Development of mammalian tissue preparation methods for in situ structural cell biology

Thesis

Visualisation of the cellular interior using electron cryotomography (cryo-ET) provides fundamental insights into the molecular basis of biological processes in situ. Performing cryo-ET in model systems that accurately recapitulate the cellular environment found in vivo (i.e. tissues) is crucial for making observations of subcellular features that more accurately reflect their native state. However, extending the application of cryo-ET into tissues is limited by technical bottlenecks. In particular, the ability to reproducibly generate artefact-free thin sections of vitreous tissue at scale remains a challenge. This thesis outlines workflow develop- ments that enable the routine preparation of mammalian brain tissue for cryo-ET. The optimisation of high-pressure freezing protocols allowed vitreous brain tissue to be obtained reproducibly. Plasma-sourced focused ion beam (PFIB) milling was adapted for cryogenic life-science samples, permitting the high-throughput fabrication of lamellae for cryo-ET analysis. The application of PFIB milling enabled the fluorescence-guided investigation of mouse hippocampus by adapting serial cryogenic lift-out approaches in high-pressure frozen samples. In addition, the use of cryogenic femtosecond pulsed laser ablation for life-science samples was explored, highlighting the potential to further increase throughput by generating large numbers of sections for cryo-ET analysis in mouse olfactory bulb. These developments demonstrate the feasibility of generating large cryo-ET datasets which could enable routine structural cell biology directly in mammalian tissue samples. Moreover, the general framework described could be extended to other tissues, including human biopsies, to investigate the molecular basis of cellular function and disease within the native tissue environment.

Authors: Smith, JLR

• DOI: 10.5287/ora-jnp6xzmz0
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English