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Introduction to Electron Microscopy Prof. David Muller, dm24@cornell.edu Rm 274 Clark Hall, 255-4065 Ernst Ruska and Max Knoll built the first electron microscope in 1931 (Nobel Prize to Ruska in 1986) T4 Bacteriophage Electron Microscopy bridges the 1 nm – 1 μm gap David Muller 2008 between x-ray diffraction and optical microscopy Tools of the Trade AFM MFM Scanned Probe Microscope (includes Atomic Force Microscope) Transmission Electron Microscope Scanning Electron Microscope David Muller 2008 Biological and Electronic Component Dimensions Biological 1 Electronic Components Logic Board Computer chip Optical Microscope Tool 10-2 SEM Size (m) 10-4 Mammalian cell 10-6 Bacterial cell Virus Transistor AFM/STM Gate Oxide Atom TEM 10-8 Gene Protein 10-10 David Muller 2008 Comparison of Optical and Electron Microscopes • Electron microscopes are operated in vacuum because the mean free path of electrons is air is short – this mean biological samples should not degas – they can either be dehydrated or frozen – pathology, not in-vivo. •Electron microscopes have higher resolution than optical microscopes – atomic resolution is possible. •Chemical imaging and spectroscopy – mapping π and σ bonds at 1nm resolution can be done. •Radiation damage is severe and limits the image quality and resolution (not as bad as x-rays or neutrons though! – see R. Henderson, Quarterly Reviews of Biophysics 28 (1995) 171-193.) David Muller 2008 Comparison of Optical and Electron Microscopes Light Microscope source 1st condenser 2nd condenser TEM SEM or STEM Viewing screen Or CCD specimen Objective lens Projector lenses CA condenser aperture OA objective aperture SA selected area aperture Image formed by scanning a small spot David Muller 2008 Viewing screen Or CCD Inside a Transmission Electron Microscope High tension cable (100-200 kV) Filament Accelerating stack Double condenser lens condenser aperture objective aperture Selected area aperture Sample sits here Viewing chamber David Muller 2008 An Electron Lens David Muller 2008 An Electron Lens David Muller 2008 Geometric Optics – A Simple Lens Focusing: angular deflection of ray α distance from optic axis θ x x Object plane David Muller 2008 front focal plane Lens at z=0 Back focal plane image plane Geometric Optics – A Simple Lens Wavefronts in focal plane are the Fourier Transform of the Image/Object θ1 θ1 x x Object plane David Muller 2008 front focal plane Lens at z=0 Back focal plane image plane X-ray and Electron Diffraction from a Silicon Crystal Bragg’s Law: nλ = d sin θ 200 keV Electrons 10 keV x-rays λ=1.54 Å David Muller 2008 λ=0.0251Å In Si d220 = 1.92 Å Electron Velocity and Wavelength De Broglie Wavelength: h λ= p Where h is Planck’s constant And p=mv are the momentum, mass and velocity of the electron If an electron is accelerated through a potential eV, it gains kinetic energy 1 2 mv = eV 2 So the momentum is mv = 2meV Electron wavelength λ= h2 1.23nm = 2meV V (V in Volts) ( relativistically correct form: David Muller 2008 h 2c 2 λ= eV ( 2m0c 2 + eV ) ) Electron Wavelength vs. Accelerating Voltage 0.05 Relativistic Non-relativistic 0.04 λ (Angstroms) Accelerating Voltage 1V v/c 0.0019784 0.0062560 0.062469 0.019194 0.54822 0.69531 0.77653 0.81352 λ (Ǻ) 12.264 1.2263 0.38763 0.12204 0.037013 0.025078 0.019687 0.0087189 0.03 100 V 1 keV 0.02 10 keV 100 keV 200 keV 300 keV 1 MeV 0.01 0 0 200 400 600 800 1000 Electron Kinetic Energy (keV) David Muller 2008 Resolution Limits Imposed by Spherical Aberration, Cs (Or why we can’t do subatomic imaging with a 100 keV electron) Lens Cs>0 Plane of Least Confusion Cs=0 d min Gaussian image plane For Cs>0, rays far from the axis are bent too strongly and come to a crossover before the gaussian image plane. For a lens with aperture angle α, the minimum blur is d min 1 = Csα 3 2 Typical TEM numbers: Cs= 1 mm, α=10 mrad → dmin= 0.5 nm David Muller 2008 Resolution Limits Imposed by the Diffraction Limit (Less diffraction with a large aperture – must be balanced against Cs) Lens d0 α0 Gaussian image plane The image of a point transferred through a lens with a circular aperture of semiangle α0 is an Airy Disk of diameter 0.61λ 0.61λ d0 = ≈ n sin α 0 α0 (0.61 for incoherent imaging e.g. ADF-STEM, 1.22 