Novel yellow- to red-emitting fluorophores: Facile synthesis, aggregation-induced emission, two-photon absorption properties, and application in living cell imaging

Article abstract of DOI:10.1016/j.dyepig.2020.108849

Four novel yellow-to red-emitting fluorophores with a 1-(2-hydroxyethyl) pyridinium core and different electron-donating terminal moieties (N-methylcarbazole, N,N-dibutylbenzenamine, N-methylpyrrole, and 2-methylfuran) were designed and facilely synthesized via the one-step Knoevenagel condensation. All these compounds (HPs) were characterized by ¹H NMR, ¹³C NMR, and HRMS. Their photophysical properties including linear absorption, one-photon excited fluorescence, two-photon absorption, and two-photon excited fluorescence, were systematically investigated in various solvents. And the density functional theory calculations were conducted to analyze the electronic structures of HPs. The two-photon absorption cross-sections (δ) values of HPs measured by the Z-scan technique were determined to be as large as 1354 (1HP-CZ), 4462 (2HP-BA), 836 (3HP-PR), and 2944 GM (4HP-FU) in DMSO. The two-photon action cross-sections (Φ × δ) values of 1HP-CZ and 2HP-BA in H₂O measured by the two-photon induced fluorescence method were about 50 GM. 1HP-CZ, 3HP-PR, and 4HP-FU also exhibited good water solubility. Meanwhile, it was found that 2HP-BA exhibited notable aggregation-induced emission characteristic in DMSO/H₂O mixture. The aggregate particle size distribution of 2HP-BA was measured by the dynamic light scattering method, and the aggregation characteristic of 2HP-BA was observed by the transmission electron microscopy. Besides, 2HP-BA also exhibited red emission. Then, HPs with relatively low cytotoxicity were used for one- and two-photon excited fluorescence imaging in living HepG2 cells. The results indicate that HPs are potential candidates in the bioimaging field due to their photophysical properties and biocompatibility.

Full text of DOI:10.1016/j.dyepig.2020.108849

Journal Pre-proof
Novel yellow-to red-emitting fluorophores: facile synthesis, aggregation-induced
emission, two-photon absorption properties, and application in living cell imaging
Qi-Ji Dong, Zhi-Bin Cai, Ling Ding, Pei-Hua Luo, Qiao-Jun He, Sheng-Li Li, Li-Jun
Chen, Qing Ye, Yu-Peng Tian
PII:
S0143-7208(20)31546-1
DOI:
https://doi.org/10.1016/j.dyepig.2020.108849
DYPI 108849
Reference:
To appear in: Dyes and Pigments
Received Date: 29 March 2020
Revised Date: 20 August 2020
Accepted Date: 9 September 2020
Please cite this article as: Dong Q-J, Cai Z-B, Ding L, Luo P-H, He Q-J, Li S-L, Chen L-J, Ye Q, Tian Y-
P, Novel yellow-to red-emitting fluorophores: facile synthesis, aggregation-induced emission, two-photon
absorption properties, and application in living cell imaging, Dyes and Pigments, https://doi.org/10.1016/
j.dyepig.2020.108849.
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Author Statement
Qi-Ji Dong: Formal analysis, Investigation, Writing-Original Draft, Writing-Review
&
Editing, Visualization
Zhi-Bin Cai: Conceptualization, Validation, Formal analysis, Writing-Original Draft,
Writing-Review & Editing, Visualization, Supervision
Ling Ding: Resources
Pei-Hua Luo: Resources
Qiao-Jun He: Resources
Sheng-Li Li: Resources
Li-Jun Chen: Investigation
Qing Ye: Investigation
Yu-Peng Tian: Resources

Novel yellow- to red-emitting fluorophores: facile synthesis, aggregation-induced
emission, two-photon absorption properties, and application in living cell
imaging
a
a,
b
b
b
c
Qi-Ji Dong , Zhi-Bin Cai *, Ling Ding , Pei-Hua Luo , Qiao-Jun He , Sheng-Li Li ,
a
a
c
Li-Jun Chen ,Qing Ye , Yu-Peng Tian
a
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou
3
10014, P. R. China
b
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
Department of Chemistry, Anhui Province Key Laboratory of Functional Inorganic
c
Materials, Anhui University, Hefei 230039, P. R. China
keywords: Two-photon absorption, Fluorescence, Aggregation-induced emission, Cell
imaging
Abstract
Four novel yellow- to red-emitting fluorophores with a 1-(2-hydroxyethyl) pyridinium
core and different electron-donating terminal moieties (N-methylcarbazole, N,N-
dibutylbenzenamine, N-methylpyrrole, and 2-methylfuran) were designed and facilely
synthesized via the one-step Knoevenagel condensation. All these compounds (HPs)
1
13
were characterized by H NMR, C NMR, and HRMS. Their photophysical
properties including linear absorption, one-photon excited fluorescence, two-photon
absorption, and two-photon excited fluorescence, were systematically investigated in
various solvents. And the density functional theory calculations were conducted to
analyze the electronic structures of HPs. The two-photon absorption cross-sections (δ)
values of HPs measured by the Z-scan technique were determined to be as large as
1
354 (1HP-CZ), 4462 (2HP-BA), 836 (3HP-PR), and 2944 GM (4HP-FU) in
DMSO. The two-photon action cross-sections (Φ×δ) values of 1HP-CZ and 2HP-BA
in H O measured by the two-photon induced fluorescence method were about 50 GM.
2
1
HP-CZ, 3HP-PR, and 4HP-FU also exhibited good water solubility. Meanwhile, it
was found that 2HP-BA exhibited notable aggregation-induced emission
characteristic in DMSO/H O mixture. The aggregate particle size distribution of 2HP-
2
1

BA was measured by the dynamic light scattering method, and the aggregation
characteristic of 2HP-BA was observed by the transmission electron microscopy.
Besides, 2HP-BA also exhibited red emission. Then, HPs with relatively low
cytotoxicity were used for one- and two-photon excited fluorescence imaging in living
HepG2 cells. The results indicate that HPs are potential candidates in the bioimaging
field due to their photophysical properties and biocompatibility.
1
. Introduction
The organic fluorophore is a chemical compound having fluorescence
emission upon light excitation [1]. Fluorophores are used prevalently in the
basic science and industry, such as bioimaging [2], intraoperative diagnosis [3],
photodynamic therapy[4], and enzyme analysis[5]. In particular, the fluorescent
marking technology serving as indispensable tools in cell and tissue imaging
has been highly focused. Comparing with other analytical methods such as
electrochemical methods[6] and capillary electrophoresis[7], the fluorescent
marking method has attracted tremendous attention[8] due to its high
sensitivity, ease of implementation, and acceptable biocompatibility. Two-
photon excited fluorescence (TPEF) imaging has demonstrated apparent
advantages compared with one-photon excited fluorescence (OPEF) imaging:
(
(
1) less absorption and scattering in tissues by using infrared light excitation;
2) lower photobleaching and photodamage due to the highly localized two-
photon excitation [9]. Therefore, TPEF imaging has gradually become the
potential imaging technology in medicine and life sciences. To realize such
application, it is vital to design and synthesize novel high-performance two-
photon absorbing fluorophores[10]. In particular, red-emitting fluorophores are
highly desirable for bioimaging. By using red-emitting fluorophores, it is
possible to obtain a higher degree of tissue penetration and resolution as the
cells and tissues will exhibit lower optical absorption and weaker interference
from autofluorescence[11]. Furthermore, fluorophores for bioimaging
applications are required to be used in aqueous solutions with consideration of
the cell growth environment. However, poor water-solubility fluorophores with
the characteristic of aggregation-induced emission (AIE) can also be used for
bioimaging[12]. These AIE-active fluorophores exhibit weak emission in
2

appropriate organic solvents with good solubility, but they become highly
emissive due to the aggregation of extremely small particles formed by the
addition of poor solvent H O[13]. For example, Tian et al. [12a] and Wang et al.
2
[
12b] reported the AIE-active fluorophores 3B and 1, respectively, which were
successfully utilized in living cell imaging. Therefore, it is highly desirable to
design novel fluorophores with large two-photon action cross-sections (Φ×δ),
low cytotoxicity, high membrane permeability, high water solubility or AIE
characteristics, and red emission.
Along this line, four novel fluorophores (1HP-CZ
, 2HP-BA, 3HP-PR, and
4
HP-FU) with the same 1-(2-hydroxyethyl) pyridinium core and different
electron-donating terminal moieties were designed (Fig. 1). First of all, the
introduction of cationic pyridinium moiety enhances not only the water
solubility of fluorophores, but also the electron-withdrawing ability. Up to now,
many pyridinium-based fluorophores used as TPEF imaging agents have been
reported[14]. Moreover, the link of 2-hydroxyethyl group to the pyridinium
core can further improve the water solubility of fluorophores (HPs) and form
strong hydrogen bonds with the other polar molecules around the
microenvironment. Secondly,
N-methylcarbazole, N,N-dibutylbenzenamine, N-
methylpyrrole, and 2-methylfuran, which possess good electron-donating and
electron-transporting ability, are introduced into HPs. These functional groups
have been widely used in optoelectronics[15]. The respective disubstitution of
pyridinium core with these functional groups not only provides structural
diversity, but also enhances intramolecular charge transfer (ICT) induced by the
formation of the D-π-A-π-D configuration. The high degree of ICT may
facilitate two-photon absorption (TPA). The One-step Knoevenagel
condensation reaction was utilized for the preparation of HPs. And they were
1
13
characterized by H NMR, C NMR, and HRMS, respectively. The detailed
synthetic procedures and characterizations of HPs were given in the
experimental section. Besides, the density functional theory (DFT) calculations
were conducted to analyze the electronic structures of HPs. Their linear and
nonlinear photophysical properties were systematically investigated in different
solvents. The AIE characteristic of 2HP-BA in DMSO/H O mixture was
2
further investigated. To evaluate their potential for bioimaging, the cytotoxicity
3

experiments as well as the living cell imaging experiments by OPEF and TPEF
microscopy were also conducted.
Fig. 1. Structures of HPs
2
2
. Results and discussion
.1. Synthesis and characterization
Scheme 1 shows the synthetic routes of HPs. 9-Methyl-9H-carbazole-3-
carboxaldehyde[16], 4-(dibutylamino)benzaldehyde[17], and 1-methyl-1H-
pyrrole-2-carboxaldehyde[18] were synthesized according to the literature
procedures. 5-Methyl-2-furancarboxaldehyde was obtained from a commercial
supplier. The novel target compounds were prepared by the one-step
Knoevenagel condensations.
1
13
HPs were fully characterized by H NMR, C NMR, and HRMS. According
1
to the H NMR spectra, as expected, the CH=CH bonds in the fluorophores are
the all-trans configurations, as evidenced by the coupling constants of 15.4-
13
1
5.7 Hz for olefinic protons. From the C NMR spectra, we can note that the
chemical shifts of hydroxyethyl appear at about 59 ppm and 53 ppm. HPs were
further characterized by HRMS and were found to be consistent with the
proposed structures.
4

Scheme 1 Synthetic routes of HPs.
2
.2. Linear photophysical properties
Table 1 shows the linear photophysical properties of HPs in different
solvents. Figs. 2 and 3 show the linear absorption and OPEF spectra of HPs in
-5
-1
DMSO with a concentration of 1×10 mol L .
As shown in Fig. 2, HPs in DMSO exhibit the absorption maxima ( ꢁ
ꢂꢄꢅ
ꢀ
ꢂꢃ) in
the range of 431-513 nm, which can be attributed to the ICT transition. Closer
inspection of Fig. 2 reveals that the absorption bands at about 328-387 nm are
ascribed to the n-π* transition of the nitrogen and oxygen atoms. The
5

absorption bands observed at the shorter wavelengths, about 296 nm for 1HP-
CZ and 295 nm for 2HP-BA, originate from the π-π* transition of the
carbazole ring and benzene, respectively. As shown in table 1, HPs exhibit the
ꢂꢄꢅ
largest
ꢀ
values in moderately polar organic solvent CH Cl and the
ꢂꢃ
2 2
ꢁ
ꢂꢄꢅ
smallest
ꢀ
values in highly polar inorganic solvent H O. It is likely that the
2
ꢁ
ꢂꢃ
solute-solvent hydrogen bonds play an important role in the blue shifts of HPs
ꢂꢄꢅ
in H O. Besides, 2HP-BA exhibits obviously red-shifted
ꢀ
in all solvents
ꢁꢂꢃ
2
relative to the other three fluorophores, which can be sequenced as 2HP-BA
HP-PR 1HP-CZ 4HP-FU
>
3
>
>
.
6

Table 1 Linear photophysical properties of HPs.
a
b
ε
ꢆ
c
Φꢆ
e
abs
-4
OPEF
max
ꢀ ꢆ
d
λ
10
λ
ν
max
Compound Solvent
-
1
-1
-1
(
nm)
465
DMSO 453
(mol L cm ) (nm)
(cm )
(%)
1
2
3
4
HP-CZ
HP-BA
HP-PR
HP-FU
CH Cl
2
2
3.11
2.71
3.55
2.46
3.99
3.87
6.15
1.10
4.26
3.68
3.56
3.08
6.39
3.32
3.53
1.37
564
563
563
583
618
602
606
632
563
560
561
557
540
541
538
3775
4313
4216
5070
2441
2882
2878
4018
3380
3510
3497
4220
4576
4718
4832
4956
17.78
3.49
16.36
3.15
6.35
0.36
2.35
2.84
3.12
0.29
1.48
0.03
7.03
1.06
6.19
0.43
EtOH
455
450
537
H O
2
CH Cl
2 2
DMSO 513
EtOH
516
504
473
f
H O
2
CH Cl
2 2
DMSO 468
EtOH
469
451
433
H O
2
CH Cl
2 2
DMSO 431
EtOH
427
426
H O
2
540
a
b
c
d
e
f
-5
-1
Maximum linear absorption wavelength,
Maximum molar absorption coefficient.
c
= 1×10 mol L .
-5
-1
Maximum one-photon excited fluorescence wavelength,
Stokes shift.
c = 1×10 mol L .
Fluorescence quantum yield.
H O containing 2 vol. % DMSO.
2

Fig. 2. Linear absorption spectra of HPs in DMSO.
In all the solvents, HPs exhibit only one fluorescence emission band, which
OPEF
can be assigned to the ICT emission. The OPEF maxima ( max ) values range
λ
from 538 to 541 nm for 4HP-FU (yellow fluorescence), from 563 to 583 nm for
1
HP-CZ and from 557 to 563 nm for 3HP-PR (orange fluorescence), and from
02 to 632 nm for 2HP-BA (red fluorescence). It is worthy of notice that red-
6
emitting fluorophores can facilitate deep-tissue imaging. The OPEF spectra of
HP-PR and 4HP-FU in different solvents display weak solvatochromism,
3
which is attributed to a fairly similar polarity between the ground state and the
excited state. Compared with 3HP-PR and 4HP-FU, the fluorescence emission
peaks for 1HP-CZ and 2HP-BA exhibit red shifts as the organic solvents
(
CH Cl , DMSO and EtOH) are changed to the inorganic solvent (H O). This
2 2 2
fact can be attributed to that the solute-solvent interaction in H O is enhanced,
2
which leads to lower the energy gap between the ground state and the excited
state[19]. In consistence with the absorption spectra, the fluorescence spectra of
2
HP-BA also show large red-shifts relative to 1HP-CZ
In addition, the fluorescence quantum yields ( ) of HPs can be sequenced as
HP-CZ 4HP-FU 2HP-BA 3HP-PR. The values of HPs (except AIE-
, 3HP-PR, and 4HP-FU.
Φ
1
>
>
>
Φ
8

active 2HP-BA) in H O tend to decrease. This behavior is due to the non-
2
radiative decay mechanism that may arise as a result of the twisted
intramolecular charge transfer (TICT) geometry[20].
Fig. 3. One-photon excited fluorescence spectra of HPs in DMSO.
2
.3. Aggregation-induced emission characteristic
To study the aggregation characteristics of HPs, their solid-state fluorescence
emission spectra were measured, which show emission peaks at 580 (1HP-CZ),
47 (2HP-BA), 595 (3HP-PR), and 670 (4HP-FU) nm, respectively. The
colors of solid-state fluorescence are orange and red (Fig. 4). The values of
6
Φ
the solids were determined to be 7.89 %, 6.75%, 0.91%, and 1.98%,
respectively. It is worthy of notice that 2HP-BA also shows an AIE
-4
-1
characteristic in the DMSO/H O mixture (c = 5×10 mol L , DMSO: good
2
solvent, H O: poor solvent). As shown in Figs. 5a and 5b, the fluorescence
2
spectrum of 2HP-BA in DMSO is basically a flat line parallel to the abscissa.
With an increase in the water fractions ( w
mixture, the spectral patterns remain unchanged. Upon further increasing the w
from 50% to 80%, the OPEF intensity increases dramatically. The higher the w
f ) from 0 to 40% in the DMSO/H O
2
f
f
9

is, the stronger the emission intensity is. The OPEF intensity of 2HP-BA in the
DMSO/H O mixture (fw = 80%) is nearly 900-folds stronger than that in DMSO.
2
To have a more accurate evaluation of the AIE characteristic, we used the
dynamic light scattering (DLS) method for studying the dimension of 2HP-BA
in the DMSO/H O mixture (fw = 80%). Besides, the transmission electron
2
microscopy (TEM) technique was used for observing the aggregation
characteristic of the aggregates. The DLS study of 2HP-BA highlighted particle
aggregation with an average size of 142 nm (Fig. 5c). Meanwhile, as shown in
Fig. 5d, the abundant rectangular aggregates were observed. Surprisingly, the
emission intensity became weaker again when the fw increased from 80% to
9
0%. This may be attributed to the effect of the aggregate size[21]. Taken
together, the above spectral, DLS, and TEM studies corroborate that 2HP-BA
is an AIE fluorophore.
1
0

Under visible light
3
65 nm UV
irradiation
Under 365 nm UV irradiation
Fig. 4. Fluorescence photographs of HPs as solids under visible light and under
65 nm UV irradiation.
3
(a)
1
1

(b)
(c)
1
2

(d)
Fig. 5. (a) One-photon excited fluorescence spectra of 2HP-BA in the
DMSO/H O mixture. (b) Plots of the emission intensity vs. the water fractions
2
of 2HB-BA. (c) Particle size distribution of 2HP-BA in the DMSO/H O
2
mixture ( w
f
= 80%) studied via DLS. (d) TEM image of 2HP-BA in the
DMSO/H O mixture (fw = 80%).
2
2
.4. Two-photon absorption properties
The femtosecond open aperture Z-scan technique was used for determining
the two-photon absorption cross-sections (δ) of HPs, as described in the
experimental section. Fig. 6 shows the open aperture Z-scan traces of 1HP-CZ
at 780 nm, 2HP-BA at 710 nm, 3HP-PR at 750 nm, and 4HP-FU at 740 nm in
DMSO, which were measured under the optimal excitation wavelengths. Their
TPA coefficients (
in table 2, the values of
BA), 836 (3HP-PR), and 2944 (4HP-FU) GM, respectively. The values of
and follow the same order as 2HP-BA 4HP-FU 1HP-CZ 3HP-PR.
β) were obtained by fitting the experimental data. As shown
δ
were calculated to be 1354 (1HP-CZ), 4462 (2HP-
β
δ
>
>
>
Besides, the electronic structures of HPs were calculated by DFT at the basis
set level of B3LYP/6-31G(d) via the Gaussian 09 program. As shown in Fig. 7,
the electron clouds of the highest occupied molecular orbitals (HOMOs) of HPs
1
3

are mainly delocalized on the electron-donating terminal moieties, while in the
lowest unoccupied molecular orbitals (LUMOs), the electron cloud density of
the pyridinium core increases significantly. The results show that HPs possess
obvious ICT characteristics, which may be favorable for the enhancement of
TPA. With consideration of the cell growth environment, the Φ×δ values of
fluorophores in H O are vital factors for the application in bioimaging. The
2
two-photon induced fluorescence method was used to determine the Φ×δ
values of 1HP-CZ
, 3HP-PR, 4HP-FU in H O, and 2HP-BA in the
2
DMSO/H O mixture (fw = 90%). As a result, the Φ×δ values of HPs are 54, 48,
2
1
8
1, and 15 GM at wavelengths of 770, 790, 770, and 750 nm, respectively (Fig.
). The results show that HPs have potential applications in bioimaging [9b].
Noticing that HPs not only have preferable Φ×δ in H O, but also emit yellow
2
to red fluorescence, we were inspired to explore their practical application in
living cell imaging.
Table 2 Open aperture Z-scan data of HPs
.
ꢆ
a
βꢆ (cm GW )
b
-1
δꢆc (GM)
1354
4462
836
Compound
λ (nm)
ex
1
2
3
HP-CZ
HP-BA
HP-PR
HP-FU
780
710
750
740
0.032
0.096
0.019
4
0.066
2944
a
Optimal excitation wavelength.
b
c
-3
-1
Two-photon absorption coefficient,
c
= 1×10 mol L .
-50
4
-1
Two-photon absorption cross-sections, 1 GM= 10 cm s photon
.
1
4

1
5

Fig. 6. Open aperture Z-scan traces (scattered dot) and fitted curves (solid line)
of HPs in DMSO.
1
6

Fig. 7. Electron density distribution of the HOMO and LUMO orbitals for HPs
.
Fig. 8. Two-photon action cross-sections of 1HP-CZ, 3HP-PR, 4HP-FU in H O and
2
2
HP-BA in the DMSO/H O mixture (f = 90%) in the 690-930 nm range.
2 w
2
.5. Cytotoxicity assay
Prior to the imaging applications, the cytotoxicity of HPs was evaluated in
the human hepatocellular carcinomas (HepG2) cells using the
thiazolyl blue tetrazolium bromide (MTT) assay. As shown in Fig. 9, the results
elucidated that HPs generally present low cytotoxicity in the living cells at low
concentrations. For example, the viability of cells incubated with HPs is 87-98%
1
7

at a concentration of 5 ꢁM. As the concentrations continue to increase, the
viability of cells incubated with 2HP-BA 3HP-PR, and 4HP-FU remains
,
above 75%. By contrast, the viability of cells incubated with 1HP-CZ is
significantly reduced, which is around 63% at 80 ꢁM. Subsequently, the
working concentration was determined to be 20 ꢁM with consideration of the
large Φ×δ and biocompatibility of HPs
.
Fig. 9. Cytotoxicity of HPs at different concentrations.
2
.5. Cell imaging
The bioimaging studies in the living HepG2 cells stained with HPs were
investigated by the OPEF and TPEF microscopy. From Fig. 10, it can be seen
that the intense fluorescence emission is from the internalized fluorophores in
the HepG2 cells, indicating that HPs successfully penetrated into the cells.
According to their bright fluorescence, the cytoplasm and cell borders can be
clearly recognized, whereas the cell nuclei are dim. Besides, the OPEF and
TPEF images of HPs indicated that the fluorescence intensity of cells stained
with 1HP-CZ and 2HP-BA was clearly stronger than that stained with 3HP-
PR and 4HP-FU
.
1
8

Bright-field
One-photon
Two-photon
Merge
Fig. 10. One and two-photon fluorescent images of the living HepG2 cells incubated
with 20 ꢁM 1HP-CZ (1a-1d), 2HP-BA (2a-2d), 3HP-PR (3a-3d), and 4HP-FU (4a-
4
d). Scale bar: 20 ꢁm.
2
.6. Photostability
Photostability is another crucial parameter for fluorescence imaging reagents.
We performed the photostability tests in the living HepG2 cells treated with
HPs for 600 s. The fluorescence microscope captured an image every 10
seconds. The time-dependent fluorescence images were shown in the
supporting information. As shown in Fig. 11, after 600 s of constant
illumination, the overall fluorescence intensity of 3HP-PR and 4HP-FU
1
9

decreases by approximately 30%, but in the case of 1HP-CZ and 2HP-BA, the
changes in the fluorescence intensity are only 6%. The results indicate that
1
HP-CZ and 2HP-BA are promising fluorophores for cell imaging.
Fig. 11. Photostability of HPs by continuous irradiation at different time.
3
. Conclusions
In conclusion, four novel TPA fluorophores with a 1-(2-hydroxyethyl)
pyridinium core and different electron-donating terminal moieties were facilely
synthesized via the one-step Knoevenagel condensation. The structures were
fully characterized. Their linear and nonlinear photophysical properties were
studied in detail. Among these compounds, 2HP-BA shows a notable AIE
characteristic and red fluorescence emission in its aggregated state. Meanwhile,
the
δ value of 2HP-BA in DMSO is 4462 GM, which is larger than those of
most reported TPA fluorophores[22]. Besides, based on their relatively low
cytotoxicity, relatively large Φ×δ in H O, and good membrane permeability,
2
HPs have been successfully applied for OPEF and TPEF imaging in living cells.
4
. Experimental
2
0

4
.1. Materials and instruments
The starting materials and the solvents were commercially available. The
melting points were measured on an X-4 micromelting point apparatus without
1
13
correction. The H and C NMR spectra were recorded on a Bruker AVANCE
III 500 spectrometer using DMSO-d6 as a solvent and all shifts were referred to
tetramethylsilane (TMS). The Mass spectrometric analyses were carried out on
a Thermo Scientific LTQ Orbitrap XL instrument.
The size distribution of the aggregates of 2HP-BA in the DMSO/H O
2
mixture (fw = 80%) was determined with the particle size analyzer (Malvern
Zetasizer Nano ZS90) by the DLS method. The morphology was observed by
the transmission electron microscopy (Hitachi HT7700 Exalens, 120 kV).
4
.2. Linear photophysical measurements
The linear absorption spectra were measured on a Shimadzu UV-2550 UV-
visible spectrophotometer. The OPEF spectra measurements were performed
using a RF-5301PC fluorescence spectrophotometer.
-1
The
Φ
was determined by using fluorescein in 0.1 mol L sodium hydroxide
= 0.69[24]) as the references,
(
Φ
= 0.9[23]) and Rhodamine B in ethanol (Φ
which was calculated according to the following Equation (1) [23-24]:
2
A n F
r
s
s
Φ ꢆ=ꢆ
s
Φrꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆ (1)
2
A n F
s
r
r
Where the subscripts s and r designate the sample and the reference,
respectively. is the absorbance at the excitation wavelength, is the refractive
is the integrated area under the corrected
A
n
index of the relevant solution, and
emission spectrum.
F
The solid emission spectra and the
Φ of the solid states were obtained by the
Edinburgh FS5 spectrofluorometer equipped with an integrating sphere.
4
.3. Nonlinear photophysical measurements
The nonlinear photophysical properties were measured by the Z-scan
measurement method and the two-photon induced fluorescence method by
using a femtosecond Ti: Sapphire laser system (Chameleon UltraⅡ , 680-1080
nm, 80 MHz, 140 fs). The open aperture Z-scan technique was used to
2
1

determine the
β. The experimental data were fitted using the following
Equation (2) [25]:
∞
ꢇꢈzꢉꢆ=ꢆ ꢊ
m=0
m
[
-q (z)]
0
ꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆ(2)
3
ꢋ
2
(
m+1)
βI0Leff
q0ꢈzꢉꢆ=ꢆ
ꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆ(3)
1
+x2
where
focus (
absorption coefficient and
the diffraction length of the beam with ω0 the beam waist radius and
β
stands for the TPA coefficient, ꢌꢍ is the intensity of laser beam at the
= 0), Leff is the effective sample length with the linear
ꢆꢎ1-e-αL
the sample length, is
z2 z02, in which z0 πω20
the
is the sample position. Based on Equation (3), the
z
ꢆ
=
ꢏ
/
α
α
L
xꢆ
=ꢆ
/
ꢆ
=ꢆ
/λ
λ
wavelength of the beam,
can be deduced.
z
β
Furthermore, the
4) [26]:
δ
of the sample was determined by the following Equation
(
hνβ
3
δꢆ=ꢆ
×10 ꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆ (4)
N c
A
where
Avogadro’s number, and
In the two-photon induced fluorescence method, the
h
is the Planck constant,
is the concentration of the sample solution.
was obtained by
ν is the frequency of incident laser, NA is the
c
δ
comparing the TPEF intensity of the sample with that of a reference compound
using the following Equation (5) [27]:
F Φ n c
s
r
r
r
δsꢆ=ꢆ
δrꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢆꢈ5)
F Φ n c
r
s
s
s
where the subscripts s and r designate the sample and the reference,
respectively. and represent the TPEF integral intensity and the fluorescence
quantum yield. and are the refractive index and the concentration of the
F
Φ
n
c
-1
solution. In this work, we selected fluorescein in 0.1 mol L sodium hydroxide
as the reference [27].
4
.4. Cytotoxicity studies
2
2

The cytotoxicity of HPs toward the HepG2 cells was studied by the MTT
assay. 10000 HepG2 cells per well were cultured in the 96-well plates and
allowed to grow in Dulbecco's modified Eagle medium (DMEM) containing 10
%
fetal bovine serum (FBS) in an atmosphere of 5% CO at 37 ºC for 24 h. HPs
2
at different concentrations (0, 5, 10 20, 40, and 80 ꢁM) were then added to the
cell suspensions. The treated cells were incubated in an atmosphere of 5% CO2
at 37 ºC for 24 h. Subsequently, the cells were treated with 5 mg/mL MTT (20
ꢁ
L/well) and incubated for an additional 4 h (37 ºC, 5% CO ). Then, the
2
solution was removed and 100 ꢁL per well of DMSO was added to dissolve the
formazan crystals, and the plate was swirled homogeneously about 10 min by
the shaker. The absorbance at 570 nm was recorded using the microplate reader
(
Thermo Scientific Multiskan Spectrum). The percent of cell survival values
was relative to the untreated control cells, which was calculated by the
following Equation (6) [28]:
OD570 (sample)
Cell viability
ꢈ
%
ꢉꢆ
=
×100%
(6)
OD570 (control)
Where OD570 (sample) represents the optical density of the wells treated with
various concentrations of HPs and OD570 (control) represents that of the well
treated with DMEM plus 10% FBS.
4
.5. Cell imaging
The HepG2 cells were grown overnight on the glass bottom dishes. The
living cells were stained with HPs (20 ꢁM) in an atmosphere of 5% CO at 37
2
ºC for 30 min. The cells were then washed with PBS three times and 1 mL of
PBS was added to each dish. The Olympus BX61W1-FV1000 two-photon
scanning microscope equipped with a 60× water immersion objective was used
for fluorescence imaging. The excitation wavelengths for 1HP-CZ
, 2HP-BA,
3
HP-PR, and 4HP-FU were 488, 488, 488, and 405 nm while the emission
wavelengths were collected at 505-605, 560-660, 505-605, and 505-605 nm,
respectively (under the one-photon excitation mode). The corresponding two-
photon fluorescence excitation wavelength was 800 nm while the emission
wavelengths were collected at 575-630, 575-630, 495-540, and 495-540 nm,
respectively.
2
3

4
.6. Photostability
For the photostability test, the living HepG2 cells treated with HPs were
continuously irradiated with the confocal laser (Olympus IX81-FV1000). The
excitation wavelengths for 1HP-CZ 2HP-BA 3HP-PR, and 4HP-FU were
88, 488, 488, and 405 nm while the emission wavelengths were collected at
20-620, 560-660, 505-605, and 505-605 nm, respectively.
,
,
4
5
4
.7. Synthesis
.7.1. 1-(2-Hydroxyethyl)-2,6-dimethylpyridinium bromide
,6-Dimethylpyridine (4.28 g, 40 mmol) and 2-bromoethanol (7.50 g, 60
4
2
mmol) were added to a round bottom flask and heated under reflux for 8 h.
Then, the mixture was cooled to room temperature and filtered. The precipitate
was washed 3 times with THF and used directly in the next step without further
purification.
4
.7.2. 2,6-bis[(1E)-2-(9-ethyl-9H-carbazol-3-yl)ethenyl]-1-(2-
hydroxyethyl)pyridinium bromide (1HP-CZ)
-(2-Hydroxyethyl)-2,6-dimethylpyridinium bromide (0.46 g, 2 mmol) was
dissolved in CH OH (20 mL) and mixed with 9-methyl-9 -carbazole-3-
1
H
3
carboxaldehyde (0.92 g, 4.4 mmol). Piperidine (0.51 g, 6 mmol) was then
added. The reaction mixture was heated under reflux for 10 h. After cooling,
the precipitate was filtered off and recrystallized from MeOH. Orange-yellow
1
crystalline powder was collected. Yield 71.3%. m.p. 297-299 ºC; H NMR
(
(
DMSO-d6, 500 MHz)
δ
: 8.67 (d,
= 7.7 Hz, 2H), 8.02 (dd, J1 = 8.7 Hz, J2 = 1.2 Hz,
= 15.7 Hz, 2H), 7.79 (d, = 15.7 Hz, 2H), 7.75 (d, = 8.7 Hz,
= 8.2 Hz, 2H), 7.55 (t, = 7.7 Hz, 2H), 7.32 (t, = 7.6 Hz, 2H),
= 4.7 Hz, 2H), 4.08 (q, = 5.4 Hz, 2H), 3.96
: 153.85, 143.82, 142.64, 141.92,
J = 1.2 Hz, 2H), 8.44 (t, J = 8.1 Hz, 1H), 8.34
d, = 8.1 Hz, 2H), 8.24 (d,
J
J
2
2
5
H), 7.99 (d,
H), 7.68 (d,
J
J
J
J
J
J
.45 (t,
J
= 5.7 Hz, 1H), 5.10 (t,
J
J
13
(
s, 6H); C NMR (DMSO-d6, 125 MHz)
δ
1
1
41.27, 126.59, 126.45, 126.30, 123.05, 122.48, 122.01, 121.30, 120.49,
19.64, 115.82, 109.76, 109.73, 59.30, 53.83, 29.29; HRMS (ESI): m/z calcd
+
for C H N O [M-Br] : 534.2545; found: 534.2537.
37
32
3
4
.7.3. 2,6-bis[(1E)-2-[4-(dibutylamino)phenyl]ethenyl]-1-(2-hydroxyethyl)
2
4

pyridinium bromide (2HP-BA)
The procedure was similar to that described for 1HP-CZ. Red crystalline
1
powder. Yield 76.8%. m.p. 245-247 ºC; H NMR (DMSO-d6, 500 MHz)
δ
: 8.20
= 15.7 Hz, 2H), 7.62 (d,
= 8.9 Hz, 4H), 5.36 (t,
= 5.4 Hz, 2H), 3.36 (t, = 7.6
= 7.4 Hz, 12H); C
: 153.76, 149.62, 142.80, 141.51, 130.47, 121.92,
21.44, 112.31, 111.27, 59.12, 53.24, 49.82, 29.02, 19.60, 13.83; HRMS (ESI):
(
t,
J
= 8.1 Hz, 1H), 8.09 (d,
= 8.9 Hz, 4H), 7.34 (d, = 15.7 Hz, 2H), 6.72 (d,
.6 Hz, 1H), 4.85 (t, = 5.0 Hz, 2H), 3.95 (q,
J = 8.1 Hz, 2H), 7.64 (d, J
J
J
J
J =
5
J
J
J
13
Hz, 8H), 1.50-1.56 (m, 8H), 1.31-1.38 (m, 8H), 0.94 (t,
NMR (DMSO-d6, 125 MHz)
J
δ
1
+
m/z calcd for C H N O [M-Br] : 582.8890; found: 582.8879.
39
56
3
4
.7.4. 1-(2-hydroxyethyl)-2,6-bis[(1E)2-(1-methyl-1H-pyrrol-2-yl)ethenyl]
pyridinium bromide (3HP-PR)
The procedure was similar to that described for 1HP-CZ. Brick-red
1
crystalline powder. Yield 72.2%. m.p. 239-241 ºC; H NMR (DMSO-d6, 500
MHz)
H), 7.08 (t,
J1 = 3.9 Hz, J2 = 2.6 Hz, 2H), 5.35 (t,
δ
: 8.21-8.27 (m, 3H), 7.66 (d,
J
J
= 15.4 Hz, 2H), 7.30 (d, J = 15.4 Hz,
2
J
= 1.9 Hz, 2H), 6.99 (dd, J1 = 3.9 Hz, J2 = 1.5 Hz, 2H), 6.21 (dd,
= 5.6 Hz, 1H), 4.82 (t,
J = 5.2 Hz, 2H),
13
3
.93 (q, J = 5.3 Hz, 2H), 3.80 (s, 6H); C NMR (DMSO-d6, 125 MHz) δ:
1
53.49, 141.57, 130.53, 130.35, 128.14, 121.77, 112.97, 112.40, 109.43, 59.11,
+
5
3.33, 33.98; HRMS (ESI): m/z calcd for C H N O [M-Br] : 334.1919;
21
24
3
found: 334.1916.
4
.7.5. 1-(2-hydroxyethyl)-2,6-bis[(1
E)-2-(5-methyl-2-furanyl)ethenyl]
pyridinium bromide (4HP-FU)
The procedure was similar to that described for 1HP-CZ. Brown needle
1
crystals. Yield 80.1%. m.p. 260-262 ºC; H NMR (DMSO-d6, 500 MHz)
δ
: 8.35
= 15.6 Hz, 2H), 7.28 (d,
= 3.3 Hz, 2H), 6.37 (dd, J1 = 3.3 Hz, J2 = 0.8 Hz,
= 5.4 Hz, 1H), 4.81 (t, = 5.1 Hz, 2H), 3.94 (q, = 5.3 Hz, 2H),
.41 (s, 6H); C NMR (DMSO-d6, 125 MHz) : 155.92, 153.12, 149.93,
(
t,
J
= 8.1 Hz, 1H), 8.19 (d,
= 15.6 Hz, 2H), 6.91 (d,
H), 5.39 (t,
J = 8.1 Hz, 2H), 7.58 (d, J
J
2
2
1
J
J
J
J
13
δ
42.74, 129.00, 123.20, 117.61, 114.32, 109.78, 58.97, 53.93, 13.73; HRMS
+
(
ESI): m/z calcd for C H NO [M-Br] : 336.1600; found: 336.1595.
21
22
3
Declaration of competing interest
2
5

There are no conflicts to declare.
Acknowledgements
We are grateful to the financial supports from the Natural Science
Foundation of Zhejiang province, China (Grant Nos. LY19B060010 and
LY15B030006) and the National Natural Science Foundation of China (Grant
No. 21103151).
2
6

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