Highly Selective Two-Photon Fluorescent Probe for Ratiometric

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Highly Selective Two-Photon Fluorescent Probe for Ratiometric Sensing and Imaging Cysteine in Mitochondria Weifen Niu, Lei Guo, Yinhui Li, Shaomin Shuang, Chuan Dong, and Man Shing Wong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04329 • Publication Date (Web): 31 Dec 2015 Downloaded from http://pubs.acs.org on December 31, 2015

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Analytical Chemistry

Highly Selective Two-Photon Fluorescent Probe for Ratiometric Sensing and Imaging Cysteine in Mitochondria Weifen Niu,† Lei Guo,‡ Yinhui Li,*,‡,§ Shaomin Shuang,† Chuan Dong*,† and Man Shing Wong*,†,‡ †

Institute of Environmental Science, College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, People’s Republic of China ‡ Department of Chemistry and Institute of Molecular Functional Materials, Hong Kong Baptist University, Hong Kong SAR, People’s Republic of China § State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, People’s Republic of China

Corresponding Authors *[email protected]; fax +852-34117348 *[email protected]; fax +86-351-7018613 *[email protected]; fax +86-731-88822523

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ABSTRACT: A novel ratiometric mitochondrial cysteine (Cys)-selective two-photon fluorescence probe has been developed based on a merocyanine as the fluorophore and an acrylate moiety as the biothiol reaction site. The biocompatible and photostable acrylatefunctionalized merocyanine probe shows not only mitochondria targeting property but also highly selective detection and monitoring of Cys over other biothiols such as homocysteine (Hcy) and glutathione (GSH) and hydrogen sulphide (H2S) in live cells. In addition, this probe exhibits ratiometric fluorescence emission characteristics (F518/F452) which is linearly proportional to Cys concentrations in the range of 0.5 - 40 µM. More importantly, the probe and its released fluorophore, merocyanine exhibit strong two-photon excited fluorescence (TPEF) with twophoton action cross-section (Φσmax) of 65.2 GM at 740 nm and 72.6 GM at 760 nm in aqueous medium, respectively which is highly desirable for high contrast and brightness ratiometric twophoton fluorescence imaging of the living samples. The probe has been successfully applied to ratiometrically image and detect mitochondrial Cys in live cells and intact tissues down to a depth of 150 µm by two-photon fluorescence microscopy. Thus, this ratiometric two-photon fluorescent probe is practically useful for an investigation of Cys in living biological systems.


