Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Imaging techniques for assaying lymphocyte activation in action

Nature Reviews Immunology volume 11, pages 21–33 (2011)Cite this article

Subjects

Key Points

  • In vivo imaging has shown that the interaction of lymphocytes and antigen-presenting cells (APCs), as well as lymphocyte motility, depend on the architecture of lymphoid organs. Lymphocyte activation is also influenced by other cells, such as stromal cells, that are part of the in vivo environment.

  • Images of T cell interactions with APCs in vitro have resolved striking morphological changes upon encounter, including the polarization of the cells towards the interface and the formation of the 'bull's eye' organization of the immunological synapse.

  • Experiments in model systems using planar substrates for activation have determined that signalling begins in small signalling clusters termed microclusters. Further studies demonstrated the dynamic nature and changing composition of microclusters.

  • Specialized techniques can be used to study molecular dynamics and interactions at the immunological synapse in real time. FRET (fluorescent resonance energy transfer), FRAP (fluorescence recovery after photobleaching) and SPT (single-particle tracking) methods have revealed complex and often unexpected dynamics of immune receptors and signalling molecules during activation.

  • Super-resolution imaging, beyond the diffraction limit of light, allows observation of the molecular organization of signalling complexes. The current techniques are limited to studies of the interfaces between live T cells and model APC membranes.

  • Technological advances will soon allow the study of lymphocyte activation in systems of increasing physiological relevance, in multiple colours and unprecedented resolution. The educated choice of imaging systems, guided by the question at hand, should allow the researcher to obtain optimal experimental results in this multidimensional space.

Abstract

Imaging techniques have greatly improved our understanding of lymphocyte activation. Technical advances in spatial and temporal resolution and new labelling tools have enabled researchers to directly observe the activation process. Consequently, research using imaging approaches to study lymphocyte activation has expanded, providing an unprecedented level of cellular and molecular detail in the field. As a result, certain models of lymphocyte activation have been verified, others have been revised and yet others have been replaced with new concepts. In this article, we review the current imaging techniques that are used to assess lymphocyte activation in different contexts, from whole animals to single molecules, and discuss the advantages and potential limitations of these methods.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Imaging techniques: hierarchy of scale.
Figure 2: Antigen-presenting cell substitutes used for lymphocyte activation.
Figure 3: Light-microscopy techniques that are widely used for cell imaging.
Figure 4: Techniques for imaging molecular dynamics and interactions.
Figure 5: High-resolution imaging techniques.

Similar content being viewed by others

References

  1. Huse, M. The T-cell-receptor signaling network. J. Cell Sci. 122, 1269–1273 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Lin, J. & Weiss, A. T cell receptor signalling. J. Cell Sci. 114, 243–244 (2001).

    CAS  PubMed  Google Scholar 

  3. Samelson, L. E. Signal transduction mediated by the T cell antigen receptor: the role of adapter proteins. Annu. Rev. Immunol. 20, 371–394 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Schwartzberg, P. L. Genetic approaches to tyrosine kinase signaling pathways in the immune system. Immunol. Res. 27, 481–488 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Kaufmann, S. H. Immunology's foundation: the 100-year anniversary of the Nobel Prize to Paul Ehrlich and Elie Metchnikoff. Nature Immunol. 9, 705–712 (2008).

    Article  CAS  Google Scholar 

  6. Stoll, S., Delon, J., Brotz, T. M. & Germain, R. N. Dynamic imaging of T cell–dendritic cell interactions in lymph nodes. Science 296, 1873–1876 (2002).

    Article  PubMed  Google Scholar 

  7. Bousso, P., Bhakta, N. R., Lewis, R. S. & Robey, E. Dynamics of thymocyte–stromal cell interactions visualized by two-photon microscopy. Science 296, 1876–1880 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Miller, M. J., Wei, S. H., Parker, I. & Cahalan, M. D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Mempel, T. R., Henrickson, S. E. & Von Andrian, U. H. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427, 154–159 (2004).

