Abstract
<jats:p>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^2. Additionally, we found beam-induced motion is effectively eliminated within single scans, suggesting conventional cryoEM's 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.</jats:p>