1. Structural Biology and Molecular Biophysics
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PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy

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Cite this article as: eLife 2019;8:e45919 doi: 10.7554/eLife.45919

Abstract

Cryo-Electron Tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

Article and author information

Author details

  1. Sergey Gorelick

    Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  2. Genevieve Buckley

    Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  3. Gediminas Gervinskas

    Ramaciotti Center for cryo-Electron Microscopy, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  4. Travis K Johnson

    School of Biological Sciences, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  5. Ava Handley

    Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  6. Monica Pia Caggiano

    Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  7. James C Whisstock

    Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  8. Roger Pocock

    Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5515-3608
  9. Alex de Marco

    Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia
    For correspondence
    alex.demarco@monash.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6238-5653

Funding

ARC centre of excellence in advanced molecular imaging (CE140100011)

  • Sergey Gorelick
  • Genevieve Buckley
  • James C Whisstock
  • Alex de Marco

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Sriram Subramaniam, University of British Columbia, Canada

Publication history

  1. Received: February 8, 2019
  2. Accepted: June 26, 2019
  3. Accepted Manuscript published: July 1, 2019 (version 1)
  4. Version of Record published: July 4, 2019 (version 2)

Copyright

© 2019, Gorelick et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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