Two-photon probes for Zn2+ ions with various dissociation constants. Detection of Zn2+ ions in live cells and tissues by two-photon microscopy

Article abstract of DOI:10.1002/asia.201000720

A series of Zn²⁺-selective two-photon fluorescent probes (AZnM1-AZnN) that had a wide range of dissociation constants (K_d ^TP=8 nm-12 νM) were synthesized. These probes showed appreciable water solubility (>3 νM), cell permeability, high photostability, pH insensitivity at pH>7, significant two-photon action cross-sections (86-110 GM) upon complexation with Zn²⁺, and can detect the Zn²⁺ ions in HeLa cells and in living tissue slices of rat hippocampal at a depth of >80 νm without mistargeting and photobleaching problems. These probes can potentially find application in the detection of various amounts of Zn ²⁺ ions in live cells and intact tissues. A-tissue a-tissue, we all fall down: A series of Zn²⁺-ion selective two-photon fluorescent probes with a wide range of dissociation constants (K_d^TP=8 nM-12 νM) were developed to be able to detect the Zn²⁺ ions in live cells and intact tissues at a depth of>80 νm without the drawbacks of mistargeting and photobleaching.

Full text of DOI:10.1002/asia.201000720

FULL PAPERS
DOI: 10.1002/asia.201000720
Two-Photon Probes for Zn2+ Ions with Various Dissociation Constants.
Detection of Zn2+ Ions in Live Cells and Tissues by Two-Photon Microscopy
Isravel Antony Danish, Chang Su Lim, Yu Shun Tian, Ji Hee Han, Min Young Kang, and
Bong Rae Cho*[a]
Abstract: A series of Zn2+-selective
two-photon fluorescent probes (AZn-
M1ꢁAZnN) that had a wide range of
dissociation constants (KdTP =8 nm-
12 mm) were synthesized. These probes
showed appreciable water solubility
(>3 mm), cell permeability, high photo-
stability, pH insensitivity at pH>7, sig-
nificant two-photon action cross-sec-
tions (86–110 GM) upon complexation
with Zn2+, and can detect the Zn2+
ions in HeLa cells and in living tissue
slices of rat hippocampal at a depth of
>80 mm without mistargeting and pho-
tobleaching problems. These probes
can potentially find application in the
detection of various amounts of Zn2+
ions in live cells and intact tissues.
Keywords: fluorescence
· fluores-
cent probes · live tissue · two-
photon microscopy · zinc
Introduction
TFLZn, QZ1 and QZ2) and fluorescein (FluoZn-3, Znpyr,
ZnAF) have been developed.[3–7] However, the use of these
probes with one-photon microscopy (OPM) requires rela-
tively short excitation wavelengths (<500 nm), which limit
their applications in tissue imaging owing to the shallow
penetration depth (<100 mm), photobleaching, and cellular
autofluorescence.
Two-photon microscopy (TPM) is an ideal tool to over-
come these problems because it utilizes two photons of
lower energy for the excitation. Combined with an appropri-
ate two-photon (TP) probe, TPM can image intact tissue for
a long period of time with minimum interference from arti-
facts of tissue preparation that can extend more than 70 mm
into the tissue slice.[8] Recently, we reported TP probes for
Zn2+ ions (AZn1 and AZn2)[9] with dissociation constants
of 1.1ꢀ0.1 and 0.50ꢀ0.04 nm, respectively. Those probes
were able to detect intracellular free Zn2+ ([Zn2+]i) in the
nanomolar range in the living tissues at tissue depths rang-
ing from 80-170 mm without interference from other metal
ions and from the membrane-bound probes. To detect [Zn2+
] over a wide range of concentrations in the live cells and
intact tissues,[10] there is a strong need for the development
of TP probes with various affinities for Zn2+ ions.
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] It plays crucial roles in the survival, growth,
and metabolism of unicellular and multicellular organisms.
In mammalian cells, the majority of zinc ions are stored in
vesicles, and the zinc-ion concentration in the cytoplasm is
approximately 1 nm. The Zn2+-ion homeostasis is main-
tained by the import of Zn2+ ions from—and export to—the
extracellular cellular space, the endoplasmic reticulum, and
intracellular vesicles. In the brain, a few millimoles of Zn2+
ion are stored in the presynaptic vesicles of specific types of
neurons. The ions are released upon synaptic activation, are
involved in the regulation of the excitatory neurotransmis-
sion, and are associated with neuronal disorders.[2] To under-
stand the role of Zn2+ ions in physiology, it is crucial to visu-
alize their distribution and transport in live cells and intact
tissues. For this purpose, a variety of one-photon fluorescent
(OPF) probes derived from quinoline (TSQ, Zinquin and
[a] Dr. I. A. Danish,+ C. S. Lim,+ Dr. Y. S. Tian, J. H. Han, M. Y. Kang,
Prof. Dr. B. R. Cho
Department of Chemistry
To address this need, we have developed a series of TP
probes for Zn2+ ions derived from 2-acetyl-6-(dimethylami-
no)naphthalene (acedan) as the reporter and N,N-di-(2-pico-
lyl)ethylenediamine (DPEN) derivatives as Zn2+ ion recep-
tors by considering the following requirements: 1) a signifi-
cant TP cross-section for a bright TPM image; 2) an appre-
ciable water solubility to stain the cells; 3) high selectivity
Research Institute for Natural Sciences
Korea University, 1-Anamdong, Seoul 136-701 (Korea)
Fax : (+82)2-3290-3121
E-mail: chobr@korea.ac.kr
[+] These two authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201000720.
