Fluorescence resonance energy transfer imaging of cell signaling from in vitro to in vivo: Basis of biosensor construction, live imaging, and image processing

Authors

  • Kazuhiro Aoki,

    Corresponding author
    1. PRESTO, Japan Science and Technology Agency (JST), Saitama, Japan
    • Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
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  • Yuji Kamioka,

    1. Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan
    2. Innovation Techno-Hub for Integrated Medical Bio-Imaging, Kyoto University, Kyoto, Japan
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  • Michiyuki Matsuda

    1. Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
    2. Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan
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Author to whom all correspondence should be addressed.

Email: k-aoki@lif.kyoto-u.ac.jp

Abstract

The progress in imaging technology with fluorescent proteins has uncovered a wide range of biological processes in developmental biology. In particular, genetically-encoded biosensors based on the principle of fluorescence resonance energy transfer (FRET) have been used to visualize spatial and temporal dynamics of intracellular signaling in living cells. However, development of sensitive FRET biosensors and their application to developmental biology remain challenging tasks, which has prevented their widespread use in developmental biology. In this review, we first overview general procedures and tips of imaging with FRET biosensors. We then describe recent advances in FRET imaging – namely, the use of optimized backbones for intramolecular FRET biosensors and transposon-mediated gene transfer to generate stable cell lines and transgenic mice expressing FRET biosensors. Finally, we discuss future perspectives of FRET imaging in developmental biology.

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