We have implemented the technique of electron tomography in a digital transmission electron microscope equipped with a post-column energy-filter in order to determine the three-dimensional arrangement and structure of supramolecular assemblies and subcellular cellular organelles. Series of energy-filtered images each containing 1024 x 1204 pixels are acquired at between sixty and eighty different tilt angles from -80 degrees to +80 degrees using a cooled CCD camera. These image series are analyzed using the IMOD program (University of Colorado) to obtain three-dimensional reconstructions. Gold particles are adsorbed onto the sections to facilitate alignment of the images in each series. We have applied electron tomography to determine the three-dimensional arrangement of proteins in post-synaptic densities (PSDs) of cultured neurons from rat brain prepared by high-pressure freezing, freeze-substitution and heavy metal staining. The structures of interest associated with the PSDs include CAM kinase II, scaffolding proteins as well as cytoskeletal fibers.

Agency
National Institute of Health (NIH)
Institute
Office of The Director, National Institutes of Health (OD)
Type
Intramural Research (Z01)
Project #
1Z01OD011058-02
Application #
6685018
Study Section
(BEPS)
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Office of the Director, NIH
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Aronova, M A; Kim, Y C; Zhang, G et al. (2007) Quantification and thickness correction of EFTEM phosphorus maps. Ultramicroscopy 107:232-44
Leapman, Richard D (2004) Novel techniques in electron microscopy. Curr Opin Neurobiol 14:591-8
Daniell, Sarah J; Kocsis, Eva; Morris, Edward et al. (2003) 3D structure of EspA filaments from enteropathogenic Escherichia coli. Mol Microbiol 49:301-8