- Email: [email protected]
Use of a fluorescence lifetime imaging microscope in an apoptosis assay of Ewing’s sarcoma cells with a vital fluorescent probe
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 367 (2007) 219–224 www.elsevier.com/locate/yabio
Use of a fluorescence lifetime imaging microscope in an apoptosis assay of Ewing’s sarcoma cells with a vital fluorescent probe Xu Li b c
a,b
, Tomohiro Uchimura
a,c,* ,
Satoshi Kawanabe a, Totaro Imasaka
a,c
a Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Nishi-ku, Fukuoka 819-0395, Japan Department of Orthopaedic Surgery, First Affiliated Hospital, China Medical University, Heping District, Shenyang 110001, China Department of Translational Research Center, Center for Future Chemistry, Kyushu University, Nishi-ku, Fukuoka 819-0395, Japan
Received 14 February 2007 Available online 27 April 2007
Abstract A fluorescence lifetime imaging microscope (FLIM) was applied to study early-stage apoptotic cells stained with a SYTO13 dye. The fluorescence lifetime of SYTO13 in healthy cells was 3.8 ± 0.3 ns but was reduced to 2.4 ± 0.4 and 1.9 ± 0.2 ns after a 3-h period of incubation with SYTO13 when doxorubicin, a known inducer of apoptosis, was added to human Ewing’s family tumor cells at final concentrations of 250 and 500 nM, respectively, in a dose–dependent experiment. On the other hand, in a time-dependent experiment, the fluorescence lifetime decreased to 2.5 ± 0.5 and 1.7 ± 0.4 ns at a doxorubicin concentration of 750 nM after 2 and 4 h, respectively. A possible explanation for these results is self-quenching induced by a change in interprobe distance that arises from the condensation of DNA during apoptosis. In this study, the FLIM system was employed to investigate early-stage apoptosis that involves only small morphological changes, suggesting the potential advantage of this method for evaluating small biological effects in living cells. 2007 Elsevier Inc. All rights reserved. Keywords: Fluorescence lifetime imaging microscopy; Apoptosis; SYTO13; Doxorubicin
In fluorescence microscopy, the fluorescence lifetime and the fluorescence intensity both have been used for characterization of cells. This technique, referred to as fluorescence lifetime imaging microscopy (FLIM)1 [1–12], provides information concerning photophysical events that are difficult, or sometimes even impossible, to obtain using currently available technology for measuring fluorescence intensity. Two approaches, time domain and frequency domain techniques, typically are employed in FLIM. In our previous study, a time domain FLIM system with a time resolution of 340 ps was developed. The system consisted of a compact dye laser with a pulse width of less than *
Corresponding author. Fax: +81 92 802 2888. E-mail address: [email protected] (T. Uchimura). 1 Abbreviations used: FLIM, fluorescence lifetime imaging microscopy; ICCD, intensified charge-coupled device; FRET, fluorescence resonance energy transfer; DMSO, dimethyl sulfoxide; EFT, Ewing’s family tumor; FBS, fetal bovine serum; ANOVA, analysis of variance; FITC, fluorescein isothiocyanate. 0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.04.032
200 ps and an intensified charge-coupled device (ICCD) camera with a minimum gate width of 200 ps [13]. In contrast to a FLIM system with a laser beam scanner, the FLIM system with a gated ICCD camera for recording time-resolved fluorescence microscope images has a distinct advantage over other scanning methods with respect to the speed of data acquisition, an advantage that is useful for measurements of a large field of view (e.g., 1024 · 1024 pixels). Moreover, when a tunable dye laser is installed, the resulting FLIM system has the potential for use in applications that require the use of various types of labeling dyes with different spectroscopic properties for the evaluation of different functions of biological cells. In fact, such investigations are performed successfully using a dye laser (e.g., measurement of the spatial distribution [mapping] of Ca2+ concentrations in biological cells) as well as in studies of fluorescence resonance energy transfer (FRET) [4,5]. Thus, the developed FLIM system may be useful in quantitative analysis and can be used in practical applications
220
Use of a FLIM in an apoptosis assay / X. Li et al. / Anal. Biochem. 367 (2007) 219–224
such as in an apoptosis assay. Apoptosis is the programmed death of cells and is genetically controlled during the ablation process. In contrast to necrosis, apoptosis is recognized as a morphological change that involves the compaction of nuclear chromatin, the shrinkage of cytoplasm, and the production of membrane-bound apoptotic bodies. Apoptosis, when induced artificially, can be recognized by genomic fragmentation and the cleavage or degradation of various cellular proteins. Because anticancer drug candidates that fail to induce apoptosis are likely to have a decreased clinical efficacy [14], the development of an apoptosis assay is very important in high-throughput drug screening in research dealing with cancer therapy. Several nucleic acid dyes have been developed to differentiate earlyand late-stage apoptotic cells from healthy cells [15]. Among various types of probes, SYTO13 has been reported to be a useful vital fluorescence probe [6]. In this study, we cultured Ewing’s sarcoma cells and induced apoptosis using the drug doxorubicin. Using the FLIM system developed in our laboratory, consisting of a picosecond dye laser and an ICCD camera, a fluorescence lifetime image of the cell during the process of apoptosis could be obtained using SYTO13 as a vital fluorescence probe. Dose- and time-dependent assays of the cells affected by the drug were examined, and the merits of the current analytical system are discussed. Materials and methods Reagent Doxorubicin was purchased from Sigma (USA). A 10-mM stock solution was prepared using distilled water and was diluted to working concentrations immediately prior to use. A fluorescent dye, SYTO13 dissolved in dimethyl sulfoxide (DMSO, 5 mM), was obtained from Molecular Probes/Invitrogen (USA). Cell culture Human Ewing’s family tumor (EFT) cell lines, SK-NMC and RD-ES, were obtained from the American Type Culture Collection (USA). A human EFT cell line, WE-68, was kindly provided by F. van Valen (University of Muenster, Germany). The RD-ES and WE-68 cell lines were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA), and the SK-N-MC cell line was cultured in DMEM supplemented with 10% FBS at 37 C and 5% CO2.
