Synthesis, crystal structure, optical properties, DNA-binding and cell imaging of an organic chromophore

Article abstract of DOI:10.1016/j.dyepig.2011.06.021

An organic chromophore (4-[N, N- bis(2′-chloroethyl)anilino] ethenyl N-methyl pyridinium-N- methyl iodide) L was synthesized and fully characterized by elemental analysis, IR, ¹HNMR spectra and single crystal X-ray diffraction analysis. One and two-photon fluorescence spectra for it were studied. Interestingly, L exhibited obvious two-photon absorption in the range from 700-850 nm in DMSO solution. The interactions between the chromophore and calf thymus DNA were investigated by absorption, fluorescence spectra and viscosity experiments. The results showed that the chromophore could interact with DNA in the mode of intercalation and be effectively used as fluorescent staining dye for cell imaging applications. Furthermore, the cell viability data for MCF-7 (Human breast carcinoma) cells indicated that the low-micromolar concentrations of the L are essentially non-toxic and can be applied in brighter two-photon fluorescent (TPF) bioimaging.

Full text of DOI:10.1016/j.dyepig.2011.06.021

Dyes and Pigments 92 (2011) 689e695
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis, crystal structure, optical properties, DNA-binding and cell imaging
of an organic chromophore
,
Shuangsheng Zhou a b, Qiong Zhang a, Xiaohe Tian c, Guiju Hu a, Fuying Hao a,
,d,e,
Jieying Wu a, Yupeng Tian a
*
a Department of Chemistry, Key Laboratory of Functional Inorganic Materials Chemistry of Anhui Province, Anhui University, Hefei 230039, PR China
b Department of Pharmacy, Anhui College of Traditional Chinese Medicine, Hefei 230031, PR China
c Department of Biomedical Science, University of Sheffield, Sheffield, UK
d State Key Laboratory of Crystal Materials, Shandong University, Jinan 502100, PR China
e State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
An organic chromophore (4-[N, N- bis(20-chloroethyl)anilino] ethenyl N-methyl pyridinium-N- methyl
iodide) L was synthesized and fully characterized by elemental analysis, IR, 1HNMR spectra and single
crystal X-ray diffraction analysis. One and two-photon fluorescence spectra for it were studied. Inter-
estingly, L exhibited obvious two-photon absorption in the range from 700e850 nm in DMSO solution.
The interactions between the chromophore and calf thymus DNA were investigated by absorption,
fluorescence spectra and viscosity experiments. The results showed that the chromophore could interact
with DNA in the mode of intercalation and be effectively used as fluorescent staining dye for cell imaging
applications. Furthermore, the cell viability data for MCF-7 (Human breast carcinoma) cells indicated that
the low-micromolar concentrations of the L are essentially non-toxic and can be applied in brighter two-
photon fluorescent (TPF) bioimaging.
Article history:
Received 19 February 2011
Received in revised form
14 June 2011
Accepted 15 June 2011
Available online 1 July 2011
Keywords:
Organic chromophore
Crystal structure
Optical properties
DNA-binding
Ó 2011 Elsevier Ltd. All rights reserved.
Cytotoxicity
Cell imaging
1. Introduction
Therefore, the challenge before us is how to design and
synthesize the chromophores bearing strong two-photon absorp-
Organic chromophores with a large two-photon absorption
(TPA) cross section have become of particular interest to chemists
and material scientists in decades years due to their potential
applications in upconverted lasing [1,2], three-dimensional fluo-
rescence microscopy [3,4], biosensing technology [5,6], optical data
storage [7,8], and 3D microfabrication [9,10]. Among these appli-
cations, bioimaging as fluorescence probes in living cells has
recently attracted a great deal of attention [11e13]. To detect cell
imaging in a living system, various fluorescent probes including
organic dyes, inorganic nanoparticles, and lanthanide coordination
complexes have been developed [11,14,15]. However, recent
investigations reveal the most probes with two-photon fluores-
cence easily exhibit quenching in high polar solvents and TPA
maximum not in the near IR range.
tion in the near-infrared (NIR) region (bio-safety window) and high
polar solvent. For this purpose, the crystal structure, one and two-
photon absorption properties, DNA-binding, and cell imaging of
a new chromophore L which was based on our previous work
[16e18] were investigated systematically in this paper.
2. Experimental
2.1. General
The 1H spectra were performed on Bruker 400 spectrometer with
TMS as the internal standard, coupling constants J are given in Hertz.
