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High- and Super-Resolution Imaging of Cell-Cell Interfaces

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The Immune Synapse

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2654))

Abstract

Physical interfaces mediate interactions between multiple types of cells. Despite the importance of such interfaces to the cells’ function, their high-resolution optical imaging has been typically limited due to poor alignment of the interfaces relative to the optical plane of imaging. Here, we present a simple and robust method to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips and bringing them into contact in a controlled and stable fashion. We demonstrate aberration-free high-resolution imaging of interfaces between live T cells and antigen-presenting cells, known as immune synapses, as an outstanding example. Imaging methods may include multiple diffraction-limited and super-resolution microscopy techniques (e.g., bright-field, confocal, STED, and dSTORM). Thus, our simple and widely compatible approach allows imaging with high- and super-resolution the intricate structure and molecular organization within a variety of cell-cell interfaces.

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References

  1. Rusu AD, Georgiou M (2020) The multifarious regulation of the apical junctional complex. https://doi.org/10.1098/rsob.190278

  2. Cavey M, Lecuit T (2009) Molecular bases of cell-cell junctions stability and dynamics. Cold Spring Harb Perspect Biol 1:a002998. 10.1101/cshperspect.a002998

    Google Scholar 

  3. Nicholls JG (2012) From neuron to brain. Sinauer Associates: Oxford University Press

    Google Scholar 

  4. Helfrich MH, Horton MA (2006) Integrins and other adhesion molecules. Dyn Bone Cartil Metab:129–151. https://doi.org/10.1016/B978-012088562-6/50009-1

  5. Minetti G, Campbell R, Barshtein G, Pretini V, Koenen MH, Kaestner L, A M Fens MH, Schiffelers RM, Bartels M, Van Wijk R (2019) Red blood cells: chasing interactions. Front Physiol 10:945. https://doi.org/10.3389/fphys.2019.00945

  6. Trautmann A, Valitutti S (2003) The diversity of immunological synapses. Curr Opin Immunol 15:249–254. https://doi.org/10.1016/S0952-7915(03)00040-2

    Article  CAS  PubMed  Google Scholar 

  7. Garnett JA, Matthews S (2012) Interactions in bacterial biofilm development: A structural perspective, Current Protein & Peptide Science 13. https://dx.doi.org/10.2174/138920312804871166

    Google Scholar 

  8. Abisado RG, Benomar S, Klaus JR, Dandekar AA, Chandler JR, Garsin DA (2018) Bacterial quorum sensing and microbial community interactions. mBio 9:e02331-17 https://doi.org/10.1128/mBio.02331-17

  9. Choquet D, Triller A (2013) The dynamic synapse. Neuron 80:691–703. https://doi.org/10.1016/J.NEURON.2013.10.013

    Article  CAS  PubMed  Google Scholar 

  10. Balagopalan L, Sherman E, Barr VA, Samelson LE (2011) Imaging techniques for assaying lymphocyte activation in action. Nat Rev Immunol 11:21. https://doi.org/10.1038/nri2903

  11. Actor JK (2012) T-cell immunity. Elsevier’s Integr Rev Immunol Microbiol 25–32. https://doi.org/10.1016/B978-0-323-07447-6.00004-1

  12. Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S (2008) Functions of natural killer cells. Nat Immunol 9:503–510. https://doi.org/10.1038/ni1582

  13. Smith-Garvin JE, Koretzky GA, Jordan MS (2009) T cell activation. Annu Rev Immunol 27:591. https://doi.org/10.1146/annurev.immunol.021908.132706

  14. Green MV, Pengo T, Raybuck JD, Naqvi T, McMullan HM, Hawkinson JE, Marron Fernandez de Velasco E, Muntean BS, Martemyanov KA, Satterfield R, Young SM, Thayer SA (2019) Automated live-cell imaging of synapses in rat and human neuronal cultures. Front Cell Neurosci 13:1–14. https://doi.org/10.3389/fncel.2019.00467

    Article  CAS  Google Scholar 

  15. Okabe S (2012) Fluorescence imaging of synapse formation and remodeling. Microscopy (Oxf) 62:51–62. https://doi.org/10.1093/jmicro/dfs083

