Two-photon fluorescent probes for detecting the viscosity of lipid droplets and its application in living cells

Article abstract of DOI:10.1039/d0ra09683k

Lipid droplets (LDs) are storage organelles at the centre of lipid and energy homeostasis, which act as vital hubs of cellular metabolism and the key to maintaining lipid and energy homeostasis. We synthesized a new two-photon fluorescent probe (CIV) that could detect the viscosity of lipid droplets. The probe is constructedviathe typical ICT system of D-π-A using carbazole as the donor and imidazole as the acceptor. With the increase in viscosity from PBS to 99% glycerol, the fluorescence intensity ofCIVincreased by 13-fold, showing sensitivity and specificity towards viscosity. In addition,CIVshowed low toxicity and excellent biocompatibility in cytotoxicity tests, and was successfully used for living cell LD imaging. Taken together, the results widen the way for the development of novel fluorescent probe-based the visualization LDs and detection in solutions, physiology and pathology.

Full text of DOI:10.1039/d0ra09683k

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Two-photon fluorescent probes for detecting the
viscosity of lipid droplets and its application in
living cells†
Cite this: RSC Adv., 2021, 11, 8250
ab
ab
ab
ab
ac
Huiying Chen, Jianzhi Zhao, Junzhi Lin, Baoli Dong, Hui Li,
Bing Geng *ac and Mei Yan*
ac
Lipid droplets (LDs) are storage organelles at the centre of lipid and energy homeostasis, which act as vital
hubs of cellular metabolism and the key to maintaining lipid and energy homeostasis. We synthesized a new
two-photon fluorescent probe (CIV) that could detect the viscosity of lipid droplets. The probe is
constructed via the typical ICT system of D–p–A using carbazole as the donor and imidazole as the
acceptor. With the increase in viscosity from PBS to 99% glycerol, the fluorescence intensity of CIV
increased by 13-fold, showing sensitivity and specificity towards viscosity. In addition, CIV showed low
toxicity and excellent biocompatibility in cytotoxicity tests, and was successfully used for living cell LD
imaging. Taken together, the results widen the way for the development of novel fluorescent probe-
based the visualization LDs and detection in solutions, physiology and pathology.
Received 14th November 2020
Accepted 31st January 2021
DOI: 10.1039/d0ra09683k
rsc.li/rsc-advances
causes a series of problems, including photobleaching, photo-
Introduction
17–19
damage and low uorescence.
Therefore, the applications of
Lipid droplets (LDs) are organelles that store lipids to produce OP probes are limited in biological samples and organisms.
1–3
energy and maintain cell homeostasis. They are ubiquitous Fortunately, the two-photon (TP) imaging uses two lower-energy
and highly dynamic in cells, which can associate with most photons as the excitation source. The long excitation wave-
other organelles through membranes and participate in length gives it numerous advantages over OP imaging, such as
numerous important physiological processes (such as cell lower background uorescence, causes less damage to biolog-
4,5
proliferation, apoptosis and migration). Abnormal viscosity in ical samples, less photobleaching, less heating effects, better 3D
20–23
LDs can destroy the interactions between different organelles in spatial positioning and deeper penetration depth.
Because
the organism and cellular metabolism, and even cause potential of these advantages, TP imaging is becoming increasingly
lipid toxicity, which can lead to diseases such as Alzheimer's popular among biologists. To date, a variety of TP uorescence
disease, high blood pressure, atherosclerosis and hyperlipid- probes have been synthesized and their applications in bio-
6
–9
24–28
29
emia. Therefore, it has become apparent to explore a fast, logical imaging have been proved.
effective and simple method to detect the viscosity in LDs. The a novel TP uorescent probe using azulene as the uorophore,
uorescence imaging technology has become the most commonly which was used for the detection of peroxynitrite and showed
Kim et al. designed

30
used non-invasive method to visualize cell components due to its excellent photostability and low toxicity. Yao has synthesized
excellent spatial–temporal resolution, good optical performance a new TP small molecule enzyme probe based on photocatalytic
1
0–16
and real-time imaging.
an excellent method to detect the viscosity of LDs.
