Preparation of aggregation-induced emission dots for long-term two-photon cell imaging

Article abstract of DOI:10.1039/c5tb00207a

Two-photon fluorescence imaging has attracted increasing interest in the biological and medical fields because of its low cell damage, high resolution, large imaging depth, and easy dynamic observation. A high-performance two-photon probe with long-term imaging capability was proposed for this imaging technology. In this work, a new two-photon probe compound was synthesized from tetraphenylethylene fluorogen with aggregation-induced emission. A phenyl-[phenyl-(1,2,5-thialdiazol)] amine group was induced to red-shift the absorption and emission wavelengths of the compound. After self-assembly, fluorescent dots with aggregation-induced emission cores and hydrophobic shells terminated by -COOH were formed. Cell experiments proved that the 4-(7-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)benzo[c][1,2,5]thiadiazol-4-yl)benzoic acid (TPECOOH) dots with red emission showed good biocompatibility and excellent two-photon imaging ability. TPECOOH dots were used successfully in direct long-term cell imaging with high efficiency. Even after twelve days, fluorescence imaging could still be observed in live HeLa cells. This journal is

Full text of DOI:10.1039/c5tb00207a

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Materials Chemistry B
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Preparation of aggregation-induced emission dots
for long-term two-photon cell imaging†
Cite this: J. Mater. Chem. B, 2015,
, 3091
3
Qiang Ye,‡ Shuangshuang Chen,‡ Dandan Zhu, Xuemin Lu and Qinghua Lu*
Two-photon fluorescence imaging has attracted increasing interest in the biological and medical fields
because of its low cell damage, high resolution, large imaging depth, and easy dynamic observation.
A high-performance two-photon probe with long-term imaging capability was proposed for this imaging
technology. In this work, a new two-photon probe compound was synthesized from tetraphenylethylene
fluorogen with aggregation-induced emission. A phenyl-[phenyl-(1,2,5-thialdiazol)] amine group was induced
to red-shift the absorption and emission wavelengths of the compound. After self-assembly, fluorescent
dots with aggregation-induced emission cores and hydrophobic shells terminated by –COOH were formed.
Cell experiments proved that the 4-(7-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)benzo[c][1,2,5]thiadiazol-
Received 28th January 2015,
Accepted 28th February 2015
4-yl)benzoic acid (TPECOOH) dots with red emission showed good biocompatibility and excellent two-
DOI: 10.1039/c5tb00207a
photon imaging ability. TPECOOH dots were used successfully in direct long-term cell imaging with high
efficiency. Even after twelve days, fluorescence imaging could still be observed in live HeLa cells.
www.rsc.org/MaterialsB
variants for genetic cell tagging, inorganic semiconductor
Introduction
2
0–24
25
quantum dots,
or organic dye-doped nanoparticles.
As a new kind of advanced nonlinear imaging method, two- Unfortunately, green fluorescent proteins possess some inherent
photon fluorescence imaging microscopy has been used exten- drawbacks, for instance, susceptibility to enzyme degradation,
1
,2
sively in live cell and tissue imaging. Compared to single small Stokes shifts, poor photostability, and interference with
2
6–28
photon fluorescence, two-photon fluorescence depends quad- normal cell functions.
ratically on the excitation intensity, with high 3D spatiotem- quantum dots, which show bright emission and a high photo-
Although inorganic semiconductor
2
9
poral resolution resulting from the fluorescent region confined bleaching threshold under imaging conditions, are used
to a volume of about a cube of the excitation wavelength widely as fluorescent probes for direct cell labelling, the release
V B l ) when excited by the focusing laser. Furthermore, of heavy metal ions results in high cytotoxicity, especially in an
3
3,4
(
it can also be excited by near-infrared light (800–1100 nm). oxidative environment, and therefore hinders their future appli-
3
0–32
Because water and blood are nearly transparent and non- cation.