addition of SYTO13 dissolved in DMSO (final concentration of 1 lM) to the cells in the medium. After 30 min of incubation, SYTO13 was washed away with probe-free medium and a series of time-resolved fluorescence intensity images were obtained, as described in the following section. Labeling with SYTO13 and irradiation of the laser beam did not induce any obvious morphological changes in the cell. FLIM measurements Fig. 1 shows a block diagram of the FLIM system used in this study. The experimental apparatus has been reported in detail elsewhere [13] and is described only briefly here. A compact picosecond dye laser (TwinstarsMini, <200 ps, Ishikawa Iron Works, Japan) pumped by the third harmonic emission of a Nd:YAG laser (Minilite I, 5 ns, 355 nm, 3.5 mJ, 10 Hz, Continuum, USA) was slightly modified to permit the laser to oscillate in a broadband mode [13]. A small fraction of the picosecond dye laser beam is reflected by a quartz plate and is detected by means of a PIN photodiode. The output is used as a trigger signal for a gated ICCD camera (C7300-10-12, minimum gate width 200 ps, Hamamatsu, Japan). The main fraction of the dye laser beam is introduced into an optical fiber (15 m, 200 lm, NA 0.2) to delay the laser pulse to be synchronized with the gate signal of the ICCD camera. The laser pulse from the optical fiber is introduced into an inverted microscope (TE-2000U, Nikon, Japan) to irradiate a sample through an objective lens (20·, NA 0.45, Nikon). The fluorescence emission is collected by the same objective lens. All fluorescence lifetime imaging experiments were performed at an excitation wavelength of 480 nm. The fluorescence was passed through a 505-nm primary beam splitter and then a 520-nm bandpass filter to reduce the scattered light. All of the time-resolved fluorescence intensity images reported here were recorded using the ICCD camera with a gate width of 200 ps. The image signal was accumulated 10 times on the CCD at a repetition rate of 10 Hz (time period of 1 s), and the delay time subsequently was changed by 200 ps to produce time-resolved
Induction of apoptosis and labeling procedures All EFT cell lines were seeded in a six-well plate (2 · 105 cells/well). After 24 h of incubation, the cells were treated with doxorubicin at concentrations of 250 to 2000 nM or with distilled water during the periods from 1 to 6 h. Healthy and apoptotic cells were labeled by the
Fig. 1. Experimental apparatus of the FLIM system.
Use of a FLIM in an apoptosis assay / X. Li et al. / Anal. Biochem. 367 (2007) 219–224
221
Fig. 2. Evaluation of apoptotic Ewing’s sarcoma cells by FLIM in a dose–dependent experiment. (A) Intensity image. (B) Lifetime image. (C) Statistical significance (results of one-factor ANOVA test). Data for WE-68 cells after 3 h of incubation are shown.
fluorescence images (typically six images) for the construction of a single FLIM picture. The fluorescence decay curve was assumed to be composed of a single component, and calculated lifetime data were used for the construction of a fluorescence lifetime image using a homemade program written by the LabVIEW (National Instruments, USA) software program. All of the calculated lifetime data were selected from the nucleus area according to the phase image. The lifetime data for five segments in which the fluorescence intensity was larger than 100 were averaged, and this value was used as the lifetime value for the construction of a cell image. The
means of lifetime values of at least 10 cells were recorded as the lifetime of one condition. The experiment was repeated at least three times, and the runs were consistent with each other. Statistical analysis Statistical analyses were performed based on a one-factor analysis of variance (ANOVA) test. A Fisher’s protected least significant difference test, where the parameter P larger than 0.05 (P < 0.05) suggested statistical significance, was used.
