Recent Advances in Activatable Organic Photoacoustic Probes for In Vivo pH‐Detection

All life forms require pH homeostasis. The disorder of pH homeostasis is associated with many severe diseases, suggesting the significance of pH‐detection for theranostic diseases. Photoacoustic imaging (PAI) technology has piqued great research interests in spatiotemporal detection of pH in vivo due to its non‐invasiveness and non‐ionization, as well as the high resolution and deep tissue penetration. This mini‐review outlines the most recent advances in activatable organic PA probes for pH‐detection with respect to molecular design, response mechanisms, and their in vivo applications. The basic understanding of PA probes is clarified. The pH‐response mechanisms of the PA probes at the molecular level are particularly emphasized to elucidate the structural design strategies. Finally, the challenges and perspectives, mainly focusing on quantitative detection, are discussed. This review aims to pave the way for further exploration of responsive organic PA probes for in vivo pH‐detection.


Introduction
Proton (H + ) plays a critical role in intracorporal acid-base equilibrium chemistry and participates in ion transportation, cell proliferation, enzymatic activity management, and so on. [1][2][3][4] pHdysregulation can lead to the inhibition of several enzymes, aberrant redox status, or neurotoxicity, which may increase the risk of numerous diseases including nephropathy, infection, fibrosis, ischemia, and inflammation. [5] Moreover, excess H + -caused acidosis is a remarkable characteristic of the tumor microenvironment (TME). The pH varies in different growth stages of tumor tissues, as well as in different organelles of tumor cells. For example, the mitochondrion shows a pH of 7.5-8.2, while the pH DOI: 10.1002/adsr.202200075 is around 7.2 for the cytoplasm, and even much lower for lysosome (in the range of 3.8-4.7) in tumor cells. There were some reported prodrugs based on the acidic TME. [6][7][8] On the other hand, the acidic TME can not only promote tumor metastasis and invasion but also inactivate some important enzymes that lead to the multidrug resistance of tumors in chemotherapy. Therefore, pH detection is of significant importance for tumor theranostics. [9] In this context, much effort has been devoted to in vivo pH detection, which could afford in situ 3D information in either a qualitative or quantitative manner, and even realize dynamic traces in real-time in a living body. Combined with in vitro detection, in vivo pH detection could be a powerful approach to comprehensively illustrate biological actions, and promote the development of precision diagnosis and treatment of diseases, especially for oncology. Based on different imaging mechanisms, diverse detection technologies have been exploited for in vivo pH detection so far including positron emission tomography (PET), [10] computed tomography (CT), [11] single photon emission computed tomography (SPECT), [11] magnetic resonance imaging (MRI), [12] ultrasound imaging (US), [13] fluorescence imaging (FI), [14] Raman mapping, [15] and photoacoustic imaging (PAI). [16] However, ionizing-based technologies, such as SPECT, PET, and CT, arouse serious safety concerns while others suffer from either low resolution or poor specificity, or high costs (Figure 2a). More information on the strengths and shortcomings of individual imaging technology can be found elsewhere. [17] By contrast, the burgeoning PAI inherits the virtues of both optical and acoustic signals, [18,19] exhibiting the desired wavelengthtunability, noninvasive and nonionized properties, good spatiotemporal resolution, and especially prominent tissue penetration depth. [16,20] The up-and-coming imaging technology has been utilized in diverse fields like ion visualization, [21] imaging of blood vessels, [22] and screening of oxygen metabolism. [23] The structural design and engineering approaches of the widely studied non-activatable PA contrast agents have been reviewed a few recently. [24][25][26][27][28][29] However, the toolbox of activatable organic PA probes for pH-detection has remained unestablished yet: the amount of such PA probes is woefully inadequate, which requires researcher's endeavor to develop new and well-performed PA probes for in vivo pH-detection. [30,31] We, herein, comprehensively summarized and reviewed the available organic PA probes for in vivo pH-detection. Of particular emphasis in this review is to systematically exemplify the pH-detection mechanisms at the molecular level, as well as demonstrate the engineering methods of organic PA probes (Figure 1). Besides the basic principle of PA technology introduced in Section 2, the challenges and outlooks were discussed at last. This review aims to provide a guide for further structure-tuning of pH-responsive PA probes to realize in vivo and real-time detection of pH, and thereby benefit disease diagnosis and therapy.

