A TP-FRET-based two-photon fluorescent probe for ratiometric visualization of endogenous sulfur dioxide derivatives in mitochondria of living cells and tissues

Article abstract of DOI:10.1039/c6cc05254a

A ratiometric two-photon fluorescent probe for SO₂ derivatives was first proposed based on acedan-merocyanine dyads via a TP-FRET strategy. It was successfully applied to visualization of the fluctuations of enzymatically generated SO₂ derivatives in the mitochondria of HepG2 cells and rat liver tissues using two-photon fluorescence microscopy imaging.

Full text of DOI:10.1039/c6cc05254a

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DOI: 10.1039/C6CC05254A
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A TP-FRET-Based Two-Photon Fluorescent Probe for Ratiometric
Visualization of Endogenous Sulfur Dioxide Derivatives in
Mitochondria of Living Cells and Tissues†
Received 00th January 20xx,
Accepted 00th January 20xx
Xiaoguang Yang,a§ Yibo Zhou,b§ Xiufang Zhang,b Sheng Yang,*a Yun Chen,b Jingru Guo,a Xiaoxuan
Li,a Zhihe Qing,a and Ronghua Yang*ab
DOI: 10.1039/x0xx00000x
www.rsc.org/
A
ratiometric two-photon fluorescent probe for SO2 restricted by one photon excitation with wavelengths in the
derivatives was first proposed based on acedan-merocyanine ultraviolet-visible (UV/Vis) spectral region, which renders them
dyads via TP-FRET strategy. It was successfully applied to can only be employed in cellular level but difficult for
visualizing of the fluctuations of enzymatically generated SO2 complicated living systems, because well absorbance, easy
derivatives in mitochondria of HepG2 cells and rat liver scattering and strong autofluorescence in the short
tissues by two-photon fluorescence microscopy imaging.
wavelength region elicits high background noise and poor
tissue penetration.9
Sulfur dioxide (SO2) was traditionally considered as a kind
of environmental pollutant,1 since studies have implied that
exposure to SO2 was usually associated with the symptoms of
neurological disorders, cardiovascular diseases, and even lung
cancer.2 However, SO2 has recently emerged as an important
gasotransmitter due to it possesses some beneficial biological
functions including antioxidation and cardiovascular
regulation.3 Interestingly, it is reported that SO2 can be
endogenously produced form sulfur-containing amino acids,
To address above issues, two-photon microscopy (TPM),
utilizing two near-infrared photons with low energy as the
exciting light, has become a potent alternative technique for
biomedical applications,10 because it provides improved three-
dimensional spatial localization with minimized background
signal, deeper tissue penetration, and reduced photodamage
to biosamples.11 Despite all these advantages, only one
example of two-photon probe for fluorescent imaging
mitochondrial SO2 derivatives in tissues has been judiciously
designed by Chao et al until recently.12 However, its detecting
signal relied on single-emission intensity changes, which would
easily suffer the external interferences from instrumental
efficiency, probe distribution, and local surrounding.13 In sharp
contrast, ratiometric probes can provide self-calibration for
the aforementioned factors and thus enable more accurate
analysis through measuring ratio variations of two emission
intensities at different wavelengths.14 It is worth pointing out
that, as far as we know, no ratiometric two-photon fluorescent
probe for visualizing mitochondrial SO2 derivatives in tissues
has been reported hitherto.
-
2-
equilibrating with sulfites/bisulfites (HSO3 /SO3
)
in
biosystems.4 Therefore, it is meaningful and valuable to
detailedly elucidate the generation and physiological roles of
SO2 and its derivatives using an effective molecular tool.
Fluorescent probe in combination with imaging instruments
offers a powerful approach for in situ and real-time visualizing
biomolecules of interest in living organisms, owing to its non-
invasiveness and high spatiotemporal resolution.5 Because
mitochondria is the main region of endogenous SO2 derivatives
formation,6
a selective, fast response and targetable
fluorescent probe for exploring SO2 biology in mitochondria is
especially appreciated. Although considerable advances in the
development of fluorescent probes for SO2 derivatives have
been made in recent years,7 so far, only a few ones are
mitochondria-targeted.8 Nevertheless, all of them are
By virtue of the dual superiorities of the TPM technique
and the ratiometric mode, herein, we firstly proposed a
mitochondria-targetable
and
ratiometric
two-photon
fluorescent probe, TP-Mito/Ratio-SO2, for SO2 derivatives
imaging via two-photon excited fluorescence resonance
energy transfer (TP-FRET) strategy. As briefly depicted in
Scheme 1, TP-Mito/Ratio-SO2 was rationally constructed based
on an acedan-merocyanine scaffold as energy transfer cassette.
