A single fluorescent probe for one- and two-photon imaging hydrogen sulfide and hydrogen polysulfides with different fluorescence signals

Article abstract of DOI:10.1016/j.dyepig.2019.107818

Reactive sulfur species (RSS), primarily containing glutathione (GSH), cysteine (Cys), hydrogen sulfide (H₂S) and hydrogen polysulfides (H₂S_n, n > 1), were crucial endogenous biological signal molecules. Among them, H

Full text of DOI:10.1016/j.dyepig.2019.107818

Journal Pre-proof
A single fluorescent probe for one- and two-photon imaging hydrogen sulfide and
hydrogen polysulfides with different fluorescence signals
Xinxin Zhao, Fangru He, Yanpeng Dai, Fulong Ma, Zhengjian Qi
PII:
S0143-7208(19)31772-3
DOI:
https://doi.org/10.1016/j.dyepig.2019.107818
DYPI 107818
Reference:
To appear in: Dyes and Pigments
Received Date: 26 July 2019
Revised Date: 16 August 2019
Accepted Date: 16 August 2019
Please cite this article as: Zhao X, He F, Dai Y, Ma F, Qi Z, A single fluorescent probe for one- and two-
photon imaging hydrogen sulfide and hydrogen polysulfides with different fluorescence signals, Dyes
and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107818.
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2019 Published by Elsevier Ltd.

A single fluorescent probe for one- and two-photon imaging
hydrogen sulfide and hydrogen polysulfides with different
fluorescence signals
a
a
a
a
a∗
Xinxin Zhao , Fangru He , Yanpeng Dai , Fulong Ma , Zhengjian Qi
a
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, School of Chemistry and
Chemical Engineering, Southeast University, Nanjing, Jiangsu 211189, PR China
Abstract
Reactive sulfur species (RSS), primarily containing glutathione (GSH), cysteine
Cys), hydrogen sulfide (H S) and hydrogen polysulfides (H S , n>1), were crucial
(
2
2 n
endogenous biological signal molecules. Among them, H S and H S played
2
2 n
extremely momentous roles in many physiological and pathological processes.
Limited by the vivo environmental diversity and complexity, it was highly desirable
to rely on the single fluorescence probe platform to visualize H S and H S according
2
n
2
different fluorescence signals. Herein, we reported a two-photon excited fluorescent
probe MC-S that had the following merits: (� ) ratiometric and colorimetric: reducing
n
detection errors and conducting visual analysis; (� ) two-photon microscope: better
performance in vivo and vitro imaging; (� ) two specific sulfide reaction sites:
studying on the mutual transformation of H S and H S in biological systems.
2
n
2
Furthermore, take advantages of low cytotoxicity (cell viability ≥ 85.99%), low limit
of detection (21 nM), significant photostability, excellent pH stability, quick response
behavior (175 seconds) and high penetration depth (110 µm), MC-S can be employed
n
to visualize endogenous and exogenous H S and H S in cells, liver tissues and
2
n
2
zebrafish.
Keywords: hydrogen sulfide; hydrogen polysulfides; two-photon microscope;
simultaneous detection
1
. Introduction
Reactive sulfur species (RSS), primarily containing glutathione (GSH), cysteine
∗
Corresponding author. E-mail address: qizhengjian@seu.edu.cn (Z. Qi).

(
Cys), hydrogen sulfide (H S) and hydrogen polysulfides (H S , n>1), were crucial
2 2 n
endogenous biological signal molecules in regulating cellular activity and maintaining
dynamic balance of redox in living organisms [1]. Among these sulfur-containing
molecules, the specific and efficient recognition methods of H S and H S were
2
2 n
receiving more and more attention because they play extremely momentous roles in
many physiological and pathological processes [2]. Sodium polysulfides (Na S , n>1)
2
n
had a wide range of applications in the tanning industry, agriculture, petroleum
refining, analytical chemistry and other fields [3]. However, excessive sodium
polysulfides not only caused enormous irreversible damage to the environmental
system but also organisms [4]. According to the literature, sodium polysulfides can be
equivalent to hydrogen polysulfides (H S , n>1) under physiological conditions [5].
2
n
Hydrogen sulfide (H S), the third endogenous gasotransmitter, played a significant
2
physiological and pathological regulation role in the cardiovascular system and the
nervous system [6]. H S and H S probably coexisted in living organisms from the
2
n
2
redox chemistry [7]. For example, hydrogen sulfide can reversibly form disulphide
species in the presence of ROS. Meanwhile, the disulphide species will fleetly
transform to other hydrogen polysulfides (1). However, the disulphide species will
rapidly undergo further redox equilibrium reactions to form other hydrogen
polysulfides, which were controlled by pH and the relative amount of the oxidized
versus reduced forms (2) [8]. In-depth understanding of the production, distribution
and physiological functions of hydrogen sulfide under normal and disease states was
of great significance for elucidating the mechanism of cell signal transduction.
However, limited by the vivo environmental diversity and complexity, it was intensely
urgent to develop a convenient and accurate detection method for H S and H S.
2
n
2
ꢁꢂ
⇌
2
H2S ꢀ H S +H O
(1)
(2)
2
2
2
-
-
-
nHS ⇌ HS
+ (n-1) HS
2
n+1
Traditional methods such as UV spectroscopic [9], titrimetry [10] and UV-vis
spectroelectrochemistry [11] have many limitations, such as high detection limit, poor
specificity and strong damage to organisms. Fluorescent probes were becoming a

