English

• Mitochondria generate energy through the respiratory chain to support numerous vital metabolic processes in cells [1, 2]. Therefore, maintaining mitochondrial homeostasis is crucial for cellular growth [3]. Once damaged, mitochondria would be selectively sequestered with the following degradation process known as mitophagy [4]. Aberration in this process can contribute to various diseases, such as neurodegenerative and cardiovascular disorders [5]. Interestingly, the occurrence of mitophagy usually results in a significant reduction in mitochondrial pH, from approximately pH 8.0 under normal physiological conditions to around pH 5.5–6.5 [6]. Therefore, developing a real-time and accurate method for detecting mitochondrial pH values holds immense significance in comprehending the pivotal functions of mitochondria under both physiological and pathological circumstances.

  Small molecule fluorescent probes possess the advantages of non-invasiveness, high sensitivity, and good specificity, which are extensively utilized in imaging physiological processes within living cells [7-10]. Currently, reported mitophagy probes primarily utilize naphthalimide or rhodamine derivatives. For example, Kim et al. developed the naphthalimide-based probe to monitor mitophagy, incorporating piperazine as a proton binding site and triphenylphosphine as the mitochondrial targeting group [11]. Zhang et al. designed a rhodamine-derived ratiometric pH fluorescent probe targeted explicitly to mitochondria for visualizing pH alterations during rapamycin-induced mitophagy [12]. To summarize, the probes employed for monitoring the mitophagy process must fulfill three fundamental requirements: (i) Excellent mitochondrial targeting capability, (ii) sensitive pH responsiveness, and (iii) emission wavelength located in the "near-infrared (NIR) window" (650–900 nm) of biological imaging [13]. Therefore, neither naphthalimide nor rhodamine derivatives are ideal mitophagy probes because most of their emission wavelengths are centered at 350–600 nm. Cyanine dyes usually exhibit instinctive capability in targeting mitochondria because of their cationic structure [14, 15], and the multi-methine structure's derivatization allows emission wavelength adjustment to the "NIR window" [16]. Nevertheless, normal cyanine dyes lack pH-responsive functional group [17, 18]. Azaindole was developed and introduced in our former works to construct the NIR photosensitizers [19]. Besides enlarging the conjugation system, the exposed indole nitrogen in azaindole is also an excellent proton-binding site for pH response [20, 21].

  As a proof of concept, an asymmetric pentamethine cyanine dye, Cy5.5-H-CyN, was constructed to continuously monitor the mitophagy process in living cells (Fig. 1A). Incorporating azaindole preserved not only the intrinsic mitochondrial targeting ability but also redshifted fluorescence emission to the "NIR window". Moreover, upon proton binding, the intramolecular charge transfer (ICT) effect of Cy5.5-H-CyN was attenuated, enabling the generation of ratiometric fluorescence signals for pH at two emission wavelengths (662 and 710 nm). Notably, Cy5.5-H-CyN exhibits a robust linear relationship within the pH range of 5.0–7.0 in both solution and cellular environments. The response of Cy5.5-H-CyN to pH variations is rapid, sensitive, and reversible. Importantly, by utilizing ratio fluorescence signals, quantitative analysis of mitochondrial pH can be achieved under diverse mitophagy stimulation conditions, such as rapamycin, thus facilitating real-time monitoring of the mitophagy process.

  Figure 1

  

  Figure 1.  (A) The chemical structure and pH sensing mechanism of Cy5.5-H-CyN in mitochondria. (B) The pH-dependent absorbance spectra of Cy5.5-H-CyN (Inset: the solution color of Cy5.5-H-CyN in PBS with different pH values). (C) The pH-dependent emission spectra of Cy5.5-H-CyN (λ_ex = 640 nm). (D) The fluorescence intensity of Cy5.5-H-CyN in PBS at different pH values (λ_ex = 640 nm). (E) The pH-dependent emission spectra of Cy5.5-H-CyN (λ_ex = 488 nm). (F) The fluorescence intensity ratio Fl. λ_{ex 640 nm}/Fl. λ_{ex 488 nm} vs. pH (4.0–9.0). (G) Linear fitting of Fl. λ_{ex 640 nm}/Fl. λ_{ex 488 nm} vs. pH (5.0–7.0).

