A highly sensitive two-photon fluorescent probe for mitochondrial zinc ions in living tissue

Article abstract of DOI:10.1039/c2cc31077e

We report a highly sensitive two-photon probe (SZn2-Mito) which shows a 70-fold two-photon excited fluorescence enhancement in response to Zn ²⁺ and can selectively detect mitochondrial Zn²⁺ in a rat hippocampal slice at a depth of 1

Full text of DOI:10.1039/c2cc31077e

View Article Online / Journal Homepage / Table of Contents for this issue
Chemical Communications
www.rsc.org/chemcomm
Volume 48 | Number 38 | 14 May 2012 | Pages 4509–4640
ISSN 1359-7345
COMMUNICATION
Cho and Kim et al.
A highly sensitive two-photon fluorescent probe for mitochondrial zinc ions in living tissue

Dynamic ArticVleiewLiAnrktiscle Online
ChemComm
Cite this: Chem. Commun., 2012, 48, 4546–4548
www.rsc.org/chemcomm
COMMUNICATION
A highly sensitive two-photon fluorescent probe for mitochondrial zinc
ions in living tissuew
a
a
Na Young Baek,z Cheol Ho Heo,z Chang Su Lim,b Goutam Masanta,a Bong Rae Cho*b
and Hwan Myung Kim*a
Received 14th February 2012, Accepted 9th March 2012
DOI: 10.1039/c2cc31077e
We report a highly sensitive two-photon probe (SZn2-Mito)
which shows a 70-fold two-photon excited fluorescence enhancement
in response to Zn2+ and can selectively detect mitochondrial
Zn2+ in a rat hippocampal slice at a depth of 100–200 lm by
using two-photon microscopy.
The zinc ion is the second-most-abundant d-block metal ion in
the human brain and is an active component in enzymes and
proteins.1,2 For proper brain functions, it is vital to maintain
Zn2+-ion homeostasis, which is controlled by the import of
intracellular Zn2+ ions ([Zn2+]i) from—and export to—the
extracellular space, the endoplasmic reticulum, and mitochondria.
While mitochondria can take up evoked [Zn2+]i rise,3,4
a
strong elevation of intra-mitochondrial Zn2+ ([Zn2+]m) can
promote mitochondrial dysfunctions.3,4 To understand the
physiology of Zn2+ in the brain, it is crucial to monitor
Scheme 1 Structures of SZn-Mito and SZn2-Mito.
receptor because it has been shown to increase the Zn2+ binding
affinity and turn-on response.11 Herein, we report that SZn2-
Mito is a much more sensitive TP probe, and can detect [Zn2+
[Zn2+
] in intact brain tissue. An attractive approach to the
m
detection of [Zn2+
]
is the use of two-photon microscopy
m
]
m
(TPM). TPM, which employs two near-infrared photons as
the excitation source, is a new technique that can visualize
biological events deep inside intact tissues (>500 mm) for
extended periods of time when combined with an appropriate
two-photon (TP) probe.5,6 Recently, we reported a targetable TP
probe (SZn-Mito) that has been successfully utilized to detect
in live cells and intact tissues at >100 mm depth with brighter
and clearer images by TPM when compared with SZn-Mito.
The preparation of SZn2-Mito is described in the ESI.w The
solubility of SZn2-Mito in buffer solution (50 mM HEPES,
100 mM KCl, pH 7.4) is approximately 2 mM, which is
sufficient to stain the cells (Fig. S2 in the ESIw). The absorption
and emission spectra of SZn2-Mito showed gradual red-shifts
with increasing solvent polarity. The shifts of emission spectra
were greater for SZn2-Mito than for SZn-Mito (Dlfl = 80 vs.
45 nm).7 The larger Stokes shift observed in SZn2-Mito
compared to SZn-Mito is likely to be due to greater stabili-
zation of the charge transfer excited state of the former
fluorophore that contains a stronger electron-withdrawing
group (Fig. S1 and Table S1 in ESIw).
[Zn2+
]
m
in live cells and tissues by TPM.7 However, this initial
design was limited by the modest turn-on response (7-fold) in the
presence of excess Zn2+. For more sensitive detection, there is a
need to develop a highly sensitive TP probe for [Zn2+]m.
