Use of a fluorescence lifetime imaging microscope in an apoptosis assay of Ewing’s sarcoma cells with a vital fluorescent probe

Li, Xu; Uchimura, Tomohiro; Kawanabe, Satoshi; Imasaka, Totaro

doi:10.1016/j.ab.2007.04.032

Cited by 9 publications

(9 citation statements)

References 19 publications

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“…Most of the organic dyes show a marked decrease in fluorescence intensity and fluorescence lifetime due to self-quenching via a variety of mechanisms, such as energy transfers discussed in Section 4.2. The decrease in fluorescence lifetime of a DNA marker SYTO 13 from 3.8 ns to ∼ 1.7 ns in tumor cells after treatment with doxorubicin has been attributed to the self-quenching of the fluorophore arising from the collapse of DNA and decrease in inter-dye distance 375. A similar concept, but with opposite trend, has been utilized in a lifetime pH sensitive nanoconstruct composed of a fluorescent NIR dye cypate aggregated on the surface of an acid degradable polymer 376.…”

Section: Exogenous Fluorescent Molecular Probes and Their Lifetimesmentioning

confidence: 99%

Fluorescence Lifetime Measurements and Biological Imaging

2010

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Section: Exogenous Fluorescent Molecular Probes and Their Lifetimesmentioning

confidence: 99%

Fluorescence Lifetime Measurements and Biological Imaging

2010

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“…65 The fluorescence lifetime of SYTO13, which is a DNA-staining dye in human Ewing's family tumor cells, was reduced as the apoptotic process was induced by doxorubicin, and the apoptosis-induced reduction of the fluorescence lifetime was explained in terms of the self-quenching of SYTO13 in DNA, which arises from a change in distance between SYTO13 dyes during apoptosis-induced DNA condensation. 66 …”

Section: ·7 Sensing Of Other Environementsmentioning

confidence: 99%

Sensing of Intracellular Environments by Fluorescence Lifetime Imaging of Exogenous Fluorophores

Nakabayashi

Ohta

2015

ANAL. SCI.

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Fluorescence lifetime imaging (FLIM) has been recognized as a powerful microscopy technique to examine environments in living systems. The fluorescence lifetime does not depend on the photobleaching and optical conditions, which allows us to obtain quantitative information on intracellular environments by analyzing the fluorescence lifetime. A variety of exogenous fluorophores have been applied in FLIM measurements to examine cellular processes. Information on the correlation between the fluorescence lifetime and the physiological parameters is essential to elucidate the cellular environments from the fluorescence lifetime measurements of exogenous fluorophores. In this review, exogenous fluorophores used for lifetime-based sensing are summarized, with the expectation that it becomes a basis for selecting the fluorophore used to investigate the intracellular environment with FLIM. Experimental results of the intracellular sensing of pH, metal ions, oxygen, viscosity, and other physiological parameters on the basis of the FLIM measurements are described along with a brief explanation of the mechanism of the change in the fluorescence lifetime.

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“…The FLIM system has been reported in detail elsewhere 3,4) and is described only briefly here. The picosecond dye laser (Twinstars Mini, Ishikawa Iron Works, Japan) pumped by the third harmonic emission of a Nd:YAG laser (Continuum, Minilite I) was used to excite the cells on the inverted microscope (Nikon, TE-2000U).…”

Section: Flim Measurementsmentioning

confidence: 99%

“…3,4) In this study, the FLIM system was applied to the monitoring of an androgen receptor (AR) with tagging green fluorescent protein (GFP) in living cells. AR is one of the steroid hormone receptors present in the cytoplasm without ligands, and it is subsequently translocated into the nucleus after binding.…”

mentioning

confidence: 99%

“…1,2) We have recently developed a fluorescence lifetime imaging microscope that consists of a picosecond dye laser and a time-gated intensified charge-coupled device (ICCD) camera. 3,4) In this study, the FLIM system was applied to the monitoring of an androgen receptor (AR) with tagging green fluorescent protein (GFP) in living cells. AR is one of the steroid hormone receptors present in the cytoplasm without ligands, and it is subsequently translocated into the nucleus after binding.…”