powerful strategy for detecting the endogenous and exogenous biothiols ascribed to
their advantages such as high sensitivity and selectivity, bioimaging ability and
performance of real-time monitoring [12]. In recent years, some probes based on
bis-electrophilic nitro-activated fluorobenzoate had been reported [13]. Unfortunately,
a ratiometric two-photon excited fluorescent probe for simultaneous detecting H2Sn
and H S had not been reported. Given to improve the understanding of mutual
2
transformation of H S and H S, we designed a novel fluorescent probe MC-S .
2
n
2
n
The ratiometric fluorescent probes detected the target analyte based on two
simultaneously varying emission peaks which can reduce detection errors caused by
the factors such as probes concentration, sample environment and instrumentation
efficiency by self-adjustment [14]. Therefore, ratiometric fluorescent probes were
more advantageous than turn on/off fluorescent probes in terms of detection accuracy
and anti-interference performance. Colorimetric fluorescent probes had the
characteristics of colorimetric measurement and visual analysis, so they had broader
application prospects [15].
One-photon microscope (OPM) had many disadvantages such as short excitation
wavelength (350~550 nm), photobleaching, photodamage and autofluorescence, thus
its application in biomedical research had been seriously limited [16]. Two-photon
microscope (TPM) produced the fluorescence through two-photon excited, exhibiting
higher spatial resolution, higher penetration depth and lower photodamage to
biological samples [17]. These advantages enabled TPM to perform better in tissue
and zebrafish imaging.
So far, fluorescent probes that can simultaneously detect and distinguish H S and
2
H S had few been reported. Simply mixing the two specific probes which aimed to
2
n
simultaneously detect both analytes by different fluorescent signals had many
limitations, such as: (� ) potential interference between two probes; (� ) larger
invasive effects; (� ) possible different localization and metabolisms of the probes
[
18]. Therefore, it was urgent and challenging to construct a single fluorescence probe
which can visualize H S and H S using different emission channels. We first
2
2 n
constructed two specific sulfide reaction sites and different fluorescence phenomena

were generated towards H S or H S . Then mutual transformation between H S and
2
2
n
2 n
H S can be real-time detected in biological systems according the different
2
fluorescence signals. This design concept should be widely applied to the construction
of biothiols or other analytes probes.
Considering the above needs and advantages, we designed a two-photon excited
fluorescent probe MC-S and had the following merits: (� ) ratiometric and
n
colorimetric: reducing detection errors and conducting visual analysis; (� )
two-photon microscope: better performance in vivo and vitro imaging; (� ) two
specific sulfide reaction sites: studying on the mutual transformation of H S and H S
2
n
2
in biological systems. In addition, low cytotoxicity, low limit of detection, significant
photostability and excellent pH stability indicted that MC-S can be employed to
n
visualize endogenous and exogenous H S and H S in cells, tissues and zebrafish by
2
n
2
one- and two-photon excitation.
2
. Experiment section
2
.1. Materials and instruments
All the materials and instruments were demonstrated in Supplementary
Information. The synthetic route of MC-S was showed in Scheme 1. The details of
n
synthetic steps were displayed in Supplementary Information.
NO2
NO2
O
O
HO
4
-dimethylaminopyridine
DMF
CH Cl
+
+
OH
Cl
Cl
Cl
2
2
CHO
CH2Cl2
F
O
F
O
1
2
3
4
F
O
Piperidine
Ethanol
O N
2
+
N
O
F
O
CHO
N
NO2
O
5
6
MC-Sn
Scheme 1. The synthetic route of MC-Sn.
2
.2. Cell culture and imaging
RAW264.7 cells from the mouse macrophage cell line were cultured in DMEM
supplemented with 10% FBS and 1% penicillin/streptomycin and incubated in air
atmosphere (5% CO ) at 37 °C. Before imaging, the cells were seeded into 12-well
2