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  Cy5.5-H-CyN was synthesized via a four-step route, and Cy5.5-H-Cy5 was prepared as a reference compound. The synthesis process was described in detail in Scheme S1 (Supporting information). Both compounds were characterized by ¹H nuclear magnetic resonance spectroscopy (NMR), ¹³C NMR, and high-resolution mass spectroscopy (HRMS). The characterizations are shown in Figs. S1–S8 (Supporting Information). Additionally, single-crystal X-ray diffraction analysis was employed to confirm the structure of Cy5.5-H-CyN, as shown in Fig. S9A (Supporting information). The Cy5.5-H-CyN exhibited an interlaced arrangement with parallel orientation in the crystal lattice (Fig. S9B and Table S1 in Supporting information).

  Firstly, the spectral behavior of molecules with varying pH was investigated. The absorbance of Cy5.5-H-CyN at 678 nm gradually decreased as the pH changed from 9.0 to 4.0, while the absorbance at 526 nm increased simultaneously, with a blue-to-pink color change of the solution (Fig. 1B). Uniform parameter settings were employed for testing to ensure consistency for subsequent intracellular fluorescence imaging. When the probe was excited at 640 nm, there was no significant change in fluorescence at 710 nm (Fl. λ_{ex 640 nm}) within a pH range of 7.0–9.0 (Fig. 1C). However, below pH 7.0, Fl. λ_{ex 640 nm} exhibited a noticeable decrease (Fig. 1D). This phenomenon can be attributed to the weakening of the ICT process following the protonation of the indole nitrogen atom in Cy5.5-H-CyN, resulting in a blue shift in molecular absorption wavelength and pronounced fluorescence quenching. These findings fully demonstrate that Cy5.5-H-CyN possesses pH-responsive capabilities and exhibits fluorescent changes within the mitophagy-relevant pH range. However, due to the absence of proton-binding sites, Cy5.5-H-Cy5 did not possess pH-responsive capability, resulting in no absorption and fluorescence changes with pH changes (Fig. S10 in Supporting information). When the probe was excited at 488 nm, a gradually intensified fluorescence peak (Fl. λ_{ex 488 nm}) was observed at 662 nm (Fig. 1E) as buffer pH decreased, corresponding to the absorption peak at 526 nm and representing the fluorescence peak of the Cy5.5-H-CyN structure after proton binding occurred, the specific absorbance values corresponding to the two wavelengths are presented in Table S2 (Supporting information). Both fluorescence emissions are located within the "NIR window", which enhances cell imaging by reducing background interference [22]. The pH and the corresponding Fl. λ_{ex 640 nm}/Fl. λ_{ex 488 nm} value can be fitted to an "S" curve based on the Henderson-Hasselbalch equation. The pK_a value of Cy5.5-H-CyN was approximately 6.5 (R² = 0.99) (Fig. 1F). Moreover, there was a robust linear relationship between Fl. λ_{ex 640 nm}/Fl. λ_{ex 488 nm} and pH values ranging from 5.0 to 7.0 (R² = 0.99) (Fig. 1G). These results demonstrated that Cy5.5-H-CyN exhibited excellent ratiometric fluorescence response for accurately monitoring pH changes.

  Water solubility is a critical parameter for bioimaging probes [23]. To demonstrate the solubility of Cy5.5-H-CyN under both mitochondrial physiological and acidic conditions, absorption spectra were measured at 678 nm (pH 8.0) and 526 nm (pH 4.0) for probes with increasing concentrations, and then the absorbance values were then correlated with the respective concentrations through fitting analysis. Remarkably, there was an excellent linear relationship between absorbance intensities and probe concentrations under both pH conditions (Fig. S11 in Supporting information). The comprehensive description of the additional photophysical properties of Cy5.5-H-CyN were provided in Tables S3 and S4 (Supporting information).