Elemental analysis was performed on PerkineElmer 240 instrument.
Single-crystal X-ray diffraction measurements were carried out on
a Bruker Smart 1000 CCD diffractometer equipped with a graphite
crystal monochromator situated in the incident beam for data
collection at room temperature. The determination of unit cell
* Corresponding author. Department of Chemistry, Key Laboratory of Functional
Inorganic Materials Chemistry of Anhui Province, Anhui University, Hefei 230039,
PR China. Tel.: þ86 551 5108151; fax: þ86 551 5107342.
parameters and data collections were performed with MoKa
ꢀ
E-mail address: yptian@ahu.edu.cn (Y. Tian).
radiation (
l
¼ 0.71073 A). Unit cell dimensions were obtained with
0143-7208/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2011.06.021

690
S. Zhou et al. / Dyes and Pigments 92 (2011) 689e695
Scheme 1. The synthetic route of the chromophore L.
least-squares refinements, and all structures were solved by direct
methods using SHELXL-97 [19]. All non-hydrogen atoms were
refined anisotropically. The hydrogen atoms were added theoreti-
cally and not refined. The final refinement was performed by full-
matrix least-squares methods with anisotropic thermal parameters
for non-hydrogen atoms on F2. Fluorescence emission spectra were
measured on PerkineElmer Lambda-850 spectrophotometer and
PerkineElmer Ls55 Spectrofluorophotometer, respectively. TriseHCl
buffer (5 mM tris(hydroxymethyl) aminomethane (Tris) hydrochlo-
ride, 50 mM NaCl, pH 7.2) was used for absorption titration, emission
titration and viscosity experiments. Solutions of DNA(Sigma.Co) in
buffer gave a ratio of UV absorbance at 260 and 280 nm of 1.8e1.9:1,
indicating that the DNA was sufficiently free of protein [20]. The
concentration of DNA was determined spectrophotometrically
assuming a molar absorption is 6600 Mꢀ1 cmꢀ1 (260 nm) [21].
mixture was stirred and refluxed for ca. 16 h at 110 ꢁC, cooled to
room temperature, and then the remains was poured into
slowly 500 cm3 ice water. The NaOH solution was added to
adjust the pH of the solution under vigorous stirring. After the
pH in the solution had reached 7.0, the beige suspension was
filtered and fully washed with distilled water three times. The
product was dried and recrystallized from EtOH. Beige needle
crystals (2) were obtained in 88% yield. Mp: 87.6e89.2 ꢁC, 1H
NMR(CDCl3):
d 9.67(s, 1H, CHO), 6.74e7.38 (m, 4H, Ph), 3.84 (t,
J ¼ 6.8 Hz, 4H, 2 ꢂ NCH2), 3.68(t, J ¼ 6.7 Hz, 4H, 2 ꢂ CH2Cl). Anal.
Calcd. for C11H13Cl2NO: C, 53.66; H, 5.32; N, 5.69; found C,
53.42; H, 5.03; N, 5.45.
2.2.2. Synthesis of 4-[N, N- bis(20-chloroethyl)anilino]ethenyl
N-methyl pyridinium iodide (L)
The chromophore 2(4.9 g, 20 mmol) and 1,4-dimethyl pyr-
idinium iodide(4.7 g, 20 mmol) were mixed in 60 mL of absolute
ethanol in the presence of 4 drops of piperidine as a catalyst. The
resulting mixture was heated to reflux overnight. After cooling, the
mixture was filtered to collect the solid followed by washing with
ethanol. The crude product was recrystallized twice from ethanol
and then dried in vacuo over P2O5 to give red crystal (L) in 52.3%
2.2. Preparation
All chemicals were available commercially and the solvents
were purified as conventional methods before using. N-phenyl-
diethanolamine (1) was synthesized by the published procedures
[18]. The target chromophore L was synthesized by following
reactions (shown in Scheme 1).
yield. Mp: 143e145 ꢁC; 1H NMR (DMSO-d6):
d3.66 (t, J ¼ 6.7 Hz, 4H,
2 ꢂ CH2Cl), 3.86 (t, J ¼ 6.8 Hz, 4H, 2 ꢂ NCH2), 4.37 (s, 3H, CH3), 6.43
(d, J ¼ 2.1 Hz, 1H), 6.59 (d, J ¼ 2.1 Hz, 1H), 6.74e7.38 (m, 8H, 4 ꢂ PhH
and 4pyridin eH); Anal. Calcd. for C18H21Cl2N2I: C, 46.66; H, 4.57; N,
6.05; found C, 46.52; H, 4.76; N, 6.21.
