High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues

Abstract

Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native states. However, the applicability of cryofixation is limited by its incompatibilities with diaminobenzidine labeling using genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation. Here we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing quality ultrastructural preservation for diverse EM applications.

Data availability

A source data file has been provided for Figure 4 (Figure 4-source data 1). The SBEM volume of a Drosophila antenna presented in this study has been deposited to the Cell Image Library. The SBEM volume, the tdTomato confocal volume and the DRAQ5 confocal volume used for 3D CLEM in a mouse brain (corresponding to Figure 5) have also been deposited to the Cell Image Library. The video of 3D CLEM in a mouse brain expressing tdTomato that corresponds to Figure 5-video supplement 1 has been deposited to the Cell Image Library.

The following data sets were generated

Article and author information

Author details

  1. Tin Ki Tsang

    Division of Biological Sciences, University of California, San Diego, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1002-106X
  2. Eric A Bushong

    Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6195-2433
  3. Daniela Boassa

    Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Junru Hu

    Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Benedetto Romoli

    Department of Psychiatry, University of California, San Diego, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Sebastien Phan

    Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Davide Dulcis

    Department of Psychiatry, University of California, San Diego, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Chih-Ying Su

    Division of Biological Sciences, University of California, San Diego, La Jolla, United States
    For correspondence
    c8su@ucsd.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0005-1890
  9. Mark H Ellisman

    Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, United States
    For correspondence
    mark@ncmir.ucsd.edu
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Institute on Deafness and Other Communication Disorders (R01DC015519)

  • Chih-Ying Su

National Institute of General Medical Sciences (P41GM103412)

  • Mark H Ellisman

Croucher Foundation

  • Tin Ki Tsang

Kavli Foundation (2015-004)

  • Chih-Ying Su
  • Mark H Ellisman

Ray Thomas Edwards Foundation

  • Chih-Ying Su

Frontiers of Innovation Scholars Program

  • Tin Ki Tsang

National Institute of General Medical Sciences (R01GM086197)

  • Daniela Boassa

Kavli Foundation (2016-038)

  • Daniela Boassa
  • Davide Dulcis

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

Reviewing Editor

  1. Moritz Helmstaedter, Max Planck Institute for Brain Research, Germany

Version history

  1. Received: January 30, 2018
  2. Accepted: May 9, 2018
  3. Accepted Manuscript published: May 11, 2018 (version 1)
  4. Version of Record published: June 5, 2018 (version 2)
  5. Version of Record updated: June 6, 2018 (version 3)

Copyright

© 2018, Tsang 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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  1. Tin Ki Tsang
  2. Eric A Bushong
  3. Daniela Boassa
  4. Junru Hu
  5. Benedetto Romoli
  6. Sebastien Phan
  7. Davide Dulcis
  8. Chih-Ying Su
  9. Mark H Ellisman
(2018)
High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues
eLife 7:e35524.
https://doi.org/10.7554/eLife.35524

Share this article

https://doi.org/10.7554/eLife.35524

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