C9940 3-Dimensional Transmission Electron Microscopy S1007 Doing structural biology with the electron microscope Tanvir (Tapu) Shaikh February 20, 2017 Syllabus Week Date Instructor Topic 1 20.2 T. Shaikh Introduction/History/Optics 2 27.2 J. Novacek Instrumentation/Tour (?) 3 6.3 J. Novacek Specimen preparation 4 13.3 T. Shaikh Image analysis I 5 20.3 T. Shaikh Image analysis II 6 27.3 J. Novacek Tomography I 7 3.4 J. Novacek Tomography I 8 10.4 T. Shaikh Image analysis III 17.4 (Easter) 9 24.4 T. Shaikh Image analysis IV 1.5 (May Day) 8.5 (Liberation Day) History of electron microscopy Munich: Ernst Ruska & Otto Scherzer Ernst (and Helmut) Ruska: timeline 1930 1940 1950 1960 1970 1980 1990 2000 2010 Milestones: 1931: Invention of the electrostatic lens magnification: 400X 1933: First electron microscope mag: 7000X (vs. LM: 2000X) 1933: Completed Ph.D. (!) 1939: First (viable) commercial EM mag: 100,000X 1939: His brother Helmut Ruska images first virus (TMV) 1952: Helmut moves from Siemens to Albany 1986: Nobel Prize in Physics Ernst Ruska http://www.biografiasyvidas.com http://ernst.ruska.de Sketch from 1929 http://ernst.ruska.dehttp://www.bluesci.org First (viable) commercial microscope (Siemens)Replica of first electron microscope Helmut Ruska (standing) next to Siemens-20 at Wadsworth Center in Albany http://www.wadsworth.org http://ernst.ruska.de First Siemens microscope, 1939 First commercial EM (1937) was Metropolitan-Vickers EM1 (EM2 shown) The first commercial electron microscope was actually by the British company Metropolitan-Vickers in 1937. However, the magnification was worse than for the light microscope, so the Siemens is considered “first.” http://emu.msim.org.uk Clarification Metropolitan Vickers eventually became AEI, which built the 1.2 million volt EM-7. http://www.wadsworth.org Otto Scherzer: timeline 1930 1940 1950 1960 1970 1980 1990 2000 2010 Milestones: 1934: First comprehensive book on electron optics 1936: Spherical aberration → resolution 50-100X the wavelength 1947-1951: Devised correction schemes for aberration correction 1949: “Can atoms be visible in the electron microscope?” “Scherzer focus” Scherzer → Harald Rose (Wadsworth, Darmstadt) → Max Haider Otto Scherzer http://www.microscopy.org First book on electron optics http://www.microscopy.org Scherzer (1949) Physikalische Blätter & Scherzer (1949) Journal of Applied Physics http://www.microscopy.org http://www.microscopy.org “Can atoms be visible in the electron microscope?” “Scherzer focus” Toronto group: E.F. Burton, James Hillier, etc. Toronto group: timeline 1930 1940 1950 1960 1970 1980 1990 2000 2010 Milestones: 1937: Grad students James Hillier, Albert Prebus designed microscope over Christmas break 1938: First images 1939: E.F. Burton et al. introduced airlock system reduced specimen prep to 300nm thickness resolution: 60Å, limited by specimen and not optics maximum mag: 180,000X Radio Corporation of America (RCA) James Hillier: timeline 1930 1940 1950 1960 1970 1980 1990 2000 2010 Milestones: 1940 February Hillier started at RCA enlisted by Vladimir Dworykin (cathode ray tube) 1940 Jul 4: Commercial EM, Model B (EMB) 1941: 300kV, for dealing with thick specimens 1947: first stigmator stigmators were iron screws tapped into the pole piece resolution → 1nm 1973 (as VP of RCA): first videodisc James Hillier http://www.museevirtuel.ca http://www.rfcafe.com ed, with Albert Prebus standing At RCA Model B, 1940 Electron microscopy in the Czech Republic