Design and synthesis of a highly sensitive off-on fluorescent chemosensor for zinc ions utilizing internal charge transfer

Article abstract of DOI:10.1002/chem.200901591

Fluorescence imaging is a powerful tool for the visualization of biological molecules in living cells, tissue slices, and whole bodies, and is important for elucidating biological phenomena. Furthermore, zinc (Zn²⁺) is the second most abundant heavy metal ion in the human body after iron, and detection of chelatable Zn²⁺ in biological studies has attracted much attention. Herein, we present a novel, highly sensitive off-on fluorescent chemosensor for Zn²⁺ by using the internal charge transfer (ICT) mechanism. The rationale of our approach to highly sensitive sensor molecules is as follows. If fluorescence can be completely quenched in the absence of Zn²⁺, chemosensors would offer a better signal-to-noise ratio. However, it is difficult to quench the fluorescence completely before Zn ²⁺ binding, and most sensor molecules still show very weak fluorescence in the absence of Zn²⁺. But even though the sensor shows a weak fluorescence in the absence of Zn²⁺, this fluorescence can be further suppressed by selecting an excitation wavelength that is barely absorbed by the Zn²⁺-free sensor molecule. Focusing on careful control of ICT within the 4-amino-1,8-naphthalimide dye platform, we designed and synthesized a new chemosensor (1) that shows a pronounced fluorescence enhancement with a blueshift in the absorption spectrum upon addition of Zn ²⁺. The usefulness of 1 for monitoring Zn²⁺ changes was confirmed in living HeLa cells. There have been several reports on 4-amino-1,8-naphthalimide-based fluorescent sensor molecules. However, 1 is the first Zn²⁺-sensitive off-on fluorescent sensor molecule that employs the ICT mechanism; most off-on sensor molecules for Zn²⁺ employ the photoinduced electron transfer (PeT) mechanism.

Full text of DOI:10.1002/chem.200901591

DOI: 10.1002/chem.200901591
Design and Synthesis of a Highly Sensitive Off–On Fluorescent Chemosensor
for Zinc Ions Utilizing Internal Charge Transfer
Kenjiro Hanaoka,[a, b] Yasuaki Muramatsu,[a, b] Yasuteru Urano,[a] Takuya Terai,[a, b] and
Tetsuo Nagano*[a, b]
Abstract: Fluorescence imaging is a
powerful tool for the visualization of
biological molecules in living cells,
tissue slices, and whole bodies, and is
important for elucidating biological
Zn2+
,
chemosensors would offer
a
ICT within the 4-amino-1,8-naphthal-
ACHTUNGTRNEiNUNG mide dye platform, we designed and
better signal-to-noise ratio. However, it
is difficult to quench the fluorescence
completely before Zn2+ binding, and
most sensor molecules still show very
weak fluorescence in the absence of
synthesized a new chemosensor (1)
that shows a pronounced fluorescence
enhancement with a blueshift in the ab-
sorption spectrum upon addition of
Zn2+. The usefulness of 1 for monitor-
ing Zn2+ changes was confirmed in
living HeLa cells. There have been sev-
eral reports on 4-amino-1,8-naphthal-
phenomena. Furthermore, zinc (Zn2+
)
is the second most abundant heavy
metal ion in the human body after
iron, and detection of chelatable Zn2+
in biological studies has attracted much
attention. Herein, we present a novel,
highly sensitive off–on fluorescent che-
mosensor for Zn2+ by using the inter-
nal charge transfer (ICT) mechanism.
The rationale of our approach to
highly sensitive sensor molecules is as
follows. If fluorescence can be com-
pletely quenched in the absence of
Zn2+
. But even though the sensor
shows a weak fluorescence in the ab-
sence of Zn2+, this fluorescence can be
further suppressed by selecting an exci-
tation wavelength that is barely ab-
sorbed by the Zn2+-free sensor mole-
cule. Focusing on careful control of
AHCTUNGTRENNiGUN mide-based fluorescent sensor mole-
cules. However, 1 is the first Zn2+-sen-
sitive off–on fluorescent sensor mole-
cule that employs the ICT mechanism;
most off–on sensor molecules for Zn2+
employ the photoinduced electron
transfer (PeT) mechanism.
