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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