1234
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 1234 – 1240

fl
max
and a wide range of affinity for Zn2+ ions; 4) large spectral
shifts in different environments for the selective detection of
[Zn2+]i ions in the cytoplasm; 5) pH resistance; and 6) high
photostability (Scheme 2). We adopted acedan from our
previous work on various TP probes[11] and DPEN deriva-
tives from the works of Nagano and co-workers[6] and Lip-
pard and co-workers.[7] Herein, we present a series of TP flu-
orescent probes for Zn2+ ions that can detect the distribu-
tion of Zn2+ ions in living HeLa cells and fresh hippocampal
slices at a depth of >80 mm without the problems of mistar-
geting and photobleaching (Scheme 1).
shifts (Dl =70 nm) with the same variations of the sol-
vent (see the Supporting Information, Table S1), thus con-
firming the utility of these molecules as polarity probes. In
addition, AZnM1ꢁAZnN was soluble in water at concentra-
tions of up to 3.0–5.0 mm, which was sufficient to stain the
cells.
When small increments of Zn2+ ions were added to a so-
lution of AZnM1ꢁAZnN in MOPS buffer (30 mm, pH 7.3,
I=0.10), the one- and two-photon excited fluorescence in-
tensity increased without affecting the absorption spectra
(see Figure 1 and the Supporting Information, Figure S1).
This outcome can be attributed
to blocking of the photo-in-
duced electron transfer (PET)
process upon complexation
with Zn2+ ions. The fluores-
cence enhancement factors
[FEF=(FꢁFmin)/Fmin] of AZ-
n1ꢁAZnN measured for the
one- and two-photon processes
ranged from 4.0–54 (Table 1).
The FEF value was always
larger for probes with a MeO
group in the receptor moieties
(12–54 vs 4.2–24) as a conse-
quence of the lower fluores-
cence quantum yield (F) in the
absence of Zn2+ ions, and the
correspondingly higher F in the
presence of excess Zn2+ ions
(Table 1). This observation has
been attributed to the dual
roles of the electron-donating
MeO group. The methoxy
moiety elevates the HOMO
level of the receptor, rendering
the PET more efficient and re-
Scheme 1. Structures of AZn1ꢁAZnN.
Scheme 2. Synthesis of AZM1ꢁAZnN. a) i) 2-bromoethanol, CaCO3, H2O; ii) CBr4, PPh3, THF; b) A, K2CO3,
KI, MeCN; c) H2/Pd/C, EtOH; d) DCC, HOBt, B, CH2Cl2. THF=tetrahydrofuran, DCC=N,N’-dicyclohexyl-
carbodiimide, HOBt=1-hydroxybenzotriazole.
Results and Discussion
The synthesis of AZnM1ꢁAZnN is summarized in
Scheme 2. Compound 2a was prepared according to a litera-
ture procedure.[6b] Compound 2b was obtained in 67% yield
by the reaction of 1b with 2-bromoethanol followed by the
bromination. Substitution of the bromide (2a,b) with the re-
ceptor moieties (A) afforded 3 in yields ranging from 30–
59%. Reduction of 3 to 4 proceeded in 82–95% yields.
Figure 1. One-photon absorption (a) and emission (b) spectra of 1 mm
AZnM2 in MOPS buffer solution (30 mm, 100 mm KCl, 10 mm NTA,
pH 7.3) in the presence of free Zn2+ ions (0–1.3 mm). NTA=nitrilotriace-
tic acid.
AZnM1ꢁAZnN were obtained by the DCC coupling of 4
with B in 35–79% yields. All products were unambiguously
1
characterized by H and 13C NMR and HRMS (see the Sup-
porting Information).
The absorption spectra of AZnM1ꢁAZnN were almost
ð1Þ
identical and showed gradual bathochromic shifts (Dl
=
duces the F value of the probes. In addition, the tighter
complexation induced by the MeO functionality also re-
duces the vibrational relaxation pathways and enhances the
F value of the complexes.[9]
max
20 nm) with the solvent polarity (ENT )[12] in the order
1,4-dioxane<N,N-dimethylformamide<ethanol<water.
The fluorescence spectra showed much-larger bathochromic
Chem. Asian J. 2011, 6, 1234 – 1240
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
1235

B. R. Cho et al.
FULL PAPERS
Table 1. Photophysical data for AZnM1ꢁAZnN.
ð1Þ
TP [d,e]
ð2Þ [f]
Compound[a]
l
/lfl [b]
F[c]
KdOP/Kd
l
dF[g]
max max
max
AZn1[h,i]
365/496
365/498
365/494
365/499
368/501
367/504
364/504
363/504
365/502
366/503
366/503
364/504
365/500
365/502
0.02
0.47
0.01
0.65
0.017
0.37
0.015
0.42
0.080
0.54
0.026
0.39
1.1/1.1 nM
0.5/0.5 nM
15/16 nM
n.d.
780
n.d.
780
n.d.
780
n.d.
780
n.d.
780
n.d.
780
n.d.
780
n.d.
