3D Electron Microscopy of the ER

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1691)

Abstract

The endoplasmic reticulum (ER) forms an extensive network in plant cells. In leaf cells and vacuolated root cells it is mainly restricted to the cortex whereas in the root meristem the cortical and cytoplasmic ER takes up a large volume throughout the entire cell. Only 3D electron microscopy provides sufficient resolution to understand the spatial organization of the ER in the root. However, high contrast staining and optimally ER specific staining is essential. Here we describe a protocol for selective ER staining that allows automated or semiautomated segmentation of the organelle in 3D datasets obtained from serial sections, Array Tomography, Serial Block Face Scanning Electron Microscopy (SBFSEM), or Focused Ion Beam (FIB) SEM.

Key words

3D EM Serial Block Face SEM Endoplasmic reticulum ZIO Automated segmentation 
This is a preview of subscription content, log in to check access.

References

  1. 1.
    Friedman JR, Voeltz GK (2011) The ER in 3D: a multifunctional dynamic membrane network. Trends Cell Biol 12:709–717CrossRefGoogle Scholar
  2. 2.
    Hawes C, Osterrieder A, Hummel E, Sparkes I (2008) The plant ER-Golgi interface. Traffic 10:1571–1580CrossRefGoogle Scholar
  3. 3.
    Seguı-Simarro JM, Austin JR 2nd, White EA, Staehelin LA (2004) Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by high-pressure freezing. Plant Cell 16:836–856CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Hamada T, Ueda H, Kawase T, Hara-Nishimura I (2014) Microtubules contribute to tubule elongation and anchoring of endoplasmic reticulum, resulting in high network complexity in Arabidopsis. Plant Physiol 166:1869–1876CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kittelmann M, Hawes C, Hughes L (2016) Serial block face scanning electron microscopy and the reconstruction of plant cell membrane systems. J Microsc 263:200–211CrossRefPubMedGoogle Scholar
  6. 6.
    Hayworth KJ, Morgan JL, Schalek R, Berger DR, Hildebrand DG, Lichtman JW (2014) Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits. Front Neural Circuits 8:68CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Domozych DS (1989) The endomembrane system and mechanism of membrane flow in the green alga, Gloeomonas kupfferi (Volvocales, Chlorophyta) II. A cytochemical analysis. Protoplasma 149:108–119CrossRefGoogle Scholar
  8. 8.
    Puhka M, Vihinen H, Joensuu M, Jokitalo E (2007) Endoplasmic reticulum remains continuous and undergoes sheet-to-tubule transformation during cell division in mammalian cells. J Cell Biol 179:895–909CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Sexton R, Hall JL (1991) Enzyme cytochemistry. In: Hawes C, Hall JL (eds) Electron microscopy of plant cells. Academic Press, London, pp 67–84Google Scholar
  10. 10.
    Shu X, Lev-Ram V, Deerinck TJ, Qi Y, Ramko EB, Davidson MW, Jin Y, Ellisman MH, Tsien RY (2011) A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol 4:e1001041CrossRefGoogle Scholar
  11. 11.
    Martell JD, Deerinck TJ, Sancak Y, Poulos TL, Mootha VK, Sosinsky GE, Ellisman MH, Ting AY (2012) Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol 11:1143–1148CrossRefGoogle Scholar
  12. 12.
    Barlow PW, Hawes C, Horne JC (1984) Structure of amyloplasts and endoplasmic reticulum in the root caps of Lepidium sativum and Zea mays observed after selective membrane staining and by high-voltage electron microscopy. Planta 160:363–371CrossRefPubMedGoogle Scholar
  13. 13.
    Hepler PK (1981) The structure of the endoplasmic reticulum revealed by osmium tetroxide-potassium ferricyanide staining. Eur J Cell Biol 26:102–111PubMedGoogle Scholar
  14. 14.
    Hawes CR, Horne JC (1983) Staining plant cells for thick sectioning: uranyl acetate, copper and lead citrate impregnation. Biol Cell 48:207–210Google Scholar
  15. 15.
    Hawes CR, Jumiper B, Horn JC (1981) Low and high voltage electron microscopy of mitosis and cytokinesis in maize. Planta 152:397–407CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Plant Cell Biology, Biological and Medical SciencesOxford Brookes UniversityOxfordUK

Personalised recommendations