A series of Zn(II) terpyridine complexes with enhanced two-photon-excited fluorescence for in vitro and in vivo bioimaging

Article abstract of DOI:10.1039/c5tb01185j

It is still a challenge to obtain two-photon excited fluorescent bioimaging probes with intense emission, high photo-stability and low cytotoxicity. In the present work, four Zn(ii)-coordinated complexes (1-4) constructed from two novel D-A and D-π-A liga

Full text of DOI:10.1039/c5tb01185j

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Z. HU, C. Brommesson, J. Wu, H. zhou, S. Li, J. Yang, Z. Sun, Y. Tian and K. Uvdal, J. Mater. Chem. B,
2015, DOI: 10.1039/C5TB01185J.
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Journal of Materials Chemistry B
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A Series of Zn(II) Terpyridine Complexes with Enhanced Two-Photon-Excited
Fluorescence for in Vitro and in Vivo Bioimaging†
Qiong Zhang,║[a,b] Xiaohe Tian,║[c] Zhangjun Hu,║[b] Caroline Brommesson,[b] Jieying Wu,*[a] Hongping Zhou,[a]
Shengli Li,[a] Jiaxiang Yang,[a] Zhaoqi Sun, *[d] Yupeng Tian*[a,e] and Kajsa Uvdal[b]
║These authors contributed equally to this work
Abstract: There has still been a challenge to obtain twoꢀphoton excited fluorescent bioimaging probes with intense emission, high photoꢀ
stability and low cytotoxicity. At present work, four Zn(II)ꢀcoordinated complexes (1ꢀ4) constructed from two novel DꢀA and DꢀπꢀA
ligands (L1 and L2) are investigated both experimentally and theoretically, aiming to explore efficient twoꢀphoton probes for bioimaging.
The molecular geometry optimization used for the theoretical calculations is obtained from the crystallographic data. Notably, the results
indicate that the complexes 1 and 2 display enhanced twoꢀphoton absorption (2PA) cross sections compared their corresponding DꢀA
ligand (L1). Furthermore, it was found that the complex 1 exhibits the advantages, including moderate 2PA cross section in the nearꢀ
infrared region, longer fluorescence lifetime, higher quantum yield, good biocompatibility and enhanced twoꢀphoton excited fluorescence.
Therefore, complex 1 is evaluated as bioimaging probe for HepG2 cells in vitro imaging, in which it is observed under twoꢀphoton
scanning microscopy that the complex 1 exhibits effective coꢀstaining with endoplasmic reticulum (ER) and nuclear membrane; as well as
for zebrafish larva in vivo imaging, in which it is observed that the complex 1 exhibits the specificity in the intestinal system.
Over the past decades, the design of fluorescent chromophore
targeting either specific intracellular structures or chemical species
has progressed rapidly.22ꢀ23 However, conventional molecules with
large 2PA cross sections tolerate high molecular weight, extended
πꢀconjugation system, low solubility, significant synthetic work
and financial costꢀscaling24ꢀ28, which are obstacles to their
extensive use for those bioꢀapplications to some extent.
Introduction
There has been considerable interest in developing
organic/inorganic hybrid materials1ꢀ4 with large twoꢀphoton
absorption (2PA) cross section (σ, expressed in GM = 1×10ꢀ50cm4
s photonꢀ1 moleculeꢀ1) in recent years, owing to their potential
applications in photodynamic therapy5ꢀ6, photochemical delivery
of biological messenger7ꢀ9
, , three
confocal microscopy10ꢀ13
dimensional data storage14ꢀ15, microꢀfabrication and optical power
limiting.16ꢀ17 These applications strongly rely on the large 2PA
cross sections of the preciselyꢀengineered organic molecules or
complexes. Several features are significant with respect to the
2PA spectra of the compounds specifically, those systems exhibit
pronounced 2PA with σ values greater than 500 GM over a broad
spectral window, in some cases ranging from 600ꢀ900 nm.18ꢀ21
Fluorescent compounds with large 2PA cross sections are
significantly important for working as twoꢀphoton fluorescent
probes, as they have increased penetration depth (greater than 500
ꢁm), localized excitation, prolonged observation time and
reducing photoꢀinduced damage. However, apart from the high
2PA cross sections, applicable 2PA materials for bioimaging
applications have to satisfy various other basic requirements, such
as water solubility and biocompatibility.
Scheme 1 Molecular structures of the ligands (L1, L2) and their
metal complexes (1-4).
[a]Department of Chemistry, Key Laboratory of Functional Inorganic
Materials Chemistry of Anhui Province, Anhui University, Hefei 230039,
P. R. China Eꢀmail: yptian@ahu.edu.cn; jywu1957@163.com
Compared to the wellꢀdeveloped studies on the 2PA organic
compounds, the research on the 2PA metalꢀcomplexes is still an
infant stage. Actually, in the recent years, there has been some
trying on multiꢀphoton bioimaging by using coordination metal
complexes as multiꢀphoton fluorescent probes.29ꢀ32 Metalꢀ
complexes can be easily synthesized in high yield by using ligand
with low molecular weight, but be conceived to show better
physical and chemical properties. This is because that the metal
ions can serve either as a multidimensional template for increasing
the molecular number density of twoꢀphoton active components (i.
e. ligands), or as an important structural control over the
intramolecular charge transfer process for modulating the σ
values.33ꢀ35 The initial investigations on the coordination metal
complexes have revealed that the control of the functional organic
ligands and the metal ion centre are interconnected, which is of
[b]
Division of Molecular Surface Physics & Nanoscience, Department of
Physics, Chemistry and Biology (IFM), Linköing University, 58183
Linköing (Sweden)
[c]
Department of Chemistry, The MRC/UCL Centre for Medical Molecular
Virology, University College London, WC1H 0AJ, London, UK
[d]
School of Physics and Material Science, Anhui University, Hefei
230601, P. R. China Eꢀmail:szq@ahu.edu.cn
[e]
State Key Laboratory of Coordination Chemistry, Nanjing University,
Nanjing 250100, P. R. China

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crucial importance to optimize physical properties of the
coordination metal complexes.36ꢀ37 Therefore, developing novel
complexes not only with large 2PA cross sections, but also with
strong fluorescence, low cytotoxicity, high stability, and low
Structures of the ligand L1 and L2 Ligands L1 and L2
crystallize in space group P21/c and triclinic group Pī,
respectively. Figure 1 and S1 show the structures of the molecules
with the atomic numbering schemes. For L1, the dihedral angle
between P1 and the phenyl plane P2 was only 24.5°, the linkage
between these two planes was conjugated with bond lengths of
1.484(5) (C4–C7), 1.291(4) (C7=C8), and 1.463(5) Å (C8–C9).