222
Use of a FLIM in an apoptosis assay / X. Li et al. / Anal. Biochem. 367 (2007) 219–224
Fig. 3. Evaluation of apoptotic Ewing’s sarcoma cells by FLIM in a time-dependent experiment. (A) Intensity image. (B) Lifetime image. (C) Statistical significance (results of one-factor ANOVA test). Data obtained for RD-ES cells at a doxorubicin concentration of 750 nM are shown.
Results and discussion Fig. 2 shows the fluorescence lifetime images obtained for apoptotic cells in the dose–dependent experiment. For healthy cells (doxorubicin, 0 nM), the fluorescence lifetime of SYTO13 was determined to be 3.8 ± 0.3 ns, as shown in Fig. 2B, in good agreement with previous values reported in the literature [6]. On the progression of apoptosis (3 h), the fluorescence lifetime of SYTO13, which is attached to the nucleus, decreases significantly and is indicative of changes in the molecular environment of the dye.
When doxorubicin was added to WE-68 cells at a final concentration of 250 nM, the fluorescence lifetime of SYTO13 decreased to 2.4 ± 0.4 ns (P < 0.001), as shown in Fig. 2B. When doxorubicin was added at a final concentration of 500 nM, the lifetime of SYTO13 decreased to 1.9 ± 0.2 ns, as shown in Fig. 2B, with a statistical significance of P < 0.001, as shown in Fig. 2C. When doxorubicin was added at a final concentration of 1000 nM, the morphology of more than 50% of the cells changed drastically and no longer could be properly labeled with the dye. Even being stained, the lifetime of SYTO13 in the apoptotic
Use of a FLIM in an apoptosis assay / X. Li et al. / Anal. Biochem. 367 (2007) 219–224
group was not significantly different from that in the nontreated group (data not shown). In the time-dependent dose experiment, the fluorescence lifetime of SYTO13 decreased from 3.7 ± 0.5 to 2.5 ± 0.5 ns (P < 0.001) 2 h after the addition of 750 nM doxorubicin to RD-ES cells, as shown in Fig. 3C. After 4 h, the fluorescence lifetime of SYTO13 further decreased to 1.7 ± 0.4 ns (P < 0.001). This tendency was disrupted when the incubation period became longer than 6 h. The same phenomenon was observed in the other cell lines that were measured (data not shown). In previous reports, some DNA probes used in investigations of early-stage apoptotic cells showed changes in fluorescence lifetime on apoptosis [16–18]. Among these vital fluorescence probes, SYTO13 is more useful because it provides a shorter fluorescence lifetime on the progression of apoptosis. This is considered to arise from strong self-quenching of the fluorescence that is caused by the change in interprobe distance as a result of the condensation of DNA [6]. In the current study, an increase in the concentration or incubation time of the drug apparently decreases the fluorescence lifetime of SYTO13 in both dose- and time-dependent experiments. When apoptosis is advanced sufficiently, however, this tendency disappears. This may be due to disruption of the nuclear membrane or to the loss of DNA. The results in this study suggest that the current analytical tool can be used as a useful means to differentiate between early-stage apoptosis and late-stage apoptosis. As reported previously, various types of fluorescent probes [19,20] have been employed to simultaneously image DNA and RNA in cells. Most of these probes, however, are toxic to cells (e.g., DRAQ5, the TOTO family, DAPI), require ultraviolet radiation for the excitation of dye molecules (e.g., the Hoechst family, DAPI), or can be applied only to dead cells (e.g., 7-AAD, propidium iodide, ethidium bromide). Some other approaches, such as the use of fluorescein isothiocyanate (FITC)-conjugated anexin V, could also differentiate between early-stage apoptosis and late-stage apoptosis [21]. The procedure is complicated, however, and the use is limited to the study of flow cytometry. In contrast, the current technique provides a noninvasive, simple, robust, and cost-effective procedure compared with other existing methods for apoptosis assay. Although further investigations (e.g., the use of other probes with different functions) will be necessary for a more conclusive discussion, a combination of the FLIM system and vital fluorescence lifetime probes provides a potentially useful analytical means for the real-time monitoring of apoptotic phenomena in a living cell. Conclusions A small-frame FLIM system was developed where image data (1024 · 1024 pixels) can be acquired at a repetition rate of 10 Hz for the construction of a FLIM image in a time period of less than 10 s. This analytical
223