2.2.1. Synthesis of N, N-bis(2-chloroethyl) phenylimino (2)
POCl3 (29.2 g, 0.18 mol) was slowly dropped into a dry
100 cm3 flask which contained 30 g DMF and was cooled with
an ice water ahead. The mixture was stirred vigorously while
N-phenyldiethanolamine (1) was added dropwise, the reaction
2.3. DNA-binding experiments
Table 1
2.3.1. Absorption measurement
Crystal data collection and structure refinement of L.
The absorptions of L were performed by using a fixed compound
concentration to which increments of the DNA stock solution were
added. The solutions were allowed to incubate for 10 min before
the absorption spectra were recorded. The operation processes
were repeated until there was no change for the spectra at least
three times, indicating binding saturation had been achieved.
CCDC No
744865
C36 H44 Cl4 I2 N4 O1
944.35
Monoclinic
C2/c
26.091(5)
11.736(5)
17.442(5)
128.708(5)
4168(2)
4
Empirical formula
Formula weight
Crystal system
Space group
ꢀ
a, b, c (A)
2.3.2. Fluorescence measurement
In the fluorescence quenching experiments, DNA was pretreated
with ethidium bromide (EB) for 30 min. The chromophore L was
b
(ꢁ)
3
ꢀ
V (A )
Z
Dcalc (g/cm3)
F(000)
1.505
1880
Table 2
ꢁ
ꢀ
Crystal size (mm)
Temperature (K)
0.30 ꢂ 0.20 ꢂ 0.20
Selected bond lengths (A) and angles ( ) for of 3.
298(2)
q
range (ꢁ)
2.00e24.99
16691
3652
227
0.0607
N2eC12
N2eC15
N2eC17
C4eC7
C7eC8
C8eC9
1.381(9)
1.490(11)
1.465(11)
1.452(11)
1.316(12)
1.471(11)
121.1(6)
121.3(6)
117.4(7)
Total no. of data
No. of unique data
No. of para refined
R1
wR2
Gof
0.2099
1.074
C
C
C
12eN2eC15
12eN2eC17
15eN2eC17
R(Fo) ¼ SkFoj ꢀ jFck/SjFoj, Rw(Fo) ¼ (SwkFoj ꢀ jFck /SjFoj )1/2, w ¼ [
s
2(Fo) þ
2
2
(0.002Fo)2]ꢀ1
.

S. Zhou et al. / Dyes and Pigments 92 (2011) 689e695
691
Fig. 1. Molecular structure with atom numbering of
L
showing 50% probability
displacement.
Fig. 3. Sigle-photon fluorescence spectra of
L
in different solvents with
1 ꢂ10ꢀ5 mol Lꢀ1
.
then added to the mixture solution and the effect on the emission
intensity was measured by keeping [DNA]/[EB] ¼ 20 ([DNA] ¼
and maintained at 37 ꢁC in an atmosphere of 5% CO2 and 95% air for
incubation 2 h. The cells were then washed with PBS (3 ꢂ 3 ml per
well) and 3 ml of PBS was added to each well. The cells were
imaged using confocal laser scanning microscopy and water
immersion lenses. Excitation energy of 760e800 nm was used and
the fluorescence emission measured from 550 nme650 nm. Co-
100
mM, [EB] ¼ 5
mM). Excited at 370 nm, the emission spectra were
observed in the range from 450 to 700 nm.
2.3.3. Determination of viscosity
Viscosity measurements were carried out using an Ubbelohde
viscometer, immersed in a water bath maintained at 30 ꢃ 0.2 ꢁC.
DNA was previously solicited for 30 min, followed by nitrogen
bubbling for 5 min. The DNA concentration was kept constant while
that of L increased. The flow time through the solution were
staining was performed using 5
in PBS.
mM SYTO9, 10 mM PI for 10 min
Microscopy: MCF-7 cells were luminescently imaged on a Zeiss
LSM 510 META upright confocal laser scanning microscope using
magnification 40ꢂ and 100ꢂ water-dipping lenses for monolayer
cultures.
measured three times to get the mean values with a digital stop-
1/3
watch. The data are presented as (
h
/h0
)
versus binding ratio
([L]/[DNA]), where
h
and h0 is the viscosity of DNA in the presence
and absence of the chromophore, respectively.