  To further investigate the adaptability of Cy5.5-H-CyN in monitoring mitophagy in living cells, pH cycle testing was conducted. As shown in Fig. S12 (Supporting information), after 4 cycles, Cy5.5-H-CyN still demonstrated satisfactory reversibility of fluorescence intensity with little loss (Fig. S12A), indicating its excellent response to pH changes for real-time pH monitoring. Subsequently, the selectivity of Cy5.5-H-CyN towards common biological disruptors, including metal cations, anions, reactive oxygen species, biothiols, and amino acids, was evaluated. At pH 7.4, the above chemicals did not interfere with the fluorescence properties of Cy5.5-H-CyN (Fig. S12B), highlighting its specific response to pH in complex biological environments, such as living cells and organelles. The fluorescence intensity of Cy5.5-H-CyN was recorded every minute for 30 min under both pH conditions, and there was no significant change (Fig. S12C), confirming its fluorescence stability. To investigate the impact of viscosity on the testing process, mixed solutions containing ethanol and 1, 5-pentanediol were prepared with different proportions. The fluorescence spectra of Cy5.5-H-CyN and Cy5.5-H-Cy5 in solutions with different viscosities were measured, and I/I₀ (the ratio of fluorescence intensity (I) to initial fluorescence intensity (I₀)) was calculated to assess the effect. As shown in Fig. S12D, the I/I₀ increase for Cy5.5-H-CyN was smaller than that of Cy5.5-H-Cy5, indicating that Cy5.5-H-CyN is insensitive to viscosity. These results demonstrated that Cy5.5-H-CyN exhibits high stability and selectivity for pH determination.

  DFT calculations were performed to further investigate the mechanism underlying the alteration of photophysical properties utilizing the M062X/def2svp functional and basis set in Cy5.5-H-CyN upon proton binding (Fig. S13A in Supporting information). Solvation effects were considered using a water medium in the SMD model. All calculations were performed using Gaussian 16 A software, with electron-hole analysis conducted using Multiwfn 3.8 [24, 25]. The frontier molecular orbitals (FMO) energy of the ground state (S₀) and singlet excited state (S₁) are depicted in Fig. S13B (Supporting information). From an energetic perspective, the protonation of Cy5.5-H-CyN (H resulted in a more stable the highest occupied molecular orbital (HOMO) arrangement than its deprotonated form, leading to an apparent energy reduction (−6.73 → −5.98 eV). However, minimal changes were observed in the lowest unoccupied molecular orbital (LUMO) energy (−2.21 → −2.00 eV). Consequently, this substantial increase in the HOMO-LUMO energy gap of Cy5.5-H-CyN (H (−3.98 → −4.52 eV) required more energy for electron transition and caused a blue shift in the absorption wavelength (Fig. 1B). The combination of protons results in a slight increase in HOMO-LUMO of the energy gap for the S₁ state, leading to a blue shift of emission wavelength consistent with experimental observations (Table S2). Therefore, the protonation induced significant alterations in the FMO, potentially influencing electronic distribution in excited states.

  The electron-hole distribution before and after proton binding was analyzed to explore electron transfer characteristics in the excited state (Fig. S14 in Supporting information). The atomic contribution heat map revealed that in S₀ coordinates, both sides of deprotonated Cy5.5-H-CyN exhibited significant contributions from indole groups to holes (58.7%) and electrons (56.5%). However, upon proton binding, this contribution decreased to 32.7% for holes and 49.6% for electrons with more concentration on the methine chain, resulting in a notable decrease in ICT as well as a more significant blue shift in wavelength. In the S₁ coordinate, regardless of proton binding, both sides of the indole groups demonstrated electron-donating properties, with hole contributions exceeding electron contributions. After proton binding occurred due to the weakened electron-donating ability of azaindole moiety, hole contribution moderately reduced (59.4% → 54.8%), weakening the ICT effect, leading to a blue shift in emission wavelength.

  Before the experiment in living cells, the toxicity of Cy5.5-H-CyN to HeLa cells was assessed using the MTT assay. Upon reaching a concentration of 8 µmol/L, the cytotoxicity of Cy5.5-H-CyN decreased to below 80% (Fig. S15 in Supporting information), indicating its favorable biocompatibility at low doses. To investigate the mitochondrial targeting ability, Cy5.5-H-CyN was co-stained with MitoTracker Green FM (a commercial dye for mitochondria) in HeLa cells. The fluorescence distribution of Cy5.5-H-CyN exhibited excellent overlap with that of MitoTracker Green FM (Pearson coefficient: 0.871) (Fig. S16 in Supporting information). Conversely, there was little overlap observed between Cy5.5-H-CyN and LysoTracker Green DND 26 (a lysosomal targeting dye) as well as Hoechst 33342 (a nuclear targeting dye) with Pearson coefficient of 0.486 and 0.076, respectively (Fig. S17 in Supporting information). This marked disparity highlights the exceptional mitochondrial specificity of Cy5.5-H-CyN.

  The decrease in mitochondrial membrane potential (MMP) is a key characteristic of mitophagy [4]. However, the reduction or disappearance of MMP can result in the leakage of normal probes, which target mitochondria based on electrostatic attraction, thereby compromising the accuracy of the test [26]. To assess the targeting ability of Cy5.5-H-CyN, a mitochondrial uncoupling experiment was conducted. CCCP, a commonly used uncoupling reagent for mitochondrial proton carriers, rapidly dissipates the H⁺ gradient across the inner membrane within 5 min, leading to acidification of mitochondria and reduction in MMP [27]. Specifically, Cy5.5-H-CyN was incubated with HeLa cells for 1 h, washed with DMEM medium three times, and subsequently co-incubated with CCCP and MitoTracker Green FM for 30 min. Rhodamine 123 (a fluorescent dye possessing mitochondrial targeting ability) was employed as a control. Surprisingly, upon the addition of CCCP, the fluorescence intensity of Cy5.5-H-CyN-treated cells only exhibited a slight decrease, and the distribution pattern of fluorescence remained comparable to that observed with MitoTracker Green FM. Notably, it continued to exhibit enrichment within mitochondria (Pearson coefficient: 0.908–0.892) (Fig. S18 in Supporting information). However, the fluorescence intensity of rhodamine 123 in the control group essentially vanished (Fig. S19 in Supporting information). Therefore, Cy5.5-H-CyN can effectively localize within mitochondria without the influence of changes in MMP. We proposed that Cy5.5-H-CyN, similar to mitochondrial respiratory inhibitors, could bind a specific protein within the mitochondrial respiratory chain [28], enhancing its affinity with mitochondria. To validate this hypothesis, we evaluated the ATP levels in HeLa cells after the addition of Cy5.5-H-CyN. As illustrated in Fig. S20 (Supporting information), introducing Cy5.5-H-CyN resulted in a noticeable reduction in ATP levels within HeLa cells compared to the control group. For example, with the introduction of 6 µmol/L Cy5.5-H-CyN, the ATP levels were approximately 70% lower than in the control group. These findings proved that Cy5.5-H-CyN indeed enhanced its ability to localize mitochondria by binding to proteins within the respiratory chain.

