Federico Forneris is a researcher at the Armenise-Harvard Laboratory of Structural Biology who studies cryo-electron microscopy (cryo-EM). Cryo-EM uses electron microscopes instead of light microscopes to image biological samples at higher resolutions. Electron microscopes require vacuum conditions and use electron beams rather than visible light. Cryo-EM flash freezes samples to image them without distortion at resolutions as high as 4 Angstroms, enabling structure determination of large biomolecules. Forneris' lab in Pavia, Italy uses cryo-EM techniques like single-particle reconstruction to study protein structures important for human health.

1. Federico Forneris, PhD
The Armenise-Harvard Laboratory of Structural Biology
http://fornerislab.unipv.it
Cryo-EM

2. The light spectrum
D≈ =
λn
(NA)2
D≈(min) =
λ
2

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
~

4. EM is microscopy with X-rays

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

9. The Electron Microscope

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

12. SEM examples

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)

15. TEM requires multiple projections for correct reconstitution

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

17. Sample preparation for EM analysis
• Vacuum
• Dehydration
• Physical damage
• Accelerated electrons
• Radiation damage
• Contrast!

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

19. Sample support for EM analysis
EM grids

20. Cryo-EM sample prep
Carbon is hydrophobic, protein solutions are hydrophilic

21. Negative Stain 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

25. The single-particle EM Experiment
5 degrees of freedom
to determine

26. Sample preparation for EM analysis

27. The lack of contrast in EM

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)

30. The single-particle EM Experiment

31. Adattato da MRC LMB Cambridge
http://www2.mrc-lmb.cam.ac.uk/research/scientific-facilities-and-support-services/electron-microscopy/
http://fornerislab.unipv.it

32. Averaging

33. Class Averaging

34. Projection matching and angular refinement

35. Density map from 2D projections

36. The single-particle EM Experiment
Collect Data 2-3 days
From days to
months
Pick Particles
Make Model
Refine
Average
Classify
Reconstitute

37. TEM requires multiple projections for correct reconstitution

38. (Cryo)Electron Microscopy
http://fornerislab.unipv.it
Adapted from A.J. Jakobi

39. (Cryo)Electron Microscopy
http://fornerislab.unipv.it
Adapted from A.J. Jakobi

40. (Cryo)Electron Microscopy
Adapted from A.J. Jakobi
http://fornerislab.unipv.it

41. EM Artefacts

42. Model Bias affecting noise
Averaging 1000 images of PURE WHITE NOISE using a model
intentionally to introduce bias
Shatsky M, J. Struct Biol, 2009