ArticleBio-protocol2022
In situ cryo-FIB/SEM Specimen Preparation Using the Waffle Method.
Article in Bio-protocol, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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18 citing papers in PubMed.
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- Characterization Standard forbioRxiv : the preprint server for biology · 2026Article
- Carbon dioxide concentration alters cyanobacterial carboxysome encapsulation and redox state in Synechococcus sp. PCC 7002.Communications biology · 2026Article
- A robust workflow for 3D imaging of human mitochondria using cryo-electron tomography.bioRxiv : the preprint server for biology · 2026Article
- Radiation-dose effects in correlative X-ray/cryo-electron microscopy of frozen-hydrated biological samples.Acta crystallographica. Section D, Structural biology · 2026Article
- [COResearch square · 2026Article
- Cationic Amino Acid Identity and Net Charge Influence Condensate Properties inBiomacromolecules · 2025Article
- Cryoprotectants-assisted plunge freezing of thick brain tissue specimens for targeted physiologically relevant cryo-imagingbioRxiv : the preprint server for biology · 2025Article
- NtLLG4-mediated unconventional polar exocytosis of NtPPME1 coordinates cell wall rigidity and membrane dynamics to control pollen tube integrity.Science advances · 2025Article
- Cryo-ET reveals the in situ architecture of the polar tube invasion apparatus from microsporidian parasites.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Quantitative Spatial Analysis of Chromatin Biomolecular Condensates using Cryo-Electron Tomography.bioRxiv : the preprint server for biology · 2024Article
- Article
- Bridging structural biology and clinical research through in-tissue cryo-electron tomography.The EMBO journal · 2024Article
- Honeycomb gold specimen supports enabling orthogonal focussed ion beam-milling of elongated cells for cryo-ET.Journal of structural biology · 2024Article
- Square beams for optimal tiling in transmission electron microscopy.Nature methods · 2024Article
- Square beams for optimal tiling in TEM.bioRxiv : the preprint server for biology · 2023Article
- CryoFIB milling large tissue samples for cryo-electron tomography.Scientific reports · 2023Article
- Recent advances in infectious disease research using cryo-electron tomography.Frontiers in molecular biosciences · 2023Review
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Authors and funding
8 authors.
Funding
Abstract
Cryo-focused ion beam (FIB) milling of vitrified specimens is emerging as a powerful method for in situ specimen preparation. It allows for the preservation of native and near-native conditions in cells, and can reveal the molecular structure of protein complexes when combined with cryo-electron tomography (cryo-ET) and sub-tomogram averaging. Cryo-FIB milling is often performed on plunge-frozen specimens of limited thickness. However, this approach may have several disadvantages, including low throughput for cells that are small, or at low concentration, or poorly distributed across accessible areas of the grid, as well as for samples that may adopt a preferred orientation. Here, we present a detailed description of the "Waffle Method" protocol for vitrifying thick specimens followed by a semi-automated milling procedure using the Thermo Fisher Scientific (TFS) Aquilos 2 cryo-FIB/scanning electron microscope (SEM) instrument and AutoTEM Cryo software to produce cryo-lamellae. With this protocol, cryo-lamellae may be generated from specimens, such as microsporidia spores, yeast, bacteria, and mammalian cells, as well as purified proteins and protein complexes. An experienced lab can perform the entire protocol presented here within an 8-hour working day, resulting in two to three cryo-lamellae with target thicknesses of 100-200 nm and dimensions of approximately 12 μm width and 15-20 μm length. For cryo-FIB/SEMs with particularly low-contamination chambers, the protocol can be extended to overnight milling, resulting in up to 16 cryo-lamellae in 24 h. Graphical abstract.
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