Fluorescence Lifetime Imaging Microscope Consisting of a Compact Picosecond Dye Laser and a Gated Charge-Coupled Device Camera for Applications to Living Cells

Uchimura, Tomohiro; Kawanabe, Satoshi; Maeda, Yuki; Imasaka, Totaro

doi:10.2116/analsci.22.1291

Cited by 8 publications

(7 citation statements)

References 13 publications

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“…Fluorescence lifetime imaging microscopy (FLIM) can measure the local microenvironment of a cell [7][8][9][10][11], which makes it possible to evaluate the efficacy of anticancer drugs [12,13]. We have developed a time-domain FLIM system that consists of a picosecond dye laser, an inverted microscope, and a gated intensified charge-coupled device (ICCD) camera [14]. Using this system, FLIM images of apoptotic cells stained by SYTO 13 were measured, and the changes in fluorescence lifetime were reported after treatment with anthracycline antibiotic doxorubicin (DXR) [15,16].…”

Section: Introductionmentioning

confidence: 99%

Applying fluorescence lifetime imaging microscopy to evaluate the efficacy of anticancer drugs

Kawanabe

Araki

Uchimura

et al. 2015

Methods Appl. Fluoresc.

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Fluorescence lifetime imaging microscopy was applied to evaluate the efficacy of anticancer drugs. A decrease in the fluorescence lifetime of the nucleus in apoptotic cancer cells stained by SYTO 13 dye was detected after treatment with antitumor antibiotics such as doxorubicin or epirubicin. It was confirmed that the change in fluorescence lifetime occurred earlier than morphological changes in the cells. We found that the fluorescence lifetime of the nucleus in the cells treated with epirubicin decreased more rapidly than that of the cells treated with doxorubicin. This implies that epirubicin was more efficacious than doxorubicin in the treatment of cancer cells. The change in fluorescence lifetime was, however, not indicated when the cells were treated with cyclophosphamide. The decrease in fluorescence lifetime was associated with the processes involving caspase activation and chromatin condensation. Therefore, this technique would provide useful information about apoptotic cells, particularly in the early stages.

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Section: Introductionmentioning

confidence: 99%

Applying fluorescence lifetime imaging microscopy to evaluate the efficacy of anticancer drugs

Kawanabe

Araki

Uchimura

et al. 2015

Methods Appl. Fluoresc.

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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.…”

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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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confidence: 99%

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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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“…Consequently, this has limited the application of FLIM in live cell studies. However, improvements in microscope design and laser technology have reduced the time for data acquisition, and FLIM images can be acquired at up to 10 frames per second on optically sectioned images [26,27]. These advancements enable the application of high-speed FLIM to measure rapid dynamic processes in live cells and serial Z-stack images from 3D models, such as invasion.…”

Section: New Applications Of Fluorescent Reporter Technologymentioning

confidence: 99%

Profiling distinct mechanisms of tumour invasion for drug discovery: imaging adhesion, signalling and matrix turnover

Carragher

2008

Clin Exp Metastasis

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Recent advances in microscopic imaging technology, fluorescent reporter reagents, 3-dimensional (3D) cell models and multiparametric image analysis have enhanced our ability to model and understand complex cell physiology. Extension of these approaches to live cell, kinetic studies allows further spatial and temporal understanding of a multitude of dynamic functional events, including tumour cell invasion. Recent in vivo and 3D in vitro studies reveal how tumour cells utilize a diverse variety of mechanisms to permit invasion through 3D tissue environments. Such high degrees of diversity and plasticity between invasion mechanisms present a significant challenge to the successful treatment of malignant cancer. This review examines how advances in time-resolved imaging has contributed to the characterization of distinct modes of invasion and their associated molecular mechanisms. Specifically, we highlight the development of fluorescent reporter molecules and their incorporation into more predictive 3D in vitro and in vivo models, to enhance mechanistic analysis of tumour invasion. We also highlight the latest advances in kinetic imaging instrumentation applicable to in vitro and in vivo models of tumour invasion. We discuss how multiparametric image analysis can be used to interpret image data generated by these approaches. We further discuss how these approaches can be integrated into drug discovery pipelines to facilitate evaluation and selection of candidate drugs and novel pharmaceutical compositions, targeting multiple invasive mechanisms.

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Fluorescence Lifetime Imaging Microscope Consisting of a Compact Picosecond Dye Laser and a Gated Charge-Coupled Device Camera for Applications to Living Cells

Cited by 8 publications

References 13 publications

Applying fluorescence lifetime imaging microscopy to evaluate the efficacy of anticancer drugs

Applying fluorescence lifetime imaging microscopy to evaluate the efficacy of anticancer drugs

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

Profiling distinct mechanisms of tumour invasion for drug discovery: imaging adhesion, signalling and matrix turnover

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