Lifetime imaging of FRET between red fluorescent proteins

Authors

  • Alexander L. Rusanov,

    1. Department of Chemistry, M. V. Lomonosov Moscow State University, Leninskie Gory, 1/3, Moscow, 119991 Russian Federation
    2. A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospekt, 33, Moscow, 119071 Russian Federation
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  • Tatiana V. Ivashina,

    1. Skryabin Institute of Biochemistry and Physiology, Russian Academy of Sciences, Pushchino, 142290 Russian Federation
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  • Leonid M. Vinokurov,

    1. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, 142290 Russian Federation
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  • Ilya I. Fiks,

    1. Institute of Applied Physics, Russian Academy of Sciences, 46 Ulyanov Str., Nizhny Novgorod, 603950 Russian Federation
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  • Anna G. Orlova,

    1. Institute of Applied Physics, Russian Academy of Sciences, 46 Ulyanov Str., Nizhny Novgorod, 603950 Russian Federation
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  • Ilya V. Turchin,

    1. Institute of Applied Physics, Russian Academy of Sciences, 46 Ulyanov Str., Nizhny Novgorod, 603950 Russian Federation
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  • Irina G. Meerovich,

    1. A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospekt, 33, Moscow, 119071 Russian Federation
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  • Victorya V. Zherdeva,

    1. A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospekt, 33, Moscow, 119071 Russian Federation
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  • Alexander P. Savitsky

    Corresponding author
    1. Department of Chemistry, M. V. Lomonosov Moscow State University, Leninskie Gory, 1/3, Moscow, 119991 Russian Federation
    2. A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospekt, 33, Moscow, 119071 Russian Federation
    • Phone: 7(495)9546512, Fax: 7(495)9528725
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Abstract

Numerous processes in cells can be traced by using fluorescence resonance energy transfer (FRET) between two fluorescent proteins. The novel FRET pair including the red fluorescent protein TagRFP and kindling fluorescent protein KFP for sensing caspase-3 activity is developed. The lifetime mode of FRET measurements with a nonfluorescent protein KFP as an acceptor is used to minimize crosstalk due to its direct excitation. The red fluorescence is characterized by a better penetrability through the tissues and minimizes the cell autofluorescence signal. The effective transfection and expression of the FRET sensor in eukaryotic cells is shown by FLIM. The induction of apoptosis by camptothecine increases the fluorescence lifetime, which means effective cleavage of the FRET sensor by caspase-3. The instruments for detecting whole-body fluorescent lifetime imaging are described. Experiments on animals show distinct fluorescence lifetimes for the red fluorescent proteins possessing similar spectral properties. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

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