Difference: CemCourseSyllabus07 (1 vs. 18)

Revision 1813 Mar 2009 - Main.DavidCowburn

 
META TOPICPARENT name="CemCourseGroup"
Cryoelectron Microscopy of Macromolecular Assemblies - 2007

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

Instructors

Changed:
<
<
>
>
  • Joachim Frank: joachim AT wadsworth.org
 

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"

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

Molecular fitting - Dec 4 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 1701 Sep 2008 - Main.DavidStokes

 
META TOPICPARENT name="CemCourseGroup"
Changed:
<
<		Cryoelectron Microscopy of Macromolecular Assemblies
>
>		Cryoelectron Microscopy of Macromolecular Assemblies - 2007
 NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals 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"

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

Molecular fitting - Dec 4 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 1620 Nov 2007 - Main.DavidStokes

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

NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals 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"

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

Changed:
<
<

Molecular fitting - Dec 6 - Hernando Sosa (Einstein)

>
>

Molecular fitting - Dec 4 - Hernando Sosa (Einstein)

 
  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 1509 Nov 2007 - Main.BillRice

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

NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals 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"

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)

Deleted:
<
<

Practical on tomography II (IMOD reconstruction)- Oct 25

 
  • 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)
Added:
>
>

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

Molecular fitting - Dec 6 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 1409 Nov 2007 - Main.BillRice

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

NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals 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"

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)

Practical on tomography II (IMOD reconstruction)- Oct 25

  • 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 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
Deleted:
<
<

Practical on 2D crystallography (2DX) - Nov 22

 

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
Added:
>
>

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

 

Molecular fitting - Dec 6 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 1309 Nov 2007 - Main.BillRice

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

NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals 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"

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)

Practical on tomography II (IMOD reconstruction)- Oct 25

  • 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 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
Changed:
<
<

Practical on single particle analysis II (refinement & reconstruction) - Nov 8

>
>

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)
Changed:
<
<

Practical on single particle analysis III (multivariate statistical analysis) - Nov 15

>
>

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

Practical on 2D crystallography (2DX) - Nov 22

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

Molecular fitting - Dec 6 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 1230 Oct 2007 - Main.BillRice

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

NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals 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"

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)

Practical on tomography II (IMOD reconstruction)- Oct 25

  • 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 30 - Joachim Frank (Wadsworth Ctr)

Changed:
<
<

Practical on single particle analysis I - Nov 1

>
>

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 (refinement & reconstruction) - 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 (multivariate statistical analysis) - 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

Practical on 2D crystallography (2DX) - Nov 22

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

Molecular fitting - Dec 6 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 1109 Oct 2007 - Main.BillRice

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

NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals 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"

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

Added:
>
>
  • 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)

Practical on tomography II (IMOD reconstruction)- Oct 25

  • 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 30 - Joachim Frank (Wadsworth Ctr)

Practical on single particle analysis I - Nov 1

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

  • Cryoelectron tomography
  • Zero-loss imaging
  • Resolution assessment

Practical on single particle analysis II (refinement & reconstruction) - 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 (multivariate statistical analysis) - 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

Practical on 2D crystallography (2DX) - Nov 22

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

Molecular fitting - Dec 6 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 1005 Oct 2007 - Main.BillRice

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

NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals 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"

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

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
Changed:
<
<

practical on tomography I (IMOD reconstruction) - Oct 18

>
>

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

 

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

Changed:
<
<

Practical on tomography II (IMOD segmentation) - Oct 25

>
>

Practical on tomography II (IMOD reconstruction)- Oct 25

 
  • 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 30 - Joachim Frank (Wadsworth Ctr)

Practical on single particle analysis I - Nov 1

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

  • Cryoelectron tomography
  • Zero-loss imaging
  • Resolution assessment

Practical on single particle analysis II (refinement & reconstruction) - 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 (multivariate statistical analysis) - 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

Practical on 2D crystallography (2DX) - Nov 22

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

Molecular fitting - Dec 6 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 905 Oct 2007 - Main.DavidStokes

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

NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals 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"

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

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 (IMOD reconstruction) - Oct 18