  16. Ng MR, Besser A, Brugge JS, Danuser G (2014) Mapping the dynamics of force transduction at cell–cell junctions of epithelial clusters. elife 3:e03282. https://doi.org/10.7554/eLife.03282

    Article  PubMed  PubMed Central  Google Scholar 

  17. Hell SW (2007) Far-field optical nanoscopy. Science 316:5828

    Google Scholar 

  18. Stender AS, Marchuk K, Liu C, Sander S, Meyer MW, Smith EA, Neupane B, Wang G, Li J, Cheng J-X, Huang B, Fang N (2013) Single cell optical imaging and spectroscopy. Chem Rev. 113:2469–2527. https://doi.org/10.1021/cr300336e

  19. Ettinger A, Wittmann T (2014) Fluorescence live cell imaging. In: Methods in cell biology, pp 77–94

    Google Scholar 

  20. Razvag Y, Neve-Oz Y, Sajman J, Reches M, Sherman E (2018) Nanoscale kinetic segregation of TCR and CD45 in engaged microvilli facilitates early T cell activation. Nat Commun 9:732. https://doi.org/10.1038/s41467-018-03127-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Nasse MJ, Woehl JC (2010) Realistic modeling of the illumination point spread function in confocal scanning optical microscopy. J Opt Soc Am A 27:295. https://doi.org/10.1364/JOSAA.27.000295

    Article  Google Scholar 

  22. Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML (1999) The immunological synapse: a molecular machine controlling T cell activation. Science 285:221–227. https://doi.org/10.1126/science.285.5425.2

  23. Bunnell SC, Barr VA, Fuller CL, Samelson LE (2003) High-resolution multicolor imaging of dynamic signaling complexes in T cells stimulated by planar substrates. Sci STKE 2003:pl8. https://doi.org/10.1126/stke.2003.177.pl8

    Article  PubMed  Google Scholar 

  24. Sajman J, Razvag Y, Schidorsky S, Kinrot S, Hermon K, Yakovian O, Sherman E (2021) Adhering interacting cells to two opposing coverslips allows super-resolution imaging of cell-cell interfaces. Commun Biol 4:439. https://doi.org/10.1038/S42003-021-01960-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Willig KI, Harke B, Medda R, Hell SW (2007) STED microscopy with continuous wave beams. Nat Methods 4:915-8. https://doi.org/10.1038/NMETH1108

  26. Khater IM, Nabi IR, Hamarneh G (2020) A review of super-resolution single-molecule localization microscopy cluster analysis and quantification methods. Patterns 1:100038. https://doi.org/10.1016/J.PATTER.2020.100038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Dertinger T, Colyer R, Iyer G, Weiss S, Enderlein J (2009) Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc Natl Acad Sci U S A 106:22287–22292. https://doi.org/10.1073/pnas.0907866106

    Article  PubMed  PubMed Central  Google Scholar 

  28. Culley S, Tosheva KL, Matos Pereira P, Henriques R (2018) SRRF: Universal live-cell super-resolution microscopy. Int J Biochem Cell Biol 101:74–79. https://doi.org/10.1016/J.BIOCEL.2018.05.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 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. https://doi.org/10.1038/nMeth.1768

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ovesný M, Křížek P, Borkovec J, Švindrych Z, Hagen GM (2014) ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30:2389–2390. https://doi.org/10.1093/bioinformatics/btu202

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Author information

Authors and Affiliations

  1. Racah Institute of Physics, The Hebrew University, Jerusalem, Israel

    Julia Sajman & Eilon Sherman

  2. Jerusalem College of technology, Jerusalem, Israel

    Julia Sajman

Authors
  1. Julia Sajman
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  2. Eilon Sherman
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Corresponding author

Correspondence to Eilon Sherman .

Editor information

Editors and Affiliations

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

    Cosima T. Baldari

  2. Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK

    Michael L. Dustin

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Sajman, J., Sherman, E. (2023). High- and Super-Resolution Imaging of Cell-Cell Interfaces. In: Baldari, C.T., Dustin, M.L. (eds) The Immune Synapse. Methods in Molecular Biology, vol 2654. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3135-5_10

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  • DOI: https://doi.org/10.1007/978-1-0716-3135-5_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3134-8

  • Online ISBN: 978-1-0716-3135-5

  • eBook Packages: Springer Protocols

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