Keywords: fluorescence
cence spectroscopy
transition metals · zinc
·
fluores-
·
sensors ·
Introduction
brain,[2] pancreas,[3] and spermatozoa.[4] Although chelatable
Zn2+ has many important roles in biological systems, its
mechanisms of action are little known in comparison with
those of other cations, such as Ca2+, Na+, and K+. There-
fore, there is considerable interest in detecting chelatable
Zn2+ in biological systems.[5]
Zinc (Zn2+) is the second most abundant heavy metal ion in
the human body after iron, and it is an essential structural
or catalytic cofactor of many proteins (e.g., carbonic anhy-
drase and zinc finger proteins).[1] In addition, there is a re-
servoir of Zn2+ that is loosely bound or chelatable in the
Several Zn2+-selective fluorescent sensor molecules have
been developed for measuring Zn2+ in living cells. Quino-
line-based fluorescent sensor molecules (6-methoxy-8-(p-tol-
uenesulfonamide)quinoline (TSQ) and its derivatives) are
the most widely used fluorescent Zn2+ chemosensors.[6] Re-
cently, several fluorescein-based sensors have also been re-
ported. These chemosensors employ the photoinduced elec-
[a] Dr. K. Hanaoka, Y. Muramatsu, Dr. Y. Urano, T. Terai,
Prof. Dr. T. Nagano
Graduate School of Pharmaceutical Sciences
The University of Tokyo
7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
Fax : (+81)3-5841-4855
tron transfer (PeT) mechanism and have been successfully
[7]
used for imaging intracellular Zn2+
.
However, the Zn2+
-
E-mail: tlong@mol.f.u-tokyo.ac.jp
[b] Dr. K. Hanaoka, Y. Muramatsu, T. Terai, Prof. Dr. T. Nagano
CREST
free sensors still retain very weak fluorescence; if the fluo-
rescence could be quenched completely in the absence of
Zn2+, a better signal-to-noise ratio could be obtained. Our
new approach to highly sensitive off–on fluorescence detec-
tion of Zn2+ is based on controlling internal charge transfer
Japan Science and Technology Agency
3-5 Sanbancho, Chiyoda, Tokyo 102-0075 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901591.
568
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Chem. Eur. J. 2010, 16, 568 – 572

FULL PAPER
(ICT) within the dye platform to promote a change in the
absorption wavelength upon Zn2+ binding.
On the basis of the above concept, we report herein the
design and synthesis of a highly sensitive off–on fluorescent
chemosensor for Zn2+ (1; Figure 1).
tions
(2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic
acid (HEPES), 100 mm, pH 7.4; Figure 2). The absorption
spectrum of 1 changed upon addition of Zn2+ (0–1.0 equiv),
with isosbestic points at l=408 and 324 nm, and then re-
mained at a plateau upon further addition of Zn2+ (Fig-
ure 2a), in accordance with the fluorescence emission spec-
Figure 1. Chemical structures of 4-amino-1,8-naphthalimide derivatives
1–3.
Results and Discussion
Design and synthesis of 1: In the design stage, we chose 4-
amino-1,8-naphthalimide as the fluorophore due to its char-
acteristic photophysical properties, such as a long emission
wavelength, a large Stokesꢁ shift, and insensitivity to pH
(e.g., Lucifer Yellow).[8] Moreover, modulation of the struc-
ture of a 4-amino donor on 1,8-naphthalimide is known to
affect ICT.[9] For example, making this substituent more
electron-deficient results in an ICT-induced blueshift in the
absorbance maxima. Therefore, we posited that modifying
the 4-amino donor to form the Zn2+ binding moiety would
provide a switch for blue-shifting the absorbance maximum.
