Lecture 2: Electron Microscope 1. Construction of the Transmission Electron Microscope 2. Interaction of Electron and Matter & Image Formation 3. Aberrations and Correctors 4. Construction of the Scanning Electron Microscope On-line cryoEM courses: Grant Jensen, CalTec: http://cryo-em-course.caltech.edu/ Sjors Scheres, MRC: http://www.ccpem.ac.uk/courses.php Eva Nogales, UCSF: https://www.youtube.com/watch?v=nkGRhYv01ag Optical vs. TEM Microscope Light Microscope schematic Source of Electrons – Electron Gun 1. Thermionic emission (Tungsten filament) 2. Shottky emission (LaB6 filament) 3. Field emission (Field Emission Gun) Thermal Emission Guns Field Emission Guns Accelerator Stacks & Coherence filament 0 kV 270 kV 240 kV 180 kV 210 kV 150 kV 120 kV 90 kV 60 kV 30 kV Wehnelt cylinder • The accelerator stacks improve both spatial and temporal coherence • Ultra-high vacuum is required for proper function of the stacks • Conditioning/baking of the gun chamber may be necessary Electromagnetic Lenses Electromagnetic Lenses Lorentz Force: Electromagnetic Lenses Electromagnetic Lenses Electromagnetic Lenses Electromagnetic Lenses Beam deflectors (tilt/shift) Beam stigmators (astigmatism) Basic TEM condenser system Sample Chamber / Vacuum System Gatan 626 side-entry holder column airlock Vacuum System 1. Rotary pump (low vacuum up to 105–10-4 Pa) 2. Diffusion pump (high vacuum 100–10-8 Pa) 3. Turbo molecular pump (high vacuum 10-2–10-8 Pa) 4. Ion getter pump (ultrahigh vacuum up to 10-9 Pa) Vacuum System 1. Rotary pump (low vacuum up to 105–10-4 Pa) 2. Diffusion pump (high vacuum 100–10-8 Pa) 3. Turbo molecular pump (high vacuum 10-2–10-8 Pa) 4. Ion getter pump (ultrahigh vacuum up to 10-9 Pa) Vacuum System 1. Rotary pump (low vacuum up to 105–10-4 Pa) 2. Diffusion pump (high vacuum 100–10-8 Pa) 3. Turbo molecular pump (high vacuum 10-2–10-8 Pa) 4. Ion getter pump (ultrahigh vacuum up to 10-9 Pa) Vacuum System 1. Rotary pump (low vacuum up to 105–10-4 Pa) 2. Diffusion pump (high vacuum 100–10-8 Pa) 3. Turbo molecular pump (high vacuum 10-2–10-8 Pa) 4. Ion getter pump (ultrahigh vacuum up to 10-9 Pa) Interaction of Electrons with Matter Interaction of Electrons with Matter Inelastic scattering (0-0.001 rad) radiation damage Elastic scattering (0-0.1 rad) small angles: phase contrast large angles: scattering contrast • Transmission EM image is formed by the elastically scattered electrons • Electrons are scattered by local EM field of both electrons and nuclei in the sample • Electrons are scattered much more than X-rays => can image individual molecules Wave–Particle Duality • Electrons, as photons, exhibit the wave-particle duality • Electrons collide with other particles (atoms) and are scattered as particles • Electrons also behave as (almost) a plane wave and are diffracted by atoms • TEM image is formed by interference of scattered and non-scattered electrons • Speed of 300 keV electrons is 0.76 c => electrons exhibit relativistic effects • At usual currents (0.1 nA), there is only one electron in the column at the time Absorption Contrast O F object lens image f Intensity A’ B’ B A hν NOT used in TEM image formation Scattering Contrast O A B F object lens + A’ B’ image hυ Scattering Contrast O A B F object lens + A’ B’ image hυ The scattering area appears dark in the image. Phase Contrast O A B F object lens + A’ B’ image hυ The image contrast is described by Phase Contrast Transfer Function. EM Image and Diffraction Formation d Thin phase object approximation: object lens F image d sin(Θ) = n λ EM Diffraction CTF and Aberrations Microscope Aberrations and CTF Microscope Aberrations and CTF Objective astigmatism: F PF ΔΦ F’ CTF Induced by Defocus CTF Induced by Defocus ¼λ ½λ ¾λ λ -0,005 -0,004 -0,003 -0,002 -0,001 0 0,001 0,002 0,003 0,004 0,005 -1 -0,5 0 0,5 1 + – CTF Induced by Spherical Aberration O lens S0 S1 S2 θ d d = Csθ3 Phase Contrast Transfer Function -0,02 -0,015 -0,01 -0,005 0 0,005 0,01 0,015 0,02 -20 -10 0 10 20 Sin[Χ(ν,θ)] = Sin[2π/λ (Csλ4ν4/4 ‒ Δz(θ) λ2ν2/2)] Scherzer focus: Sin[X(ν,θ)] = 0 Amplitude and Phase CTF CTF(ν) = B sin(Χ(ν)) + A cos(Χ(ν)) CTF(ν) = √(1-A2) sin(Χ(ν)) + A cos(Χ(ν)) Χ(ν,θ) = 2π/λ (Csλ4ν4/4 ‒ Δz(θ) λ2ν2/2) CTF with zero amplitude contrast CTF with 15% amplitude contrast Envelope Function Real CTF and Defocus Settings Projector Lenses • Magnify the image formed by the objective lens with minimal distortions. • Very small illumination angles assure almost perfect imaging. • The magnified imaged is projected to the detector. Energy Filter Detectors Detectors Backed-thinned direct electron detector Detectors Resolution • The wavelength of used radiation (visible light, 400-700 nm) is the limiting factor in the achievable resolution of images (200 nm). • In TEM, the wavelength of 300 keV electrons is 2 pm. • The limiting factor in achievable resolution of biological specimen is beam damage to the sample and consequent poor SNR of images. R = d/2 = 0.61 λ / n sin(α) = 0.61 λ / N.A. Resolution Scanning Electron Microscope Scanning Electron Microscope Secondary Electrons Secondary Electrons SE are specimen electrons that obtain energy by inelastic collisions with beam electrons. They are defined as electrons emitted from the specimen with energy less than 50 eV. Backscattered Electrons Everhart-Thornley Detector Everhart-Thornley Detector