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INTRODUCTION Intracellular reactive sulfur species (RSS) is a family of sulfur-containing biomolecules which include thiols, hydrogen sulfide, persulfides, polysulfides and S-modified cysteine adducts such as S-nitrosothiols and sulfenic acids. RSS play crucial roles in many physiological and pathological processes.1-3 Clinical studies have shown that even though the structures of these RSS are similar, they are related to different diseases. Among those, cysteine (Cys), as an important amino acid and biothiol, plays essential roles in many significant cellular functions such as protein synthesis, detoxification and metabolism.4,5 A deficiency in Cys is also implicated in many syndromes including slow growth in children, lethargy, edema, hair depigmentation, loss of muscle and fat, liver damage, skin lesions, and weakness.6-8 On the other hand, elevated levels of Cys have been associated with neurotoxicity.9 Mitochondria are a vital organelle that exists in most eukaryotic cells, which not only produce the energy of a cell but also involve in many biological processes including signalling, cellular differentiation, growth and death.10,11 In order to have a better understanding the roles of Cys in biological system, primarily in mitochondria, it is of fundamentally important to develop sensitive mitochondrial Cys-selective detection methods to monitor Cys at the cell, tissue, and organism levels. For this purpose, various fluorescent probes for Cys detection have been developed in the past decade.12-21 The Cys sensing design strategies are generally based on the strong nucleophilicity of the thiol group combined with various subsequent reaction such as cyclization, displacement of coordination, and cleavage reaction.22-24 However, due to structural similarity and comparable reactivity of RSS especially the biothiols, only few reported fluorescent probes can discriminate Cys from homocysteine (Hcy) and glutathione (GSH) so far. In fact, the discrimination among them in biological systems has been a focus and challenge for the scientific communities. Park and co-workers developed a highly selective ratiometric NIR polymethine-based cyanine probe for Cys sensing based on conjugation and removal of the specific trigger, acrylate moiety.25 Nevertheless, the small Stokes shift in this system could result in the excitation interference. Guo and coworkers reported that a chlorinated coumarin-hemicyanine fluorescent probe was exploited with three potential reaction sites which could not only simultaneously detect Cys and GSH from different emission channels but also discriminate Cys from Hcy/GSH and GSH from Cys/Hcy.26 Feng and coworkers have recently reported a naphthafluorescein-based NIR fluorescent probe for colorimetric and NIR fluorescent turn-on detection of Cys.27 Nevertheless, all these Cys-selective probes do not show mitochondria targeting property and have only been evaluated using one-photon microscopy (OPM) with relatively short excitation wavelengths, limiting their use in deep-tissue imaging because of the shallow penetration depth (500 µm) with little self-absorption and minimum interference from autofluorescence. Secondly, two-photon excitation is intrinsically localized and thus it can provide a higher spatial resolution. Thirdly, because of less energy involved in the twophoton excitation, it can minimize the photobleaching of the probes and photodamage of the biological samples.28-35 To make TPM a more versatile tool in biology and medicine, a wider variety of two-photon probes are still needed for various applications. However, progress in this field is hindered by the lack of efficient two-photon sensing probes. Considering the inherent complexity and constant evolvement of organism, ratiometric measurement is superior to a single emission intensity measurement since it can eliminate most possible effects of environmental variations, probe distribution, and instrumental performance and thus offer a more accurate analysis.36,37 In this contribution, we report herein a novel ratiometric two-photon fluorescence probe namely, ASMI, for fast responsive mitochondrial Cys-selective detection and monitoring as well as for high contrast and brightness imaging in live cells and intact tissues at the depth of 150 µm. ASMI is composed of a highly two-photon active and biocompatible merocyanine fluorophore and an acrylate moiety as a thiol reaction site. The cyanine fluorophores have been widely explored as fluorescence sensing or imaging probes because of its easily tunable organelle-targeting and large two-photon absorption properties.38-46 It has been shown that some of acrylate-functionalised probes are prone to be more reactive toward Cys over Hcy and GSH.47-50 The reaction mechanism involves the conjugate addition of Cys to acrylate to generate thioethers followed by an intramolecular cyclization to yield the merocyanine fluorophore and cyclic amide (Scheme 1).51-53 Importantly, both biocompatible and photostable ASMI and merocyanine show very large two-photon action cross-section (Φσmax) of 65.2 GM (λex = 740 nm) and 72.6 GM (λex = 760 nm), respectively, highlighting its great promise for high contrast and brightness ratiometric two-photon excited bioimaging applications. ASMI is the ratiometric fluorescent probe to exhibit highly selective detection and imaging of mitochondrial Cys with two-photon excitation mode for living cells and depth tissues applications.


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Scheme 1. The proposed sensing mechanism of ASMI with Cys.

EXPERIMENTAL SECTION Optical Measurements. Stock solutions of ASMI and merocyanine (0.01 M) were prepared in DMSO. Stock solutions (0.1 M) of the analytes (including biothiols, Na2S, amino acids and ions) were prepared in ultrapure water. For optical measurements, a solution of probe and merocyanine was obtained by diluting the stock solution to 10 µM or 20 µM in DMSO:PBS (pH 7.4) (1/1 v/v). Then, 3.0 mL of solution was poured into a quartz cell of 1 cm optical path length. The fluorescent or UV-vis spectra were then recorded upon addition of various analytes. (Excitation and emission bandwidths were both set at 1.7 nm) Determination of the Fluorescence Quantum Yield. The quantum yield of the probe was determined according to the following equation54 and the value was showed in Table S1. Φx = Φst(Dx/Dst)(Ast/Ax)(ηx2/ηst2)

(1)

Where Φst is the quantum yield of the standard, D is the area under the emission spectra, A is the absorbance at the excitation wavelength and η is the refractive index of the solvent used. x subscript denotes unknown, and st means standard. We chose quinine sulfate solution (Φ= 0.577 in 0.1 M H2SO4) as the standard. Two-Photon Fluorescence Microscopy Imaging in Cells. HeLa cells were grown in RPMI 1640 medium (Thermo Scientific HyClone) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 U/mL penicillin and 100 U/mL gentamicin at 37°C in a humidified atmosphere containing 5% CO2. The cells were firstly 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. Then, two-photon confocal ﬂuorescence imaging of HeLa cells was observed under an Olympus FV1000-MPE multiphoton laser scanning confocal microscope using a mode-locked titanium-sapphire laser source (120 fs, pulse width, 80 MHz repetition rate) set at wavelength 750 nm with laser power of 2 mW.



