Two-photon induced emissive thiophene donor-acceptor systems as molecular probes for in vitro bio-imaging: Synthesis, crystal structure, and spectroscopic properties

Article abstract of DOI:10.1039/c3ra42914h

Two new classes of organic two-photon absorption (TPA) chromophores, D-π-A (Bt₁/Tt₁) and D-π-A-π-D (Bt ₂/Tt₂), were designed and synthesized using a Sonogashira coupling reaction between 4-ethynyl-N,N-dimethylaniline and 5′-bromo-2, 2′-bithiophene/2,2′:5′,2′′-terthiophene or 5,5′-dibromo-2,2′-bithiophene/2,2′:5′, 2′′-terthiophene, respectively. At room temperature, all of the compounds had single-photon fluorescent and solvatochromatic properties, and could produce two-photon-induced luminescence upon excitation at 700 nm (Ti:sapphire femtosecond laser, 150 fs pulses). The corresponding TPA cross-section values ranged from 81 to 563 GM (10^-50 cm⁴ s photon^-1 molecule^-1). The quadrupolar-type (D-π-A-π-D) chromophores, Bt₂ and Tt₂, exhibited a larger TPA cross-section (σ₂) value than their dipolar analogues (D-π-A), Bt₁ and Tt₁, whereas the bithiophene chromophores, Bt₁ and Bt₂, exhibited a larger σ₂ value than their terthiophene counterparts, Tt₁ and Tt₂. All of the compounds were used for bio-imaging in living human cervical carcinomas (HeLa). We found that Bt₁ and Tt ₁ were able to pass through the cell membrane and to selectively stain the cell cytoplasm and lysosome, respectively. The results of these studies could provide new molecular-design strategies for two-photon imaging in vitro. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra42914h

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PAPER
Two-photon induced emissive thiophene donor–
acceptor systems as molecular probes for in vitro bio-
imaging: synthesis, crystal structure, and spectroscopic
properties3
Cite this: DOI: 10.1039/c3ra42914h
Cheuk-Fai Chow*
Two new classes of organic two-photon absorption (TPA) chromophores, D–p–A (Bt1/Tt1) and D–p–A–p–D
(Bt2/Tt2), were designed and synthesized using a Sonogashira coupling reaction between 4-ethynyl-
N,N-dimethylaniline and 59-bromo-2,29-bithiophene/2,29:59,299-terthiophene or 5,59-dibromo-2,29-bithio-
phene/2,29:59,299-terthiophene, respectively. At room temperature, all of the compounds had single-
photon fluorescent and solvatochromatic properties, and could produce two-photon-induced lumines-
cence upon excitation at 700 nm (Ti:sapphire femtosecond laser, 150 fs pulses). The corresponding TPA
cross-section values ranged from 81 to 563 GM (10250 cm4 s photon21 molecule21). The quadrupolar-type
(D–p–A–p–D) chromophores, Bt2 and Tt2, exhibited a larger TPA cross-section (s2) value than their dipolar
analogues (D–p–A), Bt1 and Tt1, whereas the bithiophene chromophores, Bt1 and Bt2, exhibited a larger s2
value than their terthiophene counterparts, Tt1 and Tt2. All of the compounds were used for bio-imaging
in living human cervical carcinomas (HeLa). We found that Bt1 and Tt1 were able to pass through the cell
membrane and to selectively stain the cell cytoplasm and lysosome, respectively. The results of these
studies could provide new molecular-design strategies for two-photon imaging in vitro.
Received 8th July 2013,
Accepted 29th July 2013
DOI: 10.1039/c3ra42914h
www.rsc.org/advances
the UV-vis excitation sources used in many single-photon
fluorescent probes.7
Introduction
Selective cellular imaging is important for advancing our
understanding of biology and clinical science.1 Single-photon
fluorescent probes have been widely used to monitor
molecular processes in cells and tissue.2 Although there are
many commercially available fluorescent probes for in vitro
imaging, most of them suffer from toxicity, high energy
excitation source, and high background noise.