for coherent or phase contrast,. E.g TEM) David Muller 2008 (for electrons, n~1, and the angles are small) Balancing Spherical Aberration against the Diffraction Limit (Less diffraction with a large aperture – must be balanced against Cs) For a rough estimate of the optimum aperture size, convolve blurring terms -If the point spreads were gaussian, we could add in quadrature: d 2 tot ⎛ 0.61λ ⎞ ⎛ 1 3⎞ ⎟ + ⎜ Csα 0 ⎟ ≈d +d =⎜ ⎜ α ⎟ ⎠ ⎝ 0 ⎠ ⎝2 2 0 2 s 2 2 100 Probe Size (Angstroms) 10 d 1 1 d 0 s Optimal aperture And minimum Spot size 1 d min = 0.66 Cs / 4λ3 / 4 David Muller 2008 α (mrad) 10 Balancing Spherical Aberration against the Diffraction Limit (Less diffraction with a large aperture – must be balanced against Cs) A more accurate wave-optical treatment, allowing less than λ/4 of phase shift across the lens gives Minimum Spot size: 1 d min = 0.43Cs / 4λ3 / 4 1 d min = 0.61Cs / 4 λ3 / 4 (Incoherent image - e.g. STEM) (coherent image - e.g. TEM) Optimal aperture: αopt ⎛ 4λ ⎞ =⎜ ⎟ ⎜C ⎟ ⎝ s⎠ 1/ 4 At 200 kV, λ=0.0257 Ǻ, dmin = 1.53Ǻ and αopt = 10 mrad At 1 kV, λ=0.38 Ǻ, dmin = 12 Ǻ and αopt = 20 mrad David Muller 2008 Electron Diffraction and Imaging a [100] Silicon Crystal Image Diffraction Pattern 220 400 λ=0.0251Å David Muller 2008 In Si d220 = 1.92 Å Depth of Field, Depth of Focus d D0 = tanα 0 For d=3nm, α=10 mrad, D0= 300 nm David Muller 2008 For d=200nm, α=0.1 mrad, D0= 2 mm! Lenses in a Transmission Microscope (and deflection coils to correct their alignment) Gun: electron source If misaligned, low intensity & other alignments may also be out Condensor: uniformly illuminate the sample If misaligned, you will lose the beam when changing magnification Objective: image sample – determines resolution. If misaligned, the image will be distorted, blurry. projector: magnifies image/ forms diffraction pattern – should not alter resolution. If misaligned, the image will be distorted, diffraction pattern may be blurry. David Muller 2008 http://www.rodenburg.org/RODENBURG.pdf Caustics in a Lens On-axis Tilted http://www-optics.unine.ch/education/optics_tutorials/aspherical_surface.html David Muller 2008 Caustics (remove extreme rays and caustics by putting in an aperture) From “Natural Focusing and Fine Structure of Light: Caustics and Wave Dislocations” by J. F. Nye David Muller 2008 Common Aberrations Astigmatism -x&y focus at different planes -fix by adjusting stigmators Bad Coma -beam is tilted off axis -fix by centering aperture Bad -Δf Δf=0 +Δf Good Good Check lens alignment by going through focus (change lens strength) David Muller 2008 Lens Alignment Correcting for a gun shift misalignment How do we align one lens, when all lenses are misaligned? Step 1: Strongly excite C1 (small spot size) cross-over moves to lens & optic axis. Use beam shift D2 to bring spot to to axis below C2 Step 2: Weaken C1 (large spot size) cross-over moves away from optic axis Use gun shift D1 to bring spot to to axis below C2. Iterate until spot stops moving David Muller 2008 http://www.rodenburg.org/RODENBURG.pdf Focusing using Fresnel Fringes 2 μm underfocus In focus 2 μm overfocus bright fringe Minimum contrast dark fringe Check lens alignment by going through focus (change lens strength) David Muller 2008 Correcting Objective Astigmatism using Fresnel Fringes Astigmatic & best focus Stigmated& focused dark fringe bright fringe David Muller 2008 Materials Microscopy Resources on Campus (http://www.ccmr.cornell.edu/facilities/) Type Atomic Force Microscopy Applications •Topographic Imaging on wafers •Accurate height measurements on flat surfaces (~ 0.5 nm vertical) •Lateral Resolution 10-20 nm •In-situ – no vacuum required •Imaging of complex structures at 120 nm resolution •X-ray mapping at 100-500 nm •In-vacuum •Clark: High spatial resolution •Snee/Bard: best x-ray mapping, OIM •1 nm (polymers) –> atomic resolution of crystals in thin samples •X-ray mapping at 1 nm •EELS at < 1 nm •Requires sample thinning (except for nanoparticles) Location •Dr. Jonathan Shu D-22 Clark Hall •Prof. Kit Umbach SB-60C Bard Hall •CNF Clean Room •Clark: Mick Thomas F3 Clark Hall •Bard/Snee: John Hunt SB56 Bard/1149 Snee Duffield: John Grazul 150 Duffield (TEM+STEM) Clark: Mick Thomas F3 Clark (STEM+EDX) Scanning Electron Microscopy Transmission Electron Microscopy