4
plates at 5×10 cell/well and incubated in air atmosphere (5% CO ) at 37 °C overnight.
2
For imaging control group, RAW264.7 cells were only incubated with MC-S (5 µM)
n
for 20 min. For imaging exogenous H S and H S , RAW264.7 cells were first
2
2 n
incubated with MC-S for 20 min and then further treated with 15 µM Na S or 15 µM
n
2
Na S for 15 min, respectively. For imaging the thiol-consumed control group,
2
2
RAW264.7 cells were incubated with NEM (2 mM) and MC-S in sequence before
n
adding Na S . For imaging H S produced by ROS and H S, RAW264.7 cells were
2
2
2
n
2
incubated with PMA (10 nM) for 30 min and then cultured with MC-S for another 15
n
min, and further cultured with Na S (15 µM) for 15 min.
2
2
.3. Two-photon imaging of liver tissues
The living liver tissue was prepared from the mouse (purchased from Nanjing
EzeRinka Biotechnology Co., Ltd.). First, the tissue was cut to slices with 300 µm
thickness by using a vibrating-blade microtome and transferred to glass-bottomed
dishes. Then the slices were first incubated with 5 µM MC-S at 37 � for 1 h and then
n
treated with 15 µM Na S . For the control experiments, the slices were only incubated
2
2
with 5 µM MC-S at 37 � for 1 h and then washed with PBS three times. The
n
two-photon fluorescence emission was collected under a two-photon confocal
microscope (ZEISS-LSM 880) in the range of 445-485, 500-540 and 580-630 nm on
excitation at 780 nm.
2
.4. Fluorescence imaging in zebrafish
The zebrafish eggs were prepared from KeyGEN BioTECH Co., Ltd. The
zebrafish were cultured in E3 medium for 3 days and were transferred to 24-well
microplates and incubated with 5 µM MC-S for 30 min as control group. After the
n
zebrafish were washed with E3M to remove the remaining MC-S , they were further
n
incubated with 15 µM Na S and Na S for another 30 min. Before being subjected to
2
2 2
TPM, the embryos were anesthetized with tricaine and embedded onto 0.5% agarose
gel. All the pictures collected were of living embryos with an observed heartbeat.
3
. Result and discussion
3
.1. Synthesis and mechanism

Benzoindole cation, which had the excellent electron-withdrawing ability, can be
used as the electron-withdrawing group of fluorophores according to the
intramolecular charge transfer (ICT) mechanism [19]. Inspired by this concept, we
chose naphthol which was linked to benzoindole cation by a C=C double bond as the
electron-donating group and a H S-responsive moiety. These two parts became a
2
complete fluorophore with showing more advantages at this time, such as two-photon
excitation, ratiometric and colorimetric property. Subsequently, we chose
2
-fluoro-5-nitro-benzoic ester as the fluorescence-quenching and H S -responsive
2 n
moiety.
The proposed response mechanisms of MC-S against H S and H S were
n
2
n
2
illustrated in Scheme 2 and 3. In our hypothesis, H S first conducted nucleophilic
2
n
aromatic substitution via replacing F atom to form an intermediate containing free
SSH group. The free −SSH group continued a spontaneous intramolecular
−
cyclization with the ester group to release the 5-Nitro-3H-1,2-benzodithiol-3-one.
Prominently, other monosubstituted mercaptans, such as Cys and GSH, can only be
nucleophilically substituted and cannot undergo subsequent intramolecular cyclization
and generate ester groups (Scheme 3) [20]. It was well-known that H S were stronger
2
n
nucleophiles than other biothiols and can react with the hemicyanine unit of MC-Sn.
The special addition reaction of H S was carried out to the polymethine chain of
2
n
1
hemicyanine unit. H NMR titration experiment was carried out with the media of
D O-DMSO-d (v/v=1/9) to illustrate the detection mechanism of MC-S with H S .
2
6
n
2 n
By comparing Scheme S2a (only MC-S ) and Scheme S2b (MC-S and H S ), we can
n
n
2 n
visually observe that a new signal of aromatic proton (H at δ 1.76 ppm) appears in the
presence of H S . The new signal was attributed to the emergence of hydrogen of the
2
n
−SSH group. We then explored the reaction site of the sulfhydryl group attack. There
were 17 aromatic protons at δ 4.6-9.0 ppm of MC-S before (Scheme S2c) and after
n
(
Scheme S2d) reaction between H S and the phenomena were consistent with
2 n
reference date [21]. This indicated that the reaction site was C=N rather than C=C.
The mass signal peak at m/z 456.14 were consistent with the reaction product (m/z
4
57.15) between MC-S and Na S , which further confirmed our hypothesis (Fig.S8).
n 2 2