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

Practical on tomography II (IMOD segmentation) - Oct 25

  • 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 30 - Joachim Frank (Wadsworth Ctr)

Practical on single particle analysis I - Nov 1

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

  • Cryoelectron tomography
  • Zero-loss imaging
  • Resolution assessment

Practical on single particle analysis II (refinement & reconstruction) - 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 (multivariate statistical analysis) - 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

Practical on 2D crystallography (2DX) - Nov 22

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
Changed:
<
<

Molecular fitting - Dec 4 - Hernando Sosa (Einstein)

>
>

Molecular fitting - Dec 6 - Hernando Sosa (Einstein)

 
  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 818 Sep 2007 - Main.DavidStokes

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

NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals 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"

Added:
>
>		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

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 (IMOD reconstruction) - Oct 18

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

Practical on tomography II (IMOD segmentation) - Oct 25

  • 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 30 - Joachim Frank (Wadsworth Ctr)

Practical on single particle analysis I - Nov 1

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

  • Cryoelectron tomography
  • Zero-loss imaging
  • Resolution assessment

Practical on single particle analysis II (refinement & reconstruction) - 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 (multivariate statistical analysis) - 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

Practical on 2D crystallography (2DX) - Nov 22

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

Molecular fitting - Dec 4 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 718 Sep 2007 - Main.RubenDiaz

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

NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals 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"

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
Changed:
<
<
    • Coulomb's Law and forces
>
>
    • 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

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 (IMOD reconstruction) - Oct 18

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

Practical on tomography II (IMOD segmentation) - Oct 25

  • 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 30 - Joachim Frank (Wadsworth Ctr)

Practical on single particle analysis I - Nov 1

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

  • Cryoelectron tomography
  • Zero-loss imaging
  • Resolution assessment

Practical on single particle analysis II (refinement & reconstruction) - 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 (multivariate statistical analysis) - 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

Practical on 2D crystallography (2DX) - Nov 22

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

Molecular fitting - Dec 4 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 617 Sep 2007 - Main.RubenDiaz

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

NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals 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"

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
Changed:
<
<
    • Faraday's Law
    • influence of a B field
    • electrostatic and electromagnetic lens
>
>
    • Coulomb's Law and forces
    • Motion of an electron in a magnetic field
    • electrostatic and magnetic lenses
 
    • deflectors
Changed:
<
<
  • electron scattering
>
>
  • electron-specimen interactions
Added:
>
>
    • particle-wave duality
 
    • scattering cross-section
    • elastic scattering
    • inelastic scattering
    • secondary electrons
    • backscattered electrons
    • x-rays
    • EELS
  • microscope column
Changed:
<
<
    • condensor, objective, intermediate, projector lenses
>
>
    • condenser, objective, intermediate, projector lenses
 
    • gun, beam, image deflector coils
    • condensor, objective, selected area apertures
  • optics
    • focal length, magnification
    • ray path diagram
    • resolution
    • aberrations
Changed:
<
<
    • coherency
>
>
    • 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

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 (IMOD reconstruction) - Oct 18

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

Practical on tomography II (IMOD segmentation) - Oct 25

  • 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 30 - Joachim Frank (Wadsworth Ctr)

Practical on single particle analysis I - Nov 1

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

  • Cryoelectron tomography
  • Zero-loss imaging
  • Resolution assessment

Practical on single particle analysis II (refinement & reconstruction) - 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 (multivariate statistical analysis) - 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

Practical on 2D crystallography (2DX) - Nov 22

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

Molecular fitting - Dec 4 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 531 Aug 2007 - Main.DavidStokes

 
META TOPICPARENT name="CemCourseGroup"
Cryoelectron Microscopy of Macromolecular Assemblies
Changed:
<
<		NYU course # G16.4408 - 3 credits
>
>		NYU course # G16.4408 - 3 credits for lectures, 1 credit for practicals
Added:
>
>		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"

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
    • 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
  • 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

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 (IMOD reconstruction) - Oct 18

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

Practical on tomography II (IMOD segmentation) - Oct 25

  • 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 30 - Joachim Frank (Wadsworth Ctr)

