MECHANISM OF IMAGE-FORMATION FOR THICK BIOLOGICAL SPECIMENS - EXIT WAVE-FRONT RECONSTRUCTION AND ELECTRON-ENERGY-LOSS SPECTROSCOPIC IMAGING

With increasing frequency, cellular organelles and nuclear structures are being investigated at high resolution using electron microscopic t omography of thick sections (0.3-1.0 mu m). In order to reconstruct th e structures in three dimensions accurately from the observed image in tensities, it is essential to understand the relationship between the image intensity and the specimen mass density. The imaging of thick sp ecimens is complicated by the large fraction of multiple scattering wh ich gives rise to incoherent and partially coherent image components. Here we investigate the mechanism of image formation for thick biologi cal specimens at 200 and 300 keV in order to resolve the coherent scat tering component from the incoherent (multiple scattering) components. Two techniques were used: electron energy-loss spectroscopic imaging (ESI) and exit wavefront reconstruction using a through-focus series. Although it is commonly assumed that image formation of thick specimen s is dominated by amplitude (absorption) contrast, we have found that for conventionally stained biological specimens phase contrast contrib utes significantly, and that at resolutions better than similar to 10 nm, superposed phase contrast dominates. It is shown that the decrease in coherent scattering with specimen thickness is directly related to the increase in multiple scattering. It is further shown that exit wa vefront reconstruction can exclude the microscope aberrations as well as the multiple scattering component from the image formation. Since m ost of the inelastic scattering with these thick specimens is actually multiple inelastic scattering, it is demonstrated that exit wavefront reconstruction can act as a partial energy filter. By virtue of exclu ding the multiple scattering, the 'restored' images display enhanced c ontrast and resolution. These findings have direct implications for th e three-dimensional reconstruction of thick biological specimens, wher e a simple direct relationship between image intensity and mass densit y was assumed, and the aberrations were left uncorrected.