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pH dependence of the fluorescence lifetime of enhanced yellow fluorescent protein in solution and cells
Journal of Photochemistry and Photobiology A: Chemistry 235 (2012) 65–71
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Journal of Photochemistry and Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem
pH dependence of the fluorescence lifetime of enhanced yellow fluorescent protein in solution and cells Takakazu Nakabayashi a, Shugo Oshita a, Ryoya Sumikawa a, Fan Sun b, Masataka Kinjo b, Nobuhiro Ohta a,∗ a b
Research Institute for Electronic Science (RIES), Hokkaido University, Sapporo 001-0020, Japan Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan
a r t i c l e
i n f o
Article history: Received 2 November 2011 Received in revised form 3 February 2012 Accepted 14 February 2012 Available online 3 March 2012 Keywords: EYFP Intracellular pH Fluorescence decay profile Fluorescence lifetime image HeLa cells
a b s t r a c t pH dependence of the fluorescence decay profile of enhanced yellow fluorescent protein (EYFP) depends on the excitation wavelength. The correlation between the fluorescence lifetime and pH in solution is discussed in terms of the acid–base equilibrium of the chromophore of EYFP. Fluorescence lifetime images of EYFP in HeLa cells have also been measured at various values of intracellular pH. A remarkable pH dependence of the fluorescence lifetime image is observed in pH 4.5–6.0, indicating that pH in a single cell can be evaluated using the fluorescence lifetime image of EYFP especially in the acidic condition. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Intracellular pH plays an important role in diverse cell functions [1–4]. The activity of most proteins highly depends on proton concentration, and the regulation of intracellular pH is important to maintain homeostasis in living systems. Furthermore, intracellular pH is not uniform in a cell and depends on the nature of cellular compartments. pH gradients between cellular compartments induce the activation of cellular processes such as ATP synthesis in mitochondria. Much effort has been therefore devoted to development of imaging techniques to monitor the spatial distribution of pH in a cell. Fluorescence microscopy with pH sensitive dyes is a very powerful method for measuring pH distribution in a cell, and a variety of fluorescent probes whose fluorescence intensity is sensitive to pH have been developed [5–12]. However, fluorescence intensity is difficult to be quantitatively evaluated with a microscope in some cases because fluorescence intensity also depends on various experimental factors such as excitation light intensity, photobleaching, dye localization, and absorption intensity of the sample, all of which are not related to pH. Either excitation ratio or emission ratio methods have been employed for quantitative evaluation of fluorescence intensity [5,6]; however, the ratiometric methods are
∗ Corresponding author. Tel.: +81 11 706 9410; fax: +81 11 706 9406. E-mail address: [email protected] (N. Ohta). 1010-6030/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotochem.2012.02.016
difficult to be combined with a microscope having z-axis resolution because of wavelength-dependent focal depth and absorption. Measurements of fluorescence lifetime of a fluorescent dye can enhance the potential of fluorescence microscopy [13–15]. Fluorescence lifetime is an inherent property of a dye molecule, and thus is independent of dye concentration, photobleaching, excitation light intensity, and other factors that limit fluorescence intensity measurements. This makes fluorescence lifetime imaging (FLIM) a powerful tool for quantitatively imaging pH in a single cell [16–21]. Neither of the ratio methods is necessary for imaging fluorescence lifetime, so that the FLIM method is compatible with confocal and multi-photon microscopes. The green fluorescent protein (GFP) has become an invaluable tool for fluorescent-imaging applications [22,23] and various kinds of GFP mutants have been developed for adding useful optical properties [9–12,22,23]. Each GFP-based protein has a characteristic chromophore structure that is responsible for absorption and fluorescence. The chromophore is rigidly encapsulated inside the barrel of the protein structure, which is thought to effectively block the non-radiative relaxation of the chromophore [22]. This unique structure results in the strong fluorescence in the visible region. The electrostatic and hydrogen-bonding interactions of the chromophore with surrounding amino acids are also responsible for fluorescence properties of GFP-based proteins. In our previous study, intracellular pH of a single cell could be imaged using FLIM of enhanced GFP (EGFP) that contains the F64L and S65T mutations [21]. The chromophore of EGFP consists of
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(HeLa) cells with varying intracellular pH, as preliminary reported in our previous paper [33]. 2. Experimental 2.1. Sample preparation
Fig. 1. The neutral and anionic forms of the chromophores of EGFP (a) and EYFP (b).
a p-hydroxybenzylidene-imidazolidinone that exists in either the neutral phenol form or the anionic phenolate form (Fig. 1a), the equilibrium of which depends on medium pH. These two forms exhibit distinct absorption and fluorescence characteristics, and the fluorescence lifetime of the anionic form is in the 2–3 ns range and that of the neutral form is in tens to hundreds of picoseconds [24–26]. It was shown in previous studies [21,25,26] that the fluorescence decay of EGFP or GFP(S65T) was observed to become faster with decreasing medium pH when the neutral chromophore was preferentially excited and the fluorescence signals both from the neutral and anionic chromophores were detected. The observed fluorescence decay can be regarded as a mixture of the decays of the excited neutral and anionic forms, and the pH-induced change in the average fluorescence lifetime was explained in terms of the acid–base equilibrium between the two forms in the ground state. The excited neutral form having a short fluorescence lifetime is preferentially formed, and the molar ratio of the neutral form increases as the medium pH decreases, which results in the reduction of the average fluorescence lifetime with lowering pH. Such a pH-induced change in average fluorescence lifetime of EGFP was observed in a single cell, which can be used as pH imaging of living systems [21]. The pH imaging using the difference in fluorescence lifetime between the neutral and anionic chromophores can also be applied for other GFP-based proteins because the neutral chromophore having a short fluorescence lifetime exists in many GFP-based proteins. It is therefore conceivable that FLIM of GFP-based proteins becomes a general method for measuring pH in a cell. The pKa between the neutral and anionic forms of EGFP is estimated to be 5.6–6.0 [21,25,27], and the significant pH-induced alteration of the average fluorescence lifetime occurs in an acidic region of pH 5–6.5. It is thus important to measure effects of pH on the average fluorescence lifetime of other GFP-based proteins having a pKa near neutral. Enhanced yellow fluorescent protein (EYFP) is one of the most widely used variants of GFP from Aequoria victoria (wild-type GFP) [23,28–32]. EYFP contains the mutations replacing serine 65 with glycine, valine 68 with leucine, serine 72 with alanine, and threonine 203 with tyrosine in wild-type GFP. EYFP has the same chromophore structure as that of EGFP, and the – interaction of the chromophore with Tyr203 induces the red shift of the fluorescence to the yellow region. The chromophore of EYFP also shows the acid–base equilibrium in the ground state (Fig. 1b), the pKa of which is 6.5–6.9 in high chloride concentrations [23,29,30] that is in the physiological range of many living systems. In the present study, effect of pH on the average fluorescence lifetime of EYFP in buffer has been examined with several excitation wavelengths. We could also measure FLIM of EYFP in human cervical carcinoma
