Novel two-photon fluorescent probe with high fluorescence quantum yields for tracking lipid droplets in biological systems

Article abstract of DOI:10.1016/j.saa.2019.02.106

Lipid droplets (LDs) played an important role in storing neutral lipids process. In this work, we developed a series of fluorescent dyes PIE1-PIE4 with high fluorescence quantum yields based on a single phenanthrenequinone imidazole fluorophore core. Amon

Full text of DOI:10.1016/j.saa.2019.02.106

Accepted Manuscript
Novel two-photon fluorescent probe with high fluorescence
quantum yields for tracking lipid droplets in biological systems
Jie Niu, Yong Liu, Weishan Wang, Weiying Lin
PII:
S1386-1425(19)30226-4
https://doi.org/10.1016/j.saa.2019.02.106
SAA 16883
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 1 November 2018
Revised date:
Accepted
date:
20 February 2019
2
5 February 2019
Please cite this article as: J. Niu, Y. Liu, W. Wang, et al., Novel two-photon fluorescent
probe with high fluorescence quantum yields for tracking lipid droplets in biological
systems, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy,
https://doi.org/10.1016/j.saa.2019.02.106
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Novel two-photon fluorescent probe with high fluorescence
quantum yields for tracking lipid droplets in biological
systems
Jie Niu, Yong Liu, Weishan Wang and Weiying Lin*
Institute of Fluorescent Probes for Biological Imaging, School of Materials Science
and Engineering, School of Chemistry and Chemical Engineering, University of Jinan
Shandong 250022, P. R. China.
*Corresponding Author.
E-mail: weiyinglin2013@163.com
Tel.: +86 0531 82769031
Fax: +86 0531 82769031

ACCEPTED MANUSCRIPT
Abstract
Lipid droplets (LDs) played an important role in storing neutral lipids process. In
this work, we developed a series of fluorescent dyes PIE1-PIE4 with high
fluorescence quantum yields based on a single phenanthrenequinone imidazole
fluorophore core. Among these compounds, we introduced diethyl aniline group on
the phenanthrenequinone imidazole core to provide the compound PIE1. It is firstly
found that PIE1 could real-color image and track lipid droplets (LDs) in living cells.
However, we modified nitrile, imidazole and methyl group on the same fluorophore
core to afford three dyes PIE2, PIE3 and PIE4, respectively. Although PIE2, PIE3
and PIE4 possessed high fluorescence quantum yields in various organic solvents,
they could not real-color image and track LDs in living cells.
Keywords
Lipid droplets; Phenanthrenequinone imidazole; Two-photon; Real-color image;
Tracking.

ACCEPTED MANUSCRIPT
1
. Introduction
The lipid droplets (LDs) are unique organelles storing neutral lipids in biological
systems [1-3]. These organelles distributed throughout the cytoplasm in different cells
lines [4]. It not only facilitated the transport of lipids to the other organelles but also
improved the transport efficiency of protein [5]. In addition, some scientists found
that virus particles were closely associated with LDs in biological systems. This result
indicated that steps of virus assembly took place around LDs [6, 7]. Moreover, some
studies found that LDs not only were simple warehouse for lipid storage, but also
were dynamic subcellular structure for regulating lipid metabolism [8, 9]. They were
closely associated with various metabolic diseases, including diabetes, cardiovascular
diseases, and hypertension and so on [10-15]. To facilitate the applications of LDs in
the biomedical research field, tracking and imaging intracellular LDs were very
important.
Fluorescent probe, as a unique tool, was attractive in detecting target molecules in
biological images field [16-22]. However, developing a fluorescent probe for selective
enrichment in lipid droplet region in biological system was very challenging. In recent
years, several classes of molecular probes have been constructed for detection of LDs
in biological imaging fields (Table S1). Some commercially available LDs probes
‘‘BODIPY493/503’’ and ‘‘Nile red’’ have been developed [23, 24]. However, research
on a low molecular weight probe that could real-color image LDs had never been
conducted. Thus, constructing a series of novel fluorescent probes for real-color
imaging and tracking lipid droplets may shed light on substantial progress in
exploring physiological function of LDs in biological systems.
As we know, phenanthrenequinone imidazole-based dyes and probes have been
developed due to their excellent physical properties [25]. In this study, we constructed
a series of fluorescent dyes PIE1-PIE4 with high fluorescence quantum yields based
on a single phenanthrenequinone imidazole fluorophore core (Fig.1). Among these
compounds, we introduced diethyl aniline group on the phenanthrenequinone
imidazole core to give the compound PIE1. It is found that PIE1 could real-color