    CAS  PubMed  Google Scholar 

  10. Miller, M. J., Wei, S. H., Cahalan, M. D. & Parker, I. Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy. Proc. Natl Acad. Sci. USA 100, 2604–2609 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bousso, P. T-cell activation by dendritic cells in the lymph node: lessons from the movies. Nature Rev. Immunol. 8, 675–684 (2008).

    Article  CAS  Google Scholar 

  12. Germain, R. N. et al. Making friends in out-of-the-way places: how cells of the immune system get together and how they conduct their business as revealed by intravital imaging. Immunol. Rev. 221, 163–181 (2008).

    Article  CAS  PubMed  Google Scholar 

  13. Mempel, T. R. et al. Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation. Immunity 25, 129–141 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Breart, B., Lemaitre, F., Celli, S. & Bousso, P. Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice. J. Clin. Invest. 118, 1390–1397 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bartholomaus, I. et al. Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature 462, 94–98 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. Beuneu, H. et al. Visualizing the functional diversification of CD8+ T cell responses in lymph nodes. Immunity 33, 412–423 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Okada, T. et al. Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biol. 3, e150 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Carrasco, Y. R. & Batista, F. D. B cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node. Immunity 27, 160–171 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Phan, T. G., Grigorova, I., Okada, T. & Cyster, J. G. Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells. Nature Immunol. 8, 992–1000 (2007).

    Article  CAS  Google Scholar 

  20. Qi, H., Egen, J. G., Huang, A. Y. & Germain, R. N. Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science 312, 1672–1676 (2006).

    Article  CAS  PubMed  Google Scholar 

  21. Junt, T. et al. Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells. Nature 450, 110–114 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Germain, R. N., Miller, M. J., Dustin, M. L. & Nussenzweig, M. C. Dynamic imaging of the immune system: progress, pitfalls and promise. Nature Rev. Immunol. 6, 497–507 (2006).

    Article  CAS  Google Scholar 

  23. Wilson, E. H. et al. Behavior of parasite-specific effector CD8+ T cells in the brain and visualization of a kinesis-associated system of reticular fibers. Immunity 30, 300–311 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bajenoff, M. et al. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25, 989–1001 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Azar, G. A., Lemaitre, F., Robey, E. A. & Bousso, P. Subcellular dynamics of T cell immunological synapses and kinapses in lymph nodes. Proc. Natl Acad. Sci. USA 107, 3675–3680 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Friedman, R. S., Beemiller, P., Sorensen, C. M., Jacobelli, J. & Krummel, M. F. Real-time analysis of T cell receptors in naive cells in vitro and in vivo reveals flexibility in synapse and signaling dynamics. J. Exp. Med. 207, 2733–2749 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Shi, M. et al. Real-time imaging of trapping and urease-dependent transmigration of Cryptococcus neoformans in mouse brain. J. Clin. Invest. 120, 1683–1693 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lee, S. J., Escobedo-Lozoya, Y., Szatmari, E. M. & Yasuda, R. Activation of CaMKII in single dendritic spines during long-term potentiation. Nature 458, 299–304 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yasuda, R. Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy. Curr. Opin. Neurobiol. 16, 551–561 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Andresen, V. et al. Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging. Curr. Opin. Biotechnol. 20, 54–62 (2009).

    Article  CAS  PubMed  Google Scholar 

  31. Herz, J. et al. Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator. Biophys. J. 98, 715–723 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hu, S., Yan, P., Maslov, K., Lee, J. M. & Wang, L. V. Intravital imaging of amyloid plaques in a transgenic mouse model using optical-resolution photoacoustic microscopy. Opt. Lett. 34, 3899–3901 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Xu, M. & Wang, L. Photoacoustic imaging in biomedicine. Rev. Sci. Instrum. 77, 041101 (2006).

    Article  CAS  Google Scholar 

  34. Dustin, M. L. Hunter to gatherer and back: immunological synapses and kinapses as variations on the theme of amoeboid locomotion. Annu. Rev. Cell Dev. Biol. 24, 577–596 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Kupfer, A., Dennert, G. & Singer, S. J. The reorientation of the Golgi apparatus and the microtubule-organizing center in the cytotoxic effector cell is a prerequisite in the lysis of bound target cells. J. Mol. Cell. Immunol. 2, 37–49 (1985).