Organic dye-doped nanoparticles have a longer cell
scattering to near-infrared (IR) light, two-photon fluorescence retention time, intense fluorescence, and a lower exocytosis
3
3
imaging has attractive advantages such as low cell damage, a rate. However, the majority of commercially available organic
high resolution ratio, a large imaging depth, and three- dyes exhibit non-specific contrast agent transfer (low Stokes
5
–9
34
dimensional imaging capability.
Current imaging techni- shift) and aggregation-caused quenching.
ques have become powerful tools to observe and research the
Direct cell labelling by organic or inorganic fluorescent
dynamic processes of live cells. However, the complexity and materials without genetic modification of cells has drawn
3
5,36
dynamic nature of biological phenomena requires fluorescent increased attention recently.
However, the currently used
probes to have continuous non-invasive long-term imaging fluorescent probes exhibit serious disadvantages of short cell
1
0–16
37
capacity.
retention time or aggregation-caused quenching problems,
Several long-term fluorescent probes for cell imaging have and hinder their application in long-term cell imaging. Recently,
1
7–19
been reported, including green fluorescent protein
and its Tang and co-workers developed a series of organic luminogens
based on tetraphenylethene (TPE) fluorogens, which showed an
School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University,
Shanghai 200240, China. E-mail: qhlu@sjtu.edu.cn
extraordinary aggregation-induced emission (AIE) feature and
may overcome the aggregation-caused quenching problem.
Inspired by their work, we designed and synthesised a new
red emissive AIE compound, 4-(7-(phenyl(4-(1,2,2-triphenyl-
vinyl)phenyl)amino)benzo[c][1,2,5]thiadiazol-4-yl)benzoic acid (TPE-
COOH), which is derived from tetraphenylethene. The introduction
3
8–40
†
Electronic supplementary information (ESI) available: NMR spectra of all the
compounds, MS spectrum of compound 9 TPECOOH, UV/Vis spectrum of
TEPCOOH in THF and the cell imaging evaluation comparison of compound 8.
See DOI: 10.1039/c5tb00207a
‡
These authors contributed equally to this work.
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of the pheny-[phenyl-(1,2,5-thialdiazol)] amine group favours the Characterization
red shift of the absorption of TPE because of molecular conjuga-
Nuclear magnetic resonance (NMR) spectra were recorded
using a MERCURY plus 400 (Varian, Inc., USA) nuclear mag-
netic resonance spectrometer in deuterated chloroform (CDCl )
3
using tetramethylsilane (d = 0) as the internal reference. High-
resolution mass-spectrometric analysis was performed on a
Fourier transform ion cyclotron resonance mass spectrometer
tion, thereby reducing the excitation wavelength and causing red
fluorescence emission. We tried to use a terminal carboxyl group
to improve the self-assembly capability of the fluorescent
compound and the stability of assembled dots in aqueous
media, and to provide a certain affinity to cell membranes.
The TPECOOH dots showed good one-photon imaging with a
large Stokes shift of 142 nm, and brilliant two-photon imaging
ability excited by light of the near-IR region (800–1100 nm).
The dots also exhibited an excellent biocompatibility and a high
efficiency in direct long-term cell imaging.
(MS) SolariX-70FT-MS (Bruker Daltonics, Germany) operating
in MALDI-mode. The ultraviolet/visible (UV/Vis) spectra were
recorded on a Lambda 20 UV/Vis spectrometer (Perkin Elmer,
Inc., USA). Fluorescence spectra were recorded using an LS 50B
spectrometer (Perkin Elmer, Inc., USA). Quantum yield mea-
surements were carried out on a QM/TM steady-state and time-
resolved fluorescence spectrofluorometer (PTI Company, USA).