However, most of the uorescent probes in the past are one- ical environments, and has good photophysical properties and
Thence, uorescent probe imaging is properties to amplify uorescent signals, which can directly
evaluate the functions of enzyme activity in complex patholog-
5,31
photon (OP) properties. OP imaging is excited by a short and deep Penetration. Peng et al. reported several TP uorescent
high-energy photon, but its penetration depth is shallow, which probes using carbazole as the donor to detect viscosity.
However, the response folds of the as-prepared probes are less
than 10-fold and the uorescence quantum yields are less than
32,33
a
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, 10%. Niu
et al. had prepared several TP uorescent probes
China. E-mail: chm_gengb@ujn.edu.cn; chm_yanm@ujn.edu.cn
using phenanthrenequinone imidazole as the uorophore core.
Niu's TP probe could only locate lipid droplets and could not
detect viscosity, reactive oxygen species (ROS) and ions etc.
In this study, a novel TP uorescence probe (CIV) with higher
viscosity response folds and uorescence quantum yields in
solution or in LDs was synthesized (Scheme 1). The probe
b
Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and
Chemical Engineering, University of Jinan, Jinan 250022, China
c
Shandong Provincial Key Laboratory of Fluorine Chemistry and Chemical Materials,
University of Jinan, Jinan 250022, China
†
Electronic supplementary information (ESI) available: Characterization details
of CIV. See DOI: 10.1039/d0ra09683k
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conforms to the design concept of traditional uorescent Synthesis of compound 2
34
probes for viscosity detection, and the typical ICT system (D–
p–A) was constructed with carbazole and imidazole in the
A
mixture of compound
mylphenylboronic acid (180 mg, 1.2 mmol), K
.03 mmol) and tetrakis(triphenylphosphine) platinum in THF
1
(409 mg,
1
mmol), 4-for-
2
3
CO (447 mg,

uorescent probe CIV, simultaneously. The carbazole used as
0
a strong electron-donating group and imidazole as electron-
accepting group are connected by a benzene ring. In the case
of low viscosity, the single bond between the benzene ring and
carbazole rotates freely, and the whole probe is not coplanar,
which hardly shows uorescence. The uorescence intensity
increased signicantly due to the suppression of the rotor free
rotation and enhancement of the conjugation length in the
system with relatively high viscosity. In addition, imidazole with
the asymmetric ve-membered heterocyclic ring endows the TP
probe with large conjugated system, which could increase the
ꢀ
(10 mL) was heated to 55 C and stirred for 12 h under nitrogen.
Aer the reaction, the solvent was removed under a reduced
pressure. The crude product was puried via silica gel column
chromatography (petroleum ether/ethyl acetate, 30 : 1) to
obtain compound 2 (yellow product; 67%)
Synthesis of CIV
Compound 2 (217.5 mg, 0.5 mmol), 1,2-diketone (105 mg, 0.5
mmol) and ammonium acetate (77 mg, 1 mmol) were taken in
the presence of L-proline in methanol (34.5 mg, 15 mol%) at
room temperature, and stirred until dissolved. The whole
mixture reacted at 60 C for 10 h. Aer completion of the
reaction, the volume of the solvent was reduced. The crude

uorescence quantum yield, stokes shi and response multi-
ples. The hexyl group on carbazole can increase the lipid
compatibility of the probe. Based on the above-mentioned
strategy, we predict that uorescent probe CIV can be used to
detect the viscosity of lipid droplets and perform TP imaging.
ꢀ
35
product was puried by silica gel column chromatography
(
(
dichloromethane/methanol, 30 : 1) to obtain compound CIV
yellow product; 74%).
Experimental
1
13
The detailed H NMR, C NMR and HRMS spectra of CIV are
Materials
shown in the Fig. S1, S2 and S3.†
All reagents and chemicals used in this article are of analytical
grade unless otherwise stated. All of the chemicals for reaction
were used directly from commercial resources and without any
further purication. Sodium hydroxide and potassium
carbonate were purchased from Damao Chemical Reagent
Factory. All other chemicals used are purchased from Aladdin
and all reagents used are from Fuyu Chemical Reagent Factory.
