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D. Li et al. / Dyes and Pigments 120 (2015) 175e183
transitions and enhanced nonlinear optical response. In the
2.2. Cell culture and incubation
nonlinear optical (NLO) material fields, thiocyanate complexes can
been optimized for NLO properties by a polarizable
hard nitrogen and the soft sulfur coordination, low-lying
p
system, the
* orbitals
HepG2 cells were seeded in 6 well plates at a density of
2 ꢁ 105 cells per well and grown for 96 h. For live cell imaging cell
cultures were incubated with the chromophores (10% PBS: 90% cell
p
and the metal-to-ligand charge-transfer (MLCT) transition [49,50].
However, few endeavor has been devoted to investigation of the
2 PA effect of the thiocyanate complexes and different d electron
configurations of transition metal complexes [51]. For pyridyl li-
gands, highly delocalized and easily polarizable system, can bind to
a wide range of transition metal ions, and have large asymmetry in
electronic distribution caused by intramolecular donoreacceptor
charge transfer while they coordinate to metal ions [52]. In the case
of the same organic ligand and anion SCNꢀ, how to choose metal
ions to improve two-photon absorption cross section effectively is a
scientific challenge, which can make contribution to the design of
two-photon active complexes. These characters are crucial to
design organic-inorganic hybrid materials with large 2 PA cross-
media) at concentrations 40 m
M and maintained at 37 ꢂC in an at-
mosphere of 5% CO2 and 95% air for incubation times ranging for
2 h. The cells were then washed with PBS (3 ꢁ 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.
2.3. Cell image
HepG2 cells were luminescently imaged on a Zeiss LSM 710
META upright confocal laser scanning microscope using magnifi-
cation 40ꢁ and 100ꢁ water-dipping lenses for monolayer cultures.
Image data acquisition and processing was performed using Zeiss
LSM Image Browser, Zeiss LSM Image Expert and Image J.
section (s).
Based on the consideration above, we have strategically
designed and synthesized a series of novel thiocyanate complexes
with high two-photon absorbing using M(SCN)2 (M ¼ Zn(II), Cd(II),
Hg(II), Ni(II), Co(II), and Mn(II)) and pyridine ligand(L) (shown in
Fig. 1). The structureeproperty relationships of these complexes
have been systematically investigated. Considering the terminal
sulfur atoms are relevant to biological systems [53,54], we singled
out Zn(SCN)2L2 (1) for biological imaging application research.
2.4. Cytotoxicity assays in cells
To ascertain the cytotoxic effect of the compounds treatment
over a 24 h period, the 5-dimethylthiazol-2-yl-2,5-diphenyl tetra-
zolium bromide (MTT) assay was performed. HepG2 cells were
trypsinized and plated to ~70% confluence in 96-well plates 24 h
before treatment. Prior to the compounds' treatment, the DMEM
was removed and replaced with fresh DMEM, and aliquots of the
compound stock solutions (500
m
M DMSO) were added to obtain
M. The treated cells
were incubated for 24 h at 37 C and under 5% CO2. Subsequently,
the cells were treated with 5 mg/mL MTT (40 L/well) and incu-
bated for an additional 4 h (37 ꢂC, 5% CO2). Then, DMEM was
removed, the formazan crystals were dissolved in DMSO (150 L/
final concentrations of 1, 3, 5,10, 20, 40, and 80
m
2. Experiments
ꢂ
m
2.1. General procedure
m
All chemicals and solvents were dried and purified by the
standard methods. Elemental analysis was performed on a Per-
kineElmer 240C elemental analyzer. IR spectra were recorded with
a Nicolet FTIR Nexus 870 instrument in the range 4000e400 cme1
by using KBr pellets. 1H NMR spectra were performed on a Bruker
av 500 MHz Ultrashield spectrometer and are reported as parts per
well), and the absorbance at 490 nm was recorded. The cell viability
(%) was calculated according to the following equation: cell viability
% ¼ OD490(sample)/OD490(control) ꢁ 100, where OD490(sample)
represents the optical density of the wells treated with various
concentration of the compounds and OD490(control) represents
that of the wells treated with DMEM þ 10% FCS. Three independent
trials were conducted, and the averages and standard deviations
are reported. The reported percent cell survival values are relative
to untreated control cells.
million (ppm) from TMS (d). The linear absorption spectra were
measured on a SPECORD S600 spectrophotometer. The single-
photon emission fluorescence (SPEF) spectra measurements were
performed using a Hitachi F-7000 fluorescence spectrophotometer.
The two-photon emission fluorescence (TPEF) spectra were
measured at femtosecond laser pulse and Ti: sapphire system
(680e1080 nm, 80 MHz, 140 fs) as the light source. The excitation
wavelengths for the complexes are 700e900 nm.
2.5. Synthesis
The synthesis of the ligand and its complexes are shown in
Supporting information.
2.6. Single-crystal structure analysis
Single-crystal 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 parameters and
data collections were performed with Mo-Ka radiation
(l
¼ 0.71073 Å). Unit cell dimensions were obtained with least-
squares refinements, and all structures were solved by direct
methods using SHELXL-97 [55]. The non-hydrogen atoms were
located in successive difference Fourier syntheses. The final
refinement was performed by full-matrix least-squares methods
with anisotropic thermal parameters for non-hydrogen atoms on
F2. The hydrogen atoms were added theoretically and riding on the
concerned atoms.
Crystallographic crystal data for complexes 1e6 are shown in
Table S1. Selected bond lengths and bond angles are listed in
Fig. 1. Synthesis route of ligand.