A benzothiazole-based near-infrared fluorescent probe for sensing SO2 derivatives and viscosity in HeLa cells

Article abstract of DOI:10.1016/j.saa.2021.119457

The unbalanced metabolism of sulfur dioxide can cause various diseases, such as neurological disorders and lung cancer. Until now, some researches revealed that the normal function of lysosomes would be disrupted by its abnormal viscosity. As a signal molecule, sulfur dioxide (SO₂) plays an important role in lysosome metabolism. However, the connection of metabolism between the SO₂ and viscosity in lysosomes is still unknown. Herein, we developed a benzothiazole-based near-infrared (NIR) fluorescent probe (Triph-SZ), which can monitor the SO₂ derivatives and respond to the change of viscosity in lysosomes through two-photon imaging. Triph-SZ present high sensitivity and selectivity fluorescence response with the addition of SO₂ derivatives based on the nucleophilic addition, and it also exhibits a sensitive fluorescence enhancement to environmental viscosity, which allows Triph-SZ to be employed to monitor the level of HSO₃^? and viscosity changes in lysosomes by the two-photon fluorescence lifetime imaging microscopy.

Full text of DOI:10.1016/j.saa.2021.119457

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 251 (2021) 119457
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A benzothiazole-based near-infrared fluorescent probe for sensing SO2
derivatives and viscosity in HeLa cells
Yibing Li a,1, Yilin Zhu c,1, Xuzi Cai d, Jimin Guo a, Cuiping Yao c, Qiling Pan b, Xuefeng Wang d,
,
⇑
Kang-Nan Wang b,
⇑
a Department of Obstetrics and Gynecology, Affiliated Shenzhen Maternity & Child Healthcare Hospital, Southern Medical University, Shenzhen 518028, Guangdong, China
b Shunde Hospital, Southern Medical University (The First People’s Hospital of Shunde), Foshan, China
c Key Laboratory of Biomedical Information Engineering of Ministry of Education, Institute of Biomedical Photonics and Sensing, School of Life Science and Technology, Xi’an Jiaotong
University, Xi’an 710049, China
d Department of Obstetrics and Gynecology, The Third Affiliated Hospital of Southern Medical University, No. 183 West Zhongshan Avenue, Guangzhou, Guangdong, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
A NIR probe Triph-SZ was developed
for detecting the SO2 derivatives and
viscosity in lysosomes.
ꢀ
Triph-SZ can response to SO2
derivatives and viscosity via two-
photon imaging and fluorescence
lifetime imaging.
ꢀ
Triph-SZ showed different
fluorescence lifetÀime with the
addition of HSO3 in lysosomes.
a r t i c l e i n f o
a b s t r a c t
Article history:
The unbalanced metabolism of sulfur dioxide can cause various diseases, such as neurological disorders
and lung cancer. Until now, some researches revealed that the normal function of lysosomes would be
disrupted by its abnormal viscosity. As a signal molecule, sulfur dioxide (SO2) plays an important role
in lysosome metabolism. However, the connection of metabolism between the SO2 and viscosity in lyso-
somes is still unknown. Herein, we developed a benzothiazole-based near-infrared (NIR) fluorescent
probe (Triph-SZ), which can monitor the SO2 derivatives and respond to the change of viscosity in lyso-
somes through two-photon imaging. Triph-SZ present high sensitivity and selectivity fluorescence
response with the addition of SO2 derivatives based on the nucleophilic addition, and it also exhibits a
sensitive fluorescence enhancement to environmental viscosity, which allows Triph-SZ to be employed
to monitor the level of HSOÀ3 and viscosity changes in lysosomes by the two-photon fluorescence lifetime
imaging microscopy.
Received 20 November 2020
Received in revised form 2 January 2021
Accepted 6 January 2021
Available online 12 January 2021
Keywords:
Near-infrared fluorescent probe
Sulfur dioxide derivatives
Viscosity of lysosomes
Two-photon fluorescence lifetime imaging
Ó 2021 Elsevier B.V. All rights reserved.