Doubly distilled water was used throughout all experiments.
Results and discussion
Fluorescence mechanism
Carbazole has become an excellent uorescent probe matrix
due to its special rigid planar structure and large conjugated
system, which endow it with high thermal and optical stability
and good photophysical properties. Therefore, we used
carbazole (donor, D) as the source, connected imidazole
36
(
acceptor, A) through the benzene ring at 3 positions of carba-
zole, and constructed a typical ICT system of D–p–A in CIV
Scheme 2). In the case of low viscosity, the free rotation of the
Synthesis of compound 1
First, 3, 6-dibromocarbazole (3.25 g, 10 mmol) and NaOH (0.8 g, 10
mmol) were dissolved in 15 mL of the mixed solvent (vTHF : vH2O
(
¼
single bond between the benzene ring and carbazole makes the
whole probe non-coplanar, so the uorescence of CIV is very
weak. The uorescence intensity increased signicantly due to
the inhibition of the free rotation of the single bond in the
system with relatively high viscosity such as glycerol. The hexyl
group at position 9 of carbazole can increase the lipophilicity of
molecules, which makes it coincide with lipid droplets better
1
: 3), and stirred at room temperature for 1.5 h. Then, bromo-
hexane (9.90 g, 60 mmol) was added and the reaction mixture was
ꢀ
heated at 55 C for 12 h. The crude product was puried via silica
gel column chromatography (petroleum ether/ethyl acetate, 30 : 1)
to obtain compound 1 (white product; 70%)
ꢀ
Scheme 1 Synthetic route of CIV. (i) NaOH, bromohexane, 55 C, 12 h;
(
ii) 4-formylphenylboronic acid, K
2
CO
3
, tetrakis(triphenylphosphine)
ꢀ
platinum, N
proline, 60 C, 10 h.
2
ꢀ
, 55 C, 12 h; (iii) 1,2-diketone, ammonium acetate, L-
Scheme 2 The mechanism of the probe CIV.
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and numerous carbazole derivatives have been designed and
conrmed as TP probes. Based on the above-mentioned
strategy, we predict that uorescent probe CIV can be used to
detect the viscosity of lipid droplets and perform TP imaging.
Absorption and emission spectroscopy
For the probe CIV, rst, we studied its optical properties. The
result of the UV absorption spectra in pure PBS and pure glyc-
erol shows an increase in the absorption intensity of CIV in
glycerol compared to that in PBS under the same concentration
(Fig. S4†). Unexpectedly, during the measurement of the uo-
rescence spectrum, the uorescence emission intensity of CIV
increased by 13-fold with the volume ratio of glycerol in the
mixed solution increasing from PBS to 99% glycerol (Fig. 1a).
This indicates that the uorescent probe CIV has a good response Fig. 2 (a) The fluorescence emission spectra of CIV (10 mM) with lem
¼
4
30 nm in the varied pH(PBS, pH ¼ 7.4); (b) fluorescence emission of
towards viscosity. To further explore the probe, we studied the
relationship between the wavelength and viscosity and obtained
the correlation coefficient of the linear relationship that reached
CIV (10 mM) in various solvents with different polarities; (c) fluores-
cence intensity of CIV at lem ¼ 430 nm in PBS buffer (pH ¼ 7.4, 10 mM)
3+
2+
2+
ꢁ
2+
2+
(
1) Al , (2) Ba , (3) Ca , (4) CNS , (5) Co , (6) Cu , (7) Cys, (8) acetic
ꢁ
2+
3+
2+
2+
0
.99 between log I430 and log h via the F ¨o rster–Hoffmann equa- acid, (9) F , (10) Fe , (11) Fe , (12) GSH, (13) H O , (14) Mg , (15) Ni
,
2 2
2+
37
ꢁ
32ꢁ
2ꢁ
3
, (19) Sn , (20) SO , (21) glycerol, (22)
tion (Fig. 1b). This indicates a good linear relationship, and the (16) NO, (17) NO
3
, (18) S
2
O
PBS; (d) the photostability of the probe CIV in glycerol and PBS with the
CIV probe is suitable for the quantitative determination of
viscosity. The uorescence quantum yields (Fig. S5†) and the
molar extinction coefficients (Fig. S6†) of CIV in glycerol were
higher than in PBS, which is consistent with the spectrum test. The
tests of the TP absorption cross-section (Fig. S7a†) and TP uo-
continuous irradiation by laser light for 60 min.
