In 2015, Chen and coworkers developed a reaction-based near-infrared (NIR) fluorescent probe (Mito-ss) for the detection of mitochondrial hydrogen polysulfides (H₂S_n, n > 1) [37]. Mito-ss consists of (i) a NIR dye based on the azo-BODIPY chromophore, (ii) a lipophilic triphenyl phosphonium group, and (iii) an H₂S_n-reactive nitrofluorobenzoate moiety (Scheme 1a). They chose a NIR fluorophore because NIR lights possess certain advantages, including deep tissue penetration, low cytotoxicity, and minimum background noise. Nitrofluorobenzoate is a commonly used functional group for the design of H₂S_n sensors [38]. Nitrofluorobenzoate bears two electrophilic sites. H₂S_n first reacts with it via nucleophilic aromatic substitution (S_NAr) to replace the F atom and form a persulfide (-SSH) intermediate, which then undergoes a spontaneous intramolecular cyclization with the ester group to uncage the fluorophore. Due to its electron with-drawing nature, nitrofluorobenzoate quenches the fluorescence of the azo-BODIPY chromophore via a donor-excited photoinduced electron transfer (d-PET) process. As such, Mito-ss is a reaction-based ‘turn-on’ sensor for H₂S_n. Mito-ss reacts rapidly (~30 s) with H₂S_n and exhibits a 24-fold fluorescence increase at an emission of 730 nm. The probe was examined with various ROS, reactive nitrogen species (RNS), and other RSS and demonstrated no fluorescence turn-on. Biothiols such as glutathione (GSH), cysteine (Cys), N-acetyl-L-cysteine, etc., could react with Mito-ss. However, as the reaction stopped at the S_NAr step, no fluorescence was observed. The limit of detection (LOD) for Mito-ss was calculated to be 25 nM. The probe was used for the real-time detection of exogenous and endogenous H₂S_n using six different cell lines. Mito-ss was also found to be suitable for the in vivo detection of exogenously injected H₂S_n in BALB/c mice.

Using the same nitrofluorobenzoate reaction site, Han et al. developed a ratiometric fluorescent probe, Mito-NRT-HP, for the detection of mitochondrial H₂S_n in 2019 [39]. The structure of Mito-NRT-HP is similar to Mito-ss, though with a two-photon responsive naphthalimide fluorophore instead of a single-photon responsive fluorophore. The naphthalimide fluorophore has the advantage of easily tunable photophysical properties by blocking and/or unblocking the internal charge transfer (ICT) process. It is highly photostable, resistant to pH interference, and possesses a large two-photon absorption cross-section. Importantly, 1,8-naphthalimide can be easily functionalized by simple synthetic tailoring [40]. The main advantages of two-photon excitation over single-photon excitation include deep tissue penetration, lesser damage, poor scattering, etc. In the case of two-photon excitation, a femto second pulsed laser is used, and the molecule can be excited only at the focal point of the laser. Three-dimensional imaging can be obtained [41]. Upon titrating with different concentrations of Na₂S₂, it was found that Mito-NRT-HP gave ratiometric responses with a changing fluorescence color from blue to green. When the solution of Mito-NRT-HP was treated with H₂S_n, the initial emission maximum at 478 nm decreased gradually, with a concomitant peak increase at 546 nm. The detection limit was 10 nM which suggests that Mito-NRT-HP could have the relevant sensitivity needed for the quantitative detection of H₂S_n under physiological conditions. The two-photon absorption cross-section values (δ) of Mito-NRT-HP and its fluorophore Mito-NRT (Scheme 1b) were recorded in a buffer using a pulsed laser, and fluorescein was used as the reference molecule. Their δ values were measured over a range of wavelengths starting from 750 nm to 825 nm. The highest δ was 290 GM [1 GM (Goeppert-Mayer) = 10⁻⁵⁰ cm⁴ s photon⁻¹] for Mito-NRT-HP and ~190 GM for Mito-NRT at 810 nm. Mito-NRT-HP was found to exhibit good cell permeability and weak cytotoxicity, which was suitable for the ratiometric imaging of endogenous H₂S₂ in cells. Mito-NRT-HP was colocalized with MitoTracker Red (MTR) and LysoTracker Red (LTR), and the colocalization coefficients were found to be 0.94 and 0.42, respectively, indicating that Mito-NRT-HP was specifically localized in the mitochondria. Using two-photon microscopy, images of the tissue slices from mice with lipopolysaccharide (LPS)-induced acute organ injury were taken and compared with the control tissues. The enhanced fluorescence in the former case was observed. In 2021, Han et al. reported a similar probe for the detection of mitochondrial H₂S_n during H₂O₂-induced redox imbalance [42]. The structure of this probe (Mito-RHP) only differed from Mito-NRT-HP in the linker between naphthalimide and the triphenylphosphonium unit. Upon the addition of Na₂S₂ to the solution of Mito-RHP, the initial emission spectra of Mito-RHP at 485 nm gradually decreased, and a continuous increase in the new peak to 550 nm was observed, along with a change in fluorescence color from blue to yellowish green. In this case, the Stokes shift was 109 nm, which was higher than that of Mito-NRT-HP. The detection limit was calculated to be 20 nM. Other properties, such as photostability, solubility, permeability, and cytotoxicity, were similar. However, the mitochondria-targeting ability of the new probe (overlap coefficient = 0.836) was not as good as that of Mito-NRT-HP (overlap coefficient = 0.94). The in vivo imaging of exogenous H₂S_n (using Na₂S₂) was performed in zebrafish using Mito-RHP.

