Sampling mismatch and correction for ptychographic single-particle analysis

Ptychographic single-particle analysis (SPA) is a promising technique for high-resolution biological imaging; however, achieving near-atomic resolution remains challenging. In this study, we identified and investigated a critical issue termed sampling mismatch. This mismatch primarily originates from inaccurate calibration of the scanning step size when applying ptychography to large-area biological specimen. Several characteristic phenomena were observed and analyzed, including pixel-size deviation, measured defocus change and phase reversal in reconstructed micrographs. In particular, the phase reversal can be described through a mismatch-induced modulation function (MIMF) imposed on reconstructed micrographs, thereby serving as a principal limiting factor for achievable resolution in subsequent ptychographic SPA. We propose a calibration strategy for the scanning step size, which can be implemented as a preprocessing step prior to subsequent ptychographic calculations. The Thermoplasma acidophilum 20S proteasome and apoferritin datasets were used to demonstrate that correction of sampling mismatch eliminates phase reversal and improves resolution by ∼1.5 Å. These results underscore the necessity of accurate scanning control to achieve optical resolution in ptychographic SPA.