Two-photon absorption (TPA) involving simultaneous
absorption of a pair of photons was theoretically calculated
in 19313 and experimentally proved in the 1960s.4 However,
two-photon fluorescent probes suitable for imaging in living
cells were not available until the 2000s.5 In recent years, two-
photon chromophores have attracted great interest due to
their advantages over their single-photon counterparts.6 The
quadratic relationship of the two-photon induced emission
with respect to the excitation power makes it a high resolution
imaging tool. The near infra-red (NIR) excitation light
penetrates cells and tissues, and is non-invasive compared to
Organic molecules are generally classified as being more
suitable for two-photon fluorescent probes than organometal-
lic and organic-coordination compounds; their low toxicity
and high flexibility in terms of structural engineering allow
researchers to develop molecules with tunable absorption.
Functional oligothiophenes have attracted comprehensive
interest among researchers all over the world and have
actually become among the most frequently used organic
active materials in electronic devices and molecular electro-
nics.8 Recently, several TPA mechanisms have been estab-
lished for oligothiophenes. Generally, two classes of
oligothiophenes have been found to be TPA chromophores: a
dipolar form with a donor–p–acceptor (D–p–A) functionality,9
and a quadrupolar mode with a D–p–D, A–p–A, D–p–A–p–D or
A–p–D–p–A motif.5b,10 Although their chemical properties are
well established, their application as two-photon fluorescent
probes remains rare.11 This study examines the relationships
among the molecular structure, TPA cross-sections, and bio-
imaging properties of functional oligothiophenes. Herein, we
report studies on the (i) linear and two-photon induced
photophysical properties; (ii) cell-permeable properties; and
(iii) in vitro bio-imaging properties of a series of novel dipolar
and quadrupolar chromophores based on 59-(4-ethynyl-
Department of Science and Environmental Studies, The Hong Kong Institute of
Education, 10 Lo Ping Road, Tai Po, New Territories, HKSAR, China.
E-mail: cfchow@ied.edu.hk; Fax: (+852) 29487676; Tel: (+852)29487671
Electronic supplementary information (ESI) available: The NMR, ES-MS spectra
3
and pKa analyses are available. See DOI: 10.1039/c3ra42914h
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2,5-diphenyltetrazolium bromide] solution (5 mg mL21) was
added to the culture medium in each well and incubated at 37
uC for 24 h. The absorption was measured at 570 nm using a
microplate reader (PowerWave XS, BioTek). Each data point
represents the ratio of the mean values between the
compounds versus the DMSO control.
Two-photon-induced emission measurements
The 850 nm pump source was from the fundamental of a
femtosecond mode-locked Ti:sapphire laser system (output
beam around 150 fs duration and 1 kHz repetition rate). The
laser was focused through an f = 10 cm lens onto an y50 mm
spot on the sample. The emitting light was collected with a
backscattering configuration into a 0.5 m spectrograph and
detected by a liquid-nitrogen-cooled CCD detector. A power
meter was used to monitor the uniform excitation.15 To further
confirm the two-photon process of the thiophene compounds,
the emission peak intensities were plotted as a function of the
incident power at 700 nm.
Scheme 1 Chemical structures of Bt0–Bt2 and Tt0–Tt2.
N,N-dimethylaniline)-2,29-bithiophene/-2,29:59,299-terthio-
phene and 5,59-di(4-ethynyl-N,N-dimethylaniline)-2,29-bithio-
phene/-2,29:59,299-terthiophene (Scheme 1).
Experimental section
Materials and general procedures
Two-photon cross-section measurements
The 5-bromo-2,29:59,299-terthiophene was prepared according
to the method given in the literature.12 2,29-bithiophene,
The theoretical framework and experimental protocol for the
two-photon cross-section measurements were outlined by
Webb and Xu.16 The two-photon excitation (TPE) ratios of
the reference and sample systems are given in eqn (i):
5-bromo-2,29-bithiophene,
5,59-dibromo-2,29-bithiophene,
2,29:59,299-terthiophene, 5,59-dibromo-2,29:59,299-terthiophene,
copper(I) iodide, bis(triphenylphosphine)-palladium(II) dichlor-
ide, 4-ethynyl-N,N-dimethylaniline, N-bromosuccinimide and
triethylamine were obtained from Aldrich. All of the solvents
used were of analytical grade.
S
S
S
:
s2 w
:
: :
CR nS F (l)
~
(i)
R
w
CS nR FR(l)
:
:
s2R
Physical measurements and instrumentation
in which w is the quantum yield, C is the concentration, n is
the refractive index, and F(l) is the integrated photolumines-
cence spectrum. The superscripts or subscripts S and R
represent the sample and reference, respectively. In our
measurements, we ensured that the excitation flux and the
excitation wavelengths were the same for both the sample and
the reference. The two-photon absorption cross-section (s2)
was determined using Rhodamine 6 G as a reference.
