Mitochondrial-Targeted Two-Photon Fluorescent Probes for Zinc Ions, H2O2, and Thiols in Living Tissues

Mitochondria provide the energy of the cells and are the primary site of oxygen consumption and the major source of reactive oxygen species. In mitochondria, metal ions and glutathione play vital roles in maintaining their structure and the redox environment. To understand their roles in mitochondria, it is crucial to monitor each of these chemical species in the mitochondria at the cell, tissue, and organism levels. An ideal tool for such purpose is the use of two-photon microscopy (TPM). Until recently, however, there has been no report on the two-photon (TP) probes suitable for such applications. In this paper, we summarize the mitochondria-targeted TP probes for Zn2+, H2O2, and thiols, as well as their bioimaging applications.


Introduction
Mitochondria provide the energy of the cells. ey are primary cellular compartments of oxygen consumption and the major source of reactive oxygen species (ROS) [1,2]. In mitochondria, metal ions and glutathione (GSH) play vital roles in maintaining their structure and the redox environment [3][4][5][6]. To understand the physiology of mitochondria, it is crucial to monitor such chemical species in mitochondria at the cell, tissue, and organism levels. For this purpose, a number of one-photon �uorescent probes, derived from �uorescein or rhodamine as the �uorophore and various receptors, have been developed [7][8][9]. However, most of these probes have been evaluated with one-photon microscopy (OPM), which uses single photon of higher energy as the excitation source (Scheme 1(a)). is requirement limited their application in live tissue imaging owing to the shallow penetration depth (less than 80 m), photobleaching, and cellular auto�uorescence.
An attractive approach to the detection of biologically important species deep inside live tissues is the use of twophoton microscopy (TPM). TPM, a new technique which employs two near-infrared photons as the excitation source (Scheme 1(a)), has become an indispensable tool in biology and medicine due to the advantages it offers. ey include deeper penetration depth (>500 m), lower tissue auto-�uorescence and self-absorption, reduced photodamage and photobleaching, in addition to the intrinsically localized excitation [13][14][15][16]. is allows molecular imaging deep inside the intact tissue for a long period of time with minimum interference from the tissue preparation artifacts which can extend >70 m into the tissue interior [17]. However, the progress in this �eld is limited by the lack of two-photon (TP) probes. As such, many biologists are using one photon �uorescent probes for TPM imaging, despite that most of them have too low two-photon (TP) cross-sections ( TPA < 50 GM) to be useful for TPM [18]. erefore, there is a pressing need to develop a variety of TP probes for speci�c applications [15,16].

Two-Photon Probes for Mitochondrial Zinc Ion
Zinc ion is the second most abundant d-block metal ion in the human brain and is an active component in enzymes and proteins [22][23][24][25][26]. For proper brain functions, it is vital to maintain Zn 2+ -ion homeostasis, which is controlled by the import of intracellular  [29,30] and TPP as the mitochondrial targeting group. SZn-Mito and SZn2-Mito are TP �uorescent turn-on probes based on the photoinduced electron transfer (PeT) process [31]. Upon addition of Zn 2+ , the �uorescence intensity of SZn-Mito and SZn2-Mito increased gradually presumably because of the blocking of the PeT upon binding with Zn 2+ . e TP �uorescence enhancement factor (FEF  max of the one-photon absorption and emission spectra in nm. [b] Fluorescence quantum yield. [c] Dissociation constants measured by one-( OP ) and twophoton ( TP ) processes, except otherwise noted. [d] Fluorescence enhancement factor, ( min )/ min , measured by one-photon processes. e number in parentheses is measured by two-photon process. [e] max and min represent the maximum and minimum ratios of yellow / blue , where yellow and blue are the �uorescence intensities measured at 425-475 nm ( blue ) and 525-575 nm ( yellow ), respectively. e number in parentheses was measured by two-photon max of the two-photon excitation spectra in nm. [g] e peak two-photon action cross-section in 10 50 cm 4 s/photon (GM). [  [32]. Moreover, they were pH insensitive in the biologically relevant pH range. Since Ni 2+ , Cd 2+ , Hg 2+ , and Cu 2+ ions rarely exist in the cells [33], these probes can detect Zn 2+ with minimum interference from other competing metal ions and pH. Moreover, the TP action cross-sections (Φ ) of SZn-Mito and SZn2-Mito were 75 and 155 GM at 760 and 750 nm, respectively, in the presence of excess Zn 2+ (Table 1). e combined results reveal that SZn2-Mito is a more sensitive TP probe which can detect mitochondrial Zn 2+ with higher sensitivity and twice as bright TPM image than SZn-Mito. e utility of SZn2-Mito in the cell imaging was con-�rmed by the following experiments. First, the TPM and OPM images of HeLa cells colabeled with SZn2-Mito and Mitotracker Red FM, a well-known OP �uorescent probe for mitochondria [8], overlapped well. e Pearson's colocalization coefficient, A, calculated by using Autoquant X2 soware, of SZn2-Mito with Mitotracker Red FM was 0.85 (Figures 1(a)-1(c)) [34]. Second, the TPEF intensity decreased dramatically when the probe-labeled cells were treated with N,N,N′,N′-tetrakis(2-pyridyl)ethylenediamine (TPEN), a membrane permeable Zn 2+ chelator that can effectively remove [Zn 2+ ] [35]. ird, SZn2-Mito showed negligible toxicity as measured by using CCK-8 kit and high photostability as indicated by the negligible change in the TP excited �uorescence (TPEF) intensity aer continuous irradiation of the fs pulses for 60 min [10]. Fourth, the TPEF intensity increased dramatically when 2,2′-dithiodipyridine (DTDP; 150 M), a reagent that promotes the release of Zn 2+ from Zn 2+ -binding proteins [36], was added to HeLa cells labeled with SZn2-Mito (Figures 1(d), 1(e), and 1(g)) and decreased abruptly upon addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP; 10 M, Figures 1(f) and 1(g)), a compound that promotes the release of intramitochondrial cations by collapsing the mitochondrial membrane potential [37].
To assess the utility of SZn2-Mito in tissue imaging, fresh hippocampal slices from a 14-day-old rat were labeled with 20 M SZn2-Mito. e TPM image of the probelabeled tissue revealed marked TPEF in the CA3 and DG regions at depths of 100-200 m (Figure 2(b)). At a higher magni�cation, the [Zn 2+ ] distribution in the DG region was clearly visualized (Figure 2(c)). Moreover, the TPEF intensity increased aer addition of DTDP and decreased upon treatment of CCCP, thereby con�rming that the bright regions re�ect the [Zn 2+ ] (Figures 2(d) and 2(e)). ese results established that SZn2-Mito is capable of detecting [Zn 2+ ] at depths of 100-200 m in living tissues by using TPM [10].

Two-Photon Probe for Mitochondrial H 2 O 2
Hydrogen peroxide (H 2 O 2 ) is a prominent member of the ROS family and plays crucial roles in physiology, aging, and disease in living organisms [38][39][40]   the H 2 O 2 -triggered boronate cleavage of the electron poor carbamate linkage would liberate the more electron rich 1-SHP, giving rise to red-shied �uorescent emission (Scheme 2) [11]. e emission spectra of a 1 M solution of SHP-Mito increased gradually at 530-600 nm ( yellow ) with a concomitant decrease at 400-470 ( blue ) nm in the presence of 1 mM H 2 O 2 in MOPS buffer [11]. is process followed pseudo 1storder kinetics with obs = 1.0-1.2 × 10 −3 s −1 and resulted in a 75-fold enhancement in the yellow / blue ratio. e detection limit of SPH-Mito for H 2 O 2 was 4.6 M. Moreover, SHP-Mito exhibited high selectivity for H 2 O 2 over competing ROS and reactive nitrogen species (RNS), as revealed by unperturbed yellow / blue ratios upon addition of 200 M concentrations of various ROS and RNS, including tertbutyl hydroperoxide (TBHP), hypochlorite (OCl − ), superoxide (O 2 − ), nitric oxide (NO), tert-butoxy radical ( • OBu ), hydroxyl radical ( • OH), and peroxynitrite (ONOO − ), and was pH insensitive at biologically relevant pH range [11]. e TP action (Φ ) spectra of SHP-Mito and 1-SHP in MOPS buffer (30 mM, pH 7.4) indicated Φ max values of 11 and 55 GM at 740 and 750 nm, respectively (Table 1). is predicts 5-fold brighter TPM images of the probe-labeled cells aer complete conversion of SHP-Mito to 1-SHP.
e utility of SHP-Mito in the live cell imaging was established by the following experiments. First, the TPM and OPM images of Raw 264.7 murine macrophage cells co-labeled with SHP-Mito and MitoTracker Red merged well with the of 0.91 [11]. Second, the ratiometric images constructed from two collection windows ranging from 400 to 470 nm ( blue ) and 530 to 600 nm ( yellow ) gave an average emission ratio of yellow / blue = 0.62 and 1.96 for SHP-Mito and 1-SHP, respectively (Figures 3(a), 3(d), and 3(e)). e ratio increased to 1.62 when the cells were preincubated for 30 min with 200 M H 2 O 2 and to 1.56 upon treatment with phorbol myristate acetate (PMA), which induces H 2 O 2 generation through a cellular in�ammation response (Figures 3(b), 3(c), and 3(e)) [45]. ese results con�rmed that SHP-Mito is responsive to the change in the H 2 O 2 concentration. ird, SHP-Mito shows nontoxicity to cells during the imaging experiments, as determined by using a CCK-8 kit [11].
e utility of SHP-Mito in the tissue imaging was established by a similar protocol as described earlier. TPM image of fresh hippocampal slices from a 2-day-old rat labeled with 20 M SHP-Mito revealed that H 2 O 2 is more or less evenly distributed in both the CA1 ( green / blue = 0.81) and CA3 ( green / blue = 0.72) regions (Figure 4(a)) at depths of 90-180 m. Upon treatment of the tissue with increasing amounts of H 2 O 2 , the ratio increased gradually to 1.57 (CA3) and 1.75 (CA1) at 1 mM H 2 O 2 , which lie between those measured in SHP-Mito-and 1-SHP-labeled tissues (Figure 4(h)). Hence, SHP-Mito is responsive to rises in H 2 O 2 in tissue. Moreover, the ratiometric image at higher magni�cation clearly showed the H 2 O 2 distribution in the individual cells in the CA3 region at a depth of 120 m (Figures 4(d)-4(f)). Hence, SHP-Mito is capable of detecting mitochondrial H 2 O 2 in live tissues using TPM [11].

Two-Photon Probe for Mitochondrial Thiols
Intracellular thiols such as cysteine (Cys), homocystein (Hcy), and glutathione (GSH) play vital roles in biology [3][4][5]. ey maintain higher-order structures of proteins and control redox homeostasis through the equilibrium between thiols (RSH) and disul�des (RSSR) [46]. In mitochondria, GSH plays a key role in maintaining the redox environment to avoid or repair oxidative damage leading to dysfunction and cell death [47]. Mitochondrial GSH (mGSH) exists predominantly in the reduced form, with the GSH : GSSG ratio being greater than 100 : 1 [48]. To detect GSH deep inside live tissues, we have developed a TP probe (SSH-Mito, Scheme 3) [12].
SSH-Mito is a TP ratiometric probe based on the internal charge transfer (ICT) process [31]. e emission spectra of SSH-Mito showed a gradual increase at 525-575 nm ( yellow ) with a concomitant decrease at 425-475 ( blue ) nm in the presence of 10 mM GSH in MOPS buffer (30 mM, pH 7.4) ( Table 1). e reaction followed 2nd-order kinetics with 2 = 2.3 ×10 −2 M −1 s −1 (Scheme 3) [12]. is indicates that the reaction proceeds by the rate-limiting attack of GSH at the disul�de bond followed by the cleavage of the C-N bond to afford 1-SSH (Scheme 3). SSH-Mito exhibited strong response toward thiols, including GSH, Cys, dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and 2-aminoethanethiol (2-AET), and a negligible response toward amino acids without thiol groups (glu, ser, val, met, ala, and ile), metal ions (Na + , K + , Ca 2+ , Mg 2+ , and Zn 2+ ), and H 2 O 2 and was pH insensitive at the biologically relevant pH range [19]. Moreover, yellow / blue , the ratio of the intensities at 425-475 nm ( blue ) and 525-575 nm ( yellow ), increased by 45-fold in the presence of 10 mM GSH (Table 1). Further, the TP action spectra of SSH-Mito and 1-SSH indicate Φ max values of 80 and 55 GM at 740 and 750 nm, respectively, which are comparable to those of existing TP probes (Table  1) [15]. ese results predict a bright ratiometric TPM image of the living specimens stained with SSH-Mito.
SSH-Mito was found to locate predominantly in mitochondria as revealed by the colocalization experiment with MitoTracker Red ( = 0.8 ) [12]. Upon 740 nm TP excitat-ion, the ratio image of the SSH-Mito-labeled HeLa cells, constructed from two collection windows, gave an average emission ratio of 1.24 (Figures 5(a) and 5(e)). More importantly, SSH-Mito was responsive to the changes in the thiol concentration; the yellow / blue ratio increased to 2.64 when the cells were preincubated for 1 day with -lipoic acid ( Figure 5(b)), which increases GSH production [49], and the value was nearly identical to those obtained with 1-SSH (2.73, Figure 5(d)). e yellow / blue ratio also decreased to 0.77 upon treatment with N-ethylmaleimide (NEM) ( Figure  5(c)), a well-known thiol-blocking reagent [50]. Further, SSH-Mito is nontoxic to cells during the imaging experiments as determined by a CCK-8 kit. ese results establish that SSH-Mito is capable of detecting mitochondrial thiol in the live cells.
e TPM image of fresh hippocampal slices from a 14day-old rat labeled with 20 M SSH-Mito revealed that thiols are more or less evenly distributed in both CA1 and CA3 regions at depths of 90-190 m ( Figure 6(b)). Moreover, the image at a higher magni�cation clearly shows the thiol distribution in the individual cells in the CA1 region with an average emission ratio of 1.66 at a 120 m depth (Figure 6(c)). Upon treatment of the tissue with 100 M NEM for 30 min, the ratio decreased to 0.85 (Figure 6(f)). It is worth noting that the changes in the emission ratios measured deep inside the tissue slice are comparable to those in the cells. Hence, SSH-Mito is clearly capable of detecting mitochondrial thiols in live tissues using TPM [12].

Conclusions
We have developed a series of mitochondrial-targeted TP probes that can selectively detect the Zn 2+ , H 2 O 2 , and thiols, respectively, in live cells and tissues. All of these probes have been developed by linking mitochondrial targeting site and speci�c receptors at the opposite ends of the TP �uorophore. ey show signi�cant TP action cross-sections, a marked turn-on or ratiometric response upon reaction with the analytes, good mitochondrial localization, low cytotoxicity, insensitivity to pH in the biologically relevant pH range, and can visualize the distribution and changes in mitochondrial Zn 2+ , H 2 O 2 , and thiols levels, respectively, in live tissues at more than 100 m depth by TPM without interference from other biologically relevant species.