Novel mono-cationic fluorescent probes based on different central π-conjugated bridges for two-photon bioimaging of cellular nuclei

Article abstract of DOI:10.1039/c6ra12193d

A series of novel pyridine mono-cationic two-photon fluorescent probes based on different central π-conjugated bridges, fluorenone (W-pyI), dibenzothiophene (S-pyI), and dibenzofuran (F-pyI), were prepared and studied. Under one-photon excitation in a N,N-dimethylformamide solution, W-pyI, S-pyI, and F-pyI displayed fluorescence quantum yields of 0.401, 0.425, and 0.09, respectively. The two-photon fluorescence performance indicated that these probes possessed two-photon absorption cross-sections of 681 GM (W-pyI), 630 GM (S-pyI), and 620 GM (F-pyI) at 800 nm femtosecond laser excitation. The luminance "turn-on" effect of W-pyI, S-pyI, and F-pyI bonding with calf thymus DNA in Tris-HCl buffer solutions displayed 58-fold, 30-fold, and 25-fold fluorescence quantum yield increments, respectively, and 350-450% two-photon absorption cross-section enhancement. The confocal fluorescence imaging showed clear one- and two-photon fluorescence imaging. The mean co-localization coefficients between these probes and Hoechst 33342 in 3T3 cells ranged from 0.89-0.92, indicating that they showed excellent nuclear targeting abilities. The counterstaining experiments showed these probes possessed good counterstaining compatibility and membrane permeability in the application of multicolor targeting. A time-dependent fluorescence intensity test under continuous femtosecond laser irradiation showed that W-pyI possessed a longer observation time (3000 seconds) and a lower fluorescence attenuation amplitude (7.1%) in the first 300 seconds than S-pyI, F-pyI and other previously reported pyridinium derivatives, demonstrating that the central π-conjugated bridge "fluorenone" played a key role in improving photostability during probe designing for two-photon bioimaging applications.

Full text of DOI:10.1039/c6ra12193d

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Novel monoꢀcationic fluorescent probes based on different
central πꢀconjugated bridges for twoꢀphoton bioimaging of
cellular nucleus
Shuheng Chia,b, Liang Lia,b, Yiqun Wua,c,*
a Key Laboratory of Material Science and Technology for High Power Lasers,
Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences,
Shanghai 201800, PR China
b University of Chinese Academy of Sciences, Beijing 100049, PR China
c Key Lab of Functional Inorganic Material Chemistry (Heilongjiang University),
Ministry of Education, Harbin 150080, PR China
*Corresponding author. Tel.: +86 021 6991 8592
Email Address: *yqwu@siom.ac.cn
Abstract
A series of novel pyridine monoꢀcationic twoꢀphoton fluorescent probes based on
different central πꢀconjugated bridges, fluorenone (WꢀpyI), dibenzothiophene (SꢀpyI),
and dibenzofuran (FꢀpyI), were prepared and studied. Under oneꢀphoton excitation in
N,Nꢀdimethylformamide solution, WꢀpyI, SꢀpyI, and FꢀpyI displayed fluorescence
quantum yields of 0.401, 0.425, and 0.09, respectively. The twoꢀphoton fluorescence
performance indicated that these probes possessed twoꢀphoton absorption
crossꢀsections of 681 GM (WꢀpyI), 630 GM (SꢀpyI), and 620 GM (FꢀpyI) at 800ꢀnm
femtosecond laser excitation. The luminance “turnꢀon” effect of WꢀpyI, SꢀpyI, and
FꢀpyI bonding with calf thymus DNA in TrisꢀHCl buffer solutions displayed 58ꢀfold,
30ꢀfold, and 25ꢀfold fluorescence quantum yield increments, respectively, and 350–
450% twoꢀphoton absorption crossꢀsection enhancement. The confocal fluorescence
imaging showed clear oneꢀ and twoꢀphoton fluorescence imaging. The mean
coꢀlocalization coefficients between these probes and Hoechst 33342 in 3T3 cells
ranged from 0.89–0.92, indicating that they showed excellent nuclear targeting
abilities. The counterstaining experiments exhibited these probes possessed good
counterstaining compatibility and membrane permeability in the application of
multicolor targeting. The timeꢀdependent fluorescence intensity test under continuous
femtosecond laser irradiation showed that WꢀpyI possessed a longer observation time
(3000 seconds) and a lower fluorescence attenuation amplitude (7.1%) in the first 300

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seconds than SꢀpyI, FꢀpyI and other previously reported pyridinium derivatives,
demonstrating that the central πꢀconjugated bridge “fluorenone” played a key role in
improving photostability during probe designing for twoꢀphoton bioimaging
applications.
Introduction
Confocal fluorescence microscopy has become a powerful tool for the observation of
cells, tissues, and vital organs. Compared with traditional biological optical
microscopy, twoꢀphoton fluorescence microscopy provides a key technique for
tracking and monitoring of living cells because it employs a nearꢀinfrared excitation
light source, and provides a low damage threshold, a high penetration depth, and
highꢀresolution threeꢀdimensional space selectivity [1ꢀ4]. However, twoꢀphoton
fluorescent probes remain an issue of widespread concern. Numerous reports have
been published on selective labeling of mitochondria, lysosomes, metal ions, and
other living tissues [5ꢀ9]; however, most of the probes show poor lipophilicity and
hydrophilicity, low photostability, and high toxicity. In addition, few studies have
been published on the effectiveness of nuclear localization using different
πꢀconjugated structural fluorescent probe molecules in twoꢀphoton fluorescence
imaging. In particular, photostability under continuous femtosecond laser irradiation
during realꢀtime monitoring and tracking of cellular nuclei in living cells has rarely
been reported. This issue needs to be further investigated.
Recently, some twoꢀphoton fluorescent probes with good performance were
prepared for twoꢀphoton bioimaging in cellular or subcelluar levels. Zheng et al. [10]
desighed a novel twoꢀphoton fluorescent cyanine, whose twoꢀphoton absorption (TPA)
crossꢀsection in glycol reached 497 GM, and the 30ꢀfold oneꢀphoton fluorescence
enhancement and 3ꢀfold enhancement of TPA crossꢀsection were achieved when
binding with calf thymus DNA in buffer solutions. Miao et al. [11] prepared two
pyridine cation derivatives for specific mitochondrial imaging. The maximum TPA
action crossꢀsection at 860 nm reached 328 GM, and the fluorescence attenuation
amplitude was 10% and 20% within 300 s, respectively. Liu et al. [12] designed two
indoleꢀbased monoꢀcationic probes for selective imaging of cellular RNA in nucleoli
and cytoplasm. The TPA action crossꢀsection of INR1 at 800 nm in the presence of
DNA and RNA in buffer solutions was 5.71 and 8.93 GM, respectively. The
fluorescence signals of INR1 and INR2 decreased by 8% and 28% after 300 s of
irradiation. Wang et al. [13] prepared a twoꢀphoton ratiometric fluorescent pH probe,
whose TPA crossꢀsection was 354 GM at pH 4.5 with good membrane permeability.
Xu et al. [14] also reported a novel series of dinuclear Ru(II) polypyridyl complexes

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for celluar cytoplasm imaging, whose maximum TPA crossꢀsections were estimated to
be 322–386 GM, and the fluorescence signals of RuL4 decreased by 38% within 300 s
under laser irradiation. The distribution percentage of the probe in the cytoplasmic
region reached 90%.
An excellent twoꢀphoton nuclear fluorescent probe should not only have a
sufficiently large twoꢀphoton absorption crossꢀsection, but also possess strong nuclear
DNAꢀtargeting ability. A probe with these two properties would exhibit good
photostability characteristics for nuclear monitoring and imaging. Therefore, the
structureꢀproperty relationship has always been the focus of attention in the design of
molecular structures [15ꢀ17]. According to several previous reports, twoꢀphoton
fluorescent probes with large TPA crossꢀsections must have the following structural
characteristics: high acceptor and donor strength, a long molecular chain, good
conjugated delocalized planarity, and structural symmetry [18ꢀ22]. However, to
possess sufficient affinity to nuclear DNA in the complex cellular microenvironment,
the probes must possess some additional important structural features [23ꢀ25]: first,
molecules should have a donorꢀacceptorꢀdonor type system for efficient charge
transfer in the sensitive bioenvironment; second, Vꢀshaped aryl arms that cause
fluorescence enhancement and the “turnꢀon” effect by restricting planar rotation; third,
the terminal units in the molecular structure should have the ability to recognize DNA
base pairs by electrostatic attraction, and the central πꢀconjugated bridge should
become embedded into the phosphate backbone of doubleꢀstranded DNA through
groove binding or intercalative binding. Based on the above theoretical guidelines, we
designed three symmetric Vꢀshaped twoꢀphoton fluorescent probes with pyridine
monoꢀcationic units and different central πꢀconjugated bridges (fluorenone,
dibenzothiophene, and dibenzofuran), referred to as WꢀpyI, SꢀpyI, and FꢀpyI (Scheme
1). The “dibenzothiophene” conjugated core had been used to connect with symmetric
pyridine rings in our previous study. To facilitate comparison of twoꢀphoton
performance, molecules with “fluorenone” and “dibenzofuran” aromatic cores were
prepared, to investigate the influences of different center conjugated bridges on the
structureꢀproperty relationships and biological fluorescence imaging performance.
Here, the oneꢀ and twoꢀphoton photophysical properties of WꢀpyI, SꢀpyI, and FꢀpyI
were studied, including calculation and analysis of TPA crossꢀsections upon excitation
using a 750–830 nm femtosecond laser source and fluorescence quantum yields in
N,Nꢀdimethylformamide (DMF) solution. The ctꢀDNA fluorescence switching
characteristics in tris(hydroxymethyl) methyl aminomethaneꢀchlorine hydride
(TrisꢀHCl) buffer solutions were researched, and the influences of the various
conjugated aromatic cores on fluorescence “turnꢀon” effect bounding with DNA were
also investigated. The oneꢀ and twoꢀphoton confocal fluorescence imaging at the

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excitation wavelength of 405 nm and 800 nm in 3T3 cells were carried out. Nuclear
coꢀlocalization staining experiments were conducted between WꢀpyI, SꢀpyI, or FꢀpyI
and Hoechst 33342 (a commercial nuclear staining reagent). The counterstaining
experiment employing WꢀpyI, SꢀpyI or FꢀpyI doubleꢀstained with MitoTracker Red
CMXRos (MTR, a commercial red mitochondrial staining reagent) were performed as
well. The variations in fluorescence intensity with time under continuous 800ꢀnm
femtosecond laser irradiation in the process of twoꢀphoton nuclear imaging were
studied, along with the effects of the three different central πꢀconjugated bridges on
photostability.
Experimental section
Materials
All solvents and reagents were of analytical grade and were purchased from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2,7ꢀDibromoꢀ9ꢀfluorenone,
2,8ꢀdibromodibenzothiophene, and 2,8ꢀdibromodibenzofuran were purchased from
Alfachem Co. (Zhengzhou, China). Methyl iodide (CH3I) and Tris(hydroxymethyl)
methyl aminomethane (Tris) were obtained from J&K Chemical (Beijing, China).
4ꢀVinylpyridine, MitoTracker Red CMXRos (MTR), Hoechst 33342, and calf thymus
DNA (ctꢀDNA) were obtained from Thermo Fisher Scientific (Waltham, USA).
Samples and ctꢀDNA dissolved in TrisꢀHCl buffer solution (Tris: 10 mM, NaCl: 100
mM, pH = 7.4) were prepared at concentrations of 10ꢀ5 M and 0–1.4×10ꢀ3 M,
respectively. All chemicals were stored at 4 °C until use.
Synthesis of WꢀpyI/SꢀpyI/FꢀpyI
The synthesis process was divided into two steps. First, 2,7ꢀdibromoꢀ9ꢀfluorenone
(4.05 g, 0.012 mol), 2,8ꢀdibromodibenzothiophene (4.10 g, 0.012 mol), or
2,8ꢀdibromodibenzofuran (4.21 g, 0.013 mol), 4ꢀvinylpyridine (0.50 g, 0.048 mmol),
tri(oꢀtolyl)phosphine (0.46 g, 1.50 mmol), palladium (II) acetate (0.03 g, 0.13 mmol),
triethylamine (20 ml, 0.14 mol), and 15 ml DMF were added and mixed uniformly in
a closed reaction vessel, which was filled with highꢀpurity nitrogen at a pressure of 3
MPa. Under the protection of the inert gas, the temperature gradually rose to 110 °C,
thermal insulation 24 h. After the reaction, the mixed liquid was fully extracted with
dichloromethane, and the crude product was obtained by vacuum distillation. Then a
red intermediate product (Wꢀpy, Sꢀpy, or Fꢀpy) was separated out via column
chromatography using ethyl acetateꢀpetroleum (4:1) and acetonitrileꢀdichloromethane
(3:1) on silica gel; second, the intermediate product Wꢀpy (3.475 g, 0.009 mol), Sꢀpy
(3.901 g, 0.010 mol) or Fꢀpy (3.367 g, 0.009 mol), methyl iodide (2.129 g, 0.015 mol)
and 40 ml of acetone were added in turn to a roundꢀbottomed flask, then the
temperature was gradually increased to 85 °C for 18 h. After cooling, an orangeꢀred
(WꢀpyI) or pale yellow (SꢀpyI, FꢀpyI) target product was separated out via vacuum

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distillation and column chromatography.
WꢀpyI: Yield: 3.85 g (80 %). Mp＞290 °C. 1H NMR (DMSO, 500 MHz): δ 8.90 (d,
4H), 8.82 (d, 1H), 8.23 (d, 4H), 8.12 (d, 1H), 7.98 (m, 8H), 4.27 (s, 3H). 13C NMR
(DMSO, 125 MHz): δ 194.14, 154.27, 147.38, 146.65, 141.23, 139.12, 137.80, 136.77,
126.66, 125.88, 125.18, 124.83, 124.53, 49.25. MALDIꢀTOFꢀMS: m/z calcd [MꢀI]+
for C28H21IN2O, 401.172; found, 401.251. Anal. calcd. for C28H21IN2O (%): C 63.65,
H 4.01, N 5.30, O 3.03; found: C 63.83, H 4.71, N 5.22, O 3.19.
SꢀpyI: Yield: 3.69 g (76 %). Mp＞290 °C. 1H NMR (DMSO, 400 MHz): δ 8.93 (d,
2H), 8.88 (m, 2H), 8.27 (t, 2H), 8.25 (m, 2H), 8.20 (d, 2H), 7.98 (m, 2H), 7.96 (m,
2H), 7.83 (s, 2H), 7.79 (t, 2H), 4.28 (m, 3H). 13C NMR (DMSO, 125 MHz): δ 153.41,
146.28, 141.92, 139.89, 135.34, 133.91, 128.02, 123.92, 122.83, 121.45, 46.88.
MALDIꢀTOFꢀMS: m/z calcd [MꢀI]+ for C27H21IN2S, 405.143; found, 405.137. Anal.
calcd. for C27H21IN2S (%): C 60.91, H 3.98, N 5.26, S 6.02; found: C 60.88, H 4.56,
N 5.63, S 5.88.
FꢀpyI: Yield: 3.58 g (71 %). Mp＞290 °C. 1H NMR (DMSO, 500 MHz): δ 8.90 (d,
4H), 8.60 (s, 2H), 8.25 (d, 4H), 8.16 (m, 2H), 7.92 (d, 2H), 7.79 (d, 2H), 7.58 (m, 2H),
4.30 (s, 3H). 13C NMR (DMSO, 125 MHz): δ 156.88, 152.32, 144.92, 140.26, 130.88,
128.50, 123.83, 123.32, 122.59, 121.02, 112.45, 46.91. MALDIꢀTOFꢀMS: m/z calcd
[MꢀI]+ for C27H21IN2O, 389.176; found, 389.263. Anal. calcd. for C27H21IN2O (%): C
62.80, H 4.10, N 5.43, O 3.10; found: C 63.41, H 4.85, N 5.76, O 2.89.
Measurements
The absorption spectra were measured using a spectrophotometer (UHꢀ5300, Hitachi,
Japan). The oneꢀphoton excited fluorescence spectra were obtained using a
spectrofluorometer (FPꢀ6500, Jasco, Japan). Rhodamine B in ethanol solution at
25 °C was used as a standard to measure the fluorescence quantum yield. The
concentration of the samples and standards used for measuring absorption and
oneꢀphoton fluorescence spectra was 10ꢀ5 M. The fluorescence quantum yield can be
calculated using Eq.(1):
ns2
nr2
F
A
s
∫
∫
r
Φs = Φr ×
×
×
(1)
A
F
s
r
The twoꢀphoton fluorescence spectra were recorded using a spectrometer
(7ISW301, Sofn Instruments, China), and excited using a modeꢀlocked Ti:sapphire
femtosecond laser system at a pulse duration of 80 fs, with a repetition rate of 80
MHz. Twoꢀphoton absorption crossꢀsections were calculated using the twoꢀphoton
induced fluorescence method, and Rhodamine B in ethanol solution with a known
value was used as the standard at a concentration of 10ꢀ4 M [26,27]. The samples were
dissolved in the DMF solvent at a concentration of 10ꢀ4 M. The TPA crossꢀsections

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were determined using Eq.(2):
Fs Φ r Cr nr
(2)
σs
=
σr
×
×
×
×
F
Φs Cs ns
r
where the subscripts
TPA crossꢀsection,
s
and
r
represent the sample and standard, respectively.
σ
is the
are
F
is the twoꢀphoton fluorescence integrated area, and
c
and
n
the concentration and refractive index of the solutions.
Cellular staining
3T3 mouse myoblasts (0.2 µl) were grown in Dulbecco’s Modified Eagle Medium
(DMEM) containing culture solution under 5% CO2 at 37 °C for 24 h. After the cells
were cultured, they were stained with WꢀpyI, SꢀpyI, or FꢀpyI at concentrations of 0.3
µM for 15 min. When completing the staining process, the coverꢀslips were taken out
and inverted on glass slides, which were sealed surrounded by glycerol/water (9:1)
solution and preserved by ventilation in the dark.
Confocal fluorescence imaging
The confocal fluorescence imaging was conducted using a Carl Zeiss LSM 710
confocal scanning microscope. For WꢀpyI, SꢀpyI and FꢀpyI, the oneꢀphoton excitation
wavelengths were set to 405 nm, and twoꢀphoton fluorescence for all samples was
excited using an 800ꢀnm femtosecond laser source; the corresponding emission
fluorescence was collected in an optical window of 525–575 nm (WꢀpyI and SꢀpyI)
and 430–490 nm (FꢀpyI). For Hoechst 33342, the oneꢀphoton excitation wavelength
was located in the range of 330–380 nm, and the twoꢀphoton imaging was excited by
a femtosecond laser at 740 nm; the corresponding emission fluorescence was
collected in an optical window of 430–490 nm. For MTR, the excitation wavelength
was 575 nm, and the emission fluorescence was recorded in the range of 580–650 nm.
Results and discussion
Oneꢀphoton photophysical properties
The absorption spectra of WꢀpyI, SꢀpyI, and FꢀpyI in DMF are shown in Fig.1. The
absorption bands of WꢀpyI and SꢀpyI were in the range of 375–410 nm and 355–415
nm, respectively, with major absorption peaks at approximately 392 nm and 397 nm.
That of FꢀpyI was between 350–390 nm, with major absorption peaks at
approximately 375 nm. Compared with FꢀpyI, the major absorption peaks of WꢀpyI
and SꢀpyI were red shifted by ~20 nm, which could be attributed to the relatively
larger intramolecular nꢀπ* transition of WꢀpyI and SꢀpyI, due to their functional

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“fluorenone” and “dibenzothiophene” aromatic cores [28]. Notably, no absorption
was observed between 500–900 nm for any of the materials, indicating that this range
could be used as a twoꢀphoton excitation window to study their twoꢀphoton
photophysical properties.
The oneꢀphoton fluorescence spectra in DMF are shown in Fig.2. The main
fluorescence emission peaks of WꢀpyI and SꢀpyI were observed at 540–545 nm, while
that of FꢀpyI was at 480 nm. The Stokes shifts of WꢀpyI and SꢀpyI were observed at
~148 nm, higher than that of FꢀpyI (~105 nm). Under the action of a central electron
acceptor, either “fluorenone” or “dibenzothiophene,” the intramolecular charge
transfer (ICT) efficiency increased, thus leading to a more stable first excited state
that existed in the solvent medium [29]. The fluorescence quantum yields of WꢀpyI,
SꢀpyI, and FꢀpyI in DMF were 0.401, 0.425, and 0.09, respectively. Moreover, based
on a previous report [30], we also knew that the fluorescence quantum yield of the
carbazole dicationic derivative (9EꢀBMVC) with symmetric pyridine rings in DMF
was 0.016. The oneꢀphoton photophysical performance data for WꢀpyI and SꢀpyI were
approximately 4.5ꢀfold and 25ꢀfold, respectively, higher than those of FꢀpyI and
9EꢀBMVC, which indicated that with the same monoꢀcationic pyridine groups, the
electronꢀwithdrawing capacity of a “fluorenone” or a “dibenzothiophene” central core
is better than that of “dibenzofuran” or “carbazole” in πꢀconjugated delocalized
molecule systems.
Twoꢀphoton photophysical properties
In order to select the optimal twoꢀphoton excitation wavelength, the TPA
crossꢀsections of WꢀpyI, SꢀpyI, and FꢀpyI generated by femtosecond laser excitation
at 750–830 nm in DMF were calculated, as shown in Fig.3. It could be clearly seen
that the TPA crossꢀsections of WꢀpyI, SꢀpyI, and FꢀpyI were largest (estimated at 681
GM, 630 GM, and 620 GM, respectively) at the wavelengths of 800 nm, 800 nm, and
790 nm, respectively. The TPA crossꢀsections of FꢀpyI at 800 nm and 790 nm were
nearly identical: 616 GM and 620 GM. Thus, to facilitate comparison, a wavelength
of 800 nm was selected as the unified twoꢀphoton excitation window to study the
twoꢀphoton photophysical properties of WꢀpyI, SꢀpyI, and FꢀpyI, especially the
influence of the three different central πꢀconjugated bridges on TPA performance.
The twoꢀphoton fluorescence spectra in DMF generated by femtosecond laser
excitation at 800 nm are shown in Fig.4. Using the twoꢀphoton induced fluorescence
method, the TPA crossꢀsections (
681 GM, 630 GM, and 616 GM, respectively while the action TPA crossꢀsections
σ×Ф) of the probes were approximately 273 GM, 267 GM, and 55 GM, respectively.
The twoꢀphoton photophysical performance of the previously reported
σ) of WꢀpyI, SꢀpyI, and FꢀpyI were estimated to be
(

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carbazoleꢀbased dicationic salt was studied as well, and showed a
σ and (σ×Ф) of
approximately 360 GM and 5.7 GM, respectively, at 800 nm [30]. Moreover,
compared with other recently reported twoꢀphoton fluorescent probes, for example,
biscarbazolylmethaneꢀbased cyanine [10],
σ
= 497 GM; ratiometric pH probes [13,
= 400 GM; DAPI [14], σ =
31], = 354 GM and 142 GM; fluorene derivatives [32],
σ
σ
0.16 GM, our new probes showed relatively higher TPA properties. Thus, as all of the
above materials had Vꢀshaped symmetric structures, the same molecular chain lengths,
and pyridinium salt electronꢀdonating arms, the central conjugate bridge plays a key
role in TPA performance. Notably, there are differences in the linkage position of
central core and two vinylꢀpyridines between SꢀpyI, FꢀpyI and WꢀpyI, which also
have a certain degree of influence on the values of TPA crossꢀsections. Therefore, the
acceptor strength and flatness of a “fluorenone” central aromatic core should be
higher than those of “dibenzothiophene,” “fluorenone,” or “carbazole,” giving WꢀpyI
stronger electronꢀwithdrawing capacity and better ICT efficiency in πꢀconjugated
delocalized systems.
DNA fluorescence switching
To investigate the DNAꢀtargeting abilities of WꢀpyI, SꢀpyI, and FꢀpyI, DNA
fluorescence switching experiments were performed in TrisꢀHCl buffer solution. The
absorption, oneꢀ and twoꢀphoton fluorescence “turnꢀon” experiments performed in the
presence of ctꢀDNA are shown in Fig. 5. Based on the trend and degree of change in
the spectra, we can determine the mode of binding between these probes and
doubleꢀstranded ctꢀDNA, and also judge the influence on probe performance and
mechanism of central conjugated bridges with different aromatic structures [33]. As
shown in Fig. 5, absorbance all gradually decreased along with slight red shifts of
absorption spectrum as the DNA concentration increased to saturation, displaying an
obvious hypochromic trend (Fig.5 I). In contrast, dramatic enhancements in both oneꢀ
and twoꢀphoton fluorescence intensity were observed as the concentration ratio
approached saturation ([DNA]:[probe] ˃ 140:1) for WꢀpyI, SꢀpyI, and FꢀpyI, which
showed an apparent hyperchromic effect (Fig.5 II and III). Based on previous research,
these Vꢀshaped conjugated probes may show intercalative binding or minor groove
binding to doubleꢀstranded DNA. Furthermore, as the DNA concentration increased to
saturation, 58ꢀfold, 30ꢀfold, and 25ꢀfold enhancements in fluorescence quantum yields
were observed for WꢀpyI, SꢀpyI, and FꢀpyI, respectively. A 350–450% increase in
TPA crossꢀsection was also observed for these new probes. The fluorescence
enhancement could be explained in the following way: when these probes recognize
nucleobases with their terminal pyridinium salt units, rotation of their pendant
vinylpyridine arms is restricted by the phosphate backbones of the DNA molecules,
greatly reducing the nonradiative decay [34,35]. We also referred to the related

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performance values of biscarbazolylmethaneꢀbased pyridinium salts bonding with
DNA in TrisꢀHCl buffer solutions, which showed an approximately 30ꢀfold
fluorescence enhancement and 3ꢀfold enhancement of the TPA crossꢀsection at
saturation [10]. Moreover, the indoleꢀbased monoꢀcationic probes INR1 and INR2 in
the presence of DNA in buffer solutions exhibited approximately 2.5ꢀfold and
2.14ꢀfold in fluorescence quantum yields, respectively [12]. Thus, WꢀpyI with a
central “fluorenone” conjugated aromatic core showed stronger DNA fluorescence
switching capacity than that with a “dibenzothiophene,” “dibenzofuran,” “carbazole”
or “indole” core, which meant that the central πꢀconjugated bridge played a leading
part in the fluorescence “turnꢀon” effect. Meanwhile, in addition to the action of the
conjugated aromatic core, the different linkage position of the central core and two
vinylꢀpyridines for these Vꢀshaped fluorescent probes had some impacts on the DNA
fluorescence switching to a certain extent as well. At present, the mechanism by
which the central πꢀconjugated bridges influence the fluorescence “turnꢀon” effect
remains unclear. According to a previous study, this phenomenon may be attributed to
the following reason [23]: when these probes bind to doubleꢀstranded DNA, they
gradually interact with the nucleobases, and the charge spacing of the “fluorenone”
aromatic core is more compatible with the interꢀphosphate distance of the DNA
molecules. Thus, compared with SꢀpyI, FꢀpyI, and the pyridine cation salts with
“carbazole” or “indole” cores, WꢀpyI had a better fluorescence effect owing to its
intercalative or minor groove binding mode.
Confocal fluorescence imaging
To explore the application of the twoꢀphoton fluorescent probes to cellular imaging,
twoꢀphoton excited fluorescence imaging of WꢀpyI, SꢀpyI, and FꢀpyI in 3T3 cells was
performed using femtosecond laser excitation at 800 nm, as shown in Fig.6. Cells
stained with WꢀpyI (Fig.6 I), SꢀpyI (Fig.6 II), and FꢀpyI (Fig.6 III) emitted bright
green and blue fluorescence, respectively. The brightness met the requirements for
monitoring and tracking in twoꢀphoton fluorescence biological imaging. In addition,
to investigate the difference between oneꢀphoton and twoꢀphoton imaging effects,
oneꢀ and twoꢀphoton fluorescence imaging of WꢀpyI, SꢀpyI, and FꢀpyI in 3T3 cells
was performed with an excitation wavelength of 405 nm and 800 nm, as exhibited in
Fig.7. Because the main emission bands for oneꢀ and twoꢀphoton fluorescence were in
the same range, to facilitate distinguishing the fluorescence strength in the images, the
twoꢀphoton fluorescence color of WꢀpyI, SꢀpyI, and FꢀpyI was set to red using builtꢀin
software. As shown in Fig.7, obvious yellow (Fig.7 Id, IId) and pink (Fig.7 IIId)
fluorescence were observed in the merged images, which indicated strong similarity
between the brightness of oneꢀ and twoꢀphoton fluorescence imaging. However, due
to relatively lower levels of background noise produced by small Rayleigh scattering

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in the twoꢀphoton absorption process, twoꢀphoton fluorescence imaging possessed
higher resolution and a better signalꢀtoꢀnoise ratio [14].
Nuclear localization and counterstaining experiments
To investigate the nuclear targeting abilities of the probes, coꢀlocalization studies
were performed using WꢀpyI, SꢀpyI, or FꢀpyI and Hoechst 33342 in 3T3 cells.
Hoechst 33342 is a commercial nuclear staining agent, and served as a baseline for
judging the accuracy of nuclear localization of WꢀpyI, SꢀpyI, and FꢀpyI by measuring
the degree of coꢀlocalization. Notably, FꢀpyI and Hoechst 33342 both emit blue
fluorescence, while the optimal oneꢀphoton excitation wavelength of Hoechst 33342
are located near 350 nm in the bioenvironment, and there is extremely weak
fluorescence emission at 405 nm for Hoechst 33342 in cells imaging. Thus, the color
of FꢀpyI fluorescence could be set to red under laser excitation at 405 nm for ease of
observation and discrimination. As shown in Fig.8, staining with WꢀpyI, SꢀpyI, or
FꢀpyI and Hoechst 33342 showed a high degree of fluorescence overlapping in the
nucleus (Fig.8 Ic, IIc, and IIIc). Using builtꢀin software, the mean coꢀlocalization
coefficients between WꢀpyI, SꢀpyI, or FꢀpyI and Hoechst 33342 were determined to
be 0.91, 0.92 and 0.89, respectively, confirming that WꢀpyI, SꢀpyI, and FꢀpyI possess
accurate nuclear localization.
To investigate counterstaining compatibility for multicolor labeling in the cellular
microenvironment, a counterstaining experiment employing WꢀpyI, SꢀpyI or FꢀpyI
coꢀstained with MTR were performed in 3T3 cells. As shown in Fig.9, red
fluorescence was emitted from the peripheral cytoplasmic regions, and the central
nuclear positions, targeted by WꢀpyI, SꢀpyI, and FꢀpyI, produced bright green (Fig.9 a
and b) and blue (Fig.9 c) fluorescence, respectively. Thus, WꢀpyI, SꢀpyI, and FꢀpyI
show good counterstaining compatibility with MTR. Moreover, the probes appear to
have entered the nuclear pores smoothly, showing their water solubility and
membrane permeability in the cellular microenvironment. Hence, they could be
applied in the study of biological imaging, which refered to physiological activity of
the nuclei to complete multicolor targeting.
Photostability studies
Photostability mainly refers to resistance of a fluorescnet probe to laser radiation
aging in biological fluorescence imaging. Under continuous excitation with a
highꢀpower laser, the molecular structure will be destroyed to a certain extent, causing
the fluorescence intensity to decrease with time, resulting in photobleaching.
Therefore, the design of the molecular structure is a key factor in photostability [36].
Here, the timeꢀdependent twoꢀphoton fluorescence intensity of WꢀpyI, SꢀpyI, and

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FꢀpyI was tested under continuous excitation with a femtosecond laser operating at
800 nm in 3T3 cells. Using the builtꢀin software, the confocal fluorescence
microscope captured an image every 30 seconds. As shown in Fig.10–Fig.13, under
excitation at the same power, the fluorescence intensity of each probe gradually
decreased until it was almostly quenched. However, the duration of fluorescence
emission varied greatly between WꢀpyI, SꢀpyI, and FꢀpyI: 3000 s, 1800 s and 540 s,
respectively. Furthermore, the fluorescence attenuation efficiency of WꢀpyI, SꢀpyI,
and FꢀpyI over the whole fluorescence decay process was 0.026%/s, 0.033%/s, and
0.081%/s, and the fluorescence attenuation amplitude (δ) was approximately 7.1%,
9.7%, and 33% in the first 300 s. Compared to some previously reported twoꢀphoton
fluorescent labels, for example, carbazoleꢀbased pyridine cation salts [11], δ = 10%
and 20%; indoleꢀbased monoꢀcationic derivatives [12], δ = 8% and 28%; dinuclear
Ru(II) polypyridyl complex [14], δ = 38%, our WꢀpyI with the “fluorenone” central
πꢀconjugated bridge showed relatively better photostability than those above probes in
twoꢀphoton fluorescence cellular imaging. This was consistent with the previous
results for TPA crossꢀsections and the DNA fluorescence switching effect. This result
can be attributed to the following two factors [37ꢀ40]: from the perspective of
molecular structure, there was a high degree of matching between the lattice structure
or charge spacing of the fluorenone aromatic core and the DNA phosphate backbone,
resulting in easier binding of the ATꢀrich regions of the minor grooves in the DNA;
from the perspective of electron energy level transition, fluorenone may show
stronger electronꢀwithdrawing capacity due to the presence of oxygen atom in the
carbonyl group, increasing the ICT efficiency between the central fluorenone bridge
and the terminal electronꢀdonating pyridinium group inside the πꢀconjugated
delocalized system. Therefore, WꢀpyI exhibited better photostability in these
homogeneous molecular structures with different central πꢀconjugated bridges.
Furthermore, these results indicate that, in the design of future twoꢀphoton
fluorescence probes, the πꢀconjugated core should be connected with atoms or groups
with high strength, good flatness, and strong electronꢀwithdrawing properties,
permitting stable electron transfer from the terminal electron donors.
Conclusions
In conclusion, a series of novel twoꢀphoton monoꢀcationic pyridinium probes with
different central πꢀconjugated aromatic cores, fluorenone, dibenzothiophene, and
dibenzofuran, were prepared. The oneꢀ and two photophysical properties showed that
WꢀpyI has the optimal electron withdrawing capacity and ICT efficiency inside the
molecular πꢀconjugated delocalized system, with a fluorescence quantum yield of
0.401 and TPA crossꢀsections of 681 GM in DMF. In the DNA fluorescence switching

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experiment, WꢀpyI showed a 58ꢀfold fluorescence enhancement and 450% TPA
crossꢀsection increasement, indicating that it had higher fluorescence “turnꢀon” effect
than the SꢀpyI, FꢀpyI, or carbazoleꢀbased fluorescent probes. Under a confocal
microscope, with excitation at wavelengths of 405 nm and 800 nm, oneꢀ and
twoꢀphoton fluorescence imaging of all of these monoꢀcationic pyridinium probes was
clear. The mean coꢀlocalization coefficient of 0.89–0.92 between these probes and
Hoechst 33342 in 3T3 cells indicated that WꢀpyI, SꢀpyI, and FꢀpyI all possess
accurate nuclear targeting abilities. The counterstain test showed that all three probes
show good counterstaining compatibility with MTR, as well as membrane
permeability in the cellular microenvironment. Finally, the fluorescence duration of
3000 s, the fluorescence attenuation efficiency of 0.026%/s, and the fluorescence
attenuation amplitude of 7.1% within 300 s illustrated that WꢀpyI shows better
photostability than the SꢀpyI, FꢀpyI, carbazoleꢀbased, and indoleꢀbased probes from
the perspective of molecular structure and electron energy level transition. Therefore,
in addition to having strong electronꢀdonating abilities and sensitive recognition units,
the excellent electronꢀwithdrawing capacity, good flatness, suitable biocompatibility
and effective binding mechnism of the central πꢀconjugated bridges are key features
in the design of twoꢀphoton fluorescent probes for nuclear imaging.
Acknowledgements
This research was supported in part by the National Natural Science Foundation of
China (Grant Nos. 61137002, 61178059 and 51172253).
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Fig.1 Normalized absorption spectra of W-pyI, S-pyI, and F-pyI in DMF, [W-pyI]=[S-pyI]=[F-pyI]=10-5 M.
148x104mm (300 x 300 DPI)

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Fig.2 Normalized one-photon fluorescence spectra of W-pyI, S-pyI, and F-pyI in DMF, [W-pyI]=[S-pyI]=[F-
pyI]=10-5 M.
148x104mm (300 x 300 DPI)

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Fig.3 Two-photon absorption cross-sections of W-pyI, S-pyI, and F-pyI in the excitation wavelength ranging
from 750 to 830 nm in DMF, [W-pyI]=[S-pyI]=[F-pyI]=10-4 M.
148x104mm (300 x 300 DPI)

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Fig.4 Two-photon fluorescence spectra of W-pyI, S-pyI, and F-pyI at excitation wavelangth of 800 nm in
DMF, [W-pyI]=[S-pyI]=[F-pyI]=10-4 M.
148x104mm (300 x 300 DPI)

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Fig.5 Absorption (I), one-photon (II) and two-photon fluorescence (III) spectra of W-pyI (a), S-pyI (b), and
F-pyI (c) as a function of different DNA concentration in Tris-HCl buffer solutions. [W-pyI]=[S-pyI]=[F-
pyI]=10-5 M, [DNA]=0–1.4×10-3 M. The arrows represent the increasing or decreasing trends.
200x150mm (300 x 300 DPI)

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Fig.6 Twoꢀphoton confocal fluorescence imaging of 3T3 cells stained with WꢀpyI (I, 0.3 µM), SꢀpyI (II, 0.3
µM) and FꢀpyI (III, 0.3 µM) for 15 min. I and II: λem = 525–575 nm; III: λem = 430–490 nm; (a) Twoꢀ
photon fluorescence images; (b) DIC pictures; (c) Overlay images of a and b. λex = 800 nm, Bar = 20 µm.
104x115mm (300 x 300 DPI)

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Fig.7 Oneꢀ and twoꢀphoton confocal fluorescence images of 3T3 cells stained with WꢀpyI (I, 0.3 µM), SꢀpyI
(II, 0.3 µM) and FꢀpyI (III, 0.3 µM) for 15 min. (a) Twoꢀphoton fluorescence images, λex = 800 nm; (b) DIC
pictures; (c) Oneꢀphoton fluorescence images, λex = 405 nm; (d) Overlay images of a and c. I and II: λem
= 525–575 nm, III: λem = 430–490 nm. Bar = 20 µm.
106x80mm (300 x 300 DPI)

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Fig.8 Confocal fluorescent images of 3T3 cells doubleꢀstained with WꢀpyI (Ia, 0.3 µM), SꢀpyI (IIa, 0.3 µM) or
FꢀpyI (IIIa, 0.3 µM) and Hoechst 33342 (b, 0.3 µM) for 15 min. (a) images of WꢀpyI (I), SꢀpyI (II) and Fꢀ
pyI (III), λex = 405 nm, I and II: λem = 525–575 nm; III: λem = 430–490 nm; (b) images of Hoechst
33342, λex = 330–380 nm, and λem = 430–490 nm; (c) overlay images of a and b. Bar = 20 µm.
80x80mm (300 x 300 DPI)

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Fig.9 Confocal fluorescence images of 3T3 cells coꢀstained with WꢀpyI (a, 0.3 µM), SꢀpyI (b, 0.3 µM), or Fꢀ
pyI (c, 0.3 µM) and MTR (a–c, 0.3 ꢁM) for 15 min. For WꢀpyI and SꢀpyI, λex = 405 nm, and λem = 525–
575 nm. For FꢀpyI, λex = 330–380 nm, and λem = 430–490 nm. For MTR, λex = 575 nm, and λem = 580–
650 nm. Bar = 20 µm.
78x26mm (300 x 300 DPI)

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Fig.10 Timeꢀdependent twoꢀphoton fluorescence imaging of 3T3 cells stained with WꢀpyI (0.3 ꢁM) for 15
min, λex = 800 nm, and λem = 525–575 nm. Bar = 50 µm.
105x93mm (300 x 300 DPI)

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Fig.11 Timeꢀdependent twoꢀphoton fluorescence imaging of 3T3 cells stained with SꢀpyI (0.3 ꢁM) for 15
min, λex = 800 nm, and λem = 525–575 nm. Bar = 50 µm.
105x95mm (300 x 300 DPI)

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Fig.12 Timeꢀdependent twoꢀphoton fluorescence imaging of 3T3 cells stained with FꢀpyI (0.3 ꢁM) for 15 min,
λex = 330–380 nm, and λem = 430–490 nm. Bar = 50 µm.
106x80mm (300 x 300 DPI)

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Fig.13 Normalized two-photon fluorescence intensity attenuation of W-pyI, S-pyI, and F-pyI as time
variation under continuous irradiation.
148x104mm (300 x 300 DPI)

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Scheme 1. Synthesis of mono-cationic fluorescent probes
77x33mm (300 x 300 DPI)