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Title

Novel pyrene-based two-photon active fluorescent dye efficiently
excited and emitting in the tissue optical window (650‒1100 nm)

Authors

Gen-ichi Konishi, Yosuke Niko

Citation

Journal of Materials Chemistry B, Vol. 3, No. 2, pp. 184‒190

Pub. date2015, 1

URL

http://pubs.rsc.org/en/content/articlelanding/2014/tb/c4tb01404a#!divAb
stractPowered by T2R2 (Tokyo Institute Research Repository)

Volume 3 Number 2 14 January 2015 Pages 161–332

Journal of

Materials Chemistry B
Materials for biology and medicine
www.rsc.org/MaterialsB

ISSN 2050-750X

PAPER
Jun Kawamata, Gen-ichi Konishi et al.
A novel pyrene-based two-photon active ﬂuorescent dye efficiently
excited and emitting in the ‘tissue optical window (650–1100 nm)’Journal of

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Cite this: J. Mater. Chem. B, 2015, 3,
184

A novel pyrene-based two-photon active
ﬂuorescent dye eﬃciently excited and emitting inthe ‘tissue optical window (650–1100 nm)’†
Yosuke Niko,a Hiroki Moritomo,b Hiroyuki Sugihara,c Yasutaka Suzuki,bJun Kawamata*b and Gen-ichi Konishi*ad
The development of two-photon (TP) active ﬂuorophores remains an important issue. Dyes that can be
excited and ﬂuoresce eﬃciently in the ‘tissue optical window’ (650–1100 nm) are especially in demand
to maximize the underlying performance of two-photon ﬂuorescence microscopy (TPFM) as anadvanced optical technique. Ideally, such dyes would be compatible with the 1050 nm femtosecond
ﬁbre laser, which has recently been developed as an inexpensive excitation source to make the TPFM
technique universal. In this work, we designed and synthesized a novel pyrene-based acceptor–p–
acceptor (A–p–A) dye, PY, which exhibited outstanding properties such as bright ﬂuorescence (lem ¼
650 nm and FFL ¼ 0.80) and a large two-photon absorption cross-section (1100 GM (1 GM ¼ 1050 cm4
per photon per molecule) at 950 nm and 380 GM at 1050 nm) in the tissue optical window. In living

Received 23rd August 2014
Accepted 17th October 2014

mitochondria, PY provided more sensitive microscopic images than current dyes and showed great

DOI: 10.1039/c4tb01404a

potential to be a building block of TP active ﬂuorescent probes for the 1050 nm ﬁbre laser. We believethat the exceptional properties of PY will be extended to other ﬂuorescent probes through further

www.rsc.org/MaterialsB

chemical modiﬁcation.

Introduction
Two-photon uorescence microscopy (TPFM) has received
considerable attention as an advanced optical technique,especially in the biomedical eld, because it can provide both invivo and in vitro three-dimensional (3D) images deep within thesamples of interest using low-energy photons, which signicantly reduces the photodamage and photobleaching of tissues
and organs.1–4 The usability of this technique strongly depends
on the performance of the probe molecules; therefore, thedevelopment of two-photon (TP) activatable uorophores
remains an urgent issue. In recent years, remarkable progresshas been made in the molecular design concepts for dyes to be
used in practical applications.1,2,5,6 For instance, TP active dyesthat function as detectors of chemical or biological events in thetarget system and can realize ratiometric imaging are highly
important.5,6 The signicance of this type of function isa

Department of Organic and Polymeric Materials, Tokyo Institute of Technology,
2-12-1, Ookayama, Tokyo 152-8552, Japan. E-mail: konishi.g.aa@m.titech.ac.jp;
j-kawa@yamaguchi-u.ac.jpbGraduate School of Medicine, Yamaguchi University, 1677-1, Yoshida, Yamaguchi
753-8512, Japanc
Graduate School of Science and Engineering, Yamaguchi University, 1677-1, Yoshida,
Yamaguchi 753-8512, Japand

PRESTO, Japan Science and Technology Agency (JST), Japan† Electronic supplementary
10.1039/c4tb01404a

information

184 | J. Mater. Chem. B, 2015, 3, 184–190(ESI)

available.

See

DOI:

apparent from the substantial amount of research on the use of
one-photon activatable molecular probes.7,8The attractive, but still challenging feature of TP active dyes
is their eﬃcient TP absorption and bright uorescence bands inthe 650–1100 nm region.5 This wavelength region is the socalled ‘tissue optical window’, where undesired absorption of
excitation light and uorescence, light scattering, and autouorescence due to biological media are minimal.4 However,the development of such dyes has been limited,6j–l as seen fromcurrent reviews.1,2 Furthermore, TP dyes would ideally be
excited with the 1050 nm femtosecond bre laser, which has
recently been developed as an inexpensive excitation source.9
One of the advantages of uorescence techniques is that they
are relatively cheaper than other diagnostic tools like singlephoton emission computed tomography (SPECT).10 Considering the expensive hardware used, this is currently not the casefor TPFM; however, the compatibility of TP dyes with such abre laser would make TPFM universally available. The
inherent advantages of the TPFM technique would be truly
maximized by employing probes that satisfy all the above
mentioned requirements.
To this end, we have developed a novel pyrene-based
acceptor–p–acceptor (A–p–A) dye based on the following rationale. Our group has previously exploited a molecular design
strategy to construct A–p–A structures (i.e. uorene-based FLW,
naphthalene-based NP, and anthracene-based AC, shown in
Fig. 1) for TPFM.5c In these structures, two pyridinium moieties,

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acting as electron acceptors (A), were linked by vinyl linkers to ap-centre consisting of a planer and rigid aromatic chromophore, typied by polycyclic aromatic hydrocarbons (PAHs). Thedyes comprising such structures exhibited large TP absorption
cross-sections (s(2)) at longer wavelengths owing to strong
intramolecular charge transfer characteristics and extended pconjugation lengths. However, the PAH-based A–p–A dyes wehave developed so far do not satisfy the above-mentioned
performance characteristics. Herein, we focused on the pyrene
chromophore as a new p-centre for A–p–A dyes. Pyrene oenhas several photophysical and structural advantages whencompared with the other PAHs, e.g. longer excitation andemission wavelengths, higher uorescence quantum yields(FFL), higher absorption extinction coeﬃcients (3), more pelectrons, and greater planarity and symmetry.11,12 These properties are thought to be related to the spectral red shi ofuorescence and TP absorption and the enhancement of s(2)values.1
In this paper, we report the synthesis of a pyrene-based A–p–A
dye PY [(4,40 -((1E,10 E)-(3,8-dibutylpyrene-1,6-diyl)bis(ethene-2,1diyl))bis(1-methylpyridin-1-ium) iodide)] and its outstanding
optical properties in dimethylsulfoxide (DMSO). We also show
the actual performance of PY in mitochondrial imaging using
the TPFM technique and compare its performance with those of
FLW, NP, AC, commercially available dyes, and other existingTP activatable dyes. These results demonstrated that PY isapplicable in biological applications, and its use could be
extended to other organic diagnostic tools, like uorescent
nanoparticles,13 through further structural modication.

Results and discussion
Synthesis of PY
The synthetic protocol of PY is outlined in Scheme 1. Firstly,compound 1 was prepared through the introduction of two nbutyl groups into the 3 and 8 positions of pyrene. Next, Friedel–Cras formylation was conducted to obtain a diformylated
compound 2. Finally, Knoevenagel condensation between 2 and
1,4-dimethylpyridin-1-ium iodide was carried out to aﬀord thenal product PY. The role of the n-butyl groups was to enhance
the solubility for the easier preparation of compound 2 and tox the two formyl groups at the 1 and 6 positions. This pattern

Molecular design of PAH-based A–p–A dyes and the role ofeach moiety.