2.4.2. Cytotoxicity assay
Establishment details of human breast adenocarcinoma cell line
(MCF-7) used for cytotoxicity testing in vitro of target derivatives
have been previously described [22]. Drug solutions of appropriate
concentration were added to a culture containing MCF-7 cells at
2.0 ꢂ 104 cells mLꢀ1 of medium and the drug exposure was pro-
tracted for 24 h. All assays were performed in triplicate, as previ-
ously described [22].
2.4. Cell image and cytotoxicity assay
2.4.1. Cell image
Cells were seeded in 6 well plates at a density of 2 ꢂ 104 cells per
well and grown for 96 h. For live cell imaging cell cultures were
incubated with L (10% PBS: 90% cell media) at concentrations 50 mM
3. Results and discussion
3.1. Structure features
The single crystals of L, suitable for the X-ray diffraction analysis,
were obtained by slow evaporation of acetonitrile at room
temperature several days later (Table 1). The selected bond
Table 3
Single- and two-photon-related photophysical properties of chromophore 3 in
several different polar solvents.
Solvents
lmax/nma
lmax/nmb
lmax/nmc
Vd
Dn/cmꢀ1e
DMF
DMSO
H2O
426
425
427
521
529
544
578
586
592
0.17
0.13
0.09
4280
4625
5036
a
Peak position of the longest absorption band.
Peak position of SPEF, exited at the absorption maximum.
Peak position of the two photon absorption band.
b
c
d
e
Quantum yields determined by using coumarin as standard.
Fig. 2. Packing diagram of L.
Stokes shift in cmꢀ1
.

692
S. Zhou et al. / Dyes and Pigments 92 (2011) 689e695
Fig. 6. Absorption spectra of L in TriseHCl buffer (NaCl, PH ¼ 7.2) upon addition of
DNA. [compound] ¼ 2 ꢂ 10ꢀ5 M, [DNA] ¼ (0, 1, 2, 3) ꢂ 10ꢀ5 M. Arrows show the
Fig. 4. Two-photon fluorescence spectra of the chromophore L in different solvents at
a concentration of 1.0 ꢂ 10ꢀ3 mol Lꢀ1
.
absorbance changing upon adding DNA concentrations.
state than the ground state in a polar solvent, suggesting that
a significant charge redistribution takes place upon excitation with
consistent of ICT (Intramolecular Charge Transfer) character [16,24].
In order to further demonstrate the influence of solvent on
fluorescence, Table 3 lists the Stokes’ shift of L in three polar
solvents. The Stokes’ shift is defined as the loss of energy between
absorption and reemission of light, which is a result of several
dynamic processes. The Lippert equation is the most widely used
equation to describe the effects of the physical properties of the
solvent on the emission spectra of fluorophores [25].
distances and angles were listed in Table 2. The structure of L,
together with the atom numbering scheme was shown in Fig. 1. For
the molecular structure of L, the least-square plane calculations
show that the dihedral angle between the two benzene rings is 8.5,
indicating they are nearly coplanar. The sum of the three CeNeC
angles taking nitrogen atom as center (C12eN2eC15, 121.1(6)ꢁ;
C
12eN2eC17, 121.3(6)ꢁ; C15eN2eC17, 117.4(7)ꢁ) is 359.8ꢁ, therefore
the trigonal NC3 is practically coplanar. The bond lengths of N2eC15
(1.490(11)) and N2eC17 (1.465(11)) are longer than that of N2eC12
(1.381(9)), confirming the two electrons on N2 are partial to the
adjacent phenyl ring. It can be seen from Table 2 that all the bond
lengths of CeC are located between the normal C]C double bond
ꢀ
ꢁ
ꢀ
ꢁ
Dn
¼
nabs
ꢀ
nem
¼
2=cha3
D
f
me
ꢀ
mg 2þconst
ꢀ
ꢀ
(1.32 A) and CeC single bond (1.53 A), which show that there is
a highly - electron delocalized system in the chromophore
molecule L, which is the necessary condition for it bearing a large
TPA cross-section [23].
Fig. 2 shows that the adjacent chromophore molecules L in the
crystal are stacked through strong interactions along c axis
where h is Planck’s constant, c is the speed of light, and a is the
radius of the cavity in which the fluorophore resides. The wave-
numbers of the absorption and emission are nabs and nem (in cmꢀ1).
The Stokes’ shifts are approximately proportional to the polarity of
the solvent (Table 3).
p
s
pep
stacked in pairs through CeH/I interactions.