    CAS  PubMed  Google Scholar 

  36. Ryser, J. E., Rungger-Brandle, E., Chaponnier, C., Gabbiani, G. & Vassalli, P. The area of attachment of cytotoxic T lymphocytes to their target cells shows high motility and polarization of actin, but not myosin. J. Immunol. 128, 1159–1162 (1982).

    CAS  PubMed  Google Scholar 

  37. Geiger, B., Rosen, D. & Berke, G. Spatial relationships of microtubule-organizing centers and the contact area of cytotoxic T lymphocytes and target cells. J. Cell Biol. 95, 137–143 (1982).

    Article  CAS  PubMed  Google Scholar 

  38. Poenie, M., Tsien, R. Y. & Schmitt-Verhulst, A. M. Sequential activation and lethal hit measured by [Ca2+]i in individual cytolytic T cells and targets. EMBO J. 6, 2223–2232 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Donnadieu, E., Bismuth, G. & Trautmann, A. Antigen recognition by helper T cells elicits a sequence of distinct changes of their shape and intracellular calcium. Curr. Biol. 4, 584–595 (1994).

    Article  CAS  PubMed  Google Scholar 

  40. Negulescu, P. A., Krasieva, T. B., Khan, A., Kerschbaum, H. H. & Cahalan, M. D. Polarity of T cell shape, motility, and sensitivity to antigen. Immunity 4, 421–430 (1996).

    Article  CAS  PubMed  Google Scholar 

  41. Brossard, C. et al. Multifocal structure of the T cell – dendritic cell synapse. Eur. J. Immunol. 35, 1741–1753 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Stinchcombe, J. C. & Griffiths, G. M. Secretory mechanisms in cell-mediated cytotoxicity. Annu. Rev. Cell Dev. Biol. 23, 495–517 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Gomez, T. S. et al. Formins regulate the actin-related protein 2/3 complex-independent polarization of the centrosome to the immunological synapse. Immunity 26, 177–190 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Fleire, S. J. et al. B cell ligand discrimination through a spreading and contraction response. Science 312, 738–741 (2006). This study uses a combination of multiple light microscopy techniques and scanning electron microscopy to reveal morphological changes in activated B cells.

    Article  CAS  PubMed  Google Scholar 

  45. Monks, C. R., Freiberg, B. A., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998). The seminal first observation of the detailed molecular organization within the immunological synapse of a 'bull's eye' pattern, termed pSMAC and cSMAC, using three-dimensional confocal imaging of cell conjugates.

    Article  CAS  PubMed  Google Scholar 

  46. Freiberg, B. A. et al. Staging and resetting T cell activation in SMACs. Nature Immunol. 3, 911–917 (2002).

    Article  CAS  Google Scholar 

  47. Potter, T. A., Grebe, K., Freiberg, B. & Kupfer, A. Formation of supramolecular activation clusters on fresh ex vivo CD8+ T cells after engagement of the T cell antigen receptor and CD8 by antigen-presenting cells. Proc. Natl Acad. Sci. USA 98, 12624–12629 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Orange, J. S. Formation and function of the lytic NK-cell immunological synapse. Nature Rev. Immunol. 8, 713–725 (2008).

    Article  CAS  Google Scholar 

  49. Carroll-Portillo, A. et al. Formation of a mast cell synapse: FcɛRI membrane dynamics upon binding mobile or immobilized ligands on surfaces. J. Immunol. 184, 1328–1338 (2010).

    Article  CAS  PubMed  Google Scholar 

  50. Trautmann, A. & Valitutti, S. The diversity of immunological synapses. Curr. Opin. Immunol. 15, 249–254 (2003).

    Article  CAS  PubMed  Google Scholar 

  51. Singleton, K. L. et al. Spatiotemporal patterning during T cell activation is highly diverse. Sci. Signal. 2, ra15 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Purtic, B., Pitcher, L. A., van Oers, N. S. & Wulfing, C. T cell receptor (TCR) clustering in the immunological synapse integrates TCR and costimulatory signaling in selected T cells. Proc. Natl Acad. Sci. USA 102, 2904–2909 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Richie, L. I. et al. Imaging synapse formation during thymocyte selection: inability of CD3ζ to form a stable central accumulation during negative selection. Immunity 16, 595–606 (2002).

