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A versatile enhanced freeze-substitution protocol for volume electron microscopy

  1. Author:
    Bélanger, Sébastien
    Berensmann, Heather
    Baena, Valentina
    Duncan, Keith
    Meyers, Blake C
    Narayan,Kedar
    Czymmek, Kirk J

  2. Author Address

    Donald Danforth Plant Science Center, Saint Louis, MO, United States., Center for Molecular Microscopy, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States., Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, United States., Division of Plant Science and Technology, University of Missouri-Columbia, Columbia, MO, United States., Advanced Bioimaging Laboratory, Donald Danforth Plant Science Center, Saint Louis, MO, United States.,
    1. Year: 2022
    2. Epub Date: 2022 08 08
  2. Journal: Frontiers in Cell and Developmental Biology
    1. 10
    2. Pages: 933376
  4. Type of Article: Article
  5. Article Number: 933376
  1. Abstract:

    Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a routine tool for understanding fundamental and applied biological questions. One of the enabling factors for its adoption has been the development of conventional fixation protocols with improved heavy metal staining. However, freeze-substitution with organic solvent-based fixation and staining has not realized the same level of benefit. Here, we report a straightforward approach including osmium tetroxide, acetone and up to 3% water substitution fluid (compatible with traditional or fast freeze-substitution protocols), warm-up and transition from organic solvent to aqueous 2% osmium tetroxide. Once fully hydrated, samples were processed in aqueous based potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide, uranyl acetate and lead acetate before resin infiltration and polymerization. We observed a consistent and substantial increase in heavy metal staining across diverse and difficult-to-fix test organisms and tissue types, including plant tissues (Hordeum vulgare), nematode (Caenorhabditis elegans) and yeast (Saccharomyces cerevisiae). Our approach opens new possibilities to combine the benefits of cryo-preservation with enhanced contrast for volume electron microscopy in diverse organisms. Copyright © 2022 Bélanger, Berensmann, Baena, Duncan, Meyers, Narayan and Czymmek.

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External Sources

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  2. DOI: 10.3389/fcell.2022.933376
  3. PMID: 36003147
  4. PMCID: PMC9393620
  5. PII : 933376

Library Notes

  1. Fiscal Year: FY2021-2022
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