Letter pubs.acs.org/ac
Dual-Color Imaging of Magnesium/Calcium Ion Activities with TwoPhoton Fluorescent Probes Xiaohu Dong,†,∥ Ji Hee Han,‡,∥ Cheol Ho Heo,§ Hwan Myung Kim,*,§ Zhihong Liu,*,† and Bong Rae Cho*,‡ †
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China ‡ Department of Chemistry, Korea University, 1-Anamdong, Seoul 136-701, Korea § Division of Energy Systems Research, Ajou University, Suwon 443-749, Korea S Supporting Information *
ABSTRACT: We report two-photon probes (FMg1 and FMg2) that can selectively detect intracellular free Mg2+ ([Mg2+]i) in live cells and tissues by twophoton microscopy. Combined with BCaM, a two-photon probe for nearmembrane Ca2+ ([Ca2+]m), FMg2 allows dual-color imaging of Mg2+/Ca2+ activities in live cells and [Mg2+]i /[Ca2+]m distributions in live tissues at a depth of 100−200 μm.
M
Scheme 1. Structures of BCaM, FMg1, FMg1-AM, FMg2, and FMg2-AM
agnesium ion is the most abundant divalent metal ion in mammalian cells and a vital component of numerous enzymes, ATP, and nucleic acids. 1−3 The total Mg 2+ concentration in the cell is approximately 10−30 mM, of which 0.2−1.0 mM exists as cytosolic free Mg2+ ([Mg2+]i).4 It modulates signal transduction, various transporters, and ion channels. Calcium ion, another alkaline earth metal ion, is an ubiquitous second messenger that controls various functions in cells.5,6 The two metal ions compete for the divalent cation binding sites such as DNA and RNA,7,8 Ca2+/Mg2+ exchange is vital to the Ca2+-regulated muscle contraction,9 and Mg2+ promotes Ca2+ influx in various tissues.10 To understand these processes in biology, it is crucial to monitor Mg2+/Ca2+ activities in live cells and tissues. An attractive approach to the detection of Mg2+ and Ca2+ deep inside live tissues is dual-color imaging with two-photon microscopy (TPM). TPM, which utilizes two photons of lower energy for the excitation, can visualize biological activities deep inside intact tissues (>500 μm) for extended periods of time.11,12 Recently, we reported a two-photon (TP) probe for near membrane Ca2+ ([Ca2+]m) (BCaM), which shows significant TP action cross section (Φδmax = 150 GM), emission maximum (λfl) at 470 nm, and a dissociation constant 13 (KTP With BCaM in hand, the dual-color d ) of 89 ± 3 μM. 2+ imaging of [Ca ]m and [Mg2+]i would be possible, if one develops a TP probe for Mg2+ with significant Φδmax, λfl > 550 nm, and KTP d ≈ 1 mM. To this end, we have developed TP probes for Mg2+ derived from 2-acetyl-7-diethylamino-9,9dimethyl-9H-fluorene as the fluorophore14 and o-aminophenolN,N,O-triacetate (APTRA) as the receptor for Mg2+(Scheme 1),15,16 respectively. We have utilized fluorene derivatives because 2-diethylamino-7-nonanoyl-9,9-dimethyl-9H-fluorene © 2012 American Chemical Society
has been shown to exhibit a Φδmax value of 400 GM with λfl at 555 nm in MeOH14 and APTRA from one-photon fluorescent probes for Mg2+ such as mag-fura-2.16,17 Herein, we report that FMg1 and FMg2 are efficient TP probes for Mg2+ and that, by using BCaM and FMg2, one can simultaneously monitor Ca2+/Mg2+ activities in live cells and detect [Ca2+]m and [Mg2+]i in live tissues at a depth of more than 100 μm for lengthy periods of time without photobleaching problems. The preparation of FMg1 and FMg2 and their acetoxylmethyl (AM) esters is described in the Supporting Information. The solubilities of FMg1-AM and FMg2-AM in Received: August 2, 2012 Accepted: September 11, 2012 Published: September 11, 2012 8110
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Table 1. Photophysical Data for FMg1 and FMg2 compda FMg1-AM FMg1 FMg1+Mg2+ FMg2-AM FMg2 FMg2+Mg2+
b λ(1) max
360 362 362 368 368 368
λflmaxb 539 540 540 553 555 555
Φc 0.011 0.0095 0.18 0.0069 0.0058 0.12
TPd KOP d /Kd
FEF/TFEFe
f λ(2) max i
1.6/1.5
18/16
1.4/1.7
20/24
nd ndi 740 ndi ndi 740
δg i
nd ndi 483 ndi ndi 633
Φδh ndi ndi 87 ndi ndi 76
All data were measured in 30 mM MOPS, 100 mM KCl, 10 mM EGTA, pH 7.2 in the absence and presence of 100 mM free Mg2+. bλmax of the one-photon absorption and emission spectra in nm. cFluorescence quantum yield, ±10%. dDissociation constants for Mg2+ in mM measured by onee TP (KOP d ) and two-photon (Kd ) processes, ±14%. Fluorescence enhancement factor, (F − Fmin)/Fmin, measured by one- (FEF) and two-photon (TFEF) processes. fλmax of the two-photon excitation spectra in nm. gThe peak two-photon cross section in 10−50 cm4s/photon (GM), ±15%. h Two-photon action cross section. iThe two-photon excited fluorescence intensity was too weak to measure the cross section accurately. a
3-(N-morpholino)propanesulfonic acid (MOPS) buffer solution (30 mM, 100 mM KCl, pH 7.2) as determined by the fluorescence method13 were approximately 3 μM, which was sufficient to stain the cells (Figure S1 in the Supporting Information). Photophysical properties of FMg1 and FMg2 were studied in MOPS buffer. FMg1 and FMg2 showed absorption maxima (λabs) at 362 nm (ε = 3.69 × 104 M−1 cm−1) and 368 nm (ε = 3.10 × 104 M−1 cm−1) with fluorescence maxima (λfl) at 540 nm (Φ = 0.0095) and 555 nm (Φ = 0.0058), respectively (Table 1). The slight bathochromic shifts observed for FMg2 is likely due to the stronger electron-withdrawing ability of the acetyl group in FMg2 than the amide group in FMg1, which would have enhanced the intramolecular charge transfer. The λabs remained nearly the same, while the λfl of FMg1 showed gradual red shifts with increasing solvent polarity (Figure S2 and Table S1 in the Supporting Information). A similar result was observed for FMg2 (Figure S3 and Table S1 in the Supporting Information), except that both λabs and λfl were slightly red-shifted presumably because of the stronger electron acceptor (see above). When Mg2+ was added to FMg1 and FMg2 in MOPS buffer, the fluorescence intensity increased dramatically without affecting the absorption spectra, presumably due to the blocking of the photoinduced electron transfer (PeT) process from APTRA to fluorophore upon complexation with Mg2+ (Figures S4a−d in the Supporting Information). Nearly identical results were observed in the TP processes (Figure 1a and Figure S4e in the Supporting Information). The fluorescence enhancement factors (FEF = (F − Fmin)/Fmin) of FMg1 and FMg2 determined for the TP processes were 16 and 24, respectively, in the presence of excess Mg2+ (Table 1). The TP dissociation constants (KOP d and Kd ) of FMg1 and FMg2 for the one- and two-photon processes were calculated from the fluorescence titration curves.18 The titration curves fitted well with a 1:1 binding model (Figures 1b and Figure S4 in the Supporting Information), the Hill plots were linear with a slope of 1.0 (Figures S4f and S5a,b in the Supporting Information),19 and the Job’s plots exhibited maxima at the mole fraction 0.50 (Figure S5c,d in the Supporting Information),20 indicating 1:1 complexation between the probes and Mg2+. The KTP d values of FMg1 and FMg2 for Mg2+ were 1.5 ± 0.1 mM and 1.7 ± 0.2 mM (Table 1), respectively, which are suitable to detect [Mg2+]i in the cells. FMg1 and FMg2 showed appreciable responses toward Ca2+ and Zn2+ and weak responses toward Fe2+, Co2+, Ni2+, Cu2+, and Mn2+ (Figure S6a,b in the Supporting Information), a result consistent with other Mg2+ ion probes with the same
Figure 1. (a) Two-photon excited fluorescence (TPEF) spectra of 1 μM FMg2 in the presence of free Mg2+ (0−100 mM). (b) One- and two-photon fluorescence titration curves for the complexation of FMg2 with Mg2+ ions at various concentrations of free Mg2+ (0−100 mM). (c) TP action spectra of FMg1 and FMg2 in the presence of 50 mM free Mg2+. These data were measured in 30 mM MOPS buffer (100 mM KCl, 10 mM EGTA, pH 7.2). (d) TPEF spectra of BCaM and FMg2-AM in HepG2 cells. The one- and two-photon excitation was provided at 368 and 740 nm, respectively.
receptor.15,16 Moreover, the KOP d values of FMg1 and FMg2 calculated from the titration curves (Figure S6c−h in the Supporting Information) for Ca2+ were 8.8 ± 0.1 and 9.8 ± 0.3 μM, while those for Zn2+ were 24 ± 2 and 16 ± 1 nM, respectively. This indicates that FMg1 and FMg2 have considerable affinity for Ca2+ and Zn2+ in aqueous buffer. Nevertheless, since [Mg2+]i is much greater than [Ca2+]i and free Zn2+ ion rarely exists in the cells,4,7 these probes can detect [Mg2+]i with minimum interference from other competing metal ions. Moreover, FMg1 and FMg2 are pH-insensitive in the biologically relevant pH range (Figure S6i,j in the Supporting Information). We then evaluated the abilities of FMg1 and FMg2 to detect [Mg2+]i in a TP mode. The TP action spectra of FMg1 and FMg2 in MOPS buffer containing excess Mg2+ indicated Φδmax values of 87 and 76 GM at 740 nm, respectively (Figure 1c), which are comparable to those of existing TP probes.12,13 These values allowed us to obtain bright TPM images of the cells and tissues that were labeled with FMg2-AM and FMg1AM (Figure 2 and Figure S7 in the Supporting Information). The two-photon excited fluorescence (TPEF) intensity 8111
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It was reported that receptors that trigger a Ca2+ influx through PLCγ1 can induce Mg2+ influx to regulate the Ca2+ influx.10 To visualize such activities, we have monitored TPM images of HepG2 cells colabeled with BCaM and FMg2-AM. The TPM images of the probe-labeled HepG2 cells clearly revealed the distribution of [Ca2+]m and [Mg2+]i (Figure 3a−c
Figure 2. (a−d) TPM images of HepG2 cells labeled with 2 μM FMg2-AM. (b) Cells were treated with 50 mM MgCl2 and 5 μM calcimycin. (c) Cells were treated with 2 mM EDTA and 5 μM calcimycin. (e) The relative TPEF intensity from the regions A−C in part d as a function of time. The digitized intensity was recorded with 2.0 s intervals for the duration of 1 h using the xyt mode. The TPEF intensities were collected at 525−600 nm upon excitation at 740 nm with a femtosecond (fs) pulse. Cells shown are representative images from replicate experiments (n = 5). Scale bar: 20 μm. Figure 3. (a−c) Dual-channel TPM images of HepG2 cells colabeled with BCaM (1 μM) and FMg2-AM (2 μM) collected at 400−450 (BCaM, Ch1) and 525−600 nm (FMg2-AM, Ch2), respectively. TPM images were obtained in PBS buffer (a), 200 s after stimulation with 5 μM calcimycin and 10 ng/mL EGF in the presence of 1.2 mM Mg2+ (b) or no Mg2+ (c). (d,e) Time course of TPEF at designated positions A (green curve) and B (red curve) in parts b and c, respectively, after stimulation. The TPEF intensities at A and B in parts b and c were measured before stimulation and normalized. Cells shown are representative images from replicate experiments (n = 5). Excitation wavelength: 740 nm. Scale bar: 15 μm.
increased (Figure 2b and Figure S7b in the Supporting Information) after treatment of the FMg2-AM-labeled cells with 50 mM MgCl2 and 5 μM calcimycin, a divalent cation ionophore which allows Mg2+ to cross the cell membrane, and decreased upon treatment with 5 μM calcimycin and 2 mM N,N,N′,N′-ethylenediaminetetraacetic acid (EDTA), a membrane permeable heavy metal ion-chelator that can effectively remove Mg2+ ions from the cell (Figure 2c and Figure S7c in the Supporting Information). These results confirmed that the bright regions in the TPM images reflect the presence of [Mg2+]i. FMg1 and FMg2 have additional benefits of high photostability as revealed by the negligible changes in the TPEF intensity in the probe-labeled HepG2 cells over 60 min (Figure 2d,e and Figure S8 in the Supporting Information) and a small decrease in cell viability at a higher probe concentration (>10 μM) as measured by a Cell Counting Kit (CCK)-8 assay (Figure S9 in the Supporting Information). We next sought to utilize FMg2-AM and BCaM as TP probes to investigate Mg2+/Ca2+ activities in HepG2 cells by dual-color imaging. Using 740 nm TP excitation in a scanning lambda mode, HepG2 cells labeled with BCaM, FMg1-AM, and FMg2-AM emitted broad spectra with λfl at 450, 470, and 525 nm (Figure S10 in the Supporting Information), respectively, which are blue-shifted from those measured in the MOPS buffer. This indicates that the polarity of the probe environment is rather homogeneous and slightly more hydrophobic than that of MOPS buffer. Moreover, the TPEF spectra of BCaM and FMg2-AM are separated by 75 nm, such that their TPEF intensities can be selectively detected by using appropriate detection windows. Since the two spectra overlapped appreciably, we have chosen half of the bands, that is, 400−450 (Ch1, BCaM) and 525−600 nm (Ch2, FMg2-AM), as the detection windows. Under this condition, the TPEF of FMg2-AM contributes 30% of the total TPEF in Ch1, while that of BCaM contributes 15% of the total TPEF in Ch2. Nevertheless, it was possible to detect [Ca2+]m and [Mg2+]i by dual-color imaging (see below).
and Figure S11 in the Supporting Information). When the cells were stimulated with 5 μM calcimycin and 10 ng/mL of epidermal growth factor (EGF), a reagent that causes a PLCγ1dependent Ca2+ influx,21 in the presence of 1.2 mM Mg2+, the two-photon excited fluorescence (TPEF) intensity increased sharply in the plasma membrane until it reached the peak intensity and then decreased to the baseline level (Figure 3d, green curve). A similar result was observed in the cytoplasm (Figure 3d, red curve), except that the rate was slower. This outcome indicates that the EGF-induced transport of Ca2+ occurs at a faster rate than that of Mg2+. When the same experiment was conducted in a Mg2+-depleted solution, the TPEF intensity increased in the plasma membrane (Figure 3e, green curve) to a lesser extent and remained nearly the same in the cytoplasm (Figure 3e, red curve). This indicates that the EGF-induced influx of Ca2+ is decreased, while that of Mg2+ is abrogated, by Mg2+ depletion, which concurred with literature results.10 Therefore, FMg2 and BCaM are clearly capable of monitoring Mg2+/Ca2+ activities by dual-color imaging. We further investigated the utility of this probe in tissue imaging. TPM images were obtained from a slice of a 14-dayold SD rat hippocampal tissue incubated with 10 μM BCaM and 20 μM FMg2-AM for 60 min at 37 °C. The slice from the brain was too large to show with one image, so two images were obtained in each plane and combined. The bright-field image revealed the CA1 and CA3 regions and also the dentate gyrus (DG; Figure 4a). As the structure of the brain tissue is known to be inhomogeneous in its entire depth, we 8112
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Letter
ACKNOWLEDGMENTS This work was supported by a grant of the KHT R&D Project, Ministry of Health & Welfare (Grant No. A111182) and NRF Grant (Grant Nos. 2010-0018921, 2011-0018396, 20120005860, and 2011-0028663) funded by the Korean Government, NSF of China (Grant No. 21075094), and the Science Fund for Creative Research Groups (Grant Nos. 20621502 and 20921062).
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Figure 4. Images of a SD rat hippocampal slice colabeled with BCaM (10 μM) and FMg2-AM (20 μM). (a) Bright-field images of the CA1CA3 regions as well as dentate gyrus (DG) at 10-fold magnification. (b) 10 TPM images collected at Ch1 and Ch2 (a) along the zdirection at the depths of approximately 100−200 μm were accumulated and then merged. (c−e) TPM images of CA1 regions collected at (c) Ch1 and (d) Ch2 at a depth of about 100 μm at 100fold magnification. (e) Merged image of parts c and d. Excitation wavelength: 740 nm. Scale bars: 30 μm (b) and 300 μm (c).