86
n.d.
95
n.d.
88
n.d.
110
n.d.
86
AZn1+Zn2+
AZn2[h,i]
AZn2+Zn2+
AZnM1[i]
AZnM1+Zn2+
AZnM2[i]
8.4/7.1 nM
0.48/0.41 mM
23/21 nM
AZnM2+Zn2+
AZnE1[i]
AZnE1+Zn2+
AZnE2[i]
n.d.
86
n.d.
89
AZnE2+Zn2+
AZnN[i]
0.018
0.29
10/12 mM
AZnN+Zn2+
[a] Data were measured in MOPS buffer solution (30 mm, 100 mm KCl,
pH 7.3) containing free Zn2+ ions (0–196 mm) and 10 mm NTA unless oth-
erwise noted. [b] lmax of the one-photon absorption and emission spectra
in nm. [c] Fluorescence quantum yield, ꢀ10%. [d] Dissociation constants
for Zn2+ ions measured by one- (KdOP) and two-photon (KdTP) processes,
Figure 2. a) Two-photon action spectra of 1 mm AZnM1ꢁAZnN in the
presence of 1.3 mm (AZnM1, ~), 1.3 mm (AZnM2, !), 3.5 mm (AZnE1,
!), 0.46 mm (AZnE2, &), 196 mm (AZnE2, *) of free Zn2+ ions.
b) Two-photon emission spectra of AZnM2 in MOPS buffer solution
(30 mm, 100 mm KCl, 10 mm NTA, pH 7.3) in the presence of free Zn2+
ions (0–1.3 mm). c) One- (*) and two-photon (*) fluorescence titration
curves for the complexation of AZnM2 with various concentrations (0–
0.13 mm) of free Zn2+ ions. d) Hill plots for the complexation of AZnM2
with free Zn2+ ions (0–0.13 mm) measured by one- (*) and two-photon
(*) processes. The excitation wavelengths for one- and two-photon pro-
cesses were 365 and 780 nm, respectively.
i
ꢀ14%. [e] The Kd values measured by the two-photon process were
9.8 nm (AZnM1), 8.7 nm (AZnM2), 0.50 mm (AZnE1), 18 nm (AZnE2),
and 9.5 mm (AZnN), respectively. [f] lmax of the two-photon excitation
spectra in nm. [g] Two-photon action cross-section in GM. [h] Referen-
ce [[16c]]. [i] Fluorescence-enhancement factor, (FꢁFmim)/Fmin, measured
by one-/two-photon (FEFop/FEFTP) processes, were 21/24 (AZn1), 54/52
(AZn2), 21/22 (AZnM1), 15/14 (AZnM2), 4.2/4.0 (AZnE1), 17/15
(AZnE2), 12/11 (AZnN), respectively.
OP
The dissociation constants (Kd
and KdTP) of AZn-
M1ꢁAZnN for the one- and two-photon processes were cal-
culated from the fluorescence titration curves.[3,6a] For all
compounds, the titration curves fitted well with a 1:1 binding
model and the Hill plots were linear with a slope of 1.0 (Fig-
ure 2c,d and the Supporting Information, Figure S1), thus
indicating a 1:1 complexation between the probes and the
showed similar selectivity except that it had an appreciable
response for Cd2+ ions (Figure 3). As Cd2+ ions are rarely
present in biological systems, AZn1ꢁAZnN can detect Zn2+
ions over a wide range of concentration without interference
from other biologically relevant metal ions.
The fluorescence intensities of AZnM1ꢁAZnN were pH
insensitive over the range of pH 3.5–8.0. In the presence of
excess Zn2+ ions, the fluorescence intensity of AZn-
OP
TP
Zn2+ ions.[13] The Kd and Kd values, which were almost
the same within experimental errors, were in the range of
8.4 nm-12 mm (Table 1). The Kd values were always smaller
for compounds with the MeO group (AZn1>AZn2,
AZnM1>AZnM2, AZnE1>AZnE2), which is in agree-
ment with its capability of inducing a tighter binding (see
above). Combined with AZn1 (KdTP =1.1 nm) and AZn2
(KdTP =0.5 nm), we now have a series of TP probes (AZ-
n1ꢁAZnN) with a wide range of binding affinities. We also
i
measured the dissociation constant (Kd) in ionophore-treat-
ed HeLa cells by TPM (see the Supporting Information, Fig-
i
ure S2).[14] These Kd values were within experimental error
of the values measured in MOPS buffer solution (Fig-
ure 1b), thus indicating that MOPS buffer solution repre-
sents the cytosolic environment reasonably well. Moreover,
these values could be applied to the quantitative measure-
i
ment
of
[Zn2+
]
ions
by
using
[Zn2+]i =Kd -
i
A
G
photon excited fluorescence (TPEF) intensities in the ab-
sence and presence of excess Zn2+ ions and the observed
TPEF intensity, respectively.[15]
Figure 3. The relative fluorescence intensity of 1 mm AZnM1ꢁAZnN in
MOPS buffer solution (30 mm, 100 mm KCl, 10 mm NTA, pH 7.3) in the
presence of 5 mm Na+, K+, Ca2+, or Mg2+ ions, or 300 mm of Fe2+, Fe3+
,
Mn2+, Co2+, Cu2+, or Cd2+ ions. Zn2+ ions were added at either 1 mm
(AZnM1, AZnM2, AZnE2) or 3 mm (AZnE1) or 200 mm (AZnN). The
data at [Zn2+]=0 mm were determined in the presence of 10 mm NTA.