Diameter analysis of the TPECOOH dots was carried out using a
ZS90 (Malvern Instruments) dynamic light scanning (DLS)
Experimental
Materials
All reagents were purchased from J&K Scientific Co. and used instrument, and all samples were measured at a scattering
without further purification. All solvents were purchased from angle of 901. Transmission electron microscopy (TEM) images
National Pharmaceutical Group Chemical Reagent Co. Analy- were recorded by using a Tecnai G2 spirit Biotwin microscope
tical grade dimethyl formamide was purified by distillation (FEI Company, USA) at an accelerating voltage of 120 kV. Cell
under an inert nitrogen atmosphere. Tetrahydrofuran (THF) imaging observations were performed on an inverted fluores-
was distilled from sodium/benzophenone. Water was purified cence microscope (IX 71, Olympus) equipped with a charge-
using a Hitech system to obtain a resistivity of above 18.2 MO cm. coupled device camera. One- and two-photon fluorescence
HeLa cells were obtained from the Cell Resource Centre of Life images were taken on a Leica TCS SP5 confocal laser scanning
Sciences in Shanghai, China. Bovine serum albumin (66 kD, microscope (Leica Microsystems, Germany).
498%), fetal bovine serum, and Dulbecco’s modified Eagle
medium were obtained from Gibco BRL. The cellular cytotoxi-
city evaluating reagent (Cell Counting Kit-8) and all other cell
Synthesis and characterization of the TPECOOH compound
culture reagents were purchased from the Beyotime Institute of The TPECOOH synthesis routes are shown in Scheme 1. The
Biotechnology.
detailed synthetic processes are as follows.
Scheme 1 Synthetic routes of TPECOOH. Conditions: (i) sulfuric acid, CH
3
OH, reflux, 24 h. (ii) bis(pinacolato)diboron, PdCl
(dba) , tri-tert-butylphosphine, sodium tert-
, toluene, reflux, 24 h. (vi) Pd(PPh , K CO (2 M, aq.), THF–ethanol–H O,
2 3
(dppf), CH COOK, DMF,
reflux, 6 h. (iii) (a) n-BuLi, THF, À5 1C, 10 h, and (b) p-toluene sulfonic acid, toluene, reflux, overnight. (iv) Pd
butoxide, toluene, reflux, 24 h. (v) Pd(OAc) , tri-tert-butylphosphine, Cs CO
0 1C, 12 h. (vii) HCl, THF–water, 70 1C, 24 h.
2
3
2
2
3
3
)
4
2
3
2
8
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Compound 2. Sulfuric acid (0.5 mL) was added to a suspen- Cs
2
CO
3
(2.93 g, 9 mmol), Pd(OAc)
2
(60.7 mg, 0.3 mmol), and tri-
sion of compound 1 (5 g) in anhydrous methanol (50 mL). The tert-butylphosphine (0.1 g, 0.5 mmol), following the same
mixture was stirred at 80 1C for 12 h and then residual procedure described for the synthesis of 6 to yield 7 as a red
1
methanol was removed under reduced pressure. The residue solid (1.05 g, 55%). H NMR (400 MHz, CDCl ) d 7.66 (d, J =
3
was recrystallized from dichloromethane (CH Cl ) to give a 8.0 Hz, 1H; Ar-H), 7.24 (dd, J = 6.1, 5.2 Hz, 2H; Ar-H), 7.17–6.96
2
2
1
white powder (4.97 g, 93%). H NMR (400 MHz, CDCl
d 8.01–7.83 (m, 2H; Ar-H), 7.67–7.51 (m, 2H; Ar-H), 3.92
dd, J = 7.9, 2.7 Hz, 3H; OCH ).
Compound 3. Pd(dppf)Cl (400 mg, 0.48 mmol) was added of potassium carbonate (2 M, aq.) mixed with 60 mL of THF
to a mixture of compound 2 (3.00 g, 14 mmol), bis- and 20 mL of ethanol, tetrakis(triphenylphosphine)palladium(0)
pinacolato)diboron (4 g, 16 mmol) and KOAc (4.1 g, 42 mmol) (72 mg, 0.063 mmol) was added under an argon atmosphere.
3
)
(m, 19H; Ar-H), 6.92–6.87 (m, 2H; Ar-H), 6.78–6.73 (m, 2H; Ar-H).
