English

• The development of imaging technology, such as fluorescent imaging, which can perform non-invasive visual diagnosis of diseases in vivo, is playing an increasingly important role in biomedicine [1-3]. Traditional fluorophores including coumarin, BODIPY, fluorescein, and rhodamine, have been widely used for fluorescence imaging and sensing. However, most of them are usually one-photon (OP) excited by a UV–visible laser, with a wavelength in the range of 350–650 nm [4-10]. Compared with OP imaging, two-photon (TP) imaging can provide deeper tissue imaging with high spatial and temporal resolution owing to its near-infrared (NIR, 700–1000 nm) excitation [11-16]. Therefore, chemists have made significant efforts to develop new TP fluorophores with desirable photophysical performance, such as longer absorption/emission wavelengths and high fluorescence quantum yields, for imaging and sensing in living systems.

  Although the excitation wavelength of TP imaging is in the NIR region, the emission wavelength of currently widely used TP fluorophores (e.g., Acedan and naphthalimide derivantives) is concentrated in the range of 450–550 nm [17-20]. Within this region, intrinsic biomolecules in the tissues such as NADH, riboflavins, retinol, and folic acid, would cause serious autofluorescence [21-24]. In recent years, several studies have been devoted to developing novel long-wavelength emitting TP fluorophores [25, 26]. Among them, π-extended Acedan analogues and DCM dyes are popular for TP probe design owing to their structural modifiability (Scheme 1A) [27-29]. However, these fluorophores still suffer from some weaknesses, such as a lack of optically tunable groups [29], sensitivity to pH, and poor solubility [30, 31]. Therefore, the development of excellent long-wavelength TP fluorophores with optically tunable groups for in vivo molecular imaging remains a challenge.

  Scheme 1

  

  Scheme 1.  (A) The reported red-emitting TP fluorophores. (B) Our design of TP fluorophores with longer emission wavelength.

  DownLoad: Full-Size Img PowerPoint

  Herein, by introducing strong electron acceptor and large conjugated group to the TPQL dye, we designed and synthesized two long-emission TP fluorophores (TPCN1 and TPCO2) with an optically tunable amino group (Scheme 1B). Importantly, TPCO2 displayed a bright red emission (λ_em = 638 nm, Φ = 0.15 in EtOH) together with a high two-photon action cross section (61 GM at 810 nm) and good water solubility, which enabled high signal-to-background ratios and deep tissue imaging. To demonstrate this versatility, we designed and synthesized two activatable TP probes: TPCO—N₃ and TPCO—NO₂. The optical properties study demonstrated that TPCO—N₃ and TPCO—NO₂ can rapidly, highly selectively, and sensitively response to H₂S and nitroreductase (NTR), respectively. Using probe TPCO—NO₂, we not only successfully distinguished the expression of NTR in cells under different hypoxic conditions, but also sensitively imaged the NTR expression levels in liver fibrosis, further realizing diagnosis of the degree of hypoxia during liver fibrosis progression.

  In a previous work, we have developed well-behaved two-photon dyes TPQLs with an optically tunable amino group [13]. However, their emission wavelengths were still short. Currently, attempts to obtain long-wavelength fluorophores have focused mainly on molecular engineering strategy by expanding the π-conjugate structure and introducing strong electron-donating (D) and electron-accepting (A) groups [32-34]. Thus, we intended to introduce malononitrile (a strong electron acceptor) and a phenol derivative (a large conjugated group) into the TPQL to afford novel long-wavelength emitting TP dyes (Scheme 1B). As design in Scheme S1 (Supporting information), TPCN1 and TPCO1 were to be synthesized by a simple one-step method. However, owing to the high rigidity of the five-membered ring (Figs. S1a and b in Supporting information), the enlarged conjugated dye TPCO1 was not successfully synthesized. Finally, we synthesized dye TPCO2 with a twisted six-membered ring (Figs. S1c and d in Supporting information). Both TPCN1 and TPCO2 were characterized by NMR analyses. Subsequently, their photophysical properties were studied in different solvents. As shown in Fig. 1, Figs. S2 and S3 (Supporting information), compared to TPQL1 (λ_abs/λ_em = 395/475 nm), by introducing a strong electron acceptor, the maximum absorption (λ_abs) and emission (λ_em) wavelengths of TPCN1 exhibited only a red-shift of approximately 50 nm, reaching 442/533 nm. However, when the π-conjugate structure was extended, the emission wavelength of TPCO2 showed a red-shift more than 150 nm, up to 638 nm. These results indicated that expanding the π-conjugation is more suitable for substantially red-shifting the wavelength of dye. This conclusion was verified by density functional theory (DFT) calculations (Fig. 1C). Furthermore, it should be noted that, with the solvent changing from dichloromethane to water, the emission wavelength of the new dyes shifted only approximately 12 nm (TPCN1) and 18 nm (TPCO2) (Fig. S4 in Supporting information), which indicated their environmental insensitivity. In addition, the high quantum yield, good water solubility, and pH insensitivity showed great potential for biological imaging (Table S1, Figs. S5 and S6 in Supporting information).

