1. Wah Chiu Basics in CryoEM Operations July 26, 2005

2. What are the major components in a TEM Vacuum System Illumination System Cold trap Specimen cryo holder Specimen tilting Objective lens (focusing and astigmatism) Magnification Recording medium

3. Vacuum System Rotary pump Diffusion pump Ion pump Turbo molecular pump Column bake-out every 6-10 months

4. Illumination and Imaging System Condenser aperture (50-100  m) Spot size : depending on microscope Brightness : specimen dose tolerance Objective aperture: 60  m

5. Cold Traps Where are they? –Between pump and column –Around specimen area How to care of the cold trap? –Daily warm up for specimen cold trap –Monthly warm up for the other traps

6. Cryo-specimen holder How to clean it –Sonicate with freon for 5 minutes How to generate the zeolite in the cryoholder How to keep it mechanically stable –O-ring –Grease –Clip ring How to trouble shoot it –Take an FFT of c-film image at 400Kx

7. How to work out specimen dose Table to relate specimen and image dose via magnification relationship – (I image = I specimen /Mag 2 ) Calibrate the reading (current density) on the microscope panel Specimen radiation tolerance level at different specimen temperatures (15-30 e/A 2 )

8. How to decide on defocus Resolution desired (1-5  m) Voltage of the microscope (100-300kV) Concept of CTF in diffraction space http://ncmi.bcm.tmc.edu/homes/wen/ctf

9. How to decide on magnification Resolution (15-130 kx) Recording medium (film vs CCD) Available scanner for film digitization

10. How to record data Photographic films CCD camera

11. How to do Low Dose Search mode in SA mode with low dose rate –Look for holes with apparent specimens Focus mode in the selected magnification –Eye balled the choice Imaging mode –Wait ~1 minute for specimen stability

12. Frequently Encountered Problems Ice too thick or no specimen Molecules do not stick to the holes Cryo holders are not pumped Specimen drift Charging Non-optimal defocus choice Astigmatism Dose not right Film camera jammed Availability of the instrument at your schedule

13. Database Usage

14. Dark room rules Check the developer and water temperature at 20C before use Reload the films with the film emulsion right- side-up and put them in dessicator Collect the films when dried Wear gloves throughout Clean up after use

15. Sign-up Rules Every Friday at 11 am Can work in the night and weekend times