HPF to enable high-quality ultrastructural analysis of biological samples

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

Observing the intricate internal structures of the cell relies on high-resolution microscopy techniques. The most powerful of these techniques, enabling the observation of the highest level of detail is electron microscopy. This technique can reveal the finest cellular details, including the internal membranes of the cell, the cell wall of plant and fungal cells, or the detailed structure of mitochondria, cellular organelles involved in energy production. EM requires the immobilisation of all cellular constituents during a sample-preparation process called fixation. During fixation, the sample needs to be handled with care. There are several fixation procedures and the selected procedure will influence how close to their native state the cellular constituents are preserved. By far the best quality of tissue preservation is observed by a technique that relies on the rapid freezing of the sample under very high pressure. This process, called high pressure freezing, prevents the formation of tiny ice crystals that would otherwise damage the structure of the biological material. High-pressure-frozen specimens imaged by electron microscopy have revealed cellular details that were not seen using more conventional fixation approaches (e.g. by the use of chemical compounds). For example, the passage of drug-containing vesicles through the cell wall of pathogenic fungi could only be visualised in samples that had been prepared by high pressure freezing. In this project, we will study cellular details by electron microscopy in a variety of samples, including fungal pathogens, plant and animal cells. To enable the highest possible quality of electron microscopy work at the University of Exeter, we propose to purchase a state-of-the-art machine for high pressure freezing. This would allow the preparation of tissue sample of the highest possible quality for electron microscopy by a large number of users.

Technical Summary

Ultrastructural analysis by EM provides the highest possible resolution to image biological specimens. EM combined with ultrathin sectioning and heavy-metal staining is able to reveal fine cellular details. Due to differences in labelling intensity during heavy metal staining, EM can visualise all cellular membranes, cell walls many other subcellular structures. This gives enormous versatility to the technique. EM is accessible for the ultrastructural study of any organism or cell type. In comparison, light microscopy often relies on species-specific reagents (e.g., transgenes or antibodies). The full potential of EM can only be realised if the best possible protocols are used for tissue preservation. By far the best quality of tissue preservation is achieved through the process of high pressure freezing followed by freeze substitution. To enable the highest possible quality of ultrastructural work at the University of Exeter, we propose to purchase a Leica high pressure freezer and a freeze-substitution unit. These instruments form a single HPF pipeline and will allow us to set up a HPF facility for the first time in Exeter. This will allow the preparation of high-quality samples for EM analysis and will significantly boost the quality and amount of ultrastructural work at the University. The equipment will add a unique and novel capacity to our research. Once installed, we plan to open up the access to the HPF facility to external academic and industrial users in order to foster new collaborations and contribute to training. We will also build on existing industrial partnerships and seek to establish new ones to increase the impact of the technology. The establishment of a HPF infrastructure in Exeter will significantly enhance the cell biological and ultrastructural research capacity and quality at the University, enhance and extend current BBSRC research and will open up new exciting collaborative opportunities that will underpin future BBSRC applications.

Planned Impact

The project will have impacts in several key areas.

- We will generate new knowledge on the detailed structure of synaptic networks in the nervous system, the structure of plant, animal and archaeal cells and fungal pathogens
- The project will benefit our established industry partnerships (Syngenta, MycoSciences Ltd. and Gilead Sciences) in the area of antifungal drugs
- The project will enable new industrial collaborations in the area of ultrastructural imaging in biomedicine
- The project will enable new research projects that will enhance the profile of the Principal Investigators. We will generate new preliminary data to underpin future grant applications and enable new collaborations in the areas of fungal biology, cell and neurobiology
- The project will contribute to expanding an electron microscopy hub in Exeter, enabling new research avenues and training opportunities in the South-West by providing access to a state-of-the-art facility and through a workshop and training course
- The Exeter-based HPF platform will expand our technical capabilities within the GW4. In this regard and through two Wellcome Trust Equipment Grants, we have recently jointly established a GW4-shared high-resolution cryoEM facility in Bristol.
- The further development of EM capacities in Exeter will create new opportunities within the GW4 partnership to develop new protocols and EM applications
- The new infrastructure installed during the project will allow the training of staff and students from and outside Exeter in state-of-the-art EM techniques
- The new pipeline will promote research excellence and high-quality publications across different fields of research
- To facilitate this impact strategy, we will organise a one-day workshop and facility open day in October 2019. During the event, we will give lectures and practical demonstrations about the technique of high-pressure freezing and serial sectioning to the scientific and affiliated industrial community.
- In June 2019, we will organise a 1-week practical course on "Tissue preparation by HPF for ultrastructural analysis". We will give theoretical and hands-on training to 12 participants.

Publications

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