Investigating T Cell Receptor Signals In Situ by Two-Photon Microscopy of Thymocytes Expressing Genetic Reporters in Low-Density Chimeras

  • Marilaine Fournier
  • Mengqi Dong
  • Heather J. MelicharEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2111)


T cell development is a dynamic process accompanied by extensive thymocyte migration, cellular interactions, and T cell receptor (TCR) signaling. In particular, thymic selection processes that ensure a functional, self-tolerant repertoire require TCR interactions with self-peptide presented by major histocompatibility complex molecules expressed by specialized thymic antigen-presenting cells. The quantity and quality of these TCR signals influence T cell fate. Two-photon microscopy, which enables live imaging of cells in intact tissue, has emerged as a powerful tool to gain insights into thymocyte migration and TCR signaling during T cell development in situ. Here we describe the generation of non-irradiated, low-density chimeric mice by neonatal injection of adult bone marrow engineered to express fluorescent TCR signaling reporters for imaging by two-photon microscopy. We also describe how the thymic lobes from chimeric mice are prepared for live imaging of thymocyte behavior and TCR signaling events. While we focus on imaging TCR signals associated with T cell development in the thymus, these techniques can also be adapted to study TCR signaling in mature T cells in peripheral lymphoid organs.

Key words

Neonatal chimera Thymus Thymocyte behavior Genetic reporters T cell receptor signaling Thymic slices Two-photon microscopy 



This work was supported by a Natural Sciences and Engineering Research Council grant (RGPIN-2019-05053) and Canadian Institutes of Health Research new investigator award (MSH-141967) to H.J.M., as well as a Fonds de recherche du Québec-Santé doctoral fellowship to M.D.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Marilaine Fournier
    • 1
  • Mengqi Dong
    • 1
    • 2
  • Heather J. Melichar
    • 1
    • 3
    Email author
  1. 1.Immunology-Oncology UnitMaisonneuve-Rosemont Hospital Research CenterMontrealCanada
  2. 2.Département de Microbiologie, Infectiologie et ImmunologieUniversité de MontréalMontrealCanada
  3. 3.Département de MédecineUniversité de MontréalMontrealCanada

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