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Two-Photon Fluorescence Microscopy Imaging in Living Tissues. A 10-week-old mouse was injected via skin-pop injection lipopolysaccharides (LPS) in peritoneal cavity, and another mouse was injected with saline as the control. After 12 hours, ASMI was injected. Then, the mice were anesthetized firstly, and then laparotomy was performed. The slices were obtained from the injection section and then were transferred to glass-bottomed dishes. Two-photon confocal fluorescence images were observed under an Olympus FV1000-MPE multiphoton laser scanning confocal microscope, with a mode-locked titanium-sapphire laser source set at wavelength of 750 nm. Cytotoxicity Assay. The cellular cytotoxicity of ASMI and its respective fluorophore, merocyanine formed after reacting with Cys towards HeLa cells as the model was evaluated using the standard MTT assay.55,56 HeLa cells were seeded into a 96-well plate at a concentration of 4 × 103 cells/well in 100 µL of MEM medium with 10% FBS. Plates were maintained at 37 °C in a 5% CO2 95% air incubator for 24 h. After the original medium was removed, the HeLa cells were incubated with ASMI and reacting of ASMI with Cys with different concentration ([ASMI] = 0, 1, 5, 10, 15 and 20 µM; [Cys] = 20 µM). The cells incubated with the culture medium only were served as the controls. The cells were washed with PBS for three times and then 100 µL MTT solution (0.5 mg/mL in PBS) was added to each well. After addition of DMSO (150 µL/well), the assay plate was allowed to shake at room temperature for 10 min. The spectrophotometric absorbance of the samples was measured by using a Tecan microplate (ELISA) reader. The cell viability was calculated based on measuring the UV-vis absorption at 570 nm using the following equation, where OD570 represents the optical density.56,57 Cell viability = [OD570(sample) − OD570(blank)] / [OD570(control)− OD570(blank)]

(2)

RESULTS AND DISCUSSION Synthesis. The synthesis of ASMI is outlined in Scheme S1. Methylation of 4-methylpyridine gave 1,4-dimethylpyridinium iodide followed by piperidine-catalyzed condensation with 4hydroxybenzaldehyde yielding stilbazolium dye, 1. Reaction of 1 and acryloyl chloride in the presence of Na2CO3 afforded ASMI, which was fully characterized by 1H NMR, 13C NMR, and HRMS. The data obtained are in good agreement with the proposed structures. It is worth mentioning that the synthesis is simple and facile without requiring tedious purification procedures. Detailed synthetic procedures and structure characterizations are provided in the Supporting Information. Optical Properties. ASMI shows good solubility in common organic solvent and water. The excitation and emission spectra of ASMI and merocyanine in DMSO:PBS (1/1 v/v) at pH 7.4 are shown in Figure S1. As one of the typical biothiols, Cys was first used to examine the sensing


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properties of ASMI. As shown in Figure 1, an addition of 100 µM of Cys to probe solution (20 µM and 10 µM for absorption and fluorescence measurements, respectively) in DMSO:PBS (1/1 v/v) at pH 7.4 resulted in fast and distinct optical changes. There is a gradual decrease in the absorption at λmax = 335 nm (εmax= 2.09 × 104 M−1·cm−1) and a simultaneously progressive increase in absorption at ~372 nm (εmax = 2.59 × 104 M−1·cm−1) with a well-defined isosbestic point at 350 nm over a period of time in the absorption spectra (Figure 1a). A notable colour change from colourless-to-orange after an addition of Cys was also observed. Meanwhile, as seen in the fluorescence spectra, the fluorescence emission intensity at 452 nm decreases gradually with a concomitant increase in fluorescence intensity at 518 nm (Figure 1b) and the emission intensity attained a saturation within 6 min indicating a faster response of reaction as compared to those of the previously reported. Moreover, the ratio of fluorescence intensity at 518 nm and 452 nm (F518/F452) was found to be linearly proportional to Cys concentration in the range of 0.5 - 40 µM, based on the linear regression equation of F518/F452 = 0.193CCyS − 0.261, with a linear coefficient of 0.998 which is more sensitive than most of the reported biothiol probes.12-21,25,27 In addition, the optical properties of ASMI in different solvents are summarized in Table S1.