instrument was used for observing apoptotic cells labeled with the vital fluorescence lifetime probe SYTO13. For early-stage apoptotic cells, the fluorescence lifetime of SYTO13 decreased significantly in dose–dependent and time-dependent experiments, a result that can be attributed to self-quenching arising from a change in interprobe distance due to the condensation of DNA. In this experiment, no morphological change occurred in the cell as a result of labeling with SYTO13 or irradiation by the laser beam. It was found that the early-stage apoptosis, which is recognized with difficulty by conventional methods, could be differentiated from the late-stage apoptosis using the current FLIM system. This analytical tool can be widely used to explain a variety of biological phenomena in living cells as well as to evaluate the clinical efficacy of apoptosis-inducing drugs. Acknowledgments This work was supported by a Grant-in-Aid for the 21st Century COE Program, ‘‘Functional Innovation of Molecular Informatics,’’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We express our gratitude to Hamamatsu Photonics, Japan, for providing us with an opportunity to use a gated ICCD camera. References [1] C.Y. Dong, T. French, P.T.C. So, C. Buehler, K.M. Berland, E. Gratton, Fluorescence-lifetime imaging techniques for microscopy, Methods Cell Biol. 72 (2003) 431–464. [2] K. Suhling, P.M.W. French, D. Phillips, Time-resolved fluorescence microscopy, Photochem. Photobiol. Sci. 4 (2005) 13–22. [3] H. Wallrabe, A. Periasamy, Imaging protein molecules using FRET and FLIM microscopy, Curr. Opin. Biotechnol. 16 (2005) 19–27. [4] A. Periasamy, P. Wodnicki, X.F. Wang, S. Kwon, G.W. Gordon, B. Herman, Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system, Rev. Sci. Instrum. 67 (1996) 3722–3731. [5] P. Urayama, W. Zhong, J.A. Beamish, F.K. Minn, R.D. Sloboda, K.H. Dragnev, E. Dmitrovsky, M.-A. Mycek, A UV–Visible–NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution, Appl. Phys. B 76 (2003) 483– 496. [6] M.A.M.J. van Zandvoort, C.J. de Grauw, H.C. Gerritsen, J.L.V. Broers, M.G.A. oude Egbrink, F.C.S. Ramaekers, D.W. Slaaf, Discrimination of DNA and RNA in cells by a vital fluorescent probe: Lifetime imaging of SYTO13 in healthy and apoptotic cells, Cytometry 47 (2002) 226–235. [7] K.P. Ghiggino, M.R. Harris, P.G. Spizzirri, Fluorescence lifetime measurements using a novel fiber-optic laser scanning confocal microscope, Rev. Sci. Instrum. 63 (1992) 2999–3002. [8] T. Oida, Y. Sako, A. Kusumi, Fluorescence lifetime imaging microscopy (flimscopy), Biophys. J. 64 (1993) 676–685. [9] D.K. Bird, K.W. Eliceiri, C.-H. Fan, J.G. White, Simultaneous twophoton spectral and lifetime fluorescence microscopy, Appl. Opt. 43 (2004) 5173–5182. [10] K. Dowling, M.J. Dayel, M.J. Lever, P.M.W. French, J.D. Hares, A.K.L. Dymoke-Bradshaw, Fluorescence lifetime imaging with picosecond resolution for biomedical applications, Opt. Lett. 23 (1998) 810–812.
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[11] J.-P. Knemeyer, D.-P. Herten, M. Sauer, Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy, Anal. Chem. 75 (2003) 2147–2153. [12] Q. Li, T. Ruckstuhl, S. Seeger, Deep-UV laser-based fluorescence lifetime imaging microscopy of single molecules, J. Phys. Chem. B 108 (2004) 8324–8329. [13] T. Uchimura, S. Kawanabe, Y. Maeda, T. Imasaka, Fluorescence lifetime imaging microscope consisting of a compact picosecond dye laser and a gated charge-coupled device camera for applications to living cells, Anal. Sci. 22 (2006) 1291–1295. [14] M. Mancini, M. Sedghinasab, K. Knowlton, A. Tam, D. Hockenbery, B.O. Anderson, Flow cytometric measurement of mitochondrial mass and function: A novel method for assessing chemoresistance, Ann. Surg. Oncol. 5 (1998) 287–295. [15] S.M. van den Eijnde, A.J.M. Luijsterburg, L. Boshart, C.I. De Zeeuw, J.H. van Dierendonck, C.P.M. Reutelingsperger, C. Vermeij-Keers, In situ detection of apoptosis during embryogenesis with annexin V: From whole mount to ultrastructure, Cytometry 29 (1997) 313–320.
[16] T. Frey, Nucleic acid dyes for detection of apoptosis in live cells, Cytometry 21 (1995) 265–274. [17] B.L. Sailer, J.A. Steinkamp, H.A. Crissman, Flow cytometric fluorescence lifetime analysis of DNA-binding probes, Eur. J. Histochem. 42 (1998) 19–27. [18] O. Kirschstein, M. Sip, L. Kittler, Quantitative and sequence-specific analysis of DNA–ligand interaction by means of fluorescent intercalator probes, J. Mol. Recogn. 13 (2000) 157–163. [19] R.P. Haugland, Handbook of Fluorescent Probes and Research Products, 9th ed., Molecular Probes, Eugene, OR, 2002. [20] P.J. Smith, M. Wiltshire, S. Davies, L.H. Patterson, T. Hoy, A novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry, J. Immunol. Methods 229 (1999) 131–139. [21] M. Eray, M. Ma¨tto¨, M. Kaartinen, L. Andersson, J. Pelkonen, Flow cytometric analysis of apoptotic subpopulations with a combination of annexin V–FITC, propidium iodide, and SYTO 17, Cytometry 43 (2001) 134–142.
