Skip to main content
SpringerLink
Log in
Book cover

The Immune Synapse pp 183–206Cite as

Super-resolution Analysis of TCR-Dependent Signaling: Single-Molecule Localization Microscopy

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1584))

Abstract

Single-molecule localization microscopy (SMLM) comprises methods that produce super-resolution images from molecular locations of single molecules. These techniques mathematically determine the center of a diffraction-limited spot produced by a fluorescent molecule, which represents the most likely location of the molecule. Only a small cohort of well-separated molecules is visualized in a single image, and then many images are obtained from a single sample. The localizations from all the images are combined to produce a super-resolution picture of the sample. Here we describe the application of two methods, photoactivation localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM), to the study of signaling microclusters in T cells.

This is a preview of subscription content, 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   129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   249.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Bunnell SC, Hong DI, Kardon JR, Yamazaki T, McGlade CJ, Barr VA et al (2002) T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J Cell Biol 158:1263–1275

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Campi G, Varma R, Dustin ML (2005) Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling. J Exp Med 202:1031–1036

    Google Scholar 

  3. Yokosuka T, Sakata-Sogawa K, Kobayashi W, Hiroshima M, Hashimoto-Tane A, Tokunaga M et al (2005) Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nat Immunol 6:1253–1262

    Google Scholar 

  4. Balagopalan L, Coussens NP, Sherman E, Samelson LE, Sommers CL (2011) The LAT story: a tale of cooperativity, coordination, and choreography. Cold Spring Harb Perspect Biol 3:89–109

    Google Scholar 

  5. Bunnell SC, Kapoor V, Trible RP, Zhang WG, Samelson LE (2001) Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT. Immunity 14:315–329

    Article  CAS  PubMed  Google Scholar 

  6. Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A (1998) Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395:82–86

    Article  CAS  PubMed  Google Scholar 

  7. Dustin ML, Chakraborty AK, Shaw AS (2010) Understanding the structure and function of the immunological synapse. Cold Spring Harb Perspect Biol 2:a002311

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Balagopalan L, Sherman E, Barr VA, Samelson LE (2011) Imaging techniques for assaying lymphocyte activation in action. Nat Rev Immunol 11:21–33

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yokosuka T, Saito T (2010) The immunological synapse, TCR microclusters , and T cell activation. In: Saito T, Batista DF (eds) Immunological synapse. Springer, Heidelberg, Berlin, pp 81–107

    Google Scholar 

  10. Sengupta P, van Engelenburg SB, Lippincott-Schwartz J (2014) Superresolution imaging of biological systems using photoactivated localization microscopy. Chem Rev 114:3189–3202

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Thorley JA, Pike J, Rappoport JZ (2014) Super-resolution microscopy: a comparison of commercially available options. In: Cornea A, Conn PM (eds) Fluorescence microscopy: super-resolution and other novel techniques. Academic, New York, NY, pp 199–212

    Google Scholar 

  12. Nienhaus K, Nienhaus GU (2016) Where do we stand with super-resolution optical microscopy? J Mol Biol 428:308–322

    Article  CAS  PubMed  Google Scholar 

  13. Sydor AM, Czymmek KJ, Puchner EM, Mennella V (2015) Super-resolution microscopy: from single molecules to supramolecular assemblies. Trends Cell Biol 25:730–748

    Article  CAS  PubMed  Google Scholar 

  14. Knight AE (2017) Super-resolution fluorescence microscopy, localization microscopy. In: Lindon J, Tranter GE, Koppenaal D (eds) Encyclopedia of spectroscopy and spectrometry, 3rd edn. Reference module in chemistry, molecular sciences and chemical engineering. Academic, New York, NY, pp 325–330

    Google Scholar 

  15. Allen JR, Ross ST, Davidson MW (2013) Single molecule localization microscopy for superresolution. J Optics 15:094001

    Article  Google Scholar 

  16. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645

    Article  CAS  PubMed  Google Scholar 

  17. Rust MJ, Bates M, Zhuang X (2006) Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution. Nat Methods 3:793–795

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. van de Linde S, Loschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M et al (2011) Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6:991–1009

    Article  PubMed  Google Scholar 

  19. Heilemann M, van de Linde S, Schuttpelz M, Kasper R, Seefeldt B, Mukherjee A et al (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl 47:6172–6176

    Article  CAS  PubMed  Google Scholar 

  20. Endesfelder U, Heilemann M (2015) Direct stochastic optical reconstruction microscopy (dSTORM). In: Verveer JP (ed) Advanced fluorescence microscopy: methods and protocols. Springer, New York, pp 263–276

    Google Scholar 

  21. Walter NG, Huang CY, Manzo AJ, Sobhy MA (2008) Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat Methods 5:475–489

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Folling J, Bossi M, Bock H, Medda R, Wurm CA, Hein B et al (2008) Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods 5:943–945

