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

SYSNO ASEP	0555839
Document Type	O - Others
R&D Document Type	Others
Title	Optimizing a workflow for cryo-TEM tomography – fabrication and transfer of frozen hydrated lamella
Author(s)	Pinkas, Dominik (UMG-J)_ORCID Záchej, S. (CZ) Havránková, J. (CZ) Raabová, Helena (UMG-J) Vlčák, Erik (UMG-J) Mimietz-Oeckler, S. (DE) Kirmse, R. (DE) Hozák, Pavel (UMG-J)_{RID, ORCID} Filimonenko, Vlada (UMG-J)_{RID, ORCID}
Year of issue	2021
Action	Microscopy Conference 2021 Joint Meeting of Dreiländertagung & Multinational Congress on Microscopy
Event date	22.08.2021 - 26.08.2021
VEvent location	Vídeň
Country	AT - Austria
Event type	WRD
Language	eng - English
Country	TR - Turkey
Keywords	Electron microscopy ; Cryo TEM tomography ; FIB lamella
Subject RIV	EB - Genetics ; Molecular Biology
OECD category	Cell biology
R&D Projects	LM2018129 GA MŠMT - Ministry of Education, Youth and Sports (MEYS)
	LTC19048 GA MŠMT - Ministry of Education, Youth and Sports (MEYS)
	EF16_013/0001775 GA MŠMT - Ministry of Education, Youth and Sports (MEYS)
Research Infrastructure	Czech-BioImaging II - 90129 - Ústav molekulární genetiky AV ČR, v. v. i.
Institutional support	UMG-J - RVO:68378050
Annotation	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. We 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.
Workplace	Institute of Molecular Genetics
Contact	Nikol Škňouřilová, nikol.sknourilova@img.cas.cz, Tel.: 241 063 217
Year of Publishing	2022

Number of the records: 1