Use of a fluorescence lifetime imaging microscope in an apoptosis assay of Ewing’s sarcoma cells with a vital fluorescent probe Xu Li b c
a,b
, Tomohiro Uchimura
a,c,* ,
Satoshi Kawanabe a, Totaro Imasaka
a,c
a Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Nishi-ku, Fukuoka 819-0395, Japan Department of Orthopaedic Surgery, First Affiliated Hospital, China Medical University, Heping District, Shenyang 110001, China Department of Translational Research Center, Center for Future Chemistry, Kyushu University, Nishi-ku, Fukuoka 819-0395, Japan
Received 14 February 2007 Available online 27 April 2007
Abstract A fluorescence lifetime imaging microscope (FLIM) was applied to study early-stage apoptotic cells stained with a SYTO13 dye. The fluorescence lifetime of SYTO13 in healthy cells was 3.8 ± 0.3 ns but was reduced to 2.4 ± 0.4 and 1.9 ± 0.2 ns after a 3-h period of incubation with SYTO13 when doxorubicin, a known inducer of apoptosis, was added to human Ewing’s family tumor cells at final concentrations of 250 and 500 nM, respectively, in a dose–dependent experiment. On the other hand, in a time-dependent experiment, the fluorescence lifetime decreased to 2.5 ± 0.5 and 1.7 ± 0.4 ns at a doxorubicin concentration of 750 nM after 2 and 4 h, respectively. A possible explanation for these results is self-quenching induced by a change in interprobe distance that arises from the condensation of DNA during apoptosis. In this study, the FLIM system was employed to investigate early-stage apoptosis that involves only small morphological changes, suggesting the potential advantage of this method for evaluating small biological effects in living cells. 2007 Elsevier Inc. All rights reserved. Keywords: Fluorescence lifetime imaging microscopy; Apoptosis; SYTO13; Doxorubicin
In fluorescence microscopy, the fluorescence lifetime and the fluorescence intensity both have been used for characterization of cells. This technique, referred to as fluorescence lifetime imaging microscopy (FLIM)1 [1–12], provides information concerning photophysical events that are difficult, or sometimes even impossible, to obtain using currently available technology for measuring fluorescence intensity. Two approaches, time domain and frequency domain techniques, typically are employed in FLIM. In our previous study, a time domain FLIM system with a time resolution of 340 ps was developed. The system consisted of a compact dye laser with a pulse width of less than *
Corresponding author. Fax: +81 92 802 2888. E-mail address: [email protected] (T. Uchimura). 1 Abbreviations used: FLIM, fluorescence lifetime imaging microscopy; ICCD, intensified charge-coupled device; FRET, fluorescence resonance energy transfer; DMSO, dimethyl sulfoxide; EFT, Ewing’s family tumor; FBS, fetal bovine serum; ANOVA, analysis of variance; FITC, fluorescein isothiocyanate. 0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.04.032
200 ps and an intensified charge-coupled device (ICCD) camera with a minimum gate width of 200 ps [13]. In contrast to a FLIM system with a laser beam scanner, the FLIM system with a gated ICCD camera for recording time-resolved fluorescence microscope images has a distinct advantage over other scanning methods with respect to the speed of data acquisition, an advantage that is useful for measurements of a large field of view (e.g., 1024 · 1024 pixels). Moreover, when a tunable dye laser is installed, the resulting FLIM system has the potential for use in applications that require the use of various types of labeling dyes with different spectroscopic properties for the evaluation of different functions of biological cells. In fact, such investigations are performed successfully using a dye laser (e.g., measurement of the spatial distribution [mapping] of Ca2+ concentrations in biological cells) as well as in studies of fluorescence resonance energy transfer (FRET) [4,5]. Thus, the developed FLIM system may be useful in quantitative analysis and can be used in practical applications
220
Use of a FLIM in an apoptosis assay / X. Li et al. / Anal. Biochem. 367 (2007) 219–224
such as in an apoptosis assay. Apoptosis is the programmed death of cells and is genetically controlled during the ablation process. In contrast to necrosis, apoptosis is recognized as a morphological change that involves the compaction of nuclear chromatin, the shrinkage of cytoplasm, and the production of membrane-bound apoptotic bodies. Apoptosis, when induced artificially, can be recognized by genomic fragmentation and the cleavage or degradation of various cellular proteins. Because anticancer drug candidates that fail to induce apoptosis are likely to have a decreased clinical efficacy [14], the development of an apoptosis assay is very important in high-throughput drug screening in research dealing with cancer therapy. Several nucleic acid dyes have been developed to differentiate earlyand late-stage apoptotic cells from healthy cells [15]. Among various types of probes, SYTO13 has been reported to be a useful vital fluorescence probe [6]. In this study, we cultured Ewing’s sarcoma cells and induced apoptosis using the drug doxorubicin. Using the FLIM system developed in our laboratory, consisting of a picosecond dye laser and an ICCD camera, a fluorescence lifetime image of the cell during the process of apoptosis could be obtained using SYTO13 as a vital fluorescence probe. Dose- and time-dependent assays of the cells affected by the drug were examined, and the merits of the current analytical system are discussed. Materials and methods Reagent Doxorubicin was purchased from Sigma (USA). A 10-mM stock solution was prepared using distilled water and was diluted to working concentrations immediately prior to use. A fluorescent dye, SYTO13 dissolved in dimethyl sulfoxide (DMSO, 5 mM), was obtained from Molecular Probes/Invitrogen (USA). Cell culture Human Ewing’s family tumor (EFT) cell lines, SK-NMC and RD-ES, were obtained from the American Type Culture Collection (USA). A human EFT cell line, WE-68, was kindly provided by F. van Valen (University of Muenster, Germany). The RD-ES and WE-68 cell lines were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA), and the SK-N-MC cell line was cultured in DMEM supplemented with 10% FBS at 37 C and 5% CO2.