    Article  PubMed  Google Scholar 

  23. Egner A, Geisler C, von Middendorff C, Bock H, Wenzel D, Medda R et al (2007) Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys J 93:3285–3290

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Sharonov A, Hochstrasser RM (2006) Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc Natl Acad Sci U S A 103:18911–18916

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Giannone G, Hosy E, Levet F, Constals A, Schulze K, Sobolevsky AI, Rosconi MP, Gouaux E, Tampe R, Choquet D, Cognet L (2010) Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys J 99:1303–1310

    Google Scholar 

  26. Curthoys NM, Parent M, Mlodzianoski M, Nelson AJ, Lilieholm J, Butler MB et al (2015) Chapter three—dances with membranes: breakthroughs from super-resolution imaging. In: Anne KK (ed) Current topics in membranes. Academic Press, New York, pp 59–123

    Google Scholar 

  27. Shelby SA, Holowka D, Baird B, Veatch SL (2013) Distinct stages of stimulated FcepsilonRI receptor clustering and immobilization are identified through superresolution imaging. Biophys J 105:2343–2354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Maity PC, Blount A, Jumaa H, Ronneberger O, Lillemeier BF, Reth M (2015) B cell antigen receptors of the IgM and IgD classes are clustered in different protein islands that are altered during B cell activation. Sci Signal 8:ra93

    Article  PubMed  Google Scholar 

  29. Lillemeier BF, Mortelmaier MA, Forstner MB, Huppa JB, Groves JT, Davis MM (2010) TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation. Nat Immunol 11:90–96

    Article  CAS  PubMed  Google Scholar 

  30. Sherman E, Barr V, Manley S, Patterson G, Balagopalan L, Akpan I et al (2011) Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor. Immunity 35:705–720

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Purbhoo MA, Liu H, Oddos S, Owen DM, Neil MAA, Pageon SV et al (2010) Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse. Sci Signal 3:ra36

    Google Scholar 

  32. Hsu C-J, Baumgart T (2011) Spatial association of signaling proteins and F-actin effects on cluster assembly analyzed via photoactivation localization microscopy in T cells. PLoS One 6:e23586

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Rossy J, Pageon SV, Davis DM, Gaus K (2013) Super-resolution microscopy of the immunological synapse. Curr Opin Immunol 25:307–312

    Article  CAS  PubMed  Google Scholar 

  34. Shtengel G, Galbraith JA, Galbraith CG, Lippincott-Schwartz J, Gillette JM, Manley S et al (2009) Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci U S A 106:3125–3130

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ober RJ, Ram S, Ward ES (2004) Localization accuracy in single-molecule microscopy. Biophys J 86:1185–1200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Abraham AV, Ram S, Chao J, Ward ES, Ober RJ (2009) Quantitative study of single molecule location estimation techniques. Opt Express 17:23352–23373

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Small A Stahlheber S (2014) Chapter 16—the role of image analysis algorithms in super-resolution localization microscopy. In: Fluorescence microscopy, Academic Press, New York, pp 227–242

    Google Scholar 

  38. Sage D, Kirshner H, Pengo T, Stuurman N, Min J, Manley S et al (2015) Quantitative evaluation of software packages for single-molecule localization microscopy. Nat Methods 12:717–724

    Article  CAS  PubMed  Google Scholar 

  39. Wiegand T, Moloney KA (2004) Rings, circles, and null-models for point pattern analysis in ecology. Oikos 104:209–229

    Article  Google Scholar 

  40. Parker J, Sherman E, van de Raa M, van der Meer D, Samelson LE, Losert W (2013) Automatic sorting of point pattern sets using Minkowski functionals. Phys Rev E Stat Nonlin Soft Matter Phys 88:022720

    Article  PubMed  Google Scholar 

  41. Subach FV, Patterson GH, Manley S, Gillette JM, Lippincott-Schwartz J, Verkhusha VV (2009) Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat Methods 6:153–159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877

    Article  CAS  PubMed  Google Scholar 

  43. Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319:810–813

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Erdelyi M, Rees E, Metcalf D, Schierle GS, Dudas L, Sinko J et al (2013) Correcting chromatic offset in multicolor super-resolution localization microscopy. Opt Express 21:10978–10988

    Article  PubMed  Google Scholar 

  45. Annibale P, Vanni S, Scarselli M, Rothlisberger U, Radenovic A (2011) Identification of clustering artifacts in photoactivated localization microscopy. Nat Methods 8:527–528

    Article  CAS  PubMed  Google Scholar 

  46. Sengupta P, Jovanovic-Talisman T, Skoko D, Renz M, Veatch SL, Lippincott-Schwartz J (2011) Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat Methods 8:969–975

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Krizek P, Raska I, Hagen GM (2011) Minimizing detection errors in single molecule localization microscopy. Opt Express 19:3226–3235

    Article  PubMed  Google Scholar 

  48. Sherman E, Barr VA, Samelson LE (2013) Resolving multi-molecular protein interactions by photoactivated localization microscopy. Methods 59:261–269

