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This effect degrades image quality rapidly at higher tilt angles and, in most cases, ~60° is the highest tilt worth expending dose on.
When high-quality diffraction was observed for an untilted crystal, the crystal was then tilted to 60° to check the diffraction quality at higher tilt angles as crystal flatness and embedding could affect the diffraction quality at higher tilt (Gonen, 2013).
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For specimens with the typical slab geometry, the longer path length of the beam through the sample at high tilt and technical constraints in tilting range of specimen holders imposes a limitation on maximum tilt angle, so that part of space is not sampled.
An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles.
These factors greatly complicate CTF correction, particularly at high tilt (Sharp et al. 2017).
The lower bound curve, reached at high tilt angles ((|theta | =pi /3)), specifically depends on the sample compression along the normal axis, while the upper bound curve, reached at zero tilt, only depends on the lateral compressions.
The progressive change of phase with dose on Volta phase plates as well as the defocus gradient at high tilt both necessitate taking phase plates significantly out of focus.
Note that the Z compression rate contribution to (n_{3,omega }) is larger at high tilt angles compared to its in-plane (X and Y) counterparts, explaining the oscillatory behavior of (n_{3,omega }) versus the micrograph index (i_omega).
Indeed, DDDs now allow measuring the defocus directly from each image in a tiltseries, even at high tilt, which is essential under experimental settings for which the actual defocus might significantly differ from the target defocus and vary widely across the images of a tiltseries.
The method has mainly been used with negative stain EM, because cryo-EM is more difficult at high tilt angles.
However, inspection of particles at various tilt angles showed that particles of linear and triangular conformations remained clearly distinguishable, even at high tilt angles.
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