With this aim in mind, we have designed a new TP probe for
[Zn2+
]m (SZn2-Mito, Scheme 1) derived from 6-(benzo[d]thiazol-
20-yl)-2-(N,N-dimethylamino)naphthalene (BTDAN) as the
reporter, a methoxy derivative of N,N-di-(2-picolyl)ethylene-
diamine (MeO-DPEN) as the Zn2+ chelator, and triphenyl-
phosphonium salt (TPP) as the mitochondrial-targeting site
(Scheme 1).8–10 MeO-DPEN has been employed as the
In metal free buffer (50 mM HEPES, 100 mM KCl, 10 mM
NTA, pH 7.4), SZn2-Mito has the absorption (lmax) and
emission maxima (lflmax) at 413 nm (e = 1.85 ꢀ 104 Mꢁ1 cmꢁ1
)
and 536 nm (F = 0.0048), respectively (Table S2 in ESIw).
Upon addition of Zn2+, the fluorescence intensity increased
dramatically with a slight blue shift in its absortion spectra
(Fig. S3a,b in ESIw), presumably due to the blocking of the
photoinduced electron transfer (PeT) process upon complexa-
tion with Zn2+. A nearly identical result was observed in the
TP process (Fig. 1a). The fluorescence enhancement factor
[FEF = (F ꢁ Fmin)/Fmin] of SZn2-Mito determined for the
a Division of Energy Systems Research and Molecular Science &
Technology Research Center, Ajou University, Suwon, 443-749,
Korea. E-mail: kimhm@ajou.ac.kr; Fax: +82-31-219-1615
b Department of Chemistry, Korea University, 145, Anamro,
Sungbuk-gu, Seoul, 136-713, Korea. E-mail: chobr@korea.ac.kr;
Fax: +82-2-3290-3544
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc31077e
z These two authors contributed equally to this work.
c
4546 Chem. Commun., 2012, 48, 4546–4548
This journal is The Royal Society of Chemistry 2012

View Article Online
Fig. 1 (a) Two-photon fluorescence spectra of 1 mM SZn2-Mito in
the presence of free Zn2+ (0–47 nM). The excitation wavelength is
750 nm with fs pulse. (b) Two-photon action spectra of SZn2-Mito in
the presence of 47 nM free Zn2+. These data were measured in 50 mM
HEPES buffer (100 mM KCl, 10 mM NTA, pH 7.4).
one-photon process was approximately 70 in the presence of
excess Zn2+ (Table S2, ESIw); the FEF of SZn2-Mito is
10-fold larger than that of SZn-Mito.7 The much larger
turn-on response determined for SZn2-Mito can be attributed
to its much smaller fluorescence quantum yield (Table S2 in
ESIw). This result underlines the importance of stabilizing the
charge transfer excited state for the design of an efficient
fluorescent probe with a large turn-on response.
Fig. 2 (a) TPM and (b) OPM images of HeLa cells co-labeled with
(a) SZn2-Mito (1 mM) and (b) Mitotracker Red FM (1 mM). (c)
Colocalized image. The wavelengths for one- and two-photon excita-
tion were 514 and 750 nm, respectively, and the emission was collected
at 450–550 (SZn-Mito) and 600–700 nm (Mitotracker Red FM),
respectively. Scale bar, 20 mm. (d–f) TPM images of 1 mM SZn2-
Mito-labeled HeLa cells, before (d) and after (e) addition of 150 mM
DTDP to the imaging solution. (f) After addition of 10 mM CCCP to
(e). (g) The relative TPEF intensity of SZn2-Mito-labeled HeLa cells
as a function of time. The TPEF intensities were collected at 450–600
nm upon excitation at 750 nm with fs pulse. Scale bar, 10 mm. Cells
shown are representative images from replicate experiments (n = 5).
