ReviewNature protocols2026
Integrated fluorescence light microscopy-guided cryo-focused ion beam-milling for in situ montage cryo-ET.
Review in Nature protocols, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed.
- Integrating in situ single-particle cryo-electron microscopy with cryo-electron tomography for high-resolution structural biology in native cellular contexts.Current opinion in structural biology · 2026Review
- Electron microscopy-based three-dimensional subcellular imaging of plant male gametophyte.Journal of integrative plant biology · 2026Review
- Cryo-FIB-enabled 3D imaging of biological specimens.Applied microscopy · 2026Review
- A robust workflow for 3D imaging of human mitochondria using cryo-electron tomography.bioRxiv : the preprint server for biology · 2026Article
- A cryo-CLEM-guided workflow for quantitative assessment and optimization of cryo-ET specimen quality.Molecules and cells · 2026Article
- Direct visualization of HIV-1 core nuclear import and its interplay with the nuclear pore.EMBO reports · 2025Article
- 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
11 authors.
Funding
Abstract
Cryogenic-electron tomography (cryo-ET) permits the in situ visualization of biological macromolecules at the molecular level. Owing to the variable thickness of cells, tissues and organisms, frozen specimens may need to be thinned by cryo-focused ion beam (FIB) milling to produce thin (<500 nm) cryo-lamellae suitable for cryo-ET. Locating regions of interest remains a challenge because untargeted milling can lead to inadvertent ablation and removal of regions of interest. Correlative light and electron microscopy, combined with cryo-FIB milling, can guide the identification of labeled targets in the cellular milieu. Multiple transfers between cryo-imaging instruments, cumbersome correlation algorithms, limited accuracy and low throughput have hindered the routine adoption of cryo-FIB milling within a multimodal correlative workflow for in situ structural biology. Here we present a workflow for 3D correlative cryo-fluorescence light microscopy-FIB-ET that streamlines fluorescence light microscopy-guided FIB milling, improving throughput while preserving both structural and contextual information. The complete integration of hardware and software described here minimizes sample contamination from cross-platform exchanges and greatly enhances the efficiency of 3D targeting in cryo-milling. We then describe procedures for implementing montage parallel array cryo-ET (MPACT), which can be easily adapted to any modern life-science transmission electron microscope. MPACT supports high-throughput cryo-ET acquisitions (10 tilt series in 1.5 h) for structure determination and comprehensive contextual understanding of macromolecules within their native surroundings. A complete session from sample preparation to MPACT data processing takes 5-7 d for an individual experienced in both cryo-EM and cryo-FIB milling.
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