Cryo-electron microscopy Tibor Füzik Lecture 5 “The structural biology continuum” Grant Jensen nm µm mmÅ How does it compare to other methods NMRX-ray crystallography Cryo-EM Nobel Prize in Chemistry 2017 The resolution revolution beta-galactosidase; Veronica Falconieri, Sriram Subramaniam, National Cancer Institute The resolution revolution Titan KRIOS • X-FEG – 300 kV • 3 condenser illumination system • no Cs corrector • autoloader for 12 samples • cooled to liquid N2 temperature • energy filter • CCD, DED The Microscope How TEM compares to optical microscope Optical microscopeTransmission electron microscope Source of photons Illumination system Sample (transparent) Objective lens Projection system Detector Source of electrons Illumination system Sample (electron transparent) Objective lens Projection system Detector bio.libretexts.org TEM schematic myscope.training Electron Gun Anode Ground Wehnelt Acceleratorstack Electron source - 0 kV - 30 kV - 60 kV - 90 kV - 120 kV - 150 kV - 180 kV - 210 kV - 240 kV - 270 kV - 300 kV - 300 kV - 0 kV Electron sources - types thermofisher.com microtonano.com snaggledworks.com Cook B.; Ultramicroscopy; 2009 Tungsten filament LaB6 crystall Schottky • Thermionic • Field emission Wehnelt Tungsten (2000 oC) LaB6 (1500oC) FEG (25oC / 1500oC) Anode Anode Anode 2 Anode 1 Thermionic Field emission Electron sources - properties • Tungsten filament • Low brightness • Inexpensive • LaB6 crystal • Higher brightness • Less energy spread • Lower lifetime mse.iastate.edu • Schottky - warm • High brightness • Lower lifetime • Higher energy spread  • Cold FEG • High brightness • Lower energy spread ☺ • Lower stability Thermionic Field emission Electron beam properties • Beam coherence • Wave-length: at 300 kV => ~2 pm = 0.002 nm = 0.02 Å • Velocity: at 300 kV => 0.76c • Magnetic momentum • Can be focused • Disadvantage: hi interaction rate (small penetration) Electron beam deflectors Gun is shifted Gun is tilted Gun is shifted and tilted Electron beam deflectors • Electron has a magnetic momentum • We can move electrons by magnetic field • We can generate magnetic fields by electric coils • The same coils controls the shift and tilt • Movement of the beam in 2D (X,Y) • Tuning the pivot points: • Tilt free shift • Shift free tilt Beam-shift Beam-tilt Pivot point Principle of convex optical lenses • Ray passing through optical axis remains unbent • Rays passing through lens are bent that they focus (converge) in focal point Principle of optical lenses magnified object image object Optical axis Central plane Focal point Image plane (front focal plane) Object distance Image distance Focal distance Principle of optical lenses Magnified object object Underfocus Principle of optical lenses Magnified object object Overfocus Alternative way of showing lenses Underfocus Overfocus How electromagnetic lenses work myscope.training Lorentz force Electromagnetic lenses - properties • Variable strength • Hysteresis • Non-homogenity of the coil current => magnetic field => aberrations B I How to correct for aberrations • Astigmatism • Defocus in one direction is different then in the other • Stigmator • Quadrupole magnetic field • Octapole stigmator (still quadrupole magnetic field) • Higher order aberrations • Non-homogeneity of the magnetic field • High frequencies affected more • Cs corrector jeol.jp wikiwand.com Ceos-gmbh.de Ceos-gmbh.de Mathematic description of aberrations Zernike polynomials Frits Zernike Nobel Prize 1953 (phase contrast microscopy) your-eye-sight.org Illumination system (Condenser system) • Condenser lenses • 2 condenser system • C1 lens – spot size • C2 lens – beam size (diameter) • 3 condenser system • C2/C3 – beam size (diameter) • Condenser apertures • Beam-shift • Deflectors • Goal for hi-res microscopy • Parallel illumination beam • Controlled dose/flux C2 C1 C1 aperture aperture C2 stigmator C2 aperture Beam-shift deflectors Spotsize C1 C2 Spot 1 Spot 2 Spot 3 Sample Area Intensity C1 C2 Converging beam Parallel beam Spreading beam Sample Area Aperture No apperture “Big” apperture “Small” apperture Sample Area C1 lens, C2 lens, C2 aperture • C1 lens • Strength changed in steps • Controlled by “Spotsize” • Major impact on electron flux • C2 lens • Strength change gradually • Controlled by “Intensity”, “Brightness” • Controls the convergence of the beam • Converging beam higher electron flux than diverging • Aperture • Cuts out part of the beam after C2 • Reduces illuminated area but not the flux C1 C1 aperture aperture C2 stigmator C2 aperture Beam-shift deflectors C2 A three-condenser system • Hi-end microscopes • Behind C2 aperture • Parallel beam across wide range of beam size • Precise setting of dose and beam size • Easier setup for phase plate usage • Calibrate the ratio between C2 and