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Cause of FSC dip and consistent in‑focus LPP imaging method

Determine the cause of the observed dip in the Fourier shell correlation near 0.16 Å−1 in nominally in‑focus laser phase plate reconstructions, verify whether subtle defocus offsets are responsible, and develop and validate a robust method for consistently recording truly in‑focus phase‑contrast datasets.

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Background

Initial in‑focus LPP experiments on apoferritin produced a reconstruction with a conspicuous FSC dip around 0.16 Å−1, hypothesized to arise from residual defocus zero‑crossings without visible Thon rings.

The authors have not reproduced the result and seek a reliable procedure for consistent in‑focus acquisition—critical for harnessing LPP’s near‑ideal CTF and improving high‑frequency information content.

References

We are not sure why this feature is present, but hypothesize that it is caused by a zero-crossing of the CTF due to the images actually being recorded at a non-zero defocus (but still too small to generate noticeable or fittable Thon rings). However, we have not been able to reproduce this result, and we are still working on developing a method for consistently recording in-focus images.

— A Laser Phase Plate for Transmission Electron Microscopy (2403.10670 - Axelrod, 15 Mar 2024) in Section 12.3, In‑focus phase contrast imaging