Two-photon fluorescent probes for intracellular free zinc ions in living tissue

Article abstract of DOI:10.1002/anie.200800929

The living image: The efficient two-photon probes AZn1 and AZn2 (see picture for rat brain tissue) show 24-to 52-fold two-photon excited fluorescence enhancement in response to Zn²⁺. They can selectively detect intracellular free Zn²⁺ ions in live cells and in living tissues at a depth of 80-150 mm without interference from other metal ions and the membrane-bound probes. (Chemical Equation Presented)

Full text of DOI:10.1002/anie.200800929

Angewandte
Chemie
DOI: 10.1002/anie.200800929
Fluorescent Probes
Two-Photon Fluorescent Probes for Intracellular Free Zinc Ions in
Living Tissue**
Hwan Myung Kim, Mun Sik Seo, Myoung Jin An, Jin Hee Hong, Yu Shun Tian, Joon Ho Choi,
Ohyun Kwon, Kyoung J. Lee, and Bong Rae Cho*
Zinc is a vital component of enzymes and proteins.[1,2] In the
brain, a few millimoles of intracellular free Zn2+ ions are
stored in the presynaptic vesicles, released with synaptic
activation, and seem to modulate excitatory neurotransmis-
sion.[2] To understand the biological roles of Zn2+, a variety of
fluorescent probes derived from quinoline (TSQ, Zinquin,
and TFLZn) and fluorescein (FluZn-3, Znpyr, ZnAF, etc.)
have been developed.[3–7] However, most of them require a
rather short excitation wavelength or suffer from pH sensi-
tivity.
membrane[14] and metal ions,[12,13] and DPEN is a well-known
receptor for Zn2+.[6] Herein, we report that AZn1 and AZn2
are capable of imaging the intracellular free Zn2+ ions in live
cells for a long period of time and in living tissue at a depth of
> 80 mm without mistargeting and photobleaching problems.
The synthesis of AZn1 and AZn2 is shown in Scheme 1.
The water solubilities of AZn1 and AZn2 are about 3.0 mm,
which is sufficient for staining the cell (see the Supporting
To visualize the biological activity deep inside living tissue
(> 80 mm) without the interference of surface preparation
artifacts,[8a] it is crucial to use two-photon microscopy (TPM),
which utilizes two photons of lower energy for the excitation.
Recently, TPM has gained much interest from biologists
because it offers a number of advantages in biological
imaging, including increased penetration depth, localized
excitation, and prolonged observation time.[8] However,
efficient two-photon (TP) probes for Zn2+ appear to be
rare.[9] Furthermore, although a few pH-resistant sensors for
Zn2+ have been reported, they require either microinjection
for cellular applications[10] or use a significant amount of
ethanol as co-solvent because of the poor water solubility.[11]
An efficient TP probe for Zn2+ should have sufficient
water solubility to stain the cells, high selectivity for Zn2+ ions,
significant TP cross section, pH resistance, and high photo-
stability. In this context, we extend our earlier work[12,13] and
present new TP probes for intracellular free Zn2+ ions (AZn1
and AZn2) derived from 2-acetyl-6-(dimethylamino)naph-
thalene (acedan) as the fluorophore[13] and N,N-di-(2-pico-
lyl)ethylenediamine (DPEN) as the Zn2+ chelator. Acedan is
a polarity-sensitive fluorophore that has been successfully
employed in the design of TP fluorescent probes for the
Scheme 1. Synthesis of AZn1 and AZn2. a) DCC (N,N’-dicyclohexylcar-
bodiimide), HOBt (1-hydroxybenzotriazole), CH2Cl2.
Information). The emission spectra showed large bathochro-
mic shifts (> 80 nm) with the solvent polarity (ETN) in the
order 1,4-dioxane < DMF < EtOH < H2O, thus indicating
the utility of these compounds as polarity probes (see the
Supporting Information).
[*] Dr. H. M. Kim, M. S. Seo, M. J. An, Dr. Y. S. Tian, Prof. Dr. B. R. Cho
Department of Chemistry, Korea University
1-Anamdong, Seoul136-701 (Korea)
When small increments of Zn2+ were added to AZn1 and
AZn2 in 3-(N-morpholino)propanesulfonic acid (MOPS)
buffer solution (30 mm, pH 7.2, I = 0.10), the one-photon
(OP) and TP excited fluorescence intensity increased grad-
ually without affecting the absorption spectra (Figure 1a and
the Supporting Information), presumably because of the
blocking of the photoinduced electron-transfer (PET) process
by the complexation with Zn2+. The fluorescence enhance-
ment factors [FEF = (FÀFmin)/Fmin] of AZn2 measured for OP
and TP processes were 2.5-fold larger than those of AZn1 as a
result of the lower fluorescence quantum yield (F) in the
absence, and higher F in the presence, of excess Zn2+ (see the
Supporting Information).
