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