Further, the chelator N,N,N’,N’-tetrakis(2-pyridylmethyl)e-
thylenediamine (TPEN) shows high selectivity for Zn2+
Figure 2. a) Absorbance and b) emission (excitation at l=408 nm) spec-
tra of an aqueous solution of 1 (5 mm; in HEPES buffer, 100 mm, pH 7.4)
upon addition of Zn2+ (added as ZnSO4; 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1, 1.2, and 2.0 equiv with respect to 1).
tra (Figure 2b). The Job plot of the fluorescence emission in-
tensity of 1 with Zn2+ also showed a maximum at a mole
fraction ([1]/([1]+[Zn2+])) of 0.5, which is indicative of the
formation of a 1:1 complex. We also measured the electron-
spray ionization (ESI) mass spectrum of the complex be-
tween 1 and Zn2+. In the mass spectrum of a solution of 1
(50 mm), ZnSO4 (50 mm), dimethyl sulfoxide (DMSO; 5%),
and H2O (5%) in methanol, three major ion peaks at m/z
679, 715, and 793 were clearly observed. These peaks were
over other metal ions found under physiological conditions,
[10]
such as Ca2+ and Mg2+
.
Accordingly, we introduced a
TPEN-based ligand for Zn2+ at the 4-amino position of 1,8-
naphthalimide. Water solubility was improved by incorpo-
rating 2-(2-aminoethoxy)ethanol into the fluorophore. We
also examined whether the absorbance and fluorescence
spectra of 1 in the absence and in the presence of 1.0 equiv-
alents of Zn2+ increase linearly between 0 and 10 mm. All
absorbance and fluorescence responses showed linear en-
hancement and it is considered, therefore, that concentra-
tion-dependent quenching does not occur in the concentra-
tion range up to 10 mm in aqueous solution (see the Support-
ing Information).
assigned as [1+Zn2+ꢀH+]
+A
(DMSO)]+ (m/z 793). These re-
z 715), and [1+Zn2+ +Clꢀ·
AHCTUNGTRENNUNG
sults support the idea that 1 forms a 1:1 complex with Zn2+
.
On the other hand, in the absence of Zn2+ 1 displayed one
major absorption band centered at l=437 nm (e=
7700 mꢀ1 cmꢀ1) with almost no fluorescence (fluorescence
quantum yield F=0.039). The complexation of 1 with Zn2+
triggered green fluorescence with characteristic absorption
(labs =380 nm, e=10100 mꢀ1 cmꢀ1) and emission (lem
=
Fluorescence and chemical properties of 1: We assessed the
spectroscopic characteristics of 1 under physiological condi-
507 nm, F=0.942) spectra. Thus, the fluorescence emission
intensity of 1 (5 mm) increased significantly (21.7 times)
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569

T. Nagano et al.
upon addition of 1.0 equiv of Zn2+, with a large blueshift of
57 nm.
We next investigated the mechanism of fluorescence in-
crease of 1. The photophysical properties of 1, 2, and 3
(Figure 1) are summarized in Table 1. The F values of 2 and
Table 1. Fluorescence emission properties of 1–3.[a]
Compound
labs [nm]
lem [nm]
F[d]
1[b]
437
380
435
441
523
507
550
546
0.039
0.942
0.016
0.397
1+Zn2+[c]
2
3
[a] All data were obtained in HEPES buffer (100 mm, pH 7.4). [b] The
data were measured in the absence of Zn2+. [c] The data were measured
in the presence of Zn2+ (1.0 equiv with respect to 1). [d] The fluorescence
quantum yields were determined by using N-butyl-4-butylamino-1,8-
naphthalimide in absolute ethanol (F=0.81) as the standard.[11]
Figure 3. Fluorescence intensity change profiles of 1 (2 mm) in the pres-
ence of various cations in HEPES buffer (100 mm, pH 7.4); excitation
l=408 nm, emission l=507 nm). Heavy metal ions (1.0 equiv relative to
1) were added as MnSO4, FeCl2, FeCl3, CoSO4, NiSO4, CuSO4, and
CdSO4. Other cations were added as ZnSO4 (2 mm), NaNO3 (100 mm),
KNO3 (100 mm), CaCl2 (5 mm), and MgSO4 (5 mm).