Fax: (+82)2-3290-3544
E-mail: chobr@korea.ac.kr
Dr. J. H. Hong, J. H. Choi, Prof. Dr. K. J. Lee
NationalCreative Research Initiative Center for Neurodynamics and
Department of Physics, Korea University (Korea)
Dr. O. Kwon
Samsung Advanced Institute of Technology (Korea)
[**] This work was supported by a KOSEFgrant funded by the Ministry of
Science and Technology (MOST) (R0A-2007-000-20027-0). J.H.H.,
J.H.C., and K.J.L. were supported by Creative Research Initiatives of
the MOST.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200800929.
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The TP action spectra of the Zn2+ complexes with AZn1
and AZn2 in buffer solutions indicated a Fd value of ca.
90 Gm at 780 nm, four- to 24-fold larger than those of TSQ
and FluZin-3 (Figure 1b and the Supporting Information).
This finding indicates that TPM images for samples stained
with AZn1 and AZn2 would be much brighter than those
stained with commercial probes.
The TPM images of 293 cells labeled with both probes
showed no two-photon excited fluorescence (TPEF) at 360–
460 nm (Figure 2b and the Supporting Information) and
appreciable TPEF at 500–620 nm (Figure 2c and the Support-
ing Information). For comparison, the TPM images of cells
labeled with Acedan-derived TP probes for Mg2+ (AMg1)[13a]
and Ca2+ (ACa1)[13b] showed TPEF in the 500–620 and 360–
460 nm regions, which has been attributed to the probes
associated with cytosol and membrane, respectively. Hence,
AZn2 appears to be predominantly located in the cytosolic
compartments, probably because of the lower molecular
Figure 1. a) TP fluorescence spectra of 2 mm AZn2 (30 mm MOPS,
100 mm KCl, 10 mm EGTA, pH 7.2) in the presence of free Zn2+ (0–
47 nm). The excitation wavelength is 780 nm. b) TP action spectra of
~
!
*
AZn1 (&), AZn2 ( ), FluZin-3 ( ), and TSQ ( ) in the presence of
1.8 mm free Zn2+. EGTA=ethylene glycol bis(2-aminoethyl ether)
N,N,N’,N’-tetraacetic acid.
DFTcalculations at the B3LYP/6-31G** level revealed
that the highest molecular orbital energies of DPEN (R1), 2-
methoxy-DPEN (R2), AZn1, and AZn2 are À4.948, À4.246,
À8.762, and À8.733 eV, respectively (see the Supporting
Information).[15] Hence, the PETfrom R2 to AZn2 might
occur more efficiently on thermodynamic grounds, thereby
decreasing the F value.[13d] On the other hand, the larger F
for AZn2–Zn2+ may be attributed to the tighter binding (see
below), which may reduce the vibrational relaxation path-
ways. Moreover, the linear Hill plots determined for Zn2+
binding with a slope of 1.0 indicated 1:1 complexation
between the probes and Zn2+ (see the Supporting Informa-
tion).[16] The optimized geometries of AZn1–Zn2+ and AZn2–
Zn2+ complexes are trigonal bipyramidal, in which Zn2+ ions
are coordinated by four nitrogen atoms and one water
molecule (see the Supporting Information).
The dissociation constants (KdOP and KdTP) for AZn1 and
AZn2 calculated from the OP and TP fluorescence titration
curves[3,6a] are (1.1 Æ 0.1) and (0.50 Æ 0.04) nm, respectively
(see the Supporting Information); the detection limits of
TP
Figure 2. a) Bright-field image. b–e) TPM images of 2 mm AZn2-labeled
293 cells collected at 360–460 (b) and 500–620 nm (c–e), before (c)
and after (d) addition of 10 mm SNOC to the imaging solution; scale
bar: 30 mm. e) After addition of 0.1 mm TPEN to (d). f) Relative TPEF
intensity of AZn1- and AZn2-labeled 293 cells collected at 500–620 nm
as a function of time. The TPEF images were collected upon excitation
at 780 nm with a femtosecond pulse. Cells shown are representative
images from replicate experiments (n=5).
these probes are in the subnanomolar range. The smaller Kd
value for AZn2 is consistent with the tighter binding between
AZn2 and Zn2+ (see the Supporting Information). Both
probes show high selectivity for Zn2+ compared with Na+,
Ca2+, Mg2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Cd2+, and are
pH-insensitive in the biologically relevant pH range (see the
Supporting Information).
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Angewandte
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weight, and thereby can detect [Zn2+]i in live cells without
interference from the membrane-bound probes.