The dissociation constants (KOd P and KdTP) of SZn2-Mito for
the one- and two-photon processes were determined from the
fluorescence titration curves (Fig. S3c,d, ESIw).12 The titration
curves fitted well with a 1 : 1 binding model (Fig. S3c, ESIw),
the Hill plots were linear with a slope of 1.0 (Fig. S3d, ESIw),
and the Job’s plot exhibited a maximum point at the mole
fraction 0.50 (Fig. S4, ESIw), indicating 1 : 1 complexation
between SZn2-Mito and Zn2+. The KdOP and KdTP values for
the SZn2-Mito/Zn2+ complex were 1.4 nM, which was 2.2-fold
smaller than that for SZn-Mito (3.1 nM).7 The stronger Zn2+
binding affinity of MeO-DPEN than DPEN was previously
reported.11 Moreover, SZn2-Mito exhibits high selectivity
a well-known OP fluorescent probe for mitochondria.16 The
TPM and OPM images were obtained by using the detection
windows at 450–550 and 600–700 nm, respectively, to selectively
detect the two signals with equal intensities (Fig. S7, ESIw).
The two images overlapped well (Fig. 2a–c), and Pearson’s
colocalization coefficient, A, calculated by using Autoquant
X2 software, of SZn2-Mito with Mitotracker Red FM was
0.85.17 Moreover, the TPEF intensity of SZn2-Mito-labeled
HeLa cells decreased dramatically upon addition of N,N,N0,N0-
tetrakis(2-pyridyl)ethylenediamine (TPEN), a membrane perme-
for 1 mM Zn2+ compared with 1 mM of Na+, K+, Ca2+
,
Mg2+, 1 mM of Mn2+, Fe2+, Co2+, Pb2+ and modest selectivity
over 1 mM Ni2+, Cd2+ (Fig. S5b, ESIw). In the presence of 1 mM
of Cu2+ and Hg2+, the fluorescence was quenched due to metal-
to-ligand electron transfer upon excitation.13 Since Ni2+, Cd2+
,
Hg2+ and Cu2+ ions rarely exist in the cells,14 this probe can
detect Zn2+ with minimum interference from other competing
metal ions. Further, SZn2-Mito is pH-insensitive in the bio-
logically relevant pH range (Fig. S5a, ESIw). The combined
results establish that SZn2-Mito can serve as a highly sensitive
turn-on probe for detecting Zn2+ with minimum interference
from other competing metal ions and pH.
able Zn2+ chelator that can effectively remove [Zn2+
]
m
(Fig. S6a,b, ESIw).3b These results confirm that the bright
regions in the TPM image reflect [Zn2+]m. Further, SZn2-Mito
showed negligible toxicity as measured by using a CCK-8 kit
(Fig. S8, ESIw), and high photostability as indicated by the
negligible decay in the TPEF intensity at a given spot on the
SZn2-Mito-labeled HeLa cells after continuous irradiation of
the fs-pulses for 60 min (Fig. S6d, ESIw).
We also evaluated the ability of SZn2-Mito to detect Zn2+
in a TP mode. The TP action spectrum of SZn2-Mito in metal
free HEPES buffer was too small to measure accurately. In the
presence of excess Zn2+, however, the Fd value increased
dramatically to 155 GM at 750 nm (Fig. 1b and Table S2 in
ESIw). Moreover, the Fd value is larger by 2-fold than 75 GM
for SZn-Mito, a result that can be attributed to the enhanced
intramolecular charge transfer (see above).15 This outcome pre-
dicts twice as bright TPM images of the cells and tissues labeled
with SZn2-Mito as those labeled with SZn-Mito (see below).
We then tested whether SZn2-Mito can monitor the changes
in [Zn2+
]
in live cells. Upon TP excitation at 750 nm, the
m
TPEF intensity increased dramatically when 2,20-dithiodipyridine
(DTDP; 150 mM), a reagent that promotes the release of Zn2+
from Zn2+-binding proteins,18 was added to HeLa cells
labeled with SZn2-Mito (Fig. 2d,e,g), and decreased abruptly
upon addition of carbonyl cyanide m-chlorophenylhydrazone
(CCCP; 10 mM, Fig. 2f,g), a compound that promotes the
release of intramitochondrial cations by collapsing the mito-
chondrial membrane potential.19 The TPEF intensity also
increased when the cells were treated with 100 mM Zn2+ and
We next sought to utilize SZn2-Mito to detect [Zn2+
]m in a
cellular environment. For this experiment, HeLa cells were
co-labeled with SZn2-Mito and Mitotracker Red FM,
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 4546–4548 4547

View Article Online
insensitivity in the biologically relevant range. Compared with
SZn-Mito, this probe shows 10-fold larger turn-on response,
2-fold larger Fd value, and 2-fold stronger Zn2+ binding affinity,
and can selectively detect [Zn2+
] in live cells and tissues at a
m
depth of 100–200 mm with a brighter and clearer TPM image.
