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Materials Chemistry
Cite this: J. Mater. Chem., 2012, 22, 17737
www.rsc.org/materials PAPER
Water-miscible organic J-aggregate nanoparticles as efficient two-photon
fluorescent nano-probes for bio-imaging†
Zhenzhen Xu,ab Qing Liao,a Yishi Wu,a Wenlu Ren,bc Wei Li,c Libing Liu,a Shu Wang,a Zhanjun Gu,*c
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Haoli Zhang*d and Hongbing Fu*a

Received 15th May 2012, Accepted 3rd July 2012
DOI: 10.1039/c2jm33081d

Many two-photon absorption (TPA) organic dyes are water insoluble and suffer from strong
fluorescence quenching in aqueous media due to the self-aggregation effect. This seriously limits their
applications as two-photon fluorescence (TPF) probes in bio-imaging. By employing a reprecipitation
method, we prepared ultrabright water-miscible organic nanoparticles (ONPs) of 1,4-dimethoxy-2,5-di
[40 -(cyano)styryl]benzene (COPV). The single-crystal structure reveals that the cooperation between
p–p stacking and hydrogen-bonding interactions drives COPV molecules into a brickwork
arrangement of J-aggregates, in which the coherent excitation delocalization reaches 2–3 molecules.
Due to the superradiance of J-aggregates, COPV ONPs are highly emissive in aqueous media with a
quantum yield >0.4; meanwhile, their TPA cross-section is greatly enhanced, probably due to exciton–
vibration coupling. As TPF probes, COPV J-aggregate ONPs are 3–4 orders of magnitude brighter
than conventional fluorescent dyes and an order of magnitude brighter than quantum dots. Moreover,
these ONPs exhibit no obvious cytotoxicity at concentrations as high as 100 mg mL1. Our results
demonstrate that ultrabright J-aggregate ONPs of COPV provide a new strategy to construct efficient
TPF nano-probes for bio-imaging.

1. Introduction fluorescence quantum yield, the development of TPF should

Two-photon fluorescence (TPF) microscopy has provided a
powerful tool for biological imaging and sensing applications,
where the use of near-IR laser light not only avoids auto-fluo-
rescence from the biological background, but also reduces
undesired tissue photo-damage due to the near-transparency of
many tissues in this spectral range.1,2 For strong two-photon
excited fluorescence (TPEF), a large TPF action cross-section
and a high dye density (c) are usually required. Since the TPF
action cross-section can be expressed as a product of d  F,3
where d represents the TPA cross-section and F is the
a
Beijing National Laboratory for Molecular Sciences (BNLMS), Institute
of Chemistry, Chinese Academy of Sciences, Beijing, 100190, P. R. China.
E-mail: hongbing.fu@iccas.ac.cn
b
Graduate University Chinese Academy of Sciences, Beijing 100049, P. R.
China
c
Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety,
Institute of High Energy Physics, Chinese Academy of Sciences, Beijing
100049, P. R. China. E-mail: zjgu@ihep.ac.cn
d
State Key Laboratory of Applied Organic Chemistry, College of
Chemistry and Chemical Engineering, Lanzhou University, Lanzhou,
730000, P.R. China. E-mail: haoli.zhang@lzu.edu.cn
† Electronic supplementary information (ESI) available: general
synthesis procedure of COPV; the relative brightness of a single COPV;
XRD data; one-photon and two-photon cell imaging. CCDC 838078.
For ESI and crystallographic data in CIF or other electronic format
see DOI: 10.1039/c2jm33081d
involve improving both d and F in order to obtain a value of
d  F > 1000 GM, which is required for practical applications. In
past decades, a myriad of TPA materials have been developed,
including inorganic quantum dots,4–6 metallic nanoparticles,7,8
and organic dyes or pigments.9,10 Inorganic probes have advan-
tages such as excellent photostability and high TPA cross-
sections.4–8 Nonetheless, their potential toxicity and the difficulty
of surface modification are major drawbacks of these inorganic
probes.11,12 On the other hand, the design and synthesis of
organic TPA dyes has achieved great successes in reaching
d > 1000 GM.9,10,13 However, many of these TPA dyes are
hydrophobic, and their fluorescence quantum yields (F) are
greatly reduced in aqueous media due to self-aggregation
induced fluorescence quenching,14 which limits the TPF action
cross-section of these TPA dyes, as it is determined by the
product of d  F. In this regard, various attempts to encapsulate
organic TPA dyes into micelles, vesicles, and silica matrices have
been reported for improving their water solubility and bio-
compatibility.15,16 In these cases, to prevent aggregation-induced
fluorescence quenching and the undesired release of excess dye
molecules, the loading density of TPA molecules needs to be
carefully adjusted at quite a low level, which is also another
limitation for efficient two-photon excitation.