HeLa cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 2 × 105 U dm−3 penicillin G, 200 mg dm−3 streptomycin sulfate, and 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37 ◦ C. HeLa cells were transfected overnight with plasmid DNA of pEYFP-C1 (Clontech, BD Biosciences, Oxford, UK) using Optifect (Invitrogen) in LAB-Tek 8well chambered coverslips (Nalge Nunc International). Calibration of intracellular pH was carried out by the so-called nigericin/high K+ method [5,16,18,34,35]. HeLa cells expressing EYFP grown on LAB-TEK chambered coverslips with eight wells were washed with KCl-rich media (125 mM KCl, 20 mM NaCl, 10 mM HEPES, 10 mM MES, 0.5 mM CaCl2 , 0.5 mM MgCl2 ) and then incubated with the same KCl-rich media containing 10 g/ml nigericin at different pH. Nigericin is a kind of K+ /H+ ionophore, which equilibrates protons across plasma membrane in the presence of a depolarizing extracellular concentration of K+ . For fluorescence decay measurements in buffer solution, EYFP was purified from E. coli carrying the plasmid DNA of pEYFP-C1 (Clontech) by nickel column chromatography (GE Healthcare). The stock solution of EYFP was then diluted in phosphate buffered saline (PBS) buffer (154 mM NaCl, 6 mM Na2 HPO4 , 1.1 mM KH2 PO4 ) at different pH. 2.2. Apparatus Measurements of fluorescence lifetime image were carried out using a confocal microscope with a four-channel time-gated detection system [36]. A full description of the experimental system was reported elsewhere [20,21]. Output pulses from a femtosecond mode-locked Ti:sapphire laser (Spectra Physics, Tsunami) were frequency doubled and used as an excitation light. The pulse duration and the repetition rate of the laser pulse were 80 fs and 81 MHz, respectively. The excitation beam was introduced into a scanning confocal microscope (Nikon, C1) and then was focused onto the sample with a 40× objective (Nikon, CFI S Fluor, NA 0.90). The fluorescence collected by the same objective was transmitted into a filter box equipped with interference filters (Nikon, BA520, EX510-560). Fluorescence was detected using a photomultiplier in a high-speed lifetime imaging module (Nikon Europe BV, LIMO). The lifetime imaging module captures the fluorescence decay into four time windows. Fluorescence lifetime at each pixel of the image was evaluated from the four time-windows signals by assuming a single exponential decay. The size of the image was 256 × 256 pixels. All of the time windows were set at 2.0 ns. The acquisition time was ∼20 min for each image. All the measurements were performed in a stage top incubator (INUG2-ONICS, Tokai Hit) with 5% CO2 at 37 ◦ C. Macroscopic measurements of fluorescence decay of EYFP in aqueous solution were carried out using a time-correlated singlephoton counting system [37]. The second harmonic of the output from the mode-locked Ti:sapphire laser was used for excitation. The repetition rate was reduced to be ∼5.8 MHz with a pulse picker (Conoptics, model 350-160). Fluorescence from the sample was dispersed by a monochromator and then detected by a microchannel-plate photomultiplier (Hamamatsu, R3809U-52). All the fluorescence decays were collected at a magic angle with respect to the vertically polarized excitation light. The instrumental response function (IRF) had a full width at half maximum (FWHM)
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Fig. 2. Absorption (dotted line) and fluorescence (solid line) spectra of EYFP in buffer solution. Excitation wavelength was 470 nm for the fluorescence measurement.
of ∼60 ps. The inverse Fourier-transform method was used for the simulation of the decay profile. The observed fluorescence decay was fitted with the convolution of the IRF with a multiexponential decay function. The convolution was mathematically done by the inverse Fourier-transform method. The simulation was performed by a home-made macro in the IGOR software (Wavemetrics). 3. Results and discussion 3.1. Absorption, fluorescence, and fluorescence decays of EYFP Fig. 2 shows absorption and fluorescence spectra of EYFP in PBS buffer at a neutral pH. The strong absorption and fluorescence bands with a peak at ∼514 and ∼530 nm, respectively, arise from the anionic chromophore of EYFP. These absorption and fluorescence intensities increase with increasing pH [29,32]. The neutral chromophore exhibits the absorption band around 400 nm that becomes dominant in acidic conditions. The broad fluorescence due to the neutral chromophore is observed around 460 nm with excitation at 400 nm [32]. In the present study, we have measured the pH dependence of the fluorescence decay of EYFP in buffer solution with the three excitation wavelengths: 470, 440, and 400 nm. The anionic and neutral forms of the EYFP chromophore are preferentially excited with the excitation wavelengths of 470 and 400 nm, respectively. Both the anionic and neutral chromophores are excited at 440 nm. The representative fluorescence decays of EYFP in buffer with the excitation wavelengths of 470, 440, and 400 nm are shown in Fig. 3a–c, respectively, where the maximum intensity is normalized to unity in all the decay profiles. The results at pH of 5.0, 6.5, and 7.5 are shown in this figure. The wavelength of the monitored fluorescence was 535 ± 5 nm in every case. In the present study, the observed fluorescence decays were fitted by the convolution of the IRF with a multi-exponential decay, i.e., i Ai exp(− t/ i ), where Ai and i denote the pre-exponential factor and the fluorescence lifetime of component i, respectively, and the obtained pre-exponential factors and the fluorescence lifetimes with the three excitation wavelengths are shown in Tables 1–3. The fluorescence lifetimes whose preexponential facthese tables. tors are more than 1% are shown in The average fluorescence lifetime ( ave ) is given by i Ai i / i Ai . Plots of ave against pH are shown in Fig. 4. 3.2. Fluorescence decays with excitation at 470 nm As shown in Table 1, three decaying components were used to fit the decay profiles with excitation at 470 nm. The component having a fluorescence lifetime of ∼3 ns, i.e., component (1), can be attributed to the excited state of the anionic chromophore formed by the excitation at 470 nm. This component was dominant in all the decay profiles, and its fluorescence lifetime, i.e.,
Fig. 3. Representative fluorescence decays of EYFP in buffer solution at pH of 5.0 (red), 6.5 (green), and 7.5 (blue) at the excitation wavelengths of 470 nm (a), 440 nm (b), and 400 nm (c). The total photon counts at each excitation wavelength are in the range of 4 × 104 –2 × 105 (400 nm), 6 × 104 –1 × 106 (440 nm), and 2 × 105 –2 × 106 (470 nm). The intensities are plotted in a linear scale. In all the decay profiles, the maximum intensity is normalized to unity. Fluorescence was observed at 535 ± 5 nm in every case. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Table 1 pH dependence of the fitting parameters of fluorescence decays of EYFP with excitation at 470 nm.a,b pH
1 (ns)
9 8 7.5 7 6.5 6 5.5 5
3.2 (1.00) 3.2 (1.00) 3.2 (1.00) 3.2 (0.97) 3.2 (1.00) 3.1 (0.94) 3.1 (0.90) 2.8 (0.78)
2 (ns)
3 (ns)
0.30 (0.03) 0.30 (0.05) 0.30 (0.09) 0.30 (0.20)
9.0 (0.01) 9.0 (0.01) 9.0 (0.02)
a Pre-exponential factor of each component, whose summation is normalized to unity in the decay, is given in parentheses. b Experimental errors of 1 , 2 and 3 are ca. ±10, ±50 and ±30%, respectively.
Table 2 pH dependence of the fitting parameters of fluorescence decays of EYFP with excitation at 440 nm.a,b pH
1 (ns)
2 (ns)
9 8 7.5 7 6.5 6 5.5 5
3.2 (0.94) 3.2 (0.93) 3.2 (0.94) 3.2 (0.92) 3.2 (0.82) 3.2 (0.10)
0.30 (0.06) 0.30 (0.06) 0.30 (0.06) 0.30 (0.07) 0.25 (0.17) 0.15 (0.04) 0.15 (0.01)
3 (ns)
4 (ns)c
9.0 (0.01) 9.0 (0.01) 9.0 (0.01) 0.001 (0.86) 0.001 (0.99) 0.001 (1.00)
a Pre-exponential factor of each component, whose summation is normalized to unity in the decay, is given in parentheses. b Experimental errors of 1 , 2 and 3 are ca. ±10, ±50 and ±30%, respectively. c 4 was fixed to be 1.4 ps.