ACCEPTED MANUSCRIPT
image and track LDs in biological systems. Although PIE2, PIE3 and PIE4
possessed high fluorescence quantum yields in various organic solvents, they could
not real-color image and track LDs in living cells.
Compared to the conventional LDs probes, PIE1 exhibits higher fluorescence
quantum yields in various solutions and photostability. Moreover, this novel probe is
capable of real-color imaging and tracking LDs in the living cells and tissues.
Fig. 1. Structures and properties of the compounds PIE1-PIE4.
2
. Material and methods
2
.1. Apparatus and chemicals
First, for all synthesis and analysis experiments, all reagents were purchased by
commercial providers. Before experiments, these reagents need to further purification.
Moreover, proton and aprotic solvents were needed further purified before experiment.
Second, for synthesis experiment, thin layer chromatography (TLC) analysis mainly
used to test reaction process of chemical reaction. In addition, separation and
purification of compounds were carried out by column chromatography; Silica gel
(mesh 200-300) was purchased from the Qingdao Ocean Chemicals. Third, for
characterization, high resolution mass spectra (HRMS) were obtained by Thermo

ACCEPTED MANUSCRIPT
Finnigan or Agilent 1100 HPLC/MSD spectrometer; Nuclear magnetic resonance
(NMR) spectra were carried out with the AVANCE III 400/500 MHz Digital NMR
spectrometer. Fourth, for analysis experiment, ultraviolet absorption (Uv) spectra
were measured using a Labtech UV Power PC spectrometer; Fluorescence emission
spectra were recorded by the HITACHI F4600 fluorescence spectrophotometer. Fifth,
for biological imaging, fluorescence imaging pictures of the cells and tissues slices
were carried out by Nikon A1MP two-photon (TP) confocal microscopy. TP and
real-color imaging were obtained by Nikon A1MP confocal microscopy. We used 40 ×
objective lens for the imaging experiments. The Mai Tai DeepSee laser offers 2.5 W
of average power and 350 nm (690-1040 nm) in useable tuning range. The pulse
width is 100fs (800 nm) and the pulse frequency of two-photon illumination is
8
0MHz. Scan resolution: ≥ 4096×4096, gray level: ≥ 12bit. Scanning speed: 20 s/scan.
Living cells fresh were cultivated using fresh medium.
2
.2. Synthesis
Chemical synthesis of the compounds PIE1- PIE4 were accomplished in one step.
The target molecule was obtained by a Knoevenagel condensation reaction. Synthetic
details of the products were given in the Supporting Information.
Synthesis of PIE1: A mixture of 9,10-phenanthroquinone (0.25 g, 1.2 mmol),
compound 2 (1 mmol) and ammonium acetate (1.54 g, 20 mmol) in glacial acetic acid
(20 mL) was heated to 100 ℃for 12 h and flushed with nitrogen. The reaction system
was cooled to 25 ℃. And then, the gray solid was obtained by filtration. The resulting
solid was washed using acetate acid, dilute sodium hydrogen carbonate solution and
water. The solid was further dried under reduced vacuum obtained the pure gray
1
product with a yield of 70 %. H NMR (400 MHz, DMSO-d ) (ppm): 8.84 (d, J =8.0
6
Hz, 2H), 8.55 (d, J =7.6 Hz, 2H), 8.12 (d, J =8.8 Hz, 2H), 7.71 (t, J =7.2 Hz, 2H),
7
.60 (t, J =7.2 Hz, 2H), 6.83 (d, J =8.8Hz, 2H), 3.44 (q, J =7.0 Hz, 4H), 1.16 (t, J =7.0
1
3
Hz, 6H). C NMR (100 MHz, DMSO-d ) δ: 174.4, 174.3, 151.0, 148.5, 128.1, 127.7,
6
+
1
27.3, 125.1, 124.2, 122.3, 117.6, 111.6, 49.1, 13.0. HRMS (m/z): [M+H] calcd for
C H N , 366.1965; found:366.1963.
25
23
3

ACCEPTED MANUSCRIPT
The compound PIE4 was synthesized by referring literature [26].
Synthesis of the compounds PIE2 and PIE3 are similar with synthetic procedure
of PIE1.
1
PIE2: H NMR (400 MHz, DMSO-d ) (ppm): 13.76 (s, 1H), 8.90 (d, J =8.4 Hz,
6
1
H), 8.86 (d, J =8.4 Hz, 1H), 8.60 (d, J =7.2 Hz, 1H), 8.55 (d, J =7.6 Hz, 1H), 8.49 (d,
13
J =8.4 Hz, 2H), 8.09 (d, J =8.4 Hz, 2H), 7.82 –7.63 (m, 4H). C NMR (100 MHz,
DMSO-d ) δ 147.6, 137.8, 134.8, 133.4, 128.8, 128.6, 128.2, 127.8, 127.6, 127.3,
6
1
27.0, 126.3, 126.0, 124.6, 124.2, 122.7, 122.6, 122.4, 119.2, 111.5. HRMS (m/z):
+
[
M+H] calcd for C H N , 320.1182; found:320.1187.
2
2
13
3
1
PIE3: H NMR (500 MHz, DMSO-d ) (ppm): 8.87 (d, J =8.0 Hz, 2H), 8.80 (s,
6
2
H), 8.69 (d, J =8.5 Hz, 2H), 8.62 (s, 1H), 7.99 (s, 1H), 7.94 (d, J =9.0 Hz, 2H), 7.74
1
3
(
t, J =7.5 Hz, 2H), 7.65 (t, J =7.5 Hz, 2H), 7.28(s, 1H). C NMR (100 MHz,
DMSO-d ) δ 148.7, 137.3, 135.9, 129.5, 129.5, 128.3, 128.1, 127.5, 125. 8, 124.3,
6
+
1
3
22.9, 121.0, 118.6. HRMS (m/z): [M+H] calcd for C H N , 361.1448; found:
2
4
16
4
61.1453.
2
.3. Preparation of Samples and Test Solution
First, to obtain 1 mM stock solution of PIE1-PIE4, the compounds of different
quantity were dissolved in DMSO. We prepared 10 μM PIE1-PIE4 to carried out
optical experiments. They were obtained by adding 50 μL the stock solution to 5.0 mL
various solutions. Fluorescence and absorption spectra were further carried out with
HITACHI F4600 fluorescence spectrophotometer and a Labtech UV Power PC
spectrometer, respectively.
Second, we further evaluated the fluorescence quantum yields of PIE1-PIE4 by the
following equation (1):
2

