1. History of cryoEM & facility tour
2. Electron microscope
3. Sample preparation techniques
4. Image analysis I
5. Image analysis II
6. 3D reconstruction
7. Single particle analysis
8. Tomography I
9. Tomography II
10. 3D visualization and segmentation
11. Hybrid methods
12. Practicals: Image analysis
13. Journal club and summary
Outline of the CryoEM Course
CryoEM Facility: Requirements
General Safety:
‐ high voltage and X‐ray shielding
‐ SF6 and oxygen detectors and exhaust
‐ precautions for using liquid nitrogen
Microscope Room:
‐ low level of vibrations and noise 
‐ low level of stray electromagnetic fields
‐ special requirements for air and heat flow
‐ cooling chillers located in an adjacent room
‐ compressed air supply for pneumatics
Factors influencing performance:
‐ mechanical or acustic vibrations
‐ electromagnetic interference (EMI)
‐ thermal fluctuations
Vibrations
Contra‐measures:
‐ the microscope room is located in the basement
‐ the microscope sits on a separated slab of concrete
‐ installations for passive dumping of vibrations (acoustic absorption panels)
Specifications: Entire spectrum  < 70dB Third‐octave bands < 55 dB
Electromagnetic Fields
Contra‐measures:
‐ microscope suite/bunker outside of the main building frame
‐ passive shielding (Faraday cage) of the microscope room
‐ active shielding compensating for EMI in real time
Specifications:
Microscope Type  Horizontal  Vertical 
Tecnai 120‐200 FEG, TEM 80 nT p‐p 95nT p‐p
Titan 80‐300 FEG, TEM  80nT p‐p  80nT p‐p
Titan 80‐300 FEG, GIF 0.3 eV 50nT p‐p  75nT p‐p
Air and Heat Circulation
• Microscope produces significant heat dissipation (~6 kW).
• Dust free, air‐conditioned room is strongly recommended.
• The microscope column should not be in the flow of air circulation.
• Cooling units (chillers) should be located in a different room.
• Chemical/oil resistant and antistatic floor covering is needed.
CryoEM Facility: Plan of the CF in CEITEC
Electron Microscopes
Titan Krios with a direct detector => determination of near-atomic structures
Tecani F20 with a 4k CCD => routine cryoEM to subnanometer resolutions
Versa3D SEM/FIB with cryo transfer chamber => FIB milling of vitrified cells