The excitation wavelength was 365 nm.
and Mg2+ ions at 5 mm, and to Mn2+, Fe2+, Co2+, Ni2+
Cu2+
1236
,
Chem. Asian J. 2011, 6, 1234 – 1240

Detection of Zn2+ Ions in Live Cells and Tissues by Two-Photon Microscopy
Figure 4. The effect of the pH value on the one-photon fluorescence in-
tensity of 1 mm AZnM1ꢁAZnN in the absence (open symbols) and pres-
ence of (closed symbols) excess Zn2+ ions in 30 mm MOPS buffer solu-
tion and 100 mm KCl at pH 7.3. The data at [Zn2+]=0 mm were deter-
mined by adding 10 mm NTA. The excitation wavelength was 365 nm.
The line is a guide to the eye.
Figure 5. TPM images of AZnM2-labeled (2 mm) HeLa cells collected at
360–620 nm (a), 360–460 nm (c), and 520–620 nm (d). b) Two-photon-ex-
cited fluorescence spectra from the intense spot (A) and homogeneous
domain (B) shown in (a), while the closed circle (*) and open square
(&) correspond to the Gaussian functions fitted to the emission spectra.
The triangle (~) and star (N) represent the two Gaussian functions
fitted to the black circle. The excitation wavelength was 780 nm. Cells
shown are representative images from replicate experiments. Scale bar,
30 mm.
M1ꢁAZnN remained nearly the same at pH>7.0, and de-
creased at lower pH values (Figure 4). The decrease was ap-
preciable for AZnE1 and significant for AZnN with the
largest Kd value. This result can be attributed to the compet-
ing pathways for protonation and chelation of Zn2+ ions by
the receptor moieties. Protonation would be the more-favor-
able event for a lower affinity probe at a lower pH value
and would reduce the effective concentration of the probe–
Zn2+ complex, thereby decreasing the fluorescence intensity.
Nevertheless, the fluorescence intensity of the Zn2+ com-
plexes with AZnM1ꢁAZnN are not subject to interference
by pH changes under near-neutral or slightly acidic condi-
tions.
two Gaussian functions with emission maxima at 445 (~)
and 485 nm (N; Figure 5b). The longer-wavelength bands
(& and N) were similar to the emission spectrum of
AZnM2 in the MOPS buffer solution, whilst the shorter-
wavelength band (~) was significantly blue-shifted. The dif-
ferences in emission maxima suggest that the probes may be
located in two regions of different polarity: a more-polar en-
vironment that is likely to be cytosol and a less-polar one
that is likely to be membrane. Moreover, the shorter wave-
length band (~) decreased to the baseline at wavelength of
<520 nm. Consistently, the TPM image collected at 520–
620 nm is homogeneous without intense spots (Figure 5d),
whereas that collected at 360–460 nm clearly shows them
(Figure 5c). Therefore, by using the detection window of
520–620 nm, we have detected cytosolic Zn2+ ions. We note
that environmental sensitivity of these probes is beneficial
to TPM where the detection window can be selected, where-
as in fluorescence microscopy without a selective detection
window, it may cause problems.
To demonstrate the utility of these probes, we have de-
tected Zn2+ ions in the cultured HeLa cells by TPM. Earli-
er, we reported a bright TPM image of 293 cells labeled
with AZn2 (Kd =0.5 nm).[9] In contrast, the TPM images of
HeLa cells labeled with AZnM2 (Kd =8.4 nm) and AZnE1
(Kd =480 nm) are dim (Figure 6a,b), presumably owing to
their lower affinities for Zn2+ ions. When the cells were
treated with Zn2+ ions and pyrithione (2-mercaptopyridine
N-oxide, which can bring Zn2+ ions into the cytoplasm),[17]
the TPEF intensity increased immediately. The increase was
The TP cross-sections of the probe–Zn2+ complexes in
MOPS buffer solutions were determined by a femtosecond
(fs) fluorescence measurement technique, as reported previ-
ously.[16] The TP action spectra of the complexes indicated
Fd values of 86–110 GM (1 GM=10ꢁ50 cm4 sphotonꢁ1) at
780 nm, values which exceeded those of TSQ and FluoZin-3
by 4~24-fold (Figure 2a and Table 1).[9] Thus, TPM images
of the cells stained with AZn1ꢁAZnN would be much
brighter than those stained with the commercial probes.
Photostability was determined under the imaging condi-
tions by monitoring the time-dependent decrease in the TP-
excited fluorescence (TPEF) intensity in HeLa cells labeled
with AZnM2 and AZnE1; measurements were made in four
individual cells chosen without bias.[11d,f] The TPEF intensity
decreased by approximately 5% within 1000 s and remained
nearly the same for approximately 1 hour (see the Support-
ing Information, Figure S3). Similar results were observed
for the other probes (data not shown). These studies showed
that AZn1ꢁAZnN has a high photostability and they are
therefore suitable for long-term imaging applications.
