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FLIM and emission spectral analysis of caspase-3 activation inside single living cell during anticancer drug-induced cell death

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Abstract

Two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM) and emission spectral imaging (ESI) are powerful tools for fluorescence resonance energy transfer (FRET) measurement. In this study, we use these two techniques to analyze caspase-3 activation inside single living cells during anticancer drug-induced human lung adenocarcinoma (ASTC-a-1) cell death. TPE-ESI of SCAT3, a caspase-3 indicator based on FRET, was performed inside single living cell stably expressing SCAT3. The TPE-ESI measurement was performed using 780 nm excitation which was considered to selectively excite the donor ECFP of SCAT3 by measuring the emission ratio of 526 to 476 nm. The emission peak at 526 nm disappeared and that of 476 nm increased after STS or bufalin treatment, but taxol treatment did not induce a significant change for the SCAT3 emission spectra, indicating that caspase-3 was activated during STS- or bufalin-induced cell apoptosis, but was not involved in taxol-induced PCD. Fluorescence lifetime of ECFP inside living cells was acquired using FLIM. The lifetime of ECFP was the same as that of the control group after taxol treatment, but increased from 1.83 ± 0.02 to 2.05 ± 0.03 and 1.90 ± 0.03 ns, respectively after STS and bufalin treatment, which agree with the results obtained using TPE-ESI. Taken together, TPE-FLIM and ESI analysis were proved to be valuable approaches for monitoring caspase-3 activation inside single living cells.

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Abbreviations

ASTC-a-1 cell:

Human adenocarcinoma cell

ECFP:

Enhanced cyan fluorescent protein

EYFP:

Enhanced yellow fluorescent protein

ESI:

Emission spectral imaging

FLIM:

Fluorescence lifetime imaging microscopy

FRET:

Fluorescence resonance energy transfer

PCD:

Programmed cell death

STS:

Staurosporine

SCAT3:

A sensor for activated caspase-3 based on FRET

TPE:

Two-photon excitation

Venus:

Mutation of EYFP

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Acknowledgments

We wish to thank Prof. M. Miura for providing us with the SCAT3 plasmid and Prof. Charles and Andrew for providing us with Bax-ECFP and Bax-EYFP plasmid. This study was supported by National Natural Science Foundation of China (Grant No. 30670507 and 60627003) and the Natural Science Foundation of Guangdong Province (F051001).

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Authors and Affiliations

  1. MOE Key Libratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, 510631, Guangzhou, China

    Wenliang Pan, Tongsheng Chen & Lei Sun

  2. Institute of Optoelectronics, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education, Shenzhen University, 518060, Shenzhen, Guangdong Province, China

    Junle Qu & Jing Qi

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  1. Wenliang Pan
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  2. Junle Qu
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  3. Tongsheng Chen
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Correspondence to Tongsheng Chen.

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W. Pan and J. Qu contributed equally to this study.

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Pan, W., Qu, J., Chen, T. et al. FLIM and emission spectral analysis of caspase-3 activation inside single living cell during anticancer drug-induced cell death. Eur Biophys J 38, 447–456 (2009). https://doi.org/10.1007/s00249-008-0390-0

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