 ( ) 
ns




Fs
Fr
r
r
s  r 




2
s (s )
nr

(1)
s and r were sample and the reference, respectively; Ф and F stand for quantum
yield and integrated emission intensity, respectively. A and n stand for absorbance and
refractive index, respectively.

ACCEPTED MANUSCRIPT
Third, two-photon absorption (TPA) cross sections of PIE1 and PIE2 were
measured using the two-photon induced fluorescence method, and the cross section
can be calculated by means of equation (2):
r c n F
r
r
s
s  r
s c n F
s
s
r
(2)
Where the subscripts s and r refer to the sample and the reference, respectively. The
terms c and n are the concentration and refractive index of the solution, respectively.
F is two-photon excited fluorescence integral intensity. Φ is the fluorescence quantum
yield. δ is the TPA cross-section of fluorescein in aqueous NaOH (pH=11) at different
r
excitation wavelength.
2
.4. Cell viability Evaluated by MTT Assays
Firstly, living cells were cultivated with 96-well plate in a 5 % CO incubator at
2
3
7 °C. Secondly, after 12 h, living cells were cultivated using fresh medium
containing different concentrations PIE1-PIE4 (1, 5, 10, 20, 30 μM). Thirdly, after 24
h, the excess medium and probes were cleared up, and then 10 μL
(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide (MTT) (5 mg/mL in
phosphate buffer solution (PBS)) was dropped into the above plate. Subsequently, the
culture medium was cleared up, and 100 μL DMSO was dropped into the dishes to
dissolve the formazan crystal product. Fourthly, 96-well plate was shaken up for a
moment. Absorbance data was obtained using multiscan spectrum. Finally, cell
viability was obtained with the equation: The cell viability (%) = (OD490 sample - OD490
blank)/(OD490 control - OD490 blank) × 100%. OD490 sample stand for the cells incubated with
probes of different concentrations, OD490 control was cells untreated with probe, OD490
was cell containing culture medium.
blank
2
.5. Colocalization experiment
Living HeLa cells were cultivated using glass coverslips in a 5 % CO incubator at
2
3
7 °C. After 2 days, two groups HeLa cells were prepared by the following
experiment procedures: Firstly, living cells were cultivated in 5 μM PIE1 and PIE2 in

ACCEPTED MANUSCRIPT
a 5 % CO cell incubator at 37 °C for 30 min, respectively; Secondly, two groups cells
2
were washed using buffer solution; Thirdly, 1mL medium containing 10 μM
BODIPY493/503 was added to two groups glass coverslips, and then incubated for 20
min in the same conditions. Fourthly, cells were washed using buffer solution. Finally,
the colocalization imaging was carried out by NIKON A1MP confocal fluorescence
microscope (40 × objective lens).
Imaging pictures of the dyes PIE1 and PIE2 were obtained between 425 and 475
nm at 405nm excitation; Fluorescence images of BODIPY493/503 were carried out
between 500 and 550 nm at 488 nm excitation.
2
.6. Real-color imaging
First, the real-color images were performed by two-photon NIKON A1MP
fluorescence microscope.
Second, the real-color images were acquired with the spectra imaging function of
NIKON A1MP fluorescence microscope. This function could separately collection of
emission in numerous short wavelength ranges.
Third, in our experiments, living Hela cells were incubated with probe PIE1 (5 μM)
for 30 min. The in situ images pictures were acquired with the spectra imaging
function of NIKON A1MP fluorescence microscope. Excitation wavelength was 760
nm; emission wavelength was 404- 643 nm. Every image picture corresponds to its in
situ emission spectral profile.
2
.7. Two-photon imaging
The living HeLa cells were cultivated with 5 μM PIE1-PIE4 for 30 min.
Subsequently, the cells were washed with buffer solution, and then transferred to
glass-bottomed dishes, and observed using confocal microscope. The fluorescence
spectral data of the cells were analyzed by Nikon analysis software NIS-Elements AR.
Tissues imaging: Firstly, we prepared slices of the liver tissues; Secondly, 400 μm
thickness slides were obtained by the vibrating-blade microtome; Thirdly, the slices
o
were incubated using 10 μM PIE1 in PBS buffer solution for 1 h at 37 C, and then