Moreover, because the fluorescence intensities of AZn1
and AZn2 increase slightly at pH < 4 (see the Supporting
Information), there is a possibility that the probes in the
acidic vesicles might partially contribute to the TPM images.
To rule out such a possibility, 293 cells and primary cortical
cultures were co-stained with AZn2 and LysoTracker Red, a
well-known OP fluorescent probe for acidic vesicles,[17] and
the images were co-localized. However, they did not merge
(see the Supporting Information). Moreover, the TPM images
taken after treatment with 100 mm N,N,N’,N’-tetrakis(2-pyr-
idyl)ethylenediamine (TPEN), a membrane-permeable Zn2+
chelator that can effectively remove Zn2+,[6,7] showed little
TPEF emission (see the Supporting Information). Further-
more, the OP microscopy images taken before and after
treatment with TPEN are nearly identical (see the Supporting
Information). Hence, AZn2 can selectively detect [Zn2+]i in
neurons by TPM without interference from the probes
associated with the acidic vesicles.
To demonstrate the utility of this probe in cell imaging, we
monitored the TPEF of AZn2-labeled 293 cells after addition
of 10 mm S-nitrosocysteine (SNOC), an endogenous NO
donor that triggers the release of Zn2+.[7b,c] The TPEF intensity
increased gradually with time and then decreased abruptly
upon addition of 0.1 mm TPEN, a membrane-permeable Zn2+
chelator that can effectively remove Zn2+ (Figure 2d–f).[7b,c]
A
similar result was observed with AZn1 except that the
response was smaller as a result of the larger Kd (Figure 2 f
and the Supporting Information). Hence, these probes are
clearly capable of detecting [Zn2+]i in live cells for longer than
1000 s.
Figure 3. TPM images of a rat hippocampalsilce stained with 10 mm
To further investigate the utility of this probe in deep-
tissue imaging, TPM images were obtained from a part of an
acute rat hippocampal slice incubated with 10 mm AZn2 for
30 min at 378C. Because the slice of a 14-day-old rat was too
big to show with one image, several TPM images were
obtained in the same plane at a depth of about 120 mm and
combined. They reveal intense fluorescence in the stratum
lucidum of CA3 and the hilus of dentate gyrus (Figure 3a).[18]
The image obtained at a higher magnification clearly shows
that [Zn2+]i is concentrated in the mossy fiber axon terminals
of pyramidal neurons in the CA3 region (Figure 3b). The
negligible TPEF after addition of TPEN provides supporting
evidence for this observation (Figure 3c). Moreover, the TPM
images obtained at a depth of 80–150 mm revealed the [Zn2+]i
distribution in the mossy fibers of dentate granule neurons
near the hilus exclusively in the given plane along the
z direction (see the Supporting Information). When 50 mm
KCl, a membrane depolarizer causing the release of Zn2+, was
added to the imaging solution, the TPEF intensity increased
and then decreased upon treatment with TPEN (Figure 3d–f
and the Supporting Information). Similar results were
reported by others.[19] These findings demonstrate that
AZn2 is capable of detecting intracellular free Zn2+ ions at
a depth of 80–150 mm in living tissues by using TPM.
AZn2. a) At a depth of ca. 120 mm with magnification 10ꢀ. Scale bar:
300 mm. b,c) Magnification at 100ꢀ in the stratum lucidum (SL) of
CA3 regions (yellow box in (a)) at a depth of ca. 100 mm before (b)
and after (c) addition of 200 mm TPEN to the imaging solution. Scale
bar: 150 mm. d–f) TPM images in the hilus (H) of dentate gyrus (DG)
regions at a depth of ca. 100 mm before (d) and after (e) addition of
50 mm KCl to the imaging solution. Scale bar: 300 mm. f) After
addition of 200 mm TPEN to (e). The TPEF images were collected at
500–620 nm upon excitation at 780 nm with a femtosecond pulse.
and (0.50 Æ 0.04) nm, respectively, and pH insensitivity in the
biologically relevant range. They also emit four- to 24-fold
stronger TPEF than TSQ and FluZin-3 upon complexation
with Zn2+. Better than the currently available probes, these
novel probes can selectively detect intracellular free Zn2+ ions
in live cells for longer than 1000 s and in living tissues at a
depth of 80–150 mm without interference from other metal
ions and the membrane-bound probes.
Received: February 26, 2008
Published online: June 4, 2008
Keywords: fluorescent probes · imaging agents · living tissue ·
.
In conclusion, we have developed TP probes (AZn1 and
AZn2) that show a 24- to 52-fold TPEF enhancement in
response to Zn2+, dissociation constants (KdTP) of (1.1 Æ 0.1)
two-photon microscopy · zinc
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