To overcome these limitations, we urgently need to develop an
approach to overcome fluorescence quenching in aggregates for a

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TPA chromophore with a large two-photon activity. Recently,
W€ urthner et al. reported a highly fluorescent J-aggregate
assembly with quantum yields of near unity according to
supramolecular design principles. If the molecules are stacked in
a parallel head-to-tail way, a J-aggregate17 is formed with several
attractive properties due to exciton–vibration coupling, such as
sharp and red-shifted absorption with giant oscillator strength,
and frequently a F that is near unity as a result of superradiance.
Oligo-phenylene vinylene (OPV) molecules are typical D–p–A
molecules with a large two-photon action cross-section (d).18
Unfortunately, a strong intermolecular p–p stacking interaction
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usually leads to H-aggregation19 of OPVs in thin films and
crystals which show blue-shifted absorption and subradiance,20,21
which generally leads to fluorescence quenching. Therefore, it is
particularly appealing to obtain J-aggregates with OPV mole-
cules, in order to combine the unique optical properties of the
J-aggregates with the nonlinear properties of OPV molecules.
Schweikart and co-workers demonstrated that the attachment of
cyano groups at the end positions of OPV leads to a high F of the
film due to the formation of J-aggregates.22 Here, we report the
preparation of water-soluble ONPs based on a oligo-phenylene
vinylene (OPV) TPA dye, namely 1,4-dimethoxy-2,5-di[40 -
(cyano)styryl]benzene (COPV). Based on single crystal data, we
found that the synergistic effect between p–p stacking and
hydrogen-bonding interactions causes a brickwork arrangement
of COPV into J-aggregates,23 yielding a high solid-state fluores-
cence quantum yield of F > 0.4. Probably due to the exciton–
vibration coupling and thus the coherent excitations involved in
J-aggregates, the TPA cross-section of the J-aggregate COPV
NPs is almost 20 times larger than that of the monomers, which
results in a 10 times stronger two-photon fluorescence (3.6  104
GM) compared to that of the monomers (2.7  103 GM).
Together with the increased dye density, the relative brightness of
a single COPV ONP is estimated to be equivalent to the
brightness of 104 ZnS-capped CdSe quantum dots. Moreover,
these ONPs exhibit good water solubility, high photostability,
and low cytotoxicity. All these features suggest a new method of
fabricating efficient TPF nano-probes for bio-imaging by using
TPF-ONPs.
2. Experimental
Materials
All reagents for organic synthesis were purchased from Sigma-
Aldrich and used as received without further purification. THF
(HPLC grade) was purchased from Beijing Chemical Agent Ltd.,
China. Ultrapure water with a resistivity of 18.2 MU cm1,
produced using a Milli-Q apparatus (Millipore), was used in all
experiments. For more details of the molecular synthesis, prep-
aration of nanoparticles and microcrystals, and the crystallog-
raphy data, see the Supplementary Information.†
Preparation
The COPV NPs were prepared by the reprecipitation method. In
a typical preparation, 0.5 mL of 1  104 M COPV/THF solu-
tion was added into a 5 mL volumetric flask, then 4.5 mL of
water was added slowly under hand-shaking. The volume ratio
of THF : water was 1 : 9. The concentration of COPV molecules
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in the mixture was 1  105 M. The mixture was left for about
12 hours. The dispersion of COPV nanoparticles in the
THF : water ¼ 1 : 9 (v/v) mixture exhibited an off-white
turbidity due to the light scattering of particles.
The bulk crystals for single crystal X-ray diffraction (SCD)
analysis were cultivated by the solvent diffusion method at the
liquid–liquid interface between CH2Cl2 and MeOH. The crys-
tallographic data have been deposited as no. 838078 at the
Cambridge Crystallographic Data Centre (CCDC).