Basis of Activatable Organic Photoacoustic Probes
Although pioneer work on the photoacoustic effect had been done by Alexander Graham Bell for several centuries, further studies on the mechanism of the PA phenomenon has been made until the 1990s, profiting from the development of laser devices, as well as ultrasound detection technology and computer science. [32] According to the Jablonski energy diagram, when a molecule absorbs light energy, the transition of an electron from the ground electronic state (S 0 ) to the singlet first excited state (S 1 ) and even the second excited state (S 2 ) occurs. Then, the photogenerated excited electrons can rapidly relax back to the S 0 state through either radiative or irradiative approaches. The former generates fluorescence or triplet state (T 1 )-based phosphorescence while the latter transforms the energy to heat via molecular vibration relaxation, followed by the emergence of PA signals (Figure 2b). Therefore, the signaling process can be concisely recapitulated as the four steps ( Figure 2c): 1) the molecule or biological tissue is excited by a nanosecond laser; 2) the irradiative relaxation of the excited molecule occurs, and the transformed thermal energy gives rise to an instantaneous high temperature, which further leads to a thermoelastic expansion and subsequently a dramatically increased pressure; 3) the pressure jump-caused ultrasound signal (called PA waves) is collected by a broadband ultrasound transducer; 4) signal reconstruction is carried out by a computer to acquire an imaging output. [33][34][35] Ultrasound wave is less scattered by tissues than light, which offers enhanced penetration and resolution while the optical signal displays higher contrast than acoustic signal. PA imaging, as an optical and ultrasound mixed imaging modality [36][37][38] inherits the superiority of both optical (high contrast and wavelength-tunability) and acoustic signal (high resolution and deep permeability), thus achieving an improved signal-to-noise ratio without tissue damage. Besides, multiscale, multispectral, and multifunctional PA imaging technology is available through the combination of diverse contrast agents to meet diverse demands in clinical applications. [39,40] PA contrast agent, which absorbs light energy and emits PA signal, is a critical factor for in vivo imaging. [41] Compared with conventional non-responsive PA probes, activatable probes interpret molecular or biological events into readable imaging data with a higher signal-to-noise ratio, due to reduced background interruption. This kind of probe normally consists of a PA signaling part (called PA sonophore) and an analyte-responsive part. PA sonophore includes endogenous (such as oxyhemoglobin, melanin, lipids, and collagen) [42] and exogenous chromophores, which can be divided into organic, inorganic, and complex materials. [29] Among them, inorganic materials are usually subject to poor biodegradation-caused long-term toxicity and complicated preparation procedures. [43,44] By contrast, organic PA materials have gained popularity recently owing to their welldefined structure, rapid metabolism, excellent biological characteristics, ease of structural modification, and thereby, feasible performance tailoring at the molecular level. Nevertheless, there is still much room for improvement in their performance when it comes to photobleaching problems and low retention ability in the target. Especially for organic polymers, the retention by the reticuloendothelial system and low accumulation of the polymeric probes in the target position will dramatically diminish the PA imaging resolution. [45][46][47][48][49] The analyte-responsive group of the PA probe determines their selectivity and sensitivity. The interaction between responsive moiety and analyte results in a variation of PA signal, endowing the probe with activatability. The changes in either maximum absorption wavelength or absorption intensity can be attributed to the structural transformation-caused chemical or physical properties alteration. In this case, the structure of the PA probe can be rationally tailored in a target analyst-oriented manner. The recognition process also can be reversible or irreversible which relies on whether the structural change is recoverable after responding to the analyte. Furthermore, activatable probes can be divided into turn-on/off and ratiometric types according to signal output variations. Turn-on/off probes show a dramatically increased/decreased signal intensity while the ratiometric type displays an absorption peak shift. Ratiometric PA probes afford relatively more accurate quantitative information than the on-off type due to less background disturbance. Therefore, there is a tendency to design ratiometric molecular probes or construct ratiometric nano-engineered probes by adopting another inner-refer molecule that has no signal change during the analyte recognition process. [50] In addition, excellent responsive specificity, high absorbance (molar extinction coefficient > 10 4 m −1 cm −1 ) in the first even second window of the near-infrared region (NIR-I or NIR-II), low quantum yield, good photothermal conversion ability, and biocompatibility are much desired for activatable PA probes. [22,51,52] To achieve this, various parameters, for example, solubility, excited state absorption, triplet state contributions, relaxation kinetics, photobleaching effect, [53][54][55] and even the number of rotatable bonds [56,57] should be comprehensively considered. Finally, it is also of much importance for the probe to well-match the laser instrument requirement.

