1. Wah Chiu Basics in CryoEM Operations June 8, 2007 wah@bcm.edu

2. Lesson 1: Introduction to basic cryo-EM Vacuum Cold trap (Anti-contaminator) Electron gun Lens system Deflectors Specimen transfer Magnification Recording camera Dose monitor Positions of all knobs

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

4. Pumps Mechanical pump : 100-0.1 Pa Diffusion pump: 0.1 – 10 -4 Pa Trubo molecular pump: 10 -7 - 10 -8 Pa Ion pump 10 -4 – 10 -7 Pa Cryo pump 10 -4 – 10 -7 Pa Mechanical and diffusion pump forces the molecules out the system Ion and cryo pump adsorbs the molecules till warm up

7. 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

13. TEM Con

18. Types of Electron Sources Tungsten filament LaB6 filament Field emission gun

19. Electron Emission J= AT 2 Exp(-  /kT) J = Emission current density A= Constant T = Temperature  = Work function K= Boltzmann constant

20. Electron Brightness Current density per unit solid angle = Number of electrons per unit time per unit area per unit solid angle (Ampere/cm 2 /strd) The smaller the solid angle, the brighter the beam can be

21. Temporal and Spatial Coherence Temporal: Monochromatic electrons (i.e. small energy spread) Spatial: Parallel electron beam (i.e. small angular spread)

32. C1, C2 spot size C3 brightness CM alpha angle OL and OM for Different mags. IS1 image shift by users IS2 image shift by engineers

41. Specimen Dose I specimen /M 2 = I image I detectpr /M 2 = I image

42. How to measure the dose rate? Current density on the screen Back calculate the dose rate on the specimen by considering the magnification

44. What magnification to use? D 0 = D i /M (Optics) D p = ½ - 1/3 D 0 (Sampling Theorem) If D 0 = 10 Å, then D p = 10/3 Å If D i = 15 x 10 4 Å (as governed by the detector), then M = 15x10 4 x3/10=45x10 3

45. Expected Resolution On The 4kx4k Gatan CCD Camera with 15  m pixels on the JEM2100