The 1H-NMR spectra were recorded using a Bruker AVANCE III
System 400 MHz NMR spectrometer. The electrospray mass
spectra (ESI-MS) were measured with an AB SCIEX API 2000
LC/MS/MS system. The elemental analyses were performed on
a Vario EL CHN analyzer. The thermal stability was measured
on a TA Instruments Thermogravimetric analyzer (TGA) Q50
from 30 to 1000 uC at a heating rate of 10 uC min21 under a N2
atmosphere. The infrared spectra in the range 500–4000 cm21
in KBr plates were recorded on a Perkin Elmer Model Frontier
FTIR spectrometer and the UV-vis spectra were measured on a
Cary 50 ultraviolet visible spectrophotometer. The emission
spectra were recorded using a Horiba FluoroMax-4 spectro-
fluorimetric with a 5 nm slit width and a 0.5 s integration time.
Luminescence quantum yields were measured by the optical
dilution method13 using an aerated aqueous solution of
[Ru(bpy)3]Cl2 (Q = 0.028, excitation wavelength at 455 nm)14
as the standard solution.
Microscopy imaging
The bio-imaging properties of Bt1, Bt2, Tt1, and Tt2 were
studied by conducting in vitro experiments using a commercial
multiphoton confocal microscope. For the two-photon ima-
ging in vitro studies, the HeLa cells in a tissue culture chamber
(5% CO2, 37 uC) were imaged using a Leica TCS SP5 spectral
confocal microscope equipped with a Ti:sapphire laser (Leica).
The cells were incubated with the probes (5 mM;
DMSO : DMEM medium v/v 0.5 : 99.5) for 30 min at 37 uC.
No washing steps were required. A laser power of 1700 mW, a
gain of 10%, and an offset of 3% were used in the image
acquisition. The excitation beam was produced by the
femtosecond laser, which was tunable in the range 680–1050
nm (lex = 700 nm, ca. 5 mW), and focused on coverslip-
adherent cells using a 406 oil-immersion objective. The
emission images were collected by a photomultiplier tube
adjusted for wavelengths 450–750 nm.
Cell culture
Human cervical carcinoma (HeLa) cells were cultured with 5%
CO2 at 37 uC in Dulbecco’s Modified Eagle Medium (DMEM)
from Gibco, combined with 10% fetal bovine serum in the
presence of antibiotic-antimycotic solution (Gibco).
MTT cell viability assay
The HeLa cells were plated in triplicates into 96-well tissue
culture plates; this procedure was repeated twice. After 24 h,
the cells were treated with Bt1, Bt2, Tt1, and Tt2 or with DMSO
as a control. After 15 min, MTT [3-(4,5-dimethylthiazol-2-yl)-
Crystal structure determination
Yellow single-plated crystals of Bt2 were grown by slow
evaporation of a mixture with a 10 : 1 ratio of a petroleum
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Compound Bt1
Table 1 Crystallographic data of Bt2
The synthesizing procedure was similar to that used with
compound Tt1 except that 5-bromo-2,29-bithiophene (2.5
mmol) was used instead of 5-bromo-2,29:59,299-terthiophene.
The procedure produced a yellow solid with the following
characteristics. Yield 75%. (400 MHz, CDCl3) d (ppm) = 3.00 (s,
6H), 6.65 (d, J = 8.8 Hz, 2H), 7.01 (t, J = 4.0 Hz, 1H), 7.03 (d, J =
4.4 Hz, 1H), 7.10 (d, J = 4.4 Hz, 1H), 7.17 (d, J = 4.0 Hz, 1H),
7.21 (d, J = 4.0 Hz, 1H), 7.38 (d, J = 8.8 Hz, 2H). ESI-MS (+ve
mode): m/z 310.2 (Bt1?H)+. IR (KBr): 2186 cm21 (CMC); 1605
cm21 and 1444 cm21 (thiophene rings). Anal. Calcd. for
C18H15NS2: C, 69.86; H, 4.89; N, 4.53. Found: C, 69.77; H, 4.88;
N, 4.54. TGA: 290 uC (2% decomposition), 380 uC (5%
decomposition).
