4D STEM Electron Ptychography

Cryo-electron microscopy (cryo-EM) has revolutionised structural biology by enabling high-resolution, three-dimensional imaging of macromolecular complexes in their native, frozen-hydrated state.

However, conventional cryo-EM and tilt-series cryo-electron tomography (cryo-ET) often face challenges such as low signal-to-noise ratios, limited contrast, and restrictions in specimen thickness, especially when imaging larger cellular environments.

An emerging complementary approach, 4D scanning transmission electron microscopy (4D-STEM) ptychography[1], offers a new way to overcome these limitations[2]. Electron ptychography is a lensless, diffraction-based phase-recovery technique that reconstructs quantitative images from overlapping diffraction patterns collected as a convergent probe scans across the specimen. This method delivers high phase sensitivity[3], low-dose efficiency [4-5], and 3D optical sectioning and tomography[8-10].

In this talk, we will introduce the principles of 4D-STEM electron ptychography and its applications to biological systems under cryogenic conditions (cryo-ePty) as shown in Fig. 1a[5]. We will present recent progress demonstrating 2D structural reconstructions of frozen Rotavirus particles, HIV virus-like particles, and resin-embedded cells, all achieved with electron doses as low as 6 e⁻/Ų. We will discuss a standardised workflow for 3D single-particle analysis (SPA)using cryo-ePty data[10], and a proof-of-concept combination ofptychography and tilted tomography for imaging 3D DNA-origami nanostructures[9]. Furthermore, we will present our latest methodological developments designed to improve the resolution and contrast of ptychographic imaging [11–12].

The seminar will conclude with an open discussion on how electron ptychography is transforming nanoscale imaging across both physical and life sciences, bridging molecular and cellular length scales while maintaining high contrast and resolution.