Compound 8 (TPECOOCH ). Into a solution of compound 7
(0.8 g, 1.25 mmol) and compound 3 (0.34 g, 1.3 mmol) in 20 mL
3
(
3
2
(
in 60 mL of dimethyl formamide under argon. The mixture was The reaction temperature was increased to 80 1C and the mixture
refluxed overnight. After being quenched with aqueous ammo- was stirred for 24 h. The solution was concentrated using a
nium chloride, the mixture was extracted with CH Cl . The rotary evaporator and extracted with CH Cl . The organic layer
2 2 2 2
combined organic layer was washed with brine, and dried over was separated and washed with brine, dried over anhydrous
anhydrous sodium sulfate. After removal of the solvent at magnesium sulfate, and evaporated to dryness under reduced
reduced pressure, the residue was purified by column chromato- pressure. The crude product was purified using a silica-gel
graphy (silica gel, ethyl acetate/petroleum ether (PE) = 1/15 v/v) to column with DCM/PE (v/v = 1/6) as the eluent. Product 8 was
1
1
obtain a white solid compound 3 (3.18 g, yield: 87%). H NMR obtained with 75% yield (0.65 g). H NMR (400 MHz, CDCl )
3
(
400 MHz, CDCl ) d 8.08–7.97 (m, 2H; Ar-H), 7.92–7.82 (m, 2H; d 8.21 (d, J = 8.4 Hz, 2H; Ar-H), 8.05 (t, J = 6.5 Hz, 2H; Ar-H), 7.66
3
Ar-H), 3.93–3.89 (m, 3H; OCH ), 1.37–1.33 (m, 12H; OC(CH ) ).
(d, J = 7.8 Hz, 1H; Ar-H), 7.32–7.28 (m, 2H; Ar-H), 7.23–7.05
Compound 5. A 2.5 M solution of n-butyllithium in hexane (m, 19H; Ar-H), 6.96 (d, J = 8.6 Hz, 2H; Ar-H), 6.84 (d, J = 8.6 Hz,
18 mL, 45 mmol) was added to a solution of diphenylmethane 2H; Ar-H), 4.00–3.98 (m, 3H; OCH ).
6.75 g, 40 mmol) in anhydrous tetrahydrofuran (50 mL) at À5 1C Compound 9 (TPECOOH). Compound 8 (0.5 g) was dissolved
3
3 2
(
(
3
under argon. After stirring for 2 h, 4-bromobenzophenone in 10 mL of THF, then 10 mL of water and 2 mL of concentrated
10.44 g, 40 mmol) was added and the reaction mixture was hydrochloric acid were added. The solution was placed in a
(
stirred for 10 h to allow the temperature to rise gradually to nitrogen atmosphere and heated to 70 1C under vigorous
room temperature. The reaction was quenched with an aqu- stirring for 48 h. The solution was concentrated using a rotary
eous solution of ammonium chloride, and the mixture was evaporator and extracted with CH Cl . The organic layer was
2
2
2 2
extracted with CH Cl . The organic layer was evaporated after separated and washed with brine, dried over anhydrous mag-
drying with anhydrous sodium sulfate and the resulting crude nesium sulfate, and evaporated to dryness under reduced
hydroxyl intermediate was dissolved in toluene (100 mL). pressure. The crude product was recrystallized from DCM to
1
p-Toluene sulfonic acid (1.0 g, 5.85 mmol) was added, and give 9 as a red powder (0.39 g, 79%). H NMR (400 MHz, CDCl
3
)
the mixture was refluxed for 12 h and cooled to room tempera- d 8.24–8.15 (m, 4H; Ar-H), 7.92 (ddd, J = 9.1, 7.0, 2.3 Hz, 2H;
ture. The mixture was evaporated and the crude product was Ar-H), 7.35–7.05 (m, 19H; Ar-H), 7.02–6.91 (m, 3H; Ar-H), 6.90–
1
3
purified by silica gel column chromatography using DCM/PE 6.82 (m, 2H; Ar-H). C NMR (101 MHz, CDCl ) d 171.34 (s),
3
(
(