Fig. 1This journal is © The Royal Society of Chemistry 2015

Journal of Materials Chemistry B

Synthetic route to PY. (i) (1) THF, n-BuLi, 1-bromobutane,
0  C, 12 h (2) n-C4H9, r.t., 3 h (78%). (ii) CH2Cl2, TiCl4, Cl2CHOCH3, 0  C,
23 h (41%). (iii) MeOH/CHCl3, piperidine, 1,4-dimethylpyridin-1-ium
iodide, reﬂux, 12 h (59%).
Scheme 1of substitution should make PY quadrupolar, which is linked tohigh s(2) values.1,12,14 The obtained PY was characterized by 1H
and 13C NMR, and high-resolution mass spectrometry (seeESI†). In addition, the solubility of PY in water was estimated tobe 2  106 mol L1 (see Fig. S1 in the ESI†), which satised
the essential requirement for living cell imaging without using
excess organic solvents.5c
One-photon absorption and uorescence properties of PY
As mentioned above, PY has suﬃcient water solubility to stain
living cells without the use of organic solvents. However, thiswater solubility is too low to reliably characterize the TP
absorption. Therefore, all spectroscopic measurements wereconducted using DMSO as the solvent, unless otherwise noted.
The one-photon absorption and uorescence spectra of PY
were recorded (Fig. 2). Interestingly, PY in DMSO exhibited anabsorption peak at 510 nm, which is at a longer wavelength thanthose observed for FLW, NP, and AC.5c The anthracene-baseddye, AC, has an absorption peak at a shorter wavelength (490
nm in DMSO), even though anthracene derivatives generally
have longer absorption wavelengths than pyrene derivatives
with similar substitution, as does the parent anthracene (labs ¼
372 nm compared with labs ¼ 330 nm for pyrene in ethanol).11b
A possible reason for this diﬀerent behaviour is that the 1 and 6
positions of pyrene may accept stronger p-conjugation from thevinyl group-linked pyridinium moieties than the 2 and 6

Fig. 2 One-photon absorption and ﬂuorescence spectra of PY in
DMSO. The tissue optical window is indicated in green.

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positions of anthracene.15 As conrmed by DFT calculations,the substitution of acceptor groups at positions 1 and 6 inpyrene and at positions 9 and 10 in anthracene is most eﬀective
to induce red-shied absorption spectra and to enhance thetransition moments (Fig. S2 and S3†). On the other hand, it isexpected that substitution at the 9 and 10 positions in anthracene by vinyl group-linked pyridinium moieties would result ina loss of planarity because of steric hindrance, which could
decrease the s(2) value. Thus, pyrene has a structural advantage
when compared with anthracene, as the pyrene p-system can beextended signicantly without losing its planar structure.
Importantly, the energy of a TP absorption allowed state is
located at around one-half the energy level of a one-photon
absorption allowed state; therefore, such a red-shied onephoton absorption wavelength for PY is preferable for our
purpose.
PY in DMSO showed a uorescence peak at 650 nm, and thespectrum extended to 850 nm, which means that PY uorescence is advantageously within the tissue optical window.
Furthermore, a FFL value of 0.80 was measured; this value issignicantly higher than those of the other PAH-based A–p–A
dyes (FLW, NP, and AC have FFL values in DMSO of 0.44, 0.36,
and 0.66, at 542 nm, 516 nm, and 640 nm, respectively).5c The
reason for the high FFL value of PY might be related to theabove-mentioned stronger perturbation from p-conjugation
with the pyrene chromophore or its rigid structure, which
decreases vibrational deactivation.16 The uorescence peak of
PY in aqueous solution was located at 650 nm. Thus, PY in water
also uoresced within the tissue optical window. The FFL value
in aqueous solution was lower (0.10); highly polarized dyes
exhibiting uorescence at longer wavelengths tend to be less
uorescent in higher polarity solvents because of charge separation, which increases internal conversion.5c,17 Consideringthat biological tissues and organs are usually less polar thanwater, such a strong decrease in the FFL value might be preferable to obtain background-free imaging.7d Thus, it was
revealed that PY exhibits one-photon absorption that is suitable
for realizing a longer wavelength TP absorption band and auorescence band with a high FFL value in the tissue optical
window.

Paper

Fig. 3Two-photon absorption spectrum of PY in DMSO.

1. Unlike FLW, PY has a quadrupolar structure, as do NP andAC.
2. PY has a larger p-electron system than the other PAH
derivatives, especially FLW and NP.
3. PY is more strongly conjugated with the vinyl group-linked
pyridinium moieties than AC, as mentioned in the section
concerning one-photon absorption.
The performance of a TP active uorescent molecule isgenerally estimated using the value of the TP action crosssection (Fs(2)), which is the product of the values of FFL and s(2).
The Fs(2) value of PY at 950 nm was 880 GM; this value is quite
large compared with the values for other TP active dyes, as
described later (see Fig. 5).
Imaging of living mitochondria with TPFM
To evaluate the actual performance of PY as a TP active uorescent probe, the imaging of living mitochondria in Hek293
cells was carried out by TPFM. It is well-known that uorophores possessing a pyridinium moiety can function as

Two-photon absorption properties of PY
The TP absorption spectrum of PY in DMSO was recorded using
the open-aperture Z-scan technique.1 The maximum s(2) valueof PY was 1100 GM (1 GM ¼ 1050 cm4 per photon per molecule)at 950 nm (Fig. 3); this value is signicantly higher than thosefor FLW, NP, and AC. PY also showed signicant TP absorption
at 1050 nm (380 GM), indicating that PY is compatible with the1050 nm bre laser source, whereas the TP absorption of FLW,
NP, and AC at wavelengths longer than 1000 nm is quite small.5c
These characteristics of PY, including the large s(2) value andlonger TP absorption wavelength, might be explained bycomparison with the other PAH-based A–p–A dyes (e.g. FLW,
NP, and AC, which show 740 GM at 780 nm, 712 GM at 695 nm,
and 687 GM at 703 nm, respectively5c) as follows:

186 | J. Mater. Chem. B, 2015, 3, 184–190

Fig. 4 Two-photon ﬂuorescence microscope images of Hek293 cells
stained with PY. This image was obtained with 950 nm excitation, andthe ﬂuorescence was collected through a 600–700 nm band-passﬁlter.

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Fig. 5 Examples of TP active ﬂuorophores: PAH-based A–p–A dyes,5c a BODIPY-based dye,5a cyanine-based dyes,5e an anthracene-based pextended dye,1,21 and ﬂuorene-based p-extended dye.2,22 These dyes were selected because they show attractive spectroscopic properties (TPabsorption and ﬂuorescence wavelengths, and Fs(2)).

mitochondrial probes.5c,d,8 A co-staining experiment withanother mitochondrial probe (FLW)5d revealed that PY could
also selectively accumulate on the mitochondria (Fig. S4†). As
shown in Fig. 4, TPFM images of the mitochondria stained by
PY were obtained with 950 nm excitation at a power of less than
3 mW. Considering that rhodamine 123,18 a commerciallyavailable mitochondrial probe, requires a power of 50 mW inthe same instruments, PY achieved extremely sensitive imaging,as expected from its high Fs(2) value. Importantly, the excitation
power of a dye is proportional to the Fs(2) value. Therefore, if a
TPFM that is equipped with an excitation light source operating
at 1050 nm is available, TPFM imaging could be measured byThis journal is © The Royal Society of Chemistry 2015

employing 8.7 mW of a 1050 nm light source. This power is
suﬃciently lower than the maximum output power of recent
1050 nm bre lasers.9 As the optical components of current
TPFMs are not optimized for excitation at 1050 nm, it is not easy
to obtain TPFM images with this excitation wavelength.
However, aer necessary optimization is realized, PY will beusable as a probe for TPFM employing the 1050 nm bre laser.
Comparison of PY with other dyes for TPFM
Finally, we compared PY with existing TP active dyes todemonstrate the superiority of our uorophore (Fig. 5). As isapparent from the above discussion, PY has many desirable

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properties for TPFM, such as a high Fs(2) value and longer TP
absorption and uorescence wavelengths, compared with other
PAH-based A–p–A dyes and commercially available mitochondrial dyes, such as green uorescent protein (GFP; Fs(2) ¼ 6 GM
at 800 nm and lem ¼ 509 nm)19 and Rhodamine 123 (Fs(2) ¼ 72
GM at 800 nm and lem ¼ 534 nm).18 There are a number ofcyanine-based near infrared region (NIR) uorescent dyes for
TPFM that have recently been reported by Berezin and coworkers, such as ICG (Fs(2) ¼ 71 GM at 1500 nm and lem ¼ 817nm) and LS-277 (Fs(2) ¼ 63 GM at 1500 nm and lem ¼ 829 nm).5e
These dyes are highly attractive owing to their uorescencewithin the tissue optical window and TP absorption activated byan ultrafast turnkey bre laser source. However, these dyes are
excited using 1500 nm light, which means their TP absorption
bands are outside the tissue optical window. Furthermore, suchdyes have relatively lower Fs(2) values at 1500 nm. Beleld et al.
recently presented a large number of TP active uorescentprobes for bioimaging.2 Although there are a few examples ofdyes with Fs(2) values at their maximum TP absorption wavelength higher than that of PY, to the best of our knowledge,
there are no dyes showing both TP absorption bands around1000 nm and uorescence bands within the optical tissue
window that are more eﬃcient than PY. In fact, most of theexisting TP-active dyes can be excited in the short-wavelength
NIR region around 700–800 nm rather than the so-called“second near-infrared window (NIR II, in the range 1000–1350nm),” where it was recently proposed that the penetration of
excitation light is ideal.20 However, this means that these dyes
are not compatible with a 1050 nm bre laser even if they
possess higher Fs(2) values than PY. As shown in Fig. 5, highly
p-extended uorene or anthracene derivatives, as well as
BODIPY derivatives, exhibit relatively similar properties to PY
with respect to Fs(2) values and uorescence wavelengths,
however, these dyes might not be suitable for bio-imaging due
to their low solubility in water. In addition, the syntheses ofthese dyes are not trivial compared to that of PY. Thus, PY canbe regarded as an exceptionally superior TP active dye with acomparatively simple and small structure. We emphasise thatthe outstanding properties of PY are strongly related to both thestructural and electronic advantages of the pyrene chromophore, i.e. high planarity and symmetry, rigidness, synthetic
accessibility for eﬃcient p-extension and derivatization, and anunderlying large p-system. Therefore, we believe that the strategic use of pyrene as a chromophore in TP active dyes will be ofgreat importance and that PY itself can be further derivatizedfor other practical applications.

Conclusions
We have developed a novel pyrene-based A–p–A dye, PY, whichshowed a TP absorption band around 1000 nm and uorescence in the tissue optical window with an outstanding Fs(2)value. Almost all the properties of PY are derived from theinherent structural and electronic advantages of the pyrene
chromophore, including planarity, symmetry, rigidness, a large
p-system, and synthetic accessibility for the eﬃcient extensionof p-conjugation. We have also demonstrated the ability of PY

188 | J. Mater. Chem. B, 2015, 3, 184–190

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to provide 3D images of the mitochondria in living Hek293 cellswith higher sensitivity than the other PAH-based dyes andcommercially available mitochondrial probes, as was expectedfrom the performance of PY in spectroscopic measurements insolution. Finally, the comparison of PY with other known TP
active uorescent probes revealed that PY is an exceptional dye
in terms of the location of its TP absorption around the NIR II
window and its high Fs(2) value, indicating that PY is a promising candidate as a diagnostic tool and its ability to be usedwith the 1050 nm bre laser will help the TPFM technique
become more universal. To date, pyrene derivatives have beendeveloped for many applications; therefore, there are a number
of well-established synthetic methodologies where pyrene isused as a building block.23 Thus, PY can be modied for many
purposes, e.g. the further improvement of the compatibility of
PY to the 1050 nm laser source by introducing other functionalgroups into the 3 and 8 positions on the pyrene chromophore,
use of ligands for active targeting,24 addition of environment
sensitivity for the detection of chemical or biological events
such as the generation of reactive oxygen species in mitochondria,8 and further derivatization for building uorescent nanoparticles.13 Based on these facts and our ndings, we believethat PY is a useful building block as a TP absorber and uorophore for TPFM probes. In fact, we are currently focusing on
the use of PY for not only in vitro cell imaging but also in vivo
deep tissue imaging, using the 1050 nm bre laser.

Acknowledgements
We thank the JSPS Fellowship for Young Scientists (to Y. N.).
This work was supported by a Grant-in-Aid for ScienticResearch (no. 23350068) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan and Yamaguchi
Prefecture Contracted R&D for the Industrial Cluster of Next
Generation from Yamaguchi Prefectural Industrial Technology
Institute, Japan.

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