For L2, the dihedral angle between P0 and plane P1 was 38.36°,
and the linkage between P0 and benzene planes (labeled P1) was
conjugated with bond lengths of 1.486 Å (C16–C18), making the
covalent bond length (1.486 Å) to be intermediate between normal
CꢀC single bond length 1.54 Å and C=C double bond length 1.38
Å. 47 To sum up, these structural characters suggest that all nonꢀ
hydrogen atoms between donor and terpyridine acceptor are
highly conjugated, being conducive to the charge transfer from
sulfide phenyl to the pyridyl unit.
molecular weight, for biomedical applications becomes
a
challengeable and meaningful work in the field of 2PA materials.
Spurred by this, we designed and synthesized two novel ligands
(L1 and L2) and their Zn (II) complexes (Scheme 1). The strategy
is based on the following considerations: (1) 2, 2': 6', 2''ꢀ
terpyridine serves as basic multidentate unit, which has electronꢀ
withdrawing (A) ability and their structural analogy with rich
coordination chemistry have been extensively studied for their
high binding affinity towards a variety of metal ions, resulting in
interesting photophysical properties;38ꢀ44 (2) tertꢀbutylthiophenyl
group works as an intermediate electronꢀdonating (D) group, in
which tertꢀbutyl group serves as a steric hindrance to diminish πꢀπ
stacking effect, as well as suitable solubility; (3) a styryl unit as an
extended πꢀconjugated system was inserted between terpyridine
and tertꢀbutylthiophenyl groups to construct L2 for comparison
with L1; (4) four complexes were synthesized by selecting closedꢀ
shell d10ꢀmetals zinc(II). The complexation would be likely to
achieve enhanced 2PA properties due to the lack of dꢀd transitions
in comparison with the others.45ꢀ46 And the photophysical
properties of all the novel complexes were systematically
investigated compared to the their free ligands. Relying on the
comprehensive studies of them, Zn (II) complex 1 was selected
for bioimging application. Bioimaging study using twoꢀphoton
scanning microscopy shows that HepG2 cells can be sufficiently
stained with 1 specifically and it is capable of specific monitoring
the fluorescence signals in the intestinal system of living zebrafish
larva at a depth of >100 ꢁm. All these results suggest that 1 is a
potential twoꢀphoton fluorescent probe for in vitro and in vivo
bioimaging.
Structures of the complexes
Type I: five-coordinated complexes
The complex 2 crystallizes in the triclinic Pī space group
(supporting Table S1). In the crystal of complex 2, there are two
crystallographic and conformational independent molecules in the
asymmetric unit cell, as shown in Figure S1.Within the complex 2,
the metal centre adopts a fiveꢀcoordinated geometry. Because of
the steric demand, the terpyridine ligands coordinate to the Zn (II)
centre with a bonding angle for N1ꢀZn1ꢀN3, which shows
deviation from the ideal values of 90ο and 180ο. The bond lengths
of ZnꢀI in 2 are in the 2.554ꢀ2.586 Å range, showing that there is a
higher degree of electron delocalization in the Znꢀtpy complex.
Type II: six-coordinated complexes
The complex 1 crystallizes in the monoclinic crystal system
(supporting Table S1). As shown in Figure 1 and S1, the structure
displays the expected geometry with both ligands coordinated in
tridentate meridional fashion to the metal centre. As expected,
there is no apparent steric hindrance to prevent the free rotation of
pyridine and further coordination with metal cations.
Results and Discussion
Figure 1 The molecular structures of L1 and 1 (H atoms and PF6 ions were omitted for clarity).
2

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a 4ꢀ5 fold escalation of the extinction coefficient of low energy
transition.
The metalꢀnitrogen bond lengths in 1 are within 2.062ꢀ2.397Å
range, whereas in related references they are in the range of 1.997ꢀ
2.234 Å.48 Otherwise, the equatorial coordination sphere
containing Zn and three N atoms in the complex 1 greatly extends
electronic delocalization rather than that of 2, thus benefiting the
excitedꢀstate charge redistribution and enhancement of 2PA
response.
One-Photon Excited Fluorescence (OPEF) Properties
The fluorescence properties of all the compounds and complexes
in five different solvents are investigated and listed in Table S3,
including fluorescence quantum yields and lifetimes
(corresponding figures S3ꢀS8 are given in the Supporting
Information as well).
