FLIM and emission spectral analysis of caspase-3 activation inside single living cell during anticancer drug-induced cell death |
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Authors: | Wenliang Pan Junle Qu Tongsheng Chen Lei Sun Jing Qi |
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Institution: | (1) MOE Key Libratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, 510631 Guangzhou, China;(2) Institute of Optoelectronics, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education, Shenzhen University, 518060 Shenzhen, Guangdong Province, China |
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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.
W. Pan and J. Qu contributed equally to this study. |
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Keywords: | FLIM Emission spectra FRET Caspase-3 Anticancer drug Programmed cell death (PCD) |
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