Difference: CemCourseSyllabus06 (13 vs. 14)

NYU course # G16.4408 - 3 credits

Instructors

• David Stokes: stokes@saturn.med.nyu.edu
• John Berriman: berriman@nysbc.org
• Bill Rice: rice@nysbc.org
• Iban Ubarretxena: iu@sci.ccny.cuny.edu
• Hernando Sosa: hsosa@aecom.yu.edu
• Joachim Frank: joachim@wadsworth.org
• Carmen Mannella: carmen@wadsworth.org

Useful Textbooks

Frank: "Three Dimensional Electron Microscopy of Macromolecular Assemblies"
Reimer: "Transmission Electron Microscopy: Physics of Image Formation and Microanalysis"
Bozzola & Russell: "Electron Microscopy: Principles and Techniques for Biologists"
Harris: "Negative Staining and Cryoelectron Microscopy"
Williams & Carter: "Transmission Electron Microscopy: A Textbook for Materials Science"

Syllabus

Intro to EM and History of 3D Reconstruction - Sept 12 - David Stokes (NYSBC/NYU)

• Basic EM techniques
  • TEM
  • SEM
  • STEM
  • Types of specimens
• Electron sources
  • tips (W, LaB6^?, FEG)
• Physics of electrons
  • Faraday's Law
  • influence of a B field
  • electrostatic and electromagnetic lens
• Interaction of electrons with matter
  • elastic scattering
  • inelastic scattering
  • secondary electrons
  • backscattered electrons
  • x-rays
  • EELS
• overview of 3D reconstruction strategies
  • 2D crystallography
  • helical reconstruction (Fourier-Bessel)
  • single particle analysis
  • tomography
• negative stain of viruses
  • model of negative stain
  • matching of projection images
  • quasiequivalence & icosahedral viruses
• optical diffraction
  • optical filtering
• bacteriorhodopsin
• acetylcholine receptor
• ribosomes
• insect flight muscle tomography

Sample preparation - Sept 19 - John Berriman (NYSBC)

Discussion leaders (Sept 26):

• tissue -> sections
  • positive stain
  • immunolabeling
• molecules -> negative stain
  • single particles
  • 2D crystals
• cryo -> no stain
  • frozen sections
  • suspensions of molecules or 2D crystals

Diffraction and Fourier transforms - Sep 26 - Iban Ubarretxena (CCNY)

• Diffraction of waves - Frauenhofer vs. Fresnel
• Sum of cosines -> Fourier transform
• Example functions (Gaussian, box, delta fcn, cosine)
• Friedel's Law
• Convolution fcn
• Correlation fcn & Convolution Thm
• Projection Thm

Single Particle Analysis I - Oct 3 - Joachim Frank (Wadsworth Ctr)

• philosophy of single-particle reconstruction
• imaging considerations
  • solid/perforated carbon
  • automated data collection
• how to get the initial model
  • random conical tilt with defined orientations
  • orientation determination using common lines
  • 3D projection matching
• model bias
• correspondence analysis
• classification and class-averages
• refinement
• defocus groups and CTF correction (Weiner filter)
• weighted backprojection, ART and direct Fourier reconstruction
• symmetry
• resolution criteria (Fourier Shell Correlation)
• overview of programs (Spider, Eman, Imagic)
• applications

Single Particle Analysis II - Oct 10 - Joachim Frank (Wadsworth Ctr)

SPA practical (Oct 17):

Crystallography - Oct 17 - Iban Ubarretxena (CCNY)

• Braggs law
• real/reciprocal lattice
  • unit cell
    • asymmetric unit
    • fractional coordinates
  • symmetry
    • symmetry operators
    • plane groups & space groups
  • Miller indices
  • reciprocal lattice symmetry (ampl and phase)
  • lattice lines
• convolution fcn -> crystal
  • convolution Thm -> reciprocal lattice
• Projection Thm and radius of Ewald Sphere
• strategy for 3D recontruction
  • missing cone
• imaging considerations
  • electron diffraction
  • spot-scan for images
• electron diffraction for amplitudes
  • radial background subtraction
  • lattice refinement
  • tilt angle/axis
  • local background sub and spot integration
  • merging
  • refinement
  • curve fitting
• imaging for phases
  • unbending
  • defocus determination
  • tilt angle/axis
  • merging, weighting
  • temperature factor compensation
• resolution criteria (point-spread fcn)

Helical reconstruction - Oct 24 - Hernando Sosa (Einstein)

• Fourier transfor in cylindrical coord
• Bessel functions
• continuous helix
• discontinuous helix
  • selection rule
  • n,l plot
  • layer lines
  • G(n,l) -> g(n,l)
• geometrical corrections
  • in-plane tilt
  • out-of-plane tilt
  • repeat distance
• averaging of G(n,l)
  • weighting
  • CTF correction
  • filtering
  • temp factor compensation
  • resolution criteria
  • symmetry groups
• Hybrid methods - unbending
  • separately refining small segments against average
  • Egelman's spider implementation for helical structures
  • real-space averaging of symmetry groups

Electron Optics - Oct 31 - David Stokes

• Physics of electrons
  • Faraday's Law
  • influence of a B field
  • electrostatic and electromagnetic lens
  • deflectors
• electron scattering
  • scattering cross-section
  • elastic scattering
  • inelastic scattering
  • secondary electrons
  • backscattered electrons
  • x-rays
  • EELS
• microscope column
  • condensor, objective, intermediate, projector lenses
  • gun, beam, image deflector coils
  • condensor, objective, selected area apertures
• optics
  • focal length, magnification
  • ray path diagram
  • resolution
  • aberrations
  • coherency

Imaging strategies and Media - Nov 7 - David Stokes

• Radiation damage
  • low dose imaging
• Electron diffraction
• Inelastic scattering
  • energy filter and zero-loss imaging
• Review of diffraction and Fourier transforms
  • scattering - Fresnel and Frauenhofer
  • sum of sines -> Fourier transform
  • Friedel's Law
  • Convolution Thm
  • Correlation function
• effect of sampling
  • discrete Fourier transform
  • box function and lattice function
  • Nyquist sampling frequency
  • boxing and floating
  • aliasing
• transfer functions and detectors (CCD and film)
  • CTF: phase vs amplitude contrast (cryo vs neg stain)
  • DQE, MTF, NTF

Tomography - Nov 14 - Carmen Mannella (Wadsworth Ctr)

• imaging considerations
  • dose fractionation
  • automated imaging and strategies for tracking
  • equal tilt increments/cosine rule
  • dual/single axis
• Alignment of images (fiducial vs. correlation)
• distortion correction (optical and mechanical)
• R-weighted backprojection vs. other reconstruction algorithms
• denoising (non-linear anisotropic diffusion vs. median filter)
• segmentation (automatic vs. manual)
• template matching and real-space averaging of subvolumes
• applications

Hybrid Methods - Nov 28 - David Stokes (NYSBC/NYU)

• Unbending of 2D crystals
• patch averaging of 2D crystals
• Real-space averaging of helical reconstructions
• SPA of helical assemblies
• SPA of tomographic volumes
• tomography of 2D crystals

Molecular fitting - Dec 5 - Hernando Sosa (Einstein)

• Boundary determination
  • isodensity contour
  • segmentation
• Automated strategies
  • Situs
  • helix hunter
• Criteria for fit

Visit Cryo EM website at http://www.nysbc.org/facilities/CEM

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-- DavidStokes - 24 Jul 2006