Activatable Organic PA Probes for In Vivo pH-Detection
As aforementioned, H + is one of the most important species that participates in the acid-base equilibrium chemistry in the living system. It has an outer valence electron configuration similar to that of alkali metal ions but is the smallest known positively charged particle. Strictly speaking, H + is formed by the separation of the nucleus of an H atom from its accompanying electron. Then, the H nucleus consists of a particle carrying a unit of positive electric charge. Owing to coulombic interaction, oppositely charged protons and electrons get attracted, and thereby, the small bare nucleus can easily interact with other particles such as electron-rich atoms and molecules.
The H + -responsive probe is closely associated with pH detection or TME-based tumor imaging. Apart from the protonation of the PA probes, in vivo H + imaging could be achieved by H +induced aggregation, ring-opening, and other complicated dualstimuli response processes of probes. Some of the representative H + -responsive probes are summarized in Table 1. In general, the involved recognition mechanisms are mainly based on the high affinity of H + to electron-rich atoms, which causes subsequent reactions.

Protonation
Protonation is a major responsive mechanism for H + detection, which usually takes place on the N, O, or other electron-rich atoms within the probes. Cyanine and its derivatives are one of the typical NIR dyes, which feature a high molar absorption coefficient, but suffer from poor water solubility. [58] To overcome the obstacle, Lin's group developed a pH-sensitive cyanine-based PA nanoprobe (LET-4) to carry out real-time in vivo gastric acid detection (Figure 3a). [59] A pH-responsive NIR PA probe (3-butene-1-amine-modified IR1061) was embedded in the hydrophobic core of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-glycol)-2000 ] (DSPE-PEG 2000 ) assembly to form LET-4, where PEG acted as a probe stabilizer and permitted better water solubility. When LET-4 was protonated in an acidic environment, the absorbance and PA signal at 808 nm enhanced as shown in Figure 3b,c (pH was in the range from 3 to 7). The mixture of LET-4 and sodium bicarbonate (NaHCO 3 ) solution (pH 8) was administered to nude mice through gavage to monitor stomach pH level in vivo, where the alkaline solution was used to neutralize the gastric juice and stimulate the secretion of gastric acid. Figure 3d demonstrates that PA 808 intensity became prominent 30 min after oral administration, reached a maximum at 90 min, and then almost disappeared after 240 min, indicating that the mice with normal gastric function could gradually excrete gastric acid to recover the normally acidic environment of the stomach (pH ≈3) within 60 min, and LET-4 could finally completely get metabolized from the stomach. The intensity of the PA 808 signal at the stomach was found to be 3.11-fold higher than that of the control group, indicating the feasibility of LET-4 nanoprobe for in vivo PAI of gastric acid in real-time.
Although cyanine derivative, represented by indocyanine green (ICG), has been explored extensively in the past few years, its poor photostability is still a big limitation in clinical practice. [58] In contrast, another class of commonly used NIR dyes, namely, croconium (Croc) derivative, exhibits sharp and strong NIR absorption and excellent photothermal stability. [60] Besides, the Croc dyes could transform from the basic anionic form to the zwitterionic acidic form, exhibiting a redshift of the absorption wavelength with decreased environmental pH, due to the protonation of the N in the molecule. The large wavelength shift and strong absorption results in high pH sensitivity. However, poor aqueous solubility significantly restricts the wide range of applications of the hydrophobic Croc dyes in biological systems. Significant research efforts have been devoted to the study of surface engineering of Croc dyes to circumvent the obstacle. Later, the same group successfully synthesized a Croc dye-anchored cell membrane vesicle (LET-5) for tumor imaging and imaging-guided photothermal therapy (PTT) (Figure 3e). [61] The hydrophobic Croc dye was first covalently bonded to the hydrophilic PEG to generate an amphiphilic Croc-PEG5K, which was further anchored to the red blood cell membrane-based (RBCm) vesicle to form biocompatible LET-5. The maximum absorption wavelength of LET-5 gradually shifted from 630 to 700 nm, and a new peak at 760 nm also appeared with increased absorbance at decreased pH, due to the protonation of the embedded Croc dye (Figure 3f). It was found that the NIR fluorescence (NIRF) NIRF 820 and PA 780 intensities of LET-5 were significantly boosted in tumor tissues compared to that in normal muscle tissues, indicating that LET-5 was activated from "off" to "on" state by acidic TME (Figure 3g). Noteworthy, nano-engineering of Croc-PEG5K with RBCm vesicles significantly reinforces the stability, extends the blood circulation time, reduces the liver uptake, and improves the renal evacuation ability of LET-5, resulting in high tumor-accumulation and probe-retention and consequently the promoted PTT effect relative to the free Croc-PEG5K probe. It indicates the significance of the nano-engineering of organic probes discussed in Section 2.
