1. Cell Biology
  2. Neuroscience

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
https://doi.org/10.7554/eLife.25916
Share this article
Cite this article
  1. C Shan Xu
  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
(2017)
Enhanced FIB-SEM systems for large-volume 3D imaging
eLife 6:e25916.
https://doi.org/10.7554/eLife.25916
  2. Download BibTeX
  3. Download .RIS

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.

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

  • 26,423
    views
  • 2,681
    downloads
  • 347
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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)

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

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

  1. C Shan Xu
  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
(2017)
Enhanced FIB-SEM systems for large-volume 3D imaging
eLife 6:e25916.
https://doi.org/10.7554/eLife.25916

Share this article

https://doi.org/10.7554/eLife.25916

Categories and tags

Further reading

    1. Cell Biology
    2. Medicine

    Disruption of the novel nested gene Aff3ir mediates disturbed flow-induced atherosclerosis in mice

    Shuo He, Lei Huang ... Jinlong He
    Research Article

    Disturbed shear stress-induced endothelial atherogenic responses are pivotal in the initiation and progression of atherosclerosis, contributing to the uneven distribution of atherosclerotic lesions. This study investigates the role of Aff3ir-ORF2, a novel nested gene variant, in disturbed flow-induced endothelial cell activation and atherosclerosis. We demonstrate that disturbed shear stress significantly reduces Aff3ir-ORF2 expression in athero-prone regions. Using three distinct mouse models with manipulated Aff3ir-ORF2 expression, we demonstrate that Aff3ir-ORF2 exerts potent anti-inflammatory and anti-atherosclerotic effects in Apoe-/- mice. RNA sequencing revealed that interferon regulatory factor 5 (Irf5), a key regulator of inflammatory processes, mediates inflammatory responses associated with Aff3ir-ORF2 deficiency. Aff3ir-ORF2 interacts with Irf5, promoting its retention in the cytoplasm, thereby inhibiting the Irf5-dependent inflammatory pathways. Notably, Irf5 knockdown in Aff3ir-ORF2 deficient mice almost completely rescues the aggravated atherosclerotic phenotype. Moreover, endothelial-specific Aff3ir-ORF2 supplementation using the CRISPR/Cas9 system significantly ameliorated endothelial activation and atherosclerosis. These findings elucidate a novel role for Aff3ir-ORF2 in mitigating endothelial inflammation and atherosclerosis by acting as an inhibitor of Irf5, highlighting its potential as a valuable therapeutic approach for treating atherosclerosis.

    1. Cell Biology
    2. Developmental Biology

    Cell Signaling: Not all dimers are equal

    Jeet H Patel, Mary C Mullins
    Insight

    Disease-causing mutations in the signaling protein BMP4 impair its secretion, but only when it is made as a homodimer.

    1. Cell Biology
    2. Genetics and Genomics

    DNA replication in primary hepatocytes without the six-subunit ORC

    Róża K Przanowska, Yuechuan Chen ... Anindya Dutta
    Research Article

    The six-subunit ORC is essential for the initiation of DNA replication in eukaryotes. Cancer cell lines in culture can survive and replicate DNA replication after genetic inactivation of individual ORC subunits, ORC1, ORC2, or ORC5. In primary cells, ORC1 was dispensable in the mouse liver for endo-reduplication, but this could be explained by the ORC1 homolog, CDC6, substituting for ORC1 to restore functional ORC. Here, we have created mice with a conditional deletion of ORC2, which does not have a homolog. Although mouse embryo fibroblasts require ORC2 for proliferation, mouse hepatocytes synthesize DNA in cell culture and endo-reduplicate in vivo without ORC2. Mouse livers endo-reduplicate after simultaneous deletion of ORC1 and ORC2 both during normal development and after partial hepatectomy. Since endo-reduplication initiates DNA synthesis like normal S phase replication these results unequivocally indicate that primary cells, like cancer cell lines, can load MCM2-7 and initiate replication without ORC.