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Abstract

A controversy exists as to whether the signal in a high resolution phase contrast electron micrograph of a particle in a thick specimen is the same irrespective of the particle's position along the beam axis. Different conceptions of inelastic scattering and its effects on wave interference have led to radically different expectations about the degree of phase contrast vs. depth. Here we examine the information available from bright field phase contrast images of small crystalline particles on the top or bottom of a thick support. The support is an aluminium foil which has strong plasmon resonances that cause a large proportion of the electron beam to lose energy in transit. Phase contrast micrographs of the atomic lattice of two ensembles of platinum particles were measured in an energy loss window corresponding to the first plasmon resonance. The signal measured for particles on top was equal to that for particles on the bottom of the foil to within a 99% confidence interval, and the measurements exclude other models of depth dependent phase contrast in the literature to >5σ. These observations are consistent with quantum theory which considers dynamical effects as independent of event sequence and is distinct from the "top-bottom effect" observed in amplitude contrast. We thus confirm that phase contrast using inelastically scattered electrons can be obtained equally well from particles within any layer of a thick specimen.

Keywords: C(c) correction; CryoEM; Inelastic phase contrast; Top bottom effect.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1

Fig. 1

Platinum particles on top of…

Fig. 1

Platinum particles on top of an aluminium foil imaged with 15 eV energy…

Fig. 1 Platinum particles on top of an aluminium foil imaged with 15 eV energy loss electrons and a 300 keV primary energy. Panel (b) is an enlargement of the particle boxed in (a), and (c) shows the Fourier transform of (b), with arrows indicating the 111 reflections from platinum (black) and aluminium (white). An electron energy loss spectrum of the specimen in (a), showing plasmon peaks of aluminium at 15 and 30 eV. The energy range selected (15 ± 2 eV) for the imaging is indicated in grey.
Fig. 2

Fig. 2

Phase contrast from particles on…

Fig. 2

Phase contrast from particles on the top and bottom of an 1100 Å…

Fig. 2 Phase contrast from particles on the top and bottom of an 1100 Å thick aluminium + 600 Å thick carbon foil. A diagram of the experiment is shown in row (a). Example phase contrast micrographs, taken under identical conditions at 15 eV energy loss and 300 keV primary energy, of platinum particles on the top and bottom are shown (b) with the power spectra of the cropped images (c) and a section through the power spectra in the directions indicated by the arrows (d). Note the low frequency peak was suppressed in (d) to aid in visualisation of the peaks.
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  • WT_/Wellcome Trust/United Kingdom
  • MC_UP_1201/17/MRC_/Medical Research Council/United Kingdom

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