Stefan Raunser - Intro to cryo-EM

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Impact Factor of IUCrJ in 2017 - 5.8.

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4. Resolution limit of a light
microscope is around 10 nm
Why can you not recognize
small details in a light
microscope?
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5. d =
0.61 λ
n sin α
Resolution criteria of Ernst Abbe:
d: The smallest still visible distance between two spots.
Numerical aperture
Wave length
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6. d =
0.61 λ
n sin α
0.61 200 nm
1.42
d = = 86 nm
Wave length
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NA = ~ 1.2 – 1.4 for a good light microscope

7. The resolution of electron microscopes is
roughly 1000x
higher than in light microscopes
Why?
Electron microscopes use electrons
instead of light.
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8. m=m0/ 1-(v/c)2
Wave
Impulse: p = h/λ
Wave length: λ
Frequency: ν
Speed: v = ν λ
Particle
Mass:
Speed: v
Impulse: p = m v
Properties of electrons
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9. Resolution of the electron microscope
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d =
0.61 λ
n sin α
100 keV,
If a numerical aperture as for
light microscopes is used
Because of strong lens aberrations smaller apertures
have to be used:
d = 2 – 3 Å at 100 keV
d < 1 Å at 1000 keV
d =
0.61 0.0037 nm
1.4
d = 0.0016 nm = 0.016 Å

10. The Millimicron
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11. Dimensions in the life sciences
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12. Electric and magnetic fields act on charged
particles in a vacuum similar to optical lenses
acting on the light beam. This was first described
and calculated by Hans Busch in 1926.
He is therefore regarded as the founder of electron
optics.
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14. Sketch of the first
electron microscope
Ernst Ruska, 1931
Nobel prize 1986
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15. By varying the current in the coil, the strength
of the magnetic field and thereby the focal
length of the lens ("refractive power") can be
changed.
A coil, which is surrounded by an iron shield,
is traversed by an electric current.
The magnetic field is concentrated by
magnetic pole shoes.
The magnetic field is rotationally symmetric.
The magnification of a magnetic lens can
therefore be continuously changed by
changing the lens current.
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16. Magnetic lenses cannot be produced with the same precision as glass lenses.
Therefore, the lenses of the electron microscope are rather poor
compared to those of the light microscope, especially at longer distances
from the optical axis.
Due to the short wavelength of the electron, the effective deflection angles of the
electron beam are very small (in the range of a few mrads (1mrad = (180/
The magnification power of a magnetic lens is quite low (50x for a typical
objective lens). High magnifications are achieved by connectin a series of
lenses that increase the first intermediate image produced by the
objective lens.
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18. Screen
Detector
Vacuum pumps
Projection chamber
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21. A field emitter is used to generate an electron beam that
has a smaller diameter,
is more coherent
and generates a current density of up to 3 orders of
magnitude larger than conventional cathodes:
better signal-to-noise ratio
better resolution
greater reliability
longer life of the cathode
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22. Coherence
The higher the coherence of the electron beam, the higher the resolution.
Incoherence leads to chromatic aberration.
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23. Envelope function
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Tungsten filament

24. Envelope function
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Field emissions gun

25. Condenser:
Combination of two magnetic lenses and two apertures: The
diameter of the beam can be condensed from 1 mm to 1 nm.
Objective lens:
Limits the resolution to ~ 0.1 - 0.4 nm, depending on the lens
parameters. The sample is inserted into the objective lens and
located between the pole pieces.
Intermediate lenses and projection lens:
Different lenses produce final magnifications from 100x to
1,000,000x. The quality of these lenses is less critical because the
deflection angle is much lower than in the objective lens.
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26. Lens aberrations
Spherical aberration, axial astigmatism, coma and chromatic aberration are the
major aberrations in electron microscopy.
Spherical aberration:
The spherical aberration reduces the focal length of
electron beams passing through outer zones of the
lens.
The spherical aberration coefficients (Cs) of objective
lenses are usually between 0.5 - 4 mm.
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27. Axial astigmatism:
Electron beams, which move in two mutually perpendicular planes,
have different focal lengths.
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Lens aberrations

28. Lens aberrations
Chromatic aberrations:
Variations of the electron energy and lens currents cause a variation
of the focal length. This means that chromatic aberrations are
generated by:
Fluctuations of the acceleration voltage
Energy distribution of the electron beam
Energy losses due to inelastic scattering
Fluctuations of the lens current.
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29. Lens aberrations
Coma or comatic aberrations:
A lens aberration occurring in the part of the image field that is some
distance from the principal axis (off-axis) of the system.
It results from different magnifications in the various lens zones
Most severe when the microscope is not properly aligned.
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Extra-axial object points
appear as short, comet-like
images (to have a tail (coma)
like a comet).

30. Next talk: Greg McMullan

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Optimal single particle cryo-EM equipment
JEOL JEM 1400 LaB6
FEI G2 Spirit
FEI Titan KRIOSFEI Talos Arctica

37. Which biological samples can you examine with a
transmission electron microscope?
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SAMPLES
Proteins/protein crystals
Protein complexes
Liposomes/proteoliposomes, viruses
Microorganisms
Sections of cells
Sections of tissue
Sections of small organisms
Size

38. Preparation of proteins, protein complexes and
liposomes/proteoliposomes
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Negative contrast EM
Cryo-EM
Preparation of proteins, protein complexes and
liposomes/proteoliposomes for electron microscopy
Single particle electron microscopy

39. Metal grids as specimen carrier
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Negative stain EM
Samples are embedded in heavy metal layer
(for example: uranyl formate, uranyl acetate,
phospho tungstate, lead citrate)
Advantages:
• Easy
• High contrast (small proteins can be visualized)
Disadvantages:
• Only surfaces of proteins.
• Particle deformation by dehydration
• Resolution is limited: ~ 20 Å

41. Negative stain EM
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42. Negative stain EM
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50 nm

43. Negative stain EM
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Presentation by Steve Ludtke

44. Cryofixation
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Advantages:
No deformation of the particles.
High to very high resolution.
Disadvantage:
Multiple exposures are difficult
Low contrast (not useful for small proteins)
1 μm
20 nm

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Sample
Cryo Preparation
Electron Microscopy and Imaging
Image Processing
Model Building and Interpretation

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54. Maps achieving given
resolution levels:
Resolution trends:
Courtesy of H. Stahlberg

55. EM squirrel X-ray squirrel
These low-resolution
Bloboligists…cryo-EM
is useless.

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Cryo-EMX-ray Crystallography
(& NMR)
Correlative light and
electron microscopy
Atomic Level Cellular Level
Light microscopy
Structural Hybrid
Approach
Cryo-EM
Cryo-ET + Subtomogram averaging

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