A family of multi-color anthracene carboxyimides: Synthesis, spectroscopic properties, solvatochromic fluorescence and bio-imaging application

Article abstract of DOI:10.1016/j.dyepig.2016.11.056

A family of anthracene carboxyimides with multi-color fluorescence have been successfully prepared, and their spectroscopic properties were investigated by UV–vis absorption spectroscopy, steady-state and time-resolved fluorescence spectroscopy. The photostability and solvatochromic fluorescence in organic solvents were also investigated. The parent anthracene carboxyimide 4a displays very strong green fluorescence (Φ_F?=?0.91 in EtOH), good pH stability and photostability, as well as a large Stokes shift. By attachment of different substituents at the 10-position of the anthracene carboxyimide, the fluorescence maxima could be effectively modulated from 500?nm to 615?nm. The 10-(picolylamine) substituted anthracene carboxyimides 5a and 5b exhibit large Stokes shifts (>80?nm) and significant positive solvatochromic fluorescence in organic solvents due to their intramolecular charge-transfer (ICT) character. Furthermore, cell staining experiments indicate that anthracene carboxyimides have excellent cell membrane permeability and good photo-bleaching resistance in living cells. Particularly, compound 5a is a specific mitochondria-targeting dye with red fluorescence. All these features enable anthracene carboxyimides to serve as promising candidates for bio-labeling/imaging applications.

Full text of DOI:10.1016/j.dyepig.2016.11.056

Accepted Manuscript
A family of multi-color anthracene carboxyimides: Synthesis, spectroscopic
properties, solvatochromic fluorescence and bio-imaging application
Junchao Xu, Guangle Niu, Xiaofang Wei, Minhuan Lan, Lintao Zeng, Joseph M.
Kinsella, Ruilong Sheng
PII:
S0143-7208(16)31027-0
10.1016/j.dyepig.2016.11.056
DYPI 5625
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 23 October 2016
Revised Date: 30 November 2016
Accepted Date: 30 November 2016
Please cite this article as: Xu J, Niu G, Wei X, Lan M, Zeng L, Kinsella JM, Sheng R, A family of multi-
color anthracene carboxyimides: Synthesis, spectroscopic properties, solvatochromic fluorescence and
bio-imaging application, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2016.11.056.
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Graphical Abstract

ACCEPTED MANUSCRIPT
A Family of Multi-Color Anthracene Carboxyimides: Synthesis,
Spectroscopic Properties, Solvatochromic Fluorescence and
Bio-imaging Application
a,#
b,#
b,#
b
a,
d
Junchao Xu , Guangle Niu , Xiaofang Wei , Minhuan Lan , Lintao Zeng *, Joseph M. Kinsella , Ruilong
c,d
Sheng *
a
Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion, School of Chemistry and
Chemical Engineering, Tianjin University of Technology, Tianjin 300384, P. R. China. E-mail: zlt1981@126.com.
(
L. Zeng)
b
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and
Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China.
c
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, P. R. China. E-mail:
rlsheng@mail.sioc.ac.cn. (R. Sheng)
d
Department of Bioengineering, McGill University, 817 Sherbrook Street, Montreal, Quebec, H3A0C3, Canada.
Abstract:
A family of anthracene carboxyimides with multi-color fluorescence have been successfully
prepared, and their spectroscopic properties were investigated by UV-vis absorption spectroscopy,
steady-state and time-resolved fluorescence spectroscopy. The photostability and solvatochromic
fluorescence in organic solvents were investigated. The parent anthracene carboxyimide 4a displays
very strong green fluorescence (Φ = 0.91 in EtOH), good pH stability and photostability, as well as
F
a large Stokes shift. By attachment of different substituents at the 10-position of the anthracene
carboxyimide, the fluorescence maxima could be effectively modulated from 500 nm to 615 nm. The
10-(picolylamine) substituted anthracene carboxyimides 5a and 5b exhibit large Stokes shifts (> 80
nm) and significant positive solvatochromic fluorescence in organic solvents due to their
intramolecular charge-transfer (ICT) character. Furthermore, cell staining experiments indicate that
anthracene carboxyimides have excellent cell membrane permeability and good photo-bleaching
resistance in living cells. Particularly, compound 5a is a specific mitochondria-targeting dye with red
fluorescence. All these features enable anthracene carboxyimides to serve as promising candidates
for bio-labeling/imaging applications.

