Polarity-dependent photophysical properties of hemicyanine dyes and their application in 2-photon microscopy biological imaging

Article abstract of DOI:10.1246/cl.2012.528

Two fluorescent hemicyanine type compounds, 1 and 2 have been developed for membrane staining, and their photophysical properties were investigated in various solvents. The demonstrated 2-photon-microscopy-biological-imaging application indicates the usefulness of 1 as a fluorescent probe.

Full text of DOI:10.1246/cl.2012.528

doi:10.1246/cl.2012.528
528
Published on the web April 28, 2012
Polarity-dependent Photophysical Properties of Hemicyanine Dyes
and Their Application in 2-Photon Microscopy Biological Imaging
Yuta Kimura,1 Atsuya Momotake,1 Noriko Takahashi,2 Haruo Kasai,2,3 and Tatsuo Arai*1
1Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577
2Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine,
Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033
3PRESTO, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0076
(Received January 20, 2012; CL-120053; E-mail: arai@chem.tsukuba.ac.jp)
Two fluorescent hemicyanine type compounds, 1 and 2 have
been developed for membrane staining, and their photophysical
properties were investigated in various solvents. The demon-
strated 2-photon-microscopy-biological-imaging application in-
dicates the usefulness of 1 as a fluorescent probe.
Aminostyryl benzimidazolium (Hemicyanine) dyes have
been used as fluorescent probes in bioimaging1-4 and have also
found application in lasers,5 nonlinear optical devices,6 materi-
als for second harmonic generation (SHG),7,8 and SHG probes
for imaging.9-11 They consist of a positively charged nitrogen-
containing heteroaromatic moiety that serves as an electron
acceptor, and another nitrogen conjugated by a polymethine unit
that acts as an electron donor. Hemicyanine exhibits charge
transfer in both the ground and excited states associated with
these electronic donor-acceptor properties. The charge-transfer
character relates to the polarity dependence of the absorption
and fluorescence spectra and fluorescence efficiency.12-14 The
fluorescence of hemicyanine is usually very weak in aqueous
solution but is enhanced in less polar media.15 In addition,
many hemicyanines have a rod-shaped amphiphilic structure
that contributes to their affinity for micelles or biomembranes,
and thus these compounds have been used for membrane
staining. Particularly, FM dyes (Molecular Probes), originally
reported by Mao,16 are widely used for live cell imaging
because of their high affinity for the cell membrane, nontoxic
nature, stability, ease of use, and availability.2,3,17-19 However
the basic photochemistry of FM dyes in conventional organic
solvents is still a relatively undeveloped area that is worthy of
study.
Figure 1. Chemical structures of FM1-43, 1, and 2.
than that of FM1-43, which may lead to significant solvato-
chromic and nonlinear optical effects.
The syntheses of FM1-43, 1, and 2 are depicted in
Supporting Information.36
It should first be noted that hemicyanine dyes can easily
form aggregates and confined specific regions (such as mono-
layers) in some solvents.27 The dyes 1 and 2 also exhibited
aggregate behavior and, therefore, the exact molar extinction
coefficient values cannot be obtained in any conventional
solvents we used. However, we could choose some solvents
which can dissolve the dyes in this experiments as “good”
solvents (Figures 2a-2c): a less polar aprotic solvent (chloro-
form), a highly polar aprotic solvent (DMSO), and protic
solvents (MeOH and phosphate buffer at pH 7.4), of which
the polarities are in the following order: phosphate buffer >
DMSO > MeOH > chloroform.
Figure 2a shows the absorption spectra of FM1-43 in good
solvents where FM1-43 should exist as a monomeric structure.
