Skip to main content
SpringerLink
Log in

Transmission Electron Microscopy Imaging to Analyze Chromatin Density Distribution at the Nanoscale Level

  • Protocol
  • First Online:
Plant Chromatin Dynamics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1675))

Abstract

Transmission electron microscopy (TEM) is used to study the fine ultrastructural organization of cells. Delicate specimen preparation is required for results to reflect the “native” ultrastructural organization of subcellular features such as the nucleus. Despite the advent of high-resolution, fluorescent imaging of chromatin components, TEM still provides a unique and complementary level of resolution capturing chromatin organization at the nanoscale level. Here, we describe the workflow, from tissue preparation, TEM image acquisition and image processing, for obtaining a quantitative description of chromatin density distribution in plant cells, informing on local fluctuations and periodicity. Comparative analyses then allow to elucidate the structural changes induced by developmental or environmental cues, or by mutations affecting specific chromatin modifiers at the nanoscale level. We argue that this approach remains affordable and merits a renewed interest by the plant chromatin community.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Leitch AR (2000) Higher levels of organization in the interphase nucleus of cycling and differentiated cells. Microbiol Mol Biol Rev 64(1):138–152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Thorn K (2016) A quick guide to light microscopy in cell biology. Mol Biol Cell 27(2):219–222

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Barlow PW (1985) Nuclear chromatin structure in relation to cell differentiation and cell activation in the cap and quiescent centre of Zea mays L. J Exp Bot 36(9):1492–1503

    Article  Google Scholar 

  4. Olszewska MJ, Bilecka A, Kononowicz AK, Kolosziejczyk P (1988) Relationship between heterochromatin and repetitive DNA content and the dynamics of nuclear DNA endoreplication in root parenchyma cells. Biol Zent Bl 107(3):311–326

    CAS  Google Scholar 

  5. Lingua G, D’Agostino G, Fusconi A, Berta G (2001) Nuclear changes in pathogen-infected tomato roots. Eur J Histochem 45(1):21

    Article  CAS  PubMed  Google Scholar 

  6. Fusconi A, Gnavi E, Trotta A, Berta G (1999) Apical meristems of tomato roots and their modifications induced by arbuscular mycorrhizal and soilborne pathogenic fungi. New Phytol 142(3):505–516

    Article  Google Scholar 

  7. Tsai Y-C, Greco TM, Boonmee A, Miteva Y, Cristea IM (2012) Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription. Mol Cell Proteomics 11(5):60–76

    Article  CAS  PubMed  Google Scholar 

  8. Nagl W, Cabirol H, Lahr C, Greulach H, Ohliger HM (1983) Nuclear ultrastructure: morphometry of nuclei from various tissues of Cucurbita, Melandrium, Phaseolus, Tradescantia, and Vicia. Protoplasma 115(1):59–64

    Article  Google Scholar 

  9. Nagl W (1979) Nuclear ultrastructure: condensed chromatin in plants is species-specific (karyotypical), but not tissue-specific (functional). Protoplasma 100(1):53–71

    Article  Google Scholar 

  10. Baluška F (1990) Nuclear size, DNA content, and chromatin condensation are different in individual tissues of the maize root apex. Protoplasma 158(1–2):45–52

    Article  Google Scholar 

  11. Cherkezyan L, Stypula-Cyrus Y, Subramanian H, White C, Cruz MD, Wali RK, Goldberg MJ, Bianchi LK, Roy HK, Backman V (2014) Nanoscale changes in chromatin organization represent the initial steps of tumorigenesis: a transmission electron microscopy study. BMC Cancer 14(1):1

    Article  Google Scholar 

  12. Belmont AS (1997) Nuclear ultrastructure: transmission electron microscopy and image analysis. Methods Cell Biol 53:99–124

    Article  Google Scholar 

  13. Almassalha LM, Tiwari A, Ruhoff PT, Stypula-Cyrus Y, Cherkezyan L, Matsuda H, Cruz MAD, Chandler JE, White C, Maneval C (2017) The global relationship between chromatin physical topology, fractal structure, and gene expression. Sci Rep 7:41061

