The SNR expressions derived in Section V. Matson, in Advances in Imaging and Electron Physics, 2002 B SNR Example If, on the other hand, the intensity in a chosen area of the specimen is much above 90% of that in an open hole, one may have to worry whether the sample has been essentially air-dried before being frozen.Ĭharles L. Either way, it can be recommended that the intensity in an area chosen for data collection should not be less than approximately 70% (and preferably between 80% and 90%) of that in an open hole in the specimen. Alternatively, a low-magnification image can be used if it is underfocused enough to give nearly the maximum contrast possible. One imaging condition employed in Search mode uses a highly defocused electron diffraction pattern, so the central spot becomes enlarged sufficiently to produce an image of a suitable size. A reasonable alternative, illustrated in Figure 1, is simply to select areas of suitable thickness based on the contrast presented by the specimen in Search mode. As a result, the loss of signal can be minimal for such thin specimens, and the shot noise associated with the inelastically scattered electrons also can be fairly minimal.Īccurate measurement of the sample thickness can be too time-consuming to perform while one is collecting image data. Because of the depth-of-field issue discussed previously, one should actually aim for a sample thickness of about less than 50 nm-i.e., approximately one-sixth of the mean-free-path for 300-keV electrons. Since the mean-free-path for inelastic scattering of 300-keV electrons in ice is estimated to be more than 300 nm, inelastically scattered electrons have a relatively small effect on image quality if the ice thickness is less than 100 nm. The combined, adverse effects of inelastic scattering, mentioned previously, nevertheless will remain fairly small if the specimen is thinner than one-third of the mean-free-path for inelastic scattering, in which case the fraction of electrons transmitted with zero energy loss is greater than 70%. It is also worth noting that a further benefit of an energy filter is to “unmask” the amplitude contrast produced by inelastic scattering, and doing so can substantially improve the SNR at low resolution ( Yonekura et al., 2006). As a result, it is always beneficial to use an energy filter to remove the background of inelastically scattered electrons. While the average value of this added constant intensity has no effect on the SNR, statistical fluctuation in this added term (i.e., shot noise) is nevertheless present, and this shot noise further degrades the SNR. This model assumes that the inelastically scattered electrons are uniformly present throughout the image and thus contribute nothing to the signal. While an accurate description of the role of the inelastically scattered electrons is too complicated to go into here, a simplified model can give at least a sense of why the SNR is further degraded. The already inferior image (as discussed in the previous paragraph) now will be degraded even further as a result of including the inelastically scattered electrons. Going beyond the loss of signal, next imagine that a zero-loss energy filter is not used (as, indeed, often is the case). The loss of electrons that occurs with a too-thick specimen has no solution, of course, because one cannot just increase the exposure to compensate for the electrons that are lost due to inelastic scattering. The effect is just as if one had intentionally used a smaller incident-electron exposure to record images for a thinner specimen. To explain why this decreases the SNR, first imagine that one were to use a zero-loss energy filter to remove all the inelastically scattered electrons. To begin with, fewer electrons make their way through the specimen without suffering an inelastic scattering event. The SNR in images of ice-embedded specimens is adversely affected in two ways when the ice thickness is greater than it needs to be. Sriram Subramaniam, in Advances in Imaging and Electron Physics, 2014 2.2.3 The SNR Is Diminished as the Fraction of Inelastically Scattered Electrons Increases
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