![]() ![]() One of the most commonly used cryo-fixation techniques is plunge freezing, where the sample is applied to the EM grid, blotted, and rapidly plunged into liquid ethane or a mixture of liquid ethane and propane. ![]() The tilt-series can then be computationally reconstructed into a 3D volume, also known as a tomogram 4.Īll cryo-EM techniques require the sample to be embedded in a thin layer of amorphous, non-crystalline, vitreous ice. These images, or tilt-series, often cover a range of +60/−60 degrees in one-to-three-degree increments. In cryo-ET, three-dimensional (3D) information is obtained by physically tilting the sample on the microscope stage and acquiring a series of images through the sample at different angles. Whereas cryo-electron tomography (cryo-ET) is uniquely suited for near-native structural and ultrastructural studies of large, heterologous objects such as bacteria, pleomorphic viruses, and eukaryotic cells 3. Single-particle cryo-EM and electron diffraction techniques are best applied to purified macromolecules in solution or in a crystalline state, respectively 1, 2. ![]() With the development, expansion, and versatility of cryo-electron microscopy (cryo-EM), researchers have examined a wide range of biological samples in a near-native state from macromolecular (~1 nm) to high (~2 Å) resolution. 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 (cryo-FIB). 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. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. During micropatterning, cell growth is directed 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. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. ![]()
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