Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography
| Autoři | |
|---|---|
| Rok publikování | 2021 |
| Druh | Článek v odborném periodiku |
| Časopis / Zdroj | Jove-Journal of Visualized Experiments |
| Fakulta / Pracoviště MU | |
| Citace | |
| www | https://www.jove.com/pdf/62351/jove-protocol-62351-preparation-cryo-fib-micromachining-saccharomyces-cerevisiae-for-cryo |
| Doi | http://dx.doi.org/10.3791/62351 |
| Klíčová slova | VITREOUS SECTIONS; IMPLEMENTATION; CELLS |
| Popis | Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples. |
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