It is well known that the strong S0-S1 absorption band of
hemicyanine, which originates from the intramolecular charge-
transfer (ICT) transition between donor (aniline site) and
acceptor (pyridinium cation site) appears around 500 nm. Like
other hemicyanines,13 FM1-43 exhibits negative solvatochrom-
ism; the absorption peak shifts to shorter wavelengths as the
solvent polarity increases, indicating a larger dipole moment in
the ground state than in the excited singlet state (Franck-Condon
region).12,28,29
In the present paper we briefly describe the photochemical
and photophysical behaviors of FM1-43 (4-[4-(dibutylamino)-
styryl]-1-[3-(triethylammonio)propyl]pyridinium
dibromide)
and the new hemicyanine dyes (Figure 1) (E)-1-[3-(triethyl-
ammonio)propyl]-4-{2-[9,9-diethyl-7-(dihexylamino)-9H-fluoren-
2-yl]vinyl}pyridinium dibromide (1) and (E)-1-[3-(triethylam-
monio)propyl]-4-{2-[7-(diethylamino)-9,9-dimethyl-9H-fluoren-
2-yl]vinyl}pyridinium dibromide (2) and demonstrate their use
as a 2-photon absorption (TPA) fluorophore to perform non-
invasive membrane staining. Although FM1-43 is a known
compound,16 its basic photophysical features in various conven-
tional solvents is still of interest because it is one of the most
widely used FM dyes for bioimaging.20-24
The negative solvatochromic shift is more prominent in
1 (Figure 2b). In chloroform, the absorption peak appeared at
545 nm, which is a longer wavelength than that for FM1-43
(530 nm), probably due to the effects of ³-extension. In an
aqueous phosphate buffer at pH 7.4, however, the peak wave-
Aminofluorene skeleton was employed for new dyes 1 and
2, because of its preferable photophysical properties including
2-photon absorption.25,26 Due to a longer donor-acceptor
distance, 1 and 2 are expected to have larger dipole moments
Chem. Lett. 2012, 41, 528-530
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529
Figure 3. a) Fluorescence spectra of FM1-43 in chloroform
(red line), methanol (orange line), DMSO (green line), and
phosphate buffer at pH 7.4 (blue line). The spectra in DMSO
and phosphate buffer at pH 7.4 are overlapped. b) Fluorescence
spectra of 1 (open circle) and 2 (solid line) in chloroform. No
fluorescence was detected in methanol, DMSO, or phosphate
buffer at pH 7.4.
Figure 2. Absorption spectra of FM1-43 (a), 1 (b), and 2 (c) in
chloroform (red line), methanol (orange line), DMSO (green
line), and phosphate buffer at pH 7.4 (blue line).
length underwent a greater blue shift to 468 nm, an even shorter
wavelength than that for FM1-43 (495 nm). These results
suggest that the ground state of 1 is stabilized by solvent
molecules more significantly than that of FM1-43, probably due
to a larger ground state dipole moment and more pronounced
localization of the HOMO electrons in 1 (Figure S136). The
ground state dipole moments were calculated by B3LYP/6-
31G* method to be 7.45 and 14.1 D for the simple model of
FM1-43 and 1, respectively.30
Despite having the same hemicyanine skeleton, the absorp-
tion bands of 2 appeared in a shorter wavelength region than
those of 1. The spectral difference is not caused by the electronic
effect of different alkyl groups, but rather is believed to be due to
the different nature of the solvent-solute interaction resulting
from the different steric bulk of the alkyl substituents.14 The
HOMO of this chromophore is located on the amine side and the
LUMO on the pyridinium side, as shown in Figure S1.31,36 The
N-alkyl chain length in 2 is shorter than that in 1, which can
permit the interaction between the anilino nitrogen and the polar
solvent molecules, resulting in stabilization of the ground state
and the corresponding larger blue shift. This is consistent with
the reported effect of the N-alkyl chain length on the absorption
band of the other hemicyanines.14
phosphate buffer, respectively. The very low fluorescence
quantum yield in aqueous solution is an important property for
membrane staining.
The fluorescence spectra of 1 appeared at a much longer
wavelength with a larger Stokes shift (4380 cm¹1) than that of
FM1-43, even in chloroform (716 nm, Figure 3b). In addition,
the fluorescence was weak in chloroform (Φf = 0.06) and could
not be detected in DMSO, MeOH, or pH 7.4 phosphate buffer.
These results indicate that the solvent relaxation in the excited
singlet state of 1 is more significant than in FM1-43. 2 exhibited
a similar fluorescence spectra and Φf value in chloroform
(709 nm, Φf = 0.08), and was also nonfluorescent in DMSO,
MeOH, and the phosphate buffer.
