A cryogenic, coincident fluorescence, electron and ion beam microscope
bioRxiv (Cold Spring Harbor Laboratory)
Abstract
Abstract Cryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualisation and structure determination of macromolecular complexes at sub-nanometre 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 fluoresence microscopy-guided FIB fabrication of a frozen-hydrated lamella to solve this problem: we built a coincident 3-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing focused ion-beam scanning electron microscope (FIB-SEM). We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope (TEM) 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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Daan B Boltje corresponding
Delft University of Technology
Affiliation as printed
Delmic B.V., Delft, The Netherlands
Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands
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Delft University of Technology
Affiliation as printed
Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands
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Delft University of Technology
Affiliation as printed
Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands
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Brigham Young University · California Institute of Technology
Affiliation as printed
Brigham Young University, Provo, UT, USA
California Institute of Technology, Pasadena, CA, USA
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Affiliation as printed
Delmic B.V., Delft, The Netherlands
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Delft University of Technology
Affiliation as printed
Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Cell and Chemical Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden, The Netherlands
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Leiden University Medical Center
Affiliation as printed
Delmic B.V., Delft, The Netherlands
Department of Cell and Chemical Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden, The Netherlands
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Delft University of Technology
Affiliation as printed
Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands
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Max Planck Institute of Biochemistry
Affiliation as printed
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Planegg, Germany
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Max Planck Institute of Molecular Physiology
Affiliation as printed
Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany
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Max Planck Institute of Molecular Physiology
Affiliation as printed
Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany
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Max Planck Institute of Molecular Physiology
Affiliation as printed
Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany
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Delft University of Technology
Affiliation as printed
Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands
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The University of Queensland · Centre for Microscopy and Microanalysis
Affiliation as printed
Centre for Microscopy and Microanalysis, The University of Queensland, Brisbane, Australia
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Affiliation as printed
Delmic B.V., Delft, The Netherlands
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