Difference: CemCourseSyllabus07 (15 vs. 16)

NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals credit also available at other institutions

Instructors

• David Stokes: stokes@saturn.med.nyu.edu
• Ruben Diaz: diaz@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"

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"

Syllabus

Intro to EM and History of 3D Reconstruction - Sept 18 - 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
• Historical overview of 3D reconstruction

Practical on negative stain sample prep - Sept 20

Optics, waves, image formation - Sept 25 - Ruben Diaz (NYSBC)

• 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
  • secondary electrons
  • backscattered electrons
  • x-rays
  • EELS
• 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
• Inelastic scattering
  • energy filter and zero-loss imaging

Practical on frozen-hydrated sample prep (Sept 27):

Diffraction and Fourier transforms - Oct 2 - 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

Practical on optical and computed FFT - Oct 4

CTF, cross-correlation, eigen vector analysis - Oct 9 - Bill Rice

• more on FT of 2D images
• 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
• brief introduction to classification

Practical on digital imaging - Oct 11

Tomography I - Oct 16 - 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

practical on tomography I (Data collection with SerialEM) - Oct 18

Single Particle Analysis I - Oct 23 - Joachim Frank (Wadsworth Ctr)

• 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)

Practical on tomography II (IMOD reconstruction)- Oct 25

Single Particle Analysis II - Oct 30 - Joachim Frank (Wadsworth Ctr)

Practical on single particle analysis I (particle picking and intro to spider) - Nov 1

Tomography II - Nov 6 - Carmen Mannella (Wadsworth Ctr)

• Cryoelectron tomography
• Zero-loss imaging
• Resolution assessment

Practical on single particle analysis II (multivariate statistical analysis) - Nov 8

Crystallography - Nov 13 - Iban Ubarretxena (CCNY)

• 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
• electron diffraction for amplitudes
• imaging for phases
  • unbending
• temperature factor compensation
• resolution criteria (point-spread fcn)

Practical on single particle analysis III (initial model by random conical tilt) - Nov 15

Helical reconstruction - Nov 20 - 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 - Nov 27 - 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

Practical on 2D crystallography (2DX) - Nov 29

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

Practical on molecular visualization (Chimera) - Dec 6

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

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-- DavidStokes - 12 Jul 2007