ACCEPTED MANUSCRIPT
washed with buffer solution; Fourthly, the tissue slices were transferred to
glass-bottomed dishes. Then, fluorescent imaging was carried out by fluorescent
microscope. The conditions of fluorescence images: Excitation wavelength: 760 nm;
Emission wavelength: 425-475 nm.
3
. Results and Discussion
3
.1. Design and synthesis of fluorescent dyes PIE1-PIE4 with high fluorescence
quantum yields
To date, two classes of LDs probes have been mainly used: AIE probes and
conventional lipid droplets probes with ACQ feature [27, 28]. For example, Yu. group
reported two-photon fluorescent lipid droplet probes N-Cy and N-Py for high-fidelity
imaging of turbid tissues and detecting fatty liver [29]. These probes with ACQ
feature exhibited low fluorescence intensity and low fluorescence quantum yields in
aqueous phase. Similarly with N-Cy, some commercial available LDs probes (ex:
‘‘Nile red’’) showed similar optical properties. Thus, generally, lipid droplets probes
showed stronger fluorescence emission intensities in organic phase than aqueous
solution.
Phenanthrenequinone imidazole derivative possessed excellent optical properties
for developing fluorescent probes. The greatest characteristic of these dyes is that they
exhibit higher quantum yields and fluorescence intensities in organic solvents than
aqueous phase. Therefore, we will construct LDs probes based on
phenanthrenequinone imidazole fluoremidine-core. In addition, alkyl chain is similar
to hydrophobic chain of lipid drops [30-32]. These compounds containing such
groups enrich in lipid drops. These properties are the basis for constructing LDs
probes. For example, in 2016, Kim et al reported a LDs probe based on pyrazolo
[
1,5-a]pyridine-fused pyrimidine [33].
In this work, we introduced diethyl aniline group on phenanthrenequinone
imidazole fluoremidine-core to afford a novel LDs probe PIE1. Because these dyes
containing hydrophobic chain should exhibit stronger fluorescence intensities in

ACCEPTED MANUSCRIPT
organic phases than aqueous phase. Other groups were modified on fluoremidine-core
could not exhibit this unique property. Thus, PIE1 was more in line with the spectral
characteristics of the conventional LDs probe than the other three dyes (Fig. 2).
Fig. 2. (A) Structures of the LDs probes Nile Red and FD13. (B) In this work:
phenanthrenequinone imidazole-based fluorescent dyes (PIE1-PIE4) with high fluorescence
quantum yields.
3
.2. Optical properties of PIE1-PIE4
With the dyes PIE1-PIE4 in hand, we set out to investigate their absorption and
emission spectra in different solvents. As shown in Fig. S1 and Tables S2-S5, for the
dyes PIE1-PIE4, our studies results demonstrated that these compounds possessed
maximum absorption and emission peaks at 345 nm and 417 nm in pure water system.
Moreover, as shown in Fig. 3A, the dye PIE1 showed high fluorescence quantum
yields in organic solvents. Especially in ether, fluorescence quantum yield of PIE1
was close to 100 %. The fluorescence quantum yields of PIE1 in organic solvents is
much higher than aqueous solution. These results are consistent with optical physical
properties of LDs probes. Similarly with PIE1, PIE2-PIE4 possessed much higher
fluorescence quantum yields in organic solvents than aqueous solution (Figs. 3B-D).
We investigated the aggregation of PIE1-PIE4 in aqueous solution by the dynamic
light scattering (DLS) technique. As shown in Fig. S2, in aqueous solution, these
compounds formed aggregates with an average size of 100 nm. We also measured the
two-photon absorption cross sections of PIE1 and PIE2 under different excitation
wavelength. As shown in Fig. S3, compared to other excitation wavelength, PIE1 and

ACCEPTED MANUSCRIPT
PIE2 showed higher two-photon absorption cross section at 760 nm excitation. The
results indicated that the dyes PIE1 and PIE2 should possess ability achieving TP
imaging in deep tissues.
Fig. 3. Fluorescence quantum yields of PIE1-PIE4 (10 μM) in various solutions. Error limit:
20%.
Next, we set out to investigate the optical properties of the compounds PIE1-PIE4
in different solvents. As shown in Fig. 4, in nonpolar solvents (Ether and DCM), the
dyes PIE1-PIE4 showed higher fluorescent emission intensities than polar solvents
(DMF and MeOH). Especially in aqueous solution, PIE1-PIE4 showed weaker
fluorescence emission intensity than nonpolar solvents. The above optical properties
might allow the rational probe design of LDs organelles/biopolymers in living cells as
unique fluorescent sensor [33].