Measurements
The compound was confirmed by high resolution mass spec-
troscopy (GCT-MS Micromass, UK) and 1H NMR. The
morphology of the COPV nanoparticles was characterized by
transmission electron microscopy (TEM, JEOL-1011). X-ray
diffraction (XRD) patterns were measured using a D/max 2400

X-ray diffractometer with Cu Ka radiation (l ¼ 1.54050 A),
operated in the 2q range from 3 to 30 , by using samples of
nanoparticles filtered on the surface of an AAO membrane.
The steady-state absorption spectra were measured on a Shi-
midazu UV-3600 UV-VIS-NIR spectrophotometer. The
stationary fluorescence spectra were measured on a Horiba
FluoroMax-4-NIR spectrophotometer equipped with an inte-
grating sphere. The relative fluorescence quantum yields of the
solutions and colloidal suspensions were measured using
9,10-diphenylanthracene as a reference (F ¼ 0.95), while the
absolute fluorescence quantum yield of COPV ONPs was
measured by using the integrating sphere equipped on the
FluoroMax-4-NIR spectrophotometer.
For measuring temporally and spectrally resolved fluorescence
spectra, the second harmonic (400 nm, 120 fs, 1 kHz) of a
regenerative amplifier (Spitfire, Spectra Physics) seeded with a
mode-locked Ti:sapphire laser (Tsunami, Spectra Physics) was
used to excite the samples (liquid sample in a 10 mm cuvette, or
crystal sample mounted on a quartz plate) at the front surface
with an incidence angle of 45 . Fluorescence collected along
the direction normal to the sample surface was dispersed by a
polychromator (250is, Chromex) and detected with a streak
camera (C5680, Hamamatsu Photonics). The spectral resolution
was 0.2 nm, and the temporal resolution was about 2 ps. For two-
photon pumped single-crystal lasing experiments, the excitation
light came directly from the fundamental (800 nm, 120 fs, 1 kHz)
of the regenerative amplifier (Spitfire, Spectra Physics) seeded
with a mode-locked Ti:sapphire laser (Tsunami, Spectra Physics)
with the excitation fluence adjusted by using a set of neutral
density filters.
TPA cross-sections were determined via a comparative
method, by measuring the two-photon excited fluorescence
(TPEF) using Rhodamine B as a reference. The fundamental of a
mode-locked Ti:sapphire laser (690–850 nm, Tsunami) was
focused into a quartz cuvette having an optical geometry, and
detected with a liquid nitrogen cooled charge-coupled device
(CCD) (SPEC-10-400 B/LbN, Roper Scientific) attached to a
polychromator (Spectropro-550i, Acton).
To assess the cytotoxicity of the COPV nanoprobes, A549 cells
(human epithelial lung cancer) were grown in the presence of
COPV nanoparticles, and the viability was measured using a
CCK-8 assay. Cells were cultured in a 96-well microplate
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(approximately 2000 cells per well), in a medium containing
various concentrations of COPV nanoparticles, for 24 h at 37  C
in a humid atmosphere of 95% air and 5% CO2. After the culture
medium was removed, 100 mL of fresh culture medium (DMEM)
containing 10% CCK-8 reagent was added to each well and the
cells were incubated at 37  C for an additional 2 h. The cell
viability was determined using an ELISA microplate reader at an
optical absorbance of 450 nm. The cell viability was calculated as
the ratio of the absorbance of the sample well to that of the
control well and expressed as a percentage.
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For cell imaging, human cervix adenocarcinoma cell line HeLa
were cultured in 25 cm2 flasks (Corning) in DMEM with 10%
FBS at 37  C under 5% CO2. After several days, the cells were put
on quartz bottom dishes, and incubated for 24 h. Next, 20 mL of
sterile filtered COPV nanoparticle dispersions (5 mg mL1) was
added to the cells and allowed to incubate for 12 h. The unbound
nanoparticles were washed away with PBS and cell samples were
fixed with 4% paraformaldehyde solution. After that, the cells
were imaged in bright field and under NIR excitation using an
fluorescence microscope (Olympus FV1000MAITAI) equipped
with a NIR laser.