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Table 3 pH dependence of the fitting parameters of fluorescence decays of EYFP with excitation at 400 nm.a,b pH
1 (ns)
2 (ns)
3 (ns)
9 8 7.5 7 6.5 6 5.5 5
3.2 (0.86) 3.2 (0.79) 3.2 (0.82) 3.2 (0.47) 3.2 (0.16) 3.1 (0.09) 3.0 (0.06) 2.8 (0.04)
0.30 (0.13) 0.15 (0.20) 0.20 (0.17) 0.30 (0.21) 0.30 (0.12) 0.35 (0.12) 0.35 (0.18) 0.35 (0.13)
9.0 (0.01) 9.0 (0.01) 9.0 (0.01) 9.0 (0.01)
4 (ns)
0.04 (0.31) 0.04 (0.72) 0.05 (0.79) 0.05 (0.76) 0.05 (0.83)
a Pre-exponential factor of each component, whose summation is normalized to unity in the decay, is given in parentheses. b Experimental errors of 1 , 2 , 3 and 4 are ca. ±10, ±50, ±30 and ±30%, respectively.
Fig. 4. Plots of the average fluorescence lifetime of EYFP in buffer solution against pH. Excitation wavelengths were 470 (green), 440 (red), and 400 nm (blue), respectively. The simulated curves at the 440- and 400-nm excitation are also shown by dotted lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
∼3.2 ns at neutral pH, is similar to those reported in previous studies [31,32]. The component having a lifetime of hundreds of picoseconds, i.e., component (2), was confirmed, and its pre-exponential factor increased with decreasing medium pH. Since the neutral chromophore is hardly excited at 470 nm, the component (2) may be ascribed to photoconverted EYFP species or the isomer of the EYFP chromophore arising from different interactions with surrounding amino acids in the barrel structure (Fig. 5). It was reported that photoconversion of EYFP occurred in the presence of green (514 or 532 nm) light [32]. It might be necessary to consider component (3) having a long lifetime of ∼9 ns to reproduce the decay in the time range longer than 10 ns; however, the pre-exponential factor of component (3) was negligibly small in neutral and alkali conditions. In pH 6.5–9.0, the lifetime of the component (1) remains almost unchanged with pH, and significant pH dependence of ave was not observed (Fig. 4). In acidic conditions, fluorescence intensity becomes weak due to the shift of the equilibrium of the
Fig. 5. The diagram of the neutral and anion species of the chromophore of EYFP and the photoconverted species.
chromophore to the neutral form. As shown in Fig. 4, the observed ave also becomes shorter with lowering pH in acidic conditions of pH 5.0–6.0, which results from the decrease in both A1 and 1 with decreasing pH. The reduction of fluorescence lifetime in acidic conditions following photoexcitation of the anionic chromophore was also observed for GFP(S65T) [26]. The alteration of 1 suggests the change in the intermolecular interaction of the anionic chromophore with amino acids in acidic conditions. 3.3. Fluorescence decays with excitation at 440 nm Both the neutral and anionic chromophores are excited with excitation at 440 nm. The component (1) having a lifetime of ∼3 ns can be assigned to the anionic chromophore in the excited state (Table 2). The component (2) having a lifetime of 0.15–0.30 ns may also be assigned to the photoconverted species or the isomer of the EYFP chromophore that is observed at the 470-nm excitation. In alkali and neutral conditions of pH 9.0–6.5, the component (3) having a long lifetime was confirmed, but the pre-exponential factor of which was 1% or less. The fast decaying component, i.e., component (4), becomes dominant in acidic conditions of pH 5–6, and its pre-exponential factor increases with lowering pH. This component is ascribed to the excited neutral chromophore formed by the excitation at 440 nm. The lifetime of the component (4) was shorter than the time-width of IRF. In the present study, therefore, the 4 value at the 440-nm excitation was assumed to be 1.4 ps, which is the reported value of the fluorescence lifetime of the neutral chromophore of EYFP [32]. As shown in Fig. 4, the correlation between the observed ave and pH at the 440-nm excitation was satisfactorily fitted by the following equation [27]: ave = a + b[1 + 10nH (pKa −pH) ]−1
(1)
where a and b are the offset and dynamic range, respectively; pKa is pH at the half of the dynamic range; nH is Hill coefficient. It should be noted that the observed fluorescence decay can be regarded as a mixture of the decays of each component, and ave was evaluated by the decay profile of the mixture. The fitted curve in Fig. 4 was obtained with pKa and nH of 6.3 and 4.5, respectively. The large Hill coefficient may arise from the very large difference between the lifetimes of the neutral and anionic chromophores. The obtained pKa is smaller than that of the neutral and anionic forms of the EYFP chromophore evaluated from the absorption and fluorescence spectra (pKa of 6.5–6.9) [23,29,30]. This difference probably arises from the difficulty in evaluation of the pre-exponential factor of the component (4), whose lifetime is shorter than the time-width of IRF. 3.4. Fluorescence decays with excitation at 400 nm The neutral form is preferentially excited by photoirradiation at 400 nm. As shown in Table 3, however, the decay profiles exhibited the component (1) having a fluorescence lifetime of ∼3 ns, which results from the excited anionic chromophore. It was reported that the photoexcited neutral chromophore of EYFP decayed with two non-radiative processes: direct non-radiative relaxation to the ground state and excited-state intramolecular proton transfer (ESIPT) to generate the excited anionic form. The direct nonradiative relaxation to the ground state was suggested to be much faster than the ESIPT to account for the very low fluorescence yield of the neutral chromophore [32]. The excited anionic chromophore observed following the 400-nm excitation is therefore considered to be generated by the direct excitation of the anionic form according to its low absorption cross section at 400 nm. As shown in Table 3, the pre-exponential factor of the anionic chromophore
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satisfactorily reproduced by Eq. (1) (Fig. 4), and pKa and nH were evaluated to be 7.0 and 1.5, respectively. 3.5. Fluorescence lifetime images of HeLa cells expressing EYFP
Fig. 6. The expansion of representative fluorescence decays of EYFP in buffer solution at pH 5.0 in the −0.2 to 0.7 ns region. The excitation wavelengths were 470 nm (green), 440 nm (red), and 400 nm (blue). The intensities are plotted in a linear scale. In all the decay profiles, the maximum intensity is normalized to unity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
decreases with lowering pH, which results from the shift of the acid–base equilibrium to the neutral form. Another fluorescence component having a lifetime of 40–50 ps, i.e., component (4), becomes dominant at pH less than 7, and its pre-exponential factor increases with lowering pH. This component can be attributed to the excited species of neutral chromophore, though its fluorescence lifetime is larger than those obtained with the 440-nm excitation (see Fig. 6). It probably comes from the presence of photoconverted species generated by the photoexcitation of the neutral choromophore of EYFP. The very short lifetime component obtained by the 440-nm excitation was not distinguished, which may be due to the interference of the existence of the component (4); both the lifetimes are shorter than the FWHM of the IRF. The correlation between ave and pH at the 400-nm excitation was
Fluorescence lifetime images of HeLa cells expressing EYFP were observed at different values of intracellular pH. Excitation wavelength was 440 nm and the fluorescence at 515–560 nm was detected. The results are shown in Fig. 7. The corresponding fluorescence intensity image is shown on the top of each fluorescence lifetime image. The intracellular pH can be evaluated by measuring the extracellular one because ionophore was added in the medium as mentioned in Experimental section. The lifetimes at different pHs showed only the small change in the intracellular pH ranging from 7.0 to 9.0. On the other hand, the remakable pH dependence of the lifetime was observed in intracellular pH less than 6.0. Fig. 7b shows the histograms of the fluorescence lifetime of HeLa cells expressing EYFP with varying intracellular pH. Plots of the average fluorescence lifetime against the intracellular pH are shown in Fig. 7c. The average fluorescence lifetime was evaluated from the peak of the histogram. It can be seen in Fig. 7c that the averaged fluorescence lifetime in cells remains almost unchanged in intracellular pH 7.0–9.0 and becomes shorter with decreasing intracellular pH especially at pH less than 6. For example, the lifetime is ∼2.52 ns at pH 6.0, and then decreases to ∼1.96 ns at pH 4.5. Thus, the significant pH dependence of the average fluorescence lifetime in cells is found in acidic conditions at the 440-nm excitation. The magnitude of the reduction of the observed lifetime is ca. 20%, when pH is changed from 6 to 4.5. From the observation of the significant pH dependence of the fluorescence lifetime of EYFP in HeLa cells, it is concluded that pH in a single cell can be evaluated using FLIM of EYFP in pH 4.5–6.0. Cells contain endogenous chromophores showing fluorescence called autofluorescence, and
Fig. 7. (a) Fluorescence intensity images (upper) and corresponding fluorescence lifetime images (lower) of HeLa cells expressing EYFP. The intracellular pH is shown on the top of each intensity image. (b) Intracellular pH dependence of the histogram of the fluorescence lifetime of HeLa cells expressing EYFP. (c) Plots of the average fluorescence lifetime of HeLa cells expressing EYFP against the intracellular pH. The average fluorescence lifetime was evaluated from the peak of the histogram in (b). Excitation wavelength was 440 nm and the fluorescence in the wavelength region of 515–560 nm was detected. pH dependence of the average fluorescence lifetime of EYFP in buffer solution at the excitation wavelength of 470 nm (this result is the same as that in Fig. 4) is also shown in (c) for comparison.