    Article  CAS  PubMed  Google Scholar 

  54. Hailman, E., Burack, W. R., Shaw, A. S., Dustin, M. L. & Allen, P. M. Immature CD4+CD8+ thymocytes form a multifocal immunological synapse with sustained tyrosine phosphorylation. Immunity 16, 839–848 (2002).

    Article  CAS  PubMed  Google Scholar 

  55. Dustin, M. L. Visualization of cell-cell interaction contacts—synapses and kinapses. Adv. Exp. Med. Biol. 640, 164–182 (2008).

    Article  CAS  PubMed  Google Scholar 

  56. Valitutti, S. & Dupre, L. Plasticity of immunological synapses. Curr. Top. Microbiol. Immunol. 340, 209–228 (2010).

    CAS  PubMed  Google Scholar 

  57. Grakoui, A. et al. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227 (1999). The first use of a glass-supported planar bilayer for real-time imaging of T cell activation with peptide–MHC molecules, revealing the formation and dynamics of TCR microclusters within the immunological synapse.

    Article  CAS  PubMed  Google Scholar 

  58. Johnson, K. G., Bromley, S. K., Dustin, M. L. & Thomas, M. L. A supramolecular basis for CD45 tyrosine phosphatase regulation in sustained T cell activation. Proc. Natl Acad. Sci. USA 97, 10138–10143 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Krummel, M. F., Sjaastad, M. D., Wulfing, C. & Davis, M. M. Differential clustering of CD4 and CD3ζ during T cell recognition. Science 289, 1349–1352 (2000).

    Article  CAS  PubMed  Google Scholar 

  60. Lee, K. H. et al. T cell receptor signaling precedes immunological synapse formation. Science 295, 1539–1542 (2002).

    Article  CAS  PubMed  Google Scholar 

  61. Cullinan, P., Sperling, A. I. & Burkhardt, J. K. The distal pole complex: a novel membrane domain distal to the immunological synapse. Immunol. Rev. 189, 111–122 (2002).

    Article  CAS  PubMed  Google Scholar 

  62. Ludford-Menting, M. J. et al. A network of PDZ-containing proteins regulates T cell polarity and morphology during migration and immunological synapse formation. Immunity 22, 737–748 (2005).

    Article  CAS  PubMed  Google Scholar 

  63. Barr, V. A. et al. Dynamic movement of the calcium sensor STIM1 and the calcium channel Orai1 in activated T-cells: puncta and distal caps. Mol. Biol. Cell 19, 2802–2817 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Oddos, S. et al. High-speed high-resolution imaging of intercellular immune synapses using optical tweezers. Biophys. J. 95, L66–L68 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Toomre, D. & Manstein, D. J. Lighting up the cell surface with evanescent wave microscopy. Trends Cell Biol. 11, 298–303 (2001).

    Article  CAS  PubMed  Google Scholar 

  66. Barr, V. A. & Bunnell, S. C. Interference reflection microscopy. Curr. Protoc. Cell Biol. 45, 4.23.1–4.23.19 (2009).

    Article  Google Scholar 

  67. Bunnell, S. C., Kapoor, V., Trible, R. P., Zhang, W. & Samelson, L. E. Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT. Immunity 14, 315–329 (2001).

    Article  CAS  PubMed  Google Scholar 

  68. Kaizuka, Y., Douglass, A. D., Varma, R., Dustin, M. L. & Vale, R. D. Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells. Proc. Natl Acad. Sci. USA 104, 20296–20301 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Ilani, T., Vasiliver-Shamis, G., Vardhana, S., Bretscher, A. & Dustin, M. L. T cell antigen receptor signaling and immunological synapse stability require myosin IIA. Nature Immunol. 10, 531–539 (2009).

