In situ X-ray assisted electron microscopy staining for large biological samples

  1. Sebastian Ströh
  2. Eric W Hammerschmith
  3. David W Tank
  4. H Sebastian Seung
  5. Adrian Andreas Wanner  Is a corresponding author
  1. Princeton University, United States
  2. Paul Scherrer Institute, Switzerland

Abstract

Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient. Here we present an in situ time-lapsed X-ray assisted staining procedure that opens the 'black box' of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in potassium ferrocyanide reduced osmium solution. X-ray assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards in silico staining experiments and simulation-guided optimization of staining protocols for large samples. Hence, X-ray assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys or humans.

Data availability

The code to analyze the X-ray projection images and to model the accumulation of heavy metals can be found on https://github.com/adwanner/XrayAssistedStaining. All X-ray data is available for download on https://www.ebi.ac.uk/empiar/ with dataset ID EMPIAR-10782.

Article and author information

Author details

  1. Sebastian Ströh

    Princeton Neuroscience Institute, Princeton University, Princeton, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8128-2617
  2. Eric W Hammerschmith

    Princeton Neuroscience Institute, Princeton University, Princeton, United States
    Competing interests
    No competing interests declared.
  3. David W Tank

    Princeton Neuroscience Institute, Princeton University, Princeton, United States
    Competing interests
    David W Tank, is an inventor of US Patent Application 16/681,028..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9423-4267
  4. H Sebastian Seung

    Princeton Neuroscience Institute, Princeton University, Princeton, United States
    Competing interests
    H Sebastian Seung, is an inventor of US Patent Application 16/681,028..
  5. Adrian Andreas Wanner

    Paul Scherrer Institute, Villigen, Switzerland
    For correspondence
    adrian.wanner@psi.ch
    Competing interests
    Adrian Andreas Wanner, is an inventor of US Patent Application 16/681,028..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5864-8577

Funding

CV Starr Fellowship in Neuroscience by the Princeton University

  • Adrian Andreas Wanner

National Institutes of Health (NS104648)

  • David W Tank
  • H Sebastian Seung

National Institutes of Health (1R01EY027036)

  • H Sebastian Seung

National Institutes of Health (U01NS090562)

  • H Sebastian Seung

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

Reviewing Editor

  1. Andreas T Schaefer, The Francis Crick Institute, United Kingdom

Ethics

Animal experimentation: Animal use procedures were approved by the Princeton University Institutional Animal Care and Use Committee (protocol number 2000) and carried out in accordance with National Institutes of Health standards (AAALAC International Institutional Number: Unit #1001, PHS assurance ID D16-00273).

Version history

  1. Preprint posted: June 20, 2021 (view preprint)
  2. Received: July 13, 2021
  3. Accepted: October 18, 2022
  4. Accepted Manuscript published: October 20, 2022 (version 1)
  5. Version of Record published: November 15, 2022 (version 2)

Copyright

© 2022, Ströh 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. Sebastian Ströh
  2. Eric W Hammerschmith
  3. David W Tank
  4. H Sebastian Seung
  5. Adrian Andreas Wanner
(2022)
In situ X-ray assisted electron microscopy staining for large biological samples
eLife 11:e72147.
https://doi.org/10.7554/eLife.72147

Share this article

https://doi.org/10.7554/eLife.72147

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