An interesting single-component multi analyte responsive NIR fluorescent probe was reported by Chen and coworkers in 2015 for the detection of the superoxide ion (O₂^•−) and H₂S_n to understand redox homeostasis in the mitochondria [43]. Both O₂^•− and H₂S_n are short-lived reactive species, and their concentrations change quickly. To solve this problem, they developed a cyanine-based NIR probe, HCy-FN. This probe consists of two different reaction sites: one for the abstraction of hydrogen to detect O₂^•− and the other for the detection of H₂S_n using nitrofluorobenzoate (Scheme 1c). Both sensing steps were monitored by two different channels. Upon reacting with O₂^•−, HCy-FN was oxidized to Cy-FN, and this transformation was monitored by an increase in the emission intensity from channel 1 at 794 nm (λ_ex = 750 nm). Next, the nitrofluorobenzoate part of Cy-FN reacted with H₂S_n to result in a decrease in the emission intensity of channel 1 followed by an increase in the emission intensity in channel 2 at 625 nm (λ_ex = 535 nm) due to the formation of Keto-Cy. They examined different ROS with HCy-FN and found that only O₂^•− was able to oxidize the probe. Similarly, the reactivity of other RSS towards Cy-FN was also evaluated, and no changes in emission spectra were noted. HCy-FN was used for the detection of exogenous and endogenous H₂S_n with the macrophage cell line RAW264.7 to monitor both sensing steps by dual channel emission. It was found that the Pearson correlation coefficient (R_r) of Cy-FN and mitochondria-localizing Rhodamine 123 was 0.98, confirming that Cy-FN was localized in the mitochondria. Moreover, HCy-FN could detect endogenously produced O₂^•−/H₂S_n in BALB/c mice. This work represents an interesting way to detect O₂^•−/H₂S_n in the biological system. However, the claim that the probe is capable of monitoring mitochondrial O₂^•−/H₂S_n may not be accurate. The authors only provided the R_r value for the intermediate compound Cy-FN and not for the actual probe. The structure of HCy-FN suggests that it may not be a suitable candidate to target the mitochondria because of the lack of a lipophilic cationic moiety.

In 2016, Chen and coworkers developed a probe (HCy-Mito) for the selective detection of superoxide anion (O₂^•−) and H₂S_n in the mitochondria [44]. The reaction sites for O₂^•− and H₂S_n were the reduced cyanine dye (similar to HCy-FN) and m-nitrophenyl ether (Scheme 1d). In the presence of O₂^•−, Hcy-Mito was oxidized to form a cyanine derivative, and the reduced nature of H₂S_n converted the nitro group to -NH₂, which terminated the d-PET process and resulted in an increase in emission intensity to 780 nm. The detection limits for O₂^•− and H₂S_n by HCy-Mito were found to be 0.1 μM and 0.2 μM, respectively. In vitro experiments with RAW264.7 cells by HCy-Mito suggest that it could image exogenous and endogenous O₂^•−/H₂S_n and localize specifically in the mitochondria (R_r = 0.93). This probe was further utilized for the in vivo detection of O₂^•− (generated from phorbol myristate acetate (PMA) and H₂S_n (via injected Na₂S₄) in BALB/c mice.

In 2019, Meng et al., used a different reaction site based on the 2-(acylthio)benzoate for the design of a mitochondria-targeted probe for H₂S_n [45]. This template utilized both the nucleophilic and electrophilic nature of H₂S_n for its recognition (Scheme 2a) [46]. Briefly, the thioester exchange between the H₂S_n and 2-(acylthio)benzoate produced a thiophenol derivative, which, in turn, reacted with H₂S_n to form an -SSH intermediate. This intermediate underwent an intramolecular cyclization to release the fluorophore. This template was attached to a red-emitting fluorophore to develop the probe, HQO-PSP. HQO-PSP itself was non-fluorescent but, upon sensing H₂S_n, exhibited a fluorescence turn-on (86-fold) at an emission of 633 nm due to the formation of the keto derivative HQO. The probe was found to be relatively fast (7 min) and highly selective to H₂S_n, with a detection limit of 95.2 nM. In vitro studies with A549 cells revealed that HQO-PSP could specifically localize within the mitochondria (R_r = 0.98) and selectively image exogenously added H₂S_n in the live cells.

In the same year, Choi et al. reported that the ratiometric probe SPS-M1 for mitochondrial H₂S_n detection was based on a two-photon excitable naphthalene fluorophore [47]. The reaction site was the same as that of HQO-PSP except for an additional self-immolating carbamate linker (Scheme 2b). The probe exhibited a blue fluorescence (λ_em = 429 nm) but produced the deprotected yellow fluorescent dye M1 (λ_em = 506 nm) upon sensing H₂S_n. Interestingly, the two-photon absorption (TPA) cross-section (δ) of SPS-M1 and M1 was found to be 11 and 108 GM, respectively, at 750 nm. The large TPA cross-section resulted from the strong ICT process in M1. SPS-M1 was found to be suitable for the quantification of H₂S_n in live cells, and the in vitro detection limit was 1 μM. The two-photon microscopic imaging with SPS-M1 for endogenous H₂S_n using the wild-type and Parkinson’s disease (PD) model neurons and brain tissues of mice revealed that H₂S_n concentrations were higher in the PD model.

Another interesting approach for the spatiotemporal detection of mitochondrial H₂S_n was reported by Han et al. in 2018 [48]. Probe H1 consisted of a fluorescein dye attached to a triphenylphosphonium group and a nitrobenzyl photoactivable protecting group (Scheme 2c). Upon irradiation with UV light (365 nm), the nitrobenzyl part produced an aldehyde derivative, which served as the H₂S_n recognition site. H₂S_n attacks the aldehyde group to form a persulfide intermediate, which then should undergo cyclization to liberate the fluorophore and generate a side product (4-hydroxybenzo[d][1,2]dithiin-1(4H)-one). H1 showed a turn-on of fluorescence at 525 nm only when it was irradiated with UV light along with H₂S₂ in the solutions. The detection limit was calculated to be 150 nM. The targeting ability of H1 was confirmed by counterstaining with MitoTracker Green (MTG) (R_r = 0.72). Although this photo-triggered probe was interesting, the authors did not provide experimental support for the proposed detection mechanism. This aldehyde-based intermediate may also possess some problems as 2-formyl carboxylate is a well-known H₂S recognition site [49,50], and the aldehyde group has a high reactivity towards free cysteine [51,52,53].