3. Results
COPV was synthesized by a one-pot method using a classical
Horner–Wadsworth–Emmons coupling reaction with a yield of
85% (Fig. 1a).24,25 COPV ONPs were prepared by a modified
reprecipitation method.26 Typically, 4.5 mL of water was slowly
added to 0.5 mL of 1.0  104 M solution of COPV in tetra-
hydrofuran (THF) under stirring. The solvent exchange induced
the nucleation and growth of ONPs. After 12 h, an off-white
colloidal suspension in the THF : H2O ¼ 1 : 9 (v/v) mixture was
obtained (the concentration of COPV is 1.0  105 M). This
colloidal suspension was stable at room temperature for several
months. Transmission electron microscopy (TEM) results
revealed that spherical ONPs were obtained with a diameter of
around 95 nm and a size distribution <10% (Fig. 1b).
Optical spectroscopic characterization
Fig. 2 presents the steady-state absorption (solid line) and fluo-
rescence (dashed line) spectra of COPV monomers (black) in the
dilute solution and ONPs (blue) dispersed in a THF : H2O ¼
1 : 9 (v/v) mixture. Table 1 summarizes the related one-photon
photophysical parameters. The lowest S0 / S1 transition of
Fig. 1 (a) Synthesis of COPV end functionalized with cyano groups, and
its single-crystal structure. (b) TEM image of COPV ONPs.
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Fig. 2 Steady-state absorption (solid line) and fluorescence (dashed line)
spectra of COPV molecules in THF solution (black) and ONPs dispersed
in a THF : water ¼ 1 : 9 (v/v) mixture (blue) with a concentration of
1.0  105 M.
COPV monomers in the dilute solution is a broad and struc-
tureless band at 413 nm with a molar extinction coefficient of 3 ¼
58 300 M1 cm1, while the S0 / S2 transition appears at
340 nm. The monomer fluorescence spectrum exhibits a vibronic
progression, with a subband spacing of 1200–1300 cm1, arising
mainly from vinyl stretching modes.20,21 The emission maximum
of the COPV monomers is at 474 nm, giving rise to a Stokes shift
of about 3100 cm1. Oelkrug et al. reported that the addition of
cyano groups at the vinyl positions of OPV causes serious fluo-
rescence quenching, because the nonplanarity enables torsional
motion induced nonradiative deactivation.27 In our case, the
COPV molecules functionalized with cyano groups at the end
positions are highly emissive in THF solution, with Fm ¼ 0.81 
0.05, similar to related compounds reported earlier.22
In striking contrast to those of the monomers, the absorption
and emission spectra of the COPV ONPs exhibit bathochromi-
cally shifted and unusually narrow bands of greater intensity
(Fig. 2). The aggregate absorption of the COPV ONPs (blue solid
line) is red-shifted to 482 nm with enhanced oscillator strength
(3 ¼ 84 500 M1 cm1), and the dipole moment of the S0 / S1
transition is increased from 3.9 D (monomer) to 4.4 D (aggre-
gate).28 This is different from the case of unsubstituted OPV, in
which the H-aggregate absorption of the ONPs is blue-shif-
ted.20,21 The fluorescence spectrum of the COPV ONPs (blue
dashed line) has a mirror-image relationship with the absorption
spectrum. The emission maximum of the COPV ONPs is at
491 nm (Table 1), yielding a Stokes shift of 380 cm1 that is much
smaller than that of the monomers (3100 cm1).
To further clarify the nature of the aggregate state, we per-
formed time-resolved fluorescence measurements (Fig. 3). The
monomer emission at 474 nm decays mono-exponentially (black
line), yielding a lifetime of sm ¼ 1.78 ns. The fluorescence decay
of the COPV ONPs at 491 nm (blue line), recorded at very low
excitation density, was also fitted mono-exponentially with a
time constant of sONP ¼ 0.40 ns. According to the equation
k ¼ F/s,29 the radiative decay rates (k) are calculated to be km ¼
0.46 ns1 and kONP ¼ 1.13 ns1 for COPV monomers and ONPs,
respectively (Table 1). Such enhanced radiative decay, known as
superradiance, is a key characteristic of J-aggregates,17,30 because
the coupling of the transition dipole moments of aggregated
J. Mater. Chem., 2012, 22, 17737–17743 | 17739

Table 1 One-photon and two-photon photophysical parameters of COPV monomers in THF solutions and ONPs in colloidal suspensions
One-photon properties
Sample labsa (nm) 3a (M1 cm1) lem (nm) F sb (ns)
Solution 413 58 300 474 0.81
ONPs 482 84 500 491 0.45
a
Molar extinction coefficient at the maximum one-photon absorption wavelength of labs. b Fluorescence lifetime. c Radiative decay rate calculated
according to kF ¼ F/s. d Two-photon absorption (TPA) cross-section (1 GM ¼ 1050 cm4 s per photon per molecule) at the maximum two-photon
absorption wavelength of lTPA. e Two-photon excited fluorescence (TPEF) cross-sections.
