1. Cell Biology
  2. Neuroscience
Download icon

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. Howard Hughes Medical Institute, University of California, Berekely, United States
  4. University of North Carolina, United States
Tools and Resources
  • Cited 88
  • Views 12,117
  • Annotations
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, Berekely, 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, Berekely, 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.

Metrics

  • 12,117
    Page views
  • 1,547
    Downloads
  • 88
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Cell Biology
    2. Chromosomes and Gene Expression
    Yu-Xuan Lu et al.
    Research Article

    Age-related changes to histone levels are seen in many species. However, it is unclear whether changes to histone expression could be exploited to ameliorate the effects of ageing in multicellular organisms. Here we show that inhibition of mTORC1 by the lifespan-extending drug rapamycin increases expression of histones H3 and H4 post-transcriptionally, through eIF3-mediated translation. Elevated expression of H3/H4 in intestinal enterocytes in Drosophila alters chromatin organization, induces intestinal autophagy through transcriptional regulation, prevents age-related decline in the intestine. Importantly, it also mediates rapamycin-induced longevity and intestinal health. Histones H3/H4 regulate expression of an autophagy cargo adaptor Bchs (WDFY3 in mammals), increased expression of which in enterocytes mediates increased H3/H4-dependent healthy longevity. In mice, rapamycin treatment increases expression of histone proteins and Wdfy3 transcription, and alters chromatin organisation in the small intestine, suggesting the mTORC1-histone axis is at least partially conserved in mammals and may offer new targets for anti-ageing interventions.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Haitao Zhang et al.
    Tools and Resources

    The human kinome comprises 538 kinases playing essential functions by catalyzing protein phosphorylation. Annotation of subcellular distribution of the kinome greatly facilitates investigation of normal and disease mechanisms. Here, we present Kinome Atlas (KA), an image-based map of the kinome annotated to 10 cellular compartments. 456 epitope-tagged kinases, representing 85% of the human kinome, were expressed in HeLa cells and imaged by immunofluorescent microscopy under a similar condition. KA revealed kinase family-enriched subcellular localizations, and discovered a collection of new kinase localizations at mitochondria, plasma membrane, extracellular space, and other structures. Furthermore, KA demonstrated the role of liquid-liquid phase separation in formation of kinase condensates. Identification of MOK as a mitochondrial kinase revealed its function in cristae dynamics, respiration, and oxidative stress response. Although limited by possible mislocalization due to overexpression or epitope tagging, this subcellular map of the kinome can be used to refine regulatory mechanisms involving protein phosphorylation.