  To quantitatively determine the pH value in mitochondria, it is necessary to calibrate the intracellular pH. HeLa cells were initially incubated with Cy5.5-H-CyN and then replaced with PBS containing nigericin (10 µmol/L) at various pH levels for a specific incubation period before cell imaging was conducted. As expected, when the pH decreased from 8.0 to 4.0, fluorescence decreased in the red channel while it increased in the green channel. The ratio in the merged channel transitioned gradually from red (pH 8.0) to orange and eventually turned green (pH 4.0). Additionally, the ratio of fluorescence signal between the red and green channels also changed progressively from red-green at pH 8.0 to blue-green until it essentially disappeared at pH 4.0 (Fig. 2A). Subsequently, the quantitative relationship between the fluorescence intensity ratio Fl. λ_{ex 640 nm}/Fl. λ_{ex 488 nm} and pH were analyzed (Fig. 2B). In HeLa cells, Cy5.5-H-CyN exhibited a linear response range (pH 5.0–7.0), consistent with the results obtained in solution (Fig. 2C). These findings indicate that Cy5.5-H-CyN can precisely track and visualize mitochondrial pH changes in live cells. When mitochondria were damaged by stress stimulation, to maintain cellular homeostasis and mitochondrial function, damaged mitochondria would undergo mitophagy and fuse with lysosomes to form autolysosomes, resulting in a pH decrease in mitochondria.

  Figure 2

  

  Figure 2.  Calibration of intracellular pH in HeLa cells with Cy5.5-H-CyN. (A) Fluorescence imaging of HeLa cells after incubation with Cy5.5-H-CyN and different pH buffers containing 10 µmol/L nigericin. Red channel: λ_ex = 640 nm, λ_em = 700–730 nm; Green channel: λ_ex = 488 nm, λ_em = 620–670 nm. Ratio channel: red channel/green channel. Scale bar: 20 µm. (B) Ratio channel fluorescence intensity diagram of different pH in HeLa cells. The data are expressed as the mean ± SD of five measurements. (C) The linear relationship between the ratio channel fluorescence intensity and pH in HeLa cells.

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  To investigate the ability of Cy5.5-H-CyN to monitor the process of mitophagy in living cells, various methods were employed to induce mitophagy. As mentioned earlier, mitochondrial uncouplers can reduce MMP, trigger mitophagy, and acidify mitochondria. However, at lower doses, mitochondrial uncouplers rapidly reduce MMP and exhibit poor ability to acidify mitochondria (for example, 10 µmol/L CCCP only reduced the pH of mitochondria from 7.8 to 7.2 [29]). Therefore, for visualization of the process of mitophagy by Cy5.5-H-CyN without effects on cell viability, HeLa cells were treated with a concentration of 50 µmol/L FCCP to trigger mitophagy. The fluorescence imaging results demonstrated a significant decrease in the fluorescence intensity in the red channel after incubation with FCCP for only 30 min, compared to the control group. Furthermore, for 120 min, the fluorescence intensity of the red channel was remarkably lowered, while the green channel exhibited obvious fluorescence signals (Figs. 3A and B). By utilizing the fluorescence ratio between two channels and referring to a pH calibration curve, it is possible to calculate that mitochondrial pH decreased from approximately 6.7 (at 30 min) to around 5.6 (at 120 min) (Fig. 3C).

  Figure 3

  

  Figure 3.  (A) Fluorescence imaging of Cy5.5-H-CyN in HeLa cells at different times after incubation with FCCP. Red channel: λ_ex = 640 nm, λ_em = 700–730 nm; Green channel: λ_ex = 488 nm, λ_em = 620–670 nm. Ratio channel: red channel/green channel. Scale bar: 20 µm. (B) The fluorescence intensity of Cy5.5-H-CyN ratio channel in HeLa cells at different times after incubation with FCCP. The data were expressed as mean ± SD of three measurements. (C) The mitochondrial pH of HeLa cells at different times after incubation with FCCP.

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  Rapamycin, a widely utilized anticancer medication, could inhibit cell proliferation through the suppression of the mammalian targets of the rapamycin signaling pathway, which can induce mitophagy in various cells [26]. Following incubation with Cy5.5-H-CyN in HeLa cells, rapamycin (5 µmol/L) was introduced to stimulate mitophagy. The imaging results demonstrated that compared to the control group, there was a significant reduction in fluorescence intensity in the red channel after 6 h and obvious fluorescence signals were observed in the green channel after 12 h (Figs. 4A and B). Based on the pH calibration curve, it can be calculated that rapamycin-induced mitophagy in HeLa cells with a mitochondrial pH change from approximately 6.0 at 6 h to around 5.6 after 12 h (Fig. 4C).

  Figure 4

  

  Figure 4.  (A) Fluorescence imaging of HeLa cells after incubation with Cy5.5-H-CyN and treatment with different mitophagy induction methods. Red channel: λ_ex = 640 nm; λ_em = 700–730 nm; Green channel: λ_ex = 488 nm, λ_em = 620–670 nm; Ratio channel: Red channel/Green channel. Scale bar: 20 µm. (B–E) The ratio fluorescence intensity of Cy5.5-H-CyN in HeLa cells was treated with different mitophagy induction methods at different times. The value is the corresponding pH value. The data are expressed as the mean ± SD of three measurements.

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  The deficiency of cell nutrients or growth under hypoxic conditions can inhibit cellular metabolism and disrupt mitochondrial function, thereby triggering mitophagy [6, 30]. Therefore, HeLa cells were incubated with Cy5.5-H-CyN and imaged using serum-free medium or hypoxic condition (1% O₂). The imaging results revealed a significant reduction in fluorescence intensity in the red channel after 6 h under both culture conditions, and the green channel exhibited noticeable fluorescence signals after 12 h (Fig. 4A). Based on the pH calibration curve, it was determined that nutrient deficiency or hypoxia resulted in mitochondrial pH levels of approximately 6.4 and 6.2 after 6 h and reached around 6.0 and 5.7 after 12 h, respectively (Figs. 4D and E). All these experimental findings demonstrated that the application of Cy5.5-H-CyN enabled quantitative visualization of mitochondrial pH to monitor the process of mitophagy.

  In the advanced stage of mitophagy, lysosomes fuse with mitophagosomes to degrade mitochondria, thereby maintaining intracellular homeostasis [4]. During this phase, the fusion of these organelles induces alterations in the corresponding co-localization coefficients as observed through cellular fluorescence imaging. To elucidate this process, Cy5.5-H-CyN was employed as a probe for monitoring mitophagy and commercially available dyes of MitoTracker Green FM and LysoTracker Red DND 99 were utilized as indicators for mitochondria and lysosomes respectively. By employing various established conditions to induce mitophagy, changes in the co-localization coefficients were observed among these three components. As shown in Figs. S21A and B (Supporting information), where Cy5.5-H-CyN, MitoTracker Green FM, and LysoTracker Red DND 99 were assigned to the blue, green, and red channels respectively for visualization purposes. The control group was maintained under normal culture conditions. Following incubation with Cy5.5-H-CyN, MitoTracker Green FM and LysoTracker Red DND 99 were subsequently added into cells. Co-localization coefficients in the blue-green and blue-red channels indicated mitochondrial localization of Cy5.5-H-CyN. Under normal conditions, a low colocalization coefficient in the green-red channel suggested distinct separation and minimal fusion between mitochondria and lysosomes. However, upon application of various mitophagy induction methods for a specific duration, there was an obvious increase in the green-red co-localization coefficient, indicating fusion events between these organelles during mitophagy process. Moreover, a significant decrease in the blue-green co-localization coefficient along with a marked increase in the blue-red co-localization coefficient further supported this phenomenon. Therefore, utilizing Cy5.5-H-CyN as a probe for long-term dynamic monitoring of mitophagy is highly feasible.