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Fig. 3 Fluorescence decay profiles of COPV monomers in THF solution
(s ¼ 1.78 ns) and ONPs in colloidal suspension (s ¼ 0.40 ns).
chromophores gives rise to a coherent excited domain. Based on
the equation N ¼ kONP/km,19,31 the size of the coherent domain
can be estimated to be N z 23 molecules in the J-aggregate
ONPs of COPV.
Crystallographic characterization and attribution
In the simple case of a parallel dimer, face-to-face or head-to-tail
interactions can lead to H- or J-aggregates, respectively.17,19,30 To
understand the molecular packing in the solid state, bulk crystals
suitable for single-crystal X-ray diffraction (XRD) analysis were
cultivated by the solvent diffusion method at the liquid–liquid
interface between CH2Cl2 and MeOH. The monoclinic COPV
crystal was determined to belong to the space group P21/c,
with cell parameters of a ¼ 8.5110(15) A,  b ¼ 14.954(3) A,  c¼
 
8.4748(15) A, a ¼ g ¼ 90 , and b ¼ 104.292(12) (Fig. 4a,
Fig. S3†).
The XRD peaks of COPV ONPs filtered onto the surface of an
alumina membrane can be perfectly indexed to the single crystal
data, but are much broader than the simulated bands (Fig. 4b).
Based on the Scherrer equation, the average crystal grain size was
estimated to be 5–10 nm (Fig. S2†), suggesting that COPV ONPs
are polycrystalline in nature. The pink dashed lines in Fig. 5a
indicate the closet intermolecular contacts between the phenyl
hydrogen C(7A)–H and the cyano nitrogen N(1) atoms, with a
separation of 2.7 A. The two molecules labelled by b and g are
connected by two CH/N hydrogen bonds in the same plane.
Extension along the b and g molecules via hydrogen bonding
forms chain-like structures that further stack into a two-dimen-
sional layer, with the shortest p–p interactions being about 3.4 A 
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Two-photon properties
kFc (ns1) lTPAd (nm) dd (GM) Fde
1.78 0.46 750 3400 2700
0.40 1.13 755 80 000 36 000
(Fig. 5a). In the a–b packing column, the pitch angle and the
 in the a–g packing
longitudinal displacement are 27.4 and 7.5 A;
column, the pitch angle and the longitudinal displacement are
13.0 and 12.8 A  (Fig. 5b).32 This is in sharp contrast to the
molecule functionalized with thiomethyl end groups (TOPV),
which forms a face-to-face H-aggregate with a negligible longi-
tudinal displacement.24 Therefore, it is the cooperation between
p–p stacking and hydrogen bonding interactions that drives the
COPV molecules to self-assemble into a brickwork arrangement
within the crystal ac plane, in which J-type coupling is preferred
(Fig. S3†).17,30
TPA measurement
We further investigated the effect of J-aggregation on the TPA
properties. First, TPA cross-sections (d) of the COPV monomers
in THF solution were determined by measuring the TPF using a
solution of Rhodamine B (dR) in ethanol as a reference,
according to the equation, dS ¼ dR(FSFRCRnS/FRFSCSnR), where
F represents the intensity of TPF, C denotes the concentration,
and n represents the refractive index of the solvent.33 As the shape
and the maximum of TPF are identical to those of one-photon
excited fluorescence (Fig. S4†), we concluded that TPF also
originated from the S1 state with the same value of F.13 As listed
in Table 1, the maximum TPA cross-section of COPV is dm ¼
3400 GM at lTPA ¼ 750 nm. Then we measured the TPF of
COPV ONPs suspended in a THF : H2O ¼ 1 : 9 mixture (inset in
Fig. 6). Since the concentrations of COPV in the THF solutions
and in the colloidal suspensions are the same at 1.0  105 M,
Fig. 4 (a) A unit cell of the monoclinic COPV crystal (CCDC no.
