Photostable fluorescent probes for 3D imaging and monitoring the metabolism of lipid droplets

Article abstract of DOI:10.1016/j.dyepig.2020.108502

In addition to being associated with energy supply, lipid droplets (LDs) play vital roles in many other important physiological and some pathological processes. Fluorescent probes are useful tools for studying the biological functions of LDs. In this study, we develop three new highly fluorescent LD-specific bioprobes, which can be excited by one- and two-photon, with favorable biocompatibility. They have little crosstalk with some commonly used commercially-available green- and red-emissive probes, such as BODIPY 493/503, MitoTracker Red CMXRos (MTR) and LysoTracker Red DND-99 (LTR), thus allowing the simultaneous multicolor imaging. Notably, one of the probes, BIP-1, has superior photostability and allows excellent 3D imaging of cellular LDs. BIP-1 can be used to monitor the number and morphological changes of LDs, and the interactions of LDs with other organelles (e.g. mitochondria) under specific physiological conditions (e.g. starvation). Overall, BIP-1 is a promising alternative fluorescent bioprobe for live-cell LD-specific imaging and long-term tracking during LD-related research.

Full text of DOI:10.1016/j.dyepig.2020.108502

Dyes and Pigments 180 (2020) 108502
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
Photostable fluorescent probes for 3D imaging and monitoring the
metabolism of lipid droplets
a,
b
c,
d
e
d
d
,
*
a,b,**
Kang-Nan Wang , Xiang-Jun Peng , Yi Li , Hai-Tao Tang , Ying-Ming Pan , Liang He
a
Key Laboratory for Biobased Materials and Energy of Ministry of Education, College of Materials and Energy, South China Agricultural University, Guangzhou, 510642,
China
b
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou, 510275, China
School of Pharmaceutical Science, Gannan Medical University, Ganzhou, 341000, China
c
d
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences of Guangxi Normal
University, Guilin, 541004, China
e
Key Laboratory of Theoretical Organic Chemistry and Function Molecule, Ministry of Education, School of Chemistry and Chemical Engineering, Hunan University of
Science and Technology, Xiangtan, 411201, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In addition to being associated with energy supply, lipid droplets (LDs) play vital roles in many other important
physiological and some pathological processes. Fluorescent probes are useful tools for studying the biological
functions of LDs. In this study, we develop three new highly fluorescent LD-specific bioprobes, which can be
excited by one- and two-photon, with favorable biocompatibility. They have little crosstalk with some commonly
used commercially-available green- and red-emissive probes, such as BODIPY 493/503, MitoTracker Red
CMXRos (MTR) and LysoTracker Red DND-99 (LTR), thus allowing the simultaneous multicolor imaging.
Notably, one of the probes, BIP-1, has superior photostability and allows excellent 3D imaging of cellular LDs.
BIP-1 can be used to monitor the number and morphological changes of LDs, and the interactions of LDs with
other organelles (e.g. mitochondria) under specific physiological conditions (e.g. starvation). Overall, BIP-1 is a
promising alternative fluorescent bioprobe for live-cell LD-specific imaging and long-term tracking during LD-
related research.
Fluorescent probe
Lipid droplet
Imidazo[1,2-a]pyridine
Metabolism
3
D imaging
1
. Introduction
Lipid droplets (LDs), which are found to be present in almost all the
Currently, commercially available LD fluorescent probes are still
limited, although some examples are frequently used in the research,
which mainly includes BODIPY derivatives [17], Nile Red [18], etc.
These commonly used LD probes often have some drawbacks that do not
meet the requirements of some experiments. For instance, BODIPY
493/503 and its derivatives possess small Stokes shifts [19]. Nile Red
has been reported to lack specificity for LDs due to its simultaneous
staining of other organelles in some cases [20,21]. Recently, numerous
efforts have been made to explore new fluorescent LD-specific probes
that are possibly suitable for various experimental conditions [22–36].