3.2. Single and two-photon excited fluorescence spectra of L
The sigle-photon fluorescence spectra of L in five different polar
solvents, including aqueous solution (as shown in Fig. 3), and exhibit
red shift with increasing of the solvent polarity. The sol-
vatochromism is indicative of a larger stabilization of the excited
Fig. 7. Fluorescence emission spectra of EB bound to DNA in the absence and presence
of different concentrations of compound L in TriseHCl buffer (NaCl, PH ¼ 7.2). [EB] ¼
5
m
M; [DNA] ¼ 100
m
M; [compound] ¼ 0
mM (a), 50 mM (b), 100 mM (c), 150 mM (d).
Inset: plots of F0/F versus [compound] for the titration of compound 3 to HS-DNA-EB
Fig. 5. TPA cross section of L in DMF and DMSO vs excitation wavelengths.
system.

S. Zhou et al. / Dyes and Pigments 92 (2011) 689e695
693
experimental results show the optimal excitation wavelengths of L
locate at 740 nm in DMSO and 760 nm in DMF (Fig. 5), the highest
cross section values of
s
are 11.4 and 14.8 GM (1GM ¼ 10ꢀ50 cm4 s
photonꢀ1), respectively. The wavelengths are suitable for bio-
imaging in the near IR range.
3.4. DNA-binding studies
3.4.1. Absorbance titration
The absorption spectra of L in the absence and presence of DNA
are given in Fig. 6. As the concentration of DNA increased, all the
absorption bands of L show slightly hypochromism. Such spectral
characters suggest that L interacts with DNA most likely through
a mode involving a stacking interaction between the dye and the
base pairs of DNA [27,28].
3.4.2. Fluorescence titration
The emission spectra of EB bound to DNA in the absence and the
presence of L are given in Fig. 7. The addition of the chromophore L
to DNA pretreated with EB causes obvious decrease in the emission
intensity, indicating that the DNA bound EB fluorophore is partially
replaced by the target molecule L. The result suggests that L
presumably intercalate into DNA. According to the classical
SterneVolmer equation [29]:
Fig. 8. Effect of increasing concentration of chromophore L on the relative viscosities
of DNA at 30.0 ꢃ 0.2 ꢁC. [L]/[DNA] ¼ 0.0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4.
The two-photon excited fluorescence spectra of L are shown in
Fig. 4 with a solution concentration of 1.0 ꢂ 10ꢀ3 mol Lꢀ1 using the
optimal excitation wavelength. From Fig. 4, it can be seen that L
exhibit strong two-photon fluorescence and there is a red shift for
the peak position of TPA fluorescence compared with that of single-
photon absorption fluorescence due to the reabsorption effect at
high concentration [24].
F0=F ¼ 1 þ K½Qꢄ
where F0 and F are of fluorescence intensities in the absence and
presence of the compound, respectively. K is a linear SterneVolmer
quenching constant. [Q] is the concentration of the quencher
compound. The fluorescence quenching curve (inset in Fig. 7)
illustrates that the fluorescence quenching of DNA e bound EB
3.3. The TPA cross-section
The two-photon absorption (TPA) cross-section
s was measured
system mixing with
L is good agreement with the linear
by comparing the TPEF intensity of the sample with that of
a reference compound by the following equation [26]:
SterneVolmer equation. In the plot of F0/F versus [L], K is given by
the ratio of the slope to intercept. The values of K for L is 3.24 ꢂ103
(R ¼ 0.99629), which also supports the intercalation of L to the
ctDNA bases (K ꢅ 1.0 ꢂ 103) [27,30].
Z
ꢂ
ꢃ ꢂ
ꢃ ꢂ
ꢃ
Fs
Fr
Arðlr
Asðls
Þ
Þ
Iðlr
Iðls
Þ
Þ
ns2
nr2
Z
FS
¼
Fr
ss
¼
F
sr Frcrnr=FrFscns
3.4.3. Viscosity studies
Fig. 8 reveals that the viscosity of DNA is affected significantly by
the addition of L. The results suggest that the title chromophore
intercalates between the base pairs of DNA [28,31], in agreement
with the above spectra experimental results.