Figure 1. The UV-vis spectral changes (a) and fluorescence spectral changes (b) of ASMI (20 µM and 10 µM, respectively) against time in the presence of Cys (100 µM) in DMSO:PBS (1/1 v/v) at room temperature. (c) Fluorescent kinetic of probe (10 µM) with 100 µM of Cys, Hcy, GSH and Na2S in DMSO:PBS (1/1 v/v) at pH 7.4. λex= 350 nm. (d) Plots of In[(Fmax-Ft)/Fmax] vs time (measured at 518 nm) for the reaction of ASMI (10µM) with Cys, Hcy, GSH, and Na2S (100 µM) by one-photon excitation. The kobs values were calculated from the slopes of the plots, respectively.

ASMI showed similar fluorescence emission spectral changes upon reacting with Hcy and GSH (Figure S2) but with very different kinetic characteristics (Figure 1c). To probe their reactivity differences, the time-dependent fluorescence ratio changes of ASMI with Cys, Hcy, GSH, and Na2S were determined. Although the kinetic properties are solvent



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dependent (Figure S3), ASMI shows a much higher reactivity resulting in a fast and distinct fluorescence ratio change for Cys under the reaction conditions as shown in Figure 1c. To gain insight into the preferentially higher reactive for Cys over other biothiols and Na2S, the reaction rate constants (Kobs) were determined from kinetic analyses of reactions between ASMI and Cys, Hcy, GSH and Na2S (Figure 1d), from which the Kobs values were estimated to be 0.75, 0.038, 0.011, and 0.012 min-1 for Cys, Hcy, GSH, and Na2S, respectively. The reaction of ASMI with Cys was approximately 20-70 times faster than that with Hcy, GSH and Na2S. As a result, it is affirmed that ASMI shows highly selective response to Cys over Hcy, GSH, and Na2S, indicating the potential application for selective detection of Cys in bio-systems. To probe the sensing mechanism of ASMI, we used mass spectroscopy and 1H NMR to analyse the product(s) from the reaction of the probe and Cys. In the mass spectrum, a base peak at m/z 212.179 corresponding to merocyanine was predominantly observed as shown in Figure S4a. Meanwhile, 1H NMR spectra after an addition of Cys taken over a period of 20 min (Figure 2) showed that ASMI underwent almost instant conjugate addition and subsequently followed by cyclization to release the merocyanine fluorophore and yield a seven-membered-ring cyclic amide which afforded a mass peak at m/z 174.0565 (Figure S4b). Indeed, the sensing reaction mechanism of ASMI with Cys as shown in Scheme 1 is followed.12,16,25,47-53

Figure 2. 1H NMR spectra of ASMI in the presence of Cys against time measured in D2O.

The pH effect of the probe in the absence or presence of Cys (Figure S5) was also investigated. The fluorescence F518/F452 ratio showed no significant change for the free probe over a wide pH range of 3.5-9.5, indicating that the probe was very stable in a relatively wide pH range. On the other hand, there was a progressive ratio enhancement with pH value greater than 6 reaching a maximum around physiological pH 7.4 when ASMI was treated with Cys. These results indicate that ASMI can work over a wide pH


