A series of carbazole cationic compounds with large two-photon absorption cross sections for imaging mitochondria in living cells with two-photon fluorescence microscopy

Article abstract of DOI:10.1007/s10895-010-0736-8

A series of carbazole cationic compounds based on donor-@II-acceptor (D-II-A) structure were synthesized and characterized. They exhibit large two-photon absorption cross sections when excited by a 810 nm a laser beam, and their photophysical properties show that the intramolecular charge transfer (ICT) character is predominant. Moreover these compounds can easily pass though the intact cell membrane of living cells, amongst, 3-(1-hydroxyethyl-4- vinylpyridium iodine)-N-butyl carbazole (9B-HVC) has been proven to be capable of accumulating within the mitochondria possessing large membrane potential and imaging this organelle in living cells by means of two-photon fluorescence microscopy. At the same time usable fluorescent photos can be obtained at lower incident excitation power (5 mW) and low-micromolar concentrations (2 μM), which does not result in significant reduction in cell viability over a period of at least 24 h.

Full text of DOI:10.1007/s10895-010-0736-8

J Fluoresc (2011) 21:497–506
DOI 10.1007/s10895-010-0736-8
ORIGINAL PAPER
A Series of Carbazole Cationic Compounds with Large
Two-Photon Absorption Cross Sections for Imaging
Mitochondria in Living Cells with Two-Photon
Fluorescence Microscopy
Xin Liu & Yuming Sun & Yuanhong Zhang & Ning Zhao &
Hongshi Zhao & Guancong Wang & Xiaoqiang Yu &
Hong Liu
Received: 5 July 2010 /Accepted: 28 September 2010 /Published online: 16 October 2010
#
Springer Science+Business Media, LLC 2010
Abstract A series of carbazole cationic compounds based
on donor- Π—acceptor (D-Π-A) structure were synthesized
and characterized. They exhibit large two-photon absorp-
tion cross sections when excited by a 810 nm a laser beam,
and their photophysical properties show that the intramo-
lecular charge transfer (ICT) character is predominant.
Moreover these compounds can easily pass though the
intact cell membrane of living cells, amongst, 3-(1-
hydroxyethyl -4-vinylpyridium iodine)-N-butyl carbazole
(9B-HVC) has been proven to be capable of accumulating
within the mitochondria possessing large membrane poten-
tial and imaging this organelle in living cells by means of
two-photon fluorescence microscopy. At the same time
usable fluorescent photos can be obtained at lower incident
excitation power (5 mW) and low-micromolar concentra-
tions (2 μM), which does not result in significant reduction
in cell viability over a period of at least 24 h.
Introduction
Extensive biochemical studies on mitochondria have
proved that they play a cardinal role on cell function and
dysfunction. Mitochondria not only contribute to the
generation of energy essential for the survival and prolif-
eration of eukaryotic cells, but also implicate in the
regulation of programmed cell death, reactive oxygen
species generation, and maintenance of calcium homeosta-
sis [1–4]. In particular, mitochondria show remarkable
plasticity, mobility, and morphological heterogeneity occur-
ring in conjunction with the metabolic state of the cell, cell
cycle, cellular development and differentiation, and patho-
logical states [5–7]. Therefore, many approaches have been
adopted to study the structure and function of mitochondria.
Fluorescence imaging technique with application of
fluorescent probes allowing high-resolution visualization
of different organelles has been considered as the valid
method and used in various studies of mitochondria [8, 9].
Currently, two-photon fluorescence microscopy (TPM), a
revolutionary development of fluorescence imaging tech-
nique, exhibits many unique capabilities in imaging of
living specimens. Adopting two-photon absorption mecha-
nism, TPM utilize the long wavelength incident beam in IR
region as exciting source thus avoiding the photodamage of
UV-vis to living specimen [10, 11]. Recently, by using
TPM, many important biological works relevant to study of
life phenomenon have been accomplished [12–16], includ-
ing those involved to mitochondria [17, 18]. However, both
of theoretical prediction and practical work have indicated
that the superiority of TPM have been hampered due to
using the conventional fluorescent probes with small two-
photon absorption cross sections (δTPA), which demanded
.
.
Keywords Two-photon microscopy Mitochondria
.
Fluorescent probe Living cells
:
:
:
:
:
:
X. Liu Y. Zhang N. Zhao H. Zhao G. Wang X. Yu (*)
H. Liu (*)
State Key Laboratory of Crystal Materials, Shandong University,
Jinan 250100, People’s Republic of China
e-mail: yuxq@sdu.edu.cn
H. Liu
e-mail: hongliu@sdu.edu.cn
Y. Sun
Optics Department, Shandong University,
Jinan 250100, People’s Republic of China

498
J Fluoresc (2011) 21:497–506
impractically high laser power [10, 19]. When these probes
are used on TPM, only a little portion of the large excitation
power could be utilized to generate fluorescence, and the
rest would cause heatdamage to the living specimen and
high-order photobleaching of substrate [20, 21]. Accord-
ingly, design and synthesis of two-photon induced fluores-
cence (TPF) probes with large δTPA have been being desired
and some excellent results on this work have been reported
[22–25]. To our best knowledge, TPF probes targeting to
mitochondria are seldom reported. Therefore, it is signifi-
cant yet practically needed to develop efficient TPF
mitochondria probes with large δTPA, which will improve
the further study of the complex organelles.
Until now, many mitochondria-targeted antioxidants,
anticancer drugs and fluorescent probes of mitochondria
have been exploited in response to the large mitochondrial
membrane potential [26–28]. These molecules commonly
are the lipophilic cationic compounds with the large ionic
radius which is inversely proportional to the enthalpy
required to move the cationics through the hydrophobic
core of the membrane [29, 30]. Upon this point, we design
and synthesize a series D-Π-A type carbazole cationics to
explore the TPF mitochondria probes (Scheme 1). As well
known, carbazole has been shown to be promising substrates
for two-photon absorption (TPA) applications due to their rigid
plane and long conjugation length [31]. In this paper, the
carbazole cationic compounds, 3-(1-methyl-4-vinylpyridium
iodine) carbazole (MVC), 3-(1-hydroxyethyl-4-vinylpyridium
iodine)carbazole (HVC), 3-(1-methyl-4-vinylpyridium
iodine)-N-butyl carbazole (9B-MVC) and 3-(1- hydroxyethyl
-4-vinylpyridium iodine)-N-butyl carbazole (9B-HVC), were
synthesized. In these compounds, carbazole was introduced
to probe molecules as the donor, and the molecular
pi-electron conjugated system was enlarged by forming—
CH=CH– in 3-position of carbazole. The cationic pyridinium
unit as acceptor groups was bound on other side of vinyl
bond [32]. Compared to MVC and HVC, the introduction of
a butyl group in place of a H atom at 9-position of carbazole
in 9B-MVC and 9B-HVC is to enhance the electron-donating
ability of carbazole, and a hydroxylethyl in HVC and 9B-
HVC in the place of the methyl in MVC and 9B-MVC to
quaternize the N-pyridinium atom is for better hydrophilicity.
In these structures, the cationic pyridinium can offer net
positive charge for the molecular, and the large conjugate
system which is composed of carbazole, vinyl and cationic
pyridinium ensures the large ionic radius. Excitedly, these
compounds not only exhibit the large δTPA, but also 9B-HVC
can exclusively label mitochondria.
Furthermore, their single- and two-photon spectroscopic
properties in various environments were investigated to
elucidate the luminescence mechanism systematically. The
imaging application of 9B-HVC for mitochondria was
carefully evaluated with wide-field fluorescence microscopy
(WFM) and TPM. In addition, the cytotoxicity was also
analyzed for choosing the suitable staining conditions.
Experimental
Synthesis
N-Butylcarbazole(1b) 13.2 g (80 mmol) of carbazole were
added into the solution of 28 g (500 mmol) KOH in 60 mL
acetone at the conditions of stirring and room temperature.
After 4 h, a solution of 1-bromobutane (13 mL, 120 mmol)
in acetone was added to the mixture above and then
maintained 24 h. The final solution was poured into 1 L
water and yellow suspended matter was obtained. The
products were filtrated and recrystallized in ethanol. White
solid were obtained, yield: 81%.1H NMR (300 MHz,
CDCl3) δ(ppm): 8.10 (d, J=7.8 Hz, 2 H), 7.44(m, 4 H),
7.22(m, 2 H), 4.3(t, J=7.2 Hz, 2 H), 1.80–1.90 (m, 2 H),
1.34–1.46 (m, 2 H), 0.94 (t, J=7.4 Hz, 3 H).
3-Bromocarbazole(2a)/3-bromo-N-butylcarbazole(2b) To
the CH2Cl2 solution of 3.56 g (20 mmol) N-bromosucci-
nimide (NBS) was added the of carbazole (3.34 g, 20 mmol)
or N-butylcarbazole (4.46 g, 20 mmol) dissolved in
CH2Cl2/acetone mixture solution, and the resulting solu-
tion was stirred at room temperature for 24 h. The mixture
was washed several times with water and saturated NaCl
solution, the organic phases was separated and the excess
organic solvent was removed by vacuum distillation. The
residue was purified by column chromatography with ethyl-
Scheme 1 Synthetic route of MVC, HVC, 9B-MVC and 9B-HVC

J Fluoresc (2011) 21:497–506
499
acetate/petroleum ether (10:1) as eluent, finally the white
solid was obtained for 3-bromocarbazole, yield: 62%. H
(1-Methyl-4-Vinylpyridium Iodine)-N-Butyl Carbazole(9B-
MVC) and 3-(1- Hydroxyethyl -4-Vinylpyridium Iodine)-
N-Butyl Carbazole(9B-HVC) After refluxing the 3-(4-
vinylpyridium)carbazole/3-(4-vinylpyridium)-N-butylcarbazole
with excess ICH3/ICH2CH2OH in acetone for overnight,
the crude product was collected as powder. After recrys-
talization from methanol, title products were obtained. For
MVC, yellow solid, yield: 85%, 1H NMR (400 MHz,
DMSO-d6) δ(ppm): 11.65 (s, 1H), 8.80(d, J=6.8 Hz, 2 H),
8.56 (s, 1H), 8.16–8.23(m, 4 H), 7.83–7.85(m, 1H), 7.43–
7.60 (m, 4H), 7.23–7.27 (m, 1H), 4.23(s, 3H). 13C NMR
(100 MHz, DMSO-d6): δ 153.6, 145.2, 143.0, 141.7,
140.8, 126.8, 126.6, 126.5, 123.5, 123.2, 122.8, 121.7,
120.8, 120.2, 120.0, 112.1, 112.0, 47.1. HRMS (m/z): [M-
I]+ calcd for C20H17N2I, 285.36; found, 285.2. Elemental
analysis calcd (%) for C20H17N2I (412.27): C 58.27, H
4.16, N 6.79; found: C 58.07, H 4.26, N 6.93.
1
NMR (300 MHz, DMSO) δ(ppm):11.4(s, 1H), 8.35(d, J=
1.8 Hz, 1H), 8.17(d, J=7.8 Hz, 1H), 7.39–7.51 (m, 4 H),
7.15–7.20 (m, 1H). White yellow oil was obtained for 3-
1
bromo-N-butylcarbazole, yield: 53%. H NMR (400 MHz,
DMSO) δ(ppm): 8.41(d, J=1.8 Hz, 1H), 8.20(d, J=7.8 Hz,
1H), 7.45–7.60(m, 4 H), 7.23(t, J=7.7 Hz, 1H), 4.30(t, J=
7.0 Hz, 2 H), 1.64–1.71(m, 2 H), 1.18–1.27(m, 2 H), 0.81(t,
J=7.3 Hz, 3 H).
3-(4-Vinylpyridium)Carbazole(3a) The 3-bromo-carbazole
(0.50 g, 2 mmol) was added into a high pressure bottle
containing the mixture of Palladium(II) acetate (4.0 mg,
0.02 mmol) and tri-o-tolyl phosphine (40.0 mg, 0.13 mmol),
then to which was added the solvent pair (triethylamine
6 mL/tetrahydrofuran 18 mL) and 4-vinylpyridine (0.42 g,
4 mmol). The bottle was sealed after bubbling 10 min with
nitrogen. After keeping the system under ~100 °C for
three days, the mixture was poured into water and orange
solid was obtained. After filtration, the precipitate was
purified by column chromatography with ethyl-acetate/
petroleum ether (1:2) as eluent, and the yellow solid was
obtained, yield: 35%.1H NMR (400 MHz, DMSO-d6)
δ(ppm): 11.42 (s, 1H), 8.53(d, J=5.9 Hz, 2 H), 8.43 (s,
1H), 8.15(d, J=7.7 Hz, 1H), 7.69–7.75 (m, 2 H), 7.56(d,
J=6.0 Hz, 2 H), 7.50–7.52(m, 2 H), 7.40–7.43(m, 1H),
7.18–7.26(m, 2 H).
1
For HVC, red solid, yield: 78%, H NMR (400 MHz,
DMSO-d6) δ(ppm): 11.65 (s, 1H), 8.82(d, J=6.8 Hz, 2 H),
8.57 (s, 1H), 8.16–8.24(m, 4H), 7.83–7.86(m, 1H), 7.43–
7.60(m, 4 H), 7.25(t, J=7.4 Hz, 1H), 5.26(s, 1H), 4.54(t,
J=4.7 Hz, 2 H), 3.87(s, 2 H). 13C NMR (100 MHz,
DMSO-d6): δ 154.1, 144.8, 143.2, 141.7, 140.8, 126.8,
126.7, 126.5, 123.5, 123.2, 122.8, 121.7, 120.8, 120.2,
120.0, 112.1, 112.0, 62.3, 60.5. HRMS (m/z): [M-I]+ calcd
for C21H19N2IO, 315.39; found, 315.2. Elemental analysis
calcd (%) for C21H19N2IO (442.29): C 57.03, H 4.33, N
6.33; found: C 56.92, H 4.39, N 6.47.
For 9B-MVC, yellow solid, yield: 89%, 1H NMR
(400 MHz, DMSO-d6) δ(ppm): 8.80(d, J=6.8 Hz, 2 H),
8.60(s, 1H), 8.18–8.23(m, 4 H), 7.88–7.90(m, 1H), 7.74(d,
J=8.6 Hz, 1H), 7.67(d, J=8.2 Hz, 1H), 7.51–7.55(m, 2 H),
7.29(t, J=7.4 Hz, 1H), 4.45(t, J=7.0 Hz, 2 H), 4.24(s, 3 H),
1.76–1.80(m, 2 H), 1.29–1.35(m, 2 H), 0.89(t, J=7.4 Hz,
3 H). 13C NMR (100 MHz, DMSO-d6): δ 153.5, 145.2,
142.8, 141.9, 141.1, 126.9, 126.7, 126.6, 123.2, 123.1,
122.5, 121.6, 120.9, 120.4, 120.1, 110.6, 110.4, 47.1, 42.7,
31.2, 20.2, 14.2. HRMS (m/z): [M-I]+ calcd for C24H25N2I,
341.47; found, 341.4. Elemental analysis calcd (%) for
C24H25N2I (468.37): C 61.54, H 5.38, N 5.98; found: C
61.76, H 5.39, N 6.17.
3-(4-Vinylpyridium)-N-Butylcarbazole(3b) 2.0 g (6.0 mmol)
of 3-bromo-N-butyl-carbazole was added into a flask
containing mixture of palladium(II) acetate (0.13 g,
0.6 mmol), tri-o-tolylphosphine (0.54 g, 1.8 mmol) and
K2CO3(4.1 g, 30.0 mmol), then to which was added the
solvent NMP (50 mL) and 4-vinylpyridine (1.68 g,
16.0 mmol). Under the protection of argon, the system
was heated at 130 °C for 2 days and dark-red suspending
solution was obtained. After pouring the reaction solution
to 1 L H2O, the mixture was extracted by ethyl acetate. The
organic phase was separated and the excess organic solvent
was removed by vacuum distillation and dark-red solution
was obtained. The solution was purified by column
chromatography with ethyl-acetate/petroleum ether (1:2)
as eluent, and the titled product was obtained as yellow
solid. Yield: 60%. 1H NMR (400 MHz, DMSO-d6) δ(ppm):
8.54(d, J=6.0 Hz, 2 H), 8.46 (d, J=0.9 Hz, 1H), 8.19(d, J=
7.7 Hz, 1H), 7.78–7.80(m, 1H), 7.72(d, J=16.4 Hz, 1H),
7.63(t, J=8.4 Hz, 2 H), 7.57(d, J=6.0 Hz, 2 H), 7.46–7.50
(m, 1H), 7.22–7.28(m, 2 H), 4.41(t, J=7.0 Hz, 2 H), 1.73–
1.78(m, 2 H), 1.28–1.34(m, 2 H), 0.89(t, J=7.4 Hz, 3 H).
For 9B-HVC, yellow solid, yield: 92%, 1H NMR
(400 MHz, DMSO-d6) δ(ppm): 8.82(d, J=6.8 Hz, 2 H),
8.59(s, 1H), 8.19–8.24(m, 4H), 7.89–7.91(m, 1H), 7.74(d,
J=8.6 Hz, 1H), 7.67(d, J=8.2 Hz, 1H), 7.50–7.56(m, 2 H),
7.29(t, J=7.4 Hz, 1H), 5.24(t, J=5.2 Hz, 1H), 4.54(t, J=
4.8 Hz, 2H), 4.45(t, J=7.0 Hz, 2H), 3.85–3.89(m, 2H), 1.78
(t, J=7.5 Hz, 2H), 1.29–1.35(m, 2H), 0.89(t, J=7.4 Hz,
3H). 13C NMR (100 MHz, DMSO-d6): δ 154.0, 144.9,
143.0, 141.9, 141.1, 126.9, 126.8, 126.7, 123.2, 123.1,
122.4, 121.6, 120.9, 120.4, 120.1, 110.6, 110.4, 62.3, 60.5,
42.7, 31.2, 20.2, 14.2. HRMS (m/z): [M-I]+ calcd for
C25H27N2IO, 371.49; found, 371.4. Elemental analysis
3-(1-Methyl-4-Vinylpyridium iodine)Carbazole(MVC), 3-(1-
Hydroxyethyl-4-Vinylpyridium iodine)Carbazole(HVC), 3-

500
J Fluoresc (2011) 21:497–506
Fig. 1 UV-vis absorption spectra (a) and single-photon excited fluorescence spectra (b) of four compounds in MeCN at an identical concentration
of 5.0×10−6
M
calcd (%) for C25H27N2IO(498.40): C 60.25, H 5.46, N
5.62; found: C 60.22, H 5.50, N 5.70.
where the subscripts s and r refer to the sample and the
reference material, respectively. δ is the TPA cross sectional
value, c is the concentration of the solution, n is the
refractive index of the solution, F is two-photon excited
fluorescence integral intensity and Φ is the fluorescence
quantum yield.
Instruments and Measurements
The UV-visible-near-IR absorption spectra of dilute sol-
utions were recorded on a UV-2550 spectrophotometer
using a quartz cuvette having 1 cm path length. One-photon
fluorescence spectra were obtained on a HITACH F-4500
spectrofluorimeter equipped with a 450-W Xe lamp. Two-
photon ones were noted on a SpectroPro300i and the pump
laser beam come from a mode-locked Ti:sapphire laser
system at the pulse duration of 200 fs, with a repetition rate
of 76 MHz (Coherent Mira900-D).
TPA cross sections have been measured using the two-
photon induced fluorescence method [33, 34], and thus
cross sections can be calculated with coumarin 307 in water
as reference [34] by means of equation (1)
Wide-Field Fluorescence Microscopy Imaging
and Two-Photon Fluorescence Microscopy Imaging
of SiHa Cells Stained with 9B-HVC
SiHa cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with penicillin/streptomy-
cin and 10% bovine calf serum with 5% CO2 at 37 °C. The
9B-HVC were dissolved in DMSO at a concentration of
1 mM and subsequently diluted in DMEM medium for
using. Cultured cells grown on glass coverslips were
incubated with 9B-HVC and/or MitoTracker Orange
(MTO, Invitrogen) in a 5% CO2 incubator at 37 °C and
then imaged with WFM and TPM.
6r cr nr Fs
WFM images were acquired with an Olympus IX71
inverted microscope coupling with a CCD and a DP
ds ¼ dr
ð1Þ
6s cs ns Fr
Table 1 Photophysical properties of the compounds in different environment
Compound
H2O
MeCN
Gly
1 1/1 2 [nm]
ε [M−1⋅cm−1
]
Φ [%]
1 1/1 2 [nm]
ε [M−1⋅cm−1
]
Φ [%]
1 1/1 2 [nm]
ε [M−1⋅cm−1
]
Φ [%]
MVC
411/561
415/564
420/568
427/570
2.70×104
3.18×104
2.78×104
3.04×104
0.31
0.31
0.48
0.39
418/561
421/558
431/570
434/570
2.64×104
2.84×104
3.70×104
3.04×104
6.47
6.69
7.45
9.66
429/552
431/553
438/562
443/563
2.40×104
2.82×104
3.26×104
2.78×104
19.08
15.63
21.68
25.30
HVC
9B-MVC
9B-HVC
1 1 and 1 2 are linear absorption, single- photon fluorescent maximum peak, respectively; ε is molar absorptivity; Φ is single-photon fluorescence
quantum yield determined using coumarin 307 (Φ=0.56) [36] as the standard

J Fluoresc (2011) 21:497–506
501
Fig. 2 Single-photon excited fluorescence spectra of compounds in glycerol/water mixtures at room temperature: a MVC, b HVC, c 9B-MVC, d
9B-HVC. Concentration: 5.0×10−6
M
controller software. The fluorescence of 9B-HVC and MTO
were excited and collected through U-MNIBA3 and U-
MWG2, respectively.
All of TPM microscopic photos were obtained with
Olympus FV 300 Laser Confocal System with a 60× water
objective (N.A.=1.25) and photomultiplier tubes. A Ti:
sapphire laser (Coherent) was used to excite specimen at
800 nm. The total power provided by laser source can
maintain stable and the incident power on samples was
modified by means of an attenuator and examined with Power
Monitor (Coherent). Fluorescence of 9B-HVC was collected
with a beam splitter DM570 and BA510-540 nm bandpass
emission filter combination. The differential interference
contrast (DIC) image was taken with 488 nm Arion laser.
Cell-Viability Assay
Fig. 3 The plots of the log(ф) versus log(η) of these compounds in
glycerol/water mixtures. Black: MVC, Red: HVC, Green: 9B-MVC
and Blue: 9B-HVC
The study of the effect of 9B-HVC on viability of cells was
carried out using the methylthiazolyldiphenyl-tetrazolium

502
J Fluoresc (2011) 21:497–506
Fig. 4 a Two-photon excited fluorescence spectra of four compounds
in MeCN at the optimal excitation wavelength of 810 nm. b Two-
photon absorption cross sections of four compounds in MeCN versus
excitation wavelengths. All concentration: 1×10–4 M. The experi-
mental uncertainty in the cross-sections values is 15%
Fig. 5 Two-photon excited fluorescence spectra of compounds in four polar solvents: a MVC, b HVC, c 9B-MVC, d 9B-HVC. Excitation
wavelength: 800 nm. All concentration: 1×10–4
M

J Fluoresc (2011) 21:497–506
503
Table 2 TPE maximum emission and TPA cross section values of compounds in various solvents
Solvent
MVC
HVC
9B-MVC
9B-HVC
1 [nm]
δ [GM]
1 [nm]
δ [GM]
1 [nm]
δ [GM]
1 [nm]
δ [GM]
MeCN
MeOH
EtOH
585
585
579
587
268
309
135
99
580
580
583
580
283
239
112
101
599
593
590
598
522
326
160
120
600
594
590
597
373
333
108
79
DMSO
1 is two-photon fluorescent maximum peak; δ is two-photon absorption cross sections determined using coumarin 307 (δ=27.7 GM) [34] at
800 nm; 1GM=10−50 cm4 ⋅s⋅photon−1 ; Concentration of samples for δ: 100 μM; error limit: 15%
bromide (MTT) assay [35]. SiHa cell cultures were grown
in 96-well plates (ca. 1×104 cells/well) in DMEM contain-
ing 10% bovine calf serum in a 5% CO2 incubator at 37 °C.
For examining the short-term cytotoxic effect, cells were
then incubated with 9B-HVC at 2 μM for 24, 48 and 72 h,
then MTT assay was used as described. Non-treated cells
were used as control. Each individual cytotoxic experiment
was repeated for three times.
the electron–acceptor and the electron–donor in carbazole
derivatives [37, 38]. In order to further elucidate whether
the ICT character is predominant in the photophysical
behavior of these compounds, the one-photon fluorescent
spectra of compounds in glycerol/water mixture, which is
often used to clarify the ICT character of fluorophores [39],
were recorded. The results show that when increasing
glycerol content, the fluorescent intensity distinctly enhan-
ces (Fig. 2). And the plots (Fig. 3) of log Ф of compounds
versus the log η, where η is viscosity of each glycerol/water
mixture obtained from the Handbook [40], show linear
relationship [39]. Thus we suggest that in these compounds,
the larger dipole moment in the excited state contributed by
the intensive ICT interacts strongly with the water, which
could lead to intramolecular twist of the vinyl group
resulting in nonradiative decay by forming twisting
intramolecular charge transfer (TICT) state. However, with
the decrease of the polarity (εglycerol=37, εwater=81.2) and
increase of the viscosity (ηglycerol=1,499, ηwater=1.005) of
solvents, the molecular torsional motions of the vinyl
groups would be restricted, consequently the fluorescence
emission of compounds restores. Carefully observing
Table 1, these compounds have lowest Ф in water, but
they can fluoresce in non-water environment such as
MeCN, furthermore the Ф of all four compounds in viscous
non-water environment such as glycerol is highest in the
three environments, which also shows the influence of the
environments on fluorescence of compounds due to the
predominant actions of ICT character. Thus, if these
Results and Disscussion
Linear Absorption and Single-Photon Excited Fluorescence
of Compounds
The UV-vis absorption (a) and one-photon fluorescence (b)
spectra of compounds in MeCN are shown in Fig. 1 and the
relevant photophysical properties of these compounds are
summarized in Table 1. These compounds exhibit the
maximum absorption at 418 nm - 434 nm and have large
εmax of 2.64×104 M−1. cm−1–3.70×104 M−1. cm−1 in
MeCN. From Fig. 1b, one can observe that 9B-HVC/9B-
MVC emit the stronger fluorescence than HVC/MVC and
the emission maximum of these compounds are at 561–
570 nm in MeCN. The higher fluorescent intensity of 9B-
HVC/9B-MVC might be attributed to the substitution of the
n-butyl for H in carbazole moiety which possesses the
electron-donating ability. It is well known that an intramo-
lecular charge transfer (ICT) state can be formed between
Fig. 6 Wide-field fluorescence
images of SiHa cancer cells
incubated with 9B-HVC (2 μM)
for overnight and with MTO (25
nM) for 30 min. a Fluorescence
of 9B-HVC, b fluorescence of
MTO, and c the merged picture

504
J Fluoresc (2011) 21:497–506
spectra have maximum emission longer than 570 nm,
which benefiting visualization of living specimen, at the
same time the two-photon excited fluorescence of com-
pounds exhibit evident solvatochromism due to their ICT
character [41, 42]. It is worth underlining that these four
compounds exhibit large δTPA in all solvents and are more
than 1 or 2 order of magnitude larger than that of many
commercial fluorophores widely used in biology, such as
fluorescein (38 GM), Indo-1 (4.5 GM), and EB (7 GM)
[26, 43].
Visualization of Mitochondria Stained with 9B-HVC
in WFM and TPM
To demonstrate the potentiality of 9B-HVC with larger δTPA
for imaging mitochondria in living cells, a standard double-
staining experiment with 9B-HVC and MTO (a commercial
mitochondria probe) has been carried out. The SiHa cells
were firstly incubated with 9B-HVC at 2 μM overnight in a
5% CO2 incubator at 37 °C and then stained with 25 nM
MTO. Subsequently, the cells were washed to remove the
excess of unbound probe and imaged by WFM immediate-
ly. As shown in Fig. 6, the fluorescent picture of 9B-HVC
(Fig. 6a) is wholly consistent with that of MTO (Fig. 6b),
and the substantial overlap between 9B-HVC and MTO in
the merged image (Fig. 6c) emits orange fluorescence.
According to Fig 6, the position, shape and amounts of the
mitochondria labeled by 9B-HVC are the same as that of
MTO. These results can confirm that 9B-HVC is a potential
fluorescent mitochondria probe. To further examine the
ability of 9B-HVC for imaging living cell on TPM, the
same cell stained by 9B-HVC was imaged with WFM, DIC
and TPM simultaneously (Fig.7). Figure 7a was obtained
with WFM and Fig. 7b was acquired by TPM using a
5 mW of beam at 800 nm, 200 fs pulses and 76 MHz (the
power is safe for living specimen) [16]. The DIC image
(Fig. 7c) of cell was taken with 488 nm Arion laser
immediately prior to the TPM imaging and Fig. 7d
presented the merged picture of Fig. 7b and c. Compared
with Fig. 7a, b show preferable contrast with high
brightness and Fig. 7d gives the clear distribution of
mitochondria, which can better overlay that from DIC
picture. Altogether these results demonstrate that 9B-HVC
can exclusively target to mitochondria and be applicable to
TPM imaging.
Fig. 7 Images of SiHa cells incubated with 2 μM 9B-HVC for
overnight. a WFM image, b TPM image, c DIC image, d merge image
of b and c
cationics can exclusively accumulate in mitochondria, they
can possess the added advantage that it is essentially
nonfluorescence in aqueous solutions and only becomes
fluorescent once it accumulates in the lipid environment of
mitochondria. Hence, their background fluorescence is low
even negligible, which enables researchers to clearly
visualize mitochondria in living cell.
Two-Photon Excited Fluorescence of Compounds
As shown in Fig. 4a, these compounds presented a bright
two-photon excited fluorescence centered at 573–591 nm in
MeCN with an optimal excitation wavelength of 810 nm
and 9B-MVC/9B-HVC exhibit higher TPF than that of
MVC/HVC which was similar with the photophysical
behavior by one-photon excitation. In order to investigate
the influence of excited wavelengths on δTPA, the laser was
tuned from 740 nm to 830 nm, and the two-photon
absorption spectra of these compounds were shown in
Fig. 4b. The maximum δTPA of these compounds occur at a
wavelength of 810 nm and their highest δTPA values are
calculated to be 268 GM (MVC), 283 GM (HVC), 522 GM
(9B-MVC) and 373 GM (9B-HVC) in MeCN, respectively.
Furthermore, the two-photon excited fluorescence spectra
of compounds in four common solvents were measured at a
wavelength of 800 nm which is an optimal excitation
wavelength provided by commercial mode-locked Ti:
sapphire laser source (Fig. 5) and the corresponding δTPA
are listed in Table 2. From Fig. 5, one can see that all
Table 3 Cytotoxicity Data (SiHa, 2 μM)a
incubate time
24 h
48 h
72 h
% cell survival
99 3
86 3
71 7
a Cell viability was quantified by the MTT assay (mean SD)

J Fluoresc (2011) 21:497–506
505
9. Kahlert S, Zündorf G, Reiser G (2008) Detection of de- and
hyperpolarization of mitochondria of cultured astrocytes and
neurons by the cationic fluorescent dye rhodamine 123. J
Neurosci Methods 171(1):87–92
10. So PTC, Dong CY, Masters BR, Berland KM (2000) Two-photon
excitation fluorescence microscopy. Annu Rev Biomed Eng 2:399–429
11. Rubart M (2004) Two-photon microscopy of cells and tissue. Circ
Res 95(12):1154–1166
12. Wang BG, König K, Halbhuber KJ (2010) Two-photon microscopy
of deep intravital tissues and its merits in clinical research. J
Microsc 238(1):1–20
13. Yuan T, Gao SS, Saggau P, Oghalai JS (2010) Calcium imaging of
inner ear hair cells within the cochlear epithelium of mice using two-
photon microscopy. J Biomed Opt 15(1):016002
Short-Term Cytotoxicity of 9B-HVC
Cytotoxicity of 9B-HVC was evaluated in SiHa cells by
MTT assay. The cell viability assay data of SiHa cells
treated with 2 μM 9B-HVC during different incubation
time was quantified (Table 3). The results show that SiHa
cells show nearly 100% viability for 24 h, and longer
incubating time can result in decreased cell survival, 86%
for 48 h and 71% for 72 h. Thus the concentrations at a
micromolar order of magnitude and short treating time for
9B-HVC could be considered as nontoxic.
14. Merlo F, Balduzzi R, Cupello A, Robello M (2004) Immunocy-
tochemical study by two photon fluorescence microscopy of the
distribution of GABA(A) receptor subunits in rat cerebellar
granule cells in culture. Amino Acids 26(1):77–84
Conclusion
15. Ghosh S, Kim D, So P, Blankschtein D (2008) Visualization and
quantification of skin barrier perturbation induced by surfactant-
humectant systems using two-photon fluorescence microscopy. J
Cosmet Sci 59(4):263–89
16. Koepsell H (2007) In vivo two-photon fluorescence microscopy
opens a new area for investigation of the excretion of cationic
drugs in the kidney. Kidney Int 72(4):387–388
17. Dedov VN, Cox GC, Roufogalis BD (2001) Visualisation of
mitochondria in living neurons with single- and two-photon
fluorescence laser microscopy. Micron 32(7):653–660
18. Mqller M, Mironov SL, Ivannikov MV, Schmidt J, Richter DW
(2005) Mitochondrial organization and motility probed by two-
photon microscopy in cultured mouse brainstem neurons. Exp
Cell Res 303(1):114–127
19. Feijό JA, Cox G (2001) Visualization of meiotic events in intact
living anthers by means of two-photon microscopy. Micron 32
(7):679–684
We have developed a series of two-photon fluorescent
compounds based on donor- Π—acceptor (D-Π-A) struc-
ture derived from carbazole. These compounds exhibit TPF
features and have larger δTPA than that of the conventional
fluorescent probes. The further study demonstrates that 9B-
HVC possesses significant potential for imaging mitochon-
dria in TPM with better specificity for mitochondria
localization and without apparent cytotoxic effects at
micromolar concentration. The results reported in this paper
provide a useful design and synthesis strategy for the two-
photon fluorescent probes of mitochondria.
Acknowledgement For financial support, we thank the National
Science Foundation of China (50673053, 50173015, 50925205,
50990303 and 50872070) and NSFC/RGC (50218001). Also Open
Project of State Key Laboratory of Supramolecular Structure and
Materials, Independent Innovation Foundation of Shandong Universi-
ty, (2009JC011) and the Program of Introducing Talents of Discipline
to Universities in China (111 programNo. b06015).
20. Chen TS, Zeng SQ, Luo QM, Zhang ZH, Zhou W (2002) High-
order photobleaching of green fluorescent proteininside live cells
in two-photon excitation microscopy. Biochem Biophys Res
Commun 291(5):1272–1275
21. Patterson GH, Piston DW (2000) Photobleaching in two-photon
excitation microscopy. Biophys J 78(4):2159–2162
22. Kim HM, Cho BR (2009) Two-photon probes for intracellular free
metal ions, acidic aesicles, and lipid rafts in live tissues. Acc
Chem Res 42(7):863–872
23. Lim CS, Kang DW, Tian YS, Han JH, Hwang HL, Cho BR
(2010) Detection of mercury in fish organs with a two-photon
fluorescent probe. Chem Commun 46(14):2388–2390
24. Lee JH, Lim CS, Tian YS, Han JH, Cho BR (2010) A two-photon
fluorescent probe for thiols in live cells and tissues. J Am Chem Soc
132(4):1216–1217
25. Morales AR, Schafer-Hales KJ, Yanez CO, Bondar MV, Przhonska
OV, Marcus AI, Belfield KD (2009) Excited state intramolecular
proton transfer and photophysics of a new fluorenyl two-photon
fluorescent probe. Chemphyschem 10(12):2073–2081
26. Fantin VR, Berardi MJ, Scorrano L, Korsmeyer SJ, Leder P
(2002) A novel mitochondriotoxic small molecule that selectively
inhibits tumor cell growth. Cancer Cell 2(1):29–42
References
1. Sanadi DR (1965) Energy-linked reactions in mitochondria. Annu
Rev Biochem 34:21–48
2. Petit PX, Susin SA, Zamzami N, Mignotte B, Kroemer G (1996)
Mitochondria and programmed cell death: back to the future.
FEBS Lett 396(1):7–13
3. Lambert AJ, Brand MD (2009) Reactive oxygen species produc-
tion by mitochondria. Methods Mol Biol 554:165–181
4. Nicholls DG (2005) Mitochondria and calcium signaling. Cell
Calcium 38(3-4):311–317
5. Boldogh IR, Pon LA (2007) Mitochondria on the move. Trends
Cell Biol 17(10):502–510
6. Rouiller C (1960) Physiological and pathological changes in
mitochondrial morphology. Int Rev Cytol 9:227–292
7. Hakenbrock CR (1968) Ultrastructural bases for metabolically
linked mechanical activity in mitochondria:II.electron transport-
linked ultrastructural transformations in mitochondria. J Cell Biol
37(2):345–369
27. Filomeni G, Piccirillo S, Graziani I, Cardaci S, Da Costa Ferreira
AM, Rotilio G, Ciriolo MR (2009) The isatin-schiff base copper
(II) complex Cu(isaepy)2 acts as delocalized lipophilic cation,
yields widespread mitochondrial oxidative damage and induces
AMP-activated protein kinase-dependent apoptosis. Carcinogenesis
30(7):1115–1124
8. Johnson LV, Summerhayes IC, Chen LB (1982) Decreased uptake
and retention of rhodamine 123 by mitochondria in a feline
sarcoma virus-transformed mink cells. Cell 28(1):7–14
28. Johnson LV, WalshML CLB (1980) Localization of mitochondria
in living cells with rhodamine 123. Proc Natl Acad Sci USA 77
(2):990–994

506
J Fluoresc (2011) 21:497–506
29. Murphy MP (2008) Targeting lipophilic cations to mitochondria.
Biochim Biophys Acta 1777(7–8):1028–1031
conjugation of the phenylacetylene linker in dipolar donor-bridge-
acceptor chromophores. J Phys Chem A 109(43):9767–9774
37. Castex MC, Olivero C, Pichler G, Adés D, Cloutet E, Siove A
(2001) Photoluminescence of donor–acceptor carbazole chromo-
phores. Synth Met 122(1):59–61
38. Adés D, Boucard V, Cloutet E, Siove A, Olivero C, Castex MC,
Pichler G (2000) Photoluminescence of donor-acceptor carbazole-
based molecules in amorphous and powder forms. J Appl Phys 87
(10):7290–7293
30. Ross MF, Kelso GF, Blaikie FH, James AM, Cochemé HM,
Filipovska A, Da Ros T, Hurd T, Smith RA, Murphy MP (2005)
Lipophilic triphenylphosphonium cations as tools in mitochondrial
bioenergetics and free radical biology. Biochemistry (Moscow) 70
(2):222–230
31. Hoegl H (1965) On photoelectric effects in polymers and their
sensitization by dopants. J Phys Chem 69(3):755–766
32. Coe BJ (2006) Switchable nonlinear optical metallochromophores
with pyridinium electron acceptor groups. Acc Chem Res 39
(6):383–393
39. Chang CC, Chu JF, Kuo HH, Kang CC, Lin SH, Chang TC
(2006) Solvent effect on photophysical properties of a fluores-
cence probe: BMVC. J Lumin 119:84–90
33. Zhang X, Yu XQ, Sun YM, Xu HY, Feng YG, Huang BB, Tao
XT, Jiang MH (2006) Synthesis, structure and nonlinear optical
properties of two new one and two-branch two-photon polymer-
ization initiators. Chem Phys 328(1–3):103–110
34. Xu C, Webb WW (1996) Measurement of two-photon excitation
cross sections of molecular fluorophores with data from 690 to
1050 nm. J Opt Soc Am B 13(3):481–491
35. Edmondson JM, Armstrong LS, Martinez AO (1988) A rapid
and simple MTT-based spectrophotometric assay for determining
drug sensitivity in monolayer cultures. J Tissue Cult Methods 11
(1):15–17
36. Lee S, Thomas KR, Thayumanavan S, Bardeen CJ (2005)
Dependence of the two-photon absorption cross section on the
40. Dean JA (ed) (1973) Handbook of chemistry. McGraw-Hill, New York
41. Bosch P, FernaÂndez-Arizpe A, Mateo JL, Lozano AE, Noheda P
(2000) New fluorescent probes for monitoring polymerisation
reactions: 1. Synthesis, solvatochromism and emission properties.
J Photochem Photobiol A Chem 133(1–2):51–57
42. Qin W, Baruah M, Sliwa M, Van der Auweraer M, De Borggraeve
WM, Beljonne D, Van Averbeke B, Boens N (2008) Ratiometric,
fluorescent BODIPY dye with aza crown ether functionality:
synthesis, solvatochromism, and metal ion complex formation. J
Phys Chem A 112(27):6104–6114
43. Malak H, Castellano FN, Gryczynski I, Lakowicz JR (1997) Two-
photon excitation of ethidium bromide labeled DNA. Biophys
Chem 67:35–41