1. Cell Biology
  2. Neuroscience
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Enhanced FIB-SEM systems for large-volume 3D imaging

  1. C Shan Xu  Is a corresponding author
  2. Kenneth J Hayworth
  3. Zhiyuan Lu
  4. Patricia Grob
  5. Ahmed M Hassan
  6. José G García-Cerdán
  7. Krishna K Niyogi
  8. Eva Nogales
  9. Richard J Weinberg
  10. Harald F Hess
  1. Janelia Research Campus, Howard Hughes Medical Institute, United States
  2. Howard Hughes Medical Institute, University of California, Berkeley, United States
  3. University of North Carolina, United States
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Cite this article as: eLife 2017;6:e25916 doi: 10.7554/eLife.25916

Abstract

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) can automatically generate 3D images with superior z-axis resolution, yielding data that needs minimal image registration and related post-processing. Obstacles blocking wider adoption of FIB-SEM include slow imaging speed and lack of long-term system stability, which caps the maximum possible acquisition volume. Here we present techniques that accelerate image acquisition while greatly improving FIB-SEM reliability, allowing the system to operate for months and generating continuously imaged volumes > 106 µm3. These volumes are large enough for connectomics, where the excellent z resolution can help in tracing of small neuronal processes and accelerate the tedious and time-consuming human proofreading effort. Even higher resolution can be achieved on smaller volumes. We present example data sets from mammalian neural tissue, Drosophila brain, and Chlamydomonas reinhardtii to illustrate the power of this novel high-resolution technique to address questions in both connectomics and cell biology.

Article and author information

Author details

  1. C Shan Xu

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    For correspondence
    xuc@janelia.hhmi.org
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8564-7836
  2. Kenneth J Hayworth

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Zhiyuan Lu

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Patricia Grob

    Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Ahmed M Hassan

    Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. José G García-Cerdán

    Department of Plant and Microbial Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Krishna K Niyogi

    Department of Plant and Microbial Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Eva Nogales

    Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9816-3681
  9. Richard J Weinberg

    Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Harald F Hess

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.

Funding

Howard Hughes Medical Institute

  • C Shan Xu
  • Kenneth J Hayworth
  • Zhiyuan Lu
  • Krishna K Niyogi
  • Eva Nogales
  • Harald F Hess

U.S. Department of Energy, Office of Science, Basic Energy Sciences, Chemical Sciences, Geosciences, and Biosciences Division (SISGRKN)

  • Krishna K Niyogi
  • Eva Nogales

Gordon and Betty Moore Foundation (GBMF3070)

  • Krishna K Niyogi

NIH (R01 NS-039444)

  • Richard J Weinberg

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

Ethics

Animal experimentation: All of the vertebrate animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#13-258.0) of UNC. UNC's PHS Assurance number is D16-00256 (A3410-01); the AALAC Unit number is 000329.

Reviewing Editor

  1. Jeremy Nathans, Johns Hopkins University School of Medicine, United States

Publication history

  1. Received: February 10, 2017
  2. Accepted: May 9, 2017
  3. Accepted Manuscript published: May 13, 2017 (version 1)
  4. Accepted Manuscript updated: May 16, 2017 (version 2)
  5. Version of Record published: June 19, 2017 (version 3)

Copyright

© 2017, Xu 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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