  Figure 1

  

  Figure 1.  Normalized absorption (A) and fluorescence emission (B) spectra of TPQL1, TPCN1 and TPCO2 in ethanol. (C) Optimized frontier molecular orbitals and orbital energies gap of TPQL1, TPCN1 and TPCO2 in the excited state.

  DownLoad: Full-Size Img PowerPoint

  The two-photon absorption cross section was a key feature of TP fluorophores. Therefore, the fluorophores were investigated in PBS buffer. As shown in Fig. S7 (Supporting information), TPCN1 and TPCO2 exhibited the broadest two-photon action cross sections of 82 and 61 GM at 810 nm, respectively. To further evaluate the TP efficiency of the new dyes, TP fluorescence imaging was compared using live tissues. The tissues were incubated with TPCO2 (10 µmol/L), TPCN1 (10 µmol/L), or Acedan-NH₂ (10 µmol/L) for 30 min. Emission signals were collected and imaged using TP microscopy. The intensity of the background fluorescence of the red light was much lower than that of the green and yellow light under the same laser power. Therefore, the imaging signal-to-background ratio of TPCO2 (15 folds) was much higher than those of Acedan-NH₂ (3 folds) and TPCN1 (5 folds). Z-scan imaging of live tissues also revealed that TPCO2 displayed deeper tissue penetration (280 µm) than Acedan-NH₂ (160 µm) or TPCN1 (120 µm) (Fig. 2).

  Figure 2

  

  Figure 2.  Confocal TP fluorescence images of liver tissues. (A) Liver tissues cultured with TPCO2, Acedan-NH₂ and TPCN1. TPCO2: λ_ex = 810 nm, λ_em = 663–738 nm; Acedan-NH₂: λ_ex = 750 nm, λ_em = 425–475 nm; TPCN1: λ_ex = 810 nm, λ_em = 500–550 nm; Scale bar = 50 µm; Laser power is 3 mW. (B) mean fluorescence intensity and signal-to-background ratio of A. (C-E) Tissue depth of Acedan-NH₂, TPCN1 and TPCO2.

  DownLoad: Full-Size Img PowerPoint

  Considering its long-emission wavelength and high QY, we chose TPCO2 for further study. First, we performed MTT assay. When the concentration of TPCO2 reached 50 µmol/L, the cells still showed a high viability (Fig. S8 in Supporting information). Next, we studied the photostability of TPCO2, and observed that TPCO2 still maintained a high brightness after 1 h of irradiation. In contrast, the brightness of Cy5 dropped to 80% of its initial value under the same conditions (Fig. S9 in Supporting information). Next, we studied the stability of TPCO2 in cells. Similar to the in vitro results, TPCO2 showed superior photostability in cells compared to Cy5. The fluorescence of Cy5 was nearly completely bleached after 10 min of illumination, however, TPCO2 remained at 50% of its original value (Fig. S10 in Supporting information). These results indicate that TPCO2 has better photostability than Cy5 and can be applied to long-term tracking imaging research.