    Article  CAS  Google Scholar 

  70. Quann, E. J., Merino, E., Furuta, T. & Huse, M. Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells. Nature Immunol. 10, 627–635 (2009).

    Article  CAS  Google Scholar 

  71. Bunnell, S. C. et al. T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J. Cell Biol. 158, 1263–1275 (2002). An extensive imaging study showing the proteins that are recruited to microclusters, the dynamics of proteins at microclusters, the sorting of proteins away from TCR microclusters and the dynamics of intracellular calcium levels.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Barda-Saad, M. et al. Dynamic molecular interactions linking the T cell antigen receptor to the actin cytoskeleton. Nature Immunol. 6, 80–89 (2005).

    Article  CAS  Google Scholar 

  73. Braiman, A., Barda-Saad, M., Sommers, C. L. & Samelson, L. E. Recruitment and activation of PLCγ1 in T cells: a new insight into old domains. EMBO J. 25, 774–784 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Huse, M. et al. Spatial and temporal dynamics of T cell receptor signaling with a photoactivatable agonist. Immunity 27, 76–88 (2007).

    Article  CAS  PubMed  Google Scholar 

  75. Yokosuka, T. et al. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nature Immunol. 6, 1253–1262 (2005).

    Article  CAS  Google Scholar 

  76. Bunnell, S. C. et al. Persistence of cooperatively stabilized signaling clusters drives T-cell activation. Mol. Cell. Biol. 26, 7155–7166 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Seminario, M. C. & Bunnell, S. C. Signal initiation in T-cell receptor microclusters. Immunol. Rev. 221, 90–106 (2008).

    Article  CAS  PubMed  Google Scholar 

  78. Harwood, N. E. & Batista, F. D. Early events in B cell activation. Annu. Rev. Immunol. 28, 185–210 (2010).

    Article  CAS  PubMed  Google Scholar 

  79. Yokosuka, T. et al. Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C θ translocation. Immunity 29, 589–601 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Kaizuka, Y., Douglass, A. D., Vardhana, S., Dustin, M. L. & Vale, R. D. The coreceptor CD2 uses plasma membrane microdomains to transduce signals in T cells. J. Cell Biol. 185, 521–534 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Barr, V. A. et al. T-cell antigen receptor-induced signaling complexes: internalization via a cholesterol-dependent endocytic pathway. Traffic 7, 1143–1162 (2006).

    Article  CAS  PubMed  Google Scholar 

  82. Varma, R., Campi, G., Yokosuka, T., Saito, T. & Dustin, M. L. T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity 25, 117–127 (2006). In this investigation, imaging of activated T cells on planar bilayers demonstrates the importance of peripheral microclusters in TCR signalling and the role of the cSMAC in its termination.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Vardhana, S., Choudhuri, K., Varma, R. & Dustin, M. L. Essential role of ubiquitin and TSG101 protein in formation and function of the central supramolecular activation cluster. Immunity 32, 531–540 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Purbhoo, M. A. et al. Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse. Sci. Signal. 3, ra36 (2010).

    Article  CAS  PubMed  Google Scholar 

  85. Balagopalan, L. et al. c-Cbl-mediated regulation of LAT-nucleated signaling complexes. Mol. Cell. Biol. 27, 8622–8636 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Balagopalan, L., Barr, V. A. & Samelson, L. E. Endocytic events in TCR signaling: focus on adapters in microclusters. Immunol. Rev. 232, 84–98 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Nguyen, K., Sylvain, N. R. & Bunnell, S. C. T cell costimulation via the integrin VLA-4 inhibits the actin-dependent centralization of signaling microclusters containing the adaptor SLP-76. Immunity 28, 810–821 (2008).

    Article  CAS  PubMed  Google Scholar 

  88. Mossman, K. D., Campi, G., Groves, J. T. & Dustin, M. L. Altered TCR signaling from geometrically repatterned immunological synapses. Science 310, 1191–1193 (2005).

    Article  CAS  PubMed  Google Scholar 

  89. Rodriguez-Fernandez, J. L., Riol-Blanco, L. & Delgado-Martin, C. What is the function of the dendritic cell side of the immunological synapse? Sci. Signal. 3, re2 (2010).

