HPF to enable high-quality ultrastructural analysis of biological samples

观察细胞复杂的内部结构依赖于高分辨率显微镜技术。这些技术中最强大的，能够观察到最高水平的细节的是电子显微镜。这项技术可以揭示最精细的细胞细节，包括细胞的细胞膜，植物和真菌细胞的细胞壁，或线粒体的详细结构，参与能量生产的细胞器。EM需要在样品制备过程中固定所有细胞成分。在固定过程中，需要小心处理样品。有几种固定程序，所选择的程序将影响细胞成分保存的接近其天然状态的程度。到目前为止，组织保存质量最好的技术是依靠在非常高的压力下快速冷冻样品。这个过程被称为高压冷冻，可以防止微小冰晶的形成，否则就会破坏生物材料的结构。高压冷冻标本通过电子显微镜成像，揭示了使用更传统的固定方法（例如通过使用化合物）无法看到的细胞细节。例如，含有药物的囊泡通过致病真菌细胞壁的过程只能在高压冷冻制备的样品中观察到。在这个项目中，我们将通过电子显微镜研究各种样品中的细胞细节，包括真菌病原体，植物和动物细胞。为了使埃克塞特大学的电子显微镜工作达到最高质量，我们建议购买一台最先进的高压冷冻机器。这将允许大量用户为电子显微镜制备最高质量的组织样品。

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.