  To demonstrate that TPCO2 can be used in the design of activatable probes, two different types of probes were prepared by incorporating a 4-nitrobenzyl carbonochloridate group and azido group into TPCO2 to detect nitroreductase (NTR) and H₂S, respectively (Scheme S2 in Supporting information). Next, we investigated its responsiveness. The fluorescence of TPCO—N₃ was negligible. When H₂S was added at room temperature, it was reduced to TPCO2 (Fig. S11 in Supporting information), resulting in strong fluorescence (Figs. 3A and B). Selectivity experiments demonstrated the specificity of TPCO—N₃'s response to H₂S (Fig. 3C and Fig. S12 in Supporting information). As shown in Fig. 3D, TPCO—NO₂ exhibits almost no fluorescence. When NTR and NADH were added, the fluorescence at 635 nm increased by 16.5 times. The selectivity of TPCO—NO₂ for the NTR was also evaluated. As shown in Fig. 3E, only when NTR and coenzyme NADH coexist, the fluorescence at 635 nm significantly increases, indicating TPCO—NO₂ has a higher selectivity for NTR. The above experiments showed that TPCO2 could be regarded as a platform for the design of activatable probes.

  Figure 3

  

  Figure 3.  (A) Structure of TPCO—N₃ and TPCO—NO₂ and its response mechanism to H₂S and NTR. (B) Fluorescence spectra of TPCO—N₃ in the presence of H₂S for 10 min. (C) Fluorescence responses of TPCO—N₃ to various species in PBS (pH 7.4) buffer solution at 37 ℃ for 10 min. (D) Fluorescence spectra of TPCO—NO₂ in the presence of NTR with 500 µmol/L NADH in PBS (pH 7.4) buffer solution at 37 ℃ for 60 min. (E) Fluorescence responses of TPCO—NO₂ to various species in PBS (pH 7.4) buffer solution at 37 ℃ for 60 min. λ_ex = 525 nm, λ_em = 570–800 nm.

  DownLoad: Full-Size Img PowerPoint

  To verify its sensing ability, the reaction kinetics of TPCO—NO₂ with NTR were monitored. When NTR was added, fluorescence at 635 nm increased and reached saturation at approximately 60 min (Fig. S13 in Supporting information). Meanwhile, the higher the NTR concentration, the more obvious the fluorescence enhancement. When the NTR concentration reached 10 µg/mL, the fluorescence at 635 nm reached saturation (Fig. 3D). When the concentration of NTR was in the range of 0–8 µg/mL, the probe showed a good linear relationship and low detection limit (0.019 µg/mL), revealing a highly sensitive response to NTR. The above results demonstrated that TPCO—NO₂ had a good specific response to NTR, and was appropriate for monitoring NTR levels in vitro.

  Hypoxia in human body leads to the overexpression of several intracellular reductases, including NTR [35]. Since TPCO—NO₂ can sensitively and selectively respond to NTR in vitro, it was used to differentiate NTR expression levels in different oxygen environments. MTT experiments showed that the probe had good biosafety and biocompatibility with HeLa cells (Fig. S8), and could be used for the imaging analysis of NTR in cells. HeLa cells were cultured in the presence of different concentrations of oxygen (21%, 10% and 1%). Then the cells were incubated with TPCO—NO₂ for 1 h, followed by fluorescence imaging (Fig. 4A). The fluorescence of the cells was weak under normoxia, indicating that the NTR was less expressed in the normal environment. As the oxygen concentration decreased, the fluorescence intensity increased, indicating that the expression of NTR increased with intensified anaerobic conditions of the cells (Figs. 4A and B). This demonstrated that TPCO—NO₂ could be applied to image the expression level of NTR under different oxygen environments. Therefore, the degree of hypoxia in organisms can be identified by analysing the expression levels of NTR.

  Figure 4

  

  Figure 4.  (A) Fluorescence imaging of HeLa cells with different pretreatments before incubation with TPCO—NO₂ (10 µmol/L) by two-photon confocal imaging. Scale bar: 10 µm. (B) Relative fluorescence intensity in HeLa cells from A. (C) Two-photon microscopy imaging of TPCO—NO₂ (10 µmol/L) in liver fibrosis mice liver tissues. (D) Depth fluorescence images of TPCO—NO₂ in 4-week CCl₄ induced mice liver tissue. λ_ex = 810 nm, λ_em = 663–738 nm. (E) Quantification of the relative mean fluorescence levels of cells from the images of A. (F) Quantification of the concentrations of HIF-1α in the normal liver and fibrosis liver tissues by ELISA Kit.