    Article  CAS  PubMed  Google Scholar 

  90. Trautmann, A. Microclusters initiate and sustain T cell signaling. Nature Immunol. 6, 1213–1214 (2005).

    Article  CAS  Google Scholar 

  91. Tolar, P., Sohn, H. W. & Pierce, S. K. The initiation of antigen-induced B cell antigen receptor signaling viewed in living cells by fluorescence resonance energy transfer. Nature Immunol. 6, 1168–1176 (2005). An important application of FRET to resolve some of the earliest steps of BCR activation on ligand binding, including clustering and conformational changes.

    Article  CAS  Google Scholar 

  92. Tolar, P., Hanna, J., Krueger, P. D. & Pierce, S. K. The constant region of the membrane immunoglobulin mediates B cell-receptor clustering and signaling in response to membrane antigens. Immunity 30, 44–55 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Xu, C. et al. Regulation of T cell receptor activation by dynamic membrane binding of the CD3ɛ cytoplasmic tyrosine-based motif. Cell 135, 702–713 (2008). The combination of live-cell FRET microscopy and NMR in this study reveals conformational changes in the TCR–CD3ɛ chain complex on receptor activation.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Gascoigne, N. R. et al. Visualizing intermolecular interactions in T cells. Curr. Top. Microbiol. Immunol. 334, 31–46 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Huppa, J. B. et al. TCR–peptide–MHC interactions in situ show accelerated kinetics and increased affinity. Nature 463, 963–967 (2010). A conceptually novel study that quantifies on-rates and off-rates of the TCR with peptide–MHC complexes at the immunological synapse using single-molecule FRET, and shows surprisingly fast dynamics between these molecules.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Hashimoto-Tane, A. et al. T-cell receptor microclusters critical for T-cell activation are formed independently of lipid raft clustering. Mol. Cell. Biol. 30, 3421–3429 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Barda-Saad, M. et al. Cooperative interactions at the SLP-76 complex are critical for actin polymerization. EMBO J. 29, 2315–2328 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Ananthanarayanan, B., Ni, Q. & Zhang, J. Molecular sensors based on fluorescence resonance energy transfer to visualize cellular dynamics. Methods Cell Biol. 89, 37–57 (2008).

    Article  CAS  PubMed  Google Scholar 

  99. Paster, W. et al. Genetically encoded Forster resonance energy transfer sensors for the conformation of the Src family kinase Lck. J. Immunol. 182, 2160–2167 (2009).

    Article  CAS  PubMed  Google Scholar 

  100. Randriamampita, C. et al. A novel ZAP-70 dependent FRET based biosensor reveals kinase activity at both the immunological synapse and the antisynapse. PLoS ONE 3, e1521 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Treanor, B. et al. Microclusters of inhibitory killer immunoglobulin-like receptor signaling at natural killer cell immunological synapses. J. Cell Biol. 174, 153–161 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Janes, P. W., Ley, S. C., Magee, A. I. & Kabouridis, P. S. The role of lipid rafts in T cell antigen receptor (TCR) signalling. Semin. Immunol. 12, 23–34 (2000).

    Article  CAS  PubMed  Google Scholar 

  103. Munro, S. Lipid rafts: elusive or illusive? Cell 115, 377–388 (2003).

    Article  CAS  PubMed  Google Scholar 

  104. Shaw, A. S. Lipid rafts: now you see them, now you don't. Nature Immunol. 7, 1139–1142 (2006).

    Article  CAS  Google Scholar 

  105. Kenworthy, A. K. Have we become overly reliant on lipid rafts? Talking point on the involvement of lipid rafts in T-cell activation. EMBO Rep. 9, 531–535 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Gaus, K., Zech, T. & Harder, T. Visualizing membrane microdomains by Laurdan 2-photon microscopy. Mol. Membr. Biol. 23, 41–48 (2006).

    Article  CAS  PubMed  Google Scholar 

  107. Rentero, C. et al. Functional implications of plasma membrane condensation for T cell activation. PLoS ONE 3, e2262 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Owen, D. M. et al. High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells. Mol. Membr. Biol. 27, 178–189 (2010).