Empirical formula
Formula weight
T (K)
C28H24N2S2
452.64
293(2)
0.71073
Monoclinic
P21/n
9.845(5), 21.819(5), 16.178(5)
90.000(5), 92.315(5), 90.000(5)
3472(2)
4
22.12
1285
5881
3719
0.0225
3719
Wavelength (Å)
Crystal system
Space group
a, b, and c Å
a, b, and c deg
Volume (Å3)
Z
m(Cu-Ka) (cm21
F(000)
)
Reflections collected
Unique reflections
Rint
Observed reflections [I . 1.50s(I)]
Final R indices [I . 1.5s(I)]
Goodness-of fit on F2
R1 = 0.0526, wR2 = 0.1863
1.192
Compound Tt2
The synthesizing procedure was similar to that of compound
Tt1 except that 5,59-dibromo-2,29:59,299-terthiophene (1.25
mmol) was used instead of 5-bromo-2,29:59,299-terthiophene
(2.5 mmol). The crude product was purified by extensive
washing with methanol, ethanol, hexane, and cold ethyl
acetate. The residue solid was purified by column chromato-
graphy (silica gel, Pet-ether/Dichloromethane v/v 10 : 1, RF =
0.25). The resulting orange solids were collected and further
washed with cold ether. The characteristics of the orange
solids were as follows. Yield 65%. (400 MHz, CDCl3) d (ppm) =
3.01 (s, 12H), 6.66 (d, J = 8.8 Hz, 4H), 7.05 (d, J = 4.0 Hz, 2H),
7.07 (s, 2H), 7.10 (d, J = 4.0 Hz, 2H), 7.39 (d, J = 8.8 Hz, 4H). ESI-
MS (+ve mode): m/z 535.0 (Tt2?H)+. IR (KBr): 2188 cm21 (CMC);
1604 cm21 and 1440 cm21 (thiophene rings). Anal. Calcd. for
C32H26N2S3: C, 71.87; H, 4.90; N, 5.24. Found: C, 71.89; H, 4.87;
N, 5.30. TGA: 303 uC (2% decomposition), 395 uC (5%
decomposition).
ether/CH2Cl2, in an open atmosphere. The geometric and
intensity data for the complexes were collected on a Bruker
Smart Apex II CCD diffractometer with Cu-Ka radiation (l =
1.54180 Å) at 293(2) K. The intensities were corrected for
Lorentz and polarization factors, and for absorption using the
multiscan method.17 All of the structures of the complexes
were solved by direct methods (SHELX-97)18 in conjunction
with standard difference Fourier techniques, and subsequently
refined with full-matrix least-square and analyzed on F2. Non-
hydrogen atoms were refined with anisotropic displacement
parameters. The hydrogen atoms were generated in their
idealized positions in each case and allowed to ride on the
respective carbon atoms (Table 1).
Compound Tt1
5-bromo-2,29:59,299-terthiophene (2.5 mmol), CuI (3 mol%),
and bis(triphenylphosphine)palladium(II) dichloride (3 mol%
relative to the 5-bromo-2,29-bithiophene used) were added to a
Compound Bt2
The synthesizing procedure was similar to that of compound
Tt2 except that 5,59-dibromo-2,29-bithiophene (1.25 mmol) was
used instead of 5,59-dibromo-2,29:59,299-terthiophene. An
orange solid with the following characteristics was collected.
Yield 68%. (400 MHz, CDCl3) d (ppm) = 3.01 (s, 12H), 6.66 (d, J
= 8.8 Hz, 4H), 7.04 (d, J = 4.0 Hz, 2H), 7.10 (d, J = 4.0 Hz, 2H),
7.40 (d, J = 8.8 Hz, 4H). ESI-MS (+ve mode): m/z 453.1 (Bt2?H)+.
