Cells to Molecules: Structural EM at Newcastle University

Light microscopes are limited in terms of the level of detail they can provide of cells and it is not possible to use them to look at the fine structure of the internal features of cells or molecules. Transmission Electron Microscopes (TEM) use high energy electrons rather than light to produce images and provide over 1000 times the level of detail possible with a light microscope. Using a TEM it is possible to look at the structures within cells to see how they form and even see the arrangement of atoms within proteins and protein machines.

In the last twenty years, the technology behind TEM instruments and the cameras used to acquire images from them have undergone significant developments. One of the major drawbacks of imaging with a TEM is the limited contrast available from biological samples and the resulting impact of noise on the images. This makes our studies difficult as it is often hard to see the structures we are looking at clearly. Modern TEMs are equipped with cameras capable of taking images with very low noise and even capturing movies to allow very fine detail to be observed that would otherwise be lost. One major advance has been the preparation of samples at very low-temperatures, this allows cells and molecules to be viewed in a naturally hydrated state and minimises the spread of sample damage caused by the electrons from the microscope. This technique is known as cryo-EM and permits cells and proteins to be imaged with a level of detail at which it is possible to see the individual amino-acid components of proteins.

Our research projects cover a wide range of subjects from understanding how the intestinal pathogen Clostridium difficile forms disinfectant resistant spores; to the production of DNA-based nanowires for use in electronics and conductive polymer materials. Other projects seek to understand how particular genes influence the formation of kidney tissues. We are also interested in how proteins called cyclin dependent kinases interact with their partner proteins to influence cell division and the production of RNA messages encoding proteins. This work has implications for the development of anti-cancer drugs.

We request funds for the purchase of a 120kV TEM with cryo-stage and tomography capabilities to enable the high-resolution imaging of biological and materials samples. This equipment will replace a 25-year old TEM that is at the end of its useful life and currently supports 40 research groups. The TEM will allow us to image samples ranging from thin-sections of cells and tissues for 3D-reconstruction of cellular ultrastructure; to protein complexes for single-particle 3D-reconstruction; and engineered nano-materials. Common to these applications is the requirement for high-resolution imaging with low readout noise and fast data-acquisition.

The applicants, supported by funding from BBSRC, the Wellcome Trust, MRC, Cancer Research UK, the MRC and the British Heart Foundation, will benefit immediately from this new equipment on projects ranging from the study of Cyclin Dependent Kinase protein complexes, human meiosis, iron storage in bacteria, sporulation in Clostridium difficile, DNA nanomaterials, and renal vascular development.

Electron microscopy, and particularly cryo-EM, are key techniques in modern cell and structural biology. The immediate impact of an upgraded TEM will be on the groups whose projects are outlined in the Case for Support. As the instrument is commissioned and data and results are disseminated within our research groupings the impact of the instrument will be highlighted to potential new users: for example, the upgraded facility will support the first substantial engagement with EMRS by groups from the Newcastle Structural Biology Laboratory beyond work done by the Marles-Wright and Lewis groups.

As users engage in conferences and meetings, and produce publications with data collected on the instrument, external impact will be realised as opportunities for new collaborations arise. This new equipment will enable users with single-particle projects to assess the quality of samples and produce the preliminary reconstructions required for access to high-end TEM resources in both regional and national centres. This will significantly impact our current projects and contribute to future funding applications.

The tomography capabilities of the instrument will complement and enhance the research currently performed on the recently acquired 3View SEM. The enhanced spatial resolution available from thin-section TEM tomography will allow researchers currently using the 3View to produce higher-quality 3D-reconstructions of tissues and cells.

With the enhanced imaging-capabilities possible with the upgraded instrument we will be able to provide better data to our commercial users, which will impact our ability to gain new commercial and industrial collaborators. The engagement with Crysalin Ltd, a new potential commercial partner, illustrates how this resource will additionally contribute to improving exchange of skills between the university and the commercial biotechnology sector.

Impact on the wider public will be realised through workshops hosted by the EMRS for A-level students as part of their curriculum. The ability to see the process of reconstructing cellular and molecular architectures from 2D-images captured using the TEM will enhance their learning experience significantly.