Electron microscopy in the Czech Republic 1930 1940 1950 1960 1970 1980 1990 2000 2010 Milestones: 1950: Ales Blaha: “Tripod” at the Institute of Theoretical and Experimentation Electrotechnology of the Technical University 1951: Tesla BS241 first Czech commercial microscope 50kV resolution: 2nm 1968: First ultrahigh vacuum system (Institute of Scientific Instruments) 1990: Delong Instruments founded 1991: TESCAN founded by engineers from Tesla (TEsla SCANing) 1997: FEI builds factory in Brno Electron microscopy in the Czech Republic http://www.isibrno.cz “Tripod,” 1950 http://www.isibrno.cz First high-vacuum system, 1961 Electron microscopy in the Czech Republic Technical Museum in BrnoTesla Factory in Brno Medical Research Council (MRC), Cambridge Aaron Klug: timeline 1930 1940 1950 1960 1970 1980 1990 2000 2010 Milestones: 1968: DeRosier & Klug – first 3D EM reconstruction (phage T4) 1982: Nobel Prize in Chemistry www.nobelprize.org Other notable events 1930 1940 1950 1960 1970 1980 1990 2000 2010 Milestones: 1934: Ladislaus Laszlo “Bill” Marton takes the first image of biological specimen: sections of a plant leaf 1937: Manfred von Ardenne (CRT) develops SEM 1948: Dennis Gabor develops electron holography (Nobel Prize in Physics, 1971) 1974: Ken Taylor & Bob Glaeser – electron crystallography of frozen hydrated catalase 1974: Walter Hoppe – 3D reconstruction of fatty acid synthase using tomography 1975: Richard Henderson – subnanometer electron crystallography 1982: Jacques Dubochet – modern cryo techniques 1987: Joachim Frank – “single particle” reconstruction of 50S ribosome 2007: Direct Electron develops first commercial direct electron detector “Oh no, not an electron microscope! We have enough trouble trying to interpret the images we get with a light microscope!” – Jules Bordet, 1934 (Nobel Prize, 1921) The Beginnings of Electron Microscopy (1985), ed. Peter W. Hawkes The basics Size ranges for structural methods 0.1nm 1nm 10nm 100nm 1µm 10µm X-ray NMR CryoEM (incl. tomography) Light microscopy ... Comparison of practical requirements CryoEM X-ray NMR Sample volume 5μL1 10μL 400μL Number of samples 5-10 1 crystal 1 (<20kDa)2 several (20-40kDa) Concentration 50 nM 50 μM3 0.5-1.0 mM Total amount of sample 0.25 pmol 500 pmol4 0.2-0.4 μmol Time for data collection 1-7 days 2h (synchotron) 48h (in-house) 1-2 weeks (<20kDa) 6-8 weeks (20-40kDa) Adapted from Raj Agrawal1 Droplet on grid, prior to blotting 2 For stable proteins of this size, a single U-13C, 15N sample is likely sufficient 3 Based on 10 mg/ml of a 100 kDa protein 4 Final amount required for the crystal. However, a multiple of this amount is required Quick review of light optics www.aplusphysics.com Right-hand rule www.education.com Right-hand rule http://www.polywellnuclearfusion.com Electron in a magnetic lens nau.edu http://www.ammrf.org.au One problem: Magnetic lenses are terrible Spherical aberration photographylife.com In electron optics, there is no lens setting* equivalent to an aspherical lens. Spherical aberration will thus be a problem (but also a benefit). Syllabus Week Date Instructor Topic 1 20.2 T. Shaikh Introduction/History/Optics 2 27.2 J. Novacek & T. Shaikh Instrumentation/Tour (?) 3 6.3 J. Novacek Specimen preparation 4 13.3 T. Shaikh Image analysis I 5 20.3 T. Shaikh Image analysis II 6 27.3 J. Novacek Tomography I 7 3.4 J. Novacek Tomography I 8 10.4 T. Shaikh Image analysis III 17.4 (Easter) 9 24.4 T. Shaikh Image analysis IV 1.5 (May Day) 8.5 (Liberation Day)