3 were 0.016 and 0.397, respectively, and the F of 1 without
Zn2+ was almost the same as that of 2. From this result, it
can be considered that variation of the electron-donating
ability of the substituent at the 4-position of 1,8-naphthali-
mide changes the F value. The effect of pH was also exam-
ined. The fluorescence emission intensity of 1 without Zn2+
at l=523 nm was measured at various pH values, with exci-
tation at l=408 nm. There was almost no effect on the
emission spectrum of 1 between pH 3.0 and 9.0 in the ab-
sence of Zn2+ (see the Supporting Information). pH 3 is suf-
ficiently low for the tertiary amine in the Zn2+ chelating
moiety of 1 (marked with an arrow in Figure 1) to be pro-
tonated because TPEN has pKa values of 7.12, 4.81, 3.30,
and 2.88.[12] In fact, a tertiary amine with similar structure to
1 had a pKa of 5.8.[13] Therefore, the finding of insensitivity
to lower pH means that the fluorescence augmentation of 1
upon addition of Zn2+ was not due to cessation of PeT from
the tertiary amine. From these results, it can be considered
that the increase of the emission intensity caused by Zn2+
addition is due to the change in the photophysical properties
of the fluorophore itself, and arises via the ICT mechanism.
The effect of adding Na+, K+, Ca2+, Mg2+, and various
heavy metal ions (Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Cd2+
) on the fluorescence emission intensity of 1 was also exam-
ined. Fluorescence emission enhancement of 1 (2 mm) was
not observed upon addition of 100 mm Na+ or K+, or 5 mm
Ca2+ or Mg2+ (these ions exist in high concentrations in bio-
logical systems) or 2 mm of various heavy metal ions, except
for Cd2+ (Figure 3). Therefore, compound 1 showed high se-
lectivity for Zn2+ over other cations except for Cd2+, which
is of little biological relevance. However, the fluorescence
intensity was weakened upon addition of Co2+ and Cu2+
(1 equiv to 1) together with Zn2+, in comparison with that
in the presence of Zn2+ alone, as shown in the Supporting
Information (Figure S4). Furthermore, the binding affinity
for Zn2+ was also assessed from the fluorescence intensity
change. The affinity of 1 for Zn2+ was measured at pH 7.4
in a high salt environment (100 mm HEPES buffer, I=0.1
(NaNO3)). The apparent dissociation constant, Kd, for Zn2+
was calculated to be 1.1 nm (see the Supporting Informa-
tion), which is sufficiently small for biological applica-
tions.[14]
Biological applications of 1: We also investigated the appli-
cation of 1 in cultured cells (Figure 4). Cultured HeLa cells
Figure 4. Bright-field transmission and fluorescence images of Zn2+ in
HeLa cells in HBSS buffer. The fluorescence at l=435–565 nm, excited
at l=385–415 nm, was measured. a) Bright-field transmission image of
cells stained with 1 (10 mm) for 30 min at RT. b) Fluorescence image of
cells stained with 1 (10 mm) for 30 min at RT. c) Fluorescence image of
cells stained with 1 and loaded with ZnSO4 (50 mm) and pyrithione (5 mm;
zinc ionophore) for 2.5 min. d) Fluorescence image of the cells following
addition of TPEN (100 mm) to the medium for 4.5 min.
were incubated with 1 (10 mm) for 30 min at room tempera-
ture, which was sufficient for intracellular accumulation of 1
to occur as judged from the fluorescence observed in the in-
tracellular regions (Figure 4b). The overlay of bright-field
transmission and fluorescence images revealed that fluores-
cence signals were localized in the perinuclear region (see
the Supporting Information). However, it is thought that
this fluorescence is not due to Zn2+, which is present in the
intracellular regions, but rather is due to the solvatochromic
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A Highly Sensitive Zinc Ion Chemosensor
FULL PAPER
sured by using a JMS-T100 LC AccuTOF spectrometer (ESI+; JEOL).