But fluorescent probes that can be used for 3D imaging and long-time
tracking of LDs are still scarce.
eukaryotic cells and store large amounts of neutral lipids, are essential
dynamic subcellular organelles [1–3]. In addition to being lipid reser-
voirs for energy generation, LDs also serve other fundamental functions,
such as membrane synthesis and trafficking, signaling, and protein
degradation [4–8]. In recent years, studies have also revealed that LDs
are closely related to a variety of physiological diseases, including fatty
liver disease, obesity, diabetes, atherosclerosis, hypertension, and can-
cers [9–15]. LDs have drawn considerable attention in recent years due
to the close relationships between LDs and lipid metabolism and
lipid-related diseases [16]. So, it is of high importance to develop suit-
able luminescent probes for the study of LDs in the relevant scientific
research and disease diagnosis and treatment.
Herein, we report three fluorescent LD probes (Scheme 1), namely
BIP-1, BIP-2 and BIP-3, which are based on the moiety of benzo [4,5]
imidazole[1,2-a]pyridine [37,38]. In this study, their photophysical
*
Corresponding author.
** Corresponding author. Key Laboratory for Biobased Materials and Energy of Ministry of Education, College of Materials and Energy, South China Agricultural
University, Guangzhou, 510642, China.
E-mail addresses: panym@mailbox.gxnu.edu.cn (Y.-M. Pan), heliang@scau.edu.cn (L. He).
https://doi.org/10.1016/j.dyepig.2020.108502
Received 2 February 2020; Received in revised form 28 April 2020; Accepted 28 April 2020
Available online 4 May 2020
0
143-7208/© 2020 Elsevier Ltd. All rights reserved.

K.-N. Wang et al.
Dyes and Pigments 180 (2020) 108502
Table 1
Photophysical properties of BIP-1, BIP-2 and BIP-3.
7
Compound
Solvent
λ
abs
λ
em
Φ
τ
k
r
À 1
(10
k
nr
a
7
(
nm)
(nm)
(ns)
s
)
(10
À 1
b
s
)
BIP-1
DMSO
PBS
380
415
388
416
394
455
482
482
484
487
485
476
0.37 ꢁ
5.3
6.0
7.1
5.2
5.3
7.3
7.0
1.0
4.6
1.2
4.7
11.9
15.7
9.4
0
.04
0.061
ꢁ
0.008
BIP-2
BIP-3
DMSO
PBS
0.33 ꢁ
0
.03
0.063
18.0
14.2
12.9
ꢁ
0.006
DMSO
PBS
0.25 ꢁ
0
.03
0.055
0.005
0.75
Scheme 1. Chemical structures of BIP-1, BIP-2 and BIP-3.
ꢁ
a
r r
The radiative rate constants (k ) were calculated according to the formula: k
spectrometer (Germany); The high resolution mass spectra (HRMS) were
recorded on an Exactive Mass Spectrometer (USA); The UV–vis spectra
were measured by a Varian Cary 300 spectrophotometer (USA); The
excitation and fluorescence spectra, and fluorescence lifetimes were
measured by an Edinburgh FLS 920 Spectrometer (UK). The fluores-
cence quantum yields of the probes were obtained by using Coumarin
¼
bΦ/
The non-radiative rate constants (knr) were calculated according to the
formula: knr ¼ (1-Φ)/
τ.
τ
.
properties have been studied in detail, and their potentials as cellular
LD-specific probes have been investigated. The bioactivity of these
selected compounds is relatively low, thus avoiding the interference of
their own bioactivity with the results of cell imaging experiments. These
probes emit cyan fluorescence (~475 nm) upon excitation at 405 nm
without crosstalk with BODIPY 493/503 or Nile Red, thus allowing the
simultaneous multicolor imaging. BIP-1 has specificity for LDs, good
biocompatibility and excellent photostability, thus allowing LDs imag-
ing and long-term tracking. Furthermore, taking advantage of these
properties, BIP-1 was successfully used to conduct 3D imaging of
cellular LDs, and to detect the number and morphological changes of
LDs under specific physiological conditions (e.g. oleic acid treatment
and starvation).
3
07 as the reference [40].
