A mitochondria-targeted ratiometric two-photon fluorescent probe for detecting intracellular cysteine and homocysteine

Author’s Accepted Manuscript A Mitochondria-targeted Ratiometric Two-photon Fluorescent Probe for Detecting Intracellular Cysteine and Homocysteine Ping Yue, Xiuli Yang, Peng Ning, Xinguo Xi, Haizhu Yu, Yan Feng, Rong Shao, Xiangming Meng www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(17)30918-9 http://dx.doi.org/10.1016/j.talanta.2017.08.085 TAL17881

To appear in: Talanta Received date: 24 May 2017 Revised date: 19 August 2017 Accepted date: 27 August 2017 Cite this article as: Ping Yue, Xiuli Yang, Peng Ning, Xinguo Xi, Haizhu Yu, Yan Feng, Rong Shao and Xiangming Meng, A Mitochondria-targeted Ratiometric Two-photon Fluorescent Probe for Detecting Intracellular Cysteine and Homocysteine, Talanta, http://dx.doi.org/10.1016/j.talanta.2017.08.085 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A Mitochondria-targeted Ratiometric Two-photon Fluorescent Probe for Detecting Intracellular Cysteine and Homocysteine Ping Yue,a Xiuli Yang,b Peng Ning,a Xinguo Xi,b Haizhu Yu,a Yan Feng,a Rong Shao,b and Xiangming Meng*a a

School of Chemistry and Chemical Engineering & Center for Atomic Engineering of Advanced Materials &

AnHui Province Key Laboratory of Chemistry for Inorganic/Organic Hybrid Functionalized Materials, Anhui University, Hefei 230601, P.R. China. b

Innovation

Protection Equipments ,

Yancheng

Jiangsu Collaborative

Center

for

Institute of

Ecological

Building

Materials

and

Environmental

Technology, Yancheng 224051, China

*Corresponding author. Fax: +86-551-63861467; Tel: +86-551-63861467 E-mail address: [email protected](Xiangming Meng).

Abstract: A novel mitochondria-targeted ratiometric two-photon fluorescent probe (Mito-MQ) for detecting intracellular cysteine (Cys) and homocysteine (Hcy) has been designed. Mito-MQ showed the ratiometric

fluorescent detection signal (the green-to-blue emissionfrom 517 nm to 460 nm) to cysteine (Cys) and

homocysteine (Hcy) over glutathione (GSH), along with the fast response rate (10 min). The detection mechanism was illustrated by 1H-NMR, ESI-MS and theoretical calculation. The co-localization coefficient of 0.87 between

Mito-MQ and MitoTracker Red revealed that the probe was predominantly present in mitochondria, therefore,

Mito-MQ was successfully applied to detect mitochondrial oxidative stress by detecting the change of Cys/Hcy. Moreover, imaging in fresh tissue slices indicated that Mito-MQ could work in deep tissue (ca. 130 μm) under

two-photon excitation. Furthermore, the measurement of Cys/Hcy detection in zebrafish showed that probe can be

used in determination of biothiols in vivo.

Keywords: mitochondria, ratiometric, two-photon fluorescent probe, deep tissue imaging, oxidative stress, in vivo.

1. Introduction Mitochondrion is an essential organelle within eukaryotic cells, utilizing oxygen to digest

carbohydrates and fats, and releasing reactive oxygen species (ROS) as well as biochemical energy.

Biothiols play important roles in regulating ROS homeostasis, which mainly occurs on mitochondria

respiratory chain[1][2][3][4]. Cysteine (Cys) and homocysteine (Hcy) in mitochondria are sensitive to the

mitochondrial oxidative stress and further result in many diseases such as liver damage, weakness and Alzheimer’s disease[5][6][7][8][9]. Therefore, real-time monitoring of Cys and Hcy level in mitochondria

is of great significance to uncover role of these amino acids in ROS homeostasis. Fluorescent probes have

been evaluated as the most powerful tools to monitor cytosolic Cys/Hcy in biological system due to their

excellent sensitivity and high selectivity. Unfortunately, most of reported Cys/Hcy fluorescent probes

failed to target the mitochondria in living cells[10][11].

