3D Visualization at the nanoscale level - WUR
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3D Visualization at the nanoscale level Electron tomography 27 October 2016 Wageningen Electron Microscopy Center (WEMC)
Transcript of 3D Visualization at the nanoscale level - WUR
PowerPoint-presentatieElectron tomography
Drawing by John O’Brien, The New Yorker Magazine (1991)
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Two ways to obtain 3D images with a TEM at the nanoscale level
Single particle cryo-electron microscopy Averaging of many images of single particles and
reconstruction of a 3D volume Electron tomography
Creating a series of tilted 2D images and recreating a 3D volume by weighted back projection after alignment of the series.
Cowpea mosaic virus
Negative stained Cryo-fixed: frozen but hydrated
Dehydrated and observed in TEM Observed in frozen state in cryo-EM
Courtesy: Dr. F. de Haas, FEI electron optics, Application Laboratory
Averages of images grouped around a particular direction of projection
Averaging and reconstruction of 3D architecture
Courtesy: Dr. F. de Haas, FEI electron optics, Application Laboratory
Courtesy: Dr. F. de Haas, FEI electron optics, Application Laboratory
Reconstruction of Cowpea mosaic virus
Courtesy of Dr. Bridget Carragher, Scripps Institute, La Jolla (CA, USA)
Tobacco Mosaic Virus reconstructed in 3D down to 10Å resolution from one data collection session (<24hrs)
Reconstruction of Tobacco mosaic virus
Two ways to obtain 3D images with a TEM at the nanoscale level
Single particle cryo-electron microscopy Averaging of many images of single particles and
reconstruction of a 3D volume Electron tomography
Creating a series of tilted 2D images and recreating a 3D volume by weighted back projection after alignment of the series.
TEM Observation of Specimen
Equipment
Compustage capable of tilting from -70° to +70°
High resolution sensitive digital camera
Software for acquisition, alignment, reconstruction and volume rendering
JEOL JEM2100 200 kV microscope
Electron Tomography What does it take?
Procedure
(Automated) Alignment of the images
3D reconstruction of a tomogram
Visualization and Analysis
1 meter 100 mm
Most cells
λ at 200 kV = 0.0028 nm d = 0.0014 nm
Current TEM specify: 0.27 nm point resolution and 0.14 nm line resolution
De Broglie
Resolution of Electron Tomography
To obtain 2.2 nm resolution of a 100 nm object
collect 140 images (from -70 to +70 each 1° tilt)
Electron tomography a matter of hours
Fully automated data collection
Aligned tilt series Tomogram
From tomogram to volume
3D Visualization of biologicals and materials at the nanoscale level
Visualization of cells and cell structures
Revealing spatial organization of membranes, organelles, protein complexes
3D localization of gold-labels
Has potential to provide 3D structural maps of cells
revealing “molecular signatures” of individual proteins
Electron tomography
EM traditionally produces 2D images
Two ways to obtain 3D images with a TEM at the nanoscale level
Singe particle cryo-electron microscopyFrozen hydrated specimen: spherical virus particles
Averaging and reconstruction of 3D architecture
Reconstruction of Cowpea mosaic virus
Reconstruction of Tobacco mosaic virus
Two ways to obtain 3D images with a TEM at the nanoscale level
TEM Observation of Specimen
Transmission electron microscopy (TEM)
Resolution of Electron Tomography
Structure of a katalase crystal
Electron tomography of shrimp virus
From tomogram to volume
Drawing by John O’Brien, The New Yorker Magazine (1991)
X
Z
Y
Two ways to obtain 3D images with a TEM at the nanoscale level
Single particle cryo-electron microscopy Averaging of many images of single particles and
reconstruction of a 3D volume Electron tomography
Creating a series of tilted 2D images and recreating a 3D volume by weighted back projection after alignment of the series.
Cowpea mosaic virus
Negative stained Cryo-fixed: frozen but hydrated
Dehydrated and observed in TEM Observed in frozen state in cryo-EM
Courtesy: Dr. F. de Haas, FEI electron optics, Application Laboratory
Averages of images grouped around a particular direction of projection
Averaging and reconstruction of 3D architecture
Courtesy: Dr. F. de Haas, FEI electron optics, Application Laboratory
Courtesy: Dr. F. de Haas, FEI electron optics, Application Laboratory
Reconstruction of Cowpea mosaic virus
Courtesy of Dr. Bridget Carragher, Scripps Institute, La Jolla (CA, USA)
Tobacco Mosaic Virus reconstructed in 3D down to 10Å resolution from one data collection session (<24hrs)
Reconstruction of Tobacco mosaic virus
Two ways to obtain 3D images with a TEM at the nanoscale level
Single particle cryo-electron microscopy Averaging of many images of single particles and
reconstruction of a 3D volume Electron tomography
Creating a series of tilted 2D images and recreating a 3D volume by weighted back projection after alignment of the series.
TEM Observation of Specimen
Equipment
Compustage capable of tilting from -70° to +70°
High resolution sensitive digital camera
Software for acquisition, alignment, reconstruction and volume rendering
JEOL JEM2100 200 kV microscope
Electron Tomography What does it take?
Procedure
(Automated) Alignment of the images
3D reconstruction of a tomogram
Visualization and Analysis
1 meter 100 mm
Most cells
λ at 200 kV = 0.0028 nm d = 0.0014 nm
Current TEM specify: 0.27 nm point resolution and 0.14 nm line resolution
De Broglie
Resolution of Electron Tomography
To obtain 2.2 nm resolution of a 100 nm object
collect 140 images (from -70 to +70 each 1° tilt)
Electron tomography a matter of hours
Fully automated data collection
Aligned tilt series Tomogram
From tomogram to volume
3D Visualization of biologicals and materials at the nanoscale level
Visualization of cells and cell structures
Revealing spatial organization of membranes, organelles, protein complexes
3D localization of gold-labels
Has potential to provide 3D structural maps of cells
revealing “molecular signatures” of individual proteins
Electron tomography
EM traditionally produces 2D images
Two ways to obtain 3D images with a TEM at the nanoscale level
Singe particle cryo-electron microscopyFrozen hydrated specimen: spherical virus particles
Averaging and reconstruction of 3D architecture
Reconstruction of Cowpea mosaic virus
Reconstruction of Tobacco mosaic virus
Two ways to obtain 3D images with a TEM at the nanoscale level
TEM Observation of Specimen
Transmission electron microscopy (TEM)
Resolution of Electron Tomography
Structure of a katalase crystal
Electron tomography of shrimp virus
From tomogram to volume