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Keywords: Anthracene carboxyimides, Fluorescence, Solvatochromic, Photostability, Bio-imaging
1. Introduction
Anthracene is a polycyclic aromatic hydrocarbon compound with excellent optical property such
as well-resolved absorption and emission band, high fluorescence quantum yield (Φ = 0.36 in
F
CH CN) and nanosecond lifetime [1, 2]. Anthracene derivatives also exhibit good fluorescence even
3
in the solid state, hence they have been widely used as blue light-emitting materials in organic
light-emitting diodes (OLEDs) [3-5]. Despite these advantages, anthracene and its derivatives still
cannot meet the criteria for bio-imaging due to some obvious defects including short emission
wavelength, poor photostability and water-solubility. For bio-imaging applications, a fluorescent dye
with water-solubility, long emission wavelength and large Stokes shift is highly desired, since good
water-solubility can enhance the cell/tissue permeability, while long emission wavelength and large
Stokes shift can minimize self-absorption and facilitate the penetration of emission light through
deep tissues [6-9].
Structurally related to anthracene, perylene and naphthalene are very important members of
polycyclic aromatic hydrocarbons, whereas they are hard to be directly used for bio-imaging due to a
short emission wavelength (perylene/naphthalene both emit in the deep blue region) [10], poor
solubility, and lack of functional groups. For decades, great efforts have been devoted to develop
new perylene/naphthalene derivatives with good solubility, long emission wavelength and large
Stokes shifts [11]. Among these derivatives, the perylene/naphthalene-based carboxyimides/diimides
show fascinating photophysical properties. The fluorescence spectra of perylene/naphthalene
carboxyimides are largely red-shifted compared to perylene/naphthalene, and the photophysical
properties can be easily modulated by introduction of electron-donating groups on certain position of
perylene/naphthalene carboxyimides to form a donor-π-conjugation-acceptor (D-π-A) structure
[
12-14]. Owing to their good chemical, thermal and photo-stability, the perylene/naphthalene-based
carboxyimides/diimides have been successfully applied in the fields of supramolecular assembly [15],
organic photovoltaics/transistors [16, 17], bio-labeling [18, 19] and chemosensors [20]. Nevertheless,
to our knowledge, anthracene-based carboxyimides/diimides are rarely explored [21-23].
Inspired by the unique properties of perylene/naphthalene-based carboxyimides/diimides, herein,
we developed a versatile approach to construct a family of anthracene carboxyimides and modulate

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their photophysical properties. In this study, we designed and synthesized a family of anthracene
dyes 4a~6 (Scheme 1) with the following features: (1) all of these anthracene dyes have a
dicarboximide moiety at the 1,9-position to extend the π-conjugation and to serve as strong
electron-withdrawing group (EWG), (2) some dyes (5a~5b) possess substituted amine groups at the
10-position as an electron-donating group (EDG). It is expected that endowing “push-pull” features
into the anthracene dyes may result in longer absorption/emission wavelength and large Stokes shifts.
The photophysical properties of these dyes were investigated by UV-vis absorption spectroscopy,
steady-state and time-resolved fluorescence spectroscopy. Furthermore, cell staining experiments
were performed to explore their abilities for bio-imaging application.
2
2
. Results and discussion
.1 Synthesis
Scheme 1 depicts the synthetic routes and molecular structures of compounds 4~6. Anthracene/
-bromoanthracene were used as the starting materials, which reacted with oxalyl chloride via
9
Friedel-Crafts acylation to produce the aceanthrylene 1,2-diones (2a and 2b). Subsequently,
aceanthrylene 1,2-dione was oxidized by oxone to give the carboxylic acid anhydrides 3a and 3b in
high yields (~ 90%). Anthracene/9-bromoanthracene have good solubility in organic solvents, while
their aceanthrylene 1,2-diones and carboxylic acid anhydrides exhibit poor solubility due to strong
π-π stacking and dipole-dipole interactions. Treatment of 3a/3b with an excess of 2-picolylamine
afforded the soluble imides 4a/5a. Similarly, treatment of 3b with 1-aminododecane produced 4b
which exhibited good solubility. These results suggest that introduction of a bulky, sterically
demanding group can improve the solubility of these anthracene carboxyimides by disruption of their
π-π stacking-induced aggregation [24]. Furthermore, we synthesized compound 5b, which was
incorporated a long alkyl group (1-dodecyl) and a bulky pyridine group to improve the solubility and
modulate the spectroscopic properties of anthracene carboxyimides. To further modulate the
photophysical property of 5a, it was reacted with acetyl chloride to afford compound 6 in 65% yield.
In this work, pyridine groups were introduced to improve the solubility and modulate the
spectroscopic properties for all molecules. As expected, compounds 4a, 5a, 5b and 6 display good
solubility in common organic solvents.
2.2 Spectroscopic properties