2
.2. Synthesis and characterization
BIP-1, BIP-2 and BIP-3 were synthesized according to the methods
reported previously by us (Scheme S1) [37]. Take BIP-1 as an example,
the synthesis process as follows: 2-methyl-1H-benzo[d]imidazole (80
mg, 0.6 mmol), 1,3-diphenylprop-2-yn-1-one (104 mg, 0.5 mmol) and
KOH (1 equiv.) were mixed in a test tube, and 1,4-dioxane (2 mL) was
�
then added. The mixture in the test tube was heated at 100 C for 6 h
under the condition of stirring. Upon completion, water (5 mL) was
added to the tube to dilute the reaction mixture, and then ethyl acetate
(
3 ꢀ 3 mL) was used to extract. The organic layers were combined,
2
. Experimental section
washed with water, then dried by magnesium sulfate (MgSO ). After
4
filtration, the solvent was evaporated under reduced pressure. Flash
column chromatography was applied to purify the obtained residue to
afford BIP-1.
2
.1. General materials and instruments
2
-methyl-1H-benzo[d]imidazole was obtained from Aladdin Re-
agents. 1,3-diphenylprop-2-yn-1- one, 3-phenyl-1-(thiophen-2-yl)prop-
-yn-1-one and 1-(furan-2-yl)-3-phenylprop-2-yn-1-one were synthe-
sized according to the reported methods [39]. Phosphate buffered saline
PBS, Sigma Aldrich). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
1
,3-diphenylbenzo [4,5]imidazo[1,2-a]pyridine (BIP-1): Compound
1
BIP-1 was obtained as a yellow powder. Yield: 0.120 g (75%). H NMR
400 MHz, CDCl
2
(
3
): δ ¼ 8.05–7.86 (m, 2H), 7.74 (d, J ¼ 7.3 Hz, 2H),
7
7
.69–7.58 (m, 5H), 7.49 (t, J ¼ 7.4 Hz, 2H), 7.45–7.38 (m, 2H),
(
1
3
.03–6.93 (m, 2H), 6.59 (d, J ¼ 8.5 Hz, 1H). C NMR (101 MHz, CDCl
3
):
trazolium bromide (MTT, Sigma Aldrich). BODIPY 493/503, Mito-
δ ¼ 149.94, 145.29, 142.01, 141.19, 138.07, 134.38, 130.20, 129.18,
Tracker™ Red CMXRos (MTR), LysoTracker™ Red DND-99 (LTR), Syto
5
1
1
29.16, 129.08, 129.03, 128.93, 127.00, 125.34, 120.53, 119.44,
9 and Hank’s balanced salt solution (HBSS) were all obtained from
þ
14.58, 113.00, 112.21. HRMS (ESI): m/z calcd for [MþH] , 321.1386;
ThermoFisher Scientific, USA. Oleic acid was bought from J&K Scien-
found: 321.1382.
The H and 13C NMR spectra were recorded on a Bruker Avance 400
tific Ltd1.
1-phenyl-3-(thiophen-2-yl)benzo
[4,5]imidazo[1,2-a]pyridine
(
BIP-2): Compound BIP-2 was obtained as a brown-yellow powder.
�
Fig. 1. (A) Emission spectra of BIP-1 (5
μ
M) measured in different solvents at 25 C. (B) Emission spectra of BIP-1 (5 M) in the mixed solvents of 1,4-dioxane and
μ
H
2
O (the percentage indicates the volume fraction).
2

K.-N. Wang et al.
Dyes and Pigments 180 (2020) 108502
Fig. 2. Molecular orbital amplitude plots of the HOMO and LUMO energy levels of BIP-1, BIP-2 and BIP-3.
Yield: 0.106 g (65%). 1H NMR (400 MHz, CDCl
): δ ¼ 7.93–7.84 (m,
H), 7.66–7.54 (m, 5H), 7.47 (dd, J ¼ 3.7, 1.0 Hz, 1H), 7.40–7.33 (m,
H), 7.09 (dd, J ¼ 5.0, 3.7 Hz, 1H), 6.97–6.89 (m, 2H), 6.53 (d, J ¼ 8.5
2.5. Detection of oleic acid-induced accumulation of LDs [27]
3
2
2
Briefly, A549 cells were pre-treated with 0, 50, 100 and 200
μM oleic
1
3
�
Hz, 1H). C NMR (101 MHz, CDCl
3
): δ ¼ 149.70, 145.55, 141.46,
acid in cell media RPMI-1640 at 37 C for 6 h. After that, cell media was
1
1
41.17, 135.14, 134.15, 130.27, 129.26, 129.20, 129.07, 128.50,
replaced by fresh media containing 2
μ
M BIP-1. After staining in cell
�
27.04, 125.36, 125.30, 120.57, 119.47, 114.46, 111.02, 110.95. HRMS
media RPMI-1640 at 37 C for 30 min, cells were immediately subjected
to cell imaging by CLSM. The fluorescence intensity of was quantified by
using ImageJ software.
