4D scanning transmission electron microscopy for structural biology

Introduction

Electron Microscopy (EM) has transformed research in the Life and Physical Sciences separately. Cryo-EM in Life Sciences allows the 3D structure determination of proteins down to 1.0 Å resolution in case they are large enough and present in high numbers in homogeneous states.

Challenges in Structure Determination

For many molecules involved in diseases like neurodegeneration, however, structure determination is still severely hampered due to their insufficient contrast when imaged in vitreous ice or in their native cellular environment.

Achievements in Physical Sciences

To date, EM in the Physical Sciences has generated utmost contrast for light atoms and established a resolution in the range of 0.2 Å, limited only by thermal motion. This record was achieved by evolving scanning transmission EM (STEM) from a 2D to a 4D imaging technique combining imaging with diffraction.

Project Objectives

In this project, we will work out 4D-BioSTEM methodologies and develop a cryo-EM tool that maximizes contrast and resolution by bringing together EM groups from Life and Physical Sciences.

Methodology

4D-STEM imaging of frozen biological specimens will be approached with:

1. Unique and specialized hardware
2. Theory and simulation
3. Development of microscope operation routines
4. Image reconstruction algorithms

Data Acquisition and Techniques

In particular, we will acquire sparse 4D data using ultrafast detectors, employ methods for direct phasing (differential phase contrast, DPC), and establish advanced, so-called ptychographic techniques to gain maximal signal from noisy 4D cryo-STEM data that are limited in their electron dose budget.

Synergies and Applications

We will make use of synergies between recently separated fields in order to make proteins smaller than 50 kDa amenable to structure elucidation.

Expansion to Tomography

Furthermore, we will expand 4D-STEM to tomography to obtain high-contrast 3D reconstructions from:

• Cellular samples
• Human brain tissue of neurodegenerative diseases
• Vitrified organic energy materials

Conclusion

A new comprehensive structural imaging framework will be put forward and benchmarked as to its utility in Life and Physical Science applications of cryo-EM.