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Wah Chiu

Wah Chiu. Basics in CryoEM Operations July 26, 2005. 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. Vacuum System. Rotary pump

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Wah Chiu

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  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 (50-100mm) • Spot size : depending on microscope • Brightness : specimen dose tolerance • Objective aperture: 60mm

  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 • (Iimage = Ispecimen/Mag2) • Calibrate the reading (current density) on the microscope panel • Specimen radiation tolerance level at different specimen temperatures (15-30 e/A2)

  8. How to decide on defocus • Resolution desired (1-5 mm) • 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

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