    Article  CAS  PubMed  Google Scholar 

  109. Wilson, B. S., Pfeiffer, J. R., Surviladze, Z., Gaudet, E. A. & Oliver, J. M. High resolution mapping of mast cell membranes reveals primary and secondary domains of FcɛRI and LAT. J. Cell Biol. 154, 645–658 (2001). An important mapping of the distribution of receptors and downstream signalling molecules at the plasma membrane of mast cells using transmission electron microscopy, revealing their organization into segregated signalling domains on receptor activation.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Wilson, B. S., Pfeiffer, J. R. & Oliver, J. M. Observing FcɛRI signaling from the inside of the mast cell membrane. J. Cell Biol. 149, 1131–1142 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Lillemeier, B. F., Pfeiffer, J. R., Surviladze, Z., Wilson, B. S. & Davis, M. M. Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton. Proc. Natl Acad. Sci. USA 103, 18992–18997 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Sohn, H. W., Tolar, P. & Pierce, S. K. Membrane heterogeneities in the formation of B cell receptor–Lyn kinase microclusters and the immune synapse. J. Cell Biol. 182, 367–379 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Glebov, O. O. & Nichols, B. J. Lipid raft proteins have a random distribution during localized activation of the T-cell receptor. Nature Cell Biol. 6, 238–243 (2004).

    Article  CAS  PubMed  Google Scholar 

  114. Douglass, A. D. & Vale, R. D. Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell 121, 937–950 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Tanimura, N. et al. Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation. J. Cell Biol. 160, 125–135 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Tolentino, T. P. et al. Measuring diffusion and binding kinetics by contact area FRAP. Biophys. J. 95, 920–930 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Andrews, N. L. et al. Small, mobile FcɛRI receptor aggregates are signaling competent. Immunity 31, 469–479 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    Article  CAS  PubMed  Google Scholar 

  119. Lillemeier, B. F. et al. TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation. Nature Immunol. 11, 90–96 (2010). The introduction of the super-resolution imaging technique PALM to study the distribution of TCR and LAT in live cells, confirming previous indications from electron microscopy on their distribution at the plasma membrane in small sub-diffraction domains that coalesce on cell activation.

    Article  CAS  Google Scholar 

  120. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  CAS  PubMed  Google Scholar 

  121. Ragan, T. et al. High-resolution whole organ imaging using two-photon tissue cytometry. J. Biomed. Opt. 12, 014015 (2007).

    Article  PubMed  Google Scholar 

  122. Dustin, M. L. Supported bilayers at the vanguard of immune cell activation studies. J. Struct. Biol. 168, 152–160 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Ahmed, F., Friend, S., George, T. C., Barteneva, N. & Lieberman, J. Numbers matter: quantitative and dynamic analysis of the formation of an immunological synapse using imaging flow cytometry. J. Immunol. Methods 347, 79–86 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. George, T. C. et al. Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow. J. Immunol. Methods 311, 117–129 (2006).

    Article  CAS  PubMed  Google Scholar 

  125. Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nature Methods 2, 905–909 (2005).

    Article  CAS  PubMed  Google Scholar 

  126. Fernandez-Suarez, M. & Ting, A. Y. Fluorescent probes for super-resolution imaging in living cells. Nature Rev. Mol. Cell Biol. 9, 929–943 (2008).

    Article  CAS  Google Scholar 

  127. Chudakov, D. M., Lukyanov, S. & Lukyanov, K. A. Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 23, 605–613 (2005).

    Article  CAS  PubMed  Google Scholar 

  128. Muik, M. et al. Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation. J. Biol. Chem. 283, 8014–8022 (2008).

    Article  CAS  PubMed  Google Scholar 

  129. Calloway, N. Vig, M., Kinet, J.P., Holowka, D. & Baird, B. Molecular clustering of STIM1 with Orai1/CRACM1 at the plasma membrane depends dynamically on depletion of Ca2+ stores and on electrostatic interactions. Mol. Biol. Cell 20, 389–399 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank R. Kortum for critically reading the manuscript. This research was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute (NCI), Center for Cancer Research (CCR).