To date, most of reported fluorescent probes are designed based on one-photon technology (OPM) with short

excitation wavelength that limits their applications in deep-tissue and in vivo, owing to their faultiness, including

photon-bleaching, photo-damage, cellular auto fluorescence as well as to the shallow penetration depth (< 80μm)[12]. Recently, two-photon microscopy (TPM) has become the leading imaging technology in biology

research for its obvious advantages over OPM, such as localized excitation, increased penetration depth, and the

reduced photo-bleaching and photo-damage. Considering the inherent complexity and constant evolution of an

organism, ratiometric measurement is superior to a single emission intensity measurement because it can eliminate

most possible effects of environmental variations, probe distribution, and instrumental performance and thus offer

a more accurate analysis[13][14][15][16][17].

Herein, we reported a novel ratiometric two-photon fluorescent probe (Mito-MQ) for detecting mitochondrial

Cys/Hcy based on 6-substituted quinoline platform, an efficient two-photon fluorophore we have developed before

(Scheme 1)[18][19]. The aldehyde group was linked directly to the quinoline skeleton as the specific reaction

point for Cys/Hcy. Triphenylphosphonium (TPP) group, which was widely used as the mitochondria targeting

group, was connected to the fluorescent group to drive the probe to locate in mitochondria[20]. Mito-MQ showed

excellent two-photon fluorescence as a result of intramolecular charge transfer (ICT)[21][22]. We believe that the

aldehyde group in Mito-MQ can form thiazolidine/thiazinane with Cys/Hcy through cyclization reaction. The ICT

process in the system will then be switched off and detection signals (i.e. the shift of the fluorescent spectrum

along with the color change) will be obtained (Scheme 1). We deem that the novel probe will specially locate in

mitochondria and exhibit good ratiometric two-photon detection signal to mitochondrial Cys/Hcy.

Scheme 1 Proposed response mechanism of Mito-MQ to Cys/Hcy.

2. Experimental Section 2.1. General procedures

All reagents and solvents were commercially purchased.

Bruker-400 MHz spectrometers and

13

1

H-NMR spectra were recorded on

C-NMR spectra were recorded on 100 MHz spectrometers.

Fluorescence spectra were obtained using a HITACHIF-2500 spectrometer. UV-vis absorption spectra

were recorded on a Tech-comp UV 1000 spectrophotometer. MS spectra were conducted by ESI mass

spectrometer. The two-photon cross section was tested in DMSO with 1 mM Mito-MQ. The test solution

of Mito-MQ (25 μM) in PBS solution (pH=7.4, DMSO/PBS, 9:1, v/v) was prepared. The solutions of

various testing species were prepared for Hcy, Cys, Phe, Gly, Leu, Ala, Ser, Arg, Glu, GSH, GSSG, Met,

Gln, Lys, DTT and 2-ME. The resulting solution was shaken well and incubated for 20 min at room

temperature before recording the spectra. 2.2. Measurement of two-photon absorption cross-section (δ)

Two-photon excitation fluorescence (TPEF) spectra were measured using femtosecond laser pulse

and Ti: sapphire system (680–1080 nm, 80 MHz, 140 fs, Chameleon II) as the light source. All

measurements were carried out in air at room temperature. Two-photon absorption cross-sections were

measured using two-photon-induced fluorescence measurement technique. The two-photon absorption cross-sections (δ) were determined in DMSO with 1 mM Mito-MQ by using optically matching solutions

of fluorescein as a standard according to the literature[23].