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UV-vis absorption and fluorescence spectra of compounds 4a~6 were measured in EtOH, and the
photophysical data were summarized in Table 1. As shown in Fig. 1, compound 4a displays a
characteristic absorption band centered at 436 nm in EtOH, and the maximum emission wavelength
is located at 500 nm, which is significantly red-shifted relative to anthracene (λ = 398 nm in EtOH,
em
Fig. 1). Interestingly, compound 4a is even more emissive (Φ = 0.91 in EtOH) than anthracene (Φ
F
F
=
0.27 in EtOH) [25], which might be attributed to the introduction of the carboxyimide moiety at
the 1,9-position of anthracene which inhibits intersystem crossing and photo-induced
dimmerization/oxidization [26]. This result suggested that the anthracene carboxyimide moiety not
only inherited some good properties of anthracene but also improved its emission efficiency and
achieved a remarkable bathochromic (red-shifted) effect on both absorption/emission spectra.
Notably, the absorption/emission profiles of 4a are separated from each other with a relatively large
Stokes shift (Fig. 2a), which may be associated with the ICT and asymmetric molecular geometry of
4a [27]. A large Stokes shift can eliminate the spectral overlap between absorption and emission and
allow reliable detection of fluorescence by reducing interference [28]. Moreover, 4a displays a long
8
−1
fluorescence lifetime (τ = 15.18 ns) and a high radiative transition rate constant (k = 0.60 × 10 s ),
F
r
which enable it to be a good material for bio-imaging.
To further modulate the spectroscopic property of anthracene carboxyimide, we synthesized
compounds 5a and 5b, which bear electron-donating (amino) groups at the 10-position of anthracene
carboxyimides. 5a exhibits a significant bathochromic shift both in the absorption and emission
spectra in comparison with 4a, whereas the fluorescence quantum yield (Φ = 0.07) was dramatically
F
decreased, which was due to the non-radiative decay caused by strong ICT and asymmetric skeleton
vibrations in the excited state. By attachment of the di(picolylamine) group at the 10-position of
anthracene carboxyimide, 5b displays moderate fluorescence quantum yield (Φ = 0.10) and a large
F
Stokes shift (~109 nm) in EtOH (Fig 2c). The fluorescence spectrum of 5b was red-shifted by 97 nm
in comparison with 4a, while blue-shifted for 18 nm relative to 5a. The blue-shift in the UV-vis
absorption and fluorescence spectra might be attributed to the electron-withdrawing character of the
di(picolyl) group. This result suggests that the spectroscopic properties of anthracene carboxyimides
can be effectively modulated by changing the substituent groups at the 10-position. To verify this
hypothesis, 5a was modified with an electron-withdrawing acetyl group at the 10-position to afford
compound 6 with an acetylamino structure. As expected, the absorption and fluorescence spectrum

