Skip to main content
SpringerLink
Log in

Three-Dimensional Structured Illumination Microscopy (3D-SIM) to Dissect Signaling Cross-Talks in Motile T-Cells

  • Protocol
  • First Online:
T-Cell Motility

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1930))

Abstract

Visualization of signal transduction events in T-cells has always been a challenge due to their miniscule size. Recent advancement in super-resolution microscopy techniques presents many new opportunities to navigate the spatial and temporal signaling cross-talks in motile T-cells. Here, we provide technical details, optimal conditions, and critical practical considerations that need to be taken into account during cell handling, sample preparation, and image acquisition of motile T-cells for performing three-dimensional structured illumination microscopy (3D-SIM).

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

Access this chapter

Log in via an institution
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
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

References

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

    Article  CAS  Google Scholar 

  2. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645

    Article  CAS  Google Scholar 

  3. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795

    Article  CAS  Google Scholar 

  4. Heilemann M, van de Linde S, Schuttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Edn 47:6172–6176

    Article  CAS  Google Scholar 

  5. Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated-emission-depletion fluorescence microscopy. Opt Lett 19:780–782

    Article  CAS  Google Scholar 

  6. Rittweger E, Han KY, Irvine SE, Eggeling C, Hell SW (2009) STED microscopy reveals crystal colour centres with nanometric resolution. Nat Photonics 3:144–147

    Article  CAS  Google Scholar 

  7. Gustafsson MG, Shao L, Carlton PM, Wang CJ, Golubovskaya IN, Cande WZ, Agard DA, Sedat JW (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94:4957–4970

    Article  CAS  Google Scholar 

  8. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682

    Article  CAS  Google Scholar 

  9. Verma NK, Dempsey E, Long A, Davies A, Barry SP, Fallon PG, Volkov Y, Kelleher D (2012) Leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction induces a novel genetic signature resulting in T-cells refractory to transforming growth factor-beta signaling. J Biol Chem 287(32):27204–27216

    Article  CAS  Google Scholar 

  10. Ball G, Demmerle J, Kaufmann R, Davis I, Dobbie IM, Schermelleh L (2015) SIMcheck: a toolbox for successful super-resolution structured illumination microscopy. Sci Rep 5:15915

    Article  CAS  Google Scholar 

  11. Xie W, Horn HF, Wright GD (2016) Superresolution microscopy of the nuclear envelope and associated proteins. Methods Mol Biol 1411:83–97

    Article  CAS  Google Scholar 

  12. Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, Kner P, Burke B, Cardoso MC, Agard DA, Gustafsson MG, Leonhardt H, Sedat JW (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320:1332–1336

    Article  CAS  Google Scholar 

  13. Schermelleh L, Heintzmann R, Leonhardt H (2010) A guide to super-resolution fluorescence microscopy. J Cell Biol 190:165–175

    Article  CAS  Google Scholar 

  14. Volkov Y, Long A, McGrath S, Ni Eidhin D, Kelleher D (2001) Crucial importance of PKC-beta(I) in LFA-1-mediated locomotion of activated T cells. Nat Immunol 2:508–514

    Article  CAS  Google Scholar 

  15. Ong ST, Freeley M, Skubis-Zegadło J, Turabe Fazil MH, Kelleher D, Fresser F, Baier G, Verma NK, Long A (2014) Phosphorylation of rab5a by PKCε is crucial for T-cell migration. J Biol Chem 289(28):19420–19434

    Article  CAS  Google Scholar 

  16. Whelan DR, Bell TD (2015) Image artifacts in single molecule localization microscopy: why optimization of sample preparation protocols matters. Sci Rep 5:7924

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by grants from Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Start-Up Grant and the Ministry of Education Singapore under its Singapore Ministry of Education Academic Research Fund (AcRF) Tier 2 Grant (MOE2017-T2-2-004) to N.K.V. 3D-SIM platform (DeltaVision OMX v4 Blaze microscope) and Institute of Medical Biology (IMB) Microscopy Unit, now renamed to the A*STAR Microscopy Platform within the Skin Research Institute of Singapore (SRIS), was funded by A*STAR, Singapore.

Author information

Authors and Affiliations

  1. Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore

    Seow Theng Ong

  2. A*STAR Microscopy Platform, Skin Research Institute of Singapore, Singapore, Singapore

    Graham D. Wright

  3. Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore

    Navin Kumar Verma

Authors
  1. Seow Theng Ong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Graham D. Wright
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Navin Kumar Verma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Seow Theng Ong .

Editor information

Editors and Affiliations

  1. Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore

    Navin Kumar Verma

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Ong, S.T., Wright, G.D., Verma, N.K. (2019). Three-Dimensional Structured Illumination Microscopy (3D-SIM) to Dissect Signaling Cross-Talks in Motile T-Cells. In: Verma, N. (eds) T-Cell Motility. Methods in Molecular Biology, vol 1930. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9036-8_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9036-8_6

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9035-1

  • Online ISBN: 978-1-4939-9036-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation