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

Publication 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.

Metrics

  • 6,753
    Page views
  • 1,140
    Downloads
  • 34
    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)

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. 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

Further reading

    1. Neuroscience
    Rong Zhao, Stacy D Grunke ... Joanna L Jankowsky
    Research Article

    Neurodegenerative diseases are characterized by selective vulnerability of distinct cell populations; however, the cause for this specificity remains elusive. Here we show that entorhinal cortex layer 2 (EC2) neurons are unusually vulnerable to prolonged neuronal inactivity compared with neighboring regions of the temporal lobe, and that reelin+ stellate cells connecting EC with the hippocampus are preferentially susceptible within the EC2 population. We demonstrate that neuronal death after silencing can be elicited through multiple independent means of activity inhibition, and that preventing synaptic release, either alone or in combination with electrical shunting, is sufficient to elicit silencing-induced degeneration. Finally, we discovered that degeneration following synaptic silencing is governed by competition between active and inactive cells, which is a circuit refinement process traditionally thought to end early in postnatal life. Our data suggests that the developmental window for wholesale circuit plasticity may extend into adulthood for specific brain regions. We speculate that this sustained potential for remodeling by entorhinal neurons may support lifelong memory but renders them vulnerable to prolonged activity changes in disease.

    1. Neuroscience
    Nace Mikus, Sebastian Korb ... Christoph Mathys
    Research Article

    Human behaviour requires flexible arbitration between actions we do out of habit and actions that are directed towards a specific goal. Drugs that target opioid and dopamine receptors are notorious for inducing maladaptive habitual drug consumption; yet, how the opioidergic and dopaminergic neurotransmitter systems contribute to the arbitration between habitual and goal-directed behaviour is poorly understood. By combining pharmacological challenges with a well-established decision-making task and a novel computational model, we show that the administration of the dopamine D2/3 receptor antagonist amisulpride led to an increase in goal-directed or ‘model-based’ relative to habitual or ‘model-free’ behaviour, whereas the non-selective opioid receptor antagonist naltrexone had no appreciable effect. The effect of amisulpride on model-based/model-free behaviour did not scale with drug serum levels in the blood. Furthermore, participants with higher amisulpride serum levels showed higher explorative behaviour. These findings highlight the distinct functional contributions of dopamine and opioid receptors to goal-directed and habitual behaviour and support the notion that even small doses of amisulpride promote flexible application of cognitive control.