Rosalind Franklin Institute researchers, working with Thermo Fisher Scientific, have developed a new approach that advances the field of electron cryo-tomography. This plasma ion sources for sample preparation required for in situ structural biology electron cryo-tomography and enables future studies into sample preparation at a faster rate.
The team used a new instrument, a prototype version of the recently announced “Arctis” microscope, to obtain the structure of a human ribosome to 4.9Å – achieving the highest resolution structure within a mammalian cell. This achievement reflects the improved preparation process and capabilities of the microscope.
The research was published this week in Nature Communications.
Electron cryo-tomography is a technique on the frontier of structural biology – as it allows the study of 3D macromolecular complexes rather than 2D. The new technology under development at the Franklin allows the study of these complexes within the context of the cell.
Dr Michael Grange, the paper’s senior author, and Group Leader at the Franklin, said, “Studying molecules in their native environment is key to understanding human disease. We hope that this improved technique will facilitate future work aimed at highlighting molecular differences in healthy and diseased cells and tissues and allow the development of improved treatments for a whole range of conditions, such as Alzheimer’s disease, heart disease, kidney malfunction and psoriasis.”
However, uptake of the technique is currently hampered by difficulties in biological sample preparation. Most biological samples are too thick to be directly imaged in a transmission electron microscope so must be thinned. Focused Ion Beam (FIB) milling is the current gold standard technique used to thin samples, generating thin sections known as lamellae. However, new methods are needed to enable it to be applied to thick specimens on reasonable timescales with high-throughput.
The team have been experimenting with the type of FIB milling to improve the production of lamellae. Typically, gallium is used as the ion source in FIB milling for structural biology but there as gallium ions can react with the sample during milling, altering the structure, new ion sources need investigating that may be sample inert and that may thin samples at greater rates.
As part of the study, the team also analysed the use of different gases to generate plasma – nitrogen, oxygen, xenon and argon – and their ability to thin cryogenic, biological samples at different rates. Combined with other work from the group, the researchers are building capacity to determine optimised strategies for FIB milling samples across imaging modalities.
The researchers also tested whether distance within the lamella from the plasma ion beam affected the structure of the ribosome. Dr Thomas Glen, one of the first authors on the paper and the Plasma FIB manager, noted “It has always been assumed that the thinner the lamellae the better, but this work indicates that the plasma beam does influence the structure of the ribosomes. So, a happy medium is possible and required – thin enough that the electrons can pass through the sample but thick enough that most of the structures within the sample are not affected by the beam.”
Berger et al. - Plasma FIB milling for the determination of structures in situ - Nature Communications (2023)