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autofluorescence in nuclei is much weaker than that in other areas, resulting in the observation of the dull nucleus region in autofluorescence images [38,39]. In the present study, the dull nucleus was not distinguishable in the intensity images (Fig. 7a), indicating that the contribution of autofluorescence was negligible. It is noted that it was difficult to measure the pH dependence of FLIM of EYFP in cells at the 400-nm excitation, owing to the very weak fluorescence intensity. In EGFP-expressed HeLa cells, FLIM images were found to depend on intracellular pH [21], which was ascribed to the pH-dependent equilibrium between the anionic and neutral forms of the EGFP chromophore in the ground state, the fluorescence lifetimes of which are in the 2–3 ns range and in tens to hundreds of picoseconds, respectively. In contrast with EGFP, the fluorescence lifetime of the neutral form of EYFP was 0.4–3 ps [32] that is too short to be detected in each timewindow (2 ns) of the present FLIM measurements. Furthermore, a part of the decaying portion following photoexcitation was cut in the FLIM measurements to exclude scattered light, and the signal at the first window was integrated from ca. 0.5 to 1 ns after photoexcitation [40]. It is therefore unlikely that the observed pH dependence of FLIM of EYFP results from a mixture of the fluorescence decays of the excited neutral and anionic chromophores. The observed fluorescence in the FLIM measurements can be therefore considered to arise from the anionic chromophore, and the pH dependence of the average fluorescence lifetime is ascribed to a change in environment surrounding the anionic chromophore induced by the change in pH. This result is consistent with the observation in buffer solution following excitation of the anion chromophore in Fig. 4: ave obtained with the 470-nm excitation became shorter with lowering pH in pH 5.0–6.0, and the magnitude of the change in ave between pH 5 and 6 was ca. 20%. It is thus concluded that the observed pH dependence of FLIM of EYFP results from the change in the protein structure due to the change in acidic property: the fluorescence lifetime of the anionic chromophore in EYFP decreases with an increase of the acidic property. In addition to the pH dependence of the fluorescence lifetime, a non-uniformity of the fluorescence lifetime of EYFP was observed in a cell, as preliminary reported [33]. At pH 4, for example, the average fluorescence lifetime at the peripheral parts of cells was longer than those inside the cells; the lifetime was ∼2.6 ns at the peripheral parts, while that was around 2.2 ns in other areas. A fluctuation of the fluorescence lifetime was also observed in EGFPexpressed HeLa cells, but the opposite relation was found: the lifetime of EGFP at the peripheral parts of cells was shorter than that in other areas [33]. Both the average fluorescence lifetimes of EYFP and EGFP become shorter with lowering pH. It is unlikely that pH at the peripheral parts of cells is higher and lower than those in other areas, respectively, in the EYFP-expressed cells and in the EGFP-expressed cells. Therefore, we consider that the origin of the variation of the fluorescence lifetime in a cell is attributed to others than the pH dependence. It has been reported that strong electric fields induce the alteration of the fluorescence lifetime and that the field effects on fluorescence lifetime opposite to each other may appear, depending on the chromophore. For example, the fluorescence lifetime of GFPuv5 was shortened by application of electric fields [41], while the fluorescence lifetime of a dimethyl derivative of p-hydroxybenzylidene-imidazolidinone, that is a model compound of the chromophore of GFP, was lengthened by application of electric fields [42]. Then, the present non-uniformity of the lifetime at the peripheral parts of cells may result from the field effects due to membranes and intracellular substances, and the fluorescence lifetime of EYFP may give the opposite electric field effect to that of EGFP.
4. Conclusion pH dependence of the fluorescence lifetime of EYFP depends on the excitation wavelength, and the pKa and nH were evaluated at the 400- and 440-nm excitation in buffer solution. The pH dependence of the fluorescence lifetime images of EYFP is also observed in HeLa cells with the 440-nm excitation. The results show that intracellular pH can be evaluated by the FLIM measurements of EYFP especially in the pH range of 4.5–6.0, which is important for acid organelles such as lysosomes and synaptic vesicles. It is also found that the fluorescence lifetime of EYFP is not uniform in each cell, which may be ascribed to different distribution of electric fields surrounding the chromophore in cells. Acknowledgement This work was supported by a Grant-in-Aid for Scientific Research in Priority Area “Molecular Nano Dynamics” from MEXT. References [1] A. Roos, W.F. Boron, Intracellular pH, Physiol. Rev. 61 (1981) 296–434. [2] W. Moody Jr., Effects of intracellular H+ on the electrical properties of excitable cells, Annu. Rev. Neurosci. 7 (1984) 257–278. [3] B.C. Gibbon, D.L. Kropf, Cytosolic pH gradients associated with tip growth, Science 263 (1994) 1419–1421. [4] S. Matsuyama, J. Llopis, Q.L. Deveraux, R.Y. Tsien, J.C. Reed, Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis, Nat. Cell Biol. 2 (2000) 318–325. [5] M. Nedergaard, S. Desai, W. Pulsinelli, Dicarboxy-dichlorofluorescein: a new fluorescent probe for measuring acidic intracellular pH, Anal. Biochem. 187 (1990) 109–114. [6] J.E. Whitaker, R.P. Haugland, F.G. Prendergast, Spectral and photophysical studies of benzo[c]xanthene dyes: dual emission pH sensors, Anal. Biochem. 194 (1991) 330–344. ´ A. Orte, E.M. Talavera, J.M. Alvarez-Pez, Photo[7] N. Boens, W. Qin, N. Basaric, physics of the fluorescent pH indicator BCECF, J. Phys. Chem. A 110 (2006) 9334–9343. [8] M.Y. Berezin, J. Kao, S. Achilefu, pH-dependent optical properties of synthetic fluorescent imidazoles, Chem. Eur. J. 15 (2009) 3560–3566. [9] J. Llopis, J.M. McCaffery, A. Miyawaki, M.G. Farquhar, R.Y. Tsien, Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 6803–6808. [10] G. Miesenböck, D.A. De Angelis, J.E. Rothman, Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins, Nature 394 (1998) 192–195. [11] G.T. Hanson, T.B. McAnaney, E.S. Park, M.E.P. Rendell, D.K. Yarbrough, S. Chu, L. Xi, S.G. Boxer, M.H. Montrose, S.J. Remington, Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application, Biochemistry 41 (2002) 15477–15488. [12] R. Bizzarri, C. Arcangeli, D. Arosio, F. Ricci, P. Faraci, F. Cardarelli, F. Beltram, Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies, Biophys. J. 90 (2006) 3300–3314. [13] H. Wallrabe, A. Periasamy, Imaging protein molecules using FRET and FLIM microscopy, Curr. Opin. Biotechnol. 16 (2005) 19–27. [14] W. Becker, A. Bergmann, C. Biskup, Multispectral fluorescence lifetime imaging by TCSPC, Microsc. Res. Tech. 70 (2007) 403–409. [15] J.A. Levitt, D.R. Matthews, S.M. Ameer-Beg, K. Suhling, Fluorescence lifetime and polarization-resolved imaging in cell biology, Curr. Opin. Biotechnol. 20 (2009) 28–36. [16] R. Sanders, A. Draaijer, H.C. Gerritsen, P.M. Houpt, Y.K. Levine, Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy, Anal. Biochem. 227 (1995) 302–308. [17] K.M. Hanson, M.J. Behne, N.P. Barry, T.M. Mauro, E. Gratton, R.M. Clegg, Twophoton fluorescence lifetime imaging of the skin stratum corneum pH gradient, Biophys. J. 83 (2002) 1682–1690. [18] H.-J. Lin, P. Herman, J.R. Lakowicz, Fluorescence lifetime-resolved pH imaging of living cells, Cytometry 52A (2003) 77–89. [19] R. Niesner, B. Peker, P. Schlüsche, K.-H. Gericke, C. Hoffmann, D. Hahne, C. Müller-Goymann, 3D-resolved investigation of the pH gradient in artificial skin constructs by means of fluorescence lifetime imaging, Pharm. Res. 22 (2005) 1079–1087. [20] H.-P. Wang, T. Nakabayashi, K. Tsujimoto, S. Miyauchi, N. Kamo, N. Ohta, Fluorescence lifetime image of a single halobacterium, Chem. Phys. Lett. 442 (2007) 441–444. [21] T. Nakabayashi, H.-P. Wang, M. Kinjo, N. Ohta, Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements, Photochem. Photobiol. Sci. 7 (2008) 668–670. [22] M. Zimmer, Green fluorescent protein (GFP): applications, structure, and related photophysical behavior, Chem. Rev. 102 (2002) 759–781.
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Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem
pH dependence of the fluorescence lifetime of enhanced yellow fluorescent protein in solution and cells Takakazu Nakabayashi a, Shugo Oshita a, Ryoya Sumikawa a, Fan Sun b, Masataka Kinjo b, Nobuhiro Ohta a,∗ a b
Research Institute for Electronic Science (RIES), Hokkaido University, Sapporo 001-0020, Japan Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan
a r t i c l e
i n f o
Article history: Received 2 November 2011 Received in revised form 3 February 2012 Accepted 14 February 2012 Available online 3 March 2012 Keywords: EYFP Intracellular pH Fluorescence decay profile Fluorescence lifetime image HeLa cells
a b s t r a c t pH dependence of the fluorescence decay profile of enhanced yellow fluorescent protein (EYFP) depends on the excitation wavelength. The correlation between the fluorescence lifetime and pH in solution is discussed in terms of the acid–base equilibrium of the chromophore of EYFP. Fluorescence lifetime images of EYFP in HeLa cells have also been measured at various values of intracellular pH. A remarkable pH dependence of the fluorescence lifetime image is observed in pH 4.5–6.0, indicating that pH in a single cell can be evaluated using the fluorescence lifetime image of EYFP especially in the acidic condition. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Intracellular pH plays an important role in diverse cell functions [1–4]. The activity of most proteins highly depends on proton concentration, and the regulation of intracellular pH is important to maintain homeostasis in living systems. Furthermore, intracellular pH is not uniform in a cell and depends on the nature of cellular compartments. pH gradients between cellular compartments induce the activation of cellular processes such as ATP synthesis in mitochondria. Much effort has been therefore devoted to development of imaging techniques to monitor the spatial distribution of pH in a cell. Fluorescence microscopy with pH sensitive dyes is a very powerful method for measuring pH distribution in a cell, and a variety of fluorescent probes whose fluorescence intensity is sensitive to pH have been developed [5–12]. However, fluorescence intensity is difficult to be quantitatively evaluated with a microscope in some cases because fluorescence intensity also depends on various experimental factors such as excitation light intensity, photobleaching, dye localization, and absorption intensity of the sample, all of which are not related to pH. Either excitation ratio or emission ratio methods have been employed for quantitative evaluation of fluorescence intensity [5,6]; however, the ratiometric methods are
∗ Corresponding author. Tel.: +81 11 706 9410; fax: +81 11 706 9406. E-mail address: [email protected] (N. Ohta). 1010-6030/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotochem.2012.02.016
difficult to be combined with a microscope having z-axis resolution because of wavelength-dependent focal depth and absorption. Measurements of fluorescence lifetime of a fluorescent dye can enhance the potential of fluorescence microscopy [13–15]. Fluorescence lifetime is an inherent property of a dye molecule, and thus is independent of dye concentration, photobleaching, excitation light intensity, and other factors that limit fluorescence intensity measurements. This makes fluorescence lifetime imaging (FLIM) a powerful tool for quantitatively imaging pH in a single cell [16–21]. Neither of the ratio methods is necessary for imaging fluorescence lifetime, so that the FLIM method is compatible with confocal and multi-photon microscopes. The green fluorescent protein (GFP) has become an invaluable tool for fluorescent-imaging applications [22,23] and various kinds of GFP mutants have been developed for adding useful optical properties [9–12,22,23]. Each GFP-based protein has a characteristic chromophore structure that is responsible for absorption and fluorescence. The chromophore is rigidly encapsulated inside the barrel of the protein structure, which is thought to effectively block the non-radiative relaxation of the chromophore [22]. This unique structure results in the strong fluorescence in the visible region. The electrostatic and hydrogen-bonding interactions of the chromophore with surrounding amino acids are also responsible for fluorescence properties of GFP-based proteins. In our previous study, intracellular pH of a single cell could be imaged using FLIM of enhanced GFP (EGFP) that contains the F64L and S65T mutations [21]. The chromophore of EGFP consists of
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(HeLa) cells with varying intracellular pH, as preliminary reported in our previous paper [33]. 2. Experimental 2.1. Sample preparation
Fig. 1. The neutral and anionic forms of the chromophores of EGFP (a) and EYFP (b).
a p-hydroxybenzylidene-imidazolidinone that exists in either the neutral phenol form or the anionic phenolate form (Fig. 1a), the equilibrium of which depends on medium pH. These two forms exhibit distinct absorption and fluorescence characteristics, and the fluorescence lifetime of the anionic form is in the 2–3 ns range and that of the neutral form is in tens to hundreds of picoseconds [24–26]. It was shown in previous studies [21,25,26] that the fluorescence decay of EGFP or GFP(S65T) was observed to become faster with decreasing medium pH when the neutral chromophore was preferentially excited and the fluorescence signals both from the neutral and anionic chromophores were detected. The observed fluorescence decay can be regarded as a mixture of the decays of the excited neutral and anionic forms, and the pH-induced change in the average fluorescence lifetime was explained in terms of the acid–base equilibrium between the two forms in the ground state. The excited neutral form having a short fluorescence lifetime is preferentially formed, and the molar ratio of the neutral form increases as the medium pH decreases, which results in the reduction of the average fluorescence lifetime with lowering pH. Such a pH-induced change in average fluorescence lifetime of EGFP was observed in a single cell, which can be used as pH imaging of living systems [21]. The pH imaging using the difference in fluorescence lifetime between the neutral and anionic chromophores can also be applied for other GFP-based proteins because the neutral chromophore having a short fluorescence lifetime exists in many GFP-based proteins. It is therefore conceivable that FLIM of GFP-based proteins becomes a general method for measuring pH in a cell. The pKa between the neutral and anionic forms of EGFP is estimated to be 5.6–6.0 [21,25,27], and the significant pH-induced alteration of the average fluorescence lifetime occurs in an acidic region of pH 5–6.5. It is thus important to measure effects of pH on the average fluorescence lifetime of other GFP-based proteins having a pKa near neutral. Enhanced yellow fluorescent protein (EYFP) is one of the most widely used variants of GFP from Aequoria victoria (wild-type GFP) [23,28–32]. EYFP contains the mutations replacing serine 65 with glycine, valine 68 with leucine, serine 72 with alanine, and threonine 203 with tyrosine in wild-type GFP. EYFP has the same chromophore structure as that of EGFP, and the – interaction of the chromophore with Tyr203 induces the red shift of the fluorescence to the yellow region. The chromophore of EYFP also shows the acid–base equilibrium in the ground state (Fig. 1b), the pKa of which is 6.5–6.9 in high chloride concentrations [23,29,30] that is in the physiological range of many living systems. In the present study, effect of pH on the average fluorescence lifetime of EYFP in buffer has been examined with several excitation wavelengths. We could also measure FLIM of EYFP in human cervical carcinoma
HeLa cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 2 × 105 U dm−3 penicillin G, 200 mg dm−3 streptomycin sulfate, and 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37 ◦ C. HeLa cells were transfected overnight with plasmid DNA of pEYFP-C1 (Clontech, BD Biosciences, Oxford, UK) using Optifect (Invitrogen) in LAB-Tek 8well chambered coverslips (Nalge Nunc International). Calibration of intracellular pH was carried out by the so-called nigericin/high K+ method [5,16,18,34,35]. HeLa cells expressing EYFP grown on LAB-TEK chambered coverslips with eight wells were washed with KCl-rich media (125 mM KCl, 20 mM NaCl, 10 mM HEPES, 10 mM MES, 0.5 mM CaCl2 , 0.5 mM MgCl2 ) and then incubated with the same KCl-rich media containing 10 g/ml nigericin at different pH. Nigericin is a kind of K+ /H+ ionophore, which equilibrates protons across plasma membrane in the presence of a depolarizing extracellular concentration of K+ . For fluorescence decay measurements in buffer solution, EYFP was purified from E. coli carrying the plasmid DNA of pEYFP-C1 (Clontech) by nickel column chromatography (GE Healthcare). The stock solution of EYFP was then diluted in phosphate buffered saline (PBS) buffer (154 mM NaCl, 6 mM Na2 HPO4 , 1.1 mM KH2 PO4 ) at different pH. 2.2. Apparatus Measurements of fluorescence lifetime image were carried out using a confocal microscope with a four-channel time-gated detection system [36]. A full description of the experimental system was reported elsewhere [20,21]. Output pulses from a femtosecond mode-locked Ti:sapphire laser (Spectra Physics, Tsunami) were frequency doubled and used as an excitation light. The pulse duration and the repetition rate of the laser pulse were 80 fs and 81 MHz, respectively. The excitation beam was introduced into a scanning confocal microscope (Nikon, C1) and then was focused onto the sample with a 40× objective (Nikon, CFI S Fluor, NA 0.90). The fluorescence collected by the same objective was transmitted into a filter box equipped with interference filters (Nikon, BA520, EX510-560). Fluorescence was detected using a photomultiplier in a high-speed lifetime imaging module (Nikon Europe BV, LIMO). The lifetime imaging module captures the fluorescence decay into four time windows. Fluorescence lifetime at each pixel of the image was evaluated from the four time-windows signals by assuming a single exponential decay. The size of the image was 256 × 256 pixels. All of the time windows were set at 2.0 ns. The acquisition time was ∼20 min for each image. All the measurements were performed in a stage top incubator (INUG2-ONICS, Tokai Hit) with 5% CO2 at 37 ◦ C. Macroscopic measurements of fluorescence decay of EYFP in aqueous solution were carried out using a time-correlated singlephoton counting system [37]. The second harmonic of the output from the mode-locked Ti:sapphire laser was used for excitation. The repetition rate was reduced to be ∼5.8 MHz with a pulse picker (Conoptics, model 350-160). Fluorescence from the sample was dispersed by a monochromator and then detected by a microchannel-plate photomultiplier (Hamamatsu, R3809U-52). All the fluorescence decays were collected at a magic angle with respect to the vertically polarized excitation light. The instrumental response function (IRF) had a full width at half maximum (FWHM)
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Fig. 2. Absorption (dotted line) and fluorescence (solid line) spectra of EYFP in buffer solution. Excitation wavelength was 470 nm for the fluorescence measurement.
of ∼60 ps. The inverse Fourier-transform method was used for the simulation of the decay profile. The observed fluorescence decay was fitted with the convolution of the IRF with a multiexponential decay function. The convolution was mathematically done by the inverse Fourier-transform method. The simulation was performed by a home-made macro in the IGOR software (Wavemetrics). 3. Results and discussion 3.1. Absorption, fluorescence, and fluorescence decays of EYFP Fig. 2 shows absorption and fluorescence spectra of EYFP in PBS buffer at a neutral pH. The strong absorption and fluorescence bands with a peak at ∼514 and ∼530 nm, respectively, arise from the anionic chromophore of EYFP. These absorption and fluorescence intensities increase with increasing pH [29,32]. The neutral chromophore exhibits the absorption band around 400 nm that becomes dominant in acidic conditions. The broad fluorescence due to the neutral chromophore is observed around 460 nm with excitation at 400 nm [32]. In the present study, we have measured the pH dependence of the fluorescence decay of EYFP in buffer solution with the three excitation wavelengths: 470, 440, and 400 nm. The anionic and neutral forms of the EYFP chromophore are preferentially excited with the excitation wavelengths of 470 and 400 nm, respectively. Both the anionic and neutral chromophores are excited at 440 nm. The representative fluorescence decays of EYFP in buffer with the excitation wavelengths of 470, 440, and 400 nm are shown in Fig. 3a–c, respectively, where the maximum intensity is normalized to unity in all the decay profiles. The results at pH of 5.0, 6.5, and 7.5 are shown in this figure. The wavelength of the monitored fluorescence was 535 ± 5 nm in every case. In the present study, the observed fluorescence decays were fitted by the convolution of the IRF with a multi-exponential decay, i.e., i Ai exp(− t/ i ), where Ai and i denote the pre-exponential factor and the fluorescence lifetime of component i, respectively, and the obtained pre-exponential factors and the fluorescence lifetimes with the three excitation wavelengths are shown in Tables 1–3. The fluorescence lifetimes whose preexponential facthese tables. tors are more than 1% are shown in The average fluorescence lifetime ( ave ) is given by i Ai i / i Ai . Plots of ave against pH are shown in Fig. 4. 3.2. Fluorescence decays with excitation at 470 nm As shown in Table 1, three decaying components were used to fit the decay profiles with excitation at 470 nm. The component having a fluorescence lifetime of ∼3 ns, i.e., component (1), can be attributed to the excited state of the anionic chromophore formed by the excitation at 470 nm. This component was dominant in all the decay profiles, and its fluorescence lifetime, i.e.,
Fig. 3. Representative fluorescence decays of EYFP in buffer solution at pH of 5.0 (red), 6.5 (green), and 7.5 (blue) at the excitation wavelengths of 470 nm (a), 440 nm (b), and 400 nm (c). The total photon counts at each excitation wavelength are in the range of 4 × 104 –2 × 105 (400 nm), 6 × 104 –1 × 106 (440 nm), and 2 × 105 –2 × 106 (470 nm). The intensities are plotted in a linear scale. In all the decay profiles, the maximum intensity is normalized to unity. Fluorescence was observed at 535 ± 5 nm in every case. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Table 1 pH dependence of the fitting parameters of fluorescence decays of EYFP with excitation at 470 nm.a,b pH
1 (ns)
9 8 7.5 7 6.5 6 5.5 5
3.2 (1.00) 3.2 (1.00) 3.2 (1.00) 3.2 (0.97) 3.2 (1.00) 3.1 (0.94) 3.1 (0.90) 2.8 (0.78)
2 (ns)
3 (ns)
0.30 (0.03) 0.30 (0.05) 0.30 (0.09) 0.30 (0.20)
9.0 (0.01) 9.0 (0.01) 9.0 (0.02)
a Pre-exponential factor of each component, whose summation is normalized to unity in the decay, is given in parentheses. b Experimental errors of 1 , 2 and 3 are ca. ±10, ±50 and ±30%, respectively.
Table 2 pH dependence of the fitting parameters of fluorescence decays of EYFP with excitation at 440 nm.a,b pH
1 (ns)
2 (ns)
9 8 7.5 7 6.5 6 5.5 5
3.2 (0.94) 3.2 (0.93) 3.2 (0.94) 3.2 (0.92) 3.2 (0.82) 3.2 (0.10)
0.30 (0.06) 0.30 (0.06) 0.30 (0.06) 0.30 (0.07) 0.25 (0.17) 0.15 (0.04) 0.15 (0.01)
3 (ns)
4 (ns)c
9.0 (0.01) 9.0 (0.01) 9.0 (0.01) 0.001 (0.86) 0.001 (0.99) 0.001 (1.00)
a Pre-exponential factor of each component, whose summation is normalized to unity in the decay, is given in parentheses. b Experimental errors of 1 , 2 and 3 are ca. ±10, ±50 and ±30%, respectively. c 4 was fixed to be 1.4 ps.
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Table 3 pH dependence of the fitting parameters of fluorescence decays of EYFP with excitation at 400 nm.a,b pH
1 (ns)
2 (ns)
3 (ns)
9 8 7.5 7 6.5 6 5.5 5
3.2 (0.86) 3.2 (0.79) 3.2 (0.82) 3.2 (0.47) 3.2 (0.16) 3.1 (0.09) 3.0 (0.06) 2.8 (0.04)
0.30 (0.13) 0.15 (0.20) 0.20 (0.17) 0.30 (0.21) 0.30 (0.12) 0.35 (0.12) 0.35 (0.18) 0.35 (0.13)
9.0 (0.01) 9.0 (0.01) 9.0 (0.01) 9.0 (0.01)
4 (ns)
0.04 (0.31) 0.04 (0.72) 0.05 (0.79) 0.05 (0.76) 0.05 (0.83)
a Pre-exponential factor of each component, whose summation is normalized to unity in the decay, is given in parentheses. b Experimental errors of 1 , 2 , 3 and 4 are ca. ±10, ±50, ±30 and ±30%, respectively.
Fig. 4. Plots of the average fluorescence lifetime of EYFP in buffer solution against pH. Excitation wavelengths were 470 (green), 440 (red), and 400 nm (blue), respectively. The simulated curves at the 440- and 400-nm excitation are also shown by dotted lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
∼3.2 ns at neutral pH, is similar to those reported in previous studies [31,32]. The component having a lifetime of hundreds of picoseconds, i.e., component (2), was confirmed, and its pre-exponential factor increased with decreasing medium pH. Since the neutral chromophore is hardly excited at 470 nm, the component (2) may be ascribed to photoconverted EYFP species or the isomer of the EYFP chromophore arising from different interactions with surrounding amino acids in the barrel structure (Fig. 5). It was reported that photoconversion of EYFP occurred in the presence of green (514 or 532 nm) light [32]. It might be necessary to consider component (3) having a long lifetime of ∼9 ns to reproduce the decay in the time range longer than 10 ns; however, the pre-exponential factor of component (3) was negligibly small in neutral and alkali conditions. In pH 6.5–9.0, the lifetime of the component (1) remains almost unchanged with pH, and significant pH dependence of ave was not observed (Fig. 4). In acidic conditions, fluorescence intensity becomes weak due to the shift of the equilibrium of the
Fig. 5. The diagram of the neutral and anion species of the chromophore of EYFP and the photoconverted species.
chromophore to the neutral form. As shown in Fig. 4, the observed ave also becomes shorter with lowering pH in acidic conditions of pH 5.0–6.0, which results from the decrease in both A1 and 1 with decreasing pH. The reduction of fluorescence lifetime in acidic conditions following photoexcitation of the anionic chromophore was also observed for GFP(S65T) [26]. The alteration of 1 suggests the change in the intermolecular interaction of the anionic chromophore with amino acids in acidic conditions. 3.3. Fluorescence decays with excitation at 440 nm Both the neutral and anionic chromophores are excited with excitation at 440 nm. The component (1) having a lifetime of ∼3 ns can be assigned to the anionic chromophore in the excited state (Table 2). The component (2) having a lifetime of 0.15–0.30 ns may also be assigned to the photoconverted species or the isomer of the EYFP chromophore that is observed at the 470-nm excitation. In alkali and neutral conditions of pH 9.0–6.5, the component (3) having a long lifetime was confirmed, but the pre-exponential factor of which was 1% or less. The fast decaying component, i.e., component (4), becomes dominant in acidic conditions of pH 5–6, and its pre-exponential factor increases with lowering pH. This component is ascribed to the excited neutral chromophore formed by the excitation at 440 nm. The lifetime of the component (4) was shorter than the time-width of IRF. In the present study, therefore, the 4 value at the 440-nm excitation was assumed to be 1.4 ps, which is the reported value of the fluorescence lifetime of the neutral chromophore of EYFP [32]. As shown in Fig. 4, the correlation between the observed ave and pH at the 440-nm excitation was satisfactorily fitted by the following equation [27]: ave = a + b[1 + 10nH (pKa −pH) ]−1
(1)
where a and b are the offset and dynamic range, respectively; pKa is pH at the half of the dynamic range; nH is Hill coefficient. It should be noted that the observed fluorescence decay can be regarded as a mixture of the decays of each component, and ave was evaluated by the decay profile of the mixture. The fitted curve in Fig. 4 was obtained with pKa and nH of 6.3 and 4.5, respectively. The large Hill coefficient may arise from the very large difference between the lifetimes of the neutral and anionic chromophores. The obtained pKa is smaller than that of the neutral and anionic forms of the EYFP chromophore evaluated from the absorption and fluorescence spectra (pKa of 6.5–6.9) [23,29,30]. This difference probably arises from the difficulty in evaluation of the pre-exponential factor of the component (4), whose lifetime is shorter than the time-width of IRF. 3.4. Fluorescence decays with excitation at 400 nm The neutral form is preferentially excited by photoirradiation at 400 nm. As shown in Table 3, however, the decay profiles exhibited the component (1) having a fluorescence lifetime of ∼3 ns, which results from the excited anionic chromophore. It was reported that the photoexcited neutral chromophore of EYFP decayed with two non-radiative processes: direct non-radiative relaxation to the ground state and excited-state intramolecular proton transfer (ESIPT) to generate the excited anionic form. The direct nonradiative relaxation to the ground state was suggested to be much faster than the ESIPT to account for the very low fluorescence yield of the neutral chromophore [32]. The excited anionic chromophore observed following the 400-nm excitation is therefore considered to be generated by the direct excitation of the anionic form according to its low absorption cross section at 400 nm. As shown in Table 3, the pre-exponential factor of the anionic chromophore
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satisfactorily reproduced by Eq. (1) (Fig. 4), and pKa and nH were evaluated to be 7.0 and 1.5, respectively. 3.5. Fluorescence lifetime images of HeLa cells expressing EYFP
Fig. 6. The expansion of representative fluorescence decays of EYFP in buffer solution at pH 5.0 in the −0.2 to 0.7 ns region. The excitation wavelengths were 470 nm (green), 440 nm (red), and 400 nm (blue). The intensities are plotted in a linear scale. In all the decay profiles, the maximum intensity is normalized to unity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
decreases with lowering pH, which results from the shift of the acid–base equilibrium to the neutral form. Another fluorescence component having a lifetime of 40–50 ps, i.e., component (4), becomes dominant at pH less than 7, and its pre-exponential factor increases with lowering pH. This component can be attributed to the excited species of neutral chromophore, though its fluorescence lifetime is larger than those obtained with the 440-nm excitation (see Fig. 6). It probably comes from the presence of photoconverted species generated by the photoexcitation of the neutral choromophore of EYFP. The very short lifetime component obtained by the 440-nm excitation was not distinguished, which may be due to the interference of the existence of the component (4); both the lifetimes are shorter than the FWHM of the IRF. The correlation between ave and pH at the 400-nm excitation was
Fluorescence lifetime images of HeLa cells expressing EYFP were observed at different values of intracellular pH. Excitation wavelength was 440 nm and the fluorescence at 515–560 nm was detected. The results are shown in Fig. 7. The corresponding fluorescence intensity image is shown on the top of each fluorescence lifetime image. The intracellular pH can be evaluated by measuring the extracellular one because ionophore was added in the medium as mentioned in Experimental section. The lifetimes at different pHs showed only the small change in the intracellular pH ranging from 7.0 to 9.0. On the other hand, the remakable pH dependence of the lifetime was observed in intracellular pH less than 6.0. Fig. 7b shows the histograms of the fluorescence lifetime of HeLa cells expressing EYFP with varying intracellular pH. Plots of the average fluorescence lifetime against the intracellular pH are shown in Fig. 7c. The average fluorescence lifetime was evaluated from the peak of the histogram. It can be seen in Fig. 7c that the averaged fluorescence lifetime in cells remains almost unchanged in intracellular pH 7.0–9.0 and becomes shorter with decreasing intracellular pH especially at pH less than 6. For example, the lifetime is ∼2.52 ns at pH 6.0, and then decreases to ∼1.96 ns at pH 4.5. Thus, the significant pH dependence of the average fluorescence lifetime in cells is found in acidic conditions at the 440-nm excitation. The magnitude of the reduction of the observed lifetime is ca. 20%, when pH is changed from 6 to 4.5. From the observation of the significant pH dependence of the fluorescence lifetime of EYFP in HeLa cells, it is concluded that pH in a single cell can be evaluated using FLIM of EYFP in pH 4.5–6.0. Cells contain endogenous chromophores showing fluorescence called autofluorescence, and
Fig. 7. (a) Fluorescence intensity images (upper) and corresponding fluorescence lifetime images (lower) of HeLa cells expressing EYFP. The intracellular pH is shown on the top of each intensity image. (b) Intracellular pH dependence of the histogram of the fluorescence lifetime of HeLa cells expressing EYFP. (c) Plots of the average fluorescence lifetime of HeLa cells expressing EYFP against the intracellular pH. The average fluorescence lifetime was evaluated from the peak of the histogram in (b). Excitation wavelength was 440 nm and the fluorescence in the wavelength region of 515–560 nm was detected. pH dependence of the average fluorescence lifetime of EYFP in buffer solution at the excitation wavelength of 470 nm (this result is the same as that in Fig. 4) is also shown in (c) for comparison.
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autofluorescence in nuclei is much weaker than that in other areas, resulting in the observation of the dull nucleus region in autofluorescence images [38,39]. In the present study, the dull nucleus was not distinguishable in the intensity images (Fig. 7a), indicating that the contribution of autofluorescence was negligible. It is noted that it was difficult to measure the pH dependence of FLIM of EYFP in cells at the 400-nm excitation, owing to the very weak fluorescence intensity. In EGFP-expressed HeLa cells, FLIM images were found to depend on intracellular pH [21], which was ascribed to the pH-dependent equilibrium between the anionic and neutral forms of the EGFP chromophore in the ground state, the fluorescence lifetimes of which are in the 2–3 ns range and in tens to hundreds of picoseconds, respectively. In contrast with EGFP, the fluorescence lifetime of the neutral form of EYFP was 0.4–3 ps [32] that is too short to be detected in each timewindow (2 ns) of the present FLIM measurements. Furthermore, a part of the decaying portion following photoexcitation was cut in the FLIM measurements to exclude scattered light, and the signal at the first window was integrated from ca. 0.5 to 1 ns after photoexcitation [40]. It is therefore unlikely that the observed pH dependence of FLIM of EYFP results from a mixture of the fluorescence decays of the excited neutral and anionic chromophores. The observed fluorescence in the FLIM measurements can be therefore considered to arise from the anionic chromophore, and the pH dependence of the average fluorescence lifetime is ascribed to a change in environment surrounding the anionic chromophore induced by the change in pH. This result is consistent with the observation in buffer solution following excitation of the anion chromophore in Fig. 4: ave obtained with the 470-nm excitation became shorter with lowering pH in pH 5.0–6.0, and the magnitude of the change in ave between pH 5 and 6 was ca. 20%. It is thus concluded that the observed pH dependence of FLIM of EYFP results from the change in the protein structure due to the change in acidic property: the fluorescence lifetime of the anionic chromophore in EYFP decreases with an increase of the acidic property. In addition to the pH dependence of the fluorescence lifetime, a non-uniformity of the fluorescence lifetime of EYFP was observed in a cell, as preliminary reported [33]. At pH 4, for example, the average fluorescence lifetime at the peripheral parts of cells was longer than those inside the cells; the lifetime was ∼2.6 ns at the peripheral parts, while that was around 2.2 ns in other areas. A fluctuation of the fluorescence lifetime was also observed in EGFPexpressed HeLa cells, but the opposite relation was found: the lifetime of EGFP at the peripheral parts of cells was shorter than that in other areas [33]. Both the average fluorescence lifetimes of EYFP and EGFP become shorter with lowering pH. It is unlikely that pH at the peripheral parts of cells is higher and lower than those in other areas, respectively, in the EYFP-expressed cells and in the EGFP-expressed cells. Therefore, we consider that the origin of the variation of the fluorescence lifetime in a cell is attributed to others than the pH dependence. It has been reported that strong electric fields induce the alteration of the fluorescence lifetime and that the field effects on fluorescence lifetime opposite to each other may appear, depending on the chromophore. For example, the fluorescence lifetime of GFPuv5 was shortened by application of electric fields [41], while the fluorescence lifetime of a dimethyl derivative of p-hydroxybenzylidene-imidazolidinone, that is a model compound of the chromophore of GFP, was lengthened by application of electric fields [42]. Then, the present non-uniformity of the lifetime at the peripheral parts of cells may result from the field effects due to membranes and intracellular substances, and the fluorescence lifetime of EYFP may give the opposite electric field effect to that of EGFP.
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