Under two-photon excitation condition, the energy transfer
dyad would fluoresce at the characteristic emission of
merocyanine due to appropriate spectrum matching between
absorption spectra of acceptor with two-photon emission
aSchool of Chemistry and Biological Engineering, Changsha University of Science
and Technology, Changsha, 410114, China.
E-mail: ysh1127@hnu.edu.cn, yangrh@pku.edu.cn. Tel/Fax: +86 731-88822523.
bState Key Laboratory of Chemo/Biosensing and Chemometrics, College of
Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
§These authors contributed equally.
†Electronic Supplementary Information (ESI) available: Experimental details,
synthesis and characterization of compound, and other data noted in the text.
See DOI: 10.1039/x0xx00000x
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spectra of donor (Fig. S1). However, the conjugation structure solution. The time-dependent processes foVirew AdrtiicflfeeOrnelinnet
of TP-Mito/Ratio-SO2 would be disturbed by nucleophilic concentrations of NaHSO3 followed firDstO-oI:r1d0e.1r03k9i/nCe6tCiCcs05w25i4thA
addition of HSO3 /SO32- toward merocyanine, impeding the TP- diverse observed rate constant k' (k'max = 1.2×10-2 s-1, Fig. S7).
-
FRET process and thus resulting in fluorescence recovery of Moreover, a linear relation between the k' with NaHSO3
acedan dye. The design rationality was supported by density passing through the origin can be found, which indicates that
functional theory (DFT) calculations (Fig. S2).
the reaction was second-order overall for TP-Mito/Ratio-SO2
and NaHSO3 with k =297 M-1s-1 (Inset of Fig. S7).16
B
A
[
NaHSO3
0
]
.60
.45
NaHSO3
3.0e+5
2.2e+5
1.5e+5
7.5e+4
NaHSO3
.30
Scheme 1 Response Mechanism of TP-Mito/Ratio-SO2 toward SO2 Derivatives
TP-Mito/Ratio-SO2 was efficiently synthesized and
characterized as described in the Electronic Supplementary
Information. With the probe in hand, we start to investigate its
200 µM
.15
0.00
350 420 490 560 630
Wavelength/nm
450 500 550 600 650
Wavelength/nm
-
photophysical properties and response abilities toward HSO3
\
/SO32-. As shown in Fig. 1A, the absorption spectra of free Fig. 1 (A) UV-vis absorption spectra of TP-Mito/Ratio-SO2 (10.0 μM) in the
probe in buffer solution exhibited strong electronic transitions presence of different amounts of NaHSO3 in PB solution (pH = 7.4, containing 20%
at 380 nm and 510 nm (ε380 = 2.1×104 M-1cm-1, ε510 = 5.3×104 EtOH), Inset: color change of TP-Mito/Ratio-SO2 after treatment with NaHSO3.; (B)
M-1cm-1), which are assigned to the characteristic absorption Fluorescence response of TP-Mito/Ratio-SO2 (5.0 μM) to NaHSO3 (0-40.0 μM) in
of acedan and merocyanine derivative respectively, indicating PB solution (pH = 7.4, containing 20% EtOH). λex =380 nm. Inset: fluorescence
that there are little or no electronic interactions between the change of TP-Mito/Ratio-SO2 after treatment with NaHSO3.