This work was supported by the Korea Healthcare technology
R&D Project, Ministry of Health & Welfare, Republic of Korea
(A111182) and the NRF grants (No. 2011-0028663) and Priority
Research Centers Program through the NRF funded by the
Ministry of Education, Science and Technology (2011-0022978),
and Ajou university research fellowship of 2011.
Fig. 3 Images of a rat hippocampal slice stained with 20 mM
SZn2-Mito for 1 h. (a) Bright-field images of the CA1–CA3 regions
as well as dentate gyrus (DG) at 10ꢀ magnification. (b) 10 TPM
images along the z-direction at the depths of approximately 100–200 mm
were accumulated. (c–e) Magnification at 40ꢀ in the DG regions
(red circle in (b)) at a depth of B100 mm (c) before and (d) after
addition of 150 mM DTDP to the imaging solution. (e) After addition of
10 mM CCCP to (d). Scale bars: (a) 300 and (e) 75 mm.
Notes and references
1 (a) B. L. Vallee and K. H. Falchuk, Physiol. Rev., 1993, 73, 79;
(b) J. M. Berg and Y. Shi, Science, 1996, 271, 1081.
2 (a) C. J. Frederickson, J. H. Koh and A. I. Bush, Nat. Neurosci.,
2005, 6, 449; (b) S. Y. Assaf and S. H. Chung, Nature, 1984,
308, 734; (c) G. A. Howell, M. G. Welch and C. J. Frederickson,
Nature, 1984, 308, 736.
3 (a) S. L. Sensi, H. Z. Yin and J. H. Weiss, Eur. J. Neurosci., 2000,
12, 3813; (b) S. L. Sensi, D. Ton-That, P. G. Sullivan, E. A. Jonas,
K. R. Gee, L. K. Kaczmarek and J. H. Weiss, Proc. Natl. Acad.
Sci. U. S. A., 2003, 100, 6157.
4 S. L. Sensi, P. Paoletti, A. I. Bush and I. Sekler, Nat. Rev.
Neurosci., 2009, 10, 780.
5 (a) F. Helmchen and W. Denk, Nat. Methods, 2005, 2, 932;
(b) W. R. Zipfel, R. M. Williams and W. W. Webb, Nat. Biotechnol.,
2003, 21, 1369.
100 mM pyrithione (2-mercaptopyridine N-oxide), a reagent
that can bring Zn2+ into the cytoplasm,20 and decreased by
the treatment with CCCP (Fig. S9, ESIw). In all cases, the
changes in TPEF intensity were larger than those observed with
SZn-Mito. These results establish that SZn2-Mito can detect
[Zn2+
] in live cells with higher sensitivity than SZn-Mito.
m
We further investigated the utility of SZn2-Mito in tissue
imaging. The TPM images were obtained from a slice of a
14 day-old rat hippocampal tissue incubated with 20 mM
SZn2-Mito for 1 h at 37 1C. The slice from the brain was
too large to show in one image, so two images were obtained in
each xy plane and combined. The bright-field image revealed
the CA1 and CA3 regions and also the dentate gyrus (DG; Fig. 3a).
As the structure of the brain tissue is known to be inhomo-
geneous in its entire depth, we accumulated 10 TPM images at
depths of 100–200 mm to visualize the distributions of the
6 (a) H. M. Kim and B. R. Cho, Acc. Chem. Res., 2009, 42, 863;
(b) H. M. Kim and B. R. Cho, Chem.–Asian J., 2011, 6, 58.
7 G. Masanta, C. S. Lim, H. J. Kim, J. H. Han, H. M. Kim and
B. R. Cho, J. Am. Chem. Soc., 2011, 133, 5698.
8 (a) E. M. Nolan and S. J. Lippard, Acc. Chem. Res., 2009, 42, 193;
(b) E. L. Que, D. W. Domaille and C. J. Chang, Chem. Rev., 2008,
108, 1517; (c) K. Komatsu, K. Kikuchi, H. Kojima, Y. Urano and
T. Nagano, J. Am. Chem. Soc., 2005, 127, 10197.
9 (a) M. P. Murphy and R. A. Smith, Annu. Rev. Pharmacol.