838078). (b) XRD pattern of COPV NPs (top) filtered on the surface of
an AAO membrane with a pore size of 20 nm. For comparison, the
simulated powder spectrum (bottom) using DIAMOND software is also
included.
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 View Article Online

Cell imaging

For the application of these ONPs as bioimaging probes, they

should be water-soluble while retaining their photophysical
properties. After frozen-vacuum-drying, re-dispersion of the
COPV ONPs in water produced a stable colloidal suspension.
The sizes and optical properties of COPV ONPs dispersed in
water remained the same after being stored at room temperature
for several months.
Measurements of the surface potential of these nanoparticles
showed that they are positively charged, and that the z-potential
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Fig. 5 (a) Brickwork arrangement of COPV molecules driven by p–p

retained a value of about 25–32 mV. The photostability of the as-
stacking and hydrogen bonding interactions, indicated by dotted lines.
synthesized COPV ONPs was studied under continuous excita-
(b) Front view of the molecular arrangement of the a, b and g molecules
shown in (a).
tion (438 nm), using a light source built in fluorometer with a
power of 2.0 mW as determined by a laser power meter.
Compared to the COPV solution in THF (black line in Fig. 7),
COPV ONPs re-dispersed in water exhibited better photo-
stability without an observable decrease in fluorescence intensity
after irradiation at 438 nm for two hours (red line in Fig. 7).
To evaluate the biocompatibility of COPV ONPs, A549 cells
were employed to test the cytotoxicities of these ONPs by using a
Cell counting Kit-8 colorimetric assay. As shown in Fig. 8, the
viability of A549 cells remained over 80% after 24 h incubation
with COPV ONPs, even at a concentration as high as 100 mg
mL1. This result shows that COPV ONPs have no obvious
cytotoxicity and are suitable for biological applications.
For examining luminescence imaging capability in living
cancer cells, HeLa cells were incubated with 20 mL of J-aggregate
ONPs of COPV in water (5.0  106 M) for 24 h and then used
for two-photon excited confocal imaging. Intense intracellular
luminescence was observed under excitation of femtosecond laser
pulses at 830 nm (Fig. 9a), which actually do not excite any
Fig. 6 Calculated TPA cross-section of COPV monomers in THF
solutions (B) and ONPs in colloidal suspensions (:). The inset shows
background fluorescence from the HeLa cells (Fig. S5†). Over-
the corresponding TPF photographs of the solution (left) and the lays of confocal luminescence and bright-field images further
nanoparticles suspension (right) for COPV under 800 nm illumination. demonstrate that the luminescence is evident in the intracellular
region (Fig. 9c). Therefore, J-aggregate ONPs of COPV are
clearly internalized into the cells rather than merely staining the
and the refractive indices of water and THF are comparable, we
membrane surface. It is reasonable to predict that the additional
roughly estimated the TPA cross-sections (dONP) of COPV
ONPs, according to dONP ¼ dm(FONPFm/FmFONP). As compared
with the TPA spectrum of COPV monomers (circles in Fig. 6),
the TPA peak of J-aggregate ONPs (triangles) was narrower and
slightly bathochromically shifted. Remarkably, we found that
the TPA cross-sections of the J-aggregate ONPs were greatly
enhanced, about 20 times larger than those of the monomers
(Table 1), probably due to the exciton–vibration coupling and
thus the coherent excitations involved in J-aggregates. It can be
seen from Table 1 that the active TPA cross-section F  d of
J-aggregate ONPs of COPV is an order of magnitude higher than
that of the monomers. To exclude the contribution of one-
photon processes, we examined the peak intensity of the two-
photon excited fluorescence spectra versus laser intensity at
800 nm for the COPV solution in THF (Fig. S6a†) and nano-
particles dispersed in the THF : water ¼ 1 : 9 (v/v) mixture
(Fig. S6b†). The log–log plots show slopes of 1.97 and 1.81,
which validate the two-photon nature of the absorptions. Fig. 7 The photo-bleaching kinetic traces of COPV molecules in THF
Significantly, these results show that, as TPF probes, COPV solution (black) and ONPs re-dispersed in water (red) with a concen-
J-aggregate ONPs are 3–4 orders of magnitude brighter than tration of 2.0  106 M, under the irradiation of monochromic light at
conventional fluorescent dyes and an order of magnitude 438 nm. The absorbance of both samples at 438 nm is 0.1 at the
brighter than quantum dots.33,34 beginning.

