Skip to main content

Preparative Methods for Imaging Plasmodesmata at Super-resolution

  • Protocol
  • First Online:
Plasmodesmata

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1217))

Abstract

Much of our knowledge of plasmodesmata has come from the ability to visualize them. Light microscopy is a popular tool for exploring subcellular structures but is limited in its resolving power due to the diffractive properties of light. At 50 nm in diameter plasmodesmata are below this limit and so cannot be resolved. Super-resolution microscopy operates beyond the limits of conventional light microscopy affording a more detailed view. Although lacking the ultrastructural resolving power of the electron microscope (EM), super-resolution microscopy helps to bridge the gap between conventional light microscopy and EM.

Here we present three preparative methods for studying plasmodesmata at super-resolution using 3D-structured illumination microscopy (3D-SIM).

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

Access this chapter

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Bell K, Oparka K (2010) Imaging plasmodesmata. Protoplasma 248:9–25

    Article  PubMed  Google Scholar 

  2. Thomas CL, Bayer EM, Ritzenthaler C et al (2008) Specific targeting of a plasmodesmal protein affecting cell-to-cell communication. PLoS Biol 6:180–190

    Article  CAS  Google Scholar 

  3. Simpson C, Thomas C, Findlay K et al (2009) An Arabidopsis GPI-anchor plasmodesmal neck protein with callose binding activity and potential to regulate cell-to-cell trafficking. Plant Cell 21:581–594

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  4. Gustafsson MGL (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198: 82–87

    Article  PubMed  CAS  Google Scholar 

  5. Dobbie IM, King E, Parton RM et al (2011) OMX: a new platform for multimodal, multichannel wide-field imaging. Cold Spring Harb Protoc 8:899–909

    Google Scholar 

  6. Schermelleh L, Carlton PM, Haase S et al (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320:1332–1336

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  7. Roberts IM, Boevink P, Roberts AG et al (2001) Dynamic changes in the frequency and architecture of plasmodesmata during the sink-source transition in tobacco leaves. Protoplasma 218:31–44

    Article  PubMed  CAS  Google Scholar 

  8. Fitzgibbon J, Bell K, King E et al (2010) Super-resolution imaging of plasmodesmata using three-dimensional structured illumination microscopy. Plant Physiol 153:1453–1463

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  9. Bell K, Mitchell S, Paultre D et al (2013) Correlative imaging of fluorescent proteins in resin-embedded plant material. Plant Physiol 161:1595–1603

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  10. Kner P, Chhun BB, Griffis ER et al (2009) Super-resolution video microscopy of live cells by structured illumination. Nat Methods 6: 339–342

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  11. Wombacher R, Heidbreder M, van de Linde S et al (2010) Live-cell super-resolution imaging with trimethoprim conjugates. Nat Methods 7:717–719

    Article  PubMed  CAS  Google Scholar 

  12. Balint S, Verdeny Vilanova I, Sandoval Alvarez A et al (2013) Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections. Proc Natl Acad Sci U S A 110:3375–3380

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  13. Knoblauch M, Peters WS (2010) Münch, morphology, microfluidics: our structural problem with the phloem. Plant Cell Environ 33: 1439–1452

    PubMed  Google Scholar 

  14. Brown R, Lemmon B, Mullinax J (1992) Immunofluorescent staining of microtubules in plant tissues: improved embedding and sectioning techniques using polyethylene glycol (PEG) and Steedman’s wax. Bot Acta 102: 54–61

    Article  Google Scholar 

  15. Baskin TI, Busby CH, Fowke LC et al (1992) Improvements in immunostaining samples embedded in methacrylate: localisation of microtubules and other antigens throughout developing organs in plants. Planta 187: 405–413

    Article  PubMed  CAS  Google Scholar 

  16. Baskin TI, Miller DD, Vos JW et al (1996) Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy. J Microsc 182: 149–161

    Article  PubMed  CAS  Google Scholar 

  17. Thompson MV, Wolniak SM (2008) A plasma membrane-anchored fluorescent protein fusion illuminates sieve element plasma membranes in Arabidopsis and Tobacco. Plant Physiol 146:1599–1610

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  18. Keene DR, Tufa SF, Lunstrum GP et al (2008) Confocal/TEM overlay microscopy: a simple method for correlating confocal and electron microscopy of cells expressing GFP/YFP fusion proteins. Microsc Microanal 14: 342–348

    Article  PubMed  CAS  Google Scholar 

  19. Hayat MA (1968) The principles and techniques of electron microscopy, vol 1. Van Nosttand Reinhold Company, New York, NY

    Google Scholar 

Download references

Acknowledgements

We would like to thank Steve Mitchell for help when developing the LR method and Dr. Alison Roberts for advice in using the vibrating microtome.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Karl Oparka .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Bell, K., Oparka, K. (2015). Preparative Methods for Imaging Plasmodesmata at Super-resolution. In: Heinlein, M. (eds) Plasmodesmata. Methods in Molecular Biology, vol 1217. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1523-1_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1523-1_4

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1522-4

  • Online ISBN: 978-1-4939-1523-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics