Mechanism of image formation for thick biological specimens: exit wavefront reconstruction and electron energy‐loss spectroscopic imaging

Abstract

SUMMARY With increasing frequency, cellular organelles and nuclear structures are being investigated at high resolution using electron microscopic tomography of thick sections (0·3–1·0 μm). In order to reconstruct the structures in three dimensions accurately from the observed image intensities, it is essential to understand the relationship between the image intensity and the specimen mass density. The imaging of thick specimens is complicated by the large fraction of multiple scattering which gives rise to incoherent and partially coherent image components. Here we investigate the mechanism of image formation for thick biological specimens at 200 and 300 keV in order to resolve the coherent scattering 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 specimens is dominated by amplitude (absorption) contrast, we have found that for conventionally stained biological specimens phase contrast contributes significantly, and that at resolutions better than ∼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 wavefront reconstruction can exclude the microscope aberrations as well as the multiple scattering component from the image formation. Since most 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 excluding the multiple scattering, the ‘restored’ images display enhanced contrast and resolution. These findings have direct implications for the three‐dimensional reconstruction of thick biological specimens, where a simple direct relationship between image intensity and mass density was assumed, and the aberrations were left uncorrected.

Improved specimen reconstruction by Hilbert phase contrast tomography

“…for thick specimens showed significant phase contrast, and this contribution dominates for resolutions better than 10 nm (Han et al, 1995). These findings are confirmed by the data shown in Fig.…”

“…Producing sharp images and strong contrast at the same time is the main advantage of the novel HPC technique over conventional defocus CET. Han et al (1995) showed that the problem of weak phase contrast persists for thick specimens (0.3-1 lm), such as ice-embedded cells or organelles as they are analyzed in CET. Objects with a thickness above 0.5 lm cause about 60% inelastic scattering which can be partially converted into amplitude contrast by energy filtering (Angert et al, 2000).…”

Image Restoration of Thick Biological Specimens for Transmission Electron Microscope Tomography

“…With increasing specimen tilt angle 0 (equivalent to an increasing effective specimen thickness following 1 / cos 9), the average image intensity decreases logarithmically (data not shown). This is in agreement with the logarithmic decrease of the elastically scattered electrons as a function of specimen thickness (Han et al, 1995c). Both of these observations are consistent with the absorption model for thick specimens.…”

“…Studies have shown that the 3D power spectrum of a through focus series (taken at equal intervals as in the exit wavefront reconstruction) can be used to evaluate the proportion of coherent electrons contributing to imaging (Han et aL, 1995c). By combining this technique with ESI, it was shown that for thick biological specimens, only the elastically (zero energy-loss) scattered electrons exhibit the linear imaging behavior through focus, which is characteristic of the coherent image component (Han et ai, 1995b).…”

Electron holography of biological samples

“…This could be circumvented by lowering the dose, however, the main drawback of this method remains that even at higher defoci the contrast-transfer function sin x will not provide a good contrast for larger spacings. Nevertheless, Han et al (1995) reported about the mechanism of image formation for thick biological specimens at 200 and 300 keV in order to resolve the coherent scattering component from the incoherent (multiple scattering) components by exit wavefront reconstruction using a throughfocus series. They found that although it is commonly assumed that image formation of thick specimens is dominated by amplitude (absorption) contrast, that for conventionally stained biological specimens phase contrast contributes significantly, and that at resolutions better than similar to 10 nm, superposed phase contrast dominates.…”