HPLC purification was performed by using a Jasco PU-1587 system fitted
with a reversed-phase column (GL Sciences (Tokyo, Japan), Inertsil
Prep-ODS 30ꢂ250 mm). UV/Vis spectra were obtained by using a V-550
UV/VIS spectrophotometer (Jasco). The fluorescence emission or excita-
character of 1. When we examined the solvatochromic prop-
erties of 1, we observed almost no fluorescence in polar
protic solvents, such as water and methanol, but a small en-
hancement of the fluorescence intensity was seen in organic
solvents, such as CH3CN, CH2Cl2, and DMSO (see the Sup-
porting Information). Moreover, 4-N,N-dimethylamino-1,8-
naphthalimide (4-DMN), which has a similar structure to 1,
has been reported to be a solvatochromic fluorophore.[15]
We believe these results are consistent with the idea that the
majority, though not all, of compound 1 inside cells exists in
relatively nonpolar aprotic environments. We then added
Zn2+ (50 mm) and a zinc-selective ionophore, pyrithione (2-
mercaptopyridine N-oxide, 5 mm), to the medium, inducing a
prompt increase of the intracellular fluorescence (Figure 4c).
This fluorescence was decreased by the extracellular addi-
tion of the cell-membrane-permeable chelator TPEN
(100 mm; Figure 4d). This result demonstrates that 1 can be
used to reversibly monitor changes in intracellular ionic
Zn2+ and has potential for biological applications.
tion spectra were recorded by using
a FP-6500 spectrofluorometer
(Jasco). The slit width was 3 nm for both excitation and emission.
UV/Vis absorption spectrum measurements: The absorption spectra of 1
(5 mm) were measured at RT in an aqueous solution buffered to pH 7.4
(100 mm HEPES buffer containing 0.25% DMSO as a cosolvent). Zn2+
was added as ZnSO4 (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,
and 2.0 equiv of Zn2+ with respect to 1).
Fluorescence emission and excitation spectral measurements: The fluo-
rescence emission spectra of 1 (5 mm) were measured in HEPES buffer
(100 mm, pH 7.4) at RT, following excitation at l=408 nm. The fluores-
cence excitation spectra of 1 (5 mm) were also measured in HEPES buffer
(100 mm, pH 7.4) at RT (fixed emission at l=507 nm). The amounts of
Zn2+ added were 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, and
2.0 equiv with respect to 1 in both emission and excitation spectral meas-
urements.
Quantum yield measurements: The fluorescence spectra were measured
by using a FP-6500 spectrofluometer (Jasco). The slit width was 3.0 nm
for both excitation and emission. The photomultiplier voltage was 400 V.
The fluorescence spectra of 1, 2, and 3 were measured in HEPES buffer
(100 mm, pH 7.4) at RT, with irradiation at l=408 nm. The quantum
yields of compounds were evaluated by using a relative method with ref-
erence to a fluorescence standard, N-butyl-4-butylamino-1,8-naphthali-
mide (F=0.81 in absolute ethanol).[11] The quantum yields of compounds
can be expressed by Equation (1),[19] in which F is the quantum yield
(“st” denotes the reference and “x” denotes the sample), A is the absorb-
ance at the excitation wavelength, n is the refractive index, and D is the
area (on an energy scale) of the fluorescence spectra. The sample and
the reference were excited at the same wavelength (l=408 nm). The
sample absorbance at the excitation wavelength was kept as low as possi-
ble to avoid fluorescence errors (Aexc <0.02).