addition of SYTO13 dissolved in DMSO (final concentration of 1 lM) to the cells in the medium. After 30 min of incubation, SYTO13 was washed away with probe-free medium and a series of time-resolved fluorescence intensity images were obtained, as described in the following section. Labeling with SYTO13 and irradiation of the laser beam did not induce any obvious morphological changes in the cell. FLIM measurements Fig. 1 shows a block diagram of the FLIM system used in this study. The experimental apparatus has been reported in detail elsewhere [13] and is described only briefly here. A compact picosecond dye laser (TwinstarsMini, <200 ps, Ishikawa Iron Works, Japan) pumped by the third harmonic emission of a Nd:YAG laser (Minilite I, 5 ns, 355 nm, 3.5 mJ, 10 Hz, Continuum, USA) was slightly modified to permit the laser to oscillate in a broadband mode [13]. A small fraction of the picosecond dye laser beam is reflected by a quartz plate and is detected by means of a PIN photodiode. The output is used as a trigger signal for a gated ICCD camera (C7300-10-12, minimum gate width 200 ps, Hamamatsu, Japan). The main fraction of the dye laser beam is introduced into an optical fiber (15 m, 200 lm, NA 0.2) to delay the laser pulse to be synchronized with the gate signal of the ICCD camera. The laser pulse from the optical fiber is introduced into an inverted microscope (TE-2000U, Nikon, Japan) to irradiate a sample through an objective lens (20·, NA 0.45, Nikon). The fluorescence emission is collected by the same objective lens. All fluorescence lifetime imaging experiments were performed at an excitation wavelength of 480 nm. The fluorescence was passed through a 505-nm primary beam splitter and then a 520-nm bandpass filter to reduce the scattered light. All of the time-resolved fluorescence intensity images reported here were recorded using the ICCD camera with a gate width of 200 ps. The image signal was accumulated 10 times on the CCD at a repetition rate of 10 Hz (time period of 1 s), and the delay time subsequently was changed by 200 ps to produce time-resolved
Induction of apoptosis and labeling procedures All EFT cell lines were seeded in a six-well plate (2 · 105 cells/well). After 24 h of incubation, the cells were treated with doxorubicin at concentrations of 250 to 2000 nM or with distilled water during the periods from 1 to 6 h. Healthy and apoptotic cells were labeled by the
Fig. 1. Experimental apparatus of the FLIM system.
Use of a FLIM in an apoptosis assay / X. Li et al. / Anal. Biochem. 367 (2007) 219–224
221
Fig. 2. Evaluation of apoptotic Ewing’s sarcoma cells by FLIM in a dose–dependent experiment. (A) Intensity image. (B) Lifetime image. (C) Statistical significance (results of one-factor ANOVA test). Data for WE-68 cells after 3 h of incubation are shown.
fluorescence images (typically six images) for the construction of a single FLIM picture. The fluorescence decay curve was assumed to be composed of a single component, and calculated lifetime data were used for the construction of a fluorescence lifetime image using a homemade program written by the LabVIEW (National Instruments, USA) software program. All of the calculated lifetime data were selected from the nucleus area according to the phase image. The lifetime data for five segments in which the fluorescence intensity was larger than 100 were averaged, and this value was used as the lifetime value for the construction of a cell image. The
means of lifetime values of at least 10 cells were recorded as the lifetime of one condition. The experiment was repeated at least three times, and the runs were consistent with each other. Statistical analysis Statistical analyses were performed based on a one-factor analysis of variance (ANOVA) test. A Fisher’s protected least significant difference test, where the parameter P larger than 0.05 (P < 0.05) suggested statistical significance, was used.
222
Use of a FLIM in an apoptosis assay / X. Li et al. / Anal. Biochem. 367 (2007) 219–224
Fig. 3. Evaluation of apoptotic Ewing’s sarcoma cells by FLIM in a time-dependent experiment. (A) Intensity image. (B) Lifetime image. (C) Statistical significance (results of one-factor ANOVA test). Data obtained for RD-ES cells at a doxorubicin concentration of 750 nM are shown.
