Advances in cryogenic transmission electron microscopy have revolutionised the determination of many macromolecular structures at atomic or nearatomic resolution. This method is based on conventional defocused phase contrast imaging. However, it has limitations of weaker contrast for small biological molecules embedded in vitreous ice, in comparison with cryo-ptychography, which shows increased contrast. Here we report a single-particle analysis based on the use of ptychographic reconstruction data, demonstrating that three dimensional reconstructions with a wide information transfer bandwidth can be recovered by Fourier domain synthesis. Our work suggests future applications in otherwise challenging single particle analyses, including small macromolecules and heterogeneous or flexible particles. In addition structure determination in situ within cells without the requirement for protein purification and expression may be possible.

PW acknowledges funding from the National Natural Science Foundation of China (11874199) and the University of Warwick Research Development Fund (RDF) 2021-22 Science Development Award. LQZ acknowledges The fellowship of China National Postdoctoral Program for Innovative Talents (No. BX2021119) and Project funded by China Postdoctoral Science Foundation (No. 2022M711564). DIS is supported by the UK Medical Research Council (MR/N00065X/1). PZ is supported by the Wellcome Trust (206422/Z/17/Z) and the European Research Council Advanced Grant (101021133). We thank Diamond Light Source for access and support in the use of the electron Physical Science Imaging Centre (Instrument E02, EM17918) that contributed to the results presented.