3D IMAGING OF WHOLE CELL ULTRASTRUCTURE USING CRYO-ELECTRON MICROSCOPIES

Abstract

Cryo-transmission electron microscopy (cryo-TEM) allows ultrastructural imaging of soft matter materials with high, and often, near-atomic resolution. Imaging whole cells with cryo-TEM, however, requires consideration of the cell’s thickness: some cells are naturally electron transparent, while others are too thick for analysis with the TEM. High-resolution imaging, therefore, relies not only on selecting the appropriate microscopy technique, but also on the inherent dimensions of the cell under examination. This dissertation presents three reports of three cell types with different dimensions. The first report explores the post-insemination phenomenological changes to the spermatozoa of Aedes aegypti mosquitoes. Because spermatozoa are naturally electron semi-transparent, we utilized cryo-energy-filtered TEM to produce high-resolution information documenting the removal of the sperm coat and outer membrane. We then describe a correlation between modifications in sperm ultrastructure and motility, and enhanced fecundity in female Aedes aegypti. The second report explores the formation of extracellular vesicles in MCF10A and hyaluronic acid synthase 3 (HAS3)-overexpressed MCF10A cells. Both cell lines are electron-opaque, and their morphologies were examined using florescence, conventional, and cryo-scanning electron microscopies. Using different microscopies elucidated the strengths and limitations of each technique, and how they could be combined to best characterize the effects of HAS3 overexpression on transformed MCF10A cells. Secreted extracellular vesicles were electron-transparent and their surface structures were examined with cryo-TEM. Microvesicles contained surface structure and exosomes did not, providing a pathway for differentiating between vesicle subpopulations in the future. The third report characterizes the chromatophores of photosynthetic bacteria Rhodobacter sphaeroides. Because whole R. sphaeroides are too thick to analyze with cryo-TEM, we thinned the cell with cryo-focused ion beam milling. Additionally, we used cryo-electron tomography to understand the 3D arrangement of chromatophores within the cell. We determined that chromatophores adopt both isolated and connected morphologies. Connected chromatophores exist as either budded structures, or linked to neighboring chromatophores through ~10 nm long extrinsic linkers. Cryo-TEM provides ultrastructural information of cells and cellular components. However, the exact approach depends on the dimensions of the cell and the feature of interest. When carefully chosen, the techniques described in this dissertation provide invaluable insight into cell structure.