Difference: CemCourseSyllabus09 (1 vs. 8)

Revision 809 Nov 2009 - Main.DavidCowburn

 
META TOPICPARENT name="CemCourseGroup"
Cryoelectron Microscopy of Macromolecular Assemblies - 2009

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

Instructors

Changed:
<
<
>
>
 

Useful Textbooks

Glaeser, Downing, DeRosier, Chiu, Frank:
"Electron Crystallography of Biological Macromolecules"

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:

Syllabus

Intro to EM, Optics, image formation and Diffraction - Sept 15 - 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 22 - 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

Tomography I - Sept 29 - 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

Single Particle Analysis I - Oct 6- 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 13 - Joachim Frank (Columbia University)

Tomography II - Oct 20 - Carmen Mannella (Wadsworth Ctr)

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

Single Particle Analysis III - Oct 27 - Joachim Frank (Columbia University)

Practicals - Nov 2-6

  • 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 10 - Ruben Diaz (NYSBC)

  • 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 17 - Iban Ubarretxena (Mt. Sinai)

Helical reconstruction - Nov 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 - Dec 1 - 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 8 - 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 - 14 Sep 2009

Revision 719 Oct 2009 - Main.BillRice

 
META TOPICPARENT name="CemCourseGroup"
Cryoelectron Microscopy of Macromolecular Assemblies - 2009

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

Instructors

Useful Textbooks

Glaeser, Downing, DeRosier, Chiu, Frank:
"Electron Crystallography of Biological Macromolecules"

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:

Syllabus

Intro to EM, Optics, image formation and Diffraction - Sept 15 - 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 22 - 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
Changed:
<
<

Single Particle Analysis I - Sept 29 - Joachim Frank (Columbia University)

>
>

Tomography I - Sept 29 - Carmen Mannella (Wadsworth Ctr)

Added:
>
>
  • 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

Single Particle Analysis I - Oct 6- 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)

Changed:
<
<

Single Particle Analysis II - Oct 6 - Joachim Frank (Columbia University)

>
>

Single Particle Analysis II - Oct 13 - Joachim Frank (Columbia University)

 
Changed:
<
<

Tomography I - Oct 13 - Carmen Mannella (Wadsworth Ctr)

>
>

Tomography II - Oct 20 - Carmen Mannella (Wadsworth Ctr)

Deleted:
<
<
  • 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

Single Particle Analysis III - Oct 20 - Joachim Frank (Columbia University)

Tomography II - Oct 27 - Carmen Mannella (Wadsworth Ctr)

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

Single Particle Analysis III - Oct 27 - Joachim Frank (Columbia University)

 

Practicals - Nov 2-6

  • 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 10 - Ruben Diaz (NYSBC)

  • 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 17 - Iban Ubarretxena (Mt. Sinai)

Helical reconstruction - Nov 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 - Dec 1 - 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 8 - 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 - 14 Sep 2009

Revision 614 Sep 2009 - Main.DavidStokes

 
META TOPICPARENT name="CemCourseGroup"
Changed:
<
<		Cryoelectron Microscopy of Macromolecular Assemblies - 2008
>
>		Cryoelectron Microscopy of Macromolecular Assemblies - 2009
 NYU course # G16.4408 - 4 credits - credit also available at other institutions

Instructors

Useful Textbooks

Glaeser, Downing, DeRosier, Chiu, Frank:
"Electron Crystallography of Biological Macromolecules"

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:

Syllabus

Intro to EM, Optics, image formation and Diffraction - Sept 15 - 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 22 - 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 29 - 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 6 - Joachim Frank (Columbia University)

Tomography I - Oct 13 - 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

Single Particle Analysis III - Oct 20 - Joachim Frank (Columbia University)

Tomography II - Oct 27 - Carmen Mannella (Wadsworth Ctr)

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

Practicals - Nov 2-6

  • 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 10 - Ruben Diaz (NYSBC)

  • 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 17 - Iban Ubarretxena (Mt. Sinai)

Helical reconstruction - Nov 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 - Dec 1 - 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 8 - 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 - 14 Sep 2009

Revision 514 Sep 2009 - Main.DavidStokes

 
META TOPICPARENT name="CemCourseGroup"
Cryoelectron Microscopy of Macromolecular Assemblies - 2008

NYU course # G16.4408 - 4 credits -

Changed:
<
<		[[CemCourseOrganization07][credit also available at other institutions]
>
>		credit also available at other institutions
 

Instructors

Useful Textbooks

Glaeser, Downing, DeRosier, Chiu, Frank:
"Electron Crystallography of Biological Macromolecules"

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:

Syllabus

Intro to EM, Optics, image formation and Diffraction - Sept 15 - 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 22 - 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 29 - 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 6 - Joachim Frank (Columbia University)