Results and discussion Fig. 2 shows the fluorescence lifetime images obtained for apoptotic cells in the dose–dependent experiment. For healthy cells (doxorubicin, 0 nM), the fluorescence lifetime of SYTO13 was determined to be 3.8 ± 0.3 ns, as shown in Fig. 2B, in good agreement with previous values reported in the literature [6]. On the progression of apoptosis (3 h), the fluorescence lifetime of SYTO13, which is attached to the nucleus, decreases significantly and is indicative of changes in the molecular environment of the dye.
When doxorubicin was added to WE-68 cells at a final concentration of 250 nM, the fluorescence lifetime of SYTO13 decreased to 2.4 ± 0.4 ns (P < 0.001), as shown in Fig. 2B. When doxorubicin was added at a final concentration of 500 nM, the lifetime of SYTO13 decreased to 1.9 ± 0.2 ns, as shown in Fig. 2B, with a statistical significance of P < 0.001, as shown in Fig. 2C. When doxorubicin was added at a final concentration of 1000 nM, the morphology of more than 50% of the cells changed drastically and no longer could be properly labeled with the dye. Even being stained, the lifetime of SYTO13 in the apoptotic
Use of a FLIM in an apoptosis assay / X. Li et al. / Anal. Biochem. 367 (2007) 219–224
group was not significantly different from that in the nontreated group (data not shown). In the time-dependent dose experiment, the fluorescence lifetime of SYTO13 decreased from 3.7 ± 0.5 to 2.5 ± 0.5 ns (P < 0.001) 2 h after the addition of 750 nM doxorubicin to RD-ES cells, as shown in Fig. 3C. After 4 h, the fluorescence lifetime of SYTO13 further decreased to 1.7 ± 0.4 ns (P < 0.001). This tendency was disrupted when the incubation period became longer than 6 h. The same phenomenon was observed in the other cell lines that were measured (data not shown). In previous reports, some DNA probes used in investigations of early-stage apoptotic cells showed changes in fluorescence lifetime on apoptosis [16–18]. Among these vital fluorescence probes, SYTO13 is more useful because it provides a shorter fluorescence lifetime on the progression of apoptosis. This is considered to arise from strong self-quenching of the fluorescence that is caused by the change in interprobe distance as a result of the condensation of DNA [6]. In the current study, an increase in the concentration or incubation time of the drug apparently decreases the fluorescence lifetime of SYTO13 in both dose- and time-dependent experiments. When apoptosis is advanced sufficiently, however, this tendency disappears. This may be due to disruption of the nuclear membrane or to the loss of DNA. The results in this study suggest that the current analytical tool can be used as a useful means to differentiate between early-stage apoptosis and late-stage apoptosis. As reported previously, various types of fluorescent probes [19,20] have been employed to simultaneously image DNA and RNA in cells. Most of these probes, however, are toxic to cells (e.g., DRAQ5, the TOTO family, DAPI), require ultraviolet radiation for the excitation of dye molecules (e.g., the Hoechst family, DAPI), or can be applied only to dead cells (e.g., 7-AAD, propidium iodide, ethidium bromide). Some other approaches, such as the use of fluorescein isothiocyanate (FITC)-conjugated anexin V, could also differentiate between early-stage apoptosis and late-stage apoptosis [21]. The procedure is complicated, however, and the use is limited to the study of flow cytometry. In contrast, the current technique provides a noninvasive, simple, robust, and cost-effective procedure compared with other existing methods for apoptosis assay. Although further investigations (e.g., the use of other probes with different functions) will be necessary for a more conclusive discussion, a combination of the FLIM system and vital fluorescence lifetime probes provides a potentially useful analytical means for the real-time monitoring of apoptotic phenomena in a living cell. Conclusions A small-frame FLIM system was developed where image data (1024 · 1024 pixels) can be acquired at a repetition rate of 10 Hz for the construction of a FLIM image in a time period of less than 10 s. This analytical