Except for the differences on the linear absorption, the
enhanced oneꢀphoton excited fluorescence of complex 1 compared
with L1 were also observed (Figure 2b). No similar trends of
fluorescence changes, however, were found for their
corresponding DꢀπꢀA systems of complex 3 compared with L2
(Figure S9). It may be inferred that the directly conjugated
terpyridine system and the closedꢀshell d10ꢀmetal centres (Zn) are
both responsible for the thus enhanced fluorescence.50 The largely
enhanced fluorescence was only observed for complex 1 against
L1. It can be concluded that such coordination between Zn (II) and
L1 could be enable the fluorescence enhancement of d10ꢀmetal
complexes,51 and the possibility that the heavy atom (Iodine)
consumes energy from the excited state molecule could be applied
Figure 2 (a) OPA spectra of L1 and 1 (c = 1.0 × 10−5 mol L−1) in DMF
(b) OPEF spectra of L1 and 1 in DMF
Photophysical Properties
The linear absorption and fluorescence properties for all the
compounds in the different solvents were investigated, which
were summarized in Table S3.
to interpret that 2 only shows very modest fluorescence
enhancement against L1. The dual emission of L1 (Figure
S3)apparently arises from the twisted structure of the DꢀA
configuration, while dualꢀemission were not seen for either
complex 1 or 2, which further suggests that the characteristic dualꢀ
fluorescence feature is from a twisted intramolecular charge
transfer (TICT) transition.52ꢀ53
Linear absorption and computation details The UVꢀvis
absorption spectral data of the both two ligands L1 and its relevant
complex 1 in DMF are given in Figure 2. As shown in Figure 2a,
L1 mainly presents a broad absorption band around 276 nm and a
shoulder around 315 nm in absorption spectra. The TDꢀDFT {[6ꢀ
31G(d)]} calculations (Figure 3 and Table 1) indicate that the high
energy transition presents marked charge transfer (CT) character,
with the HOMO and the LUMO being mainly located on the 4 ꢀ (
tert ꢀ butylthio ) phenyl unit (denoted as πD) and S–phenyl unit
(denoted as π*S–phenyl) with moderate contribution from terpyridine
unit (denoted as π*A), respectively. The remaining one band (λmax
= 289.4 nm, f = 0.76) is assigned mainly to a πD→π*A transition as
a result of the HOMO–1→LUMO+1 transition with some
contributions from an ICT transition [πD→π*S–phenyl, HOMOꢀ2→
LUMO]. The lowest energy excitation band of L1 at 323.8 nm (f =
0.16) is assigned as the intramolecular charge transfer (ICT)
transition [πD→π*S–phenyl & π*A] because of the HOMOꢀ1→LUMO
Figure 3 Molecular orbital diagram for L1 (B3LYP/6ꢀ31G(d)) and 1
(B3LYP/LANL2DZ) (the two colors of electron clouds is representative of
the square of the wave function of atomic orbitals).
Singletꢀstate lifetime The fluorescence lifetimes (τ) of the
complexes 1-4 are a little longer than those of the ligand L1 and
L2 when they are all under the excitation wavelength of 360 nm
(supporting Table S3). The short singletꢀstate lifetime in the
complexes is consistent with interꢀsystem crossing being very
rapid because of the strong spinꢀorbital coupling induced by
mixing of the ligand (π and π*) and metal dπ orbitals.
Fluorescence quantum yield The fluorescence quantum yields (Φ)
of all the compounds in different solvents are determined using
coumarin as a standard. The quantum yields of the ethylenic
conjugated system in each compound show the sequence of L2
(0.70) > 4 (0.48) > 3 (0.32) in DMF, while another system
follows the order of 1 (0.34) > L1 (0.16) > 2 (0.04). Since the
fluorescence quantum yield process is affiliated with an
energetically unstable state, it can be sensitive to a great variety of
internal factors defined by the chromophore’s structure and
external surrounding factors including polarity. Obviously, in
DMF, 2 shows much lower quantum yields compared to those of
transition (Table 1). Complex
1 shows a weak positive
solvatochromic (4−16 nm) absorption behavior, as a result of no
significant permanent groundꢀstate dipole moment in polar
solvents.49 For 1, the most intense transitions (339.0 nm) (Figure
2a) was contributed by the excitation involving several molecular
orbitals, with a predominant weight of excitations from HOMOꢀ
1→LUMO with the HOMOꢀ1 and the LUMO being mainly
located on sulfur atom π character and π* orbitals of terpyridine
unit with minor Zn dyz, respectively (Figure 3). These transitions
are in keeping with the calculation results (337.4 nm, Table 1),
which are contributed to two classes of transitions. One
corresponds mainly to CT transition from the sulfide group to the
terpyridine part and zinc centre, the complex 1 can arise from a
locally excited (LE) transition as singleꢀelectronic transition
localized in the DꢀA system.52 Furthermore, it is accompanied by
3

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the other complexes, the results can mainly be explained by the
photoꢀisomerization, viz, the molecular planarity is reduced due to
the rotating of the flexibleꢀcenter when the molecules accept
energy in the dilute solution.
LippertꢀMataga plot
The magnitudes of dipole moment change (µeꢀµg) are
proportional to the one photon oscillator strengths, so their values
Two-Photon Excited Fluorescence (2PEF) Spectroscopy
and Two-Photon Absorption (2PA) Cross-Sections
As shown in Figures S3 to S8, there is no linear absorption in
the wavelength range 680ꢀ900 nm for all the compounds in DMF,
which indicates that there is no energy level corresponding to an
electron transition in this spectral range. If frequency upconverted
fluorescence appears upon excitation with a tunable laser in this
range, it should be safely attributed to multiphoton excited
fluorescence (Figure S11). Detailed experiments revealed that the
peak positions of the 2PEF spectra of these cpmpounds are
independent of the excitation wavelengths, but the emission
intensities of the 2PEF are dependent on the excitation
wavelengths. The electrons can be pumped to the different excited
states by 2PA due to the different selection rule; however, they
would finally relax to the same lowest excited state via internal
conversion and/or vibrational relaxation.
can be calculated from the linear absorption spectrum.However,
(µeꢀµg) is rarely determined experimentally, so the design of
efficient 2PA compounds has proceeded empirically, with
guidance from theoretical calculations of transition moments. As
seen in Table S3, the fluorescence spectra showed moderate
Stokes shifts depending on the solvent polarity. Further, the
solvatochromism is observed, suggesting a large change in dipole
moment between ground and excited states. The LippertꢀMataga
equation is the most widely used to evaluate the dipole moment
changes of the dyes with photoexcitation: 54ꢀ55
2ꢀf
ꢀ
υ
=
(
ꢁe −
ꢁ
)2 + b
g
4
πε 0
hca3
(1)
ε
2ε
−1
+1
n2 −1
2n2 +1
ꢀf =
−
(2)
in which ∆ν = νabs ꢀ νem stands for Stokes shift, νabs and νem are
absorption and emission (cmꢀ1), h is the Planck’s constant, c is the
velocity of light in a vacuum, a is the Onsager radius and b is a
constant. ∆f is the orientation polarizability, µe and µg are the
dipole moments of the emissive and ground states, respectively,
and ε0 is the permittivity of the vacuum. (µeꢀµg)2 is proportional to
the slope of the Lippert ꢀMataga plot.
Plots of the Stokes shifts as a function of the solvent polarity
factor ∆f are shown in Figure S10. Only the data involving the
aprotic solvents are shown because application of this analysis
with solvents where specific soluteꢀsolvent interactions are
present is not appropriate. The slope of the bestꢀfit line is related
to the dipole moment change between the ground and excited
states (µeꢀµg). As shown in Figure S10, the slopes of L1 and 1, 2
are ꢀ19563, ꢀ7198 and ꢀ8718, respectively. So the values of µeꢀµg
were calculated as 3.22 D for L1, 4.35D for 1, 3.29 D for 2,
respectively (eq 1). The µeꢀµg values of the complexes indicate
that the molecule in the excited state has an extremely polar
structure,56 an enhanced 2PA response of the complexes was
found compared with L1, which is proved by the crystal structures
(L1, 1 and 2) and in good agreement with their nonlinear optical
properties as shown in Figure. 4.
Among the occupied states of 1 based on Zn orbitals, the
calculated results exhibit that HOMOꢀ1 is comprised of sulfur
atom π character while the HOMO is localized in one of the
terpyridine units with a small contribution from Zn dxz. Also
LUMO distribution is localized in the π* orbitals of the other
terpyridine unit with minor Zn dyz. Therefore, there are relatively
strong electron interactions between the ligand molecules and the
zinc centre, and the electron density in each atom of the ligand is
very similar, it can be inferred that there are more electron density
on average at the metal ion centre in the charge transfer (CT)
state.57
Figure 4 2PA spectra of L1 and related complex 1
The 2PA cross sections (σ) of all compounds were obtained by
a comparison of the 2PEF spectra of individual compound with
fluorescein in a calibration standard.58 By tuning the pump
wavelengths incrementally from 680 to 900 nm, keeping the input
power fixed, almost no 2PEF signal of ligand L1 was detected.
The 2PEF intensities of complexes 1 and 2 are much increased
compared to those of their free ligands L1 (supporting Figure
S12a).
Twoꢀphoton absorption crossꢀsection values σ of the L2 ligand
have been shown in Figure S13b. The value at 720 nm (139×10ꢀ50
cm4 s photonꢀ1, 139 GM) can be compared to the values of the
other two complexes (around 95ꢀ128 GM) at 710 nm. This
comparison depends on the wavelength. It was found that almost
no increase of the 2PA crossꢀsections was observed on going from
the ligand to its complexes. Such trend is unexpected since it is
commonly known that the increase of the conjugation length leads
to an increase of the 2PA activity. Since coordination to Zn (II)
centre leads to increased acceptor properties of the terpyridine
rings of pushꢀpull dipolar ligands, it reasonably modifies the
charge transfer form affecting the ground electronic state, as a
result of the ligands bonded to the metal ion to form an large
extended πꢀconjugated system in the coordination compound
molecules, preventing electron flowing within the whole molecule,
in agreement with the observed decrease of the 2PA crossꢀ
sections59ꢀ60
.
However, it is interesting to point out that the efficiency of
pushꢀpull dipolar (DꢀA) systems has been described by large
increasing tendency. As shown, in the measured region, the 2PA
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crossꢀsection
σ
values of complex 1 were significantly enhanced
(Figure 1, S2 and Table 2). The observed 2PA band is moderately
strong: σ = 65 GM in 1 and even stronger (99 GM) in 2. This
should imply a nonrealistically large change of permanent dipole
moment because of nonsymmetrical vibration.62 Significantly, by
comparing those with its free L1, 1 bears moderate active σ value,
as well as it exhibits higher quantum yield, longer lifetime in
ethanol (supporting Table S3), and smaller molecular volume,
which stimulated us further to explore its potential application in
biological imaging.
compared to their free ligand L1 in high polarity solvent DMF.
Importantly, the peak 2PA crossꢀsections appear in the nearꢀ
infrared range. This large 2PA response comes from the marked
core to periphery charge redistribution in complexes upon
photoexcitation.61 Therefore, complexation with Zn (II) enhances
the electronꢀacceptor character of terpyridine group, which
converts the ligand to a more strongly polarized DꢀA unit, which
may favor the ICT, causing an enhanced twoꢀphoton absorption.