FM1-43 has already been reported to provide insights
into endocytosis, exocytosis,3 and vesicular release by single
synaptic terminals,32 and also has been used for 2-photon
imaging.23 Thus, we next tested the performance of 1 as a
fluorescent dye for 2-photon excitation microscopy and com-
pared the fluorescence signal with that from FM1-43.
Figure 4 shows the 2-photon fluorescence images of INS-1
cells that were immersed in solutions containing each of the
three dyes at a concentration of 10 ¯M. The brightness of each
dye was evaluated at two wavelengths (830 and 910 nm). Bright
reticulation structures represent the plasma membranes of the
cells within a cluster of INS-1 cells and are very similar to the
¢-cells in the islets in the previous studies using FM1-43.33-35
We found that 1 successfully labeled the plasma membrane
in the living INS-1 cells, as did FM1-43. Importantly, little
fluorescence was found in the cytosol of the cells, indicating that
1 did not penetrate the plasma membrane, as is the case with
FM1-43. At an excitation wavelength of 830 nm, FM1-43 was
brighter, whereas 1 exhibited brighter fluorescence at 910 nm.
These findings suggest that the larger TPA cross-section of 1
at 910 nm offsets its lower fluorescence efficiency, whereas at
830 nm, FM1-43 still have higher TPA efficiency.
The fluorescence peaked at 589 nm in chloroform and was
red-shifted to around 630 nm in the highly polar solvents
DMSO, MeOH, and the pH 7.4 phosphate buffer (Figure 3a).
The fluorescence red shift is caused by the solvent relaxation
of the excited singlet state of FM1-43 that has a strong
intramolecular charge-transfer (ICT) character. The Stokes shift
¹1
values were 1890, 3500, 4490, and 4300 cm in chloroform,
MeOH, DMSO, and phosphate buffer, respectively, almost
increasing monotonically with increasing solvent polarity. The
fluorescence quantum yield (Φf) of FM1-43 was high in
chloroform (Φf = 0.42) and decreased in highly polar or protic
solvents: 0.03, 0.005, and 0.002 in DMSO, MeOH, and
Chem. Lett. 2012, 41, 528-530
© 2012 The Chemical Society of Japan
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7
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Figure 4. Two-photon excitation live images of clusters of
INS-1 cells which were stained with either 1 or FM1-43. Each
dye was applied from the extracellular medium at a concen-
tration of 10 ¯M. The excitation wavelength was 830 or 910 nm,
as indicated. The fluorescence intensities were represented by
the pseudocolor code (inset), where AU stands for arbitrary unit.
The mean AU values of the plasma membranes at 830 nm were
200 and 2600 AU for 1 and FM1-43, and those at 910 nm were
900 and 300 AU for 1 and FM1-43, respectively. The scale bar
indicates 10 ¯m.
In conclusion, we have developed the new hemicyanine-
type fluorescent dyes 1 and 2 and evaluated their photophysical
properties. Compared to FM1-43, 1 and 2 exhibit a more
significant solvatochromic shift in both their absorption and
fluorescence spectra and a lower fluorescence quantum yield.
The demonstrated 2-photon microscopy biological imaging
application indicates the usefulness of 1 as a fluorescent probe,
especially for 2-photon imaging in the longer wavelength
region. More detailed studies of the structure-photophysical
property relationships of the new dyes and their potential
application for SHG imaging are under way.
This work was supported by a Grant-in-Aid for Scientific
Research in a Priority Area “New Frontiers in Photochromism”
(No. 471),
(No. 23350075), for young scientists (B) (No. 22750143 to
AM) and Grant-in-Aid for Specially Promoted Areas
a
Grant-in-Aid for Scientific Research (B)
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(No. 2000009 to HK) from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT), Japan. This study was
also supported by the Japan Foundation for Applied Enzymol-
ogy, the Sankyo Foundation of Life Science, and the Human
Frontier Science Program Organization. INS-1 was kindly
provided by C.B.Whollheim.
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Chem. Lett. 2012, 41, 528-530
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/