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

Kód výsledku v IS VaVaI

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Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10601 - Cell biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2021

Kód důvěrnosti údajů

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