A cryogenic, coincident fluorescence, electron, and ion beam microscope
eLife, vol. 11
Abstract
Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.
Authors 16
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Delft University of Technology
Affiliation as printed
Delmic B.V
Department of Imaging Physic, Delft University of Technology
Delmic B.V; Department of Imaging Physic, Delft University of Technology
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Delft University of Technology
Affiliation as printed
Department of Imaging Physic, Delft University of Technology
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Delft University of Technology
Affiliation as printed
Kavli Institute of Nanoscience, Delft University of Technology
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Brigham Young University · California Institute of Technology
Affiliation as printed
Brigham Young University
California Institute of Technology
Brigham Young University; California Institute of Technology
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Affiliation as printed
Delmic B.V
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Delft University of Technology
Affiliation as printed
Department of Imaging Physic, Delft University of Technology
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Leiden University Medical Center
Affiliation as printed
Department of Cell and Chemical Biology, Leiden University Medical Center
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Leiden University Medical Center
Affiliation as printed
Delmic B.V
Department of Cell and Chemical Biology, Leiden University Medical Center
Delmic B.V; Department of Cell and Chemical Biology, Leiden University Medical Center
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Delft University of Technology
Affiliation as printed
Kavli Institute of Nanoscience, Delft University of Technology
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Max Planck Institute of Biochemistry
Affiliation as printed
CryoEM Technology, Max Planck Institute of Biochemistry
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Max Planck Institute of Molecular Physiology
Affiliation as printed
Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology
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Max Planck Institute of Molecular Physiology
Affiliation as printed
Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology
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Max Planck Institute of Molecular Physiology
Affiliation as printed
Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology
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Delft University of Technology
Affiliation as printed
Department of Imaging Physic, Delft University of Technology
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The University of Queensland · Centre for Microscopy and Microanalysis
Affiliation as printed
Centre for Microscopy and Microanalysis, University of Queensland
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Affiliation as printed
Delmic B.V
Cited by 40 stored of 40
Cited by patents worldwide 4 (Lens.org)
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METHOD FOR CREATING A SAMPLE FOR USE IN A CHARGED PARTICLE MICROSCOPEEP4679056A1 2026-01-14 Pending
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METHOD AND SYSTEM FOR SAMPLE PREPARATIONWO2024254007A1 2024-12-12 Pending
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Method for localizing a region of interest in a sample and micromachining the sample using a charged particle beamNL2032640B1 2024-02-06 Active
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METHOD FOR LOCALIZING A REGION OF INTEREST IN A SAMPLE AND MICROMACHINING THE SAMPLE USING A CHARGED PARTICLE BEAMWO2024025410A1 2024-02-01 Pending