    Article  CAS  PubMed  Google Scholar 

  49. Fricke F, Beaudouin J, Eils R, Heilemann M (2015) One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy. Sci Rep 5:14072

    Article  PubMed  PubMed Central  Google Scholar 

  50. Lee S-H, Shin JY, Lee A, Bustamante C (2012) Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM). Proc Natl Acad Sci U S A 109:17436–17441

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Almada P, Culley S, Henriques R (2015) PALM and STORM: Into large fields and high-throughput microscopy with sCMOS detectors. Methods 88:109–121

    Article  CAS  PubMed  Google Scholar 

  52. Holm T, Klein T, Loschberger A, Klamp T, Wiebusch G, van de Linde S et al (2014) A blueprint for cost-efficient localization microscopy. Chemphyschem 15:651–654

    Article  CAS  PubMed  Google Scholar 

  53. Olivier N, Keller D, Rajan VS, Gönczy P, Manley S (2013) Simple buffers for 3D STORM microscopy. Biomed Opt Express 4:885–899

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Olivier N, Keller D, Gönczy P, Manley S (2013) Resolution doubling in 3D-STORM imaging through improved buffers. PLoS One 8:e69004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Shcherbakova DM, Sengupta P, Lippincott-Schwartz J, Verkhusha VV (2014) Photocontrollable fluorescent proteins for superresolution imaging. Annu Rev Biophys 43:303–329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Allen JR, Ross ST, Davidson MW (2013) Sample preparation for single molecule localization microscopy. Phys Chem Chem Phys 15:18771–18783

    Article  CAS  PubMed  Google Scholar 

  57. Wang S, Moffitt JR, Dempsey GT, Xie XS, Zhuang X (2014) Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging. Proc Natl Acad Sci U S A 111:8452–8457

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Dempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X (2011) Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods 8:1027–1036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Wolter S, Schuttpelz M, Tscherepanow M, Van De Linde S, Heilemann M, Sauer M (2010) Real-time computation of subdiffraction-resolution fluorescence images. J Microsc 237:12–22

    Article  CAS  PubMed  Google Scholar 

  60. Deschout H, Zanacchi FC, Mlodzianoski M, Diaspro A, Bewersdorf J, Hess ST et al (2014) Precisely and accurately localizing single emitters in fluorescence microscopy. Nat Methods 11:253–266

    Article  CAS  PubMed  Google Scholar 

  61. Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Ha T, Tinnefeld P (2012) Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. Annu Rev Phys Chem 63:595–617

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Vaughan JC, Jia S, Zhuang X (2012) Ultrabright photoactivatable fluorophores created by reductive caging. Nat Methods 9:1181–1184

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Schmied JJ, Gietl A, Holzmeister P, Forthmann C, Steinhauer C, Dammeyer T et al (2012) Fluorescence and super-resolution standards based on DNA origami. Nat Methods 9:1133–1134

    Article  CAS  PubMed  Google Scholar 

  65. Banterle N, Bui KH, Lemke EA, Beck M (2013) Fourier ring correlation as a resolution criterion for super-resolution microscopy. J Struct Biol 183:363–367

    Article  CAS  PubMed  Google Scholar 

  66. Nieuwenhuizen RPJ, Lidke KA, Bates M, Puig DL, Grunwald D, Stallinga S et al (2013) Measuring image resolution in optical nanoscopy. Nat Methods 10:557–562

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Erdélyi M, Sinkó J, Kákonyi R, Kelemen A, Rees E, Varga D et al (2015) Origin and compensation of imaging artefacts in localization-based super-resolution microscopy. Methods 88:122–132

    Article  PubMed  Google Scholar 

  68. Patterson G, Davidson M, Manley S, Lippincott-Schwartz J (2010) Superresolution imaging using single-molecule localization. Annu Rev Phys Chem 61:345–367

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgment

We thank Eilon Sherman for generating the algorithms used for our PALM analysis and continued advice on imaging methods. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute (NCI), and Center for Cancer Research.

Author information

Authors and Affiliations

  1. Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, 20892-4256, USA

    Valarie A. Barr, Jason Yi & Lawrence E. Samelson

Authors
  1. Valarie A. Barr
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jason Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lawrence E. Samelson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lawrence E. Samelson .

Editor information

Editors and Affiliations

  1. Department of Life sciences, University of Siena, Siena, Siena, Italy

    Cosima T. Baldari

  2. University of Oxford, Kennedy Institute of Rheumatology, Headington, Oxford, United Kingdom

    Michael L. Dustin

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Barr, V.A., Yi, J., Samelson, L.E. (2017). Super-resolution Analysis of TCR-Dependent Signaling: Single-Molecule Localization Microscopy. In: Baldari, C., Dustin, M. (eds) The Immune Synapse. Methods in Molecular Biology, vol 1584. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6881-7_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6881-7_13

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6879-4

  • Online ISBN: 978-1-4939-6881-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation