A two-photon fluorescent probe for viscosity imaging in vivo

Article abstract of DOI:10.1039/C7TB00136C

A new two-photon fluorescent probe (MCN) for viscosity imaging was developed based on a 6-substituted quinoline framework. MCN showed an excellent “off-on” fluorescence response (ca. 90-fold enhancement) with viscosity increasing in the glycerol-water viscosity system. MCN showed great sensitivity to viscosity (R² = 0.98, x = 0.65), which gave rise to cell imaging for micro-viscosity or real-time cell imaging during apoptosis with low cytotoxicity under two-photon excitation (λ_ex = 800 nm). Fluorescence lifetime imaging (FLIM) of living HeLa cells stained with MCN revealed that the intracellular average viscosity value was 73.45 ± 21.55 cP in cytosol. Imaging in living tissue slices indicated that MCN can work in deep tissue (～130 μM) under two-photon excitation. Moreover, MCN also showed the capacity for in vivo imaging viscosity in zebrafish with obvious fluorescence emission.

Full text of DOI:10.1039/C7TB00136C

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ISSN 2050-750X
PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
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A Two-Photon Fluorescent Probe for Viscosity Imaging in vivo
Peng Ning,a Pengyu Dong,b Qian Geng,c Lei Bai,d Yaqi Ding,d Xiaohe Tian,c Rong Shao,b* Lin Lid and
Xiangming Menga*
Received 00th January 20xx,
Accepted 00th January 20xx
A new two-photon fluorescent probe (MCN) for viscosity imaging was developed based on 6-substituted quinoline
framework. MCN showed excellent “off-on” fluorescence response (ca. 90-fold enhancement) with viscosity increasing in
glycerol-water viscosity system. MCN showed great sensitivity to viscosity (R2 = 0.98, x = 0.65), which gave rise to cell
imaging for micro-viscosity or real-time cell imaging during apoptosis with low cytotoxicity under two-photon excitation
(λex = 800 nm). Fluorescence lifetime imaging (FLIM) of living HeLa cells stained with MCN revealed that the intracellular
average viscosity value was 73.45 ± 21.55 cP in cytosol. Imaging in living tissue slices indicated that MCN can work in deep
tissue (~130 μm) under two-photon excitation. Moreover, MCN also showed the capacity for in vivo imaging viscosity in
zebrafish with obvious fluorescence emission.
DOI: 10.1039/x0xx00000x
www.rsc.org/
fluorescence microscopy, such as higher spatial resolution,
deeper penetration depth (more than 100 μm) and lower
phototoxicity.16-22 Therefore, we are interested in developing
Introduction
Intracellular viscosity plays a critical role during cellular
diffusion-controlled processes, including the transportation
and interaction of bio-molecules.1, 2 Abnormal fluctuations in
intracellular viscosity are related to many diseases, such as
two-photon fluorescent probes for in vitro or in vivo micro-
viscosity mapping.
Herein, we report a new two-photon fluorescent probe
4
(MCN) for viscosity detection based on 6-substituted quinoline
Alzheimer’s disease and atherosclerosis.3, Therefore, it is
group, an efficient two-photon fluorophore from our group
(Scheme 1).23-25 Dicyanovinyl group was conjugated to the
quinoline framework through a C-C bond.26, 27 We envisage
that in non-viscous or low-viscosity environment, the
dicyanovinyl group will rotate freely around the C-C bond that
results in non-radiative quenching of the excited state of the
probe, which will quench the fluorescence of the probe.
Whereas, in high-viscosity environment, the rotation is
restricted, the fluorescence of probe is expected to be
restored. Thus, the “off-on” two-photon fluorescent response
important to monitor viscosity at cellular level. Unfortunately,
traditional viscosity measuring devices can’t be applied to
monitor micro-environmental viscosity in living cells because
they are designed for macroscopic fluids. Therefore, new
methods are warranted to monitor intracellular micro-
viscosity.
Fluorescent probes have been proven an efficient way to
monitor micro-viscosity due to their high sensitivity, simplicity
for implementation, real-time detection, and good
compatibility for bio-samples.5-10 Most of reported fluorescent
probes are designed based on one-photon fluorescence
technology. However, the short excitation wavelength limits
their application in bio-sample and in vivo owing to the shallow
penetration depth, photobleaching, photo-damage, and
for viscosity will be achieved by the probe MCN
.
Results and discussion
cellular
auto-fluorescence.11-15
Recently,
two-photon
Synthesis of MCN
fluorescence microscopy has become popular in biological
research, owing to its advantages over one-photon
As shown in Scheme 2, MCN can be easily synthesized in a four
steps process from readily affordable materials. The structures
of MCN and the intermediates were confirmed by 1H NMR, 13
C
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excitation action cross section value (δΦmax) at 800 nm
enhanced from 17.23 to 82.08 GM, which is suitable for
imaging in deep tissues and in vivo.
Theoretical calculation
DFT calculations were employed to study the rotation
mechanism of MCN. All the quantum-chemical calculations
were done with the Gaussian 09 suite.38 The potential-energy
curves of MCN with different torsion angles φ values of the C-C
bond between the dicyanovinyl group and the quinoline group
was investigated (Fig. 2a). Then, the energy gap between the
Scheme 1 The structure and proposed mechanism of MCN for
viscosity response.
Scheme 2. Synthesis route of MCN.
Fig. 1 (a) Changes of fluorescence emission spectra of MCN (10 μM)
with the variation of solution viscosity in the water/glycerol system
(λex = 400 nm). Inset: photo of MCN (10 μM) in water (left) and
glycerol (right) under a UV lamp (λex = 365 nm). (b) Linear
relationship of log I470 nm and log η, R2 = 0.98, x = 0.65. (c)
Fluorescence lifetime spectra of MCN (10 μM) with the variation of
solution viscosity in the water/glycerol system (λem = 470 nm). Inset:
linear relationship of log τ and log η, R2 = 0.99, x = 0.35. (d) Two-
photon excitation action cross-section of MCN in glycerol-water
system with different viscosities. Two-photon excitation: 680-880
nm.
NMR and ESI mass spectra.
One/two-photon fluorescence response to viscosity
As shown in Fig. 1a, MCN exhibited negligible fluorescence in
water. However, as the increasing viscosity in glycerol-water
system from 1.03 to 956.00 cp (the volume ratio of glycerol to
water increased), the emission intensity at λ = 470 nm of MCN
was enhanced by ca. 90-fold upon excitation at 400 nm (Fig.
S1, ESI†). The obvious “off-on” fluorescence response of MCN
could be observed by the naked eye (inset of Fig. 1a). In
addition, as shown in Fig. 1b, MCN fits the linear relationship
well between log I470 nm and log η (R2 = 0.98) (Equation 2) that
the value of x is as high as 0.65, which presents the possibility
for robust detection of viscosity in vitro. As shown in Fig. 1c,
the fluorescence lifetime τ of MCN also fits well with solvent
viscosity η (R2 = 0.99, x = 0.35) (Equation 3). The UV-Vis spectra
and the fluorescence emission spectra for different solvents
with different polarity show that the effect of solvent polarity
on MCN is insignificant (Fig. S2, ESI†). All these results
indicated that MCN can serve as an excellent fluorescent
probe for viscosity.
S0 and S1 states of MCN with different φ values calculated (Fig.
2b). From these results, it was observed that the energy gap
between the S0 and S1 states is lower in coplanar
conformations (C-C, φ=0o) and maximum in orthogonal
conformations (C-C, φ=90o), which indicates that for
orthogonal conformation, maximum non-radiative decay
phenomena were observed and minimum for the coplanar
conformation.
pH stability
We further investigated the pH titration curve of MCN in
water-glycerol system (3:7, v/v, 10 mM PBS buffer) (Fig. S3,
ESI†). The pH titration curve revealed that MCN kept a
The two-photon action cross section of MCN was further
determined using the two-photon induced fluorescence
measurement technique.37, 40 As shown in Fig. 1d, the two-
photon absorption spectra of MCN showed apparent
enhancement with the viscosity of system increasing, these
may be attributed to block of the rotation of dicyanovinyl
group along the C-C bond. With the volume ratio of glycerol to
water increased from 60% to 99%, the maximum two-photon
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Fig. 2 (a) Potential-energy curves of MCN at the S0 and S1
states with the dihedral angle φ around the C-C bond between
the dicyanovinyl group and the quinoline group. (b) The energy
gap between the S0 and S1 states of MCN with the S0 and S1
states with the dihedral angle φ around the C-C bond.