As shown in Fig.S9, the C−F signal of MC-S appeared at -96.68 ppm. When an
n
excess of H S was added, the C−F signal disappeared due to the F atom was
2
n
substituted by H S . These results indicated that 2-fluoro-5-nitro-benzoic ester as a
2
n
bis-electrophilic group can be used to specific recognition of H S . Thus, we chose
2
n
2
-fluoro-5-nitro-benzoic ester and hemicyanine unit as two reaction sites for detecting
H S .
2
n
Scheme 2. The proposed response mechanism of MC-S against H S and H S .
n
2
2 n
F
O
O
N
SH
NO
2
2
Specific addition
O
O
OH
OH
reaction of H
2
S
n
N
N
NO
2
N S
SH
O
S
2
O N
H
2
S
S
n
HS
O
S
O
N
NO2
Scheme 3. The proposed response mechanism of MC-S against H S and H S .
n
2
2 n
Hydrogen sulfide was the reduced form of H S and its structure was very similar
2
n
to H S , which seriously affected the accuracy of MC-S to detect H S in vivo.
2
n
n
2 n
Subsequently, we studied the proposed response mechanism of MC-Sn against H S.
2
+
According to mass signal peak at m/z 593.3 ([TM+H] ) of the reaction product
between MC-S and the H S, we found that the −SH group were introduced into
n
2
MC-S by addition reaction (Fig.S10). We speculated that H S only react with
n
2

hemicyanine unit by an addition reaction because it had stronger nucleophilic ability
than other biothiols, such as Cys, Hcy, GSH and so on.
3
3
.2. Spectral properties of MC- Sn
.2.1. Reaction Kinetics of exogenous H S and H S formed by ROS and H S
2
n
2
n
2
The fluorescence response time of MC-S towards Na S (a common H S donor)
n
2
2
2 n
was first optimized. After incubation with 15 µM Na S in 10 mM HEPES buffer at
2
2
pH 7.4, the fluorescence intensity was recorded every 25 seconds. By comparing the
black and red curves in Fig.1, we concluded that the fluorescence intensity enhanced
significantly after adding Na S and reached its plateau within 175 seconds and then
2
2
remained at a stable value. It’s beneficial for the real-time and rapid detection of H2Sn
in vitro or vivo with such quick response behavior. Subsequently, we tested the
reaction kinetics of H S generated by Na S (a common H S donor) and H O (a
2
n
2
2
2
2
common ROS). A relatively weak fluorescence enhancement (blue curves) was
emerged when 15 µM Na S was premixed with H O . Since the fluorescence intensity
2
2
2
was proportional to the concentration of H S , the weak fluorescence enhancement
2
n
proved that the system can form H S but in a small amount. Encouraged by the fact
2
n
that glutathione peroxidase (GSH-Px) scavenged reactive oxygen species (ROS) by
converting reduced biothiol (RSH) to oxidized biothiol (RSSR), we suspected that
GSH-Px may be involved in the biosynthesis of H S . As expected, the fluorescence
2
n
intensity was extremely increased with GSH-Px, indicating that GSH-Px can
efficiently catalyze the conversion of H O and H S to H S . In summary, benefit
2
2
2
2 n
from the quick and real-time response behavior, MC-S can detect not only exogenous
n
H S but also H S formed by ROS and H S.
2
n
2
n
2

