ArticleCell reports methods2025
Thickness- and quality-controlled fabrication of fluorescence-targeted frozen-hydrated lamellae.
Article in Cell reports methods, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
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Who cites it
2 citing papers in PubMed.
- Beyond structure: spectroscopic imaging in cryogenic electron microscopy.Current opinion in structural biology · 2026Review
- Workflow for Fluorescence-Targeted Lamella Milling From Vitrified Cells With a Coincident Fluorescence, Electron, and Ion Beam Microscope.Bio-protocol · 2025Article
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Authors and funding
6 authors.
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Abstract
Cryogenic focused ion beam (FIB) milling is essential for fabricating thin lamella-shaped samples out of frozen-hydrated cells for high-resolution structure determination. Structural information can only be resolved at high resolution if the lamella thickness is between 100 and 200 nm. While the lamella fabrication workflow has improved significantly since its conception, quantitative, live feedback on lamella thickness, quality, and biological target inclusion remains lacking. Using coincident light microscopy integrated into the FIB scanning electron microscope (FIB-SEM), we present three strategies that enable accurate, live control during lamella fabrication. First, we combine four-dimensional (4D) STEM with fluorescence microscopy (FM) targeting to determine lamella thickness. Second, with reflected light microscopy (RLM), we screen target sites for ice contamination and monitor lamella thickness and protective Pt coating integrity during FIB milling. Third, we exploit thin-film interference for fine-grained feedback on thickness uniformity below 500 nm. Finally, we present a fluorescence-targeted, quality-controlled workflow for frozen-hydrated lamellae, benchmarked with excellent agreement with energy-filtered transmission electron microscopy (EFTEM) measurements and tomograms from electron cryotomography.
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