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bioRxiv · 10.64898/2026.07.25.736262

Electron counting enables cryo-electron ptychography for near-atomic-resolution cryo-electron microscopy

Li, S.·Shen, B.·Yan, Z.·Liu, J.·Tang, C.·Wang, Z.·Deng, Z.·Li, X.
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Abstract

Cryo-electron ptychography is an emerging technique for studying radiation-sensitive biological specimens, developed from four-dimensional transmission electron microscopy (4D-STEM). Although ptychography has achieved ultrahigh resolution beyond conventional transmission electron microscopy limits for radiation-resistant samples, its application to frozen hydrated biological specimens currently remains at sub-nanometer resolution. Here we overcome this limitation by implementing electron counting with a hybrid-pixel detector, establishing key technical foundations for near-atomic-resolution cryo-ptychography. This counting approach significantly improves weak signal detection in convergent-beam electron diffraction, enabling ptychographic reconstruction at doses below 1 e-/[A]{superscript 2}. Additionally, we found beam-induced motion is effectively eliminated within single scans, suggesting conventional cryoEMs dose-fractionation approach may need reevaluation. Demonstrating high contrast under both low-dose and tilted conditions, along with achieving 3.59 [A] resolution for the [~]700 kDa T20S proteasome, we validate cryo-electron ptychography as a viable general imaging modality that could complement or surpass conventional phase-contrast cryoEM methods.

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Li, S., Shen, B., Yan, Z., Liu, J., Tang, C., Wang, Z., Deng, Z., Li, X.. 2026-07-28. Electron counting enables cryo-electron ptychography for near-atomic-resolution cryo-electron microscopy. https://doi.org/10.64898/2026.07.25.736262

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