Advertisement

Large Volumes in Ultrastructural Neuropathology Imaged by Array Tomography of Routine Diagnostic Samples

  • Irene WackerEmail author
  • Carsten Dittmayer
  • Marlene Thaler
  • Rasmus Schröder
Protocol
  • 63 Downloads
Part of the Neuromethods book series (NM, volume 155)

Abstract

Routine samples in pathology and neuropathology are usually prepared according to certified standard sample preparation protocols that do not necessarily introduce the large amounts of heavy metals required to generate optimized contrast and to render the final resin block conductive. Imaging of such samples by volume electron microscopy (EM) methods such as serial block face scanning electron microscopy (SBFSEM) or focused ion beam scanning electron microscopy (FIB-SEM) can thus be challenging due to both contrast and charging issues. Array tomography on the other hand, where hundreds of ultrathin serial sections are deposited on conductive substrates and imaged in a modern field emission scanning electron microscope (FESEM) does not encounter such problems: Section arrays may be poststained with heavy metals leading to superior imaging contrast even from weakly stained blocks. Using a sample from a patient with a congenital myopathy (nebulin-related myopathy) characterized by the so-called electron-dense nemaline rods in muscle fibers we describe preparation of section arrays and how they are imaged in a FESEM in an automated way using a typical, commercially available software platform. We further demonstrate how we can target individual cells by hierarchical imaging cascades. Alignment/registration of image stacks using freeware packages such as Fiji and its TrakEM2 plugin and semiautomated single plane-based segmentation of the nemaline rods using IMOD are also explained.

Key words

Volume electron microscopy Array tomography (AT) Serial sections FESEM 3D pathology Muscle pathology Myopathy Nemaline 

Supplementary material

Supplementary Movie S1

Hierarchical imaging using Atlas 5 AT. Starting from an overview of the entire array zooming in stepwise first to one section, then to one cell, finally to the branched nemaline rod at high magnification and moving from there to a region with distorted filaments (MP4 14,098 kb)

References

  1. 1.
    Micheva KD, Smith SJ (2007) Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55:25–36CrossRefGoogle Scholar
  2. 2.
    Wacker I, Schröder RR (2013) Array tomography. J Microsc 252:93–99CrossRefGoogle Scholar
  3. 3.
    Wacker IU, Veith L, Spomer W et al (2018) Multimodal hierarchical imaging of serial sections for finding specific cellular targets within large volumes. J Vis Exp 133:e57059.  https://doi.org/10.3791/57059CrossRefGoogle Scholar
  4. 4.
    Wacker I, Chockley P, Bartels C et al (2015) Array tomography: characterizing FAC-sorted populations of zebrafish immune cells by their 3D ultrastructure. J Microsc 259:105–113CrossRefGoogle Scholar
  5. 5.
    Wacker I, Spomer W, Hofmann A et al (2016) Hierarchical imaging: a new concept for targeted imaging of large volumes from cells to tissues. BMC Cell Biol 17:38CrossRefGoogle Scholar
  6. 6.
    Stirling JW, Curry A (2009) Quality standards for diagnostic electron microscopy. Ultrastruct Pathol 31:365–367.  https://doi.org/10.1080/01913120701638660CrossRefGoogle Scholar
  7. 7.
    Malfatti E, Lehtokari V-L, Böhm J et al (2014) Muscle histopathology in nebulin-related nemaline myopathy: ultrastructural findings correlated to disease severity and genotype. Acta Neuropathol Commun 2:44CrossRefGoogle Scholar
  8. 8.
    Malfatti E, Romero NB (2016) Nemaline myopathies: State of the art. Rev Neurol 172:614–619CrossRefGoogle Scholar
  9. 9.
    Richardson KC, Jarett L, Finke EH (1960) Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol 35:313–325CrossRefGoogle Scholar
  10. 10.
    Reynolds ES (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 16:208–212CrossRefGoogle Scholar
  11. 11.
    Cardona A, Saalfeld S, Schindelin J et al (2012) TrakEM2 software for neural circuit reconstruction. PLoS One 7:e38011CrossRefGoogle Scholar
  12. 12.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682CrossRefGoogle Scholar
  13. 13.
    Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76CrossRefGoogle Scholar
  14. 14.
    Blumer MJF, Gahleitner P, Narzt T et al (2002) Ribbons of semithin sections: an advanced method with a new type of diamond knife. J Neurosci Methods 120:11–16CrossRefGoogle Scholar
  15. 15.
    Mollenhauer HH (1987) Contamination of thin sections: some observations on the cause and elimination of “embedding pepper”. J Electron Microsc Tech 5:59–63CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Irene Wacker
    • 1
    Email author
  • Carsten Dittmayer
    • 2
  • Marlene Thaler
    • 3
  • Rasmus Schröder
    • 1
    • 4
  1. 1.Cryo EM, Centre for Advanced Materials (CAM)Universität HeidelbergHeidelbergGermany
  2. 2.Department of NeuropathologyCharité-Universitätsmedizin BerlinBerlinGermany
  3. 3.Carl Zeiss Microscopy GmbHOberkochenGermany
  4. 4.Cryo EM, BioQuantUniversitätsklinikum HeidelbergHeidelbergGermany

Personalised recommendations