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of compound 6 (Fig. 2d) showed dramatic blue-shift (λ /λ = 437/509 nm) compared to that of 5a
abs em
(
λ /λ = 533/615 nm), and the quantum yield (Φ = 0.35) was greatly enhanced.
abs em F
To further understand the fluorescence emission properties of 4a~6, the fluorescence decay traces
were fitted by using a monoexponential function (Fig. 3), and the fluorescence lifetimes were
determined to be 15.18 (4a), 3.56 (5a), 5.52 (5b) and 1.66 (6) ns, respectively. The monoexponential
nature of the excited state decays gave evidence for the absence of aggregation of these compounds,
which was consistent with the absorption and emission spectra without any sign of aggregates.
Importantly, both 5a and 5b have shorter excited state lifetimes and lower fluorescence quantum
yields than 4a, which suggests that the non-radiative deactivation of the excited state in 5a and 5b
were increased by attachment of some electron-donating groups at the 10-position of anthracene
carboxyimides. Analogue 6 exhibited very similar absorption/fluorescence feature (λ /λ
=
abs em
4
37/509 nm) to that of 4a (λ /λ = 436/500 nm, τ = 15.18 ns) but with a shorter fluorescence
abs em F
lifetime (τ = 1.7 ns), which elicited that 6 might undergo some non-radiative energy attenuation due
F
to the attached functional groups on the 10-position.
2.3 Solvatochromism fluorescence
To gain an insight into the solvatochromic behavior of these anthracene carboxyimides, the
fluorescence and UV-vis absorption spectra were measured in different polarity organic solvents
from hexane to EtOH (shown in Fig. 4-5 and Fig. S1-2), and the photophysical properties of
anthracene carboxyimides were shown in Table 2. As shown in Fig. 4, the UV-vis absorption
spectrum of 4a is less impressionable to the solvent polarity, whereas the fluorescence spectrum
exhibits a significantly positive solvatochromism (red-shift) with increase of the polarity of solvents.
This remarkable solvatochromism in fluorescence spectra might be due to an ICT effect. It is well
established that ICT compounds usually have large dipole moments in the excited state. Such a
polarized excited-state would be stabilized by polar solvents because the dipole-dipole interactions
between solvent and ICT compound would reduce the energy of the excited state [29]. For 5a,
significant red-shifts in absorption and emission maxima were observed when the solvent changed
from hexane to EtOH. This observation might be due to the strong ICT and intramolecular
hydrogen-bond interaction [30] between the -NH- and the pyridyl in 5a. Likewise, 5b showed a

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pronounced red-shift with the increase of solvent polarity, as shown in Fig. 5. 5a and 5b are typical
electron donor-π-conjugation-electron acceptor (D-π-A) structured fluorescent dyes with strong ICT
characters, which make great contribution to their prominent solvatochromic effects. Owing to the
strengthening of dipole-dipole interactions between solute and the solvent, the positive
solvatochromism significantly indicated that ICT is involved in all the 4a~6 anthracene
carboxyimides.
2.4 Photostability.
Photostability of the fluorescent dye/imaging agent is an important parameter for bio-imaging
applications [8]. To investigate the photostabilities of these anthracene carboxyimides, the samples in
EtOH were irradiated with a 35 W Xe lamp for 120 min. Fig. 6 shows the photobleaching of 4a~6
and the commercially available dye fluorescein, which was monitored by steady-state fluorescence
spectroscopy. As shown in Fig. 6, compounds 4a~6 lose less than 2.2% of their fluorescence
intensity under the irradiation from a 35 W Xe lamp for 120 min, while the commercial fluorescein
loses 6.1%. It is likely that introduction of electron-withdrawing carboxyimide moiety at 1,9-position
of anthracene would decrease the HOMO and LUMO energy level, thus inhibits its photo-induced
dimmerization/oxidization (photodimerized under UV irradiation) [31]. The good photostabilities of
4a~6 make feasible their use as long-term bio-imaging agents for in situ visualizing biological events
in living cells.
2.5 pH stability
The pH stabilities of these anthracene carboxyimides were evaluated in various pH buffer
solutions (50 mM, pH 3~10.5) containing 10% EtOH. As shown in Fig. 7, the fluorescence intensity
of 5a and 6 almost remain stable over a wide range of pH values (3.0~9.0), which were attributed to
the insensitive protonation/de-protonation features of aromatic-ring attached amino groups. The
anthracene carboxyimides with good pH stability features are favorable for intracellular analysis and
imaging because they are less impressionable to the pH inside various subcellular organelles.
2.6. Cell imaging
The excellent fluorescence properties of these anthracene carboxyimides prompted us to test their