Another example of the importance of nano-engineering is given by Yang's group. Polyaniline (PANI) is a type of commercial pH-responsive stable polymer. It can undergo a structural transformation from emeraldine base (EB) to emeraldine salt (ES) state in strong acid conditions due to the protonation of imine moieties. However, the required transformation pH is much lower than that of TME (5.4-7), badly confining the application of PANI in cancer theranostics. To overcome the limitations of poor aqueous solubility and low-pH protonation for PANI, they developed bovine albumin (BSA)-engineered pH-responsive nanoprobe, namely, BSA-PANI. [62] The monomer aniline (ANI) was first polymerized to PANI assisted by BSA and Fe 3+ ions simultaneously, and then the hydrophilic BSA and hydrophobic PANI were self-assembled into BSA-PANI NPs with excellent water dispersibility and biocompatibility. More importantly, the isoelectric point of BSA (4.7) was significantly elevated after forming BSA-PANI assemblies (5.61), reflecting the strong interaction between BSA and PANI. This results in a self-doping effect by intermolecular acid-base reactions between carboxyl groups on BSA and imine groups on PANI, thereby reducing the degree of deprotonation of PANI and promoting the conversion from EB to ES state at pH < 7, as demonstrated in Figure 4a. This process resulted in the obvious redshift of the absorption peak from visible (570 nm) to NIR region (800 nm) and the generation of PA signal when the pH changed from 8 to 3 (Figure 4b). The in vivo  [59] Copyright 2019, American Chemical Society; e-g) Reproduced with permission. [61] Copyright 2021, Elsevier B.V.
PAI capacity of the nanoparticle was examined by using a 4T1 cell-treated mouse model (Figure 4c). It was found that the PA intensity of the tumor was four times higher than that of muscle tissues at 5 min post-injection. Besides, the PA signal of the tumor was dramatically amplified at 12 h post-injection (i.v.), and then significantly attenuated after 24 h, while the control group always exhibited a constant but weak PA signal, illustrating that the assemblies were activated under tumor pH.
Except for the protonation of the N atom as discussed above, the protonation of the O atom could also be utilized to construct pH-responsive PA probes. On the other hand, single-channel PA probes are often limited by fluctuations in the machine itself and the environment. In order to achieve more accurate imaging, Pu's group elaborately designed a multicomponent ratiometric H + -sensitive PA nanoprobe SON 50 , wherein a pH-responsive boron-dipyrromethene deriva-tive (pH-BDP) and an inert PA matrix (F-DTS) were entrapped in an amphiphilic copolymer, namely, poly(ethylene glycol)b-poly(propylene glycol)-b-poly(ethylene glycol) (PEG-b-PPG-b-PEG). [63] The hydroxyl groups on the pH-BDP backbone could be protonated in acidic conditions. The pH-BDP worked as a pH indicator and also an electron acceptor, while the pH-inert internal reference semiconducting oligomer F-DTS was used as an electron donor to facilitate the intraparticle photoinduced electron transfer (PET). The PET-caused fluorescence quenching of F-DTS in turn elevated the PA signal intensity of SON 50 by ≈3.1fold at 680 nm compared with the pH-BDP non-doping SON 0 , indicating the signal amplification effect of pH-BDP within the ingenious nanoprobe (Figure 5a). Moreover, the super-strong stability was impressed for the assemblies SON 50 , whose size remained almost unchanged for more than 24 days for solutions at different pH, as nano-doping of the planar pH-BDP could well  stabilize the nanoprobe, which was possibly due to the strong intramolecular -stacking and hydrophobic interaction. For in vivo performance, PA 680 intensity (from pH-inert F-DTS) remained unchanged, while PA 750 intensity (generated by pH-BDP) diminished dramatically with the decrease of pH, and a linear relationship between the ratio of PA 680 /PA 750 and pH could be well established for in vivo real-time H + imaging in tumor tissues (Figure 5b,c). The ratiometric PA intensity (ΔPA 680 /ΔPA 750 ) in the tumor was around 1.9-fold of that in muscle. Given the above-mentioned discussion, it was found that it is feasible to design H + imaging PA probes by introducing protonable atoms, in particular, N atom on the skeleton of dyes.