accumulated 10 TPM images at depths of 100−200 μm to visualize the distributions of the [Ca2+]m and [Mg2+]i ions (Figure 4b). The TPM images collected from Ch1 and Ch2 revealed the [Ca2+]m and [Mg2+]i distributions, which did not merge (Figure 4b−e and Figure S12b−d in the Supporting Information). This outcome confirms that BCaM and FMg2 can independently detect [Ca2+]m and [Mg2+]i. The images taken at a higher magnification at a tissue depth of about 100 μm clearly revealed [Ca2+]m and [Mg2+]i distributions in the pyramidal neuron layer composed of cell bodies in the CA1 region (Figure 4c,d). Thus, dual-channel imaging of [Ca2+]m and [Mg2+]i is clearly possible at a 100−200 μm depth in live tissues by TPM using BCaM and FMg2 as the probes. In conclusion, we have developed TP probes (FMg1 and FMg2) that show 16−24-fold TPEF enhancement in response to Mg2+, high selectivity for Mg2+, maximum TP action cross section values of 76−87 GM in the presence of excess Mg2+, dissociation constants (KTP d ) of (1.5−1.7) ± 0.2 mM, and pH insensitivity in the biologically relevant range. Combined with BCaM, FMg2 allows dual-color imaging of Ca2+/Mg2+ activities in live cells and [Mg2+]i/[Ca2+]m distributions in live tissues at a depth of 100−200 μm without photobleaching artifacts.
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REFERENCES
(1) Magnesium and the Cell; Birch, N. J., Ed.; Academic Press: San Diego, CA, 1993. (2) Cowan, J. A. Biometals 2002, 15, 225−235. (3) Yang, W.; Lee, J. Y.; Nowotny, M. Mol. Cell 2006, 22, 5−13. (4) Romani, A. M. Front. Biosci. 2007, 12, 308−331. (5) Orrenius, S.; Zhivotovsky, B.; Nicotera, P. Nat. Rev. Mol. Cell Biol. 2003, 4, 552−565. (6) Rizzuto, R.; Pozzan, T. Physiol. Rev. 2006, 86, 369−408. (7) Hogan, P. G.; Lewis, R. S.; Rao, A. Annu. Rev. Immunol. 2010, 28, 491−533. (8) Davis, J. P.; Rall, J. A.; Reiser, P. J.; Smillie, L. B.; Tikunova, S. B. J. Biol. Chem. 2002, 277, 49716−49726. (9) Finley, N.; Dvoretsky, A.; Rosevear, P. R. J. Mol. Cell Cardiol. 2000, 32, 1439−1446. (10) Li, F.-Y.; Chaigne-Delalande, B.; Kanellopoulou, C.; Davis, J. C.; Matthews, H. F.; Douek, D. C.; Cohen, J. I.; Uzel, G.; Su, H. C.; Lenardo, M. J. Nature 2011, 475, 471−477. (11) Helmchen, F.; Denk, W. Nat. Methods 2005, 2, 932. (12) (a) Kim, H. M.; Cho, B. R. Acc. Chem. Res. 2009, 42, 863. (b) Kim, H. M.; Cho, B. R. Chem. Asian J. 2011, 6, 58. (13) Kim, H. J.; Han, J. H.; Kim, M. K.; Lim, C. S.; Kim, H. M.; Cho, B. R. Angew. Chem., Int. Ed. 2010, 49, 6786−6789. (14) Kucherak, O. A.; Didier, P.; Mely, Y.; Klymchenko, A. S. J. Phys. Chem. Lett. 2010, 1, 616−620. (15) Kim, H. M.; Jung, C.; Jung, S.-Y.; Hong, J. H.; Ko, Y.-K.; Lee, K. J.; Cho, B. R. Angew. Chem., Int. Ed. 2007, 46, 36460−3463. (16) Hauglang, P. R. The Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 10th ed.; Molecular Probes: Eugene, OR, 2005. (17) (a) Trapani, V.; Schweigel-Rontgen, M.; Cittadini, A.; Wolf, F. I. Methods Enzymol. 2012, 505, 421−444. (b) London, R. E. Annu. Rev. Physiol. 1991, 53, 241−258. (18) Long, J. R.; Drago, R. S. J. Chem. Educ. 1982, 59, 1037−1039. (19) Connors, K. A. Binding Constant; Wiley: New York, 1987. (20) Huang, C. Y. Methods Enzymol. 1982, 87, 509−525. (21) Xie, Z.; Peng, J.; Pennypacker, S. D.; Chen, Y. Biochem. Biophys. Res. Commun. 2010, 399, 425−428.
ASSOCIATED CONTENT
* Supporting Information S
Synthesis, photophysical, and imaging experiments and additional figures and images. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (B.R.C.);
[email protected] (Z.L.);
[email protected] (H.M.K.). Author Contributions ∥
These authors contributed equally.
Notes
The authors declare no competing financial interest. 8113
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