Author information

Author notes

  1. Lakshmi Balagopalan and Eilon Sherman: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Lakshmi Balagopalan, Eilon Sherman, Valarie A. Barr & Lawrence E. Samelson

Authors
  1. Lakshmi Balagopalan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eilon Sherman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Valarie A. Barr
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lawrence E. Samelson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lawrence E. Samelson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information S1 (Box)

FRET measurements in cells (PDF 151 kb)

Glossary

Diffraction limit of light

This refers to the physical impossibility of focusing light that is emitted from a point source into a single point owing to diffraction, which limits optical resolution to a distance of about half of the light wavelength (∼200 nm for green light).

Confocal microscopy

A technique in which light that is emitted by fluorescent targets is passed through a pinhole, thus removing out-of-focus light and allowing accurate volume observation by the sequential acquisition of x–y images along the z axis.

Two-photon laser scanning microscopy

A technique in which an image is formed by scanning a sample with a high-power pulsed laser. A spot of excitation is produced where the combined energy from the simultaneous absorption of two low-energy photons is sufficient to excite a fluorophore.

Transmission light microscopy

A technique that uses light to enlarge and image objects by passing the light through a set of lenses and subsequently detecting it by eye or with a detector.

Differential interference contrast microscopy

A phase-imaging technique that produces contrast from differences in refractive indices at various parts of the sample.

Epifluorescence microscopy

A technique that captures the fluorescence coming from the entire emitting volume of the sample.

Transmission electron microscopy

A technique that produces an image from a beam of electrons that are transmitted through a thin specimen containing electron-dense material to create an image with a very high resolution of several Angstroms.

Scanning electron microscopy

A technique that images the surface of a solid sample with high-energy electrons and detects features on its surface with a resolution of several nanometres.

Deconvolution

A computational image restoration technique that removes the out-of-focus blur that is typical of epifluorescence images and improves both lateral and axial resolution.

Optical trapping

A technique that uses a focused laser beam to exert small mechanical forces to trap cells or other microscopic objects in suspension, thus restricting or directing their motion and orientation and allowing their subsequent study by light microscopy.

Total internal reflection fluorescence microscopy

A technique that uses an evanescent wave, which is generated when the excitation beam is completely reflected from the coverslip, to excite fluorescent molecules in a thin layer within about one hundred nanometres of the coverslip.

Interference reflection microscopy

A technique that uses the interference of reflected rays of light to produce an image that contains only the regions of close contact between the cell and the contact surface (0–200 nm).

Mechanical trapping

The use of nanometre-scale structures built into lipid bilayers that act as barriers and inhibit the movement of T cell receptor microclusters.

Lipid raft

An ordered sphingolipid- and cholesterol-rich membrane domain. These domains are thought to reside within the more diffusive and unordered pool of lipids of the plasma membrane.

Fluorescence recovery after photobleaching

A technique that involves photobleaching fluorescent molecules in a region of a cell and then measuring the recovery of fluorescence that is due to the repopulation of the bleached area by diffusion of unbleached molecules.

Anisotropy

A method that measures the loss of correlation in polarization between the polarized excitation light and the light emitted from a rotating probe; this can be used to indicate changes in rotation speed caused by binding of the labelled molecule.

Plasma membrane sheets

The part of the plasma membrane of an adherent cell that remains on the adhering surface after the rest of the cell is removed during preparation for subsequent electron microscopy imaging.

Photoactivatable fluorophores

Fluorophores (fluorescent proteins or synthetic fluorophores) that change their spectral properties on the absorption of light, providing a unique method for the optical labelling and tracking of molecules.

Fluorescence cross-correlation spectroscopy

A spectroscopy method that correlates the fluctuations in intensity of two types of probes that diffuse through a small illumination volume, thus reporting on their binding.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Balagopalan, L., Sherman, E., Barr, V. et al. Imaging techniques for assaying lymphocyte activation in action. Nat Rev Immunol 11, 21–33 (2011). https://doi.org/10.1038/nri2903

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nri2903

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing