A red fluorescence two-photon absorption probe for sensitive imaging of live mitochondria

Article abstract of DOI:10.1246/cl.140447

We synthesized two-photon absorption probes based on naphthalene and anthracene: 1,1′-dimethyl-4,4′-(2,6-naphthylenedi- 2,1-ethenediyl)dipyridinium diiodide (NP) and 1,1′-dimethyl- 4,4′-(2,6-anthrylenedi-2,1-ethenediyl)dipyridinium diiodide (AC). These two probes were successfully accumulated in mitochondria with different fluorescence colors: yellow (NP) and red (AC). Two-photon absorption cross sections (σ⁽²⁾) were 712 and 685GM (1GM = 10^-50cm⁴s photon^-1molecule^-1), respectively. These values are nine times larger than the σ⁽²⁾of the commercially available mitochondria-selective probe with efficient two-photon absorption property (rhodamine 123).

Full text of DOI:10.1246/cl.140447

CL-140447
Received: May 1, 2014 | Accepted: May 22, 2014 | Web Released: May 28, 2014
A Red Fluorescence Two-photon Absorption Probe for Sensitive Imaging of Live Mitochondria
Makoto Tominaga,1 Shuhei Mochida,2 Hiroyuki Sugihara,2 Koichiro Satomi,1 Hiroki Moritomo,1
Akinari Fuji,1 Arina Tomoyuki,3 Yasutaka Suzuki,*1 and Jun Kawamata1
1Graduate School of Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8512
2Graduate School of Science and Engineering, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8512
3Faculty of Science, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8512
(E-mail: ysuzuki@yamaguchi-u.ac.jp)
We synthesized two-photon absorption probes based on
naphthalene and anthracene: 1,1¤-dimethyl-4,4¤-(2,6-naphthyl-
enedi-2,1-ethenediyl)dipyridinium diiodide (NP) and 1,1¤-di-
methyl-4,4¤-(2,6-anthrylenedi-2,1-ethenediyl)dipyridinium di-
iodide (AC). These two probes were successfully accumulated
in mitochondria with different fluorescence colors: yellow (NP)
and red (AC). Two-photon absorption cross sections (·(2)) were
712 and 685 GM (1 GM = 10¹50 cm4 s photon¹1 molecule¹1),
respectively. These values are nine times larger than the ·(2)
of the commercially available mitochondria-selective probe with
efficient two-photon absorption property (rhodamine 123).
comprises a fluorene core and pyridinium groups.7 The cationic
pyridinium moiety was designed to adsorb onto the negatively
charged mitochondrial membrane through electrostatic inter-
action. The FLW showed a large two-photon absorption
¹50
(TPA) cross section (·(2)) with 750 GM (1 GM = 10
cm4 s photon¹1 molecule¹1) at 730 nm. This value is larger than
those of typical mitochondria-selective probes, for example,
green fluorescent protein (GFP; 8 GM)8 and rhodamine 123
(80 GM).9 Although efficient TPA compounds are usually
insoluble in the aqueous medium because of their hydrophobic
character, the two cationic pyridinium moieties of FLW contrib-
ute to the increase of water solubility. FLW was soluble in water
at a concentration on the order of 10¹4 mol L¹1 and stained cells
without using any organic solvent. This advantage allowed long-
time imaging of live cells for more than 24 h because toxicity due
to organic solvents was not a concern. However, the fluorescence
maximum of FLW was at 545 nm, which is not in the range of
the tissue optical window as well as conventional TPFM probes.
In this study, we aimed to develop our molecular design for
a two-photon fluorescence probe that exhibits both excitation
and fluorescence in the tissue optical window. Polycyclic
aromatic hydrocarbons are known as efficient fluorophores.
Fluorescence wavelengths of these fluorophores are reported to
be longer than that of fluorene.10 Actually, the fluorescence
wavelengths corresponding to the largest energy gap of fluorene,
naphthalene, and anthracene are 300, 311, and 382 nm,
respectively.10 Therefore, we expected that fluorescence wave-
lengths of the analogs of FLW bearing the naphthalene or
anthracene moiety instead of the fluorene moiety are longer
than that of FLW. Here, we designed and synthesized two
compounds: the naphthalene derivative 1,1¤-dimethyl-4,4¤-
(2,6-naphthylenedi-2,1-ethenediyl)dipyridinium diiodide (NP)
(Figure 1b) and the anthracene derivative 1,1¤-dimethyl-4,4¤-
(2,6-anthrylenedi-2,1-ethenediyl)dipyridinium diiodide (AC)
(Figure 1c). The synthetic procedures for both NP and AC are
described in Supporting Information.
Two-photon fluorescence microscopy (TPFM) is known as
an advanced technology for three-dimensional (3D) imaging of
tissues and organs.1 This technique enables 3D imaging of blood
flow,2 neural network,3 neutrophil movement,4 local oxygen
concentration in the bone,5 and so forth. In a typical TPFM, the
excitation wavelength is ca. 800 nm and the observed fluores-
cence is between 400 to 550 nm. Transparency for biological
tissues in the wavelength region from 400 to 550 nm is
insufficient. Therefore, imaging at a point deeper than 1 mm
cannot be achieved using the conventional TPFM probe. In
contrast, a wavelength region from 600 to 1300 nm is known as
the “tissue optical window”.6 Biological materials usually have
low absorption in this window, so that light penetrates deeper
inside the tissue. By employing the probe that can be both
excited and emitted in the tissue optical window, deeper
observation of tissue and organs by TPFM can be achieved.
We have developed an efficient two-photon fluorescence
probe: 1,1¤-dimethyl-4,4¤-(9,9¤-diethyl-2,7-fluorenediyl-2,1-eth-
enediyl)dipyridinium diperchlorate (FLW, Figure 1a). This probe
(a)
Absorption and fluorescence spectra of FLW, NP, and AC
are shown in Figure 2. The absorption peak of FLW was
observed at 420 nm. The wavelength of the absorption peak of
NP was observed at 424 nm and shoulders were observed at 381
and 410 nm. Absorption peaks of AC were observed at 338, 356,
391, 413, and 477 nm. The cutoff wavelengths of the absorption
bands of FLW, NP, and AC were 480, 470, and 520 nm,
respectively. The cutoff wavelength of AC was observed at
the longest wavelength among three compounds. Contrary to
expectation, the cutoff wavelength of NP was shorter than that of
FLW. Absorption peaks of nonsubstituted fluorene and naph-
thalene were reported at 206-301 and 221-320 nm, respectively,
showing a longer wavelength of the absorption band of
(b)
(c)
Figure 1. Chemical structure of FLW (a), NP (b), and AC (c).
1490 | Chem. Lett. 2014, 43, 1490–1492 | doi:10.1246/cl.140447
© 2014 The Chemical Society of Japan

Figure 2. Absorption and fluorescence spectra of FLW (dotted
line), NP (solid line), and AC (dashed line) in dimethyl
sulfoxide.
naphthalene than of fluorene.11 However, the absorption band of
NP was shorter than that of FLW. In FLW, NP, and AC, terminal
pyridinium groups and central aromatic rings are categorized
as electron acceptor (A) and electron donor (D) groups,
respectively. The Hammett’s substituent constant (·p) value,
which represents electron donation ability, of the 2-fluorenyl
and 2-naphthyl groups are ¹0.49 and ¹0.135, respectively.12
Therefore, the intramolecular charge transfer of FLW would be
larger than that of NP. This is the most probable reason for the
absorption wavelength of NP being shorter than that of FLW.
On the other hand, the absorption peaks of nonsubstituted
anthracene were reported at 252-375 nm; the wavelength of the
absorption band of nonsubstituted anthracene was longer than
that of nonsubstituted fluorene or naphthalene. The wavelength
of the absorption band of AC was the longest among the three
compounds, as expected.
As can be seen from Figure 2, the wavelength of the
fluorescence maximum of FLW, NP, and AC were 542, 516, and
640 nm, respectively. The fluorescence maximum of FLW was at
a longer wavelength than that of NP, reflecting a longer cutoff
wavelength of the absorption band. The fluorescence maximum
wavelength of AC was the longest among the three compounds
and was within the tissue optical window. The fluorescence
quantum yields of FLW, NP, and AC in dimethyl sulfoxide were
0.44, 0.36, and 0.66, respectively, whereas the values in water
were 0.33, 0.31, and 0.08, respectively. For aqueous solutions,
fluorescence quantum yields of compounds exhibiting fluores-
cence at a longer wavelength region tend to be low.13 Thus, the
decrease of the quantum yield of AC in water compared to that
in an organic solvent should be considered to be a typical
behavior of a fluorescence dye emitting in a longer wavelength
region. The fluorescence quantum yield of AC in water was
comparable to that of other red-fluorescence probes in water and
thus, the fluorescence property of AC is thought to be sufficient
for using it as a bio-imaging probe.
Figure 3. Two-photon absorption spectra of FLW (a), NP (b),
and AC (c) in dimethyl sulfoxide.
tissue optical window. In the case of AC, both excitation and
fluorescence were within the tissue optical window.
To image living cells for a long time, use of organic solvents
should be avoided in the staining procedure. Therefore, a
¹1
probe, of which water solubility is higher than 10¹7 mol L
,
is required. The water solubility of NP and AC was estimated to
be 2 © 10¹4 and 6 © 10¹6 mol L¹1, respectively. Although these
probes have relatively larger π-electron systems, the water
solubility was enough for introducing them into living cells as
an aqueous solution.
The ·(2) values of the compounds treated in this study were
estimated by the open aperture Z-scan technique.14 The TPA
spectra of FLW, NP, and AC are shown in Figures 3a-3c. The
maximum values of ·(2) of FLW, NP, and AC were 740 GM at
780 nm, 712 GM at 695 nm, and 687 GM at 703 nm, respec-
tively. The ·(2) of FLW, NP, and AC were comparable to each
other. Thus, NP and AC maintained the efficient ·(2) of FLW.
Furthermore, TPA bands of all probes were observed within the
The “one-photon” fluorescence images of HEK293 cells
stained by NP and AC are shown in Figures 4a and 4b. The
costaining fluorescence images of rhodamine 123, which is a
mitochondria-selective probe, and FLW are shown in Figures 4c
and 4d. The merged image of NP and rhodamine 123 is shown
in Figure 4e and the merged image of AC and FLW is shown
in Figure 4f. To avoid the overlap of the fluorescence color, a
costaining experiment was conducted by using rhodamine 123 in
Chem. Lett. 2014, 43, 1490–1492 | doi:10.1246/cl.140447
© 2014 The Chemical Society of Japan | 1491

source required to obtain TPFM images of NP, AC, and
rhodamine 123 were 5, 5, and 50 mW, respectively. Reflect to
efficient TPA properties of NP and AC, sensitive observation
can be accomplished by employing these probes.
In summary, we synthesized two novel water-soluble TPA
probes based on naphthalene or anthracene: NP and AC.
Although NP exhibited a shorter wavelength for fluorescence
(516 nm) than the tissue optical window, greater TPA efficiency
and water solubility were observed. In the case of AC, we
successfully controlled the fluorescence wavelength within the
tissue optical window with enough water solubility, efficient
TPA property, and mitochondria selectivity. Although introduc-
tion of polycyclic aromatic hydrocarbons much larger than
anthracene to the present molecular back-bone would result
in improved two-photon efficiency and a longer fluorescence
wavelength, such structural modification would decrease water
solubility. Therefore, AC is considered to be one of the most
promising candidates for an efficient TPFM probe exhibiting
both red fluorescence and sufficient water solubility.
Supporting Information is available electronically on J-STAGE.
References
1
2
W. Denk, J. H. Strickler, W. W. Webb, Science 1990, 248,
73.
J. Lecoq, A. Parpaleix, E. Roussakis, M. Ducros, Y. G.
Houssen, S. A. Vinogradov, S. Charpak, Nat. Med. 2011, 17,
893.
3
4
5
R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S.
Sato, H. Yokoyama, T. Nemoto, Sci. Rep. 2013, 3, Article
number 1014.
T. Lämmermann, P. V. Afonso, B. R. Angermann, J. M.
Wang, W. Kastenmüller, C. A. Parent, R. N. Germain,
Nature 2013, 498, 371.
J. A. Spencer, F. Ferraro, E. Roussakis, A. Klein, J. Wu,
J. M. Runnels, W. Zaher, L. J. Mortensen, C. Alt, R.
Turcotte, R. Yusuf, D. Côté, S. A. Vinogradov, D. T.
Scadden, C. P. Lin, Nature 2014, 508, 269.
R. Weissleder, Nat. Biotechnol. 2001, 19, 316.
S. Tani, K. Nakagawa, T. Honda, H. Saito, Y. Suzuki, J.
Kawamata, M. Uchida, A. Sasaki, M. Kinjo, Curr. Pharm.
Biotechnol. 2012, 13, 2649.
Figure 4. One-photon fluorescence microscope images of
HEK293 cells. (a) shows cells stained with NP. (c) shows cells
stained with rhodamine 123. (e) is a merged image of (a) and (c).
(b) shows cells stained with AC. (d) shows cells stained with
FLW. (f) is a merged image of (b) and (d).
6
7
8
9
P. T. C. So, C. Y. Dong, B. R. Masters, K. M. Berland, Annu.
Rev. Biomed. Eng. 2000, 2, 399.
R. Wolleschensky, T. Feurer, R. Sauerbrey, U. Simon, Appl.
Phys. B: Lasers Opt. 1998, 67, 87.
Figure 5. Two-photon fluorescence microscope images of
HEK293 cells stained with NP (a) and AC (b). These images
are obtained by 750 nm excitation and fluorescence were
collected through a 500-600 nm band-pass filter.
10 Chemical Society of Japan, Kagaku Binran Kisohen II,
Maruzen, pp. 759-760.
11 Chemical Society of Japan, Kagaku Binran Kisohen II,
Maruzen, pp. 754-755.
the case of NP. The merged image (Figures 4e and 4f) revealed
that the structures stained with NP and AC were very similar to
those stained with rhodamine 123 and FLW. Thus, NP and AC
were thought to accumulate in the mitochondria in HEK293
cells.
The TPFM images of HEK293 cells stained with NP and
AC are shown in Figures 5a and 5b. Under this experimental
condition, the averaged incident power of the excitation light
12 Chemical Society of Japan, Kagaku Binran Kisohen II,
Maruzen, p. 380.
13 a) M. C. Basheer, U. Santhosh, S. Alex, K. G. Thomas, C. H.
Suresh, S. Das, Tetrahedron 2007, 63, 1617. b) L. Wang, J.
Fan, X. Qiao, X. Peng, B. Dai, B. Wang, S. Sun, L. Zhang,
Y. Zhang, J. Photochem. Photobiol., A 2010, 210, 168. c) B.
Song, Q. Zhang, W.-H. Ma, X.-J. Peng, X.-M. Fu, B.-S.
Wang, Dyes Pigm. 2009, 82, 396.
1492 | Chem. Lett. 2014, 43, 1490–1492 | doi:10.1246/cl.140447
© 2014 The Chemical Society of Japan