Fluorescence imaging of the CIV probe in living cells
rescence curves (Fig. S7b†) in PBS and glycerol showed that CIV Cytotoxicity test is one of the criteria for testing whether the
was a TP uorescent probe.
uorescent probe CIV can be used for living cell imaging.
The intracellular environment is complex, and probe CIV Therefore, we tested the cytotoxicity of the probe by the stan-
may respond to different environments or heterogeneity, which dard MTT analysis. As shown in Fig. 3, when the living cells
affects its response towards viscosity. Therefore, we simulated were incubated for 24 h in CIV concentration range of 0–50 mM,
the potential performance variations of the probe in different the cell survival rate was still above 90%, which indicates that
pH, polar environments and in the presence of cations, anions, CIV has great biocompatibility and low cytotoxicity.
amino acids, and active oxygen by uorescence spectroscopy.
Then, we veried the specic targeting ability of the uo-
First, the uorescence changes of CIV in buffer solutions with rescent probe CIV towards lipid droplets. Co-localization
38
different pH values of 4–10 were tested, and the results showed experiments were performed with commercial dye Nile Red.
that it was stable without signicant changes (Fig. 2a and S8†). As shown in Fig. 4, the cells were observed under a confocal
Second, we examined the impact of different polar solvents, and microscope. The probe showed green uorescence in the FITC
the nal result showed that it has little effects on the probe channel (Fig. 4b) and Nile Red showed red uorescence in the
(Fig. 2b). The test results in the presence of numerous ions showed
that these heterogeneities had negligible effects on the probe
compared to viscosity (Fig. 2c). Finally, CIV showed excellent light
stability both in PBS and glycerol aer irradiating the CIV solution
and solids by a certain light source (Fig. 2d and S9†).
Fig. 1 (a) The fluorescence emission spectra of 10 mM$of CIV with lex
¼
340 nm in the varied viscosity media of the PBS/glycerol (v/v)
mixtures; (b) linear relationship of log I430 and log h.
Fig. 3 The cytotoxicity of the probe CIV in HeLa cells.
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when the cells were incubated for 30 min only with the CIV
probe, they emitted weak green uorescence (Fig. 5a), while the
cells stimulated by monensin and nystatin emitted strong

uorescence(Fig. 5b and c). Obviously, as the intracellular
viscosity increases, the uorescence intensity of the probe CIV
will increase, which is consistent with the experimental results
of uorescence spectroscopy. The most important thing is that
the effect of the TP imaging is clearer than that of one-photon
(Fig. 5a4–c4). We conrmed again that the TP uorescence in
cells was caused by the probe CIV rather than intracellular
riboavin, avin etc. (Fig. S11†). This shows that CIV has TP
performance, which is less damaging to cells and superior
penetration depth.
Conclusions
Fig. 4 Co-localization fluorescence images of the probe CIV in HeLa
cells; (a) the merged image of (b) and (c); (b) CIV (10 mM) stain, lex
¼
In summary, we synthesized a novel TP uorescent probe (CIV)
for detecting the viscosity of lipid droplets. CIV shows excellent
sensitivity towards viscosity and the uorescence emission
intensity increased by 13-fold with the increase in the volume
ratio of glycerol in the mixed solution, from PBS to 99% glycerol.
In addition, CIV also has low toxicity, excellent biocompatibility
and good TP performance. The probe can accurately target lipid
droplets in living cells and detect viscosity. The TP uorescent
probe synthesized by us may be a potential candidate for
viscosity detection in solutions and complex biological systems.
4
05 nm, collected at 500–550 nm; (c) Nile Red stain, lex ¼ 561 nm,
collected at 570–620 nm; (d) intensity scatter plots of the probe CIV
across the HeLa cells in the red and green channels. (e) Intensity profile
of the two channels.
TRICT channel (Fig. 4c). The experiments that can exclude the
interference of CIV in the FRICT channel and Nile red in the
FRIT channel at 405 nm excitation had been nished
(Fig. S10†). The merged image (Fig. 4a) showed that the two
channels overlap very well, and the high overlap coefficient of
0.928 is obtained.
Monensin and Nystatin as external stimuli can induce
abnormalities, including swelling or structural changes of cells,
Conflicts of interest
and ultimately increase intracellular viscosity, in the intracel- There are no conicts of interest to declare.
25,39
lular environment.
Therefore, they are oen used as iono-
phores to externally interfere with changes in the intracellular
viscosity. As observed from the confocal microscope images,
Acknowledgements
This work was supported by Young Taishan Scholars tsqn
[Grant No. 20161036] and the National Science Foundation of
China [Grant No. 21304037], National Science Foundation of
China [Grant No. 21675064], Major Natural Science Foundation
of Shandong Province [Grant No. ZR2017ZC0529].
Notes and references
1
J. A. Olzmann and P. Carvalho, Nat. Rev. Mol. Cell Biol., 2019,
0, 137–155.
2
2
3
R. V. Farese Jr and T. C. Walther, Cell, 2009, 139, 855–860.
Y. Li, M. Zhang, X. Chen, J. Liang, D. Chen, M. Gao and
L. Ren, Tetrahedron Lett., 2019, 60, 1880–1884.
4
5
J. Y. Rui Guo, Y. Ma, G. Li, Q. Wang and W. Lin, Sens.
Actuators, B, 2018, 271, 321–328.
M. Peng, J. Yin and W. Lin, New J. Chem., 2018, 42, 18521–
1
8525.
Fig. 5 (a1–a4) Confocal fluorescence images of the HeLa cells incu-
bated with 10 mM CIV for 30 min; (b1–b4) confocal fluorescence
images of the HeLa cells incubated with 10 mM CIV + 10 mM monensin
for 1 h; (c1–c4) confocal fluorescence images of the HeLa cells
incubated with 10 mM CIV + 10 mM nystatin for 1 h. OP: lex ¼ 405 nm;
TP: lex ¼ 820 nm; 500–550 nm were collected.
6
7
M. A. Welte, Curr. Biol., 2015, 25, R470–R481.
A. S. Greenberg, R. A. Coleman, F. B. Kraemer,
J. L. McManaman, M. S. Obin, V. Puri, Q.-W. Yan,
H. Miyoshi and D. G. Mashek, J. Clin. Invest., 2011, 121,
2102–2110.
©
2021 The Author(s). Published by the Royal Society of Chemistry
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8
9
P. S. Guenther Boden, M. Mozzoli, P. Cheung, K. Gumireddy, 25 L. Hou, P. Ning, Y. Feng, Y. Ding, L. Bai, L. Li, H. Yu and
P. Reddy, Z. Luo and N. Ruderman, Diabetes, 2005, 54, 3458–
465.
C. Koutsari and M. D. Jensen, J. Lipid Res., 2006, 47, 1643–
650.
X. Meng, Anal. Chem., 2018, 90, 7122–7126.
26 R. Huo, X. Zheng, W. Liu, L. Zhang, J. Wu, F. Li, W. Zhang,
C.-S. Lee and P. Wang, Chem. Commun., 2020, 56, 10902–
10905.
3
1
1
1
1
1
1
0 Z. Yang, J. Cao, Y. He, J. H. Yang, T. Kim, X. Peng and 27 X. Wei, X. Yang, Y. Feng, P. Ning, H. Yu, M. Zhu, X. Xi, Q. Guo
J. S. Kim, Chem. Soc. Rev., 2014, 43, 4563–4601. and X. Meng, Sens. Actuators, B, 2016, 231, 285–292.
1 C. Zeng, X. Zhang and L. Pu, Chem.–Eur. J., 2017, 23, 2432– 28 W. Song, Y. Chen, Q. Xu, H. Mu, J. Cao, J. Huang and J. Su,
438. ACS Appl. Mater. Interfaces, 2018, 10, 24689–24698.
2 L. Bu, B. Shen and Z. Cheng, Adv. Drug Delivery Rev., 2014, 76, 29 L. C. Murn, M. Weber, S. J. Park, W. T. Kim, C. M. Lopez-
2
2
1–38.
Alled, C. L. McMullin, F. Pradaux-Caggiano, C. L. Lyall,
G. Kociok-Kohn, J. Wenk, S. D. Bull, J. Yoon, H. M. Kim,
T. D. James and S. E. Lewis, J. Am. Chem. Soc., 2019, 141,
19389–19396.
3 K. Suhling, P. M. French and D. Phillips, Photochem.
Photobiol. Sci., 2005, 4, 13–22.
4 W. Zhang, X. Liu, P. Li, W. Zhang, H. Wang and B. Tang,
Trends Anal. Chem., 2020, 123, 115742.
30 L. Qian, L. Li and S. Q. Yao, Acc. Chem. Res., 2016, 49, 626–
634.
1
5 B. del Rosal and A. Benayas, Small Methods, 2018, 2, 1800075.
1
6 M. Ye, W. Hu, M. He, C. Li, S. Zhai, Z. Liu, Y. Wang, H. Zhang 31 M. Peng, J. Yin and W. Lin, Spectrochim. Acta, Part A, 2020,
and C. Li, Chem. Commun., 2020, 56, 6233–6236. 224, 117310.
7 Q. Ma, J. Xu, X. Zhang, L. Zhou, H. Liu and J. Zhang, Sens. 32 J. Niu, Y. Liu, W. Wang and W. Lin, Spectrochim. Acta, Part A,
Actuators, B, 2016, 229, 434–440. 2019, 216, 35–44.
8 P. Ning, P. Dong, Q. Geng, L. Bai, Y. Ding, X. Tian, R. Shao, 33 Y. Liu, J. Niu, W. Wang, B. Dong and W. Lin, J. Mater. Chem.
L. Li and X. Meng, J. Mater. Chem. B, 2017, 5, 2743–2749. B, 2017, 5, 7801–7808.
9 L. Zhou, D.-Q. Lu, Q. Wang, S. Hu, H. Wang, H. Sun and 34 S.-C. Lee, J. Heo, H. C. Woo, J.-A. Lee, Y. H. Seo, C.-L. Lee,
X. Zhang, Sens. Actuators, B, 2017, 238, 274–280. S. Kim and O. P. Kwon, Chem.–Eur. J., 2018, 24, 13706–13718.
0 B. R. C. Hwan Myung Kim, Acc. Chem. Res., 2009, 42, 863– 35 S. Samai, G. C. Nandi, P. Singh and M. S. Singh, Tetrahedron,
72. 2009, 65, 10155–10161.
1 H. W. Liu, Y. Liu, P. Wang and X. B. Zhang, Methods Appl. 36 J. Yin, Y. Ma, G. Li, M. Peng and W. Lin, Coord. Chem. Rev.,
Fluoresc., 2017, 5, 012003. 2020, 412, 213257.
2 W. Denk, J. H. Strickler and W. W. Webb, Science, 1990, 248, 37 Y.-G. Choi and G. Kwak, Eur. Polym. J., 2019, 110, 307–312.
1
1
1
2
2
2
2
2
8
73.
38 E. P. M. Phillip Greenspan and S. D. Fowler, J. Cell Biol.,
1985, 100, 965–973.
3 S. Yan, H. Guo, J. Tan, J. Jiang, J. Liang, S. Yan and H. Xiao,
Talanta, 2020, 218, 121210.
4 M. Jiang, X. Gu, J. W. Y. Lam, Y. Zhang, R. T. K. Kwok,
K. S. Wong and B. Z. Tang, Chem. Sci., 2017, 8, 5440–5446.
39 S. P. S. L. J. Mandel, J. Membr. Biol., 1986, 94, 153–161.
8254 | RSC Adv., 2021, 11, 8250–8254
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