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Fig. 8 Viability of A549 cells at various concentrations of COPV ONPs.
advantages of J-aggregate ONPs of COPV for tissue imaging
also include superior resolution, as well as deep penetration
offered by two-photon near-infrared laser excitation.7,8
4. Conclusions
In summary, by employing a reprecipitation method, we
prepared ultrabright water-soluble organic nanoparticles
(ONPs) of 1,4-dimethoxy-2,5-di[40 -(cyano)styryl]benzene
(COPV). Single crystal data revealed that the cooperation
between p–p stacking and hydrogen bonding interactions causes
the brickwork arrangement of the COPV into J-aggregates, in
which the coherent excitation delocalization reaches 2–3 mole-
cules. Due to the superradiance of J-aggregates, COPV ONPs are
highly emissive in aqueous media with a quantum yield >0.4;
meanwhile, their TPA cross-section is greatly enhanced. As TPF
probes, COPV J-aggregate ONPs exhibit a TPF cross-section of
d  F ¼ 3.6  104 GM, and are 3–4 orders of magnitude brighter
than conventional fluorescent dyes and an order of magnitude
brighter than quantum dots. Moreover, these ONPs exhibit
no obvious cytotoxicity, even at a concentration as high as
100 mg mL1. We suggest that these ONPs, having strong two-
photon fluorescence, good water solubility, high photostability
and low cytotoxicity, are promising as efficient two-photon
fluorescent nano-probes for bio-imaging.
Fig. 9 (a) TPF image of HeLa cells incubated with J-aggregate ONPs of COPV upon excitation at 800 nm. (b) The bright-field image of HeLa cells
incubated with J-aggregate ONPs of COPV. (c) The overlaid picture of TPF and the corresponding bright-field images.
17742 | J. Mater. Chem., 2012, 22, 17737–17743
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (no. 20925309, 21190034, 21073079,
J1103307), the National Basic Research Program of China (973)
2011CB808402, 2012CB933102, the Specialized Research Fund
for the Doctoral Program of Higher Education (SRFDP,
20110211130001), and the Fundamental Research Funds for the
Central Universities and 111 Project.
Notes and references
1 W. Denk, J. H. Strickler and W. W. Webb, Science, 1990, 248, 73.
2 W. R. Zipfel, R. M. Williams and W. W. Webb, Nat. Biotechnol.,
2003, 21, 1369.
3 F. He, L. L. Tian, X. Y. Tian, H. Xu, Y. H. Wang, W. J. Xie,
M. Hanif, J. L. Xia, F. Z. Shen, B. Yang, F. Li, Y. G. Ma,
Y. Q. Yang and J. C. Shen, Adv. Funct. Mater., 2007, 17, 1551.
4 D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark,
M. P. Bruchez, F. W. Wise and W. W. Webb, Science, 2003, 300, 1434.
5 U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke and
T. Nann, Nat. Methods, 2008, 5, 763.
6 M. Green, Angew. Chem., Int. Ed., 2004, 43, 4129.
7 X. Huang, S. Neretina and M. A. El-Sayed, Adv. Mater., 2009, 21,
4880.
8 H. F. Wang, T. B. Huff, D. A. Zweifel, W. He, P. S. Low, A. Wei and
J. X. Cheng, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 15752.
9 M. Albota, Science, 1998, 281, 1653.
10 F. Terenziani, C. Katan, E. Badaeva, S. Tretiak and M. Blanchard-
Desce, Adv. Mater., 2008, 20, 4641.
11 R. Hardman, Environ. Health Perspect., 2006, 114, 165.
12 V. Mail€ander and K. Landfester, Biomacromolecules, 2009, 10, 2379.
13 G. S. He, L.-S. Tan, Q. Zheng and P. N. Prasad, Chem. Rev., 2008,
108, 1245.
