3. Electron Microscopes were developed due to limitations
of Light Microscopes.
Scientific desire to see the fine details of the interior
structures of organic cells.
10,000x plus magnification is not possible using Light
Microscopes.
History of EM
Decrease
wavelength range
Increase
resolution limit
λ = 0.00251 nm (200 keV) R 0.2–0.3 nm
~
5. Light Microscope Electron Microscope
Light Source Visible Light Electrons
Lens Type Glass Electromagnets
Magnificatio
n Method
Lens Movement
Current changes through
lens coil
Sample
viewing
Eyepiece
Camera
Fluorescent screen
X-ray camera
Vacuum No Yes
Electron vs Light Microscopy
6. Physics of electron microscopy: the electron source
Electron sources:
• W
• LaB6
• Field Emission Gun
7. Physics of electron microscopy: the need for vacuum
- Enable the electron beam to travel in straight lines (10-5 mbar)
- Tungsten filaments burn out in air
- Columns must be kept dust free
2-fold pumping
- mechanical pump, stage-1
- membrane diffusion pump (or turbo pump), stage-2
8. electrons are charged, and are therefore
deflected when they cross a magnetic field
Physics of electron microscopy: the electron lenses
electron beam
soft iron pole piece
electrical coil
10. Cryo-EM in Pavia
ThermoFisher Glacios (FEG 200 kV, Cryo-TEM)
Typical image Pixel size 0.1 Å
JEOL JEM-1200-EX (LaB6 120 kV)
Typical image Pixel size 7 Å
11. Electron microscopes: TEM vs SEM
Detection of
electrons scattered by
the specimen
Detections of back-
scattered and
generated electrons
TEM Resolution is 10X higher than SEM
13. EM images are in contrast scale ONLY
Colors are a prerogative of visible light
14. Structural Biology with CryoEM
O’Reilly et al., Science (2020)
Patel et al., Science (2021) Ke et al., Nature (2020)
Abdella et al., Science (2021)
Wagner et al., Nature (2020)
16. Techniques for image reconstitution
• Single-particle reconstruction
• Slow
• Simpler setup
• Higher resolution
• Requires multiple objects
• Tomography
• Fast
• Complex Setup
• Lower Resolution
• All on a single object
TEM requires multiple orientations of the same object
to obtain complete structural reconstruction
18. • High contrast image
• No special temperature control
• Essentially no radiation damage
• Particle distorted
• Image = stain “shell” around the
particle
• Low resolution: 20-15 Å
• Great choice for initial sample
screening
• Low contrast image
• Sample maintained at cryogenic
temperature (85 K)
• High radiation damage
• Particle undistorted
• Image is of the actual particle
• Higher resolution obtained: 15-4 Å
• Best choice for reconstruction
Negative stain vs. Cryo-EM
22. Adattato da Science Magazine
http://www.sciencemag.org/news/2017/10/cold-clear-view-life-wins-chemistry-nobel
http://fornerislab.unipv.it
23. Cryo-EM sample prep
deposit blot plunge
• 3-4 µL of highly pure sample in solution
• Decide humidity, blot force, blot time
• Manipulation after plunge-freezing to prepare
sample holder cassette
24. Adattato da MRC LMB Cambridge
http://www2.mrc-lmb.cam.ac.uk/research/scientific-facilities-and-support-services/electron-microscopy/
http://fornerislab.unipv.it
28. • Collect image set (20-100 images, vary focus)
• Correct motion, astigmatism, defocus
• Perform contrast-transfer-function (CTF) correction for
each image
• Pick Particles (4000-100,000)
• Center, align, classify, make “class averages”
The single-particle EM Experiment
29. Fast readout, better quality
Software-based motion correction can (partically) account for molecular drift
Direct electron detectors are game-changers
Current frame rate (Gatan K3: 1500 frames/sec)