The TPM image of the HeLa cells labeled with AZnM2
revealed intense spots (A) and homogeneous domains (B;
Figure 5a). The homogeneous domain (B) could be fitted to
a single Gaussian function (&) with an emission maximum
at 495 nm, whereas the intense spot (A) could be fitted to
Chem. Asian J. 2011, 6, 1234 – 1240
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
1237

B. R. Cho et al.
FULL PAPERS
Figure 6. TPM images of HeLa cells incubated with a) AZnM2 and
b) AZnE1 (2 mm) for 20 min at 378C. (c, d) Time courses of TPEF in indi-
vidual cells as designated by the * for c) sample AZnM2 (from (a)) and
d) sample AZnE1 (from (b)) after treatment with pyrithione/Zn2+
(1.0:0.5, 5.0:2.5, 5.0:5.0, 25:25 mm) and then with TPEN (0.1 mm) at inter-
vals of 5 min. Images were collected at 520-620 nm with 1.6 s intervals
using an excitation wavelength at 780 nm. Scale bar, 30 mm.
qualitatively proportional to the concentration of added
Zn2+ ions, reaching the maximum concentration after the
addition of 5.0 and 25 mm for the cells labeled with AZnM2
and AZnE1, respectively (Figure 6c,d). Also, the fluores-
cence intensity decreased abruptly to the baseline level
upon addition of 0.1 mm N,N,N’,N’-terakis(2-pyridyl)ethyle-
nediamine (TPEN), a membrane-permeable Zn2+ ion chela-
tor that can effectively remove Zn2+ ions (Figure 6c,d).[6,7]
Thus, the response time is very short and the TPEF is owing
to the presence of probe–Zn2+ complexes in the cells.
To further assess the utility of these probes, we have de-
tected Zn2+ ions deep inside live tissues. The bright field
image of a part of fresh hippocampal slices from the postna-
tal 2-week-old rat incubated with 20 mm AZnM2ꢁAZnN
for 1 hour at 378C showed the CA1 and CA3 regions as
well as the dentate gyrus (DG); the TPM images revealed
the Zn2+ ion distribution in the same regions at a depth of
110 mm (see the Supporting Information, Figure S4). The
TPM images of the slice labeled with AZnM2 showed in-
tense fluorescence in the stratum lucidum (SL) of the CA3
region (Figure 7a). The image taken at a higher magnifica-
tion clearly reveals the Zn2+ ion distribution in the same
region (Figure 7b). The increase in the TPEF intensity after
the addition of 50 mm KCl (Figure 7c,g) as a membrane de-
polarizer causing the release of Zn2+ ions, and the decrease
in the TPEF upon treatment with TPEN (Figure 7d,h), pro-
vide supporting evidences for this observation.[18] When the
tissue was labeled with AZnE2, the image was dim except
in the SL where Zn2+ ions were more-concentrated, as pre-
dicted from the lower affinity (Figure 7a,e). Consistently,
the TPM images of the tissue slices labeled with lower affin-
ity probes became progressively dimmer (see the Supporting
Information, Figure S4). Notably, the lower affinity probes
Figure 7. TPM images of slices of rat hippocampal labeled with 20 mm
AZnM2 (a–d) and AZnE2 (e–h), respectively, for 1 h at 378C. a, e) TPM
images at a depth of approximately 110 mm with magnification ꢁ10. Scale
bar, 300 mm. b–d, f–h) Magnification at ꢁ100 in stratum lucidum (SL) of
CA3 regions (white box) at a depth of approximately 120 mm before (b,
f) and after addition of 50 mm KCl (c, g) to the imaging solution. d,
h) After addition of 200 mm TPEN to (c) and (g), respectively. Scale bar,
30 mm. All images were collected at 520–620 nm and the excitation wave-
length was 780 nm with fs pulses.
can highlight the Zn2+-rich regions. Moreover, the TPM
images of the slices labeled with AZnM2 revealed the Zn2+
ion distribution in the given xy plane of the CA3 region at
80–170 mm depth, thus indicating the sectioning capability of
TPM (see the Supporting Information, Figure S5). These re-
sults demonstrate the capacity of AZnM1ꢁAZnN to detect
1238
www.chemasianj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 1234 – 1240

Detection of Zn2+ Ions in Live Cells and Tissues by Two-Photon Microscopy
Table 2. The calculated [Zn2+] free concentration of each solution.
Zn2+ ions over a wide range of concentrations in live tis-
sues.
A
]
]
]
]
total [mM]
free [nM]
total [mM]
free [nM]
1.0
0.85
6.9
2.0
1.9
7.1
19
3.0
3.3
8.0
30
4.0
5.1
8.4
41
5.0
7.6
9.0
65
5.5
9.2
9.4
130
6.0
11
9.9
AHCTUNGTRENNUNG
N
A
17
1300
Conclusions
In conclusion, we have developed a series of TP Zn2+
probes (AZnM1-AZnN) with various Kd values ranging
from sub-nanomolar to sub-millimolar. They show apprecia-
ble water solubility, cell permeability, significant TP action
cross-sections when complexed with Zn2+ ions, high selectiv-
ity for Zn2+ ions, high photostability, pH insensitivity at
pH>7, and short response times. By using AZnM2 and
AZnE1, different responses to changes in extracelluar Zn2+
ion concentration were observed. Moreover, the distribution
of Zn2+ ions in the living rat-brain hippocampal slices could
be detected by TPM at a depth of >80 mm by using AZn-
M1ꢁAZnN. Combined with the existing TP probes for Zn2+
ions (AZn1 and AZn2), AZn1ꢁAZnN will find useful ap-
plications in the detection of various amounts of Zn2+ ions
in live cells and intact tissues.