The enhanced trend observed in the experiments was consistent
with above structural investigation and theoretical calculations
Table 1 Ab initio calculation data of linear absorption spectra, the experimental data of linear absorption spectra, and oscillator strengths of all the
compounds in DMSO
L1
L2
1
2
289.4
323.8
368.4
302.1
337.4
335.7
λ
abꢀOPA (nm)
(calculated) a
abꢀOPA (nm)
276.0
315.0
335.0
282.0
339.0
283.0
321.0
281.0
λ
(experiment, DMF)
0.76
0.16
1.68
0.24
0.32
0.65
f b
103 (Hꢀ2)→106 (L) (0.42)
104 (Hꢀ1)→107 (L+1) (0.54)
104 (Hꢀ1)→106 (L) (0.58)
132(H)→133(L) (0.55)
132(H)→135(L+2) (0.62)
131(Hꢀ1)→134(L+1) (0.19)
OI and Tcc
284(Hꢀ1)→286(L) (0.60)
115(Hꢀ3)→ 123 (L+3)(0.598)
a The TDꢀDFT calculation data of oneꢀphoton absorption (OPA) spectra with the peak position of the maximum absorption band (nm), Oscillator
b
Strengths (f) c The excited states computed by TDDFT Method with the Orbitals Involved (OI) in the Excitations Including Transition Coefficients (Tc)
A coꢀstaining experiment with Mitotracker® (Figure 5f and g)
was performed to further determine that whether was
Biological Imaging Application of 1
In order to evaluate their application in intracellular imaging, Zn
(II) complex 1 was selected due to stronger fluorescence intensity
in DMSO/H2O, higher quantum yield, moderate 2PA crossꢀ
section and longer fluorescent lifetime (Table S3). We have
conducted cytotoxicity assays in HepG2 cells. Figure S14 shows
the cell viability data for HepG2 cells treated with 1 as quantified
by the MTT assay, an established method of probing cell death.
These data show that HepG2 cells show near 100% viability
following 24 h of treatment with 20 ꢁM 1. Higher concentrations
result in decreased cell survival, with 65% and 85% viability
observed following 24 h of treatment with 40 ꢁM L1 and 1. These
cytotoxicity tests show that subꢀ and lowꢀmicromolar
concentrations of 1 are low toxic over a period of at least 24 h and
could be safely used for further biological studies. Micrographs of
oneꢀphoton confocal microscopy and twoꢀphoton microscopy of
HepG2 cells labeled with 1 were captured. 1 was first dissolved in
DMSO to 2 mM and then diluted by PBS (phosphate buffer
solution) to 40 ꢁM as working concentration. HepG2 cells firstly
incubated with 40 ꢁM 1 for 30 minutes and imaged directly
without fixation. The live HepG2 cells images (Figure 5a, 63×
magnification) exhibited effective cellular uptake (Figure 5a)
within the short incubation period. It also showed remarkable
stability of intracellular fluorescence as we switch from oneꢀ
photon excitation (Figure 5b) to twoꢀphoton excitation (Figure
5d). It is interesting to note that although 1 indicated similar
cellular distribution pattern under oneꢀphoton and twoꢀphoton
excitation, TP excited cells (Figure 5c) showed much less
background noise and resulting in an autoꢀfluorescence free
micrograph compare to OP (Figure 5e) excited cells.
1
internalized with membraneꢀrich organelles mitochondria. The
merged micrographs (Figure 5a) clearly indicated overlapping
either with OP or TP excited channel. It was not unexpected that
such complex could effectively uptake by cells due to its small
molecular weight, and its relatively good hydrophilic nature and
positive charge would imply the uptake mechanism is likely to be
endocytosis. Therefore, the above results suggested that 1 was not
coꢀlocalized with intracellular mitochondria and might stained
with other endocytotic compartments.
Figure 5 (a) Merged and DIC merged image of HepG2 cells
incubated with 40 ꢁM 1 after 30 min of incubation, washed by PBS
buffer. (b) Oneꢀphoton image of HepG2 cells incubated with 40 ꢁM 1
after 30 min of incubation, washed by PBS buffer. λex = 405 nm
5

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(emission wavelength from 440 to 480 nm). (c, e, g)
Fluorecense intensity profile of complex 1 cross a single cells corr
esponding to the line segment from b, d and f. (d) Twoꢀphoton
image of HepG2 cells incubated with 40 ꢁM 1 after 30 min of
incubation, washed by PBS buffer. λex
= 700 nm (emission
wavelength from 543 to 606 nm). (f) Oneꢀphoton image of HepG2
cells incubated with Mitotracker. λex = 633 nm (emission wavelength
from 650 to 700 nm). (h) Twoꢀphoton image of HepG2 cells
incubated with 40 ꢁM 1, λex = 700 nm (emission wavelength from 543
to 606 nm) and Oneꢀphoton image of HepG2 cells incubated with ER
tracker, λex = 488 nm (emission wavelength from 500 to 550 nm) .
.
Figure 7 (a) Oneꢀphoton image of 72 hꢀzebrafish larva incubated with
30 ꢁM 1 after 4 h of incubation, washed by PBS buffer. λex = 405 nm
(emission wavelength from 440 to 480 nm); Twoꢀphoton image of 72
hꢀzebrafish larva incubated with 30 ꢁM 1 after 4 h of incubation,
washed by PBS buffer. λex = 700 nm (emission wavelength from 543
to 606 nm); The overlay of 1PA and 2PA images. (b) The overlay of
bright field and (a). (c) Twoꢀphoton excited fluorescence 3D images
of zebrafish larva reconstructed from selected region from (b) (d) One
and two photon fluorescence intensity of 72 hꢀzebrafish larva
incubated with 30 ꢁM 1, from selected segment from (a). The scale
bars represent 100ꢁm.
can be found in peripheral areas in ratiometric image. The overlay
of TP, OP and brightꢀfield images discloses one of the bright
regions located in the intestinal system (Figure 7b). Moreover, the
TP signal again showed much less background noise, higher
intensity and more specified staining within intestinal system
(Figure 7d). Comparison between the ratiometric images of L1
treated zebrafish larva and the 1 treated larva suggests that
zebrafish incubated with control ligand L1 indicated expected
intestinal system localization (Supporting Information, Figure
S15). Both oneꢀ and twoꢀphoton fluorescence microscopies
provided similar imaging results to 1. However, the fluorescence
intensity of L1 is significantly weaker than 1 (compare with Figure
7). The 2PM images revealed that 1 distributed in intestinal region
at 128.2 ꢁm depth (Figure 7c). Control experiments were carried
out with zebrafish larva treated with L1, which was shown to
detect fluorescence signals of the tissues only at a depth of up to
55 ꢁm (Figure S15). This much deeper penetration, coupled with
the significantly lower background fluorescence and better clarity
observed in the 2PM images, clearly demonstrated the potential
application of these newly developed, twoꢀphoton probes for
tissueꢀbased imaging experiments in future.
To further prove the stability of the complex 1, the variable
temperature 1H NMR of 1 has been measured (supporting Figures
S16). The result indicates that the well resolved peaks of the
spectra exhibit almost no change in the temperature range of
273~303K, which proves that the solid structure of
[L1ZnL1]2+cation is still kept in solution. Additionally, their
crystal structures were further confirmed by single crystal Xꢀray
diffraction analysis. The unusual twoꢀphoton luminescent
properties and the low cytotoxicity of 1 make it a potential
candidate as novel luminescence material for live cell imaging.
Conclusions
Figure 6 Schematic representation of Zn (II) complex crossing the
cell membrane via endocytosis and penetrating into ER and cell
nucleaus (Endo: Endosome, CM: cell membrane, ER: endoplasmic
reticulum. N: nucleus, NP: nuclear pores, NIM: nuclear innerꢀ
membrane, NOM: nuclear outerꢀmembrane).
Therefore we then captured
a higher resolution liveꢀcell
micrograph (100×) stained with 1 under TP excitation and coꢀ
stained with ER tracker and DAPI. Figure 5h showed much more
intracellular details under TP confocal microscopy and clearly
indicated nice (red merge green make yellow) coꢀlocalization
between endoplasmic reticulum (ER) and 1 at certain region. It
was not surprised; as ER closely associated with cell membrane,
endosomal system and cell nucleus, play a crucial role in
endosomal cargo sorting and protein synthesis. It is noteworthy
that 1 staining with other endomembrane system including nuclear
membrane (Figure 5h, arrows) and even nuclear region (Figure
5h, stars) also observed. Since nuclear region lack of
endosomal/lysosomal compartment, this might suggest the free
release of 1 from endocytotic pathway from ER and uniformly
distributed on the dynamic nuclear membrane, and consequently
the positively charger Zn (II) complex partially penetrated into
negatively charged cell nuclear (Figure 6) DNA. In addition,
cytotoxicity is a potential side effect of 1 that must be controlled
when dealing with living cells or tissues.
Besides the imaging ability in living cells of 1, the first
ratiometric in vivo imaging in 3ꢀdayold zebrafish larva was also
investigated via staining the larva by 1 (30 ꢁM, 4 h). As shown in
Figure 7a, the OPM images of the larva head exhibit mainly two
regions of bright fluorescence. The 2PM image obtained from
band paths 543ꢀ606 nm displays the red regions on the same
location, indicating the higher 1 level in these two bright spots
than that in the rest of field. Only very minor green and red spots
Four novel Zn (II) complexes with L1 and L2 as ligands have
been synthesized and determined. The results showed that the
complexes based on two types of terpyridine ligands (DꢀπꢀA and
6

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Journal of Materials Chemistry B
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DOI: 10.1039/C5TB01185J
DꢀA) can arouse different optical properties. It was notable that
7.98 (d, J = 8.00 Hz, 2H), 8.04ꢀ8.05 (d, J = 4.00 Hz, 2H), 8.68ꢀ
8.70 (d, J = 8.00 Hz, 3H), 8.75ꢀ8.78 (t, J = 12.00 Hz, 3H); 13C
NMR (DMSOꢀd6, 100 MHz): (ppm) = 155.68, 154.93, 149.31,
137.45, 131.51, 130.26, 130.13, 129.98, 129.52, 128.83, 127.55,
127.25, 126.85, 124.52, 120.94, 117.61, 45.96, 30.65; FTꢀIR
(KBr, cmꢀ1): v = 2956 (w), 2919 (w), 1717 (w), 1701 (w), 1685
(w), 1651 (w), 1602 (s), 1581 (s), 1564 (s), 1541 (m), 1511 (m),
1467 (s), 1440 (m), 1416 (m), 1389 (s), 1361 (m), 1263 (m), 1094
(s), 1037 (m) 989 (m), 964 (s), 947 (w), 876 (w), 834 (s), 792 (s),
736 (s), 678 (m), 659 (m), 620 (m), 577 (w) 541 (s) 508 (w).
C50H46ZnF12N6P2S2: Calcd. C 52.20, H 4.03, N 7.31. Found: C
52.18, H 4.05, N 7.29. HRMS (GCTꢀMS), C50H46ZnN6S2: 858.25,
found: 860.0 [M+H, 100%].
Preparation of [L1-Zn-L1] [PF6]2 (1): Complex 1 was prepared
by a procedure similar to that for 3. 0.044 g (0.2 mmol) in place of
Zn (OAc)2·2H2O. 1H NMR (DMSOꢀd6, 400 MHz): (ppm) =9.44 (s,
2H), 9.18ꢀ9.20 (d, J=4.00, 3H), 8.52ꢀ8.53 (d, J=4.00, 3H), 8.31 (s,
3H), 7.99ꢀ8.03 (t, J=8.00, 6H), 7.73ꢀ7.75 (d, J=8.00, 3H), 7.52ꢀ
7.58 (m, 8H), 1.30 (s, 18H); 13C NMR (DMSOꢀd6, 100 MHz):
(ppm) = 154.36, 149.45, 147.80, 141.27, 139.81, 137.29, 134.34,
129.84, 128.60, 127.52, 123.53, 120.62, 46.05, 30.68; FTꢀIR
(KBr, cmꢀ1): v = 1796 (m), 1735 (w), 1600 (s), 1573 (s), 1544 (s),
1477 (w), 1421 (m), 1363 (m), 1248 (m), 1017 (s), 974 (m), 832
(s), 791 (m), 732 (m), 682 (m), 659 (m), 638 (m), 557 (s), 542
(m). C50H46ZnF12N6P2S2: Calcd. C 52.20, H 4.03, N 7.31. Found:
C 52.18, H 4.05, N 7.29. HRMS (GCTꢀMS), C50H46ZnN6S2:
858.25, found: 860.0 [M+H, 100%].
