Interrogating the behavior of macromolecules within a native cellular context will allow us to gain an enhanced understanding of how biological molecules function inside cells, which is vital to combating disease. Cryo-electron microscopy (cryo-EM) such as single particle analysis (SPA) and computer tomography (CT) provides key 3D structural information and bridges the gap between individual molecules and the whole cell. Nevertheless, both of these two powerful approaches rely on the use of conventional phase contrast TEM imaging. Unstained biological samples embedded in thin vitreous ice are essentially pure phase objects that are extremely radiation sensitive, resulting in images with low signal-to-noise ratios and low contrast except at high defocus. Therefore, it is more challenging for heterogeneous samples, specimens at low concentration, structures with low symmetry, and small (<150 kDa), or flexible molecules. For these reasons, methods that improve information transfer over a wide range of spatial frequencies are required.
An alternative strategy called Cryo-electron ptychography (Cryo-EPty)  is an emerging computational microscopy technique for acquiring images with resolutions beyond the limits imposed by lenses. This approach scans a sample with a convergent probe in an array and records a series of two-dimensional diffraction patterns as a function of probe position forming a four-dimensional (4D) dataset. Using this 4D dataset of diffraction patterns, quantitative phase data with the highest possible spatial resolution can be recovered using one of several iterative phase retrieval algorithms. In recent our work, we successfully resolved the 2D structure of frozen Rotavirus virus, HIV-VLP and resin embedded cells, across a range of biological molecular sizes, using as little as 6 e-/Å2 . We further extended to combine ptychography with tomography to simultaneously image both the organic and inorganic components in a 3D DNA-origami framework hybrid nanostructure  with high contrast at the room temperature.
Furthermore, a pulsed-source cryo-electron microscope recently developed in the Rosalind Franklin Institute is equipped with an electron dose modulator, which can turn the beam on and off with variable pulse widths a nanosecond. This instrument provides us with a unique capability of adjusting the beam intensity or dose without changing the image conditions during the experiment. This optical configuration is essential to systemically study dose-dependent contrast mechanisms at the ns time scale in terms of potential ultrafast imaging application for probing biomolecular structural dynamics.
In this project, we will develop new cryo-ptychographic imaging for whole-cell imaging towards single molecular resolution enhanced with artificial intelligence (AI). By combining the 3D reconstruction pipelines of SPA and CT, we will further develop completely new 3D reconstruction schemes based upon cryogenic ptychography (Ptycho-SPA and Ptycho-CT) and establish standardized protocols to enable high-resolution imaging. Finally, we will further incorporate these methodologies to the state-of-the-art pulsed-source cryo-electron microscope to systemically study the dose-dependent contrast mechanism as a function of a pulse-time down to a nanosecond time resolution, which will lay the critical foundations for future time-resolved imaging to reveal previously hidden rapid dynamic events in biology.
- Zhou, L., … & Wang* P.Low-dose phase retrieval of biological specimens using cryo-electron ptychography. Nature Communications, 11, 2773 (2020).
- Ding, Z.,.... & Wang*, P.Three-dimensional electron ptychography of organic-inorganic hybrid nanostructures. Nature Communications 13, 4787, (2022).
BBSRC Strategic Research Priority: Understanding the rules of life – Systems Biology.
Techniques that will be undertaken during the project:
Cryo-EM, Ptychography, Iterative Phase Restoration, Single Particle Analysis, Electron Tomography