IR (KBr): 2190 cm21 (CMC); 1599 cm21 and 1438 cm21
(thiophene rings). Anal. Calcd. for C28H24N2S2: C, 74.30; H,
5.34; N, 6.19. Found: C, 74.41; H, 5.29; N, 6.22. TGA: 300 uC
(2% decomposition), 390 uC (5% decomposition).
two-neck round-bottomed flask equipped with
a reflux
condenser and a stir bar and the whole setup was degassed
and back-filled three times with a gaseous hydrogen. THF (20
mL) was introduced via a syringe followed by triethylamine (3
ml). The reaction mixture was set to about 60 uC and a solution
of the 4-ethynyl-N,N-dimethylaniline (3 mmol) in 15 mL THF
was introduced under the reducing atmosphere. The reaction
was followed by a TLC analysis. The disappearance of the
amine staring material happened at around hour 48. The
solvents were then dried and the crude product was purified
by
column
chromatography (silica
gel,
Pet-ether/
Dichloromethane 20 : 1, RF = 0.2). A yellow solid was obtained
(Yield 78%). The characteristics of the solid were as follows.
(400 MHz, CDCl3) d (ppm) = 3.01 (s, 6H), 6.66 (d, J = 8.8, Hz,
2H), 7.03 (m, 2H), 7.08 (m, 2H), 7.10 (d, J = 4.0, 1H), 7.19 (d, J =
4.0, Hz, 1H), 7.23 (d, J = 4.0, Hz, 1H), 7.39 (d, J = 8.8 Hz, 2H).
ESI-MS (+ve mode): m/z 392.0 (Tt1?H)+. IR (KBr): 2190 cm21
(CMC); 1607 and 1443 cm21 (thiophene rings). Anal. Calcd. for
C22H17NS3: C, 67.48; H, 4.38; N, 3.58. Found: C, 67.18; H, 4.22;
N, 3.50. TGA: 291 uC (2% decomposition), 382 uC (5%
decomposition).
Results and discussion
Synthesis and characterization
This study of the structure-TPA relationship compared
different donor and acceptor functionalities containing a
conjugated backbone. Two different systems, D–p–A and D–p–
A–p–D, were designed and synthesized using a Sonogashira
coupling reaction. In the systems, dimethylamino and
oligothiophene functionalities were used as the donor and
acceptor respectively, whereas phenylacetylene functionality
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Linear photophysical properties of Bt0–Bt2 and Tt0–Tt2
The thiophenes with donor and acceptor functionalities
displayed intriguing photophysical properties in solutions.
Table 3 summarizes the spectroscopic and spectrofluorometric
properties of Bt0–Bt2 and Tt0–Tt2 in DMSO (50 mM). The
absorption maximum, lmax, and molar absorptivity (e, M21
cm21) of Bt0 were 305 nm (15 200 M21 cm21), whereas those of
Tt0 were 359 (20 600 M21 cm21). All of the absorption of Bt0
and Tt0 was tentatively assigned as p A p* transitions. Upon
the covalent linking of one donor, dimethylamino, via the
conjugated phenylacetylene linkage, to the oligothiophene
backbone, a D–p–A system was achieved. The lmax of Bt1 and
Tt1 were red-shifted compared to that of the corresponding Bt0
and Tt0. The lmax of Bt1 shifted to 382 nm (35 100 M21 cm21),
whereas that of Tt1 shifted to 411 nm (56 700 M21 cm21).
Further incorporation of the two donors to the oligothiophene
backbone resulted in Bt2 and Tt2 becoming a more pro-
nounced ICT system, D–p–A–p–D; this lead to a further red-
shift of their absorption bands. In the case of Bt2, the lmax 422
nm (30 900 M21 cm21), whereas that of Tt2 shifted to 442 nm
(13 800 M21 cm21). All of the absorption of Bt1, Bt2, Tt1, and
Tt2 was tentatively assigned as intramolecular charge transfer
(ICT) transitions.21 The UV-vis absorption properties of the
Bt0–Bt2 and Tt0–Tt2 are summarized and shown in Fig. 2a and
3a, respectively.
Fig. 1 Perspective view of Bt2 with numbering scheme adopted. Hydrogen
atoms were omitted for clarity. (S in yellow; C in grey; N in blue).
acted as the p conjugated link. Bt1 and Tt1 were classified as
the D–p–A system, and Bt2 and Tt2 were classified as the D–p–
A–p–D system. Bt0 and Tt0 were purchased and used solely as
controls (Scheme 1). All of the products, Bt1, Bt2, Tt1, and Tt2,
were isolated as air-stable solids with a reasonable yield, and
they were thoroughly characterized by H-NMR, IR spectro-
scopy, microanalysis, and thermogravimetric analysis.