ACCEPTED MANUSCRIPT
Fig. 4. (A) Structure and optical properties of PIE1 (10 μM) resulting from solvents polarity; (B)
Structure and optical properties of PIE2 (10 μM) in different solvents polarity; (C) Structure and
optical properties of PIE3 (10 μM) in different solvents polarity; (D) Structure and optical
properties of PIE4 (10 μM) in different solvenst polarity.
3
.3. LDs imaging in living cells.
To prove the above assumption, we verify PIE1-PIE4 whether could locate
selectively to LDs in biological systems. Before achieving cells images, cell
viabilities of PIE1-PIE4 were evaluated by the standard MTT experiment [34]; The
MTT experiment results demonstrated that PIE1-PIE4 exhibited low cytotoxicity for
living cells (Table S6-S9). Thus, these probes were suitable of imaging living cells.
Interestingly, PIE1 and PIE2 exhibited clear fluorescence images in living cells
(Figs. 5A-F). However, PIE3 and PIE4 cannot present clear fluorescence images due
to their weak fluorescence intensities or high background signals (Figs. 5G-L). To

ACCEPTED MANUSCRIPT
further verify whether PIE1 and PIE2 can image LDs in the living cancer cells,
co-localization experiments were carried out. HeLa cells were incubated with PIE1
and commercially available lipid droplets indicator BODYPI 493/503, and then
fluorescence imaging was carried out. Co-localization experiments showed that the
fluorescence of PIE1 and BODYPI 493/503 merged well. The Mander’s overlap
coefficient and Pearson’s colocalization coefficient were determined as 0.92 and 0.87,
respectively. View of this, PIE1 predominantly located to lipid droplets in living cells
(Figs. 6A-E). However, the Mander’s overlap coefficient and Pearson’s colocalization
coefficient for probe PIE2 with BODYPI 493/503 only were 0.41 and 0.43,
respectively (Figs. 6F-J). The results indicated that PIE2 could not image lipid
droplets in living cells.
Fig. 5. Confocal images of PIE1-PIE4 (5 µM) in living HeLa cells. (A, D, G, J): Bright-field
images; (B, E, H, K): Fluorescence images of PIE1-PIE4 (λ = 405 nm; λ = 425-475 nm); (C, F,
ex
em
I, L): Merged pictures. Scale bar =20 μm.

ACCEPTED MANUSCRIPT
Fig. 6. Confocal images of cells treated with PIE1 and PIE2 (5 μM) for 30 min and
BODYPI493/503 (10 μM) for 20 min. Fluorescence images of PIE1 (A) and PIE2 (F) collected
between 425 and 475 nm upon excitation at 405 nm; (B, G): Fluorescence images of
BODYPI493/503 collected between 500 and 550 nm upon excitation at 488 nm; (C and H):
Merged images of (A and B) and (F and G). Intensity profile of the linear region of the interest
(white arrow) across the cells co-incubated with BODYPI493/503 and PIE1 (D) and PIE2 (I); (E,
J): Correlation plot of BODYPI493/503 and probes and intensities. Scale bar = 20 μm.
Through the above study, PIE1 containing diethyl aniline group is capable of
imaging LDs in living cells. Although PIE2 is capable of achieving fluorescent
imaging in living cells, PIE2 containing nitrile group could not locate to LDs in living
cells. PIE3 and PIE4 could not achieve cells images in living cells. Thus, the dyes
PIE1, PIE2, PIE3 and PIE4 exhibit dramatical distinctions in optical properties and
cell imaging utilities due to subtle differences of structures. This finding may explore

ACCEPTED MANUSCRIPT
novel sense site for constructing LDs fluorescent probes with high fluorescence
quantum yields.
3
.4. Two-photon real-color imaging and tracking lipid droplets in living biological
systems
We set out to investigate whether PIE1 could real-color image LDs in living
biological systems with fluorescence microscope. First, HeLa cells were incubated
with probe PIE1 (5 μM) for 30 min. The imaging results demonstrated that PIE1
showed strong fluorescence intensity or low background signals than PIE2 (Fig. 7).
Second, TP real-color imaging was further obtained. We separately collected emission
spectra of PIE1 in different short wavelength ranges (405-645 nm) by TP in situ
software. As shown in Fig 8A, the fluorescent images with 405-645 nm exhibited blue
in situ fluorescence signal at 760 nm excitation. The merge of all these images gave a
real-color imaging picture and showed in situ fluorescence spectra (Fig. 8B). The
results indicated that TP in situ emission spectra was consistent with one-photon
spectra in vitro. Thus, PIE1 was capable of in situ imaging lipid droplets in living
cells by two-photon fluorescence microscope. Similarly with PIE2, we further
obtained in situ imaging and in situ emission spectra (Fig. S4).
Fig. 7. Two-photon images of PIE1 and PIE2 (5 µM) in living HeLa cells. (A, C): Bright-field
images; (B, D): Fluorescence images of PIE1 (B) and PIE2 (D) (λ = 760 nm; λ = 425-475 nm).
ex
em
Scale bar = 20 μm.

ACCEPTED MANUSCRIPT
Fig. 8. TP in situ fluorescence images and spectra of the HeLa cells incubated with PIE1 (5 µM)
by the Nikon infrared spectra detector (IR SD). (A) TP fluorescence images of HeLa cells
collected between 404 and 643 nm upon excitation at 760 nm. (B) TP fluorescence spectra of the
HeLa cells collected between 404 and 643 nm upon excitation at 760 nm.
Photostability was a key criterion for developing excellent fluorescent probe
[
35-37]. Fluorescence images of PIE1 were acquired at different times under
successive excitation (Fig. 9). Furthermore, TP mean intensities of blue channel at
different times were obtained. The above results demonstrated that PIE1 showed high
two-photon photostability at different times under successive excitation. However, the
compound PIE1 showed lower one-photon photostability at different times under
successive excitation than TP (Fig. S5). The probe PIE1 is capable of tracking
dynamic process of LDs in living cells in a long period at 760 nm excitation (Video
S1). In addition, the probe PIE2 possesses one- and two-photon photostability in
living cells (Figs. S6 and S7).

ACCEPTED MANUSCRIPT
Fig. 9. (A) Two-photon fluorescence images of living cells incubated with PIE1 acquired at
different times under successive two-photon excitation. (B) Two-photon fluorescent intensities of
the cells incubated with PIE1 (5 μM) at different times. Excitation wavelength: 760 nm; Emission
filter: 425-475 nm.
3
.5. Two-photon imaging in living tissue
We have demonstrated that PIE1 can image LDs in living cells. These favorable
results further encouraged us to study LDs imaging in deep living tissues. First, we
incubated liver tissues with the probe PIE1, and then TP fluorescence images were
carried out (Fig. 10). As shown in Fig. 10A and Fig. S8, strong blue fluorescence
signals were acquired in tissues. We prolonged the incubation time and added the
concentration of the probe. As shown in Fig. S9, strong blue fluorescence signals were
still observed in deep tissue (90 μm) at 760 nm excitation. However, fluorescence
signals couldnot be observed in deep tissue at 405 nm excitation. We further obtained
the 3D imaging pictures from superposition of tissue section (Fig. 10B). Because we
have proved that PIE1 could image LDs in living cell. Thus, we envisioned that PIE1
could image LDs in living tissues. To prove PIE1 can sense LDs in tissues,
colocalization experiments were carried out. The results indicated PIE1 stained

ACCEPTED MANUSCRIPT
selectively LDs in tissues (Figs. S10-S11).
To get in-depth understand imaging characteristics of PIE1 to LDs in tissues, we
obtained the fluorescence intensities of each slide. Through this study, it was found
that fluorescence signal of probe presented tendency of increases firstly and then
decreases (Fig. 10C). It provided an important reference for investigating distribution
characteristics of LDs in living tissues. In addition, it was found that PIE2 exhibited
clear TP fluorescence 3D imaging results in living tissue (Fig. 11 and Figs. S12-S13).
We have proved the PIE2 couldnot image LDs in living cells. Thus, PIE2 couldnot
image LDs in living tissues.
Fig. 10. Two-photon fluorescence images of the living tissues treated with PIE1 (10 μM) for 1 h.
(A and B) 3D fluorescence images (λex = 760 nm, λem = 425-475 nm); (C) Mean fluorescence
intensities of blue channel at different depth.

ACCEPTED MANUSCRIPT
Fig. 11. Two-photon fluorescence images of the living tissues treated with PIE2 (10 μM) for 1 h.
(A and B) 3D fluorescence images (λex = 760 nm, λem = 425-475 nm); (C) Mean fluorescence
intensities of blue channel at different depth.
4
. Conclusion
In summary, we constructed a series of fluorescent dyes PIE1-PIE4 with high
fluorescence quantum yields. We introduced diethyl aniline group on the
phenanthrenequinone imidazole core to afford a new LDs probe PIE1. By
investigating one- and two-photon imaging properties of PIE1, it is firstly found that
this novel dye was capable of real-color imaging and tracking LDs in living cells by
TP fluorescence microscope. Compared to conventional LDs probes, the probe PIE1
possessed much higher fluorescence quantum yields in various solution; Although the
compounds PIE2, PIE3 and PIE4 exhibited higher fluorescence quantum yields in
various solutions, they could not real-color image and track LDs in living cells. The
main reasons were as follows: Similarly with hydrophobic chain of lipid drops, dyes
containing alkyl chains possessed strong hydrophobicity property. These compounds
containing hydrophobic chain often sense lipid drops as novel LDs probes. Moreover,
these probes exhibited stronger fluorescence emission intensities in organic phase

ACCEPTED MANUSCRIPT
than aqueous phase. Thus, PIE1 was more in line with the spectral characteristics of
the conventional LDs probes than PIE2-PIE4. This research will provide important
clues for constructing new LDs probes.
Acknowledgements
Funding was partially provided by NSFC (21472067, 51503077, 21672083);
Taishan Scholar Foundation (TS 201511041), doctor start up fund of University of
Jinan (XBS1512), NSFSP (ZR2015PE001), and the startup fund of the University of
Jinan (309-10004).
References
[
1] B. Wölk, W. M. D. Heiner, Hepatitis C virus living off the fat of the land,
Hepatology 47 (2008) 343-345.
[
2] Y. Miyanari, K. Atsuzawa, N. Usuda, K. Watashi, T. Hishiki, M. Zayas, R.
Bartenschlager, T. Wakita, M. Hijikata, K. Shimotohno, The lipid droplet is an
important organelle for hepatitis C virus production, Nat. Cell Biol. 9 (2007)
1
089-1097.
3] S. Vanni, Intracellular Lipid Droplets: From structure to function, Lipid Insights
2017) 10.
[
[
(
4] M. P. Sheetz, S. L. Dabora, E Steuer, T. A. Schroer, Control of organelle
movements and endoplasmic reticulum extension powered by kinesin and
cytoplasmic dynein, Current Topics in Membranes & Transport 36 (1990)
1
17-128.
[
5] Y. Ding, L. Yang, S. Zhang, Y. Wang, Y. Du, J. Pu, G. Peng, Y. Chen, H. Zhang,
J. Yu, H. Hang, P. Wu, F. Yang, H. Yang, A. Steinbüchel, P. Liu, Identification
of the major functional proteins of prokaryotic lipid droplets, J. Lipid Res. 53
(2012) 399-411.
[
[
6] J. Hendry, A. Kulyavtsev, D. Litvintsev, A. Moibenko, S. Naymola, G. Oleynik,
Solving Small Files Problem in Enstore (Abstract for CHEP 2013), Nat. Cell Biol.
9
(2013) 1089-1097.
7] J. Mclauchlan, Hepatitis C virus: viral proteins on the move, Biochem. Soc. T. 37
2009) 986-990.
(

ACCEPTED MANUSCRIPT
[
[
[
[
8] M. Suzuki, Regulation of lipid metabolism via a connection between the
endoplasmic reticulum and lipid droplets, Anat. Sci. Int. 92 (2017) 50-54.
9] K. Liu, M. J. Czaja, Regulation of lipid stores and metabolism by lipophagy, Cell
Death & Differentiation 20 (2013) 3-11.
10]W. J Shen, S. Patel, D. Jue, Z. Yu, F. B. Kraemer, Changes of expression of lipid
droplet associated proteins with high fat feeding, Diabetes 56 (2007) 354.
11]P. M. Mcdonough, R. M. Agustin, R. S. Ingermanson, P. A. Loy, B. M. Buehrer,
J. B. Nicoll, N. L. Prigozhina, I. Mikic, J. H. Price, Quantification of lipid
droplets and associated proteins in cellular models of obesity via
high-content/high-throughput microscopy and automated image analysis, Assay
Drug Dev. Technol. 7 (2009) 440-460.
[
[
12]A. Paul, L. Chan, P. E. Bickel, The PAT family of lipid droplet proteins in heart
and vascular cells, Curr. Hypertens Rep. 10 (2008) 461-466.
13]Y. L. Perov, Y. V. Postnov, Lipid droplets of interstitial medullary cells of intact
rat kidney with 2 kidney goldblatt hypertension, Virchows Archiv B: Cell
Pathology 22 (1976) 163-172.
[
[
14]A. A. Elamin, M. Stehr, M. Singh, Lipid droplets and mycobacterium leprae
infection, Journal of pathogens 2012 (2012) 1-10.
15]H. Liu, J. Bai, X. Weng, T. Wang, M. Li, Amelioration of insulin resistance in rat
cells by Astragalus polysaccharides and associated mechanisms, Exp. Ther. Med.
7
(2014) 1599-1604.
[
[
[
[
16]X. Guan, W. Lin, W. Huang, Development of a new rhodamine-based fret
2
+
platform and its application as a cu probe. Org. Biomol. Chem. 12 (2014)
944-3949.
3
17]H. Chen, B. Dong, Y. Tang, W. Lin, A unique "integration" strategy for the
rational design of optically tunable near-infrared fluorophores. Acc. Chem. Res.
5
0 (2017) 1410 -1422.
18]L. Yuan, W. Lin, L. Tan, K. Zheng, W. Huang, Lighting up Carbon Monoxide:
Fluorescent Probes for Monitoring Carbon Monoxide in Living Cells, Angew
Chem. Int. Edit. 52 (2013) 1628-1630.
19]P. Ning, W. Wang, M. Chen, Y. Feng, X. Meng, Recent advances in
mitochondria- and lysosomes-targeted small-molecule two-photon fluorescent
probes, Chin. Chem. Lett. 28 (2017) 1943-1951.