Preparation of L1ZnI2 (2): Complex 2 was prepared by a
procedure similar to that for 1. 1H NMR (DMSOꢀd6, 400 MHz)
(ppm) = 9.43 (s, 1H), 9.16ꢀ9.19 (d, J=12.00, 2H), 9.06ꢀ9.07 (d,
J=8.00 1H), 8.96 (s, 1H), 8.33ꢀ8.45 (d, J = 8.00 Hz, 2H), 8.30 (s,
2H), 7.95ꢀ7.96 (d, J=4.00 2H), 7.86ꢀ7.87 (d, J=4.00, 1H), 7.78ꢀ
7.80 (d, J=8.00, 1H), 7.50ꢀ7.52 (t, J=4.00, 1H), 1.38 (s, 9H).13C
NMR (DMSOꢀd6, 100 MHz): (ppm) = 156.43, 155.11, 148.65,
146.41, 141.18, 137.34, 137.28, 128.38, 127.68, 123.43, 121.27,
120.63, 46.47, 30.76; FTꢀIR (KBr, cmꢀ1): v = 2958 (w), 2922 (w),
1786 (w), 1773 (w), 1747 (w), 1735 (w), 1717 (w), 1700 (w),
1685 (w), 1651 (w), 1612 (s), 1573 (m), 1560 (m), 1541 (s), 1523
(w), 1509 (w), 1474 (s), 1459 (m), 1423 (m), 1393 (m), 1363 (w),
1340 (w), 1297 (w), 1249 (w), 1161 (m), 1096 (w), 1067 (w),
1013 (s), 895 (w), 829 (m), 792 (s) 732 (w), 677 (w), 658 (w), 637
(w), 531 (w). C25H23I2ZnN3S: Calcd. C 41.89, H 3.23, N 5.50.
more intensive OPEF and 2PEF, and significant increased twoꢀ
photon cross section values were obtained for the Zn(II)
complexes compared to those of their free ligand L1. It was
proposed that the increased fluorescence could be contributed by
closedꢀshell d10 feature of Zn(II). And an extended conjugated
bridge, facilitating the πꢀelectron contribution from the metal ions
in the L1 system within the complexes, may favor the ICT, thus
causing enhanced twoꢀphoton absorption. As was expected, the
complex 1 was successfully applied as 2PA probe for in vivo and
in vivo bioimaging. ER and nuclear membrane staining in live
cells and the uptake mechanism were discussed in detail.
Furthermore, complex 1 has been utilized to visualize the
intestinal system of live zebrafish larva in vivo . The results favor
that 1 may be a potential biocompatible and longꢀterm in vivo
imaging agent.
Experimental Section
Preparation
of
4'-(4-(tert-butylthio)phenyl)-2,2':6',2''-
terpyridine (L1): tꢀBuOK (1.6 g, 13.9 mmol) was dissolved in 30
mL THF, and 2ꢀaceylpyridine (1.1 g, 8.95 mmol) was added to the
solution. After the reaction mixture was stirred at room
temperature for 2 h, a (0.73g, 4 mmol) was added dropwise. After
the reaction mixture was stirred at room temperature for 8 h, the
ropy mixture was added to a stirred solution of ammonium acetate
(12.0 g, excess) in ethanol (250 mL). The reaction was heated at
reflux for 5 h, affording a dark red solution. Cooled to the room
temperature, bright yellow product was crystallized from the
filtered solution. The yellow granular product was recrystallized
1
from ethanol and dried in vacuum to give T with yield 68%. H
NMR (DMSOꢀd6, 400 MHz): (ppm) = 1.31 (s, J = 9H), 7.53ꢀ7.56
(t, J = 6.00 Hz, 2H), 7.69ꢀ7.71 (d, J = 8.00 Hz, 2H), 7.94ꢀ7.96 (d,
J=8.00, 2H), 8.03ꢀ8.07 (t, J = 7.60 Hz, 2H), 8.68ꢀ8.69 (d, J = 4.0
Hz, 2H), 8.73 (s, 2H), 8.77ꢀ8.78 (d, J = 4.00 Hz, 2H); 13C NMR
(DMSOꢀd6, 100 MHz): (ppm) = 155.72, 154.79, 149.33, 148.65,
137.79, 137.62, 137.51, 133.73, 127.22, 124.60, 120.94, 117.94,
46.18, 30.70; FTꢀIR (KBr, cmꢀ1): v = 2958 (s), 2921 (w), 1796
(w), 1747 (w), 1735 (w), 1717 (w), 1700 (w), 1685 (w), 1672 (m),
1651 (s), 1583 (s), 1540 (s), 1510 (w), 1492(w), 1470 (s), 1440
(m), 1403 (w), 1384 (s), 1364 (m), 1265 (m), 1167 (s), 1142 (w),
1090 (w), 1073 (m), 1037 (w), 1017 (w), 993 (w), 891 (w) 834
(m), 790 (s), 734 (s), 661 (m), 641 (m), 620 (m), 580 (w), 528 (w).
C25H23N3S: Calcd. C 75.53, H 5.83, N 10.57. Found: C 75.68, H
5.81, N 10.60. HRMS (GCTꢀMS) calcd for C25H23N3S: 397.54;
Found, 398.09.
Found:
C 42.01, H 3.24, N 5.48. HRMS (GCTꢀMS),
C25H23I2ZnN3S: 714.90, found: 587.13 [MꢀI, 100%].
