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

0555836 - ÚMG 2022 RIV AT eng O - Ostatní výsledky
Pinkas, Dominik - Záchej, S. - Havránková, J. - Raabová, Helena - Vlčák, Erik - Mimietz-Oeckler, S. - Kirmse, R. - Hozák, Pavel - Filimonenko, Vlada
Optimizing a workflow for cryo-TEM tomography – fabrication and transfer of frozen hydrated lamella.
2021
Grant CEP: GA MŠMT(CZ) LM2018129; GA MŠMT LTC19048; GA MŠMT(CZ) EF16_013/0001775
Výzkumná infrastruktura: Czech-BioImaging II - 90129
Institucionální podpora: RVO:68378050
Klíčová slova: Electron microscopy * Cryo TEM tomography * FIB lamella
Obor OECD: Cell biology

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.
Trvalý link: http://hdl.handle.net/11104/0330295