The stability constant for the Zn2+ complex of NTA (KZnꢁNTA) was taken
from Ref. [22].
Thus, for NTA (pH 7.3, 0.1m KCl, 258C), pK1 =9.73, pK2 =2.49, pK3 =
1.89, log KZnꢁNTA =10.66. All protonation constants must be corrected
upward by 0.11 when determined in 0.1m ionic-strength solutions.
[NTA]total was set at 10 mm, and [Zn2+
]
was varied from 0–9.9 mm.
total
The calculated [Zn2+
]
concentration of each solution is shown in
free
Table 2.
When [Zn2+
]
was over 9.9 mm in the NTA buffer system, various ali-
total
quots of 1–10 mm aqueous ZnSO4 solutions were directly added to 30 mm
MOPS buffer solution (0.1m KCl, pH 7.3), which was treated with
Chelex resin (Bio-Rad, manufacturer protocol).
To determine the apparent dissociation constants for the Zn2+ complex
of probes, the fluorescence titration curves (Figure 1b) were obtained
and fitted to Equation (2) (see Figure 2c).[6a,22]
½Zn2þꢂfree
ð2Þ
F ¼ F0 þ ðFmax ꢁ F0Þ
Kd þ ½Zn2þꢂfree
As shown in Equation (2), F is the fluorescence intensity, Fmax is the max-
imum fluorescence intensity, F0 is the fluorescence intensity in the ab-
Experimental Section
Spectroscopic Measurements
sence of Zn2+ ions, and [Zn2+
]
is the free-Zn2+-ion concentration. The
free
Kd value that best fits the titration curve (Figure 2c) with Equation (2)
was calculated by using the Excel program.
Absorption spectra were recorded on a Hewlett–Packard 8453 diode
array spectrophotometer, and fluorescence spectra were obtained with
Aminco-Bowman series 2 luminescence spectrometer with a 1-cm stan-
dard quartz cell. The fluorescence quantum yield was determined with
Coumarin 307 as the reference using a literature method.[18]
TP
To determine the Kd for the two-photon process, the TPEF spectra
were obtained with a DM IRE2 Microscope (Leica) by using the xyl
mode at a scan speed of 800 Hz. The samples were excited by a mode-
locked titanium-sapphire laser source (Coherent Chameleon, 90 MHz,
200 fs) set at a wavelength of 780 nm and an output power of 1180 mW,
which corresponded to an average power in the focal plane of approxi-
mately 10 mW. The TPEF titration curves (Figure 2b) were obtained and
fitted to Equation (2), as shown in Figure 2c.
Water Solubility
A small amount of dye was dissolved in N,N-dimethylsulfoxide to pre-
pare the stock solutions (1.0ꢁ10ꢁ3 m). The solution was diluted to (6.0ꢁ
10ꢁ3 ~6.0ꢁ10ꢁ5 m) and added to a cuvette containing 3.0 mL of H2O
using a microsyringe. In all cases, the concentration of DMSO in H2O
was maintained at 0.2%.[19] The plots of fluorescence intensity against
the dye concentration were linear at low concentration and showed
downward curvature at higher concentrations. The maximum concentra-
tion in the linear region was taken as the solubility. The water solubilities
of AZnM1ꢁAZnN were in the range of 3.0–5.0 mm (AZnM1, 5.0 mm;
AZnM2, 4.0 mm; AZnE1, 3.0 mm; AZnE2, 5.0 mm; AZnN, 4.0 mm).
Measurement of Two-Photon Cross-Sections
The two-photon cross-section (d) was determined by using a femtosecond
(fs) fluorescence measurement technique, as described before.[16] The
probes were dissolved in 30 mm MOPS buffer solution (100 mm KCl,
pH 7.3) at concentrations of 5.0ꢁ10ꢁ6 m and then the TPEF intensity was
measured at 740–940 nm by using fluorescein (8.0ꢁ10ꢁ5 m, pH 11) as the
reference, whose two-photon property has been well-characterized in the
literature.[23] The emission intensities of the TPEF spectra of the refer-
ence and the sample were determined at the same excitation wavelength.
The two-photon absorption (TPA) cross-section was calculated by using
Determination of Apparent Dissociation Constants
A series of MOPS (4-morpholinepropanesulfonic acid) buffer solutions
(30 mm, pH 7.3, 0.1m KCl) containing various amounts of ZnSO4 (0~
d=drACHTUNGRTENN(GU SsFrfrcr)/HCAUTNGTERN(NUGN SrFsfscs) where the subscripts s and r stand for the
9.9 mm) and 10 mm of NTA (nitrilotriacetic acid) were prepared. The
sample and reference molecules. The intensity of the signal collected by a
CCD detector was denoted as S, the fluorescence quantum yield as F,
and the overall fluorescence collection efficiency of the experimental ap-
paratus as f. The number density of the molecules in solution was denot-
ed as c, whilst dr represents the TPA cross-section of the reference mole-
cule.
