Amplus – Large Volume Tomography
Large Volume Tomography with Electron Microscopy
High resolution large volume tomography with electron microscopy has the potential to transform our understanding of life, by giving researchers access to the atomic and molecular structure of protein complexes in their biological context – the cell.
This offers opportunities in our basic understanding of human health, to pathogen research and drug development.
Amplus comes from the Latin word meaning large, magnificent and important – a fitting name for a technique with the possibility to show life at a cellular level in a completely new way.
Like existing cryo-EM, a technique which has revolutionised our ability to see the molecular structures of life, large volume tomography uses frozen samples, with ice in a glass-like ‘vitreous’ state. These are analysed with an electron beam, giving insight into the atomic and molecular structures of the sample. Using specially prepared samples and analysis, a 3D image of a whole cell or collection of cells could be obtained.
The samples are prepared using a first of the Cryo-Plasma-FIB (Cryo-PFIB), which is now being released by Thermo Fisher Scientific as the Arctis. The development of this instrument was a collaboration between us, Thermo Fisher Scientific and Diamond Light Source. The team at the Franklin have recently utilised this microscope in HTP lamellae production from human cells in an automated process (Berger et al, 2022), providing the first in-situ atomic structure of the human ribosomes.
To aid with the development of the physical technologies, the team has developed a digital twin software pipeline, known as parakeet.
Parakeet has been used to evaluate the effect of different experimental parameters on tomographic reconstructions. As a practical example, parakeet has allowed them to “quantify the effects of the missing wedge and sample geometry” (Parkhurst et al, 2021).
The team are interested initially in using the technique to explore three major challenges in human biology;
- Understanding intracellular bacterial pathogens, which are becoming increasingly antibiotic resistant. Observing the bacteria inside the human cell could help researchers understand its life cycle and develop novel drugs to combat it. The tiny size of intracellular bacterial pathogens makes them a perfect first candidate for this technique.
- Viral replication. The ability to see a viral infection of a cell at different stages, and observe the whole life cycle of a virus, rather than studying purified particles, offers huge leaps in our understanding of viral infection.
- Protein folding. Many diseases are caused by misfolded proteins, either with a genetic cause, such as cystic fibrosis, or caused by multiple factors, such as Alzheimer’s disease. In both cases, the ability to see a protein associated with a disease in context in the cell will enable better understanding of drug action, and of disease mechanism.
Professor James Naismith (Franklin) and Professor Dave Stuart (Diamond)
Project team members at The Franklin:
Diamond Light Source
The Rosalind Franklin Institute (funded by the UK Government through UK Research and Innovation’s Engineering and Physical Sciences Research Council)