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Fluorescence Lifetime Imaging Microscopy for the Monitoring of Green Fluorescent Protein-Tagged Androgen Receptors in Living Cells

Miyake

Uchimura

et al. 2013

CHEMICAL & PHARMACEUTICAL BULLETIN

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Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the interaction between androgen receptor (AR) tagging of a green fluorescent protein (GFP) and the ligands in living cells. The fluorescence lifetime of the AR-GFP without ligands was ca. 3.1 ns, which was reduced to ca. 2.5 ns after treatment with agonist 5α-dihydrotestosterone. On the other hand, the fluorescence lifetime of AR-GFP was not changed after treatment with antagonist hydroxyflutamide. The reaction kinetics was simulated in the present study, and the obtained results indicated the possibility of the presence of an intermediate complex during the reaction. FLIM can be used to record the ratio of the AR as it reacts with an agonist, and, therefore, it is useful for acquiring information concerning the interaction between AR and ligands in living cells.Key words androgen receptor; 5α-dihydrotestosterone; hydroxyflutamide; fluorescence lifetime imaging microscopy; green fluorescent protein Fluorescence intensity imaging microscopy has been widely used in cytology and histology. However, fluorescence intensity images cannot quantitatively be compared with each other, since fluorescence intensity is deduced as a relative value. On the other hand, fluorescence lifetime is measured as an absolute, and is influenced by the characteristics of a fluorophore's microenvironment, such as temperature, viscosity, and pH. Therefore, it is possible to use fluorescence lifetime imaging microscopy (FLIM) to evaluate biological phenomena in living cells.1,2) We have recently developed a fluorescence lifetime imaging microscope that consists of a picosecond dye laser and a time-gated intensified charge-coupled device (ICCD) camera. 3,4) In this study, the FLIM system was applied to the monitoring of an androgen receptor (AR) with tagging green fluorescent protein (GFP) in living cells. AR is one of the steroid hormone receptors present in the cytoplasm without ligands, and it is subsequently translocated into the nucleus after binding.5) Until now, the location of AR in living cells has been observed primarily through the use of fluorescence intensity images. [6][7][8] We confirmed the intracellular location of AR-GFP after treatment with 5α-dihydrotestosterone (DHT) and a hydroxyflutamide (OH-FLU) by using the FLIM system. Moreover, the results of the dose-dependent experiment were also simulated in the present study. ExperimentalCell Culture and Transfection of AR-GFP COS-7 cells were obtained from the Japanese Collection of Research Bioresources, and were maintained in Dulbecco's minimal essential medium (DMEM; Invitrogen, U.S.A.) supplemented with 10% fetal bovine serum (FBS; Gibco, U.S.A.) and AntibioticAntimycotic at 37°C and 5% CO 2 .The AR-GFP plasmids (pEGFP-N2) were kindly provided by H. Iwamoto (Kyushu University). The cells were cultured in 35 mm glass-bottom dishes (Mat-Tek) (5×10 4 cells/dish) on the day before transfection. The AR-GFP plasmids and transfection reagent (Superfect transfection reagent, Invitrogen) were added to 100 µL DMEM ...

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Use of a fluorescence lifetime imaging microscope in an apoptosis assay of Ewing’s sarcoma cells with a vital fluorescent probe

Cited by 9 publications

References 19 publications

Fluorescence Lifetime Measurements and Biological Imaging

Fluorescence Lifetime Measurements and Biological Imaging

Sensing of Intracellular Environments by Fluorescence Lifetime Imaging of Exogenous Fluorophores

Fluorescence Lifetime Imaging Microscopy for the Monitoring of Green Fluorescent Protein-Tagged Androgen Receptors in Living Cells

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