ACCEPTED MANUSCRIPT
[
[
[
[
[
20]B. Zhang, X. Yang, R. Zhang, Y. Liu, X. Ren, M. Xian, Y. Ye, Y. Zhao, A
lysosomal-targeted two-photon fluorescent probe to sense hypochlorous acid in
live cells, Anal. Chem. 89 (2017) 10384-10390.
21]Y. Lin, W. Lin, Y. Yang, J. Song, A fast-responsive fluorescent probe for
detection of gold ions in water and synthetic products, Chem. Commun. 47 (2011)
4
703-4705.
22]L. Yuan, W. Lin, Y. Feng, A rational approach to tuning the pK values of
a
rhodamines for living cell fluorescence imaging, Org. Biomol. Chem. 9 (2011)
1
723-1726.
23]E. E. Spangenburg, S. J. P. Pratt, L. M. Wohlers, R. M. Lovering, Use of
BODIPY (493/503) to Visualize Intramuscular Lipid Droplets in Skeletal Muscle,
J. Biomed. Biotechnol. 2011 (2011) 598358 (8 pages).
24]M. Romek, B. Gajda, E. Krzysztofowicz, M. Kepczynski, Z. Smorag, New
technique to quantify the lipid composition of lipid droplets in porcine oocytes
and pre-implantation embryos using Nile Red fluorescent probe, Theriogenology
5
3 (2005) 5766-5771.
[
[
25]F. C. Krebs, H. Spanggaard, An exceptional red shift of emission maxima upon
fluorine substitution, J. Org. Chem. 67 (2002) 7185-7192.
26]W. Lin, L. Long, L. Yuan, Z. Cao, B. Chen, W. Tan, A ratiometric fluorescent
probe for cysteine and homocysteine displaying a large emission shift, Org. Lett.
1
0 (2008) 5577-5580.
[
27]M. Kang, X. Gu, R. T. K. Kwok, C. W. T. Leung, J. W. Y. Lam, F. Li, B. Z.
Tang, A near-infrared AIEgen for specific imaging of lipid droplets, Chem.
Commun. 52 (2016) 5957-5960.
[
[
28]P. Greenspan, E. P. Mayer, S. D. Fowler, Nile Red: A Selective Fluorescent Stain
for Intracellular Lipid Droplets, J. Cell Biol. 100 (1985) 965-973.
29]L. Guo, M. Tian, R. Feng, G. Zhang, R. Zhang, X. Li, Z. Liu, X. He, J. Z. Sun, X.
Yu, Interface-targeting strategy enables two-photon fluorescent lipid droplet
probes for high-fidelity imaging of turbid tissues and detecting fatty liver, ACS
Appl. Mater. Interfaces 10 (2018) 10706−10717.
[
30]E. Kim, S. Lee, S. B. Park, A Seoul-Fluor-based bioprobe for lipid droplets and
its application in image-based high throughput screening, Chem. Commun. 48
(2012) 2331–2333.

ACCEPTED MANUSCRIPT
[
[
[
[
[
[
31]X. Zheng, W. Zhu, F. Ni, H. Ai, C. Yang, A specific bioprobe for
super-resolution fluorescence imaging of lipid droplets, Sens. Actuators B 255
(2018) 3148–3154.
32]M. Jiang, X. Gu, J. W. Y. Lam, Y. Zhang, R. T. K. Kwok, K. S. Wong, B. Z.
Tang, Two-photon AIE bio-probe with large Stokes shift for specific imaging of
lipid droplets, Chem. Sci. 8 (2017) 5440–5446.
33]H. Kim, A. Jo, J. Ha, Y. Lee, Y. S. Hwanga, S. B. Park, A pyrazolo[1,5-a]
pyridine-fused pyrimidine based novel fluorophore and its bioapplication to
probing lipid droplets, Chem. Comm. 52 (2016) 7822-7825.
34]J. H. Park, L. Gu, M. G. Von, E. Ruoslahti, S. N. Bhatia, M. J. Sailor,
Biodegradable luminescent porous silicon nanoparticles for in vivo applications,
Nat. Mater. 8 (2009) 331-336.
35]R. A. Haack, K. M. Swift, Q. Ruan, R. J. Himmelsbach, S. Y. Tetin, The
photostability of the commonly used biotin-4-fluorescein probe, Anal. Biochem.
5
31 (2017) 78-82.
36]A. L. Capodilupo, V. Vergaro, F. Baldassarre, A. Cardone, G. A. Corrente, C.
Carlucci, S. Leporatti, P. Papadia, G. Gigli, G. Ciccarella, Thiophene-based
fluorescent probes with low cytotoxicity and high photostability for lysosomes in
living cells, BBA - General Subjects 1850 (2015) 385-392.
[
37]C. W. T. Leung, Y. Hong, S. Chen, E. Zhao, J. W. Y. Lam, B. Z. Tang, A
photostable AIE luminogen for specific mitochondrial imaging and tracking, J.
Am. Chem. Soc. 135 (2013) 62.

ACCEPTED MANUSCRIPT
Graphical Abstract
In this work, we developed a series of fluorescent dyes PIE1-PIE4 with high
fluorescence quantum yields based on a single phenanthrenequinone imidazole
fluorophore core. Among these compounds, the compound PIE1 was given by
modification of diethyl aniline group on the phenanthrenequinone imidazole core. It is
firstly found that PIE1 could real-color image and track LDs in living cells at 760 nm
excitation.

ACCEPTED MANUSCRIPT
Highlights:



We developed a series of fluorescence dyes PIE1-PIE4 with high fluorescence
quantum yields.
It is found that PIE1 could real-color image LDs in biological systems under 760
nm excitation.
Two-photon probe PIE1 is capable of tracking LDs in living cells.