MC-S
n
1000
MC-S +Na S
n
2 2
MC-S +Na S
n
2
MC-S +Na S+H O
2 2
n
2
8
6
4
2
00
00
00
00
0
MC-Sn+Na S+H2O2+GPx
2
0
50
100
150
200
250
Time (s)
Fig.1 Fluorescence intensity at 468 nm of MC-S (5 µM) towards blank (black), Na S (15 µM) (red), NaHS (15
n
2 2
µM) (green), NaHS (15 µM) + H O (50 µM) (blue) and NaHS (15 µM) + H O (50 µM) + GSH-Px (50 U/L) (cyan)
2
2
2
2
as a function time in HEPES buffer solution (10 mM, pH 7.4). (λ =410 nm)
ex
3
.2.2. Spectral response of MC-S with H S
n 2 n
The UV absorption spectrum illustrated the changes of MC-S before and after
n
incubation with Na S and Na S (Fig.2). After treatment with Na S and Na S, the
2
2
2
2
2
2
intrinsic absorption of MC-S centered at 465 nm disappeared. These results indicated
n
that Na S and Na S can both react with hemicyanine unit by an addition reaction.
2
2
2
MC-S (5 µM) itself had a relatively weak emission intensity at λ =606 nm, thus
n
em
exhibiting dark orange fluorescence under light (365 nm) (Fig.3a). Fluorescence
titration experiments were performed to research the spectral response of MC-S with
n
H S As Fig.3 illustrated, MC-S reacted with H S induced a dramatically decrease
2
n.
n
2 n
in the emission intensity at λ =606 nm, accompanied by the formation of a new
em
emission peak at 468 nm. Since the emission peak at 606 and 468 nm were changed
synergistically, the ratiometric detection of H S was completed with the emission
2
n
ratio at 606 and 468 nm. MC-S as a ratiometric fluorescence probe was more
n
sensitive and accurate attributed to two simultaneously varying emission bands and
can reduce detection errors due to factors such as probes concentration, sample

environment and instrumentation efficiency by self-adjustment. The emission
intensity ratios at 468 and 606 nm (I /I ) increased by about 194-fold and revealed
4
68 606
an excellent linear relationship with the H S concentration in the range of 3-10 µM
2
n
2
(
R =0.9978). There were noticeable changes in color from yellow to light pink under
natural lights, which further supported the colorimetric fluorescence response. The
−1
detection limit of MC-S was calculated to be 21 nM based on IUPAC (CDL = 3S m )
n
b
according to 10 blank measurements. These results indicated that MC-S was capable
n
of qualitatively and quantitatively detecting H S under simulated physiological
2
n
conditions and had the potential to apply it to bioimaging.
0.6
0.5
0.4
0.3
0.2
0.1
0.0
MC-S
n
MC-S +Na S
n
2 2
MC-S +Na S
n
2
400
500
600
700
Wavelength (nm)
Fig.2 Absorption spectra of MC-Sn (5 µM) towards blank (black), Na S (15 µM) (red), Na S (15 µM) (blue) in
2
2
2
HEPES buffer solution (10 mM, pH 7.4).
20
1000
(
a)
(b)
8
6
4
2
00
00
00
00
0
15
1
0
5
0
Y= 2.2742x-2.058
2
R
= 0.9978
4
50
500
550
600
650
700
750
0
4
8
12
16
Wavelength (nm)
Conc. of Na S /µM
2 2
Fig.3 (a) Fluorescence intensity of MC-S (5 µM) in HEPES buffer solution (10 mM, pH 7.4) before and after
n
incubation with different concentrations of Na2S2 (0-16 µM) at 37 � for 3 min. Insets from left to right:
photographs of MC-S without or with Na S (15 µM) under light (365 nm). (b) The ratiometric fluorescence
n
2 2

spectra (I468/I606) as a function of the concentrations of Na S in the range of 0-16 µM. The linear relationship of
2
2
the ratiometric fluorescence spectra (I468/I606) versus the concentration of Na S in the range of 3-10 µM. Insets
2
2
from left to right: photographs of MC-S without or with Na S (15 µM) under natural light. (λ =410 nm)
n
2
2
ex
Subsequently, we measured the fluorescence intensity of MC-S against H S
n
2
(
Fig.4). Similarly, the emission intensity significantly decreased at λ =606 nm, but a
em
new emission peak was formed at 519 nm. The emission intensity ratios at 519 and
06 nm (I /I ) increased by about 37-fold and showed a good linear relationship
6
5
19 606
2
with the H S concentration in the range of 0-11 µM (R =0.9958). The detection limit
2
of MC-S was calculated to be 34 nM. Although both H S and H S can attenuate the
n
2
n
2
emission intensity at λ =606 nm, H S induces new emission peak at 468 nm and
em
2 n
H S at 519 nm. Therefore, MC-S could distinguish H S and H S by different
2
n
2
n
2
fluorescence emission signals.
4
3
3
2
2
1
1
00
50
00
50
00
50
00
8
7
6
5
4
3
2
1
0
(
a)
(b)
Y= 0.6158x + 0.6752
2
R
= 0.9958
5
0
0
500
550
600
650
700
750
0
5
10
15
20
Wavelength (nm)
Conc. of NaHS/µM
Fig.4 (a) Fluorescence intensity of MC-S (5 µM) in HEPES buffer solution (10 mM, pH 7.4) before and after
n
incubation with different concentrations of Na S (0-20 µM) at 37 � for 3 min. Insets from left to right:
2
photographs of MC-S without or with Na S (15 µM) under light (365 nm). (b) The ratiometric fluorescence
n
2
spectra (I519/I606) as a function of the concentrations of Na S in the range of 0-20 µM. The linear relationship of the
2
ratiometric fluorescence spectra (I519/I606) versus the concentration of Na S in the range of 0-11 µM. Insets from
2
left to right: photographs of MC-S without or with Na S (15 µM) under natural light. (λ =410 nm)
n
2
ex
3
.2.3. detecting performance of MC-Sn
Specificity was a major property of fluorescence probe, so we proceeded to
measure the selectivity of MC-S toward H S by testing the fluorescence responses to
n
2 n
2
−
3−
−
RSS (GSH, Cys, Hcy, Na S, Na S , Na S ), RNS (NO , NO , NO, ONOO ) and
2
2
2
2 4
−
−
2
•
1
ROS (H O , ClO , O , OH, O ) (Fig.5). Upon addition of Na S and Na S , the
2
2
2
2
2
2 4

emission intensity ratios at 468 and 606 nm (I /I ) increased by about 194-fold and
4
68 606
2
16-fold, respectively. It had been reported that when the concentration of H S was
2
high (50 µM), H S can be stored in the sulfane sulfur pool in the form of H S [22].
2
2 n
Therefore, after adding regular and high concentrations of Na S, the emission
2
intensity ratios at 468 and 606 nm (I /I ) increased by about 13-fold and 48-fold,
4
68 606
respectively. These results revealed that MC-S could detect H S with high
n
2 n
selectivity under simulated physiological conditions.
Fig.5 The ratiometric fluorescence spectra (I468/I606) of MC-S (5 µM) in HEPES buffer solution (10 mM, pH 7.4)
n
2−
3−
−
−
− •
towards 15 µM RNS, ROS and RSS (1: MC-S alone; 2-5: NO , NO , NO, ONOO ; 6-10: H O , ClO , O , OH,
n
2
2
2
1
O ; 11-17: GSH, NaHS, NaHS (50 µM), Cys, Hcy, Na S , Na S ), respectively. (λ =410 nm)
2
2
2
2
4
ex
3
.2.4. photostability and pH effect
Inspired by the quick response behavior, quantitatively detecting performance
and high selectivity, we tested the photostability and pH effect to prove whether
MC-S was appropriate for physiological detection. First, the fluorescence intensity of
n
MC-S and fluorescein (a common organic dye) was recorded every 25 min under the
n
same condition, respectively. As illustrated in Fig.6, the fluorescence intensity
(
I /I ) of MC-S was almost unchanged, but the fluorescence intensity of
468 606 n
fluorescein varied greatly. Subsequently, we examined the fluorescence intensity
I /I ) when the pH value ranging from 4 to 10. As illustrated in Fig.7, the
(
4
68 606
fluorescence intensity (I /I ) of MC-S treated with or without Na S was stable at
4
68 606
n
2 2
different pH values. These results revealed that MC-S possessed significant
n
photostability and excellent pH stability and had the potential to apply it to biological

imaging.
100
95
90
85
80
75
MC-Sn
Fluorescein
0
50
100
150
200
250
300
350
Time (min)
Fig.6 Time-based fluorescence spectra of MC-S and fluorescein over a course of 6 h.
n
1
1
1
1
1
8
6
4
2
0
8
6
4
2
0
MC-Sn
MC-S + Na S
n
2
2
4
5
6
7
8
9
10
pH
Fig.7 The ratiometric fluorescence spectra (I468/I606) of MC-S (5 µM) in the absence (black square) and presence
n
(
red cycle) versus different pH from 4 to 10 in HEPES buffer solution (10 mM, pH 7.4). (λ =410 nm)
ex
3
.3. Fluorescence imaging studies of MC- Sn
.3.1. Imaging H S and H S in cells
3
2
n
2
Based on the excellent specificity and sensitivity of MC-S in vitro assay, the
n
probe was revealed to have the potential application to respond to H S and H S in the
2
n
2
mouse macrophage cell line RAW264.7 cells. A cytotoxicity test was conducted to
evaluate whether MC-S could be used in cell assays. As illustrated in Fig.S11, cell
n
viability decreased slowly with increasing the concentration of MC-S , but even at a
n
very high concentration (100 µM), cell viability remained at a high level (cell viability
≥
85.99%). These results indicated that MC-S can be applied to detect H S and H S
n 2 n 2

in RAW264.7 cells.
First, RAW264.7 cells were only incubated with MC-S (5 µM) for 20 min,
n
while the control group exhibited distinct red fluorescence (Fig.8A). After the same
treatment according to the control group, we further treated with 15 µM Na S and 15
2
µM Na S for 15 min, respectively. With the addition of Na S, the green fluorescence
2
2
2
grew brighter and the red fluorescence gradually darkens until it disappears (Fig.8B).
The red fluorescence also disappeared after adding Na S , but blue fluorescence grew
2
2
brighter instead of green fluorescence (Fig.8C). By comparing the fluorescence
imaging added with the same concentrations of Na S and Na S , we found that blue
2
2 2
fluorescence (Fig.8C � ) was much brighter than green fluorescence (Fig.8B � ),
which was consistent with in vitro fluorescence. In the control experiment,
RAW264.7 cells were incubated with N-ethylmaleimide (2 mM) (NEM, RSS
blocking agent) and MC-S in sequence for 15 minutes before adding Na S . An
n
2 2
interesting phenomenon had emerged that the blue fluorescence disappeared and the
red fluorescence reappeared (Fig.8D). This was because NEM competitively inhibits
the binding of the MC-S to Na S , so that the MC-S can only release its own red
n
2
2
n
fluorescent signal. In summary, we confirmed that MC-S can be used to detect
n
exogenous Na S and Na S according to the changes in fluorescent channels before
2
2 2
and after adding detection substances.
Although the biosynthetic pathway of H S in vivo had not been elucidated, there
2
n
was some evidence that H S can be converted to H S in the presence of ROS.
2
2 n
Through in vitro testing, we demonstrated that H S can be formed when H S reacts
2
n
2
with intracellular oxidants such as superoxide, H O and hypochlorous (HOCl).
2
2
Inspired by the theory and experiment, we next attempted to image H S in
2
n
RAW264.7 cells, which was produced by ROS and H S. First, RAW264.7 cells
2
incubated with phorbol 12-myristate 13-acetate (PMA, 10 nM) for 30 minutes to
release excess ROS. Then, the cells cultured with MC-S (5 µM) for 15 minutes.
n
Subsequently, the cells were washed with DMEM to remove excess MC-S and
n
further incubated with Na S for another 15 minutes. By comparing Fig.8A and Fig.8E,
2
it was confirmed that MC-S can detect H S produced from ROS and H S according
n
2
n
2

to the fluorescent channels changed from red channel to blue channel. Credited to the
catalysis GSH-Px, which the RAW264.7 cells released it to clear ROS by converting
reduced biothiol (RSH) to oxidized biothiol (RSSR), MC-S can perform the detection
n
of H S in a relatively short period of time. To sum up, these results demonstrated that
2
n
MC-S can be applied to the detection of H S generated by ROS and H S in
n
2
n
2
RAW264.7 cells.
Fig.8 Confocal fluorescence microscopy images of RAW264.7 cells visualizing H S and H S level changes using
2
n
2
MC-S (5 µM). A: RAW264.7 cells incubated with MC-S for 30 min at 37 °C; B: RAW264.7 cells incubated with
n
n
MC-S for 30 min and then further incubated with 15 µM Na S at 37 °C; C: RAW264.7 cells incubated with
n
2
MC-S for 30 min and then further incubated with 15 µM Na S at 37 °C; D: RAW264.7 cells were pretreated with
n
2 2
2
mM NEM for 15 min and then incubated with MC-S for another 15 min before adding Na S at 37 °C; E:
n 2 2
RAW264.7 cells cultured with 10 nM PMA for 30 min and incubated with MC-S for 15 min. Subsequently, the
n
cells were washed with DMEM, then the cells were incubated with Na S for 15 min at 37 °C. (λ =405 nm)
2
ex
3
.3.2. Imaging H S in liver tissues
2 n
Two-photon microscopy was gradually becoming an indispensable tool in
biomedical research due to its high spatial resolution, high penetration depth, low

photodamage to biological samples and Z-scanning confocal imaging of biological
samples. Inspired by the superior performance of MC-S in living cell imaging, we
n
studied the ability of MC-S to detect biothiols in tissues by TPM. Fresh liver tissue
n
(
thickness at 300 µm) was pre-incubated with 5 µM MC-S at 37 � for 1 h and then
n
treated with PBS buffer alone, 15 µM Na S or Na S. As displayed in Fig.S12, the
2
2
2
liver tissue only incubated with MC-S which showed red fluorescence. Significant
n
fluorescence signal was observed changing from red to blue after treating with Na2S2
and the TPM imaging of tissues reached a penetration depth up to 110 µm (Fig.9),
which no obvious OPM fluorescence could be observed when the penetration depth
up to 80 µm (Fig.S13). As the Fig.10 showed, the fluorescence signal changed from
red to green after treating with Na S. These results indicated that MC-S can visualize
2
n
Na S and Na S according to the changes in fluorescent channels. Taken together,
2
2 2
these results further indicated that MC-S could be applied to detect biothiols in liver
n
tissues by using Z-scanning confocal imaging.
Fig.9 Z-scanning confocal imaging of liver slice treated with MC-S (5 µM) and then treated 15 µM Na S at the
n
2 2
penetration depth 0-120 µm by using two-photon microscope. The TPEF were collected at 445-485 nm by using
excitation 780 nm.
Fig.10 Z-scanning confocal imaging of liver slice treated with MC-S (5 µM) and then treated 15 µM Na S at the
n
2
penetration depth 0-120 µm by using two-photon microscope. The TPEF were collected at 500-540 nm by using

excitation 780 nm.
3
.3.3. Imaging H S in zebrafish
2 n
To further highlight the merits of MC-S in two-photon microscope and different
n
fluorescence signals for Na S and Na S, we examined whether it can be used to
2
2
2
detect biothiols in living animals and zebrafish was used as the animal model. The
-day-old zebrafish stained with MC-S alone for 30 min as control group and a red
3
n
fluorescence was appeared (Fig.11A). This was similar with that observed in living
cells and liver tissues. After pre-incubating with Na S and Na S , the MC-S -stained
2
2
2
n
zebrafish showed significant green and blue fluorescence, respectively (Fig.11B and
Fig.11C). These results imply that MC-S can be used to visualize Na S and Na S in
n
2
2 2
vivo.
Fig.11 Confocal microscopy images of exogenous Na S and Na S detection in zebrafish larvae. (A) zebrafish
2
2 2
incubated with MC-S (5 µM); (B) zebrafish treated with 15 µM Na S followed by incubating of with MC-S ; (C)
n
2
n
zebrafish treated with 15 µM Na S followed by incubating of with MC-S . Images were obtained with 780 nm
2
2
n
excitation, and the emission was collected in the range of 445-485, 500-540 and 580-630 nm.
4
. Conclusion
In summary, we developed a two-photon excited fluorescent probe MC-S to
n
study the mutual transformation of H S and H S. MC-S had the following merits: (� )
2
n
2
n
ratiometric and colorimetric: reducing detection errors and conducting visual analysis;
� ) two-photon microscope: better performance in vivo and vitro imaging; (� ) two
specific sulfide reaction sites: studying on the mutual transformation of H S and H S
(
2
n
2

in biological systems. In addition, these advantages, such as low cytotoxicity (cell
viability ≥ 85.99%), low limit of detection (21 nM), significant photostability,
excellent pH stability, quick response behavior (175 seconds) and high penetration
depth (110 µm) indicted that MC-S can successfully visualize endogenous and
n
exogenous H S and H S in cells, liver tissues and zebrafish. And it will help us to
2
n
2
clarify the physiological and pathological regulation role of hydrogen sulfide and
hydrogen polysulfides in both the cardiovascular system and the nervous system. The
greatest highlight of probe design concept was the construction of two specific sulfide
reaction sites in a single fluorescence probe, and the mutual transformation between
H S and H S can be real-time visualize with different fluorescence signals in
2
n
2
biological systems. These results of this work suggested that MC-S will be a
n
powerful molecular tool for meeting various biological application.
Conflicts of interest
There are no conflicts to declare.
Acknowledgments
This research work was financially supported by Research and Application of
Common Technologies in National Quality Foundation (2018YFF0215204) and the
Science and Technology Achievements Transformation Special Fund of Jiangsu
Province (BA2017035). Furthermore, we were grateful for Wang Liang form
Shiyanjia Lab (www.shiyanjia.com) for the analysis.
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Highlights
>
Ratiometric and colorimetric: reducing detection errors and conducting
visual analysis
>
>
Two-photon microscope: better performance in vivo and vitro imaging
two specific sulfide reaction sites: studying on the mutual transformation
of H2Sn and H2S in biological systems
Low cytotoxicity (cell viability 85.99%), low limit of detection (21 nM),
significant photostability, excellent pH stability, quick response behavior
175 seconds) and high penetration depth (110 m)
>
≥
(
µ