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instrument was used for observing apoptotic cells labeled with the vital fluorescence lifetime probe SYTO13. For early-stage apoptotic cells, the fluorescence lifetime of SYTO13 decreased significantly in dose–dependent and time-dependent experiments, a result that can be attributed to self-quenching arising from a change in interprobe distance due to the condensation of DNA. In this experiment, no morphological change occurred in the cell as a result of labeling with SYTO13 or irradiation by the laser beam. It was found that the early-stage apoptosis, which is recognized with difficulty by conventional methods, could be differentiated from the late-stage apoptosis using the current FLIM system. This analytical tool can be widely used to explain a variety of biological phenomena in living cells as well as to evaluate the clinical efficacy of apoptosis-inducing drugs. Acknowledgments This work was supported by a Grant-in-Aid for the 21st Century COE Program, ‘‘Functional Innovation of Molecular Informatics,’’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We express our gratitude to Hamamatsu Photonics, Japan, for providing us with an opportunity to use a gated ICCD camera. References [1] C.Y. Dong, T. French, P.T.C. So, C. Buehler, K.M. Berland, E. Gratton, Fluorescence-lifetime imaging techniques for microscopy, Methods Cell Biol. 72 (2003) 431–464. [2] K. Suhling, P.M.W. French, D. Phillips, Time-resolved fluorescence microscopy, Photochem. Photobiol. Sci. 4 (2005) 13–22. [3] H. Wallrabe, A. Periasamy, Imaging protein molecules using FRET and FLIM microscopy, Curr. Opin. Biotechnol. 16 (2005) 19–27. [4] A. Periasamy, P. Wodnicki, X.F. Wang, S. Kwon, G.W. Gordon, B. Herman, Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system, Rev. Sci. Instrum. 67 (1996) 3722–3731. [5] P. Urayama, W. Zhong, J.A. Beamish, F.K. Minn, R.D. Sloboda, K.H. Dragnev, E. Dmitrovsky, M.-A. Mycek, A UV–Visible–NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution, Appl. Phys. B 76 (2003) 483– 496. [6] M.A.M.J. van Zandvoort, C.J. de Grauw, H.C. Gerritsen, J.L.V. Broers, M.G.A. oude Egbrink, F.C.S. Ramaekers, D.W. Slaaf, Discrimination of DNA and RNA in cells by a vital fluorescent probe: Lifetime imaging of SYTO13 in healthy and apoptotic cells, Cytometry 47 (2002) 226–235. [7] K.P. Ghiggino, M.R. Harris, P.G. Spizzirri, Fluorescence lifetime measurements using a novel fiber-optic laser scanning confocal microscope, Rev. Sci. Instrum. 63 (1992) 2999–3002. [8] T. Oida, Y. Sako, A. Kusumi, Fluorescence lifetime imaging microscopy (flimscopy), Biophys. J. 64 (1993) 676–685. [9] D.K. Bird, K.W. Eliceiri, C.-H. Fan, J.G. White, Simultaneous twophoton spectral and lifetime fluorescence microscopy, Appl. Opt. 43 (2004) 5173–5182. [10] K. Dowling, M.J. Dayel, M.J. Lever, P.M.W. French, J.D. Hares, A.K.L. Dymoke-Bradshaw, Fluorescence lifetime imaging with picosecond resolution for biomedical applications, Opt. Lett. 23 (1998) 810–812.
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[11] J.-P. Knemeyer, D.-P. Herten, M. Sauer, Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy, Anal. Chem. 75 (2003) 2147–2153. [12] Q. Li, T. Ruckstuhl, S. Seeger, Deep-UV laser-based fluorescence lifetime imaging microscopy of single molecules, J. Phys. Chem. B 108 (2004) 8324–8329. [13] T. Uchimura, S. Kawanabe, Y. Maeda, T. Imasaka, Fluorescence lifetime imaging microscope consisting of a compact picosecond dye laser and a gated charge-coupled device camera for applications to living cells, Anal. Sci. 22 (2006) 1291–1295. [14] M. Mancini, M. Sedghinasab, K. Knowlton, A. Tam, D. Hockenbery, B.O. Anderson, Flow cytometric measurement of mitochondrial mass and function: A novel method for assessing chemoresistance, Ann. Surg. Oncol. 5 (1998) 287–295. [15] S.M. van den Eijnde, A.J.M. Luijsterburg, L. Boshart, C.I. De Zeeuw, J.H. van Dierendonck, C.P.M. Reutelingsperger, C. Vermeij-Keers, In situ detection of apoptosis during embryogenesis with annexin V: From whole mount to ultrastructure, Cytometry 29 (1997) 313–320.
[16] T. Frey, Nucleic acid dyes for detection of apoptosis in live cells, Cytometry 21 (1995) 265–274. [17] B.L. Sailer, J.A. Steinkamp, H.A. Crissman, Flow cytometric fluorescence lifetime analysis of DNA-binding probes, Eur. J. Histochem. 42 (1998) 19–27. [18] O. Kirschstein, M. Sip, L. Kittler, Quantitative and sequence-specific analysis of DNA–ligand interaction by means of fluorescent intercalator probes, J. Mol. Recogn. 13 (2000) 157–163. [19] R.P. Haugland, Handbook of Fluorescent Probes and Research Products, 9th ed., Molecular Probes, Eugene, OR, 2002. [20] P.J. Smith, M. Wiltshire, S. Davies, L.H. Patterson, T. Hoy, A novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry, J. Immunol. Methods 229 (1999) 131–139. [21] M. Eray, M. Ma¨tto¨, M. Kaartinen, L. Andersson, J. Pelkonen, Flow cytometric analysis of apoptotic subpopulations with a combination of annexin V–FITC, propidium iodide, and SYTO 17, Cytometry 43 (2001) 134–142.