Preparation of (E) - 4' - (4 - ( 4 - ( tert - butylthio ) styryl )
phenyl ) - 2, 2' : 6', 2'' - terpyridine (L2): 4ꢀ(2, 2': 6', 2''ꢀ
Terpyridylꢀ4')ꢀbenzyltriphenylphosphonium bromide46 (2.0 g, 3.0
mmol), 4ꢀFormylꢀSꢀtertꢀbutylphene (0.55 g, 3.0 mmol) and tꢀ
BuOK (1.3 g, 12.3 mmol) were placed into a dry mortar and
milled vigorously for about 20 mins, and monitored by TLC until
reaction completion. The mixture was dispersed in 100 mL
ethanol. The residual solid was filtered and recrystallized from
water/ethanol, giving pale yellow crystals of L with yield 77%.1H
NMR (DMSOꢀd6, 400 MHz): (ppm) =1.27 (s, 9H), 7.30ꢀ7.32 (d, J
= 8 Hz, 1H), 7.42 (s, 3H), 7.51ꢀ7.54 (t, J = 6.00 Hz, 3H), 7.67ꢀ
7.87 (d, J = 8.00 Hz, 1H), 7.83ꢀ7.84 (d, J = 4.00 Hz, 2H), 7.96ꢀ
Preparation of [L2-Zn-L2] [PF6]2 (3): L2 (0.200g, 0.4 mmol) and
Zn(OAc)2·2H2O 0.053 g (0.2 mmol) were dissolved in MeOH (10
mL). The mixture was refluxed for 2 h at 70 °C and then 10mL
MeOH containing NH4PF6 0.065 g (0.4 mmol) was added in. The
mixture stirred for 30 min to give a clear yellow solution. Yellow
crystals suitable for Xꢀray diffraction analysis were obtained after
two weeks by slow evaporation of the methanol solution at room
temperature. 1H NMR (DMSOꢀd6, 400 MHz): (ppm) =9.44 (s, 4H),
9.18ꢀ9.20 (d, J=8.00, 4H), 8.52ꢀ8.54 (d, J=8.00, 4H), 8.29ꢀ8.33 (t,
J=8.00, 4H), 7.98ꢀ8.04 (m, 8H), 7.73ꢀ7.51 (d, J=8.00, 4H), 7.51ꢀ
7.59 (m, 2H), 1.30 (s, 18H). 13C NMR (DMSOꢀd6, 100 MHz):
(ppm) = 154.38, 149.46, 147.74, 141.28, 139.63, 137.37, 137.30,
7

Journal of Materials Chemistry B
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DOI: 10.1039/C5TB01185J
References
136.98, 134.35, 131.88, 129.86, 128.87, 128.60, 128.57, 127.67,
127.53, 126.98, 46.05, 30.68; FTꢀIR (KBr, cmꢀ1): v = 2957 (w),
2920 (w), 1796 (m), 1700 (w), 1651 (w), 1579 (s), 1572 (m), 1541
(m), 1475 (s), 1521 (w), 1459 (m), 1400 (m), 1424 (m), 1361 (m),
1247 (m), 1161 (s), 1096 (w), 1012 (s), 967 (m), 874 (m), 836
(m), 788 (s),681 (m), 540 (s). C66H58ZnF12N6P2S2: Calcd. C 58.52,
H 4.32, N 6.20. Found: C 58.34, H 4.33, N 6.22. HRMS (GCTꢀ
MS), C66H58ZnN6S2: 1062.35, found: 1062.10 [M, 100%].
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methanol solution at room temperature. H NMR (DMSOꢀd6, 400
MHz): (ppm) = 1.30 (s, 9H), 7.43ꢀ7.46 (d, J=12.00, 1H), 7.48ꢀ
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J=4.00, 1H), 9.05ꢀ9.07 (d, J=8.00, 1H), 9.17 (s, 1H), 9.20ꢀ9.22 (d,
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131.96, 131.90, 131.66, 129.58, 128.35, 128.31, 127.23, 126.71,
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(w), 1796 (w), 1772 (w), 1734 (w), 1716 (w), 1699 (w), 1650 (m),
1598 (s), 1613 (s), 1573 (m), 1542 (m), 1521 (m), 1474 (s), 1425
(m), 1402 (s), 1363 (m), 1248 (m), 1160 (m), 1067 (w), 1013 (m),
968 (m), 874 (m), 836 (m), 789 (s), 729 (m), 682 (m), 636 (m),
541 (m). C33H29I2ZnN3S: Calcd. C 48.40, H 4.32, N 5.13. Found:
C 48.32, H 4.37, N 5.14. HRMS (GCTꢀMS), C33H29I2ZnN3S:
816.95, found: 817.11 [M+H, 100%].
Acknowledgment This work was supported by grants from the
National Natural Science Foundation of China (51372003,
21271004, 51272001, 51472003, 51432001 and 21271003),
Ministry of Education Funded Projects Focus on returned overseas
scholar, Program for New Century Excellent Talents in University
(China) and Doctoral Program Foundation of Ministry of
Education of China (20113401110004), China Postdoctoral
Science Foundation (2015M571912), the Swedish Research
Council (VR) (621ꢀ2013ꢀ5357) and Swedish Government
strategic faculty grant in material science (SFO, MATLIU) in
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Supporting Information Available: †Crystallographic date
reported in this paper has been deposited with the Cambridge
Crystallographic Date Center and allocated the deposition
numbers CCDC 890201(1), 890203(2), 890205(L2), 890206(L1).
Copies of these information may be obtained free of charge from
the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK
(fax: +44ꢀ1223/336ꢀ033; eꢀmail: deposit@ccdc.cam.ac.uk).
Synthetic procedures and detailed experimental information; Xꢀ
ray crystallographic data (CIF); computation studies; optical
measurements; twoꢀphoton excited fluorescence (2PEF)
spectroscopy and twoꢀphoton absorption (2PA) crossꢀsection; cell
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9

Journal of Materials Chemistry B
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DOI: 10.1039/C5TB01185J
Table of Contents entry:
TPA cross sections are enhanced for the complexes containing D-A type ligand L1. 1 exhibits
specificity in two-photon fluorescent imaging.