v/v = 1/10) as the eluent to yield compound 5 as a white powder 154.59 (s), 151.13 (s), 147.23 (s), 145.70 (s), 143.97 (s), 143.56
13.6 g, 83%). H NMR (400 MHz, CDCl ) d 7.25–7.18 (m, 2H; (d, J = 18.9 Hz), 142.84 (s), 140.92 (s), 140.54 (s), 140.02 (s),
3
1
Ar-H), 7.12 (ddd, J = 6.6, 5.3, 3.3 Hz, 9H; Ar-H), 7.06–6.98 (m, 6H; 139.16 (s), 132.26 (s), 131.70–131.12 (m), 130.52 (s), 129.27 (s),
Ar-H;), 6.93–6.87 (m, 2H; Ar-H). 128.97 (s), 128.21 (s), 127.67 (d, J = 2.7 Hz), 127.42 (s), 126.45
Compound 6. Tri-tert-butylphosphine (26.3 mg, 0.13 mmol), (d, J = 7.2 Hz), 124.47 (s), 123.56 (d, J = 48.8 Hz), 123.31–123.13
(dba) (0.1 g, 0.11 mmol), and sodium tert-butoxide (0.96 g, (m), 122.55 (s). High-resolution MS (MALDI, m/z): [M+] calcd for
0 mmol) in dry toluene (30 mL) were added to a mixture of C H N O S: 677.81; found: 677.212816.
Pd
2
3
1
4
5
31 3 2
compound 5 (3.28 g, 8 mmol) and aniline (0.94 g, 10 mmol)
under argon. The mixture was stirred at 110 1C for 24 h. After
Preparation of TPECOOH dots
solvent removal, water (50 mL) and DCM (100 mL) were added. The as-prepared TPECOOH (1 mg) was dissolved in 0.5 mL of
À1
The organic layer was separated and washed with brine, dried THF, and then 0.5 mL of the sample solution (2 mg mL ) was
over anhydrous magnesium sulfate and evaporated to dryness added dropwise into 5 mL of water under intensive stirring. The
under reduced pressure. The crude product was purified by mixture was stirred vigorously at room temperature for 24 h.
column chromatography on silica gel using DCM/PE (v/v = 1/6) The TPECOOH molecules aggregated to form AIE dots that were
as the eluent to yield 6 as a pale yellow solid (2.1 g, 62%). then suspended in water.
1
H NMR (400 MHz, CDCl ) d 7.25–7.20 (m, 2H; Ar-H), 7.16–6.99
3
Cell culture
(m,17H; Ar-H), 6.94–6.86 (m, 3H; Ar-H), 6.80 (d, J = 8.6 Hz, 2H;
Ar-H), 5.65 (s, 1H; NH).
HeLa cells were cultured in Dulbecco’s modified Eagle medium
Compound 7. Compound 7 was prepared using 6 (1.26 g, containing 10% fetal bovine serum and antibiotics (50 units per
mmol), 4,7-dibromo-2,1,3-benzothidiazole (2.6 g, 9 mmol), mL penicillin and 50 units per mL stretomycin) at 37 1C in a
3
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humidified atmosphere containing 5% CO
seeded into 6-well plates at 10 cells per well and cultured for
2
. The cells were
5
6
h to reach confluence. TPECOOH dots dispersed in water
À1
(5 mg mL ) were added for cell imaging. Twelve hours later, after
medium removal and washing twice with phosphate buffer solution,
cell imaging was recorded using a fluorescence microscope.
Cytotoxicity study
The cytotoxicity of the TPECOOH dots was determined using
a cell counting kit (CCK)-8. The HeLa cells were seeded into
4
9
6-well plates at 10 cells per mL. After cell spreading for 80%
confluence, TPECOOH dots were added at 0.01, 0.1, 1, 5, 10, and
À1
5
0 mg mL . The well without TPECOOH dots was set as a control.
To eliminate the influence of TPECOOH dots, the same TPECOOH
dot solution was added into the well without cells. After incubating
the cells for different periods of time, the absorbance at 450 nm
was recorded using a microplate reader (BIO-RED).
