PRACTICAL IMAGE-RESTORATION OF THICK BIOLOGICAL SPECIMENS USING MULTIPLE FOCUS LEVELS IN TRANSMISSION ELECTRON-MICROSCOPY

Three-dimensional electron tomographic studies of thick specimens such as cellular organelles or supramolecular structures require accurate interpretations of transmission electron micrograph intensities. In ad dition to microscope lens aberrations, thick specimen imaging is compl icated by additional distortions resulting from multiple elastic and i nelastic scattering. Extensive analysis of the mechanism of image form ation using electron energy-loss spectroscopy and imaging as well as e xit wavefront reconstruction demonstrated that multiple scattering doe s not contribute to the coherent component of the exit wave (Han et al ., 1996, 1995). Although exit wavefront restored images showed enhance d contrast and resolution, that technique, which requires the collecti on of more than 30 images at different focus levels, is not practical for routine data collection in 3D electron tomography, where usually o ver 100 projection views are required for each reconstruction, Using a 0.7-mu m-thick specimen imaged at 200 keV, the accuracy of reconstruc tions using small numbers of defocused images and a simple linear filt er (Schiske, 1968) was assessed by comparison to the complete exit wav e restoration. We demonstrate that only four optimal focus levels are required to effectively restore the coherent component (deviation 5.1% ). By contrast, the optimal single image (zero defocus) shows a 25.5% deviation to the exit wave restoration. Two pairs of under-and over-de focus images should be taken: one pair at quite high defocus (>10 mu m ) to differentiate the coherent (single elastic scattering) from the i ncoherent (multiple elastic and inelastic scattering) components, and the second pair to optimize information content at the highest desired resolution (e.g., 5 mu m for (2.5 nm)(-1) resolution). We also propos e a new interpretation of the restored amplitude and phase components where the specimen mass-density is proportional to the logarithm of th e amplitude component and linearly related to the phase component, Thi s approach should greatly facilitate the collection of high resolution tomographic data from thick samples. (C) 1997 Academic Press.