Practical on single particle analysis I - Nov 1

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

  • Cryoelectron tomography
  • Zero-loss imaging
  • Resolution assessment

Practical on single particle analysis II (refinement & reconstruction) - 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 (multivariate statistical analysis) - 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

Practical on 2D crystallography (2DX) - Nov 22

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

Molecular fitting - Dec 4 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 431 Aug 2007 - Main.DavidStokes

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

NYU course # G16.4408 - 3 credits

Deleted:
<
<		Contents
 

Instructors

Useful Textbooks

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

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 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
    • 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
  • 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

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 (IMOD reconstruction) - Oct 18

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

Practical on tomography II (IMOD segmentation) - Oct 25

  • 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 30 - Joachim Frank (Wadsworth Ctr)

Practical on single particle analysis I - Nov 1

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

  • Cryoelectron tomography
  • Zero-loss imaging
  • Resolution assessment

Practical on single particle analysis II (refinement & reconstruction) - 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 (multivariate statistical analysis) - 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

Practical on 2D crystallography (2DX) - Nov 22

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

Molecular fitting - Dec 4 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 324 Aug 2007 - Main.DavidStokes

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

NYU course # G16.4408 - 3 credits

Contents

Instructors

Useful Textbooks

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

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 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
    • 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
  • 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

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 (IMOD reconstruction) - Oct 18

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

Added:
>
>

Practical on tomography II (IMOD segmentation) - Oct 25

 
  • 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 23 - Joachim Frank (Wadsworth Ctr)

Practical on tomography II (IMOD segmentation) - Oct 25

>
>

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

Practical on single particle analysis I - Nov 1

 

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

  • Cryoelectron tomography
  • Zero-loss imaging
  • Resolution assessment

Practical on single particle analysis II (refinement & reconstruction) - 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 (multivariate statistical analysis) - 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

Practical on 2D crystallography (2DX) - Nov 22

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

Molecular fitting - Dec 4 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 212 Jul 2007 - Main.DavidStokes

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

NYU course # G16.4408 - 3 credits

Contents

Instructors

Useful Textbooks

Changed:
<
<
Glaeser, Downing, DeRosier, Chiu, Frank:
"Electron Crystallography of Biological Macromolecules" 		frank.jpg
>
>		Glaeser, Downing, DeRosier, Chiu, Frank:
"Electron Crystallography of Biological Macromolecules"
 
Changed:
<
<
Frank:
"Three Dimensional Electron Microscopy of Macromolecular Assemblies" 		frank.jpg
Reimer:
"Transmission Electron Microscopy:
Physics of Image Formation and Microanalysis" 		Reimer.jpg
Bozzola & Russell:
"Electron Microscopy: Principles and Techniques for Biologists" 		Bozzola.jpg
Harris:
"Negative Staining and Cryoelectron Microscopy" 		harris.jpg
Williams & Carter:
"Transmission Electron Microscopy: A Textbook for Materials Science" 		WilliamsCarter.jpg
>
>		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"

Added:
>
>		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
    • 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
  • 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

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 (IMOD reconstruction) - 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)

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

Practical on tomography II (IMOD segmentation) - Oct 25

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

  • Cryoelectron tomography
  • Zero-loss imaging
  • Resolution assessment

Practical on single particle analysis II (refinement & reconstruction) - 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 (multivariate statistical analysis) - 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

Practical on 2D crystallography (2DX) - Nov 22

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

Molecular fitting - Dec 4 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

Revision 112 Jul 2007 - Main.DavidStokes

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

NYU course # G16.4408 - 3 credits

Contents

Instructors

Useful Textbooks

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

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

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
    • 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
  • 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

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 (IMOD reconstruction) - 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)

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

Practical on tomography II (IMOD segmentation) - Oct 25

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

  • Cryoelectron tomography
  • Zero-loss imaging
  • Resolution assessment

Practical on single particle analysis II (refinement & reconstruction) - 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 (multivariate statistical analysis) - 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

Practical on 2D crystallography (2DX) - Nov 22

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

Molecular fitting - Dec 4 - Hernando Sosa (Einstein)

  • 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

  • Set ALLOWTOPICVIEW =

-- DavidStokes - 12 Jul 2007

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