One- and two-photon fluorescence cellular imaging
For one- and two-photon fluorescence imaging, HeLa cells were
À1
seeded onto the coverslip for 6 h and were added with 5 mg mL
TPECOOH dots. After being incubated for 12 h, samples were
fixed with 2.5% glutaraldehyde for 5 min, and the coverslip was
sealed onto the glass slide. Two-photon fluorescence images
were taken using a confocal laser scanning microscope (Leica
TCS SP5) upon excitation at 880 nm. For comparison, one-
photon fluorescence images were also measured upon excita-
Fig. 1 (a) UV/Vis absorption spectrum of TPECOOH dots suspended in
tion at 488 nm. Cells were exposed to the laser for 20 min to water. (b) Photoluminescence spectra (PL) of TPECOOH in THF–water
À5
mixtures with different water fractions (concentration of TPECOOH: 10 M,
evaluate the resistance to photobleaching.
excitation wavelength: 510 nm).
Results and discussion
concentration. This phenomenon can be attributed to the AIE
Synthesis and characterization of TPECOOH
effect of TPECOOH. The inset in Fig. 1b shows the fluorescence
emission of TPECOOH in THF and THF–water (10/90) solution,
respectively, at 365 nm irradiation using a commercially available
UV lamp. Compared with the absorption peak at 488 nm in the UV/
Vis spectra, the TPECOOH suspension has a maximum emission at
TPECOOH was synthesized as shown in Scheme 1. NMR and
MS were used to confirm the molecular structure of TPECOOH.
1
13
The H NMR, C NMR, and high-resolution MS results
for TPECOOH in Fig. S7–S9 (ESI†), respectively, show that
TPECOOH was synthesized successfully.
630 nm with a large Stokes shift of 142 nm. The TPECOOH
suspension showed a fluorescence quantum yield (F ) of B30%.
f
Optical properties of TPECOOH
The optical properties of TPECOOH were studied by UV/Vis and Fabrication and characterization of TPECOOH dots
fluorescence spectra. Fig. 1a shows the UV/Vis absorption
spectrum of the TPECOOH dots suspended in water, which
addition of TPECOOH in THF into the aqueous media resulted
was similar to that of the TPECOOH solution in THF (see
The fabrication of TPECOOH dots is illustrated in Fig. 2. The
Fig. S10, ESI†). TPECOOH exhibited two absorption peaks at
310 and 488 nm, respectively. Fig. 1b shows the one-photon
luminescence spectra of the TPECOOH molecule dissolved in
THF and THF–water mixtures with different water proportions.
Almost no fluorescence emission was detected when the
concentration of water, as a poor solvent, was less than 30%.
When the water concentration exceeded 50%, TPECOOH aggrega-
tion occurred and the free rotation of tetraphenylethene inside the
aggregate was restricted. Therefore, the non-radiative decay chan-
nels were blocked, which leads to obvious fluorescence emission at
Fig. 2 Schematic illustration of the fabrication of AIE-dots for application
6
30 nm. The emission intensity increased with an increase in water in long-term cell imaging.
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0.26 (Fig. 3b). Because of the formation of a hydration layer on the
dots, the diameter of the dots obtained from DLS measurements
was slightly larger than that obtained from TEM.
Cytotoxicity and photostability
The biocompatibility of fluorescent dots is of vital significance
in biomedical applications. A cytotoxicity evaluation of the
TPECOOH dots was performed using a cell counting kit
Fig. 3 (a) TEM image of TPECOOH dots, inset: magnified image. (b) Diameter
distribution of TPECOOH dots obtained by DLS. Polydispersity index
(CCK)-8. The results are shown in Fig. 4a. Cell viabilities over
(PDI): B0.260, and average size (Da): B68 nm.