þ
(
ESI): m/z calcd for [MþH] , 327.0950; found: 327.0943.
3
-(furan-2-yl)-1-phenylbenzo [4,5]imidazo[1,2-a]pyridine (BIP-3):
Compound BIP-3 was obtained as a brown-yellow powder. Yield: 0.105
1
g (68%). H NMR (400 MHz, CDCl
3
): δ ¼ 7.99 (s, 1H), 7.89 (d, J ¼ 8.2
2.6. Monitoring of the dynamics of LDs during starvation
Hz, 1H), 7.70–7.55 (m, 6H), 7.40 (t, J ¼ 7.6 Hz, 1H), 7.03–6.92 (m, 2H),
1
3
6
.87 (d, J ¼ 3.4 Hz, 1H), 6.60–6.47 (m, 2H). C NMR (101 MHz, CDCl
3
):
The cells in the control group were further incubated in fresh com-
plete cell media, and the starved cells were further incubated in HBSS at
δ ¼ 151.44, 149.53, 144.93, 144.00, 141.30, 134.18, 131.43, 130.28,
�
1
1
3
29.21, 129.08, 125.40, 120.69, 119.23, 114.47, 112.32, 109.18,
37 C for 4, 8, 16, 24 h. The control and starved cells were stained with
þ
08.95, 108.92. HRMS (ESI): m/z calcd for [MþH] , 311.1179; found:
BIP-1 (2
μ
M) and MTR (100 nM) or LTR (100 nM) in complete cell media
�
11.1175.
and HBSS, respectively, at 37 C for 30 min. Afterward, the cells were
subjected to cell imaging by CLSM immediately.
2
.3. Cell culture and colocalization assay
Human lung cancer cells (A549) were cultured as described before
[
41,42]. A549 cells were co-incubated with 2
μ
M BIP-1, BIP-2 or BIP-3
�
and 1
μg/mL BODIPY493/503 in cell media RPMI-1640 at 37 C for 30
min. After staining, the cell media was removed, and the cells were
washed three times with PBS and then kept in PBS. Cell fluorescence
imaging was performed immediately by confocal laser scanning micro-
scopy (CLSM). BIP-1, BIP-2 and BIP-3 were excited at 405 nm (one--
photon) or 780 nm (two-photon) and their fluorescence was collected at
4
75 ꢁ 20 nm. BODIPY493/503 was excited at 488 nm, and its fluores-
cence was collected at 520 ꢁ 20 nm. The other co-localization experi-
ments followed the same procedure as this one.
2
.4. Cytotoxicity
The cytotoxicity of BIP-1, BIP-2 and BIP-3 towards A549 cells was
determined by an MTT method according to previously reported liter-
ature [41,42].
Fig. 3. Cell viability of A549 cells after incubation with BIP-1, BIP-2 and BIP-3
(
0, 5 and 10 M) for 48 h.
μ
3

K.-N. Wang et al.
Dyes and Pigments 180 (2020) 108502
Fig. 4. Confocal fluorescence images of live A549 cells co-stained with BIP-1, BIP-2 or BIP-3 (2
μ
M) and BODIPY 493/503 (1
μ
g/mL) for 30 min λex ¼ 405 (OPM)
and 780 nm (TPM) for BIP-1, BIP-2 and BIP-3 (green). λex ¼ 488 nm for BODIPY 493/503 (red). Overlay 1: Merged images of the BODIPY signal and OPM signals of
BIP derivatives. Overlay 2: Merged images of the BODIPY signal and TPM signals of BIP derivatives. All panels share the same scale bar: 10 m. (For interpretation of
μ
the references to color in this figure legend, the reader is referred to the Web version of this article.)
