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Book Sections Year : 2021

Micropatterning of cells on EM grids for efficient cryo-correlative light electron microscopy

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Abstract

Cryo-transmission electron microscopy (cryo-TEM) provides access to high resolution information of adherent cell ultrastructures in a close to native environment but only volumes of less than 500 nm can be imaged. Cryo-focused ion beam (FIB) milling overcomes this obstacle through the generation of thin lamella of less than 200 nm. These lamellas can be imaged by cryo-electron tomography (cryo-ET) giving access to ultrastructural data within the volume of the imaged cells. Nevertheless, a lack of control on the positioning of the samples on the electron microscopy (EM) grids drastically constrains its throughput. The use of custom-designed micropatterned EM grids bypasses these issues by accurately positioning cells in areas that allow FIB milling followed by cryo-ET. Combined with fluorescent light microscopy in correlative light electron microscopy (CLEM) pipelines to pinpoint specific events and automated FIB milling, micropatterning of cells on EM grids has the potential of dramatically accelerating the workflow of cryo-ET. Here, a detailed description is provided of the key steps necessary to implement photomicropatterning of EM grids for improved CLEM pipelines.
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Dates and versions

pasteur-03263636 , version 1 (17-06-2021)

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Léa Swistak, Anna Sartori-Rupp, Matthijn Vos, Jost Enninga. Micropatterning of cells on EM grids for efficient cryo-correlative light electron microscopy. Methods in Microbiology, 48, Elsevier, pp.95-110, 2021, ⟨10.1016/bs.mim.2020.11.001⟩. ⟨pasteur-03263636⟩
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