such a wide concentration range had no obvious change after
À1
24 h of incubation. Even when cultured for 48 h at 50 mg mL
,
the cell viability was more than 85%, which indicates that the
TPECOOH dots have a low cytotoxicity and good biocompat-
ibility. Most probe molecules are photobleached and are
quenched easily under laser irradiation, which limits their
application. In this study, the photostability of the TPECOOH
dots was investigated. Fig. 4b shows the change in fluorescence
intensity of the TPECOOH dots that were exposed to continuous
laser irradiation of 488 nm for 20 min. Only a slight decrease
in fluorescence occurred, but no obvious photobleaching was
Fig. 4 (a) Cell viability of TPECOOH dots at different concentrations observed, which implies excellent photostability. The good bio-
cultured for 24 h. (b) Photostability of TPECOOH dots under laser irradia-
compatibility and photostability endow TPECOOH dots with
tion at 488 nm for 20 min (emission filter wavelength was 620 nm when
great potential for cell imaging and long-term tracing.
excited by 488 nm with a laser power of 2 mW), inset: initial confocal
images taken before (left, 0 min) and after (right, 20 min) irradiation,
Live cell tracing
respectively. Scale bar = 25 mm.
The requirement of long-term fluorescent probes has been
recognized in recent years for applications in clinical cancer
in the hydrophobic–hydrophilic balance force promoting diagnosis. Here, HeLa cells were cultured with TPECOOH at a
À1
TPECOOH molecular aggregation and the formation of concentration of 5 mg mL . After being cultured for 12 h, the
TPECOOH dots with hydrophilic shells terminated by a –COOH culture medium was removed and rinsed twice with phosphate
moiety, which exhibited strong fluorescence. The morphology buffer solution to remove the non-adsorbed TPECOOH dots.
of the AIE dots is shown by the TEM images in Fig. 3a, which The new culture medium was added again to provide nutrition
exhibit a spherical structure with good monodispersity and for cells. Fluorescence images taken using normal fluorescence
40–60 nm diameter. The inset image shows a magnification of the microscopy are shown in Fig. 5. Fig. 5a taken after 2 days,
TPECOOH dots. DLS measurements also show that the TPECOOH shows super bright fluorescence. The fluorescence intensity
À1
dots at 0.1 mg mL in aqueous solution had a hydrodynamic decreased with time because of cell proliferation, in which the
diameter of approximately 68 nm with a polydispersity index of TPECOOH dots divide into daughter cells. Fluorescence can
Fig. 5 Long-term cell tracing images of TPECOOH dots (from 2 to 12 days). Excitation wavelength: 545 nm. Scale bar = 100 mm.
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fabricated easily by aggregation of TPECOOH molecules dis-
persed in a THF–water mixture. They showed good mono-
dispersity, significant aggregation-induced emission properties
in aqueous solution, and were able to enter cells because of
the good affinity of the carboxyl group to the cell membrane.
Our studies show that the TPECOOH dots showed red emis-
sion, good two-photon imaging ability, low toxicity, good
photostability, large Stokes shift, and biocompatible properties.
Furthermore, they performed very well in direct long-term cell
imaging. The AIE dots may have great potential in direct long-
Fig. 6 Two-photon confocal laser scanning microscopy images (excited term cell imaging.
at 880 nm). (a) Scale bar = 50 mm. (b) Magnification of (a), and scale bar =
2
5 mm.
Acknowledgements
This research was supported by 973 Projects (2012CB933803,
still be observed after 12 days, which indicates that the
TPECOOH dots are appropriate for long-term tracing.
2014CB643605), the National Science Fund for Distinguished
Young Scholars (50925310), the National Science Foundation of
China (21374060, 51173103), and Excellent Academic Leaders
of Shanghai (11XD1403000).
For comparison, the cell imaging capability of compound 8
was also evaluated. However, the hydrophobic dots of com-
pound 8 cannot be used to label the cell even after being
cultured for 48 h. The hydrophobic nanoparticles were not
taken up into the cell, possibly because of the lack of aqueous
stability that results in precipitation and a low affinity to the
cell membrane (Fig. S11, ESI†). This comparison verified our
hypothesis that for cell imaging, the intrinsic balance between
hydrophobicity and hydrophilicity of the fluorescent molecule
plays a crucial rule in labeling and tracing cells, and the good
affinity of TPECOOH dots to cell membranes originates from
the hydrophilic carboxyl group.
Notes and references
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1
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