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ability for bio-imaging in living cells. Cell cytotoxicity of anthracene carboxyimide dyes were
examined by Cell Counting Kit-8 (CCK-8), and the results indicated that these dyes have very low
cytotoxicity (Fig. S3). HeLa cells were incubated with dye 4a (10 ꢀM) and a nucleus-specific
staining probe Hoechst (1 ꢀM) for 30 min, and then washed with PBS for three times to remove the
excessive dye. As shown in Fig. 8c, the nuclei with strong blue fluorescence were observed after
incubation with Hoechst. At the surrounding area of nuclei, a clear cell profile with green
fluorescence was clearly observed after incubation with 4a, suggesting that 4a has permeated into the
HeLa cells in a short period. The good cell membrane permeability and fluorescence property of 4a
enable it to be used as a dye/staining agent for bio-imaging in living cells.
As mentioned before, dye 4a showed good photostability under the irradiation from a Xe lamp.
Herein, we also evaluated the long-term imaging capability of 4a by measuring its photobleaching
resistance capability in HeLa cells. As shown in Fig. 9, a clear HeLa cell profile with green
fluorescence was observed after incubation with 4a (10 ꢀM) for 30 min, then the cells were steadily
irradiated using a 200 W laser (at 488 nm) for 5 min and the cell images were taken by fluorescence
microscope. It could be observed that the fluorescence intensity of HeLa cells was not obviously
changed after exposure to UV irradiation for 5 min. The photobleaching resistance feature made 4a
attractive for long-term bio-imaging applications.
The bio-imaging ability of dye 5a was also examined. HeLa cells were co-incubated with 5a (10
ꢀ
M) and Hoechst (1 ꢀM) for 30 min. As shown in Fig. 8g, nuclei with strong blue fluorescence were
observed, and a clear cell profile with red fluorescence was observed at the surrounding area of
nucleic. To determine the subcellular localization of 5a, a commercially available mitochondrial dye
(
MitoTracker Green FM) was used for a co-localization study. HeLa cells were co-incubated with 5a
10 ꢀM) and MitoTracker Green FM (200 nM) at 37 °C for 30 min. Fig. 10d shows the bright field
(
image of HeLa cells with good cell morphology after incubation with 5a and MitoTracker Green FM.
As shown in Fig. 10b, the cells exhibited a clear mitochondria profile with strong green fluorescence
after incubation with MitoTracker Green FM for 30 min. A similar mitochondria profile with red
fluorescence was observed from the red channel after incubation with 5a for 30 min (Fig. 10a). The
red fluorescence signal originated from 5a overlaid very well with the green fluorescence of
MitoTracker Green FM (Fig. 10c and f). In addition, Pearson's co-localization coefficient, that
describes the correlation of the intensity distribution between two channels, was calculated to be 0.96

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(Fig. 10e), confirming that 5a was site-specifically stained in mitochondria of living cells. This
observation might be due to protonation of pyridyl (pKb for pyridine is ~8.8) inside the cancer cells,
which facilitates it to accumulate in the mitochondria of living cells. The detailed mechanism of the
mitochondria localization effect of 5a is under investigation.
3. Conclusions
In summary, a family of anthracene carboxyimides has been successfully prepared from
anthracene/9-bromoanthracene. Their fluorescence maxima could be effectively modulated from 500
nm to 615 nm by attaching various substituent groups to the 10-position of anthracene carboxyimides.
These compounds display moderate to high fluorescence quantum yields, good pH stability as well
as large Stokes shifts. Additionally, these compounds offer positive solvatochromic fluorescence and
good photo-bleaching resistance capability in organic solvents. Cell staining experiments indicate
that anthracene carboxyimides have excellent cell membrane permeability and good photo-bleaching
resistance in living cells. Particularly, compound 5a is a specific mitochondria-targeting dye with red
fluorescence, which enables it to be a potential mitochondria-staining agent. This research not only
developed a versatile approach to construct a family of anthracene carboxyimides with multi-color
fluorescence, but also opened an avenue to utilize them for bio-imaging.
Acknowledgments
We gratefully acknowledge the financial support from National Science Foundation of China (No.
21203138, 21372251) and the Opening funding from Key Laboratory of Photochemical Conversion
and Optoelectronic Materials, TIPC, Chinese Academy of Sciences. Dr. Ruilong Sheng also
appreciates the Chinese Academy of Sciences (CAS) for the CAS-Canada Young Visiting Scientist
Scholarship and Youth Innovation Promotion Association (YIPA, No2012204) Scholarship.
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Table 1 Photophysical properties of anthracene carboxyimides 4~6 in EtOH.
Compound λabs
ε
λem
ΦF
ꢀν
τF
kr
knr
−
1
−1
−1
−1
−1
(
nm) (M ·cm ) (nm)
(cm ) (ns)
(s )
(s )
3
7
6
4
a
436 9.60 × 10
500 0.91 2936 15.18 5.99 × 10
5.93 × 10
―
3
4
3
4
4
b
438 9.87 × 10
533 1.02 × 10
488 9.61 × 10
437 1.08 × 10
505 0.88 3029
615 0.07 2502
597 0.10 3741
509 0.35 3237
―
―
7
7
8
8
8
8
5
a
3.56 1.97 × 10
5.52 1.81 × 10
1.66 2.11 × 10
2.61 × 10
1.63 × 10
3.92 × 10
5
b
6