Crystal structure determination
Single crystals of Bt2 (orange plates) were grown from the slow
evaporation of a concentrated complex of petroleum ether/
CH2Cl2 mixture at a 10 : 1 ratio, in an open atmosphere. A
perspective view of the crystal structure of Bt2, with atom
labeling, is shown in Fig. 1. The bithiophene unit in Bt2
showed a planar orientation. Within the bithiophene unit, the
C1–C2 and C3–C4 bond-lengths [1.368(7) and 1.370(7) Å,
respectively] were significantly shorter than those of C2–C3
[1.397(7) Å]. This was consistent with the normal bonding
picture for bithiophene.19 The similar disparity found in the S–
C bond lengths [S1–C1, 1.728(5) Å and S1–C2, 1.732(5) Å] was
also consistent with the literature.20 The –CMC– bridges were
slightly bent from linearity with bond angles of 175.2u at C(7)–
CMC and 176.6u at CMC–C(4). There were no significant
hydrogen-bonding interactions in the crystal structure, which
was expected to be stabilized by van der Waals interactions.
Crystal data and other X-ray crystallographically experimental
details are summarized in Table 1. Selected bond distances
and angles are summarized in Table 2.
Upon excitation at 318 nm, Bt0 displayed a broad emission
band with lmax at 369 nm in DMSO at room temperature.
Upon excitation at 350 nm in the same condition, Tt0
displayed an emission band with lmax at 411 nm. As
mentioned, because both the D–p–A and D–p–A–p–D systems
induced an effective ICT, pronounced red-shifts in their
emission bands were expected. Upon excitation at 350 nm in
DMSO at room temperature, Bt1, Tt1, Bt2, and Tt2 displayed
emission bands with lmax at 511, 552, 549, and 563 nm,
respectively. The emissions of Bt1, Tt1, Bt2, and Tt2 were
tentatively assigned to mixed triplet intramolecular charge
transfer (3ICT) and intraligand (3IL) excited states, whereas
3
those of Bt0 and Tt0 were assigned as IL excited states solely.
The quantum yield of the emission bands of all structures in
DMSO at room temperature were measured using an integrat-
ing sphere with their corresponding linear excitation. The
luminescent properties of the Bt0–Bt2 and Tt0–Tt2 are
summarized in Fig. 2b and 3b, respectively.
Spectrofluorometric studies of the oligothiophenes at
various pH values revealed that only the amino-pending
oligothiophenes, Bt1, Tt1, Bt2, and Tt2, were pH-dependent.
The relationship between their absorbance and pH is plotted
Table 2 Selected bond lengths (Å) and angles (deg) from Bt2
Selected Bond Lengths (Å)
in S.Fig. 1, ESI. The pK of Bt and Tt were found to be 1.7
3
a
1
1
C1–C2
C2–C3
S1–C4
C6–C7
N1–C13
1.368(7)
1.397(7)
1.732(5)
1.426(7)
1.449(7)
C3–C4
S1–C1
C5–C6
N1–C10
N1–C14
1.370(7)
1.728(5)
1.208(7)
1.370(7)
1.426(7)
and 1.8, respectively, whereas those in Bt2 and Tt2 were found
to be 2.0 and 2.1, respectively. The values are coherent with the
normal acidity of substituted anilines.22
Two-photon induced photophysical properties of Bt1, Bt2, Tt1,
and Tt2
Selected Bond angles (deg)
C(5)–C(6)–C(7)
C(1)–S(1)–C(4)
C(10)–N(1)–C(14)
175.2(5)
92.2(2)
120.8(5)
C(4)–C(5)–C(6)
C(10)–N(1)–C(13)
C(13)–N(1)–C(14)
176.6(5)
120.4(4)
118.1(4)
The two-photon absorption cross-sections (TPA) of the studied
compounds were determined using the up-converted fluores-
cence emission technique.23 The emission maxima, band
shape, and band width of Bt1, Bt2, Tt1, and Tt2 upon linear
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Table 3 Absorption properties, luminescent properties and two-photon absorption cross sections (s2) of Bt0–Bt2 and Tt0–Tt2 in DMSO (50 mM)
Absorption lmax [nm] and (e in M21 cm21
)
Emissiona lem [nm] and (w)b
pKa
s2 [GM]d
c
Bt0
Bt1
Bt2
Tt0
Tt1
Tt2
305 (15 200)
369 (0.04)
511 (1.20)
549 (0.67)
411 (0.24)
552 (0.62)
563 (0.34)
—
0
308 (17 300), 382 (35 100)
299 (19 800), 422 (30 900)
359 (20 600)
290 (24 700), 411 (56 700)
300 (8400), 442 (13 800)
1.7
2.0
—
1.8
2.1
432
563
0
81
380
a
b
Emission quantum yields of Bt0: lem = 320–620 nm, lex = 318 nm; while the others: lem = 400–700 nm, lex = 350 nm. w were measured
c
d
using an aerated aqueous solution of [Ru(bpy)3]Cl2 as the standard solution. pKa were measured in buffer : DMSO v/v 2 : 1. TPA cross-
section (GM = 10250 cm4 s photon21 molecule21, lex = 700 nm).