14 S. Kim, Q. Zheng, G. S. He, D. J. Bharali, H. E. Pudavar, A. Baev and
P. N. Prasad, Adv. Funct. Mater., 2006, 16, 2317.
15 M. Velusamy, J. Y. Shen, J. T. Lin, Y. C. Lin, C. C. Hsieh, C. H. Lai,
C. W. Lai, M. L. Ho, Y. C. Chen, P. T. Chou and J. K. Hsiao, Adv.
Funct. Mater., 2009, 19, 2388.
16 A. Burns, H. Ow and U. Wiesner, Chem. Soc. Rev., 2006, 35, 1028.
17 F. W€urthner, T. E. Kaiser and C. R. Saha-M€ oller, Angew. Chem., Int.
Ed., 2011, 50, 3376.
18 M. Albota, D. Beljonne, J. L. Bredas, J. E. Ehrlich, J. Y. Fu,
A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder,
D. McCord-Maughon, J. W. Perry, H. Rockel, M. Rumi,
C. Subramaniam, W. W. Webb, X. L. Wu and C. Xu, Science,
1998, 281, 1653.
19 F. C. Spano, Acc. Chem. Res., 2010, 43, 429.
20 F. C. Spano, Annu. Rev. Phys. Chem., 2006, 57, 217.
This journal is ª The Royal Society of Chemistry 2012

21 J. Gierschner, M. Ehni, H. J. Egelhaaf, B. Milian Medina,
D. Beljonne, H. Benmansour and G. C. Bazan, J. Chem.Phys.,
2005, 123, 144914.
22 K. H. Schweikart, M. Hanack, L. Luer and D. Oelkrug, Eur. J. Org.
Chem., 2001, 293.
23 S. J. Yoon, J. W. Chung, J. Gierschner, K. S. Kim, M. G. Choi,
D. Kim and S. Y. Park, J. Am. Chem. Soc., 2010, 132, 13675.
24 F. Gao, Q. Liao, Z. Z. Xu, Y. H. Yue, Q. Wang, H. L. Zhang and
H. B. Fu, Angew. Chem., Int. Ed., 2010, 49, 732.
25 D. S. Seferos, D. A. Banach, N. A. Alcantar, J. N. Israelachvili and
G. C. Bazan, J. Org. Chem., 2004, 69, 1110.
26 H. B. Fu and J. N. Yao, J. Am. Chem. Soc., 2001, 123, 1434.
27 D. Oelkrug, A. Tompert, J. Gierschner, H. J. Egelhaaf, M. Hanack,
Published on 04 July 2012. Downloaded by Heriot Watt University on 03/10/2014 01:22:29.
M. Hohloch and E. Steinhuber, J. Phys. Chem. B, 1998, 102, 1902.
28 Transition dipole moments were calculated according to a report
method: W. Liptay, R. Wortmann, H. Schaffrin, O. Burkhard,
W. Reitinger and N. Detzer, Chem. Phys., 1988, 120, 429.
This journal is ª The Royal Society of Chemistry 2012
View Article Online
29 R. H. Friend, R. W. Gymer, A. B. Holmes, J. H. Burroughes,
R. N. Marks, C. Taliani, D. D. C. Bradley, D. A. Dos Santos,
J. L. Bredas, M. Logdlund and W. R. Salaneck, Nature, 1999, 397,
121.
30 T. Kobayashi, J-Aggregates, World Scientific, Singapore,
1996.
31 T. Kaiser, H. Wang, V. Stepanenko and F. W€ urthner, Angew. Chem.,
2007, 119, 5637.
32 M. D. Curtis, J. Cao and J. W. Kampf, J. Am. Chem. Soc., 2004, 126,
4318.
33 C. Xu and W. W. Webb, J. Opt. Soc. Am. B, 1996, 13, 481.
34 Based on the method reported in the reference: E. B. Cho,
D. O. Volkov and I. Sokolov, Adv. Funct. Mater., 2011, 21, 3129,
the relative brightness of a single COPV ONP for one-photon
excitation is estimated to be about 3.5  105 R6G dye-molecule
brightness, equivalent to the brightness of 104 ZnS-capped CdSe
quantum dots (Fig. S1†).
J. Mater. Chem., 2012, 22, 17737–17743 | 17743