ACCEPTED MANUSCRIPT
Table 2 Photophysical properties of anthracene carboxyimides 4a~6 in different solvents.
Compound Solvent
λabs
ε
λem
ΦF
ꢀν
−
1
−1
−1
(
nm) (M ·cm ) (nm)
(cm )
4
4a
5a
5b
6
Hexane 427 1.21 × 10
Toluene 435 9.50 × 10
465 0.61 1914
483 0.69 2285
490 0.93 2475
485 0.83 2530
500 0.91 2936
594 0.04 3286
606 0.08 2841
611 0.10 2609
609 0.07 2810
615 0.07 2502
555 0.06 2815
573 0.06 2873
587 0.12 2964
583 0.07 2968
597 0.10 3741
486 0.59 2572
491 0.52 2517
497 0.48 2658
497 0.36 2815
509 0.35 3237
3
4
3
3
3
4
4
3
4
3
3
3
3
3
4
4
DCM
THF
437 1.01 × 10
432 9.50 × 10
436 9.60 × 10
EtOH
Hexane 497 9.87 × 10
Toluene 517 1.18 × 10
DCM
THF
527 1.44 × 10
520 9.10 × 10
533 1.02 × 10
EtOH
Hexane 480 9.35 × 10
Toluene 492 8.42 × 10
DCM
THF
500 9.22 × 10
497 8.12 × 10
488 9.61 × 10
EtOH
Hexane 432 1.07 × 10
Toluene 437 1.15 × 10
4
4
DCM
THF
439 1.11 × 10
436 1.19 × 10
437 1.08 × 10
4
EtOH

ACCEPTED MANUSCRIPT
O
X
O
O
O
O
i)
ii)
v)
X
X
X=H 1a
X=Br 1b
X=H 2a
X=Br 2b
X=H 3a
X=Br 3b
iii)
from 3b
from 3a
from 3b
N
N
N
C10H21
C10H21
O
N
O
O
N
O
O
N
O
O
N
O
O
N
O
vi)
iv)
O
N
N
HN
N
Br
N
4
a
4b
N N
5b
6
5a
Scheme 1 Synthetic routes for anthracene carboxyimides. i) oxalyl chloride, AlCl ; ii) oxone; iii)
3
1
-aminododecan; iv) 2,2'-dipicolylamine; v) 2-picolylamine; vi) acetyl chloride, K CO .
2 3

ACCEPTED MANUSCRIPT
(
a)
(b)
anthracene
anthracene
1
0
0
0
0
0
.0
.8
.6
.4
.2
1.0
0.8
0.6
0.4
0.2
0.0
4a
5a
5b
6
4a
5a
5b
6
.0
3
00 350 400 450 500 550 600 650
350 400 450 500 550 600 650 700
Wavelength (nm)
Wavelength (nm)
(c)
(d)
4a
5a
5b
6
4a
5a
5b
6
Fig. 1 Normalized UV-vis absorption (a) and fluorescence (b) spectra of anthracene and anthracene
carboxyimide dyes in EtOH. Photographs of anthracene carboxyimide dyes under daylight (c) and
365 nm UV lamp (d).

ACCEPTED MANUSCRIPT
(
a) 4a
Abs 1.0
FL
(b) 5a
1
0
0
0
0
0
.0
.8
.6
.4
.2
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
Abs
FL
0.8
0.6
0.4
0.2
.0
3
0.0
50
420
490
560
630
700
350
420
490
560
630
700
Wavelength (nm)
Wavelength (nm)
(
c) 5b
Abs 1.0
FL
(d) 6
1
0
0
0
0
0
.0
.8
.6
.4
.2
1.0
0.8
0.6
0.4
0.2
0.0
Abs 1.0
FL
0.8
0.6
0.4
0.2
0.8
0.6
0.4
0.2
.0
3
0.0
0.0
50
420
490
560
630
700
350
420
490
560
630
700
Wavelength (nm)
Wavelength (nm)
Fig. 2 Normalized UV-vis absorption and fluorescence spectra of 4a (a), 5a (b), 5b (c) and 6 (d) in
EtOH.

ACCEPTED MANUSCRIPT
4
3
2
■ 4a
10
10
10
■
5a
5b
6
■
■
0
20
40
60
80
100
120
Time (ns)
Fig. 3 Time-resolved fluorescence decay traces recorded for 4a~6 in EtOH at 25 °C. Excitation
wavelength: 480 nm.

