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

Wah Chiu

Basics in CryoEM Operations

June 8, 2007

wah@bcm.edu

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

Vacuum System

• Rotary pump

• Diffusion pump

• Ion pump

• Turbo molecular pump

• Column bake-out every 6-10 months

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

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

TEM Con

Types of Electron Sources

• Tungsten filament

• LaB6 filament

• Field emission gun

Electron Emission

J= AT2Exp(-/kT)

J = Emission current densityA= Constant

T = Temperature = Work function

K= Boltzmann constant

Electron Brightness

Current density per unit solid angle

= Number of electrons per unit time per unit area per unit solid angle (Ampere/cm2/strd)

The smaller the solid angle, the brighter the beam can be

Temporal and Spatial Coherence

• Temporal: Monochromatic electrons (i.e. small energy spread)

• Spatial: Parallel electron beam (i.e. small angular spread)

C1, C2 spot sizeC3 brightnessCM alpha angle

OL and OM forDifferent mags.

IS1 image shift by usersIS2 image shift by engineers

Specimen Dose

• Ispecimen /M2 = Iimage

• Idetectpr/M2 = Iimage

How to measure the dose rate?

• Current density on the screen

• Back calculate the dose rate on the specimen by considering the magnification

What magnification to use?

• D0 = Di /M (Optics)

• Dp = ½ - 1/3 D0 (Sampling Theorem)

• If D0 = 10 Å, then Dp = 10/3 Å

• If Di = 15 x 104 Å (as governed by the detector),

then M = 15x104x3/10=45x103

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