Here, V is the quantum yield, n is the refractive index, I(
relative intensity of the exciting light, A( ) is the absorbance of the
solution at the exciting wavelength , and F is the integrated area
l) is the
l
l
under the corrected emission spectrum, c is the concentration of
the solution in mol Lꢀ1. The sr value of reference was taken from the
literature [16,24]. Subscripts s and r refer to the sample and refer-
ence solutions, respectively. The experimental errors are estimated
to be ꢃ10% from sample concentrations and instruments. The
3.5. Cell image and cytotoxicity assay
To examine the live cellular image properties of the chromo-
phore L for two-photon microscopy (TPM), MCF-7 (human breast
Fig. 9. Chromophore L (red, 2 h) with Live/Dead staining by using SYTO9 (green)/Propidium Iodide(PI) and overlay image (the scale bar represents 10
references to color in this figure legend, the reader is referred to the web version of this article.).
mM) (For interpretation of the

694
S. Zhou et al. / Dyes and Pigments 92 (2011) 689e695
Table 4
Cell viability (%) of MCF-7 cells after incubation for 24 h with different concentration of the chromophore 3.a
Concentration (
Viability (%)
m
M)
5
10
20
40
80
97.14 ꢃ 1.23
88.43 ꢃ 1.52
73.56 ꢃ 1.46
58.27 ꢃ 1.64
40.06 ꢃ 1.28
a
Cell viability was quantified by the MTT assay (n ¼ 3, mean ꢃ s).
Fig. 10. (a) The bright-field image of MCF-7 cells incubated with 5 mM L. (b) Two-photon microscopy (TPM) image of the same cells with excitation at 780 nm. (c) Overlay image.
carcinoma) cells were incubated and stained with 50
m
M L by co-
near IR range in DMSO solution and can obviously interact with
DNA via intercalating interaction as well. Significantly, L exhibits
brighter two-photon fluorescent bioimaging which make it
successfully applied to a two-photon fluorescent staining dye in
live cells. Further work involving co-localization fluorescence study
and 3D cell image are in progress. These explorations for the
chromophore may provide a useful guide to the design of novel TPA
materials.
stain with two commercially available dye-SYTO9 and Propidium
Iodide (PI), which perform as cell-permeable nuclear acid dye in
live cells and non-cell-permeable DNA dye in dead cells respec-
tively for live/dead staining. After 2 h incubation, the cells was
washed by PBS (phosphate buffer solutions) three times and
directly move to two-photon microscopy without fixation. As seen
from Fig. 9, greenchannel from SYTO9 (left) and purple channel
from PI (middle) indicated almost all the cells keep healthy even
suffering long incubation time and high concentration. The red
channel clearly reveal MCF-7cells successfully uptake L, interest-
ingly, the luminescence located in cell cytosol as well as cell
nuclear. The luminescences which come from the cytosol suppose
that the chromophore permeated the phospholipids bilayers of
cellular mitochondria and bounded with mitochondrial DNA hence
fluorescence come out eventually. The cellular image result
demonstrated that L would be able to functionalize as cellular DNA
that both exist in cell nuclear and mitochondria.
Acknowledgments
This work was supported by a grant for the National Natural
Science Foundation of China (21071001, 50873001, 20875001),
Department of Education Committee of Anhui Province
(KJ2010A030, KJ2010A222), and the Foundation for Scientific
Innovation Team of Anhui Province (2006KJ007TD). The 211 Project
of Anhui University, Ministry of Education funded projects focus on
retired overseas scholar.
Cytotoxicity is a potential side effect of L that must be controlled
when dealing with living cells or tissues. We have conducted
cytotoxicity assays in MCF-7 cells before microscopy studies.
Table 4 lists the cell viability data for MCF-7 cells treated with L as
quantified by the MTT assay. These data indicated that MCF-7 cells
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show nearly w100% viability following 24 h of treatment with 5 mM
L (Table 4). Although obvious decrease of cell viable was observed
when incubated reach high concentration. These cytotoxicity tests
showed that sub- and low-micromolar concentrations of L are
essentially nontoxic over a period of 24 h and could safely be used
for further biological studies.
To further demonstrate the potential applications of L for two-
photon microscopy (TPM) imaging in living cells, MCF-7 cells
were cultured and stained with L. A bright-field image (Fig. 10a) of
each cell was taken immediately prior to the two-photon micros-
copy (TPM) imaging. The results showed that L is clearly capable of
detecting the cytoplasm section in MCF-7 cells. The record TPA
coefficient of L and its application for in vivo two-photon imaging
highlight the potential of the chromospheres (Fig. 10b, c).
4. Conclusions
An organic chromophore L has been synthesized and charac-
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