app
ZnꢁNTA
[Zn2+
]
was calculated from the K
, [NTA]total, and [Zn2+
]
by
free
total
using Equation (1).[3, 20]
½Zn2þꢂfree ¼ ½Zn2þ
ꢂ
total=ðaZn ꢃ KZapnpꢁNTA ꢃ ½NTAꢂfree
Þ
ð1Þ
Where,
Cell Culture and Imaging
KZapnpꢁNTA =KZnꢁNTA/aZnaNTA
,
HeLa human cervical carcinoma cells (ATCC, Manassas, VA, USA)
were cultured in DMEM (WelGene Inc, Seoul, Korea) supplemented
with heat-inactivated 10% FBS (WelGene Inc, Seoul, Korea), penicillin
(100 unitsmLꢁ1), and streptomycin (100 mgmLꢁ1) at 378C in an incubator
with a humidified atmosphere of 5% CO2 and 95%. Three days before
imaging, the cells were detached and were replaced on glass-bottomed
dishes (MatTek). For labeling, the cells were rinsed with phosphate-buf-
fered saline solution (DPBS; WelGene Inc) and incubated with 2 mm of
aaZn ¼ 1 þ 10ðpHꢁpK Þ þ 10ð2pHꢁpK
Þ þ 10ð3pHꢁpK
1 ꢁpK2
1ꢁpK2ꢁpK3
Þ:::
1
aaNTA ¼ 1 þ 10ðpK ꢁpHþ0:11Þ þ 10ðpK
ꢁ2pHþ0:22Þ þ 10ðpK
1 þpK2
1 þpK2þpK3
ꢁ3pHþ0:33Þ:::
1
and
G
ꢁ
Thus,
KZnꢁNTA ð1þ10ðpK
ꢁpHÞ Þ
ZnꢁNTA
app
ZnꢁNTA
K
¼
ð1þ10ðpHꢁpK ÞÞð1þ10ðpK ꢁpHÞ þ10ðpK
1 þpK2
ꢁ2pHÞÞ
Zn
1
Chem. Asian J. 2011, 6, 1234 – 1240
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
1239

B. R. Cho et al.
FULL PAPERS
probe for 20 min at 378C. After washing with DPBS three times, the cells
were mounted on the microscope stage and following addition of the in-
dicated concentrations of ZnSO4/pyrithione, they were imaged.
[6] a) T. Hirano, K. Y. Urano, T. Nagano, J. Am. Chem. Soc. 2002, 124,
6555–6562; b) K. Komatsu, K. Kikuchi, H. Kojima, Y. Urano, T.
Nagano, J. Am. Chem. Soc. 2005, 127, 10197–10204; c) K. Komatsu,
Y. Urano, H. Kojima, T. Nagano, J. Am. Chem. Soc. 2007, 129,
13447–13454.
Two-Photon Fluorescence Microscopy
[7] a) S. C. Burdette, C. J. Frederickson, W. Bu, S. J. Lippard, J. Am.
Chem. Soc. 2003, 125, 1778–1787; b) C. J. Chang, J. Jaworski, E. M.
Nolan, M. Sheng, S. J. Lippard, Proc. Natl. Acad. Sci. USA 2004,
101, 1129–1134; c) E. M. Nolan, J. W. J. Jaworski, R. P Feazell, M.
Sheng, S. J. Lippard, J. Am. Chem. Soc. 2006, 128, 15517–15528.
[8] a) R. M. Williams, W. R. Zipfel, W. W. Webb, Curr. Opin. Chem.
Biol. 2001, 5, 603–608; b) W. R. Zipfel, R. M. Williams, W. W.
Webb, Nat. Biotechnol. 2003, 21, 1369–1377; c) F. Helmchen, W.
Denk, Nat. Methods 2005, 2, 932–940.
[9] H. M. Kim, M. S. Seo, M. J. An, J. H. Hong, Y. S. Tian, J. H. Choi,
O. Kwon, K. J. Lee, B. R. Cho, Angew. Chem. 2008, 120, 5245–5248;
Angew. Chem. Int. Ed. 2008, 47, 5167–5170.
[10] C. J. Frederickson, M. A. Klitenick, W. I. Manton, J. B. Kirkpatrick,
Brain Res. 1983, 273, 335–339.
TPM images of the probe-labeled HeLa cell and tissues were obtained
with spectral confocal and multiphoton microscopes (Leica TCS SP2)
with ꢁ100 oil and ꢁ10 dry objective, numerical aperture (NA)=1.30 and
0.3, respectively. The TPM images were obtained with a DM IRE2 Mi-
croscope (Leica) by exciting the probes with a mode-locked titanium-sap-
phire laser source (Coherent Chameleon, 90 MHz, 200 fs) set at wave-
length 780 nm and output power 1180 mW, which corresponded to ap-
proximately 10 mW average power in the focal plane. To obtain images
at the 360~620 nm, 360~460 nm, and 500~620 nm ranges, internal PMTs
were used to collect the signals in an 8 bit unsigned 512ꢁ512 pixels at
400 Hz scan speed.
Preparation and Staining of Fresh Rat-Hippocampal Slices
All experiments were performed in accordance with the guidelines estab-
lished by the Committee of Animal Research Policy of Korea University
College of Medicine. Rats that were 2 weeks old were sacrificed by cervi-
cal dislocation within 2 hours of arrival. Slices were prepared from the
hippocampi of 2-week-old rats (SD). Coronal slices (400 mm thick) were
cut by using a vibrating-blade microtome in artificial cerebrospinal fluid
(ACSF; 124 mm NaCl, 3 mm KCl, 26 mm NaHCO3, 1.25 mm NaH2PO4,
10 mm d-glucose, 2.4 mm CaCl2, and 1.3 mm MgSO4). Slices were incubat-
ed with 20 mm of the probes in ACSF bubbled with 95% O2 and 5%
CO2 for 1 hour at 378C. Slices were then washed three times with ACSF
and transferred to glass-bottomed dishes (MatTek) and observed by
using a spectral confocal multiphoton microscope.