2.3. Cytotoxicity assay

MTT (5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide) assay was performed as previously

reported to test the cytotoxic effect of the probe in cells. MCF-7 cells were passed and plated to ca. 70% confluence in 96-well Plates 24 h before treatment. Prior to Mito-MQ treatment, DMEM (Dulbecco’s

Modified Eagle Medium) with 10% FCS (Fetal Calf Serum) was removed and replaced with fresh

DMEM, and aliquots of Mito-MQ stock solutions (1 mM DMSO) were added to obtain final concentrations of 10, 20 and 30mM respectively. The treated cells were incubated for 24 h at 37 ◦C under

5%CO2. Subsequently, cells were treated with 5 mg/mL MTT (40 mL/well) and incubated for an additional 4 h (37 ◦C, 5% CO2). Then the cells were dissolved in DMSO (150 mL/well), and the absorbance at 570 nm was recorded. The cell viability (%) was calculated according to the following

equation:

Cell viability % = OD570 (sample)/OD570 (control) × 100 % where OD570(sample) represents the optical density of the wells treated with various concentration of Mito-MQ and OD570(control) represents that of the wells treated with DMEM containing 10% FCS. The percent of cell survival values is relative to untreated control cells.

2.4. Cell culture and two-photon fluorescence microscopy imaging

For two-photon imaging, MCF-7 cells were cultured in DMEM supplemented with 10% FCS, penicillin (100 μg/mL), and streptomycin (100 μg/mL) at 37◦C in a humidified atmosphere with 5% CO 2 and 95% air. Cytotoxicity assays show that Mito-MQ is safe enough for two-photon bio-imaging at low

concentrations. The cells were first washed with PBS, incubated with probe, N-ethylmaleimide (NEM),

thiol and H2O2 respectively in the incubator at 37 °C and then rinsed for three times with PBS. Cells imaging was carried out on a confocal microscope (Zeiss LSM 510 Meta NLO). Two-photon fluorescence

microscopy images of labeled cells were obtained by exciting the probe with a mode-locked

titanium–sapphire laser source at 740 nm.

2.5. Two-photon fluorescence microscopy imaging in living tissues

For two-photon bio-imaging, slices were prepared from the liver of 7-day-old mouse. Slices were cut to 180 μm thickness by using a vibrating-blade microtome in 10mM PBS buffer (pH=7.4). Slices were incubated with 25 μM Mito-MQ in PBS buffer bubbled with 95% air and 5% CO 2 for 1 h at 37℃ . Then, slices were washed three times with PBS buffer, transferred to glass-bottomed dishes. Mouse liver slices

imaging was carried out on a confocal microscope (Zeiss LSM 710 Meta NLO). Two-photon fluorescence

microscopy images of fresh mouse liver slices were obtained by exciting the probe with a mode-locked

titanium–sapphire laser source at 740 nm.

2.6. Preparation of zebrafishes and two-photon fluorescence imaging

For two-photon bio-imaging in vivo, 5-day-old zebrafishes were prepared

with probe,

N-ethylmaleimide (NEM), thiol respectively in PBS buffer at 28 oC for 1 h. All the fishes were terminally

anaesthetized using MS222, and images were carried out on a confocal microscope (Zeiss LSM 710 Meta

NLO). Two-photon fluorescence microscopy images of zebrafishes were obtained by exciting the probe

with a mode-locked titanium–sapphire laser source at 740 nm.

2.7. Theoretical calculations

We carried out density functional theory(DFT)calculations with MB3LYP/6-311+G(d) level using the

Gaussian 09 program for the geometry optimizations and molecular orbitals calculations [24][25]. The remote

TPP group was simplified with methoxyl group as the end substituted group hardly influence the optical

spectra in DFT calculations.

3. Results and discussion 3.1. Synthesis of Mito-MQ

Mito-MQ can be easily synthesized in a five-step process with an overall yield of 60% from

crotonaldehyde (Fig.S1). The structures of Mito-MQ and the intermediates were all confirmed by 1

H-NMR, 13C-NMR and ESI mass spectra.

3.2. 1H-NMR titration and ESI-TOF MS spectra analysis The reaction of Mito-MQ with Cys was monitored by the partial 1H-NMR spectra. As shown in Fig.