ACCEPTED MANUSCRIPT
(
a)
(b)
Hexane
Toluene
DCM
THF
EtOH
1
0
0
0
0
0
.0
.8
.6
.4
.2
1.0
0.8
0.6
0.4
Hexane
Toluene
DCM
THF
EtOH
0.2
0.0
.0
3
50
375
400
425
450
475
500
450
475
500
525
550
575
600
Wavelength (nm)
Wavelength (nm)
Fig. 4 Normalized UV-vis (a) and fluorescence (b) spectra of 4a in different solvents.

ACCEPTED MANUSCRIPT
(
a)
(b)
Hexane
Toluene
DCM
THF
EtOH
1
0
0
0
0
0
.0
.8
.6
.4
.2
1.0
0.8
0.6
Hexane
Toluene
DCM
THF
EtOH
0.4
0.2
0.0
.0
3
50
400
450
500
550
600
525 550 575 600 625 650 675 700
Wavelength (nm)
Wavelength (nm)
Fig. 5 Normalized UV-vis (a) and fluorescence (b) spectra of 5b in different solvents.

ACCEPTED MANUSCRIPT
1
0
0
0
0
0
.00
.98
.96
.94
.92
.90
4
5
6
a
a
Fluorescein
0
20
40
60
80
100 120
Irradiation Time (min)
Fig. 6 Changes of fluorescence intensity (c = 10 ꢀM) under the continuous irradiation from a 35 W
Xe lamp.

ACCEPTED MANUSCRIPT
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
4
5a
6
a
3
4
5
6
7
8
9
10
pH
Fig. 7 Changes of fluorescence intensity (c = 10 ꢀM) at different pH values in buffer solution (50
mM, pH 3~10.5, with 10% EtOH).

ACCEPTED MANUSCRIPT
Bright field
a)
Dye
Hoechst
Overlay
(
(b)
(f)
(c)
(g)
(d)
(h)
4
5
a
a
(e)
Fig. 8 Confocal fluorescence images of HeLa cells stained with Hoechst (1 ꢀM) and dye 4a/5a (10
M). (a, e) Bright field image. (b) Image from dye 4a (green channel: λ = 488 nm, λ = 500–530
ꢀ
ex
em
nm). (c, g) Image from Hoechst (blue channel: λ = 405 nm, λ = 425–475 nm). (d) Merged image
ex
em
of (b) and (c). (f) Image from dye 5a (red channel: λ = 543 nm, λ = 560–610 nm). (h) Merged
ex
em
image of (f) and (g). Scale bar: 10 ꢀm.

ACCEPTED MANUSCRIPT
Bright field
Dye 4a
Overlay
0
s
(
a)
(b)
(e)
(c)
(f)
3
00 s
(d)
Fig. 9 Confocal fluorescence images of HeLa cells stained with dye 4a (10 ꢀM) at different time
points. (a, d) Bright field image. (b, e) Image from dye 4a (green channel: λ = 488 nm, λ
=
em
ex
500–530 nm). (c) Merged image of (a) and (b). (f) Merged image of (d) and (e). Scale bar: 10 ꢀm.

ACCEPTED MANUSCRIPT
(
a)
d)
(b)
(e)
(c)
(
(f)
Fig. 10 Confocal fluorescence images of HeLa cells stained with (a) 200 nM MitoTracker Green FM
green channel: λ = 488 nm, λ = 500–530 nm) and (b) 10 ꢀM dye 5a (red channel: λ = 543 nm,
(
ex
em
ex
λem = 560–610 nm). (c) Merged image of (a) and (b). (d) Bright field image. (e) Correlation plot of
MitoTracker Green FM and dye 5a intensities. (f) Intensity profile of regions of interest (ROI) across
HeLa cells. Scale bar: 10 ꢀm.

ACCEPTED MANUSCRIPT
Highlights
ꢀ
ꢀ
ꢀ
ꢀ
A family of anthracene carboxyimides with multi-color fluorescence have been
successfully prepared.
The steady-state, time-resolved and solvatechromic fluorescence of the anthracence
carboxyimides could be modulated by changing the substitution groups.
The anthracene carboxyimides shows high photostability and good cell staining
properties.
Red fluorescent 5a shows a specific mitochondria-targeting effect.