Whole-cell cryo-electron tomography of cultured and primary eukaryotic cells on micropatterned TEM grids [article]

Bryan S Sibert, Joseph Y Kim, Jie E Yang, Elizabeth R Wright
2021 bioRxiv   pre-print
Whole-cell cryo-electron tomography (cryo-ET) is a powerful technique that can provide nanometer-level resolution of biological structures within the cellular context and in a near-native frozen-hydrated state. It remains a challenge to culture or adhere cells on TEM grids in a manner that is suitable for tomography while preserving the physiological state of the cells. Here, we demonstrate the versatility of micropatterning to direct and promote growth of both cultured and primary eukaryotic
more » ... lls on TEM grids. We show that micropatterning is compatible with and can be used to enhance studies of host-pathogen interactions using respiratory syncytial virus infected BEAS-2B cells as an example. We demonstrate the ability to use whole-cell tomography of primary Drosophila neuronal cells to identify organelles and cytoskeletal stuctures in cellular axons and the potential for micropatterning to dramatically increase throughput for these studies. During micropatterning, cell growth is targeted by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design make micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (FIB-SEM).
doi:10.1101/2021.06.06.447251 fatcat:cm6gb4z3c5czhe7uawjiq6wsya