Optimizing a workflow for cryo-TEM tomography – fabrication and transfer of frozen hydrated lamella

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F68378050%3A_____%2F21%3A00555832" target="_blank" >RIV/68378050:_____/21:00555832 - isvavai.cz</a>

Alternative codes found

RIV/68378050:_____/21:00555836 RIV/68378050:_____/21:00555837 RIV/68378050:_____/21:00555839

Result on the web

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DOI - Digital Object Identifier

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Result language

angličtina

Original language name

Optimizing a workflow for cryo-TEM tomography – fabrication and transfer of frozen hydrated lamella

Original language description

Electron microscopy provides unique insight into the ultrastructure of cells and tissues. Preservation of sensitive biological samples in close-to-native state is crucial for obtaining quality data. Vitrification with subsequent observation in cryo conditions in an electron microscope is a valuable approach. Cryo-electron tomography is a method of choice to gain volume information about the object. A serious technical limitation is the thickness of the object. While small objects, like bacteria, viruses, isolated cellular organelles, or thin areas of cytoplasm at the edge of a eukaryotic cell can be imaged directly, bigger parts of cells or tissues need to be thinned before observation. Fabrication of a thin FIB-milled lamellae from a frozen hydrated sample with subsequent cryo-transfer and tilt series acquisition in cryoTEM is currently the best workflow introducing minimal artefacts into the sample compared to other available techniques. The workflow is technically challenging and needs significant skills and optimization of all steps to produce homogeneously thin lamella and to avoid heat damage, mechanical damage, and surface contamination of the lamella. nWe demonstrate optimization of the semi-automated cryo TEM lamella preparation workflow on yeast, mammalian and plant samples using TESCAN FIB-SEM Cryo AMBER system equipped with the Leica VCT500 cryo transfer stage for operation in cryogenic conditions.  The use of a side-entry TEM cryoholder makes the workflow more universal and accessible for a broader range of microscopy workplaces, compared to autoloader-equipped systems that are currently used in most cases.

Czech name

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Czech description

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Type

O - Miscellaneous

CEP classification

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OECD FORD branch

10601 - Cell biology

Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2021

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů