Coherent Diffractive Imaging in Biological Transmission Electron Microscopy

Electron ptychography revolutionized materials science microscopy by achieving record-breaking resolutions through phase retrieval algorithms, but its application to radiation-sensitive biological specimens remains largely unexplored. This thesis pioneers the application of 4D-STEM ptychography to frozen hydrated biological specimens, where electron doses must remain substantially low to prevent radiation damage, orders of magnitude lower than materials science applications. We developed an automated 4D-STEM acquisition pipeline and collected datasets from proteins in single particle configurations and thick cellular specimens requiring tomographic reconstruction. For single particles, ptychographic reconstruction achieved 5.8 Å resolution for apoferritin, far below the 1.09 Å obtained by conventional TEM from the same specimen. For tomography: multi-slice ptychography, designed to correct multiple scattering in thick specimens, yielded only 40Å resolution at 6-10e/Ų per tilt, while the theoretically less-sophisticated parallax method achieved approximately 20 Å. This outcome exposes a fundamental limitation, that at extreme low doses, sophisticated iterative ptychography algorithms fail to converge while simpler direct methods remain stable. The probe reconstruction, critical for ptychographic success, either stagnates or evolves toward unphysical solutions when working with too few electron counts. These findings demonstrate that biological electron microscopy operates in a regime where algorithmic stability matters as much as theoretical sophistication. Future progress requires developing fundamentally new approaches that treat extreme dose limitation as the central constraint rather than a secondary consideration.