Correlative 4D-STEM Ptychography and EELS for Cryogenic Single-Particle Analysis

Cryogenic electron microscopy has enabled atomic resolution structural determination of biological macromolecules. However, phase contrast imaging alone does not reveal elemental composition or other valuable chemical information. Electron energy loss spectroscopy (EELS) provides rich vibrational and chemical details, integrating it with high-resolution structural imaging for correlative analysis of biological specimens remains a major challenge, as conventional acquisition modes are often mutually exclusive. Here we propose two optical geometries that enable correlative 4D-STEM with EELS: (1) a sequential acquisition with a post-specimen beam deflector, and (2) a simultaneous acquisition with a hollow detector. We compared the two setups for biological specimens, and focused on the hollow detector geometry which allows simultaneous acquisition of diffraction patterns and EELS spectra in a single dose budget. Using an apoferritin dataset, we demonstrate sub-nanometre 3D resolution in single particle analysis, even with a hollow angle as of 55% of the convergence angle and approximately 30% of the total dose allocated to EELS. Finally, we propose a workflow that integrates ptychography with EELS for correlative, energy-resolved 3D mapping of biological macromolecules, paving the way toward unified structural, chemical, and vibrational analysis of biological specimens.