Conclusions
In conclusion, we have designed and synthesized a novel,
highly sensitive fluorescent molecule (1) for sensing Zn2+ by
using 4-amino-1,8-naphthalimide as a fluorophore and a
TPEN derivative as a Zn2+ binding moiety. Compound 1
showed pronounced fluorescence enhancement, with a blue-
shift in the absorption spectrum upon addition of Zn2+
.
There have been many reports on 4-amino-1,8-naphthali-
mide-based ratiometric or off–on fluorescent sensor mole-
cules for Zn2+, sugars, Hg2+, Pd2+, pH, anions, Ca2+, H2O2,
Na+, K+, and Cu2+ by using the PeT or ICT mecha-
nism.[13,16] However, compound 1 is the first Zn2+-sensitive
off–on fluorescent sensor molecule based on the ICT mech-
anism on a 4-amino-1,8-naphthalimide platform, though a
similar ICT working principle was reported with 7-nitro-
benz-2-oxa-1,3-diazole (NBD).[17] Our design strategy should
be applicable to a range of off–on fluorescent sensor mole-
cules for Zn2+ and other molecules of interest in biological
applications.
2
Fx=Fst ¼ ½Ast=Axꢁ½nx2=nst ꢁ½Dx=Dstꢁ
ð1Þ
Apparent dissociation constant (Kd) measurements: Upon addition of
various concentrations of Zn2+, the fluorescence intensity and the ab-
sorbance of 1 linearly changed up to a 1:1 [Zn2+]/[1] molar ratio, and the
fluorescence and absorption spectra remained at a plateau with further
addition of Zn2+. Furthermore, the Job plot analysis revealed that maxi-
mum fluorescence intensity was obtained at a 1:1 ratio. The ESI mass
spectrum also showed the ion peaks of a 1:1 complex between Zn2+ and
1. These data suggested that 1 forms a 1:1 complex with Zn2+. So, the ap-
parent dissociation constant, Kd, was determined from the fluorescence
intensity in HEPES buffer (100 mm, pH 7.4, I=0.1 (NaNO3)) at RT
(lexc =408 nm). [Zn2+
] was controlled by using ZnSO4/NTA (0–
10 mm:10 mm) systems.[20] The fluorescence intensity data were fitted to
Equation (2), in which F is the fluorescence intensity, Fmax is the maxi-
mum fluorescence intensity, F0 is the fluorescence intensity with no addi-
tion of Zn2+, and [Zn2+]f is the free Zn2+ concentration. The value of Kd
was determined from the fittings for the fluorescence intensity.
Experimental Section
All reagents and solvents were of the highest commercial quality and
were used without purification, except for ethanol, which was used after
distillation. DMSO, HEPES, nitrilotriacetic acid (NTA), TPEN, 2-mor-
pholinoethanesulfonic acid (MES), and N-cyclohexyl-2-aminoethanesul-
fonic acid (CHES) were purchased from Dojindo Laboratories (Japan).
4-(n-Butylamino)naphthalic-1,8-N-butylimide was prepared according to
a literature procedure.[18] All other reagents and solvents were purchased
from Tokyo Chemical Industry Co. (Japan), Wako Pure Chemical Indus-
tries (Japan), or Aldrich Chemical Co. (USA). Silica gel column chroma-
tography was performed by using Chromatorex-NH (Fuji Silysia Chemi-
cal, Kasugai, Japan), Silica Gel 60N (spherical, neutral), or Silica Gel 60
(spherical) (Kanto Chemical Co., Tokyo, Japan).
F ¼ F0 þ ðFmaxꢀF0Þð½Zn2þꢁfÞ=ðKd þ ½Zn2þꢁfÞ
ð2Þ
Effect of pH on the fluorescence intensity: The following buffers were
used: ClCH2COOH–ClCH2COONa buffer (100 mm, pH 3.0 and 3.6),
AcOH–AcONa buffer (100 mm, pH 4.2, 4.8 and 5.4), MES buffer
(100 mm, pH 5.7, 6.1 and 6.5), HEPES buffer (100 mm, pH 7.0, 7.4 and
8.0), and CHES buffer (100 mm, pH 8.5 and 9.0). The fluorescence inten-
sity (excitation l=408 nm, emission l=523 or 507 nm in the absence or
presence of Zn2+ (2 mm), respectively) of each sample of 1 (2 mm) was
plotted.