Ring-Opening
Nano-engineering of probes offers an effective pathway for performance optimization, yet still inevitably suffers from tedious synthesis, multistep integration procedures, and risk of leakage. Significantly more efforts have been devoted to the synthesis of biocompatible molecular PA probes. Recently, Ohe Kouichi's group reported a pH-sensitive probe, CypHRGD, with a peptide grafted in the skeleton. [64] The ICG derivate was applied in the NIRF/PA dual-modal imaging of tumors. The introduction of short peptide c(RGDfK) (cRGD) aimed at improving tumortargeting, as well as enhancing biocompatibility, by avoiding dye agglomeration. The substituted bulky phenyl group was perpendicular to the -conjugated plane, which further eliminated the probe stacking, while the sulphonate group of the probe was designed to increase the solubility in a physiological environment. The nucleophilic thioether bond on the side chain was gradually cleaved to form a ring-opened structure with a decrease in the pH (pH ranging from 6 to 4), accompanied by a significant increase in the NIR absorption (800 nm) and emission (825 nm). Moreover, the PA brightness of CypHRGD at pH = 4.2 was 5.7-fold of that at pH = 7.2, indicating the robust pH responsiveness of the nanoprobe between the two pH conditions. Overall, the incorporated cRGD prominently facilitated the tumor recognition by shortening the time for specific tumor imaging (6 h post-injection) than the control group without cRGD (24 h postinjection), though the probe also accommodated in the liver.

Dechelation
Unlike the above-stated research, excellent water solubility and blood compatibility could also be achieved by constructing a zwitterionic structure. Recently, Xing's group designed such a pHresponsive PA probe (Fe-ZDS). [65] It is a water-soluble Fe(III)catechol complex as displayed in Figure 6a. H + could trigger the dechelation of ligands toward metal ions. Therefore, the saturated tris-coordination form of Fe-ZDS could convert into a bisor mono-coordination form in an acidic environment, accompanied by a redshift of the light absorption even to the NIR region. The structural transformation of Fe-ZDS could significantly influence the ligand-to-metal charge transfer bands, which resulted in the change of the light absorption and switching off of the PA signal. Specifically, the probe exhibited a maximum light absorption at 520 nm at pH 7.4, which shifted to 720 nm at pH 5 with a more than three-time increment in absorption coefficient at 808 nm (Figure 6b). The higher NIR light absorption of Fe-ZDS in a slightly acidic environment than in a neutral condition boosted the PAI and PTT toward the tumor. After direct injection of Fe-ZDS in tumor and muscle tissues, respectively, a twofold stronger PA signal at 808 nm than that at 680 nm was observed in the tumor site, while in the muscle region, a conspicuous PA signal was recorded only at 680 nm, confirming that Fe-ZDS was activated in the tumor, but not in muscle. Another advantage of the zwitterionic form is rapid renal clearance, which is of much importance for declining the risks of long-term toxicity. The Fe (III)-dopa core . Reproduced with permission. [66] Copyright 2021, American Chemical Society.
could also work as an MRI contrast, thus leading to the achievement of MRI/PAI dual-modal imaging-escorted PTT for cancer treatment (Figure 6c). However, the maximum molar extinction coefficient in acidic conditions was calculated to be around 2500 m −1 cm −1 at 808 nm, much lower than that of ICG (≈1 × 10 5 m −1 cm −1 at 780 nm).
Briefly, the ring-opening or dechelation process of the probes is intrinsically attributed to the high affinity of H + to electronrich S and O atoms, similar to the protonation mechanism. However, the affinity dramatically weakens the original C─S covalent bond or metal-ligand coordinate bond to finally form quite different NIR-absorber structures, while the protonation of probes does not get involved in remarkable skeleton transformation. It provides a possible strategy to design PA probes for H + imaging, that is, introducing nucleophilic groups with relatively weak bond strength in a pre-NIR dye structure.

