Difference: CemCourseSyllabus08 (12 vs. 13)

NYU course # G16.4408 - 4 credits - credit also available at other institutions

Instructors

• DavidStokes: stokes@saturn.med.nyu.edu
• RubenDiaz: diaz@nysbc.org
• BillRice: rice@nysbc.org
• IbanUbarretxena: iu@sci.ccny.cuny.edu

• JoachimFrank: jf2192@columbia.edu
• CarmenMannella: carmen@wadsworth.org

Useful Textbooks

Frank:
"Three Dimensional Electron Microscopy of Macromolecular Assemblies"

Steward:
"Fourier Optics: An Introduction"

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"

Requirements

The following will be required for students desiring credit for this course:

• complete a number of homework assignments during the class
• present a paper at one of the discussion sessions
• attend all of the practical sessions
• write a final paper at the end of the class

Syllabus

Intro to EM, Optics, image formation and Diffraction - Sept 16 - David Stokes (NYSBC/NYU)

• Basic EM techniques (TEM, SEM, STEM)
• Interaction of electrons with matter
• Image formation
• overview of 3D reconstruction strategies
  • 2D crystallography
  • helical reconstruction (Fourier-Bessel)
  • single particle analysis
  • tomography
• Physics of electrons
  • Faraday's Law and Lorenz force
  • Motion of an electron in a magnetic field
  • electrostatic and magnetic lenses
  • deflectors
• electron-specimen interactions
  • particle-wave duality
  • scattering cross-section
  • elastic scattering
  • inelastic scattering
    • energy filter and zero-loss imaging
    • EELS
  • secondary electrons
  • backscattered electrons
  • x-rays
• microscope column
  • condenser, objective, intermediate, projector lenses
  • gun, beam, image deflector coils
  • condensor, objective, selected area apertures
• optics
  • focal length, magnification
  • ray path diagram
  • resolution
  • aberrations
  • coherence
• Radiation damage
  • low dose imaging
• Electron diffraction
• Diffraction of waves - Frauenhofer vs. Fresnel
• Sum of cosines -> Fourier transform

Diffraction, Fourier transforms, CTF, cross-correlation - Sept 23 - Bill Rice (NYSBC)

• Sum of cosines -> Fourier transform
• Example functions (Gaussian, box, delta fcn, cosine)
• Friedel's Law
• Convolution fcn
• Correlation fcn & Convolution Thm
• Projection Thm
• relative importance of amplitude and phase
• Nyquist limit
• Convolution and convolution theorem
• Correlation
• Effect of aperture in diffraction plane; point spread function
• image formation in the microscope
• weak phase approximation
• lens aberrations, defocus : CTF function
• Envelope functions
• Effect of CTF on images
• CTF correction

Single Particle Analysis I - Sept 30 - Joachim Frank (Columbia University)

• alignment
• classification and class-averages
• how to get the initial model
  • random conical tilt with defined orientations
  • orientation determination using common lines
  • 3D projection matching
• model bias
• refinement
• multivariable statistical analysis (MSA)
• defocus groups and CTF correction (Weiner filter)
• weighted backprojection, ART and direct Fourier reconstruction
• symmetry
• resolution criteria (Fourier Shell Correlation)

Single Particle Analysis II - Oct 7 - Joachim Frank (Columbia University)

Single Particle Analysis III - Oct 14 - Joachim Frank (Columbia University)

Tomography I - Oct 21 - 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

Tomography II - Oct 28 - Carmen Mannella (Wadsworth Ctr)

• Cryoelectron tomography
• Zero-loss imaging
• Resolution assessment
• Motif averaging
• Missing wedge compensation

Practicals - Nov 3-7

• Day 1 (Nov. 3): Introduction to SPIDER and EMAN
  • basic operations
  • manual particle picking with boxer
  • Chimera tutorial (homework for the week)

• Day 2 (Nov. 4): Generation of initial model by Random Conical Tilt
  • use SPIDER

• Day 3 (Nov. 5): Generation of initial model by Common Lines
  • use both SPIDER and EMAN

• Day 4 (Nov. 6): Model generation and refinement using Projection Matching
  • SPIDER
  • CTF determination using EMAN (ctfit) and SPIDER

• Day 5 (Nov. 7): Tomography
  • Generation of tomograms with eTomo

Crystallography I - Nov 11 - Iban Ubarretxena (Mt. Sinai)

• Braggs law
• real/reciprocal lattice
  • unit cell
  • symmetry
  • Miller indices
  • reciprocal lattice symmetry (ampl and phase)
  • lattice lines
• convolution fcn -> crystal
• Projection Thm and radius of Ewald Sphere
• strategy for 3D recontruction
  • missing cone
• imaging considerations
  • electron diffraction
  • spot-scan for images
• Unbending of 2D crystals
• electron diffraction for amplitudes
• imaging for phases
  • unbending
• temperature factor compensation
• resolution criteria (point-spread fcn)

Crystallography II - Nov 18 - Iban Ubarretxena (Mt. Sinai)

Helical reconstruction - Nov 25 - 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 - Dec 2 - David Stokes (NYSBC)

• 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 9 - 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

• Set ALLOWTOPICVIEW =

-- DavidStokes - 04 Aug 2008