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

Swistak, Léa; Sartori-Rupp, Anna; Vos, Matthijn; Enninga, Jost

doi:10.1016/bs.mim.2020.11.001

Cited by 6 publications

(3 citation statements)

References 25 publications

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“…125 However, the monitoring of sectioning and the site selectivity of ultramicrotomy can be challenging, 122 and more importantly, the mechanical sectioning steps may introduce residual stress, impacting the determination of high resolution information. 126 The FIB method has been explored for slicing off an electron-permeable membrane from a particular region of interest in bulk samples. 127,128 The FIB method performs well in slicing specimens with different morphologies and components, such as silicon and ZnSe optical ceramics.…”

Section: Technological Innovations For Low Dose Imagingmentioning

confidence: 99%

Atomic-level imaging of beam-sensitive COFs and MOFs by low-dose electron microscopy

Zhan,

Liu,

Wang

et al. 2024

Nanoscale Horiz.

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Section: Technological Innovations For Low Dose Imagingmentioning

confidence: 99%

Atomic-level imaging of beam-sensitive COFs and MOFs by low-dose electron microscopy

Zhan,

Liu,

Wang

et al. 2024

Nanoscale Horiz.

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“…An ideal sample for cryo-FIB/cryo-ET would have a repeatable and uniform distribution of cells situated in the centers of the grid squares. We and others have published studies demonstrating the positioning of individual adherent cells (7)(8)(9)(10)(11) or adherent cell pairs (12,13) on EM grids micropatterned with extracellular matrix (ECM) proteins in preparation for cryo-ET. Another approach for providing a uniform distribution of cells for cryo-FIB/cryo-ET, applicable to both adherent and non-adherent cell types, is the Waffle Method (14).…”

Section: Introductionmentioning

confidence: 99%

Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes

Engel

Zaoralova

Dunn

et al. 2023

Preprint

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Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T-cells and Jurkat cells, for cryo-FIB and cryo-ET. Our affinity capture system facilitated the nanoscale imaging of Jurkat cells, revealing extracellular filamentous structures. It improved workflow efficiency by consistently producing grids with a sufficient number of well-positioned cells for an entire cryo-FIB session. Affinity capture can be extended to facilitate high resolution imaging of other adherent and non-adherent cell types with cryo-ET.

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“…In this protocol, we describe the details of a combined workflow with several newly available technological upgrades. i) Micropatterning the electron microscopy (EM) grids prior to cell culture using PRIMO: the customized patterns result in improved control over cell shape and spatial distribution over the entire grid ( Engel et al, 2019 ; Swistak et al, 2021 ). By covering the EM grid in an anti-fouling layer and selectively removing it through UV light, the cell can only grow on selected areas of the grid.…”

Section: Introductionmentioning

confidence: 99%

Correlative Light and Electron Cryo-Microscopy Workflow Combining Micropatterning, Ice Shield, and an In-Chamber Fluorescence Light Microscope

Berkamp,

Daviran,

Smeets

et al. 2023

BIO-PROTOCOL

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In situ cryo-electron tomography (cryo-ET) is the most current, state-of-the-art technique to study cell machinery in its hydrated near-native state. The method provides ultrastructural details at sub-nanometer resolution for many components within the cellular context. Making use of recent advances in sample preparation techniques and combining this method with correlative light and electron microscopy (CLEM) approaches have enabled targeted molecular visualization. Nevertheless, the implementation has also added to the complexity of the workflow and introduced new obstacles in the way of streamlining and achieving high throughput, sample yield, and sample quality. Here, we report a detailed protocol by combining multiple newly available technologies to establish an integrated, high-throughput, optimized, and streamlined cryo-CLEM workflow for improved sample yield. Key features • PRIMO micropatterning allows precise cell positioning and maximum number of cell targets amenable to thinning with cryo focused-ion-beam–scanning electron microscopy. • CERES ice shield ensures that the lamellae remain free of ice contamination during the batch milling process. • METEOR in-chamber fluorescence microscope facilitates the targeted cryo focused-ion-beam (cryo FIB) milling of these targets. • Combining the three technologies into one cryo-CLEM workflow maximizes sample yield, throughput, and efficiency. Graphical overview

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

Cited by 6 publications

References 25 publications

Atomic-level imaging of beam-sensitive COFs and MOFs by low-dose electron microscopy

Atomic-level imaging of beam-sensitive COFs and MOFs by low-dose electron microscopy

Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes

Correlative Light and Electron Cryo-Microscopy Workflow Combining Micropatterning, Ice Shield, and an In-Chamber Fluorescence Light Microscope

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