Multi-Level Process
H + imaging can also be realized by cascade processes as the ion-triggered cleavage or protonation of probes may induce further chemical reactions or physical processes, such as charge inversion, aggregation, or size transformation of the probe, which varies the PA signals. For instance, water-soluble organic carboxylate could convert to insoluble carboxyl after being protonated in a slightly acidic medium, indicating the pH-responsiveness of the group. Keeping this in mind, 3,4-ethylenedioxythiophene-alt-3,4-ethylenedioxythiophene copolymer-bearing carboxylate side chains (PPE) were developed as a pH-activatable PA probe. [66] The light absorption of PPE was easily red-shifted to the second near-infrared (NIR-II) region by oxygen doping in an ambient condition without any assisted oxidants (Figure 7a). Moreover, the carboxyl groups on side chains led to significant improvement in the solubility at a neutral physiological pH and played a key role in the aggregation of PPE in an acidic environment. With the protonation of carboxylate side chains at pH < 7, the solubility of PPE is reduced, causing the aggregation of the polymer to large NPs (Figure 7b). It led to 3.1-and fivefold enhancement of the PA 1100 signal at pH 6.5 and 5.5 (vs pH 7.4), respectively. For in vivo performance, the PA signal of PPE was much stronger in tumor tissue than in muscle (Figure 7c), with a 3.4-fold enhancement obtained. The study offered an easy and surfactant-free strategy to construct aqueous compatible pH-responsive polymeric NIR-II PA probes with excellent stability (keeping unchanged for up to 18 days) and deep tissue imaging depth (4.4 cm with PPE concentration of 0.5 mg mL −1 ).
To overcome the hurdle of the variability of machines and the environment, much effort has been devoted to the development of the aforementioned ratiometric probes. [28] However, the achievement of accurate detection is still a challenging task due to the sophisticated environment of cells. For example, some inflammatory tissues could also over-express certain stimuli similar to tumor tissues. This may cause the undesirable premature activation of the single-stimulus responsive probe during blood circulation, curtailing the tumor-specificity of PA probes and even conveying false imaging information. In this case, more attention has been paid to dual-stimuli responsive PA probes, which could undergo a complicated activation process triggered by two analytes in a synergistic manner. To date, a few pHsensitive dual-stimuli responsive PA probes have been developed. Recently, Zhang's group synthesized an exquisite ratiometric adenosine triphosphate (ATP)-H + dual-stimuli responsive PA probe. [67] It was constructed using an ATP-H + receptor unit and a fluorescence resonance energy transfer (FRET) dyad, in general. The FRET dyad consisted of a NIR silicon rhodamine (Si-Rh) as an energy donor and a dye (CS) as an energy acceptor.  [67] Copyright 2021, American Chemical Society; e-f) Reproduced with permission. [68] Copyright 2020, American Chemical Society.
Moreover, piperidine-4-carboxylic acid was used as a linker of the dyad to strengthen the steric hindrance and thereby prevented Si-Rh from being interfered with by glutathione (GSH) in the tumor. The result indicated that the spirolactone opening of CS dye only occurred in the presence of ATP under tumor pH, indicating the synergistic effect of H + and GSH during the probe activation (Figure 8a). The FRET from the Si-Rh donor to the dye acceptor resulted in the redshift of the fluorescence emission and the emergence of a new absorption peak in the NIR region (Fig-ure 8b). The PA signal at 680 nm was gently strengthened due to the probe enrichment in the tumor, while that at 740 nm increased rapidly with the accumulation and activation of the probe, leading to the enhancement of the PA 740 /PA 680 ratio over time (Figure 8c,d).
The citraconic group and cysteine (Cys) precursor (1,2aminothiol derivative) are known as H + and GSH recognizable functional groups, respectively. They were both linked to a 2-cyanobenzothiazole (CBT) motif to obtain an H + -GSH Highly relied on the affinity of proton to electron-rich atoms.
Ring-opening Similar to the protonation mechanism, which is intrinsically attributed to the high affinity of H + to electron-rich S and O atoms. But finally form quite different NIR-absorber structures, while the protonation of probes does not get involved in remarkable skeleton transformation.
A universal response unit, and thus easy to achieve structural variability.
Elaborate skeleton design of the dye precursors is extremely important.

Dechelation
Excellent water solubility and blood compatibility could also be achieved by constructing a zwitterionic structure.
Usually confined to be metal complexes.

Multi-level process
Dual-stimuli responsive probes Multiple responsive moieties in the structure to realize synergistic functions.
