Real-time interactive two-photon photoconversion of recirculating lymphocytes for discontinuous cell tracking in live adult mice

Authors

  • Tatyana Chtanova,

    Corresponding author
    1. Immunological Diseases Division, Garvan Institute of Medical Research, Sydney, Australia
    2. St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia
    • Phone: +61 2 92958414 or +61 2 92958404, Fax: +61 2 92958404===

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    • These authors contributed equally to this work.

  • Henry R. Hampton,

    1. Immunological Diseases Division, Garvan Institute of Medical Research, Sydney, Australia
    2. St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia
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  • Louise A. Waterhouse,

    1. Immunological Diseases Division, Garvan Institute of Medical Research, Sydney, Australia
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  • Katherine Wood,

    1. Immunological Diseases Division, Garvan Institute of Medical Research, Sydney, Australia
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  • Michio Tomura,

    1. Center for Innovation in Immunoregulative Technology and Therapeutics, Kyoto University Graduate School of Medicine, Kyoto, Japan
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  • Yoshihiro Miwa,

    1. Department of Molecular Pharmacology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan
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  • Charles R. Mackay,

    1. Immunology Department, Monash University, Melbourne, Australia
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  • Robert Brink,

    1. Immunological Diseases Division, Garvan Institute of Medical Research, Sydney, Australia
    2. St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia
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  • Tri Giang Phan

    1. Immunological Diseases Division, Garvan Institute of Medical Research, Sydney, Australia
    2. St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia
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    • These authors contributed equally to this work.


Abstract

The potential usefulness of intravital two-photon microscopy for fate mapping is limited by its inability to track cells beyond the confines of the imaging volume. Therefore, we have developed and validated a novel method for in vivo photolabelling of spatially-restricted cells expressing the Kaede optical highlighter by two-photon excitation. This has allowed us to optically mark a cohort of follicular B cells and track their dissemination from the original imaging volume in the lymph node to the spleen and contralateral lymph node. We also present the first demonstration, to our knowledge, of in vivo photoconversion of a freely moving single cell in a live adult animal. This method of `discontinuous' cell tracking therefore significantly extends the fate mapping capabilities of two-photon microscopy to delineate the spatiotemporal dynamics of cellular processes that span multiple anatomical sites at the single cell level. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

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