Toxicol., 2007, 47, 629; (b) L. F. Yousif, K. M. Stewart and
S. O. Kelley, ChemBioChem, 2009, 10, 1939.
10 (a) B. C. Dickinson, D. Srikun and C. J. Chang, Curr. Opin. Chem.
Biol., 2010, 14, 50; (b) B. C. Dickinson and C. J. Chang, J. Am.
Chem. Soc., 2008, 130, 9638.
11 H. M. Kim, M. S. Seo, M. J. An, J. H. Hong, Y. S. Tian,
J. H. Choi, O. Kwon, K. J. Lee and B. R. Cho, Angew. Chem.,
Int. Ed. Engl., 2008, 47, 5167.
12 (a) H. J. Kim, J. H. Han, M. K. Kim, C. S. Lim, H. M. Kim and
B. R. Cho, Angew. Chem., Int. Ed. Engl., 2010, 49, 6786;
(b) M. K. Kim, C. S. Lim, J. T. Hong, J. H. Han, H. Y. Jang,
H. M. Kim and B. R. Cho, Angew. Chem., Int. Ed. Engl., 2010,
49, 364.
[Zn2+
] (Fig. 3b). They reveal marked TPEF in the CA3 and
m
DG regions (Fig. 3b).11 At a higher magnification, the [Zn2+
]
m
distribution in the DG region is clearly visualized (Fig. 3c). As
observed in the HeLa cells, the TPEF intensity increased after
addition of DTDP and decreased upon treatment of CCCP,
thereby providing a strong evidence that the bright regions
reflect the [Zn2+
]
(Fig. 3d,e). Moreover, the TPM images
m
obtained at a depth of 100–200 mm revealed the [Zn2+
]
m
13 E. U. Akkaya, M. E. Huston and A. W. Czarnik, J. Am. Chem.
Soc., 1990, 112, 3590.
distribution in the given plane along the z direction in the
14 T. D. Rae, P. J. Schmidt, R. A. Pufahl, V. C. Culotta and
T. V. O’Halloran, Science, 1999, 284, 805.
DG region (Fig. S10, ESIw). Hence, SZn2-Mito is clearly
capable of detecting [Zn2+
]
at a depth of 100–200 mm in
m
15 H. M. Kim and B. R. Cho, Chem. Commun., 2009, 153.
16 A Guide to Fluorescent Probes and Labeling Technologies, ed.
R. P. Haugland, Molecular Probes, Eugene, OR, 10th edn, 2005.
17 K. Emmerson and K. Roehrig, Comp. Biochem. Physiol., B:
Biochem. Mol. Biol., 1992, 103, 663.
18 E. Aizenman, A. K. Stout, K. A. Hartnett, K. E. Dineley,
B. McLaughlin and I. J. Reynolds, J. Neurochem., 2000, 75, 1878.
19 T. Caporale, D. Ciavardelli, C. Di Ilio, P. Lanuti, D. Drago and
S. L. Sensi, Exp. Neurol., 2009, 218, 228.
living tissues by using TPM. Further, the TPM images shown
in Fig. 3b,c are brighter and clearer than those stained with
SZn-Mito (Fig. S11, ESIw),7 demonstrating the advantage of
SZn2-Mito.
In conclusion, we have developed a mitochondrial-targeted
and highly sensitive TP probe (SZn2-Mito) that shows a
70-fold TPEF enhancement in response to Zn2+, a maximum
TP action cross section value of 155 GM in the presence of
excess Zn2+, a dissociation constant (KTdP) of 1.4 nM, and pH
20 (a) B. L. Barnett, H. C. Kretschmar and F. A. Hartman, Inorg.
Chem., 1977, 16, 1834; (b) C. J. Chandler and I. H. Segel, Antimicrob.
Agents Chemother., 1978, 14, 60.
c
4548 Chem. Commun., 2012, 48, 4546–4548
This journal is The Royal Society of Chemistry 2012