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

Abstract

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 cells 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).

Recent advances in infectious disease research using cryo-electron tomography

“…Sample preparation methods for cryo-ET have revolutionized the field in the last few years. Micropatterning is a useful tool to target cells directly on the grids at a precise location ( Sibert et al, 2021a ; Sibert et al, 2021b ). Sample thinning methods like serial lift-out and waffle method utilize HPF demonstrate the potential of handling multicellular and tissue samples ( Bobe et al, 2022 ; Schiøtz et al, 2023 ).…”

Cryo-Electron Tomography: The Resolution Revolution and a Surge of In Situ Virological Discoveries

“…Finally, cells are seeded on the grid, and their growth will accurately follow the micropatterned areas (111). Micropatterning techniques have been further developed to facilitate the formation of thin intercellular contacts suitable for direct cryo-ET imaging (25), as well as to investigate virus-host interactions, such as RSV release from infected cells (99). To improve milling speed and reduce the risks of lamella damage, a Waffle method suitable for large lamella preparation was proposed.…”

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

“…Micropatterning has been successfully applied to a number of projects within the laboratory 19 . Here, a micropatterning protocol is presented, including specific adaptations to study cultured HeLa cells, respiratory syncytial virus (RSV)-infected BEAS-2B cells, and primary larval Drosophila melanogaster neurons 20 .…”