Metal ion selectivity measurements: The fluorescence emission enhance-
ment of 1 was measured in HEPES buffer (100 mm, pH 7.4) at RT (exci-
tation l=408 nm, emission l=507 nm). Heavy metal ions (2 mm) were
Instruments: 1H and 13C NMR spectra were recorded by using a JNM-
LA300 or a JNM-LA400 (JEOL) spectrometer. Mass spectra were mea-
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T. Nagano et al.
added as MnSO4, FeCl2, FeCl3, CoSO4, NiSO4, CuSO4, and CdSO4. Other
cations were added as ZnSO4 (2 mm), NaNO3 (100 mm), KNO3 (100 mm),
CaCl2 (5 mm), and MgSO4 (5 mm).
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Preparation of cells: HeLa cells were cultured in Dulbeccoꢁs modified
Eagle medium (DMEM; Invitrogen, Carlsbad, USA) supplemented with
10% fetal bovine serum (Invitrogen), 1% penicillin, and 1% streptomy-
cin (Invitrogen) at 378C in a CO2/air (5:95) incubator. The cells were
grown on an uncoated 35 mm diameter glass-bottomed dish (MatTek,
Ashland, USA), and washed twice with Hanksꢁ balanced salt solution
(HBSS) buffer (Invitrogen), then the medium was replaced with HBSS
buffer before imaging.
Microscopy and imaging methods: The imaging system was comprised of
an inverted microscope (IX71; Olympus) and a cooled CCD camera
(Cool Snap HQ; Roper Scientific, Tucson, USA). The microscope was
equipped with a xenon lamp (AH2-RX; Olympus), a ꢂ40 objective lens
(Uapo/340, N.A. 1.35; Olympus), a dichroic mirror (t2003 DM; Olym-
pus), an excitation filter (XF1076 400AF30; OMEGA), and an emission
filter (XF3088 435ALP; OMEGA). The whole system was controlled
using MetaFluor 7.1 software (Molecular Devices, Meta Imaging Soft-
ware, PA, USA). HeLa cells were grown on an uncoated 35 nm diameter
glass-bottomed dish (MatTek, Ashland, USA) and washed twice with
HBSS buffer; then the cells were incubated with 1 (10 mm) in HBSS
buffer for dye loading for 30 min at RT, which was long enough for intra-
cellular accumulation of 1 to occur, as judged from the fluorescence seen
in the intracellular regions (Figure 4). The stained cells were washed
twice with HBSS buffer, and the medium was replaced with HBSS buffer
before imaging (0 min). We then added zinc sulfate (50 mm) and pyri-
thione (5 mm) to the medium on the microscope stage, inducing a prompt
increase of intracellular fluorescence at 4.5 min. This fluorescence de-
creased upon extracellular addition of the cell-membrane-permeable che-
lator TPEN (100 mm) to cell samples on the microscope stage at 9.5 min.
The fluorescence images were measured every 30 s.
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Dye loading conditions: The cells were cultured in DMEM supplemented
with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin at
378C in a CO2/air (5:95) incubator. The cells were washed with HBSS
buffer, and then incubated with 1 (10mm) in HBSS buffer for dye loading
for 30 min at RT. The stained cells were washed twice with HBSS buffer,
and the medium was replaced with HBSS buffer before imaging.
Acknowledgements
This research was supported in parts by the Ministry of Education, Cul-
ture, Sports, Science and Technology of Japan (20689001, 19890047, and
21659024 to K.H.). K.H. was also supported by the Mochida Memorial
Foundation for Medical and Pharmaceutical Research and Sankyo Foun-
dation of Life Science.
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Received: June 11, 2009
Published online: November 13, 2009
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