  In summary, an azaindole-based asymmetric pentamethine cyanine dye Cy5.5-H-CyN was designed and easily synthesized, exhibiting NIR fluorescence, excellent mitochondrial targeting, and sensitive and reversible pH response-ability. Linear relationships were obtained within the pH range of 5.0–7.0 in both aqueous solution and intracellular media. By employing various mitophagy induction methods, real-time and quantitative ratiometric fluorescence imaging of mitochondrial pH changes was successfully achieved during mitophagy in HeLa cells. Therefore, Cy5.5-H-CyN not only provides a promising tool for monitoring pH changes during mitophagy but also offers an effective means to investigate the role of mitophagy in diverse intracellular pathological processes. This asymmetric multi-methine structure enables further extension of the emission wavelength by elongating the methine chain, thereby enhancing imaging depth and minimizing autofluorescence interference.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Tiancong Shi: Writing – original draft, Validation, Methodology, Data curation, Conceptualization. Xi Chen: Software, Formal analysis. Xiao Zhou: Software. Hongyi Zhang: Resources. Fuping Han: Methodology. Lihan Cai: Resources. Wen Sun: Methodology, Formal analysis. Jianjun Du: Writing – review & editing, Visualization, Supervision, Conceptualization. Jiangli Fan: Supervision, Project administration, Funding acquisition. Xiaojun Peng: Supervision, Project administration, Funding acquisition.

  Acknowledgments

  This work was financially supported by the Fundamental Research Funds for the Central Universities (Nos. DUT23YG137 and DUT22LAB601), Liaoning Binhai Laboratory (No. LBLB-2023-03), Liaoning Province Science and Technology Joint Fund (Nos. 2023JH2/101800039 and 2023JH2/101800037), National Natural Science Foundation of China (Nos. 21925802, 22090011, and 21878039).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110408.

  
  
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  Figure 1  (A) The chemical structure and pH sensing mechanism of Cy5.5-H-CyN in mitochondria. (B) The pH-dependent absorbance spectra of Cy5.5-H-CyN (Inset: the solution color of Cy5.5-H-CyN in PBS with different pH values). (C) The pH-dependent emission spectra of Cy5.5-H-CyN (λ_ex = 640 nm). (D) The fluorescence intensity of Cy5.5-H-CyN in PBS at different pH values (λ_ex = 640 nm). (E) The pH-dependent emission spectra of Cy5.5-H-CyN (λ_ex = 488 nm). (F) The fluorescence intensity ratio Fl. λ_{ex 640 nm}/Fl. λ_{ex 488 nm} vs. pH (4.0–9.0). (G) Linear fitting of Fl. λ_{ex 640 nm}/Fl. λ_{ex 488 nm} vs. pH (5.0–7.0).

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  Figure 2  Calibration of intracellular pH in HeLa cells with Cy5.5-H-CyN. (A) Fluorescence imaging of HeLa cells after incubation with Cy5.5-H-CyN and different pH buffers containing 10 µmol/L nigericin. Red channel: λ_ex = 640 nm, λ_em = 700–730 nm; Green channel: λ_ex = 488 nm, λ_em = 620–670 nm. Ratio channel: red channel/green channel. Scale bar: 20 µm. (B) Ratio channel fluorescence intensity diagram of different pH in HeLa cells. The data are expressed as the mean ± SD of five measurements. (C) The linear relationship between the ratio channel fluorescence intensity and pH in HeLa cells.

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  Figure 3  (A) Fluorescence imaging of Cy5.5-H-CyN in HeLa cells at different times after incubation with FCCP. Red channel: λ_ex = 640 nm, λ_em = 700–730 nm; Green channel: λ_ex = 488 nm, λ_em = 620–670 nm. Ratio channel: red channel/green channel. Scale bar: 20 µm. (B) The fluorescence intensity of Cy5.5-H-CyN ratio channel in HeLa cells at different times after incubation with FCCP. The data were expressed as mean ± SD of three measurements. (C) The mitochondrial pH of HeLa cells at different times after incubation with FCCP.

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  Figure 4  (A) Fluorescence imaging of HeLa cells after incubation with Cy5.5-H-CyN and treatment with different mitophagy induction methods. Red channel: λ_ex = 640 nm; λ_em = 700–730 nm; Green channel: λ_ex = 488 nm, λ_em = 620–670 nm; Ratio channel: Red channel/Green channel. Scale bar: 20 µm. (B–E) The ratio fluorescence intensity of Cy5.5-H-CyN in HeLa cells was treated with different mitophagy induction methods at different times. The value is the corresponding pH value. The data are expressed as the mean ± SD of three measurements.

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