excitation were reproduced by near infra-red (NIR) excitation
through two-photon absorption at 700 nm by a femtosecond
laser. The two-photon induced emission spectra of Bt1 and Bt2
and those of Tt1 and Tt2 are shown in Fig. 4. The number of
photons absorbed to induce the emission under the excitation
at 700 nm was studied in the power-dependence experiments
(inset of Fig. 4). In every case, the output intensity of the two-
photon excited fluorescence was linearly dependent on the
square of the input incident laser intensity, thus confirming
their two-photon absorptions.
To explore the possibility of using the studied compounds
as tools to visibly image an in vitro specimen, the TPA cross-
sections of the compounds in solution were evaluated.24 The
TPA cross-sections of Bt1, Bt2, Tt1, and Tt2 at 700 nm were
measured and compared to that of Rhodamine 6 G (The TPA
cross-section value is 38.67 GM)5,25 using eqn (i).26 The TPA
cross-sections of Bt1, Bt2, Tt1, and Tt2 are compiled in Table 3.
All of the compounds were found to have significantly greater
Fig. 2 (a) Absorption and (b) normalized luminescence spectra of Bt0, Bt1 and
Bt2 in DMSO (5.0 6 1025 M) at room temperature. Bt0: lex = 318 nm; while Bt1
and Bt2: lex = 350 nm.
Fig. 3 (a) Absorption and (b) normalized luminescence spectra of Tt0, Tt1, and
Tt2 in DMSO (5.0 6 1025 M) at room temperature. lex = 350 nm.
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Fig. 4 Two-photon induced emission spectra of (upper) Bt1 and Bt2 and (lower)
Tt1 and Tt2 in DMSO. The inset shows the power-dependence experiments with
Bt1, Bt2, Tt1, and Tt2 [1 6 1024 M; lex = 700 nm].
TPA values (ranging from 81 to 563 GM) than suggested by
Furuta et al. for bio-imaging (.0.1 GM) in live specimens;27
moreover, the values obtained were also highly comparable
with those of most organic compounds used for in vitro
imaging (10–100 GM).28
The bithiophene chromophores, Bt1 (432 GM) and Bt2 (563
GM), exhibited a larger s2 value than their terthiophene
counterparts, Tt1 (81 GM) and Tt2 (380 GM). This can be
explained by the higher fluorescence quantum yields of the
bithiophene compounds relative to their corresponding
terthiophene compounds. Furthermore, the symmetrical
quadrupolar structures (D–p–A–p–D) chromophores, Bt2 and
Tt2, exhibited a larger TPA cross-section (s2) value than their
dipolar analogues (D–p–A), Bt1 and Tt1. This may be due to the
higher coplanarity of the molecular structures in quadrupolar
structures. In addition, the cancellation of the D–A dipoles in
the structures of Bt1 and Tt1 tends to induce charge transfer
quenching of two photon fluorescence and hence lower their
TPA cross-section.29 These results concur with previous
studies.30
Fig. 5 Normalized luminescence spectra of (a) Bt1, (b) Bt2, (c) Tt1, and (d) Tt2 in
solvents of differing polarity at room temperature (5.0 6 1025 M). lex = 350 nm.