S2, upon the addition of Cys to solution (in DMSO-d6) of Mito-MQ, the peak at 10.13 ppm (a) assigned to the aldehyde proton disappeared along with the appearance of two new peaks at 6.31 ppm, (b) and 6.23

ppm (c) assigned to the protons of the thiazolidine. These results suggested the formation of thiazolidine

after the addition of Cys to Mito-MQ solution[26][27]. As shown in Fig. S3, ESI MS spectra analysis also

confirmed the cyclization reaction between Cys and Mito-MQ. Mito-MQ has a peak at m/z = 590.2227.

Upon the addition of Cys, a peak at m/z = 693.2300 (assigned to the thiazolidine) appeared along with the

disappearance of the peak at m/z = 590.2227.

3.3. Amino acids selectivity and pH stability

All spectroscopic measurements were carried out under simulated physiological conditions in

buffered solution (pH=7.4, PBS/DMSO, 1:9, v/v). The optical responses of Mito-MQ to various amino

acids were investigated by detection of the emission spectroscopy. The solutions of various testing species

were prepared for Hcy, Cys, Phe, Gly, Leu, Ala, Ser, Arg, Glu, GSH, Met, Gln, Lys, DTT and 2-ME. As shown in Fig.S4, among the tested amino acids (100μM), only Hcy and Cys induced the blue-shift of the

fluorescence. Moreover, the color change of fluorescence of Mito-MQ upon the addition of Cys/Hcy

could be easily observed by naked eyes under hand-held UV-lamp (Fig.S11). The pH-dependent

experiment results indicated that the ratio fluorescent signal (I460nm/I517nm) of Mito-MQ to Cys was almost pH insensitive in the pH range of 5.6-10 (Fig.S5). The results suggested that Mito-MQ could served as a

Cys/Hcy-selective fluorescent probe in biological system.

3.4. UV–vis and fluorescence spectra responses

The fluorescence and absorption titration experiments of Mito-MQ with the addition of Cys were

carried out in DMSO/PBS buffer (9/1, v/v, pH 7.4). As shown in Fig.1a, the UV-vis absorption spectra of

Mito-MQ exhibited a maximum absorption at 360 nm. Upon the addition of Cys, the maximum

absorption was gradually blue-shifted to 320 nm. The ratio of absorbances (A320nm /A360nm) increased from 0.6 to 1.3 with the increase of Cys molar concentrations and reached its equilibrium in 10 minutes.

Meanwhile,the emission spectra of Mito-MQ showed a larger blue-shift from 517 nm to 460 nm with

addition of Cys. The ratio of fluorescent intensities (I460nm/I517nm) increased from 0.4 to 1.2 (Fig.1b). The blue shift of UV-vis and fluorescence spectra was caused by blocking of the ICT process in molecular

system after the formation of thiazolidine (Scheme 1). Similar behavior was also observed for Hcy

(Fig.S6, S7). For clearance, Cys was used in the following experiment. These results suggested that

Mito-MQ was suitable for detection of Cys/Hcy with ratiometric fluorescent signal.

Fig.1 (a) Absorption spectral changes of Mito-MQ (25 μM) after addition of Cys (100 μM) in pH 7.4 PBS/DMSO (1:9, v/v, measured each 1min). Inset: Ratiometric calibration curve (A320nm/A360nm) as a function of the time of additional Cys. (b) Emission spectra of Mito-MQ (25 μM) after addition of Cys (0-100 μM) λex = 360 nm. Inset: Ratiometric calibration curve (I460nm/I517nm) as a function of the concentration of Cys. Spectra were recorded for 20 min after Cys addition.

Fig.2(a) Two-photon absorption cross-sections of Mito-MQ and Mito-MQ+Cys. The maximum two-photon absorption cross-sections decreased from 112 GM to 59 GM excited by 740 nm. (b) The square relationship of two-photon excited fluorescence intensity (Iout) of Mito-MQ (1 mM) with the addition of Cys (2 mM) and input power (Iin = 200-800 mW), and excitation carried out at 740 nm.

3.5. Two-photon absorption properties study

The two-photon absorption cross-sections of Mito-MQ and Mito-MQ + Cys were determined using

the two-photon induced fluorescence spectra. As shown in Fig.2a, the maximum two-photon absorption cross-section (λmax) value of Mito-MQ is 112 GM at 740 nm. Upon addition of Cys, the λ max value at 740 nm was decreased to 59 GM. The result suggested that Mito-MQ can potentially be used for

TPM-assisted bio-imaging for Cys in living systems. The power-squared dependence of the two-photon

excited fluorescence intensity at 740 nm versus the incident laser power was carried out. As shown in Fig. 2b, the logarithmic plots of the fluorescence integral (Iout) versus input power (Iin = 200-800 mW) with a

slope of 2.14 for Mito-MQ (1 mM) with the addition of Cys (2 mM) at 740 nm, suggesting the

luminescence arose from a two-photon absorption process.

3.6. Theoretical calculations

To further understand the change of the photophysical properties, density functional theory (DFT)

calculations of the energy gaps between HOMO (the highest occupied molecular orbitals) and LUMO (the

lowest unoccupied molecular orbitals) of Mito-MQ and Mito-MQ + Cys were performed.

As shown in Fig.S8, the energy gapes between HOMO and LUMO of Mito-MQ and Mito-MQ + Cys were

calculated to be 3.183 eV and 3.699 eV, respectively, which agreed well with the blue-shift of the absorption

spectra. The unanimous result was also obtained for Hcy (Fig.S9). These and previous results[28][29] suggested

that directly decorating the fluorescent core with aldehyde group would be an efficient design strategy for

ratiometric fluorescent probes for Cys/Hcy.

3.7. Cytotoxicity assay

Cell cytotoxicity assays were conducted using MCF-7 cells to test the cytotoxicity of Mito-MQ. As shown in Fig. S10, the cell viability remained more than 80% after treated with 25 μM Mito-MQ for 24 h. The result

indicated that Mito-MQ is little cytotoxicity for long period incubation at low concentration and should be safe for

two-photon bio-imaging[30].

Fig.3 Two-photon and one-photon images of MCF-7 cells costained with Mito-MQ (25 μM) and Mitotracker red (1 μM) for 30 min at 37 °

C. The wavelengths for two-photon and one-photon excitation were 740 and 579 nm, respectively. From left to right: (a) λem = 475−550

nm (Mito-MQ) (b) λem =580-600 nm (Mitotracker red) (c) overlay (d) Intensity profile of ROIs across MCF-7 cells. Scale bar: 20 μm.

Fig.4 Two-photon confocal microscopy fluorescence images of Cys/Hcy in living MCF-7 cells. (A) MCF-7 cells incubated with Mito-MQ (25 μM) for 30 min. (B) MCF-7 cells pretreated with NEM (1.0 mM) for 30 min and then incubated with Mito-MQ (25 μM) for 30min. (C, D, E) MCF-7cells pretreated with NEM (1.0 mM) for 30 min and then incubated with Cys, Hcy, and GSH (100 μM) for 1 h, respectively, and finally incubated with Mito-MQ (5 μM) for 30 min. From left to right: Bright Field, Channel 1: λem = 440-480 nm, Channel 2: λem = 475-550 nm, Overlay. λex = 740 nm. Scale bar: 20 μm.

3.8. Two-photon excited fluorescence imaging in live cells and tissues To determine the sub-cellular localization property of this probe, a co-localization study of Mito-MQ was

conducted in MCF-7 cells with Mitotracker red (Fig.3), a well-known one-photon fluorescent commercial dye for

mitochondria targeting. As shown in Fig.3, the fluorescent images of Mito-MQ and Mitotracker red overlapped very well with each other. The Pearson’s colocalization coefficient (calculated using Autoquant X2 software) of

Mito-MQ with Mitotracker red was calculated as 0.87. The changes in the intensity profile of linear regions of

interest (ROIs) of Mito-MQ and Mitotracker red were almost synchronous. These results indicated that Mito-MQ

was specifically driven to mitochondria in living cells.

Fig.5 Two-photon confocal microscopic fluorescence images in live MCF-7 cells. (A) MCF-7 cells incubated with Mito-MQ (25 μM) for 30 min. (B) MCF-7 cells pretreated with H2O2 (200 μM) for 30 min and then incubated with Mito-MQ (25 μM) for 30 min. From left to right: Channel 1: λem = 440−480 nm, Channel 2: λem = 470−550 nm, Bright Field. λex = 740 nm. Scale bar: 20 μm.

Fig.6 Top photograph: two-photon confocal fluorescence images of a fresh rat liver slice treated with 25 μM Mito-MQ at depths of approximately 0 to 130 μm with a magnification of 40 ×, λex = 740 nm, emission wavelength from 475 nm to 550 nm. Bottom photograph: Two-photon confocal fluorescence images of a fresh rat liver slice treated with 100 μM Cys and 25 μM Mito-MQ at depths of approximately 0 to 130 μm, emission wavelength from 440 nm to 480 nm. Scale bars: 100 μm

Fig.7 Two-photon confocal microscopy fluorescence images of zebrafish. From left to right: zebrafish incubated with Mito-MQ (25 μM) for 30min. zebrafish pretreated with NEM (1.0 mM) for 30 min and then incubated with Mito-MQ (25 μM) for 30 min. zebrafish pretreated with NEM (1.0 mM) for 30 min and then incubated with Cys, Hcy, and GSH (100 μM) for 30min, respectively, and finally incubated with Mito-MQ(25 μM) for 30 min. (A) Channel 1: λem = 440−480 nm, (B) Channel 2: λem = 470−550 nm, (C) Bright Field, (D) Overlay. λex = 740 nm. Scale bar: 400 μm

To demonstrate the practical use as a ratiometric two-photon fluorescent probe in biological samples,

the two-photon images of the MCF-7 cells labeled with Mito-MQ were monitored by dual emission channels: (1) blue (λem = 440-480 nm, channel 1) and (2) green (λem = 475-550 nm, channel 2) channels (Fig. 4) upon excitation at 740 nm with femtosecond laser pulses. After the cells were incubated with Mito-MQ (25 μM) for 30 min, weak blue two-photon excited fluorescence (TPEF) in channel 1 as well as

bright green fluorescence in channel 2 were observed resulting from the presence of endogenous Cys/Hcy

in living cells (Fig. 4A). To assess the potential selective response of Mito-MQ toward Cys/Hcy over

other thiols in the live cell environment, a series of control experiments were carried out. When the

MCF-7 cells were pretreated with N-ethylmaleimide (NEM, 1.0 mM), a well-known thiol-blocking agent for the depletion of intracellular thiol species for 30 min before incubating with Mito-MQ (25 μM), a

strong TPEF in the green channel was observed, but there was no emission detected in the blue channel

(Fig. 4B), indicating that thiol species were completely reacted by NEM. Upon the addition of Cys, Hcy, or GSH (100 μM) to the NEM-pretreated MCF-7 cells for 1 h followed by an incubation with Mito-MQ (25 μM), marked variations of fluorescence responses in different channels were observed. With the

treatment of Cys/Hcy, the green TPEF in channel 1 was diminished accompanying with a sharp increase

in TPEF in the blue channel (Fig. 4C and Fig. 4D, channel 2), On the contrary, the treatment of GSH

could not induce any blue TPEF emission at all(Fig. 4E). Such findings were consistent with the fact that

Mito-MQ showed reactive and selective ratiometric fluorescence responses to Cys/Hcy in living cells.

These also further supported that Mito-MQ could be used as a ratiometric two photon fluorescent probe

for selective bioimaging of mitochondrial Cys/Hcy in living cells.

As intracellular thiols concentration is known to associate with oxidative stress level, an investigation of the

probe in response to the change of the Cys/Hcy concentration in living cells was also demonstrated by altering the redox balance. When MCF-7 cells were pretreated with H2O2 (200 μM) for 30 min before incubating with Mito-MQ (25 μM) probe, there was a obvious decrease in blue TPEF (Fig. 5, channel 2) accompanied by a strong

increase in green TPEF observed (Fig. 5, channel 1) as compared with those without H2O2 pretreatment. Such a change in fluorescence was responsive to the decrease of the intracellular Cys/Hcy concentration induced by H 2O2 oxidation of Cys/Hcy. Therefore, these findings suggested that Mito-MQ probe could be used as a tool to detect

mitochondrial oxidative stress by detecting the change of Cys/Hcy.

We further investigated the capability of the probe Mito-MQ of two-photon fluorescence imaging in

the thick living tissues (Fig.6), utilized three-dimensional fluorescence imaging of two-photon fluorescence microscopy. Fresh tissues slices (a thickness of 180 μm) of the rat liver were prepared for

two-photon fluorescence microscopic studies. One of the living tissue slices was incubated with Mito-MQ (25 μM) in 10 mM PBS buffer for 30 min. By comparison, the other one was prepared with 100 μM Cys for 30 min, then treated with Mito-MQ (25 μM) in 10 mM PBS buffer for another 30 min at 37 oC. As

shown in Fig.6, three-dimensional fluorescence imaging showed that the penetrative depth was up to 130 μm with a green-to-blue change. This result indicated that Mito-MQ was capable of imaging Hcy/Cys at

depths in living tissues using two-photon fluorescence microscopy, which was consistent with its good

two-photon fluorescence properties.

3.9. Two-photon fluorescence imaging in vivo

In addition to the cell-based studies, we investigated the capability of this probe to monitor Cys/Hcy in vivo.

As is shown in Fig.7, the phenomena indicated that Mito-MQ is highly promising for monitoring of Cys/Hcy in

vivo and could serve as a useful research tool for thiols-related studies.

4. Conclusions In summary, we have successfully developed a ratiometric two-photon fluorescent probe Mito-MQ

for Cys/Hcy detection. Mito-MQ showed not only a selectively marked green-to-blue emission to

Cys/Hcy with short response time (10 min) but also excellent mitochondrial-targeting ability (0.87) in

living cells. Mito-MQ has been successfully applied to monitor mitochondrial oxidative stress level

change in living cells under two-photon excitation. Furthermore, Mito-MQ could be utilized to image Cys/Hcy in vivo and living tissues with deep tissue penetration (ca. 130 μm) by two-photon fluorescence

microscopy. We deem that this ratiometric two-photon fluorescent probe would be practically useful and

greatly beneficial to investigate and monitor Cys/Hcy in living biological systems.

Acknowledgements This work was supported by National Natural Science Foundation of China (21372005). The research

fund of Jiangsu Collaborative Innovation Center for Ecological Building Materials and Environmental

Protection Equipments, and Finance support from Anhui University.

Appendix A. Supplementary material

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Grapical abstract

We designed a novel mitochondria-targeted ratiometric two-photon fluorescent probe (Mito-MQ) for detecting intracellular thiols and applied it to monitor mitochondrial oxidative stress.

Hightlights 1、 Mito-MQ showed obvious ratiometric fluorescent detection signal in to cysteine (Cys) and homocysteine (Hcy) over GSH. 2、 Mito-MQ was successfully applied to detect mitochondrial oxidative stress by detecting the change of Cys/Hcy. 3、 Imaging in living tissue slices indicated that Mito-MQ could work in deep tissue (ca. 130μm) under two-photon excitation. 4、 Mito-MQ can be used in determination of biothiols in vivo.