| MORAVCOVÁ, Jana, Matyáš PINKAS, Radka HOLBOVÁ and Jiří NOVÁČEK. Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography. Jove-Journal of Visualized Experiments. CAMBRIDGE: JOURNAL OF VISUALIZED EXPERIMENTS, 2021, NOV, No 177, p. „e62351“, 16 pp. ISSN 1940-087X. Available from: https://dx.doi.org/10.3791/62351. |
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@article{1836561,
author = {Moravcová, Jana and Pinkas, Matyáš and Holbová, Radka and Nováček, Jiří},
article_location = {CAMBRIDGE},
article_number = {177},
doi = {http://dx.doi.org/10.3791/62351},
keywords = {VITREOUS SECTIONS; IMPLEMENTATION; CELLS},
language = {eng},
issn = {1940-087X},
journal = {Jove-Journal of Visualized Experiments},
title = {Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography},
url = {https://www.jove.com/pdf/62351/jove-protocol-62351-preparation-cryo-fib-micromachining-saccharomyces-cerevisiae-for-cryo},
volume = {NOV},
year = {2021}
}
TY - JOUR
ID - 1836561
AU - Moravcová, Jana - Pinkas, Matyáš - Holbová, Radka - Nováček, Jiří
PY - 2021
TI - Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography
JF - Jove-Journal of Visualized Experiments
VL - NOV
IS - 177
SP - „e62351“
EP - „e62351“
PB - JOURNAL OF VISUALIZED EXPERIMENTS
SN - 1940087X
KW - VITREOUS SECTIONS
KW - IMPLEMENTATION
KW - CELLS
UR - https://www.jove.com/pdf/62351/jove-protocol-62351-preparation-cryo-fib-micromachining-saccharomyces-cerevisiae-for-cryo
N2 - 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.
ER -
MORAVCOVÁ, Jana, Matyáš PINKAS, Radka HOLBOVÁ and Jiří NOVÁČEK. Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography. \textit{Jove-Journal of Visualized Experiments}. CAMBRIDGE: JOURNAL OF VISUALIZED EXPERIMENTS, 2021, NOV, No~177, p.~„e62351“, 16 pp. ISSN~1940-087X. Available from: https://dx.doi.org/10.3791/62351.
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