energy transfer dyad in the ground state. Upon addition of
To evaluate the selectivity performance of this new probe,
increasing concentrations of NaHSO3, the probe’s absorption fluorometric titrations for TP-Mito/Ratio-SO2 were extended to
peak gradually attenuated concomitant with a color change various bio-relevant species including common anions (F−, Cl−,
−
from red to colourless (inset of Fig. 1A), revealing that it could Br−, I−, AcO−, HCO3 , SCN−, CN−, SO42−), representative reactive
response to SO2 derivates. Mass data (new peak appeared at oxygen and nitrogen species (H2O2, NO), and other reactive
m/z 715) and disappearance of the resonance signal sulfur species (S2O32−, HS-, Cys, Hcy, and GSH). Fig. S8 depicted
corresponding to the alkene proton in partial 1H NMR also that no significant variation could be found in the presence of
-
2-
provide the evidence of addition reaction between TP- aforementioned competitive substrates other than HSO3 /SO3
Mito/Ratio-SO2 and NaHSO3 (Fig. S3,S4). indicating that TP-Mito/Ratio-SO2 could selectively response
To evaluate the sensing performances of TP-Mito/Ratio- toward SO2 derivates. Furthermore, competition experiments
,
SO2, fluorometric titration of the probe toward different depicted that this probe could specifically react with SO2
concentrations of NaHSO3 in buffer solution were first carried derivates in the presence of other potentially competitive
out. As shown in Fig. 1B, under one-photon (λex = 380 nm) species, which is helpful in validation of the designed probe to
excitation, the free probe exhibits slight characteristic meet the demands of selectivity for SO2 derivates assay in
emission peak of acedan but strong fluorescence emission of biological applications.
merocyanine at 590 nm. With the increasing concentration of
Effect of pH on the fluorescence properties of TP-
NaHSO3, there is significant decrease in characteristic emission Mito/Ratio-SO2 and its response toward SO2 derivates were
of merocyanine and a blue-shifted center around 500 nm subsequently investigated (Fig. S9). The free probe was pH
appears with around 90 nm emission shift and a clear iso- insensitive in the broad pH range (4.0-10.0), demonstrating
emission point at 560nm. Observably, a signal to background that the probe was steady at physiological pH conditions. Upon
-
ratio, S/B (S/B = [F500/F590]n/[F500/F590]0),15 was increased from treatment with HSO3 , the maximal emission intensity ratio
1.0 to 6.5 when treated with 40.0 μM NaHSO3 (Fig. S5). was observed in the pH range of 8.0–11.0, suggesting that the
Moreover, there was a well linear relationship between the two-photon ratiometric probe functions properly at
S/B and the concentrations of NaHSO3 in the range from 0 to physiological pH. There was a drastic downward trend in
10.0 μM with a detection limit (3σ/slope) of 50 nM, response signal at pH values changing from 8.0 to 6.0, which
-
demonstrating that TP-Mito/Ratio-SO2 is potentially suitable was in accordance with the pKa2 of HSO3 (pKa2 = 7.20),17
-
-
for quantitative determination of HSO3 /SO32- concentrations.
indicating that protonation of HSO3 lessen its nucleophility to
Real time fluorescence records and kinetic studies of TP- the acceptor unit of TP-Mito/Ratio-SO2 under acidic condition.
Mito/Ratio-SO2 in response to different concentrations of The two-photon excitation action cross-section (Φδ) is one
NaHSO3 were then performed. As shown in Fig. S6, the of pivotal parameter for a admirable two photon bioimaging
emission intensity ratio, F500/F590, gradually increased with probe. Particularly noteworthy is the fact that the maximum
time and reached a maximum plateau in about 3 min after Φδ values of both TP-Mito/Ratio-SO2 and its reaction product
treatment with 40.0 μM NaHSO3 solution, such a rapid with NaHSO3 were near 100 GM at 760 nm (Fig. S10),
response is appealing for real-time detection. However, the demonstrating that the maximum two-photon excitation
free probe showed no detectable change with time, wavelength of the energy donor portions remains stationary,
demonstrating that the probe was stable under aqueous which is significantly superior for TP ratiometric imaging than
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ICT based ones.18 Meanwhile, under the optimum two-photon show tendency to synchrony (Fig. 2d). Moreover,VtiehweAirtnictleeOnnsliitnye
DOI: 10.1039/C6CC05254A
irradiation (760 nm), two-photon fluorescence emission scatter plot is high correlationship with Pearson’s
spectra of the probe and its reaction product with NaHSO3 are colocalization coefficient 0.95 (Fig. 2e). The above
similar as their one-photon based ones (Fig. S11), impressively, investigations confirmed that TP-Mito/Ratio-SO2 could
emission intensity at 590 nm of TP-Mito/Ratio-SO2 is about 5- specifically accumulate in mitochondria.
fold brighter than that of free acceptor, confirming that TP-
FRET process from acedan to merocyanine occurs.
Mitochondria localization feature of TP-Mito/Ratio-SO2
actuate us to test its imaging performances toward
The excellent two-photon properties and response endogenous SO2 derivates. As we known, SO2 derivates can be
performances in vitro of probe encourage us to explore endogenously generated from reaction of Na2S2O3 and GSH
whether TP-Mito/Ratio-SO2 could function in mitochondria of catalyzed by thiosulphate sulphurtransferase (TST) in
living systems. Before bioimaging experiments, the mammals. Previous studies also demonstrated that liver injury
cytotoxicities of TP-Mito/Ratio-SO2 on HepG2 cells were arised from oxidative stress can lead to depletion of abundant
primarily evaluated by standard MTT assay (Fig. S12). After GSH.19 Therefore, to testify our assumption, the human
cells were treated with TP-Mito/Ratio-SO2 (0−30 μM) for 24 h, hepatoma cell line (HepG2) were treated with exogenous GSH
a high cell survive rate (﹥90%) can be found. The low and Na2S2O3 as TST substrates in the absence and presence of
cytotoxic effect of the candidate probe is well suitable for TST inhibitor SNAP for cellular imaging experiments20. One can
bioimaging application. Subsequently, TPM imaging of living find in Fig. 3 that, in comparison with the free probe loaded
cells were carried out on confocal laser scanning microscopy. cells, similar imaging patterns could be observed from probe-
As descried in Fig. S13, HepG2 cells incubated with 5.0 μM TP- loaded cells in the presence of Na2S2O3 and GSH individually.
Mito/Ratio-SO2 show fluorescence in red channels, but there is By contrast, there was obviously increasing fluorescence signal
negligible fluorescence signal through green channels. On the in green channels concomitant with decrease of fluorescence
contrary, the probe-loaded cells appear clear cellular profiles signal in red channels for the probe-treated cells with the
with brighter green fluorescence when treated with the presence of both Na2S2O3 and GSH, resulting in prominent
increase of NaHSO3 for 10 min, the original fluorescence signal fluorescence ratio signal change (Fig.3D). Moreover, even
in the red channel gradually weakened accordingly. These though simultaneous treatment with Na2S2O3 and GSH, there
imaging results indicate that TP-Mito/Ratio-SO2 shows good was still no significant fluorescence change when probe-loaded
cell-permeability and can be applied to dual-colour monitor HepG2 cells were pre-incubated with TST inhibitor SNAP (Fig.
the variations in concentrations of SO2 derivatives in living cells. 3E), confirming that the fluorescence changes were indeed
initiated from endogenously generated HSO3 /SO32-. These
results strongly suggest that that TP-Mito/Ratio-SO2 was
suitable for visualizing changes of endogenous SO2 derivates
inside HepG2 cells.
-
b)
c)
a)
D
E
B
C
A
2-
2-
2-
+ GSH/S2O3
SNAP
Ch 1
Ch 2
Control
+ GSH
+ S2O3
+ GSH/S2O3
e)
d)
f)
0
3
6
9
12
Length (µm)
Fig.2 Fluorescence images of TP-Mito/Ratio-SO2 colocalizes to the mitochondria
in HepG2 cells. Cells were stained with (a) TP-Mito/Ratio-SO2 (5.0 μM, Channel 1
(red), λex = 405 nm; λem = 580-630 nm) and (b) MitoTracker Green FM (1.0 μM,
Channel 2 (green), λex = 488 nm; λem = 500-530 nm) simultaneously. (c) The
merged pattern of a and b. (d) intensity profile of ROI cross the HepG2 cell
costained with TP-Mito/Ratio-SO2 and MitoTracker Green FM. (e) Intensity
scatter plot of two channels. (f) bright field pattern of cells. Scale bar: 10 μm.
similar as well-known mitochondria-targetable cationic dyes
including cyanine and rhodamine, TP-Mito/Ratio-SO2 also
possess an overall positive charge, speculating that the Fig.3 Two-photon ratiometirc fluorescence images of endogenous SO2 derivates
designed probe would accumulated in the mitochondria. In in mitochondria of HepG2 cells using 5.0 μM TP-Mito/Ratio-SO2 under different
order to determine it’s subcellular localization, colocalization conditions: (A) the probe only. (B) in the presence of 500 μM GSH. (C) in the
experiments were conducted by costaining HepG2 cells with a presence of 250 μM Na2S2O3. (D) in the presence of TST substrates 250 μM/500
commercially mitochondrial tracker MitoTracker Green FM μM Na2S2O3/GSH. (E) 100 nM SNAP solution was pre-incubated with HepG2 cells,
and TP-Mito/Ratio-SO2 (Fig. 2). These cells appear red and and then treated with the probe in the presence of 250 μM Na2S2O3 and 500 μM
green fluorescences in Channel 1 (Fig. 2a) and Channel 2 (Fig. GSH. Scale bar: 20 μm.
2b), respectively. The merged image (Fig. 2c) reveals that the
To further display the advantage of TPM, immediately after
staining of TP-Mito/Ratio-SO2 overlays well with that of cellular imaging experiments, we proceeded to investigate the
MitoTracker Green FM. The variations in intensity profile of utility of the probe for monitoring endogenous SO2 in deep-
linear regions of interest (ROI) across cell in the two channels tissue imaging. Because high levels of GSH are conserved in
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normal hepatocytes,20 two-photon ratiometric imaging of TP- systems with respect to the enzymatic reaction of Na2S2O3 and
View Article Online
DOI: 10.1039/C6CC05254A
Mito/Ratio-SO2 in fresh rat liver tissue slices were carried out GSH, demonstrating that the newly proposed probe will be an
under different conditions from cellular ones. As is shown in effective molecule tool to study metabolism of sulfur-
Fig. 4A, the tissue treated with probe showed weak containing species in biomedical research.
fluorescence signal in green channels but strong fluorescence
The authors would like to acknowledge the financial support
signal in red channels. Moreover, Z-scan mode revealed that from NSFC (21505006, 21135001, 21575018, 21521063) and
the fluorescence signals were collected at different tissue the Open Funds of State Key Laboratory of Chemo/Biosensing
depths (0−200 μm), as depicted in Fig. S14, suggesting that TP- and Chemometrics of Hunan University (2015011).
Mito/Ratio-SO2 could be successfully applied for deep-tissue
imaging. Difference from the free probe-loaded tissue, the
fluorescence intensity in green channel slightly enhanced
Notes and references
along with faint signal decrease in the red channel for probe-
loaded cells in the presence of Na2S2O3, thus a certain change
of ratiometric pattern could be observed (Fig. 4B). In sharp
contrast, the fluorescence ratio of the control ones increased
dramatically upon treatment with α-lipoic acid (GSH stimulator)
and decreased significantly when dealing with N-
ethylmaleimide (NEM, a well-known biothiol scavenger) (Fig.
4C,D), meaning that enzymatically generated SO2 derivates in
tissues really originate from the interaction of Na2S2O3 and
endogenous GSH. What is more, when the tissue was pre-
treated with SNAP to invalidate catalytic activity of TST,
ratiometrical fluorescence changes would disappear (Fig.4E).
These imaging findings demonstrate that TP-Mito/Ratio-SO2
can effectively enable the determination of endogenous SO2
fluctuation at depths in rat live tissues using TPM.
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Fig.4 Two-photon fluorescence images of endogenous SO2 derivates in
mitochondria of fresh rat liver tissues at depth of 120 μm using 50 μM TP-
Mito/Ratio-SO2 under different conditions: (A) the probe only. (B) in the presence
of 2.5 mM Na2S2O3. (C) in the presence of 2.5 mM Na2S2O3 and 5.0 mM α-lipoic
acid. (d) in the presence of 2.5 mM Na2S2O3 and 10.0 mM NEM. (e) For the
control experiments, 2 μM SNAP solution was pre-incubated with tissue, and
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lipoic acid. Scale bar: 50 μm.
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In summary, we have constructed a mitochondria-targetable
and ratiometric two-photon fluorescence imaging probe for
SO2 derivates based on acedan-merocyanine dyads for the first
time. This probe, designed with the aim of modulating two-
photon fluorophore with energy acceptor via TP-FRET process,
exhibits a significant red-to-green fluorescence color change in
response to SO2 derivates with excellent features including
high TP cross section, fast response, good selectivity, and
robust staining ability of mitochondria, thereby allowing
ratiometrical visualization of the variation of SO2 derivates in
mitochondria of living cells and deep tissues. More importantly,
the ratiometric TPM imaging studies clearly reveal the
endogenous SO2 derivates within mitochondria of living
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