3
. Results and discussion
close to those in DMSO, respectively. The energy gaps between the
HOMO and LUMO in water are calculated to be 3.782, 3.567 and 3.581
eV for BIP-1, BIP-2 and BIP-3, respectively. The corresponding calcu-
lated absorption spectra of BIP-1, BIP-2 and BIP-3 in DMSO and PBS are
mostly consistent with the experimental absorption spectra. (Fig. S10).
3
.1. Chemical synthesis, characterization and photophysical properties
Compounds BIP-1, BIP-2 and BIP-3 were synthesized in good yields
by a reported method developed by us recently [37]. All the compounds
were fully characterized by HRMS, 1H and C NMR spectroscopy
13
3
.2. Cytotoxicity, cellular localization and photostability
(
Figs. S1–S6).
The photophysical properties of these compounds were first studied
Before cell imaging experiments, the cytotoxicity of the compounds
in dimethyl sulfoxide (DMSO) and PBS. The results are illustrated in
Fig. S7, and the photophysical data are summarized in Table 1. Upon
excitation at 405 nm, all the compounds exhibited cyan (~475 nm)
fluorescence. It was worth mentioning that, the fluorescence quantum
yields of these probes showed dramatic polarity dependence, increasing
significantly with the decrease of solvent polarity (Fig. 1A). For instance,
there was an 8-fold increase in the emission intensity in ethyl acetate
compared with that in PBS for BIP-1 at 475 nm. To further compare and
analyze the relationship between spectral properties and solvent polar-
was assessed in A549 cells. Cell viability was measured by an MTT
method after cells were incubated with 5 and 10 M BIP-1, BIP-2 and
μ
BIP-3 for different periods (Fig. 3). The results showed that BIP-1, BIP-2
and BIP-3 did not cause any significant inhibition of cell viability at
concentrations up to 5 M for 48 h, indicating the good biocompatibility
μ
of BIP-1, BIP-2 and BIP-3.
We then investigated the cellular localization of these compounds by
CLSM with the excitation of both one-photon and two-photon. As
illustrated in Fig. 4 and Fig. S11, colocalization experiments with a
commercial available LD-specific probe, BODIPY 493/503, showed that
the fluorescence of BIP-1, BIP-2 and BIP-3 was well merged with that of
BODIPY 493/503, with Pearson’s colocalization coefficients (PCCs) of
ity, the emission spectra of BIP-1 (2
μ
M) were tested in a 1,4-dioxane/
H
2
O system (Fig. 1B). As the polarity increased, the fluorescence in-
tensity of BIP-1 at 475 nm decreased obviously with the fluorescence
quantum yields decreased from 43.29% to 6.13%. Furthermore, the
fluorescence intensity of BIP-1 showed solvent viscosity dependence
0
.935, 0.902 and 0.916, respectively. Little colocalization (PCC < 0.20)
was observed for BIP-1, BIP-2 and BIP-3 with mitochondria-specific
probe MTR, lysosome-specific probe LysoTracker Green DND-26 (LTG)
and nuclear probe Syto 59 in A549 cells (Fig. 6A and Figs. S12–S14).
These results demonstrate that the probes can specifically label the LDs,
with little staining for other organelles, such as mitochondrion, lyso-
some and nucleus. It is worth mentioning that, the intracellular distri-
butions of BIP-1, BIP-2 and BIP-3 could be clearly detected by both one-
photon microscopy (OPM) and two-photon microscopy (TPM, λex ¼ 780
nm). Furthermore, the Z-stack images, excited by either one- or two-
(
Fig. S8), which indicated it could be possible that BIP-1 may act as a
fluorescent molecular rotor. However, the fluorescence intensity of BIP-
in the pH range of 4.5–8.5 in PBS solution exhibited small changes
Fig. S9), which indicated that BIP-1 was mostly pH insensitive at
1
(
physiological pH. The fluorescence lifetimes of these probes were
measured to be between 5.2 and 7.3 ns in DMSO and PBS. The radiative
and non-radiative rate constants were also calculated and the results are
shown in Table 1. And then, the HOMO and LUMO energy levels of these
dyes were evaluated in water and DMSO. As shown in Fig. 2, both the
HOMO and LUMO energy levels of BIP-1, BIP-2 and BIP-3 in water were
photon, of A549 cells stained with BIP-1 (2
μM) were obtained
(
Fig. S15). As compared with the OPM, the TPM of BIP-1 has deeper
4

K.-N. Wang et al.