1. Introduction
Sulfur dioxide (SO2), in forms of sulfite (SO23À) and bisulfite
(HSOÀ3 ) in living organisms, can be formed physiologically during
methionine and cysteine metabolism or oxidation of hydrogen sul-
fide in the cellular environments [1–4]. As a biological gasotrans-
mitter, many studies have proven that the endogenous SO2
⇑
Corresponding authors.
E-mail addresses: douwangxuefeng@163.com (X. Wang), wangkn3@mail2.sysu.
edu.cn (K.-N. Wang).
1
Yibing Li and Yilin Zhu contributed equally.
https://doi.org/10.1016/j.saa.2021.119457
1386-1425/Ó 2021 Elsevier B.V. All rights reserved.

Y. Li, Y. Zhu, X. Cai et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 251 (2021) 119457
derivatives show unique bioactivities, such as inflammatory
responses, maintaining the balance of biological sulfur and relax
blood vessels in the body. [5–8] It is reported that SO2 derivatives
play an important role in inflammation-induced cervical cancer.[9]
However, the generation and function of SO2 in many biological
processes still remain unclear. On the other hand, lysosomes, as
the recycling centers in living cells, contain various hydrolases
for hydrolyzing many cellular biomacromolecules. The lysosome
is an essential organ in the cells as a ‘‘stomach”, which plays a crit-
ical role in physiological activities, such as cell signaling, cell
migration, tissue remodeling, and initiation of apoptosis [10–12].
Moreover, the dysfunction of lysosomes will induce numbers of
diseases like lysosomal storage diseases (LSDs), tuberculosis, even
cardiovascular diseases [13,14].
results indicated the right structure and high purity of the
compound.
1H NMR (500 MHz, DMSO) d 8.38 (d, J = 8.0 Hz, 1H), 8.18 (d,
J = 8.5 Hz, 1H), 8.12 (d, J = 15.6 Hz, 1H), 7.92 (d, J = 8.8 Hz, 2H),
7.84 (t, J = 7.8 Hz, 1H), 7.76 (d, J = 21.0, 11.6 Hz, 2H), 7.43 (t,
J = 7.8 Hz, 4H), 7.24 (t, J = 7.4 Hz, 2H), 7.20 (d, J = 7.6 Hz, 4H),
6.91 (d, J = 8.8 Hz, 2H), 4.29 (s, 3H). 13C NMR (126 MHz, DMSO)
d 172.17 (s), 151.83 (s), 149.17 (s), 145.98 (s), 142.48 (s), 132.33
(s), 130.51 (s), 129.65 (s), 128.49 (s), 127.86 (s), 126.65 (s),
125.95 (s), 124.54 (s), 119.46 (s), 116.97 (s), 110.59 (s), 36.58 (s).
ESI-MS (m/z): calcd for [MÀI]+ (C28H23N2S+): 419.19; found:
419.36.
2.2. Absorption and fluorescence emission spectra measurement
With regard to viscosity, as one of the primary factors, inten-
sively impacts intracellular chemical signals transportation, bio-
molecules interaction, and the diffusion of reactive metabolites
within living systems. And as we all know, the viscosity of lysoso-
mal is an important benchmark for lysosome condition and reflects
the organelles’ status and function [15,16]. Changes of lysosomes
viscosity have been shown to be associated with many diseases
at the cellular level [17,18]. Therefore, a deep understanding of
lysosomal viscosity is of great benefit to disease diagnosis and
treatment. Thus, developing efficient detection tools for tracing
the viscosity in subcellular organelles is of great importance for
investigating and diagnosing related diseases. Many reports point
out the connection between SO2 derivatives and mitochondrial vis-
cosity [19,20], but we still know little about the relationship
between SO2 derivatives and lysosomal viscosity.