[11] a) H. M. Kim, B. R. Cho, Acc. Chem. Res. 2009, 42, 863–872; b) P. S.
Mohan, C. S. Lim, Y. S. Tian, W. Y. Roh, J. H. Lee, B. R. Cho,
Chem. Commun. 2009, 5365–5367; c) Y. S. Tian, Lee, H. Y. , C. S.
Lim, J. M. Park, H. M. Kim, Y. N. Shin, E. S. Kim, H. J. Jeon, S. B.
Park, B. R. Cho, Angew. Chem. 2009, 121, 8171–8175; Angew.
Chem. Int. Ed. 2009, 48, 8027–8031; d) M. K. Kim, C. S. Lim, J. T.
Hong, J. H. Han, H. Y. Jang, H. M. Kim, B. R. Cho, Angew. Chem.
2010, 122, 374–377; Angew. Chem. Int. Ed. 2010, 49, 364–367;
e) J. H. Lee, C. S. Lim, Y. S. Tian, J. H. Han, B. R. Cho, J. Am.
Chem. Soc. 2010, 132, 1216–1217; f) C. S. Lim, D. W. Kang, Y. S.
Tian, J. H. Han, H. L. Hwang, B. R. Cho, Chem. Commun. 2010, 46,
2388–2390.
[12] C. Reichardt, Chem. Rev. 1994, 94, 2319–2358.
[13] K. A. Connors, Binding Constants, Wiley, New York, 1987.
[14] H. M. Kim, P. R. Yang, M. S. Seo, J.-S. Yi, J. H. Hong, S.-J. Jeon, Y.-
G. Ko, K. J. Lee, B. R. Cho, J. Org. Chem. 2007, 72, 2088–2096.
[15] R. E. London, Annu. Rev. Physiol. 1991, 53, 241–258.
[16] S. K. Lee, W. J. Yang, J. J. Choi, C. H. Kim, S.-J. Jeon, B. R. Cho,
Org. Lett. 2005, 7, 323–326.
[17] a) B. L. Barnett, H. C. Kretschmar, F. A. Hartman, Inorg. Chem.
1977, 16, 1834–1838; b) C. J. Chandler, I. H. Segel, Antimicrob.
Agents Chemother. 1978, 14, 60–68.
Acknowledgements
This work was supported by a National Research Foundation (NRF)
grant funded by the Korean Government (No. 2010-0018921), and by a
Priority Research Centers Program through the NRF funded by the Min-
istry of Education, Science and Technology (No. 2010-0020209). I.A.D.,
C.S.L., J.H.H., and M.Y.K. were supported by a BK21 scholarship.
[18] J. N. Demas, G. A. Crosby, J. Phys. Chem. 1971, 75, 991–1024.
[19] H. M. Kim, H.-J. Choo, S.-Y. Jung, Y.-G. Ko, W.-H. Park, S.-J. Jeon,
C. H. Kim, T. Joo, B. R. Cho, ChemBioChem 2007, 8, 553–559.
[20] M. Taki, J. L. Wolford, T. V. OꢂHalloran, J. Am. Chem. Soc. 2004,
126, 712–713.
[21] A. E. Martell, R. M. Smith, NIST Critical Stability Constants of
Metal Complexes. NIST Standard Reference Database 46, Version
7.0, 2003.
[1] a) B. L. Vallee, K. H. Falchuk, Physiol. Rev. 1993, 73, 79–118;
b) J. M. Berg, Y. G. Shi, Science 1996, 271, 1081–1085.
[2] a) C. J. Frederickson, J.-H. Koh, A. I. Bush, Nat. Neurosci. 2005, 6,
449–462; b) S. Y. Assaf, S. H. Chung, Nature 1984, 308, 734–736;
c) G. A. Howell, M. G. Welch, C. J. Frederickson, Nature 1984, 308,
736–738.
[3] C. J. Fahrni, T. V. OꢂHalloran, J. Am. Chem. Soc. 1999, 121, 11448–
11458.
[22] T. Hirano, K. Kikuchi, Y. Urano, T. Higuchi, T. Nagano, J. Am.
Chem. Soc. 2000, 122, 12399–12400.
[4] K. Kikuchi, K. Komatsu, T. Nagano, Curr. Opin. Chem. Biol. 2004,
8, 182–191.
[23] C. Xu, W. W. Webb, J. Opt. Soc. Am. B 1996, 13, 481–491.
[5] D. W. Domaille, E. L. Que, C. J. Chang, Nat. Chem. Biol. 2008, 4,
168–175.
Received: October 4, 2010
Published online: February 17, 2011
1240
www.chemasianj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 1234 – 1240