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wilson SM, Bacic A (2012) Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes. Nat Protoc 7(9):1716–1727

    Article  CAS  PubMed  Google Scholar 

  15. Matsko N, Mueller M (2005) Epoxy resin as fixative during freeze-substitution. J Struct Biol 152(2):92–103

    Article  CAS  PubMed  Google Scholar 

  16. Kaech A, Ziegler U (2014) High-pressure freezing: current state and future prospects. Methods Mol Biol 1117:151–171

    Article  CAS  PubMed  Google Scholar 

  17. Studer D, Humbel BM, Chiquet M (2008) Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution. Histochem Cell Biol 130(5):877–889

    Article  CAS  PubMed  Google Scholar 

  18. Feder N, Sidman RL (1958) Methods and principles of fixation by freeze-substitution. J Biophys Biochem Cytol 4(5):593–602

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17(1):208–212

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Mirny LA (2011) The fractal globule as a model of chromatin architecture in the cell. Chromosom Res 19(1):37–51

    Article  CAS  Google Scholar 

  21. Lebedev DV, Filatov MV, Kuklin AI, Islamov AK, Kentzinger E, Pantina R, Toperverg BP, Isaev-Ivanov VV (2005) Fractal nature of chromatin organization in interphase chicken erythrocyte nuclei: DNA structure exhibits biphasic fractal properties. FEBS Lett 579(6):1465–1468

    Article  CAS  PubMed  Google Scholar 

  22. Bancaud A, Lavelle C, Huet S, Ellenberg J (2012) A fractal model for nuclear organization: current evidence and biological implications. Nucleic Acids Res 40(18):8783–8792

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Rowley JC, Moran DT (1975) A simple procedure for mounting wrinkle-free sections on formvar-coated slot grids. Ultramicroscopy 1(2):151–155

    Article  PubMed  Google Scholar 

  24. Huxley HE, Zubay G (1961) Preferential staining of nucleic acid-containing structures for electron microscopy. J Biophys Biochem Cytol 11(2):273–296

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Bernhard W (1969) A new staining procedure for electron microscopical cytology. J Ultrastruct Res 27(3–4):250–265

    Article  CAS  PubMed  Google Scholar 

  26. Hendzel MJ, Kruhlak MJ, Bazett-Jones DP (1998) Organization of highly acetylated chromatin around sites of heterogeneous nuclear RNA accumulation. Mol Biol Cell 9(9):2491–2507.

    Google Scholar 

Download references

Acknowledgment

This work was supported by The Swiss Initiative in Systems Biology SystemsX.ch (MechanX grant 145676 to CR and TNF), the Swiss National Science Foundation (grant 31003A_149974 to CB and the University of Zürich). We are grateful for the technical support of Dr. Andres Kaech and from the Center for Microscopy and Image Analysis of the University of Zürich.

Author information

Authors and Affiliations

  1. Department of Plant and Microbial Biology, Basel-Zürich Plant Science Center, University of Zürich, 107 Zollikerstrasse, 8008, Zürich, Switzerland

    Tohnyui Ndinyanka Fabrice, Christoph Ringli & Célia Baroux

  2. Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL, 60208, USA

    Lusik Cherkezyan

Authors
  1. Tohnyui Ndinyanka Fabrice
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lusik Cherkezyan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christoph Ringli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Célia Baroux
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Célia Baroux .

Editor information

Editors and Affiliations

  1. Department of Molecular Biology, Wageningen University & Research, Wageningen, The Netherlands

    Marian Bemer

  2. Department of Plant and Microbial Biology, Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland

    Célia Baroux

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Fabrice, T.N., Cherkezyan, L., Ringli, C., Baroux, C. (2018). Transmission Electron Microscopy Imaging to Analyze Chromatin Density Distribution at the Nanoscale Level. In: Bemer, M., Baroux, C. (eds) Plant Chromatin Dynamics. Methods in Molecular Biology, vol 1675. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7318-7_34

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7318-7_34

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7317-0

  • Online ISBN: 978-1-4939-7318-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation