Practical image restoration of thick biological specimens using multiple focus levels in transmission electron microscopy

Journal of Structural Biology
K F HanD A Agard

Abstract

Three-dimensional electron tomographic studies of thick specimens such as cellular organelles or supramolecular structures require accurate interpretations of transmission electron micrograph intensities. In addition to microscope lens aberrations, thick specimen imaging is complicated by additional distortions resulting from multiple elastic and inelastic scattering. Extensive analysis of the mechanism of image formation using electron energy-loss spectroscopy and imaging as well as exit wavefront reconstruction demonstrated that multiple scattering does not contribute to the coherent component of the exit wave (Han et al., 1996, 1995). Although exit wavefront restored images showed enhanced contrast and resolution, that technique, which requires the collection of more than 30 images at different focus levels, is not practical for routine data collection in 3D electron tomography, where usually over 100 projection views are required for each reconstruction. Using a 0.7-micron-thick specimen imaged at 200 keV, the accuracy of reconstructions using small numbers of defocused images and a simple linear filter (Schiske, 1968) was assessed by comparison to the complete exit wave restoration. We demonstrate that only four optimal fo...Continue Reading

References

Sep 1, 1978·Journal of Dentistry·C R Cowell, C A Saxton
Oct 1, 1992·Ultramicroscopy·A J KosterD A Agard
Jun 1, 1993·Ultramicroscopy·A J Gubbens, O L Krivanek

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Citations

Aug 2, 2003·Journal of Structural Biology·Hanspeter Winkler, Kenneth A Taylor
Jan 22, 2003·Traffic·François Képès
Apr 17, 2001·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·B F McEwen, M Marko

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