Fig. 4 (a) – (j) Two-photon confocal images of HeLa cells
incubated with MCN (10 μM) at different time points after
addition of 2.0 μM etoposide, λex = 800 nm, emission
wavelength from 450 nm to 490 nm. (k) Plot of relative
fluorescence intensities of MCN against time with (square,
black line) and without (circle, red line) etoposide. The
measurement range of the relative emission intensity is 0-1000.
Scale bars: 20 μm.
Fig. 3 Two-photon confocal images of HeLa cells incubated with
dimethylthi-azol-2-yl-2,5-diphenyltetrazoliu-m bromide) assay
was performed according to the reported method. HeLa cells
were incubated with MCN (0, 5, 10, 15, 20 and 25 μM) for 24 h.
The results were illustrated in Fig. S4 (ESI†). The cell viability
remained 92% under the treatment of MCN (10 μM), which
indicated that the cytotoxicity of MCN to cells is low and
suitable for cell imaging. In addition, we cultured HeLa cells
with MCN and PI (Propidium Iodide) in working concentration
(10 μM).28 The PI was a general nucleic acid dye for dead cells
or cells in terminal apoptosis, which could present
fluorescence emission in red channel. Approximately, 95% of
the HeLa cells were still alive upon incubation with MCN (up to
10 uM) for 30 min (Figure S5, ESI†).
o
o
MCN (10 μM), after 30 min of incubation at (a) 25 C and (d) 37 C,
respectively. Then, washed with 10 mM PBS buffer. λex = 800 nm,
emission wavelength from 450 nm to 490 nm; (b), (e) bright-field of
HeLa cells; (c) the overlay of panels (a) and (b); (f) the overlay of
panels (d) and (e). Scale bars: 20 μm.
constant minimal value at pH 3-10, which indicated that the
probe could work at
physiological studies.
a broad range of pH values in
Cell cytotoxicity and live cell imaging
Cytotoxicity may limit the potentials of a probe in live cell
imaging. To ascertain the cytotoxic effect of MCN, the MTT (5-
In order to demonstrate the imaging ability of MCN in living
cells, the HeLa cell line was incubated with MCN (10 μM) at
different temperatures to obtain different viscosities in living
cells.29 The images of the cells under two-photon excitation
were collected. As shown in Fig. 2, the fluorescence of the cells
at 25 oC is stronger than that at 37 oC due to the decreasing of
viscosity. Confocal co-localized images of HeLa cells with MCN
and NucRed® Live 647 ReadyProbes® Reagent showed that
MCN could be localized in cytoplasm (Fig. S6, ESI†). The above
results suggested that MCN could be used for monitoring
micro-viscosity change in cytoplasm of living cells.
Considering that micro-viscosity of the cells will change
greatly during the apoptosis process, which is an apparent
mode of cell death.30 MCN was used to monitor the real-time
changes of viscosity during apoptosis of HeLa cells induced by
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etoposide. The two-photon fluorescence images were The living tissue slices (a thickness of 180 μm) of the liver were
collected at different time during the apoptosis. As shown in prepared for two-photon fluorescence microscopic analyses.
Fig. 3, upon addition of etoposide, the fluorescence intensity The living tissues slices incubated with MCN (20 μM) at 37 oC
of the cells increased greatly during the apoptosis. In contrast,
the fluorescence intensity of MCN remained unchanged
without etoposide (Fig. S7, ESI†). These results indicated that
MCN could be used to monitor intracellular viscosity change
during apoptosis process.
Two-photon Fluorescence Lifetime Imaging (FLIM) in living cells
We further carried out fluorescence lifetime imaging (FLIM)31,
32 of MCN for quantitatively detecting viscosity distribution (Fig.
4) in living HeLa cells. As shown in Fig. 4a, the distribution of
MCN was mainly in living HeLa cells cytosol (Fig. S7, ESI†) with
Fig. 7 Two-photon fluorescence images of 5-day-old zebrafish
incubated with MCN (10 μM), after 60 min of incubation,
washed with 10 mM PBS buffer. From top to bottom, Cyan
channel, Bright field, Merge. λex 800 nm, emission
wavelength from 450 nm to 490 nm. Magnification is 20 ×,
scale bars: 400 μm.
Fig. 5 Fluorescence intensity (a) and FLIM images (b) for HeLa
cells incubated with MCN (10 µM) for 30 min. Two-photon
excitation: λex = 800 nm, emission wavelength from 450 nm to
490 nm. Scale bars: 20 μm.
=
in 10 mM PBS buffer for 30 min, and exhibited bright
fluorescence under two-photon excitation at 800 nm. As
shown in Fig. 5, relatively significant fluorescence was
observed up to 130 μm. This result indicated that MCN was
capable of imaging viscosity at depth in living tissues using
two-photon fluorescence microscopy, meanwhile, this was
consistent with its good two-photon fluorescence properties.
Two-photon fluorescence imaging in vivo
We further explored the capability of MCN for mapping micro-
viscosity in vivo under two-photon excitation. The survival rate
of zebrafishes was up to 92 % treated with MCN (10 μM) in 96
hours (Fig. S9, ESI†). As shown in Fig. 6, when five-day-old
Fig. 6 Two-photon confocal fluorescence images of a fresh rat liver
slice treated with MCN (20 μM) at depths of approximately 0 to 130
μm with a magnification of 20 ×, λex = 800 nm, emission wavelength
from 450 nm to 490 nm. Scale bars: 200 μm.
o
zebrafish was prepared with MCN (10 μM) for 60 min at 28 C
in 10 mM PBS buffer, a bright fluorescence was observed.
Moreover, the merge images revealed that bright fluorescence
distributed almost the whole body of zebrafish. This result
indicated that two-photon fluorescent probe MCN possessed
good two-photon fluorescence properties. MCN is also a
potential candidate of two-photon fluorescent probe for
monitoring micro-viscosity in vivo.
punctuate distribution. FLIM imaging (Fig. 4b) with detailed
analysis (Fig. S8, ESI†) revealed that the lifetime of MCN (10
μM) was distributed over a wide range from ca. 1400 to 2500
ps under two-photon excitation at 800 nm. According to the
experiment results in vitro, it could be calculated that the
intracellular viscosity value was from 29.80 cP to 156.36 cP.
The average viscosity distribution was mainly found to be
73.45±21.55 cP in cytosol, which was larger than that in
droplets (40.95±10.95 cP). Thus, MCN could be used as a
quantitative viscosity probe by two-photon fluorescence
lifetime imaging for the viscosity distribution in living cells.
Two-photon fluorescence imaging in living tissues
Conclusion
In summary, we have developed
a new two-photon
fluorescent probe (MCN) for viscosity detection on basis of 6-
substituted quinoline framework. MCN showed great “off-on”
fluorescence response (ca. 90-fold enhancement) with
viscosity increasing in glycerol-water viscosity system. MCN
We further investigated the capability of the probe MCN of
two-photon fluorescence imaging in the thick living tissues.
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exhibited excellent sensitivity to viscosity and could detect the
in which η is the viscosity, I is the emission intensity, C is a
real-time viscosity change of the cells during apoptosis with constant, and x represents the sensitivity of the fluorescent
low cytotoxicity. Fluorescence lifetime imaging (FLIM) of living probe to viscosity.
log τ = C + x log η
(3)
HeLa cells stained with MCN revealed that the intracellular
average viscosity value was 73.45 ± 21.55 cP in cytosol. MCN
also showed the ability to image viscosity in deep tissue (~130
μm) and in living zebrafish under two-photon excitation. Thus,
MCN is a potential candidate of two-photon fluorescent probe
for monitoring micro-viscosity in vivo.
in which η is the viscosity, τ is the fluorescence lifetime, C is
a constant, and x represents the sensitivity of the fluorescent
probe to viscosity.
Measurement of two-photon absorption cross-section (δ)
Two-photon excitation fluorescence (TPEF) spectra were
measured using femtosecond laser pulse and Ti: sapphire
system (680~1080 nm, 80 MHz, 140 fs, Chameleon II) as the
light source. All measurements were carried out in air at room
temperature. Two-photon absorption cross-sections were
Experimental
General procedures
All reagents and solvents were commercially purchased. All
reactions were magnetically stirred and monitored by thin
layer chromatography (TLC). Flash chromatography (FC) was
measured
using
two-photon-induced
fluorescence
measurement technique. The Two-photon absorption cross
sections (δ) are determined by comparing their TPEF to that of
fluorescein in different solvents, according to the following
equation:
1
performed using silica gel 60 (200-300 mesh). H NMR spectra
were recorded on Bruker-400 MHz spectrometers and 13C
NMR spectra were recorded on 100 MHz spectrometers.
Fluorescence spectra were obtained using a HITACHIF-2500
spectrometer. UV-vis absorption spectra were recorded on a
Tech-comp UV 1000 spectrophotometer. MS spectra were
Φref cref nref
F
δ=δref
(4)
Fref
Φ
c
n
Here, the subscripts ref stands for the reference molecule. δ
is the TPA cross-section value, c is the concentration of
solution, n is the refractive index of the solution (because of
the low concentrations of the solutions, the refractive indices
of the solutions were replaced with those of the solvents), F is
the TPEF integral intensities of the solution emitted at the
exciting wavelength, and Φ is the fluorescence quantum yield.
The δref value of reference was taken from the literature.37
Theoretical calculation
recorded on
a
ESI-MS spectrometer. Time resolved
fluorescence spectra were recorded on Edinburgh Instruments
FLSP920 Steady State and Transient State Fluorescence
Spectrometer. The viscosity was measured with a NDJ-8S
rotational viscometer. The fluorescence lifetime imaging (FLIM)
studies were carried out using an inverted-type laser scanning
confocal microscope with a 100× objective.
Measurement of fluorescence quantum yield (Φ)
UV-vis absorption spectra were recorded on a Tech-comp UV
1000 spectrophotometer. Fluorescence spectra were obtained
using a HITACHIF-2500 spectrometer. The relative fluorescence
quantum yields were determined with quinine sulfate as a
standard33, 34 and calculated using the following equation:
All the quantum-chemical calculations were done with the
Gaussian 09 suite.38 The geometry optimizations of the dyes
were performed by using density functional theory (DFT) with
the Becke three-parameter hybrid exchange function with the
Lee–Yang–Parr gradient-corrected correlation functional (B3-
LYP functional) and 6–31G** basis set. No constraints to
bonds/angles/dihedral angles were applied in the calculations
and all the atoms were free to optimize. The potential energy
curves for the S0 state were qualitatively scanned by
constrained optimizations, with the dihedral angles kept fixed
at a series of values. The potential-energy curves for the S1
state were obtained by calculating the Frank–Condon
transition energies for the ground-state optimized structures
at fixed dihedral angles by using the time-dependent (TD)-DFT
method.
2
)
Aref λref
F
n
Φ=Φref
(
(1)
Fref
A
λ
nref
Here,
Φ is the quantum yield; F is the integral intensities of
the solution emitted at the exciting wavelength; A is the
absorbance at the excitation wavelength ( 0.05); is the
≤
λ
excitation wavelength; n is the refractive index of the solution
(because of the low concentrations of the solutions, the
refractive indices of the solutions were replaced with those of
the solvents).35 The subscripts ref stands for the reference
molecule.
Cytotoxicity assays
The Forster-Hoffmann equation
The Forster-Hoffmann equation36 was utilized to correlate the
relationship between the fluorescence emission intensity of
MCN and the solvent viscosity or between the fluorescence
lifetime of MCN and the solvent viscosity.
MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide)
assay was performed as previously reported to test the
cytotoxic effect of the probe in cells. HeLa cells were passed
and plated to ca. 70% confluence in 96-well plates 24 h before
treatment. Prior to MCN treatment, DMEM (Dulbecco’s
Modified Eagle Medium) with 10% FCS (Fetal Calf Serum) was
removed and replaced with fresh DMEM, and aliquots of MCN
stock solutions (1 mM DMSO) were added to obtain final
concentrations of 0, 5, 10, 15, 20 and 25 μM respectively. The
log I = C + x log η
(2)
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o
treated cells were incubated for 24 h at 37 C under 5% CO2.
The cytotocity for zebrafish was prepared with 5-day-old
Subsequently, cells were treated with 5 mg/mL MTT (40 zebrafishes. Four hundred zebrafishes was divided into four
o
μL/well) and incubated for an additional 4 h (37 C, 5% CO2). groups, each group was fed with different concentrations MCN
Then the cells were dissolved in DMSO (150 μL/well), and the (0, 5, 10, 15 μM) respectively. The total cultured time reached
absorbance at 570 nm was recorded. The cell viability (%) was up to 96 hours, and the survival of zebrafishes was counted
calculated according to the following equation:
Cell viability %=OD570(sample)/OD570(control)×100
per 12 hours.28
Animals
(5)
All procedures involving animals were approved by and
conformed to the guidelines of the Anhui University Animal
Care Committee, School of life science. We have taken great
efforts to reduce the number of animal used in these studies
and also taken effort to reduce animal suffering from pain and
discomfort.
where OD570(sample) represents the optical density of the
wells treated with various concentration of MCN and
OD570(control) represents that of the wells treated with DMEM
containing 10% FCS. The percent of cell survival values is
relative to untreated control cells.39
Synthesis of compound 1
A mixture of 4-iodobenzenamine (18.0 g, 82.18 mmol), HCl (6
o
N, 100 mL) was heated to 105 C, then crotonaldehyde (8.6 g,
Cell culture and two-photon fluorescence microscopy imaging
For two-photon bio-imaging, HeLa cells were cultured in
DMEM supplemented with 10% FCS, penicillin (100 μg/mL),
and streptomycin (100 μg/mL) at 37 oC in a humidified
atmosphere with 5% CO2 and 95% air. Cytotoxicity assays show
that MCN is safe enough for two-photon bio-imaging at low
concentrations, and HeLa cells were cultured and stained with
MCN (10 μM) within 30 min and washed by PBS buffer. Cells
imaging was carried out on a confocal microscope (Zeiss LSM
710 Meta NLO). Two-photon fluorescence microscopy images
of labeled cells were obtained by exciting the probe with a
mode-locked titanium–sapphire laser source set at wavelength
800 nm.
122.70 mmol) was added slowly, the result mixture was
refluxed until TLC shows no raw material exist. After cooling to
room temperature, 200 mL H2O was added, the mixture was
extracted with EtOAc (100 mL × 2) to remove the un-reacted
crotonaldehyde. The aqueous phase was neutralized with
ammonia water and then extracted with EtOAc (50 mL × 2).
The organic phase were dried over Na2SO4 and evaporated to
give crude residue. The residue was recrystallized in acetic
ether/petroleum ether (1:20) to the 16.0 g product (59.46
mmol, 72.35%).
1H NMR (400 MHz, CDCl3, ppm): δ 8.13 (s, 1H), 7.90 (d, J =
8.8 Hz, 2H), 7.73 (d, J = 8.9 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 2.72
(s, 3H). 13C NMR (100 MHz, CDCl3, ppm): δ 159.70, 146.77,
138.08, 136.20, 134.93, 130.39, 128.23, 122.69, 90.87, 25.44.
Synthesis of compound 2
Two-photon Fluorescence Lifetime Imaging (FLIM) in living cells
HeLa cells were cultured and stained with MCN (10 μM) within
30 min and washed by PBS buffer. The fluorescence lifetime
imaging (FLIM) studies were carried out using an inverted-type
laser scanning confocal microscope (Zeiss LSM 880 Meta NLO)
with a 100× objective.
A solution of compound 1 (10.0 g, 37.16 mmol) in dioxane (200
o
mL) was heated to 60 C. SeO2 (7.43 mmol, 0.82 g) was added
to this solution per ten minutes, totally 4.95 g. Then the
reaction temperature was increased to 80 oC. After 4 h, the
mixture was cooled to room temperature. Precipitates were
filtered off and washed with 1,4-dioxane (25 mL×2). The
organic phase were combined and concentrated to give a
crude product. The crude material was purified by column
chromatography (DCM as the eluent) to give product 6.7 g
(23.75 mmol, 63.91%).
Preparation of fresh mouse liver slices and two-photon fluoresce-
nce imaging
For two-photon bio-imaging, slices were prepared from the
liver of 7-day-old mouse. Slices were cut to 180 μm thickness
by using a vibrating-blade microtome in 10 mM PBS buffer
(pH=7.4). Slices were incubated with MCN (20 μM) in PBS
buffer bubbled with 95% air and 5% CO2 for 1 h at 37 oC. Then,
slices were washed three times with PBS buffer, transferred to
glass-bottomed dishes. Mouse liver slices imaging was carried
out on a confocal microscope (Zeiss LSM 710 Meta NLO). Two-
photon fluorescence microscopy images of fresh mouse liver
slices were obtained by exciting the probe with a mode-locked
titanium–sapphire laser source set at wavelength 800 nm.
Preparation of zebrafishes and two-photon fluorescence imaging
1H NMR (400 MHz, CDCl3, ppm): δ 10.20 (s, 1H), 8.32 (s, 1H),
8.20 (d, J = 8.5 Hz, 1H), 8.05 (t, J = 9.1 Hz, 1H), 7.96 (d, J = 8.9
Hz, 1H).13C NMR (100 MHz, CDCl3, ppm): δ 193.36, 152.86,
146.82, 139.40, 136.72, 136.15, 131.84, 131.39, 118.14, 95.77.
Synthesis of compound 3
The mixture of compound
2 (4 g, 14.1 mmol), 1-ethynyl-4-
methoxybenzene (3.86 g, 16.9 mmol), Pd(PPh3)2Cl2 (0.1 g, 0.14
mmol), Et3N (3.66 g, 36.2 mmol), CuI (0.5 g, 2.6 mmol) and THF
(20 mL) was heated at room temperature for 12 h. Then, the
mixture was filtered to remove salts. The result was
evaporated to yield a crude product, then recrystallized in
For two-photon bio-imaging in vivo, 5-day-old zebrafishes
were prepared. Zebrafishes were fed with MCN (10 μM) in PBS
buffer at 28 oC for 1 h. All the fishes were terminally
anaesthetized using MS222, and images were carried out on a
confocal microscope (Zeiss LSM 710 Meta NLO). Two-photon
fluorescence microscopy images of zebrafishes were obtained
by exciting the probe with a mode-locked titanium–sapphire
laser source set at wavelength 800 nm.
EtOAc/PE (1:5) to give compound 3, 3.08 g (10.72 mmol, 76%).
1H NMR (400 MHz, DMSO-d6, ppm): δ 10.13 (s, 1H), 8.60 (d,
J = 8.4 Hz, 1H), 8.34 (s, 1H), 8.23 (d, J = 8.7 Hz, 1H), 8.04 (d, J =
8.5 Hz, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.59 (d, J = 8.1 Hz, 2H), 7.04
6 | J. Name., 2012, 00, 1-3
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(d, J = 8.0 Hz, 2H), 3.82 (s, 3H). 13C NMR (100 MHz, CDCl3,
ppm): δ 193.42, 160.16, 152.58, 147.07, 136.93, 133.55,
133.22, 130.44, 130.35, 129.92, 124.86, 118.01, 114.61,
114.18, 92.72, 87.59, 77.35, 77.03, 76.71, 55.36.
Synthesis of MCN
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The mixture of compound 3 (3.6 g, 10.73 mmol), malononitrile
(1.24 g, 18.77 mmol), alkaline Al2O3 (0.38 g, 3.73 mmol) and
100 ml DCM was heated at room temperature for 4 h. Then,
the mixture was filtered. the filtrate was evaporated to yield a
crude product, then the crude material was purified by column
chromatography (DCM as the eluent) to give MCN 2.12 g (6.32
mmol, 58.90%).
1H NMR (400 MHz, DMSO-d6): δ 8.72 (s, 1H), 8.59 (d, J = 8.5
Hz, 1H), 8.29 (s, 1H), 8.05 (d, J = 8.9 Hz, 1H), 7.96 (d, J = 8.8 Hz,
1H), 7.88 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.04 (d, J =
8.6 Hz, 2H), 3.82 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 160.38,
156.99, 148.44, 147.31, 136.87, 133.82, 133.44, 130.40,
130.07, 128.97, 125.50, 122.81, 114.59, 114.29, 113.69,
112.15, 93.67, 87.72, 87.60, 55.40. ESI-MS m/z: [M + H]+
C22H14N3O+ calcd, 336.1131; found, 336.1132.
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237-243.
,
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Acknowledgements
This work was supported by National Natural Science
Foundation of China (21372005), The research fund of Jiangsu
Collaborative Innovation Center for Ecological Building
Materials and Environmental Protection Equipments, and
Finance support from Anhui University.
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Journal of Materials Chemistry B
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DOI: 10.1039/C7TB00136C
Graphic Abstract
A quinoline-based two-photon fluorescent probe (MCN) was developed for viscosity
imaging in vivo.