter pump ( up to 10-9
Pa)
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Vacuum system
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TEM
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TEM
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TEM
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TEM
Differential pumping apperture
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TEM
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TEM
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SEM
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SEM
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Dual beam FIB/SEM
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SEM - detection
- Everhart-Thornley Detector (ETD)
- Ion Convertion to Electron Detector (ICE)
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SEM - detection
- Concentric Backscatter detector