Elaborate skeleton design of the dye precursors is extremely important.
dual-stimuli responsive probe Cy-1 which was activated within the tumor only. [68] A NIR dye Cy7 was also covalently connected to the structure for signaling and PTT. According to the pHresponsive multi-level process, the tumor-overexpressed H + and reductive GSH induced the hydrolysis of the citraconic motif and the cleavage of the disulfide bond. In sequence, an active intermediate Cy-1-Core was acquired, followed by an instantaneous intermolecular condensation between CBT and Cys to obtain an amphiphilic cyclized dimer. Then, the dimer assembled into large NPs (Cy-NPs) via strong intramolecular -interaction, which promoted the enrichment and retention of the probe in the tumor (Figure 8e). Therefore, the PA signal of Cy-1 in acidic conditions (pH 6.5, GSH = 10 mm) was brighter than that in neutral buffer, and the increasement was quantified as high as 2.9-fold. For in vivo imaging performance, Cy-1 was distinctly activated in the tumor region, compared with the reference probe Cy-Scr, which could be reduced by GSH but could not be hydrolyzed by acid, as illustrated in Figure 8f.
Tables 1 and 2 provide a general conclusion that H + recognition is mainly based on the high affinity of proton to electron-rich atoms, and the protonation of probes is an efficient and extensively explored strategy for H + imaging. Besides, to design an H + -activatable probe by bond cleavage to form NIR dye, an elaborate skeleton design of the dye precursors is extremely important. On the other hand, some probes show a relatively narrow or unmatched pH response range or unsatisfied biocompatibility as required, rational nano-engineering of probes may provide wonderful access to solutions or performance optimization as desired. Among the above-listed probes, the Croc derivative features excellent photostability and intrinsic H + -sensitivity. Undeniably, a lot more effort could be put into its chemical structure modification to tailor properties such as the longer wavelength of light absorption. As far as the dual-stimuli responsive probes are concerned, more consideration is necessary for the rational embedding of multiple responsive moieties in the structure to realize synergistic functions.

Challenges and Outlook
The organic PA probe-mediated pH-detection is meaningful for in situ imaging of pathological tissues and in the real-time trac-ing of physiological activities. However, the reported organic PA probes for in vivo pH-detection are quite inadequate. Current problems mainly lie in the following three aspects: i) the dearth of high-performed organic PA probes; ii) the lack of quantitative detection methods; and iii) the instrumental limitations. Specifically, owing to the extremely low intracorporal concentration of H + and serious interference for pH-detection in living systems, the sensitivity and selectivity of the reported PA probes are unsatisfactory. The poor water solubility and low tissue retention time are also required to be well-addressed. At last, accurate quantitative detection of pH and instrumental limitations should also be settled urgently.
To make further progress, both the exploration of novel organic PA probes and the rational structural modification of the existing molecules are useful approaches. First of all, it is necessary to unveil the relationship between chemical structures and properties of PA probes. Noteworthy i) the photophysical properties (photostability, strong NIR region absorbance), ii) chemical properties (chemical stability, sensitivity, selectivity, and watersolubility, high photothermal conversion efficiency), iii) biological characteristics (long blood circulation, targeting ability, low toxicity, biocompatibility, biodegradability), and iv) economic cost should be comprehensively considered and evaluated. For example, strong absorption of PA probes in the NIR-II region is desirable, as the NIR-II absorption window (1000-1700 nm) shows much lower tissue absorption and scattering, and suppressed self-luminescence from tissues with regard to the NIR-I region. Therefore, NIR-II PA probes could achieve deeper tissue penetration, better spatial resolution, and higher contrast of imaging outcomes. However, the strong NIR absorption of the PA probe usually involves large structural conjugation, which can lead to high molecular rigidity and consequently poor water solubility. To improve the biological performance, increasing the targeting capability of the probes will effectively increase their enrichment in the target and thus boost the S/N ratio for PAI. Moreover, lengthening the blood circulation time and increasing the biostability of the probes could likely offer better PA performance. Second, new detection methods or responsive mechanisms for optimal in vivo pH-detection or quantitative detection are highly desired, for example, i) reversible pH-detection to achieve dynamic imaging; moreover, it could eliminate the disturbance of the activated probes after they leave the target sites; ii) multi-lock probes; and iii) ratiometric molecular probes to avoid component-leakage, which is necessary for high-precision pH-detection. Third, it is required to overcome instrumental limitations such as poor resolution, narrow dynamic range, and confined laser wavelength for boosted resolution and contrast imaging outcomes. [24,28,69,70]