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range and the best at physiological pH, holding a promise for biological applications. Furthermore, the photostability of the probe was studied by continuously monitoring of the fluorescence intensity over a period of 2 h. Figure S6 shows the time course of fluorescence intensity of the probe in DMSO:PBS buffer (1/1 v/v) at pH 7.4 at room temperature indicating that the probe possesses good photostability. Selectivity Studies. Besides biothiols and Na2S, interferences from other biologically relevant analytes were evaluated. The ratio of fluorescence intensity (F518/F452) was measured in the presence of Cys, Hcy, GSH, Na2S, amino acids (Lys, Ser, Leu, Arg, Pro, Phe, Gly, His, Met, Ala, Try, Val, Asp, Thr, Trp, Glu), essential metal ions (Na+, K+, Ca2+, Mg2+, Zn2+, Fe3+, Al3+, Cu2+), and anions (SO42-, SO32-, SCN-, AcO-, CO32-, NO3-, NO2-, Cl-, Br-). As depicted in Figure S7, the fluorescence ratio of F518/F452 increased more than 10-fold upon an addition of Cys; in sharp contrast, the intensity ratio change was found to be insignificant for all the other tested analytes. Results of these competition experiments clearly and further demonstrated that the reaction of ASMI with Cys was much more reactive than other thiols which could be applied to selectively detect cellular Cys without any interference from other biologically relevant analytes. Two-Photon Excited Fluorescence Imaging in Live Cells. To explore the potential of the probe for two-photon excited imaging applications, the two-photon action cross-sections (Φσ) of ASMI and its respective fluorophore, merocyanine formed after reaction with Cys were determined by the two-photon induced fluorescence method using a femtosecond pulsed laser in the range of 690-840 nm with rhodamine 6G as the reference. Figure S8 shows the two-photon action cross-section excitation spectra of ASMI and merocyanine. The Φσmax of ASMI and merocyanine were determined to be 65.2 GM at 740 nm and 72.6 GM at 760 nm (1 GM = 10-50 cm4 s photon-1 molecule-1) in DMSO:H2O (1:5), respectively.58 A probe with such large twophoton action cross-section values in aqueous medium would be highly favourable for high contrast and brightness two-photon images of biological samples. Since ASMI is a positively charged fluorophore, it would have a high tendency to localize in mitochondria of a cell due to the large negative membrane potential of the inner mitochondrial membrane.59 To determine the subcellular localization property of this probe, a colocalization study of ASMI was conducted in HeLa cells with Mitotracker red (Figure 3), a well-known one-photon fluorescent probe for mitochondria targeting. The two-photon images indicated that the probe was efficiently taken up by HeLa cells and overlapped well with the one-photon images of Mitotracker red. This finding demonstrated that ASMI was capable of targeting mitochondria in living cells.



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Figure 3. Two-photon and one-photon images of HeLa cells co-stained with ASMI (5 µM) and Mitotracker red (1 µM) for 30 min at 37 oC. The wavelengths for two-photon and one-photon excitation were 750 and 580 nm, respectively, and the corresponding emissions were collected at 430470 nm (AMSI) and 620-660 nm (Mitotracker red). Scale bar: 20 µm.

To demonstrate the practical use as a ratiometric two-photon excited fluorescent probe in biological samples, the two-photon images of the HeLa cells labelled with ASMI were monitored by dual emission channels; (1) blue (λem = 430-470 nm) and (2) green (λem = 490-530 nm) channels (Figure 4) upon excitation at 750 nm with femtosecond laser pulses. After the cells were incubated with ASMI (5 µM) for 30 min, weak blue twophoton excited fluorescence (TPEF) in channel 1 as well as bright green fluorescence in channel 2 (Figure 4A) were observed resulting from the presence of endogenous Cys in living cells. To assess the potential selective response of ASMI towards Cys over other thiols in live cell environment, a series of control experiments were carried out. When the HeLa cells were pre-treated with N-ethylmaleimide (NEM, 1.0 mM), a well-known thiolblocking agent for the depletion of intracellular thiol species60 for 30 min before incubating with ASMI (5 µM), a strong TPEF in the blue channel was observed but there was no emission detected in the green channel (Figure 4B) indicating that thiol species were completely reacted by NEM. Upon an addition of Cys, Hcy or GSH (100 µM) to the NEM-pre-treated HeLa cells for 1 h followed by an incubation with ASMI (5 µM), marked variations of fluorescence responses in different channels were observed. With the treatment of Cys, the blue TPEF in channel 1 was diminished (Figure 4C, channel 1) concomitant with a sharp increase in TPEF in the green channel (Figure 4C, channel 2). On the contrary, the treatment of Hcy could only afford a very weak green fluorescence in channel 2 (Figure 4D) and the treatment of GSH could not induce any green TPEF emission at all (Figure 4E). Such findings were consistent with the fact that ASMI showed reactive and selective Cys-induced ratiometric fluorescence responses in living cells. These also further supported that ASMI could be used as a ratiometric two-photon fluorescent sensing probe for selective bioimaging of mitochondrial Cys in living cells. Furthermore, ASMI and merocyanine formed after the reaction of ASMI and Cys were found to be non-cytotoxic to cells during the imaging experiments as further confirmed by MTT viability assays (Figure S9).


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Figure 4. Two-photon confocal microscopy fluorescence images of Cys in living HeLa cells. (A) HeLa cells incubated with ASMI (5 µM) for 30 min. (B) HeLa cells pre-treated with NEM (1.0 mM) for 30 min, and then incubated with ASMI (5 µM) for 30 min. (C, D, and E) HeLa cells pre-treated 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 ASMI (5 µM) for 30 min. From left to right: Bright Field, Channel 1: λem = 430-470 nm, Channel 2: λem = 490-530 nm, overlay. λex = 750 nm. Scale bar: 20 µm.

As intracellular Cys concentration is known to associate with oxidative stress level, an investigation of the probe in response to the change of the Cys concentration in living cells was also demonstrated by altering the redox balance. When HeLa cells were pre-treated with H2O2 (200 µM) for 30 min before incubating with ASMI (5 µM) probe, there was a dramatic decrease in green TPEF (Figure 5B, channel 2) accompanied with a strong increase in blue TPEF observed (Figure 5B, channel 1) as compared with those without H2O2 pre-treatment. Such a change in fluorescence was responsive to the decrease of the intracellular Cys concentration induced by H2O2 oxidation of Cys. Therefore, these findings suggested that ASMI probe could be used as a tool to monitor the level of intracellular Cys.

Figure 5. Two-photon confocal microscopic fluorescence images in live HeLa cells. (A) HeLa cells incubated with ASMI (5 µM) for 30 min. (B) HeLa cells pre-treated with H2O2 (200 µM) for 30 min



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and then incubated with ASMI (5 µM) for 30 min. From left to right: Bright Field, Channel 1: λem = 430-470 nm, Channel 2: λem = 490-530 nm, overlay. λex = 750 nm. Scale bar: 20 µm.

Two-Photon Excited Fluorescence Imaging in Living Tissue. In addition to the cellbased studies, we investigated the capability of this probe to monitor Cys in living tissue. A mouse suffered an acute inflammatory response in peritoneal cavity by means of skinpop injection of 200 µL lipopolysaccharides (LPS, 1.0 mg·mL-1) which act as endotoxins to elicit strong immune responses such as inflammation and the control mouse was injected with saline (200 µL). ASMI (50 µM) was applied via skin-pop injection 12 hours after LPS-treatment. Then, the tissue slices were obtained from the injection section for imaging studies (Figure 6). A strong TPEF signal upon excitation at 750 nm with femtosecond laser pulses in green (λem = 490-530 nm) channel and a relatively weak blue fluorescence signal (λem = 430-470 nm) in channel 1 were observed in tissues treated with saline. On the contrary, the TPEF intensity in the LPS-treated tissues diminished sharply in the green channel and increased intensely in blue channel (Figure 6). These phenomena indicated that LPS induced immune response to cause a significant drop in Cys level in vivo. It is worth mentioning that the changes in the emission profile measured deep inside the tissue slices are consistent to those observed in the cells. These further ascertain that ASMI is highly promising for monitoring of Cys in living tissue and could serve as a useful research tool for Cys-related studies.

Figure 6. Two-photon confocal microscopic fluorescence images in tissue slices injected ASMI (50 µM) after pre-treated with saline (200 µL) and LPS (200 µL, 1.0 mg·mL-1), respectively. From left to right: Channel 1: λem = 430-470 nm, Channel 2: λem = 490-530 nm, overlay. λex = 750 nm.

To further demonstrate the deep tissue imaging capability of ASMI, two 1.0 mm thick tissue slices were obtained from the ASMI injection section after treated with LPS and saline, respectively. Then, the fluorescence images were obtained by two-photon confocal microscopy and the images of normal and inflamed tissues were recorded at both of the blue and green emission channels, respectively. The 3D reconstitution of confocal XYZ scanning micrographs were obtained from 50 confocal Z-scan two-photon fluorescence imaging sections (Figure 7A, C), which indicated that the probes were evenly distributed in tissues and showed a high signal-to-noise ratio. The confocal Z-scan two-photon imaging sections at different penetration depths exhibited bright TPEF signal down to 150


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µm of penetration depth (Figure 7B, D), indicating that the probe showed the deep tissue imaging capability.

Figure 7. The 3D reconstitution of confocal XYZ scanning micrographs from 50 confocal Z-scan twophoton imaging sections (A) LPS-treated slice (λem = 430-470 nm) and (C) saline-treated slice (λem = 490-530 nm). (B, D) The confocal Z-scan two-photon imaging sections at different penetration depths for 0, 10, 20, 50, 100 and 150 µm of (A) and (B), respectively. λex = 750 nm.

CONCLUSION In summary, we have developed highly selective and sensitive, ratiometric two-photon fluorescent probe namely, ASMI, for high contrast and brightness two-photon excited cellular and tissue imaging of mitochondrial cysteine. The probe is composed of a biocompatible merocyanine as the fluorophore and an acrylate moiety as the biothiol reaction site. This acrylate-functionalized merocyanine shows not only mitochondrial targeting ability but also a selectively marked blue-to-green emission change upon detecting/reacting with Cys over other biothiols including Hcy and GSH and H2S in live cells. Furthermore, this probe has been found to show low cytotoxicity, good photostability and excellent biocompatibility. More importantly, the probe and its respective fluorophore, merocyanine possess very large two-photon action cross-sections of 65.2 GM (λex = 740 nm) and 72.6 GM (λex = 760 nm) in aqueous medium, respectively, which are highly favourable for high contrast and brightness ratiometric TPEF imaging. ASMI has been successfully applied to selectively detect mitochondrial Cys and monitor Cys concentration change in live cells and living tissues down to a depth of 150 µm by two-photon fluorescence microscopy. As a result, this ratiometric two-photon fluorescent sensing probe would be practically useful and greatly beneficial to investigate and monitor Cys in living biological systems. ASSOCIATED CONTENT Supporting Information. Materials and apparatus, synthesis and characterization of fluorescent probe, photophysical properties of ASMI, cytotoxicity assay, pH effect, photostability,



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selectivity, two-photon action cross-section excited spectra. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *[email protected]; fax +852-34117348 *[email protected]; fax +86-351-7018613 *[email protected]; fax +86-731-88822523 Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No. 21475080, No. 21575084 and No. 21305036), the Hundred Talent Program of Shanxi Province, Shanxi Province Scholarship Council of China and Natural Science Foundation of Chongqing Municipal Education Commission (No. KJ1401210). This work was also financially supported by GRF (HKBU 203212), Hong Kong Research Grant Council, FRG (FRG2/13-14/059), 2014 Hong Kong Scholars Program, and Institute of Molecular Functional Materials which was supported by a grant from the University Grants Committee, Areas of Excellence Scheme (AoE/P-03/08). REFERENCES (1) Gruhlke, M. C. H.; Slusarenko, A. J. Plant Physiol. Biochem. 2012, 59, 98-107. (2) Giles, G. I.; Jacob, C. Biol. Chem. 2002, 383, 375-388. (3) Giles, G. I.; Tasker, K. M.; Jacob, C. Free Radical Biol. Med. 2001, 31, 1279-1283. (4) Reddie, K. G.; Carroll, K.S. Curr. Opin. Chem. Biol. 2008, 12,746-754. (5) Paulsen, C.E.; Carroll, K.S. Chem. Rev. 2013, 113, 4633-4679. (6) Droge, W.; Eck, H. P.; Mihm, S. Immunol. Today, 1992, 13, 211-214. (7) Lieberman, M. W.; Wiseman, A. L.; Shi, Z. Z. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 79237926. (8) Shahrokhian, S. Anal. Chem. 2001, 73, 5972-5978. (9) Wang, X. F.; Cynader, M. S. J. Neurosci. 2001, 21, 3322-3331. (10) Levine, B.; Kroemer, G. Cell. 2008, 132, 27-42. (11) McBride, H. M.; Neuspiel, M.; Wasiak, S. Curr. Biol. 2006, 16, 551-560.


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For TOC only


Highly Selective Two-Photon Fluorescent Probe for Ratiometric

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