Fluorescence microscopy provides higher contrast than classical
microscopic methods, has become one of the most powerful tools
for investigating the physiological process on cellular scales. More-
over, specific fluorescent dyes offer the unique even within single
cells. One of the fluorescence imaging technology may be fluores-
cence lifetime imaging (FLIM), which can not only present the
dynamic changes of intracellular environment (such as ion content,
viscosity, pH, etc.) but also quantify the information changes in liv-
ing cells with high accuracy, because the fluorescence lifetime of
probes is dependent on the microenvironment rather than on the
probe concentration. [21] As one of the essential technologies in
fluorescence imaging, it is also very suitable for SO2 derivative
detection and lysosomal viscosity imaging [22].
The required measurement concentration of Triph-SZ solution
was achieved by diluting with DMSO. At room temperature, the
absorption spectrum was collected with a Varian Cary 300 spec-
trophotometer, while the fluorescence emission spectrum was col-
lected with an Edinburgh FLS 920 spectrometer. Stock solutions of
anions (10 mM, including HSOÀ3 , SO32À, SO24À, ClOÀ, FÀ, ClÀ, BrÀ, IÀ,
CNÀ, HPO4À, CO32À, and SCNÀ, they used as sodium or potassium
salts) and thiols (10 mM, including Cys, GSH, and Hcy) were pre-
pared in deionized water. The stock solution of the probe Triph-
SZ (10
lM) was prepared in DMSO/H2O (1/1, v/v). The optical
selectivity experiments were implemented through the addition
of the same amounts of anions and HSOÀ3 into the Triph-SZ testing
solution. For the titration assays, the different amount of HSO3À
were added into the Triph-SZ solution and measured the visible
changes.
2.3. Determination of viscosity
The solvents were prepared by mixing the methanol-glycerol
system in different proportions. The solutions of Triph-SZ of differ-
ent viscosity were obtained by adding the stock solution to 10 mL
of solvent mixture systems to make the final concentration of the
Triph-SZ (10 mM). After standing for 1 h at a constant temperature,
the solutions were detected in a fluorescence spectrophotometer.
2.4. Cells culture and imaging
In this work, a benzothiazole-based near-infrared (NIR) fluores-
cent probe Triph-SZ was applied to detect the SO2 derivatives and
respond to the change of viscosity in lysosomes through the two-
photon imaging. Triph-SZ presented high sensitivity and selectiv-
ity fluorescence response with the addition of HSOÀ3 based on the
nucleophilic addition. In contrast, the fluorescence intensity was
obviously increased as the viscosity increases since the free two
phenyl and benzothiazole bring about the twisting intramolecular
charge transfer (TICT) effect. Triph-SZ possesses an excellent
lysosome-targeting property and can be applied for monitoring
the viscosity changes induced by the unbalanced metabolism of
sulfur dioxide in lysosomes through the two-photon fluorescence
lifetime imaging microscopy (TP-FLIM).
HeLa cells were offered by the Cell Bank of Type Culture Collec-
tion of the Chinese Academy of Sciences (Shanghai, China). The
HeLa cells were propagated in DMEM medium modified by Dul-
becco and supplemented using 10% fetal bovine Serum (FBS) in a
fully humidified incubator (containing 5% CO2, 37 °C). Before imag-
ing, HeLa cells were seeded in 25-Petri dishes and adhere for 48 h.
Followed by washing with PBS, the cells were incubated using the
probe Triph-SZ (10
l
M) at 37 °C for 30 mins. Next, HSOÀ3 was
added, and the HeLa cells were continually incubated for another
30 mins. After that, the cells were washed three times by PBS prior
to imaging. Meanwhile, the HeLa cells incubated only with the
probe Triph-SZ (10 lM) for 30 mins were employed as the control.
Then, the same set of cells was employed for the following fluores-
cence image measurement.
2. Experimental section
2.5. Cellular localization assay
2.1. Synthesis and characterization of Triph-SZ
For the colocalization, the cells were first treated with Triph-SZ
The structure and synthetic route of Triph-SZ is presented in
Scheme S1. NMR data spectra were recorded from a Mercury
Plus-500 NMR spectrometer. And the ESI-MS data was recorded
from an LCMS-2010 system. The characterizations (ESI-MS, 1H
NMR, and 13C NMR) of Triph-SZ are described in Figs. S1–S3. These
(10 lM) for 30 mins, and further cultured in the discoloration med-
ium containing Lyso-Tracker Deep Red (LTDR, 100 nM) at 37 ℃.
After incubation for 30 mins, cells were rinsed by PBS and immedi-
ately imaged under the confocal microscope. The images were
taken under excitation at 488 nm.
2

Y. Li, Y. Zhu, X. Cai et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 251 (2021) 119457
2.6. Tracking of viscosity in lysosomes by TP-FLIM
could be used for the biological samples testing in a wide pH range.
The selectivity of Triph-SZ toward HSOÀ3 was important in com-
plexed bioenvironment, so the selectivity evaluation of probe
was performed. Triph-SZ was incubated with different anions
and thiols in PBS/DMSO (1/1, v/v) system. As shown in Fig. 2, the
competition experiments were implemented by the addition of a
mixture of 1.5 equivalent of HSOÀ3 and 5.0 equivalent of different
anions and thiols, which showed that these competitive anions
and thiols have nearly no influence on the detection of HSOÀ3 . These
results indicated that Triph-SZ could be employed potentially to
quantitatively detect HSOÀ3 with a high selectivity.
35 mm culture dishes were used for Hela cells in exponential
growth for 48 h. After that, the culture medium was removed,
and replaced with Triph-SZ (10
lM) solution before imaging.
Under two-photon excitation, the fluorescence intensity and fluo-
rescence lifetime of Triph-SZ were detected, respectively. Using a
Becker & Hickl SPC- imaging analysis software to get the fluores-
cence lifetime data. kex = 850 nm (TPM); kem = 650 20 nm.
3. Results and discussion
3.1. Spectroscopic property of Triph-SZ and its response to HSO3À
3.3. Proposed recognition mechanism
To further understand the fluorescence ‘‘witch-off” behavior of
Triph-SZ under the HSOÀ3 conductions, the Job’s plots were mea-
sured. We verified that the stoichiometry of the reaction product
between Triph-SZ and HSOÀ3 was found to be 1:1 (Fig. S9). We
speculated that the conjugate system was breaking after reaction
with HSOÀ3 , so Triph-SZ showed a noticeable fluorescence signal
change at 682 nm under 488 nm excitation, which is consistent
with the conclusion of the reported work. [30,31].
Firstly, the spectroscopic evaluation of Triph-SZ (10
the reaction between Triph-SZ and HSOÀ3 was performed in the
PBS/DMSO (1/1, v/v, 10 M, pH 7.4) system. The maximum UV/
Vis absorption and emission wavelength of Triph-SZ are at
295/510 nm and 682 nm, respectively (Fig. 1a and Fig. S4).
Triph-SZ showed an intense absorption band centered at 510 nm
lM) and
l
in combination with another less intense
p ? p* transitions at
295 nm (Fig. S4). After its reaction with HSOÀ3 , the absorbance
intensity of HSOÀ3 at 510 nm was gradually decrease, accompanied
by a ratiometric increase at 295 nm and produced a large blue-
shift, which resulted in obviously color changing that complete
bleaching of the original reddish-pink color of the receptor
(Fig. S5). The fluorescence spectrum of Triph-SZ showed a clear
emission wavelength of 682 nm (Fig. 1b), which was owned to
the intramolecular charge transfer (ICT) from the triphenylamine
to benzothiazole (Fig. S6). After adding 1.4 equivalent of HSOÀ3 ,
an obvious fluorescence quenching can be monitored in Fig. 1b.
We speculated that the observed bleaching of the color after the
treatment with HSOÀ3 may arise from the nucleophilic addition
reaction of HSOÀ3 at the unsaturated C@C double band of Triph-
SZ that the conjugation interrupted, which resulted in the descent
of the fluorescence intensity of 682 nm. And the limit of detection
3.4. Viscosity response
The triphenylamine unit is considered to be a typical rotor
group, and is widely used in the development of viscosity-
responsive imaging reagents. Therefore, the absorption spectrum
and fluorescence spectra of Triph-SZ in different ration glycerol-
methanol mixture were further investigated its viscosity response
characteristics. Even though the Triph-SZ exhibits an inert absorp-
tion response to viscosity (Fig. S10), as shown in Fig. 3a, with the
increasing fraction of glycerol, the fluorescence intensity of
Triph-SZ increased gradually. In consequence, a good linear rela-
tionship excited between Log (Imax) and percentage of glycerin
with an excellent coefficient of 0.995 (Fig. 3b). The sensitive
response of Triph-SZ to viscosity is also reflected in the fluores-
cence lifetimes (Fig. 3c), while it does not respond to pH and sol-
vent polarity (Fig. S11). Due to the intramolecular rotation of the
triphenylamine and benzothiazole, the formation of the twisted
intramolecular charge transfer (TICT) state leads to strong fluores-
cence (Fig. 3d). All of those results were indicating that Triph-SZ
could be applied to measure the solution viscosity quantitatively.
(LOD) for HSOÀ3 was calculated as 1.88 nM based on 3
means the standard deviation for the fluorescence intensity of
r/k (in which,
r
the blank test in the absence of HSOÀ3 , and k is the slope of the lin-
ear equation) (Fig. S7), which indicates that Triph-SZ has outstand-
ing sensitivity toward HSOÀ3 for its applications. In general, the
effect of reaction time on the fluorescence intensity of the system
was also investigated. The fluorescence intensity of Triph-SZ
decreases with increasing reaction time upon the reaction time
within 40 s. (Fig. 1c). As an important biochemical parameter,
the fluorescence lifetime can reflect the important information of
the microenvironment in living cells. As shown in Fig. 1d, with
the addition of HSOÀ3 (0–1.2 eq.), the fluorescence lifetime
decreases from 2.52 ns to 0.79 ns in PBS/DMSO (pH 7.4, 1:1, v/v)
solution, which indicated that Triph-SZ can monitor the HSO3À
changes of a microenvironment through fluorescence lifetime. This
result could be ascribed to the unsaturated C@C double bond to
offer specific recognized sites for HSOÀ3 so that the ICT processes
were inhibited and the fluorescence intensity was decreased [23–
29].
3.5. Fluorescence imaging in living cells
The biocompatibility of Triph-SZ was firstly evaluated via its
cytotoxicity toward HeLa cell lines by MTT assay. To investigate
the cytotoxic of Triph-SZ, HeLa cells were incubated with different
concentrations of Triph-SZ. After 24 h of incubation, more than
75% of Hela cells still survived in a solution with a concentration
of 50
lM (Fig. S12), which indicated that Triph-SZ had negligible
cytotoxicity in the 10
lM working solution. Inspired by the excel-
lent performance of Triph-SZ toward the detection of HSO3À
in vitro, the abilities of Triph-SZ for intracellular detection and
the imaging of HSOÀ3 were further investigated in HeLa cells. After
the living cells were incubated with Triph-SZ for 30 mins, strong
fluorescence in HeLa cells can be detected. Moreover, the fluores-
cence intensity gradually increased along with the increase of
Triph-SZ concentration (Fig. S13). To further explored the subcel-
lular localization ability of Triph-SZ, the colocalization study of
Triph-SZ was also investigated with a commercially available lyso-
some dye, LTDR. As a result, Triph-SZ is mainly located in lyso-
somes in the cell with a high overlap Pearson’s coefficient of 0.81
(Fig. 4), indicating the excellent cell permeability and reasonable
3.2. Effects of pH and selectivity evaluation
The sensing responses of most fluorescence probes are signifi-
cantly affected by the pH value of the solution system, which
may affect the monitoring of SO2 derivatives and viscosity in the
intracellular environment. Thus, the pH effect on the fluorescence
intensity of Triph-SZ was investigated in detail. As shown in
Fig. S8, the fluorescence intensity of Triph-SZ was stable and even
barely affected from pH 4.5 to 9.0, which indicating that Triph-SZ
3

Y. Li, Y. Zhu, X. Cai et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 251 (2021) 119457
Fig. 1. UV–vis absorption spectra (a) and fluorescence spectra (b) of Triph-SZ (10
l
M) treated with HSOÀ3 (0–1.4 eq.) at 25 °C (kex = 488 nm) in PBS/DMSO (1:1, v/v, pH = 7.4).
(c) Fluorescence time response of Triph-SZ (10 l lM) in mixture of PBS buffer/DMSO (pH 7.4, 1:1, v/v) with the
M) to HSOÀ3 (1.2 eq). (d) Fluorescence lifetime of Triph-SZ (10
addition of HSOÀ3 (0–1.2 eq).
experiments could find that TPM does show deeper penetration
(Fig. S15). Those results demonstrated that Triph-SZ is suitable
for exploring the microenvironmental changes of the lysosome of
living cells through TPM, the results also lay the foundation for fur-
ther TP-FLIM of this probe. After the imaging experiments, HeLa
cells were cultured and stained with Triph-SZ with 30 mins,
washed it three times by using PBS buffer solution, and then trea-
ted with HSOÀ3 for another 30 mins, and a noticeable fluorescence
disappeared can be observed (Fig. 5), which indicated that Triph-
SZ has the potential of being employed to visualize HSOÀ3 level.
These consequences also demonstrate the capacity of Triph-SZ
for the imaging and detection of HSOÀ3 in living cells.
Fluorescence lifetime imaging have no concern with concentra-
tion but depends on its molecular environment, and the fluores-
cence lifetime imaging microscopy (FLIM) can reveal the
alterations with higher accuracy [36,37]. Moreover, TP-FLIM using
excitation by the near-infrared (NIR) light with deep penetration
depth, helps eliminating the background fluorescence virtually
possible, and reflects the molecular environment changes of the
chromophore in a quantitative way [38–40]. Thus, the TP-FLIM
was used to investigate the distribution of probe in living cells
(Fig. 6). As expected, the probe displays strong fluorescence in
the red channel, and there is almost no fluorescence produced
upon the treatment with HSOÀ3 . More attractively, the fluorescence
lifetime of Triph-SZ in the lysosome can reach almost 790 ps. It
also showed the different fluorescence lifetime distributions
(500–790 ps) of different regions in the lysosome, which repre-
sents the change in lysosomal viscosity at the corresponding posi-
tion. However, the fluorescence lifetime of Triph-SZ achieves
almost 2000 ps with the addition of exogenous HSOÀ3 , which can
Fig. 2. The competitive selectivity of Triph-SZ (10 l
M) in solution to HSOÀ3 /SO32À in
the presence of various analytes at room temperature.
localization specificity of Triph-SZ at the cellular lysosomes in liv-
ing cells.
In contrast to the traditional one-photon fluorescence imaging
(OPM), two-photon fluorescence imaging (TPM) have some advan-
tages, such as lower tissue autofluorescence, increased penetration
depth, and reduced photodamage, which avoids the shortcomings
of conventional fluorescent dyes with short excitation wavelengths
[22,32–35]. As can be seen from Fig. 5 and Fig. S14, Triph-SZ can
not only image the lysosomes of living cells in OPM, but also the
TPM can also be performed by the probe. Further 3D scanning
4

Y. Li, Y. Zhu, X. Cai et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 251 (2021) 119457
Fig. 3. (a) Fluorescence spectra of Triph-SZ (10
lM) in the different concentrations of glycerin (Gly) in MeOH (kex = 488 nm); (b) linear fitting curve of the fluorescence
intensity at 680 nm of Triph-SZ (10
lM) vs. the viscosity of Gly in MeOH solution; (c) fluorescence lifetime spectra of Triph-SZ (10 lM) in the different concentrations of Gly
in MeOH (kex = 488 nm); (d) simulated molecular rotation of Triph-SZ in solutions of low to high viscosity.
Fig. 4. Lysosome co-localization imaging of HeLa cells stained with Triph-SZ (10.0
kex: 633 nm, kem: 650 20 nm for LDTR. Scale bar: 20 m.
lM) and LTDR (100 nM, pseudo-green) at 37 °C. kex: 488 nm, kem: 650 20 nm, for riph-SZ;
l
indicate that the addition of HSOÀ3 could induce the increasing vis-
cosity in the lysosome.
the level of HSOÀ3 and viscosity changes in lysosomes with a high
colocalization coefficient. As promising, this lysosomes-targeted
fluorescence probe exhibits promising potential in practical appli-
cations of the connection between the HSOÀ3 -mediated and
viscosity-caused biological researches.
4. Conclusions
In summary, we have presented a benzothiazole-based NIR flu-
orescent probe Triph-SZ to detect the SO2 derivatives, and respond
to the change of viscosity in lysosomes via TPM and TP-FLIM. The
probe Triph-SZ displayed high sensitivity and selectivity fluores-
cence response with the remarkable decrease of fluorescence
intensity toward HSOÀ3 . Moreover, the fluorescence spectra of
Triph-SZ have significant fluorescence enhancement with the
changes of the viscosity of the MeOH-Gly solution. Furthermore,
cell imaging proved that Triph-SZ could be utilized to monitor
5. Novelty statement
(1) A lysosome-targarted near-infrared fluorescent probe Triph-
SZ was developed for the sulfur dioxide SO2 derivatives and
viscosity.
(2) Triph-SZ can response to HSO-3 and viscosity via two-photon
imaging and fluorescence lifetime imaging microscopy.
5

Y. Li, Y. Zhu, X. Cai et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 251 (2021) 119457
Fig. 5. Confocal fluorescence images of Hela cells. The fluorescence images were captured at 650 20 nm from OPM (kex = 488 nm), and TPM (kex = 850 nm). Scale bar: 20 mm.
Fig. 6. TPM confocal images and fluorescence lifetime images of Triph-SZ (10
at 650 20 nm under excitation at 850 nm for TPM. Scale bar: 20 mm.
l
M) before and after the living cells treated with HSOÀ3 . The fluorescence images were captured
(3) Triph-SZ showed a different fluorecence lifetime toward
Declaration of Competing Interest
HSO-3 and viscosity in lysosomes.
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
CRediiT authorship contribution statement
Yibing Li, and Yilin Zhu contributed equally to this work. Kang-
Nan Wang and Xuefeng Wang conceived and directed the project.
Yilin Zhu designed, synthesized, and characterized the compounds.
Yibing Li, Yilin Zhu, and Cuiping Yao performed photophysical
properties. Yilin Zhu and Jimin Guo carried out confocal imaging
experiments. Yibing Li and Xuzi Cai discussed and analyzed the
data. Yibing Li and Yilin Zhu wrote the paper under the direction
of Cuiping Yao, Qiling Pan and Kang-Nan Wang.
Acknowledgements
We are grateful for the financial support from the National Nat-
ural Science Foundation of China (No. 61775178) and the China
Postdoctoral Science Foundation (2019M662968), and Guangdong
Basic and Applied Basic Research Foundation (2019A1515110356).
6

Y. Li, Y. Zhu, X. Cai et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 251 (2021) 119457
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Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2021.119457.
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