Tomography I - Oct 13 - 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

Single Particle Analysis III - Oct 20 - Joachim Frank (Columbia University)

Tomography II - Oct 27 - Carmen Mannella (Wadsworth Ctr)

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

Practicals - Nov 2-6

  • 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 10 - Ruben Diaz (NYSBC)

  • 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 17 - Iban Ubarretxena (Mt. Sinai)

Helical reconstruction - Nov 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 - Dec 1 - 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 8 - 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 - 14 Sep 2009

Revision 414 Sep 2009 - Main.DavidStokes

 
META TOPICPARENT name="CemCourseGroup"
Cryoelectron Microscopy of Macromolecular Assemblies - 2008

NYU course # G16.4408 - 4 credits -

Changed:
<
<		credit also available at other institutions
>
>		[[CemCourseOrganization07][credit also available at other institutions]
 

Instructors

Useful Textbooks

Glaeser, Downing, DeRosier, Chiu, Frank:
"Electron Crystallography of Biological Macromolecules"

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:

Syllabus

Intro to EM, Optics, image formation and Diffraction - Sept 15 - 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 22 - 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 29 - 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 6 - Joachim Frank (Columbia University)

Tomography I - Oct 13 - 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

Single Particle Analysis III - Oct 20 - Joachim Frank (Columbia University)

Tomography II - Oct 27 - Carmen Mannella (Wadsworth Ctr)

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

Practicals - Nov 2-6

  • 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 10 - Ruben Diaz (NYSBC)

  • 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 17 - Iban Ubarretxena (Mt. Sinai)

Helical reconstruction - Nov 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 - Dec 1 - 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 8 - 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 - 14 Sep 2009

Revision 314 Sep 2009 - Main.DavidStokes

 
META TOPICPARENT name="CemCourseGroup"
Cryoelectron Microscopy of Macromolecular Assemblies - 2008

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

Instructors

Useful Textbooks

Glaeser, Downing, DeRosier, Chiu, Frank:
"Electron Crystallography of Biological Macromolecules"

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:

Changed:
<
<
>
>
 
Changed:
<
<
>
>
 

Syllabus

Intro to EM, Optics, image formation and Diffraction - Sept 15 - 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 22 - 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 29 - 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 6 - Joachim Frank (Columbia University)

Tomography I - Oct 13 - 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

Single Particle Analysis III - Oct 20 - Joachim Frank (Columbia University)

Tomography II - Oct 27 - Carmen Mannella (Wadsworth Ctr)

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

Practicals - Nov 2-6

  • 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 10 - Ruben Diaz (NYSBC)

  • 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 17 - Iban Ubarretxena (Mt. Sinai)

Helical reconstruction - Nov 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 - Dec 1 - 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 8 - 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 - 14 Sep 2009

Revision 214 Sep 2009 - Main.DavidStokes

 
META TOPICPARENT name="CemCourseGroup"
Cryoelectron Microscopy of Macromolecular Assemblies - 2008

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

Instructors

Useful Textbooks

Glaeser, Downing, DeRosier, Chiu, Frank:
"Electron Crystallography of Biological Macromolecules"

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:

Changed:
<
<
  • 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 15 - 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 22 - 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 29 - 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 6 - Joachim Frank (Columbia University)

Tomography I - Oct 13 - 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

Single Particle Analysis III - Oct 20 - Joachim Frank (Columbia University)

Tomography II - Oct 27 - Carmen Mannella (Wadsworth Ctr)

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

Practicals - Nov 2-6

  • 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 10 - Ruben Diaz (NYSBC)

  • 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 17 - Iban Ubarretxena (Mt. Sinai)

Helical reconstruction - Nov 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 - Dec 1 - 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 8 - 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 - 14 Sep 2009

Revision 114 Sep 2009 - Main.DavidStokes

 
META TOPICPARENT name="CemCourseGroup"
Cryoelectron Microscopy of Macromolecular Assemblies - 2008

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

Instructors

Useful Textbooks

Glaeser, Downing, DeRosier, Chiu, Frank:
"Electron Crystallography of Biological Macromolecules"

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 15 - 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 22 - 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 29 - 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 6 - Joachim Frank (Columbia University)

Tomography I - Oct 13 - 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

Single Particle Analysis III - Oct 20 - Joachim Frank (Columbia University)

Tomography II - Oct 27 - Carmen Mannella (Wadsworth Ctr)

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

Practicals - Nov 2-6

  • 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 10 - Ruben Diaz (NYSBC)

  • 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 17 - Iban Ubarretxena (Mt. Sinai)

Helical reconstruction - Nov 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 - Dec 1 - 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 8 - 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 - 14 Sep 2009

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