Dyes and Pigments 180 (2020) 108502
Fig. 5. (A) Confocal fluorescence images of live A549 cells stained with BIP-1 (2
μ
M, 30 min) and BODIPY 493/503 (1
μ
g/mL, 30 min) after indicated laser scans. All
panels share the same scale bar: 10
μm. (B) Photobleaching curves the fluorescence of BIP-1 and BODIPY 493/503 after laser scanning for the same times. λex ¼ 405
nm (for BIP-1) and 488 nm (for BODIPY 493/503).
penetration, with the fluorescence of BIP-1 still being detected by TPM
Supplementary data related to this article can be found at https
://doi.org/10.1016/j.dyepig.2020.108502.
in a depth of ca. 15 m, which suggested that BIP-1 might be a potential
μ
probe for tissue imaging by TPM.
The photostability of BIP-1 was evaluated. As shown in Fig. 5, after
20 laser scans, the fluorescence intensity of BIP-1 only decreased
slightly, remaining at a level of above 90%, while that of BODIPY 493/
503 markedly reduced, dropping to ca. 15%, proving that BIP-1 has
better photostability than BODIPY 493/503.
Besides, because LDs are dynamic organelles, we also used BIP-1 to
observe the movement of LDs in A549 cells by TPM (Movie S1), whose
fluorescence co-localized with that of BODIPY 493/503 throughout the
process indicated by the yellow dots, suggesting that BIP-1 has speci-
ficity for LDs and can be used to observe the dynamics of LDs. The
specificity of these probes for LDs may result from the lipophilic nature
of the compounds and the nonpolar lipophilic environment of LDs. The
fluorescence characteristics, biocompatibility and LD specificity of the
three probes were of little difference. Probe BIP-1 was selected as the
representative for subsequent imaging studies.
3.3. 3D imaging
As we know, cells and their internal organelles have complex three-
dimensional structures. A conventional CLSM image of cells can only
capture single layer cellular information by one time laser scanning. Co-
5

K.-N. Wang et al.
Dyes and Pigments 180 (2020) 108502
Fig. 6. (A) Co-localization images and (B and C) 3D images of live A549 cells after incubation with BIP-1 and MTR (100 nM, 30 min) or Syto 59 (200 nM, 30 min).
ex ¼ 405, 543 and 633 nm for BIP-1, MTR and Syto 59, respectively. Overlay: Overlay of the BIP-1 and MTR or Syto 59. Scale bars, A: 10 m; B and C: 5 m.
λ
μ
μ
staining images of BIP-1 with MTR or Syto 59 could only show the
relative positions between LDs and mitochondria/nucleus at one single
layer (Fig. 6A). 3D imaging of cells by CLSM can be achieved by scan-
ning different layers of the cells in the same direction (z-axis) and the
reconstruction of the images obtained per scan. Obviously, multiple
scans are involved in this process, which requires the probe to have high
photostability and bright fluorescence. Significantly, BIP-1 allowed
excellent 3D imaging of LDs, which showed the cellular localization of
LDs and the interrelations with other organelles more clearly, such as
mitochondria and nucleus (Fig. 6B and C).
were newly formed in cells upon oleic acid pre-treatment. These results
suggest that BIP-1 can be used to monitor the accumulation of cellular
LDs by CLSM.
3
.5. Monitoring of LDs changes under starvation
LDs contain large amounts of fatty acids, which provide cellular
energy under starvation conditions [6]. To get more insight into the
dynamic changes of cellular LDs during starvation, we further used
BIP-1 to monitor the distribution and abundance of LDs in A549 cells
under starvation. Cells were starved in HBSS for different times (0, 4, 8,
16, 24 h), and then cellular LDs were labeled by BIP-1. As shown in
Fig. 8, starvation in HBSS induced a rapid increase in the number of
intracellular LDs, and the newly produced LDs were distributed in
clusters. These observations are consistent with literature reports
[44–46]. As to why the LDs, serving as a major nutrient reservoir for
cells, will increase in response to starvation, the main point of view is
that when cells feel the pressure of survival following the initial phases
of starvation, they will activate relevant mechanisms (e.g. autophagy) to
synthesize large amounts of energy substances stored in LDs to meet the
3
.4. Detection of oleic acid-induced accumulation of LDs
The application of BIP-1 to detect the accumulation of LDs after oleic
acid treatment was investigated. We used oleic acid to stimulate the
formation of intracellular LDs [43]. A549 cells were treated with 0, 50,
1
00 and 200
μ
M oleic acid for 6 h, and then stained with 2 M BIP-1 for
μ
3
0 min. The results showed that the fluorescence intensity of BIP-1 and
the number of fluorescent dots increased markedly after oleic acid
treatment (Fig. 7 and Fig. S16), indicating that a large number of LDs
6

K.-N. Wang et al.
Dyes and Pigments 180 (2020) 108502
Fig. 7. Confocal fluorescence images of live A549 cells upon incubation with BIP-1 (5
μ
M, 30 min) after cells were pre-treated with oleic acid at concentrations of (a)
0
μ
M, (b) 50
μ
M, (c) 100
μM and (d) 200
μM for 6 h λex ¼ 405 nm. Scale bar, 20
μ
m.
needs of future survival [47]. Moreover, co-localization experiments
with MTR, a mitochondria-specific probe, or LTR, a lysosome-specific
probe, under starvation were performed. It turned out that LDs were
often observed near mitochondria but not lysosomes (Fig. 8 and
Fig. S17), probably to allow LDs to provide energy substances (e.g. fatty
acids) to the mitochondria [44–46,48], further revealing that BIP-1
could be used to investigate the interactions of LDs with other important
cellular organelles (e.g. mitochondria and lysosomes) under specific
physiological conditions. These results collectively demonstrate that
BIP-1 can serve as a useful fluorescent tool for the study of the cell
biology of LDs.
moiety of benzo [4,5]imidazole[1,2-a]pyridine were developed. They
can be applied in both OPM and TPM. These fluorophores emit cyan
fluorescence (~475 nm) upon excitation at 405 nm with high quantum
yields and great photostability. The fluorescence intensity of these
probes increases with decreasing polarity, thus endowing them with
specificity for LDs due to low polarity inside the LDs. They have little
crosstalk with some commonly used commercially-available green- and
red-emissive probes, such as BODIPY 493/503, MTR and LTR, thus
allowing the simultaneous multicolor imaging. These properties of
BIP-1 allowed obtaining high-quality CLSM images of LDs, as well as
multicolor 3D images. Moreover, BIP-1 was used to successfully detect
the number and the dynamics of cellular LDs, and the interactions of LDs
with other important cellular organelles (e.g. mitochondria) under
specific physiological conditions (e.g. oleic acid treatment and starva-
tion). Overall, these probes, especially BIP-1, are attractive candidates
4
. Conclusions
To sum up, three new fluorescent LD-specific bioprobes based on the
7

K.-N. Wang et al.
Dyes and Pigments 180 (2020) 108502
Fig. 8. Starvation in HBSS induced a rapid increase in the amount of intracellular LDs. Confocal fluorescence images of live A549 cells incubated in HBSS for 0, 4, 8,
6 and 24 h, respectively, followed by staining with BIP-1 (2
M, 30 min) and MTR (100 nM, 30 min). λex ¼ 405 and 543 nm for BIP-1 and MTR, respectively. All
panels share the same scale bar: 20 m.
1
μ
μ
for LD imaging and long-term tracking in LD-associated research.
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Kang-Nan Wang: Conceptualization, Investigation, Writing - orig-
inal draft. Xiang-Jun Peng: Methodology, Investigation. Yi Li: Formal
analysis, Funding acquisition. Hai-Tao Tang: Resources. Ying-Ming
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