Solvatofluorescence of Bt1, Bt2, Tt1, and Tt2
The luminescence spectra of Bt1, Bt2, Tt1, and Tt2 in solvents
of differing polarity at 293 K are illustrated in Fig. 5. Their
emission spectra showed strong solvatofluorescence. A large
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Fig. 6 In vitro staining microscopy with (a) Bt1 (50 mM; green, two-photon
excitation lex = 700 nm, green emission lem = 500–550 nm); (b) brightfield
image; (c) Hoechst 33342 (50 mM; specific dye for nucleus; single-photon
excitation lex = 405 nm, blue emission lem = 435–485 nm) and (d) their
overlapping images in HeLa cells.
Fig. 7 In vitro staining microscopy with (a) Tt1 (50 mM; two-photon excitation
lex = 700 nm, green emission lem = 500–550 nm); (b) brightfield image; (c)
LysoTracker1 Red (50 mM; specific dye for lysosome; single-photon excitation
lex = 543 nm, red emission lem = 580–620 nm) and (d) their overlapping images
in HeLa cells.
red shift was observed when the system was solvated from
non-polar to polar solvent (i.e., from benzene, THF, or
acetonitrile to DMSO). The bathochromic shift of the ICT
emission band with increasing solvent polarity occurred
because the polarity of the excited state was larger than that
of the ground state of the donor–acceptor system.31 In spite of
this, a larger bathochromic shift of the emission band was
observed in the D–p–A systems, Bt1 and Tt1 (a 76 nm-red shift
was found in between the lem of benzene and that of DMSO)
than in that of the D–p–A–p–D systems, Bt2 and Tt2, (a 62 nm-
red shift was found in between the lem of benzene and that of
DMSO). We attribute this to the different geometries in the
ground (S0) and excited electronic (S1) states of the systems.
confocal luminescence, counterstains (Hoechst 33342 for
nucleus) and brightfield images further demonstrated that
the luminescence was evident in the cytoplasm over the
nucleus and membrane (Fig. 6b–6d). Tt1 revealed granular
appearance located in the lysosome of the HeLa cells (Fig. 7a).
Overlays of the confocal, LysoTracker1 Red counterstains
(specific dye for lysosome) and brightfield images showed the
lysosome of a typical HeLa cell have been co-stained by the
fluorescent dye and Tt1 (Fig. 7b–7d).
For Bt2 and Tt2, no emissions were observed in the cells
from the tissue culture medium. This may be due to the results
of the difference in their cell permeability. Since in the pH 7.4
cell culture medium, Bt1, Bt2, Tt1, and Tt2 are neutrally
charged molecules (pKa of Bt1, Bt2, Tt1, and Tt2 = 1.7, 2.0, 1.8
and 2.1 respectively), the more lipophilic Bt1 and Tt1 could be
a favor for them to penetrate, via the cell membrane, in the
cell; while the more hydrophilic Bt2 and Tt2 could be a hinder
for them to enter the cell. Toxicity tests were performed on the
Bt1 and Tt1 compounds, and no evidence of cytotoxicity on the
HeLa cells was found upon exposure to Bt1 and Tt1 (50 mM) for
24 h (Fig. 8). The MTT assays on cells exposed to 50 mM of Bt1
and Tt1 for prolonged periods of time revealed no significant
decrease in the number of viable cells.
In vitro imaging and cytotoxicity studies
Given the high TPA cross-sections of Bt1, Bt2, Tt1, and Tt2 and
their positively charged properties in the physiological condi-
tion, we explored the application of the studied compounds as
organic molecular probes for multiphoton confocal imaging.
The cellular internalization characteristics of Bt1, Bt2, Tt1, and
Tt2 and their affinity for cellular organelles were tested on
living mammalian cells, specifically, human cervical carci-
noma cells (HeLa) (Fig. 6). The most interesting finding was
our molecular probes’ NIR-excited induced visible photophy-
sical properties. Bt1 and Tt1 produced strong green emissions
in the in vitro imaging under TPA excitation at 700 nm (y5
mW).
Conclusions
Fig. 6 and 7 show the intracellular localization of Bt1 and
Tt1. Bt1 showed a diffused staining of the whole cytoplasm of
the HeLa cells. The nucleus displayed no emission, which is
indicative of negligible nuclear uptake (Fig. 6a). Overlays of the
We developed two new classes of organic TPA chromophores, D–
p–A (Bt1 and Tt1) and D–p–A–p–D (Bt2 and Tt2), based on 59-[4-
ethynyl-N,N-dimethylaniline]-2,29-bithiophene/2,29:59,299-terthio-
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This work was financially supported by an ECS grant (HKIEd
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