C3 C1 C2 C3 Objective lens Objective Stigmator Sample Objective aperture Objective lens • Objective lens – highest magnification 50x • Strength set by “Focus” • Objective stigmator • Remove two-fold astigmatism • Objective aperture • Remove electrons with high angle scattering angle • Improves contrast • Has influence on resolution (cut-off) • Has influence on astigmatism Objective lens system Sample stage • Moving in 3 dimensions (X, Y, Z) • Eucentric height • Rotation (tilt) • Sample centered • goniometer • Cooled at liquid nitrogen temp • Stability crucial for hi-res • Cryo-decontamination box Upper Objective lens Lower Objective lens Pole piece Pole piece Projector system Intermediate lens Projector lens 1 Projector lens 2 Difraction stigmator Image deflectors Selected area aperture• Intermediate lens and Projector lens • Intermediate magnification 10-20x • Final magnification of the image • Rotation free lenses • Magnification modes • LowMag • SA • EFTEM • Image deflectors • Shifts the beam-shifted image back to detector Stage-shift vs image-shift (beam-shift) Original position Stage-shift Image-shift V V Vgun detector sample grid Beam deflectors Image deflectors Stage-shift vs beam-shift/image-shift Stage-shift • Mechanical • Takes time to stabilize (~15 s) • Large movements (+- grid surface) • Small precision Beam-shift/Image-shift • Electro-magnetic • Faster stabilization (~5 s) • Small movements (~5 µm) • High precision • Introduces beam-tilt Optic system of the microscope Electromagnetic lens Deflectors Stigmators Apertures Condenser Objective Projector Energy filter • Elastic electron scattering • Inelastic electron scattering • In column (Omega filter) • Post column ƴ Prism Energy filter – electromagnetic prism Jeol.jp Omega filter Post-column filter Gatan Inc. In-column / post-column filter thruanelectronlens.wordpress.com Detectors • Fluorescent screen • Zinc sulfide • Robust • Film • Silver reduced from silver halide • Linear response • Digitalization of films • No drift correction • CCD – charge couple device • Direct electron detector twitter.com/difluorine CCD camera • Convert e- to light and detect by CCD • Sensitive to cosmic rays • Scattering between scintillator and fiber optics e- scintillator Optic fiber CCD Readout line by line http://meroli.web.cern.ch Direct electron detector • Directly detects electrons (no conversion to photons) • Non-sensitive to cosmic rays • Fast readout (movie mode) • Back thinning reduce e- backscattering • Every pixel own readout electronics • Linear (integrative) mode • Counting mode franklab.cpmc.columbia.edu schemanticscholar.org DQE - detective quantum efficiency McMullan et al, 2014, Paul Mooney Direct electron detector ThermoFisher – Falcon 3 ThermoFisher – Falcon 4 Gatan K2 summit Gatan K3 Location in the microscope Vacuum system • TEM requires high vacuum • Rotary pump (low vacuum up to ~10-4Pa) • Oil diffusion pump (high vacuum ~10-8Pa) • Turbomolecular pump (high vacuum ~10-8Pa) • IGP – ion getter pumps (ultrahigh vacuum up to 10-9Pa) • Mechanical pump • Produce vibrations • Low vacuum • Used as backing pump • Dry scroll pumps Rotary pump vacaero.com Turbomolecular pump • High vacuum • Requires intermediate vacuum • Need backing pump wikipedia.org Oil diffusion pump ~100 000 RPM wikipedia.org wikipedia.org wikipedia.org Ion getter pump • Ultra-High vacuum • Slow • Requires high-vacuum • Turbomolecular pump backed Vacuum system • Combination of pumps • Isolated chambers • Pressure gauges • Buffer tank Sample preparation • Preparation of the perfect sample to image • The better sample the better result • 1st most difficult step • molecular biology / microbiology / biochemistry • Preparative methods – proteins / protein complexes • Vitrification • Embed the sample into vitreous ice • Vitrified sample storage • Keep the sample vitrified • EM grids • Copper • Gold • Grid coating layer • Carbon coated • Lacey carbon • Holey carbon • Carbon coated holey carbon • Holey Gold (UltrAuFoil) • Hole parameters • Hole shape – circular, hexagonal • Hole size/spacing – 2/1; 2/2; 1.3/1.6 etc…. Types of EM grids edeninstruments.com agarscientific.com emresolutions.com emresolutions.com Sample preparation - vitrification Murata et al., 2018 Jacque Dubochet Vitrification - instrumentation • Immobilization • Why to freeze the samples • Vitreous ice • Beam induced damage Vitrobot Mark IV Sample preparation - challenges Murata et al., 2018 Spottiton, Vitrojet, Shake-it-off • New approaches to sample • Preparation • Commercial vs open source • Not widely used yet What we have learned….. • Cryo-EM not a complimentary method any more • Principles of optic parts of TEM • Detection devices and their properties • Non-optic parts of electron microscope • Sample preparation The end ThermoFisher Jeol