  DownLoad: Full-Size Img PowerPoint

  Liver fibrosis develops in any etiology of chronic liver injury [36]. Currently, there is no effective treatment for liver fibrosis, which severely affects the life of patients. Recent studies have shown that hypoxia is an important factor in liver fibrosis [37]. Therefore, exploring the relationship between liver fibrosis and hypoxia can provide a theoretical basis for the analysis and treatment of this disease. Given the excellent sensitivity and selectivity of TPCO—NO₂, we used it to monitor the degree of hypoxia during liver fibrosis by detecting NTR. The liver fibrosis model was established by intraperitoneal injection of CCl₄ as previously described (Animal experiments were approved by the Animal Ethics Committee of College of Biology (Hunan University)). To monitor the degree of hypoxia during liver fibrosis, we set up two experimental groups, treated with CCl₄ for 2 and 4 weeks, respectively. All liver tissues were stained with TPCO—NO₂ (10 µmol/L) for two-photon microscopy imaging. Liver tissues from normal mice exhibited negligible fluorescence, reflecting the relatively low NTR expression in healthy mice (Fig. 4C). Meanwhile, the fluorescence intensity of the liver tissue in mice with liver fibrosis was significantly stronger (Fig. 4C). With the aggravation of liver fibrosis, fluorescence intensity significantly increased, indicating that hypoxia worsened as fibrosis progressed. Moreover, the penetration depth of TPCO—NO₂ reached 160 µm in a 4-week CCl₄-induced mice liver tissue (Fig. 4D). When the NTR inhibitor dicoumarol was added to liver fibrosis tissues, the tissues exhibited significantly weak fluorescence, indicating that TPCO—NO₂ can monitor the NTR activity and assess the degree of hypoxia (Figs. 4C and E). Considering the essential role of HIF-1α (hypoxia-inducible factor) in the progression of liver fibrosis [38-42], we further monitored the levels of HIF-1α in liver fibrosis tissues via ELISA. As shown in Fig. 4F, with the progression of liver fibrosis, HIF-1α level significantly increased. This data is consistent well with the fluorescence result, indicating that TPCO—NO₂ has the potential to be used for monitoring the liver fibrosis by hypoxia imaging.

  In summary, we developed a novel red-emitting TP dye with an optically tunable amino group. TPCO2 displayed a bright red emission (λ_em = 638 nm, Φ = 0.15) together with a high two-photon action cross section and good water solubility, which enabled high signal-to-background ratios and deep tissue imaging. Based on the favourable photophysical properties of TPCO2, we designed two different types of probes. The probe TPCO—NO₂ showed good selectivity, high sensitivity, and selectivity to NTR, and could realize the imaging of NTR expression in cells under different hypoxic conditions. More importantly, TPCO—NO₂ was successfully applied to high signal-to-background ratio imaging of NTR in liver fibrosis, further realizing the diagnosis of the degree of hypoxia during liver fibrosis. Therefore, TPCO2 can be used as a platform for designing activated TP probes.

  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.

  Acknowledgments

  This work is supported by the National Natural Science Foundation of China (Nos. 22074036, 22004033, 21877029) and Special Funds for the Construction of Innovative Provinces in Hunan Province (No. 2019RS1031).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2022.107835.

  
  
• 1. [1]

     T.B. Ren, W. Xu, W. Zhang, et al., J. Am. Chem. Soc. 140 (2018) 7716–7722. doi: 10.1021/jacs.8b04404

  2. [2]

     X. Fan, T. Ren, W. Yang, X. Zhang, L. Yuan, Chem. Commun. 57 (2021) 8644–8647. doi: 10.1039/d1cc02999a

  3. [3]

     G. Jiang, T.B. Ren, E. D'Este, et al., Nat. Commun. 13 (2022) 2264. doi: 10.1038/s41467-022-29547-3

  4. [4]

     W. Xu, Z. Zeng, J.H. Jiang, Y.T. Chang, L. Yuan, Angew. Chem. Int. Ed. 55 (2016) 13658–13699. doi: 10.1002/anie.201510721

  5. [5]

     X. Chen, F. Wang, J.Y. Hyun, et al., Chem. Soc. Rev. 45 (2016) 2976–3016. doi: 10.1039/C6CS00192K

  6. [6]

     D. Cao, Z. Liu, P. Verwilst, et al., Chem. Rev. 119 (2019) 10403–10519. doi: 10.1021/acs.chemrev.9b00145

  7. [7]

     J. Bai, J. Zhou, X. Ji, W. Zhao, et al., Chin. Chem. Lett. 33 (2022) 4175–4178. doi: 10.1016/j.cclet.2022.02.017

  8. [8]

     G. Hu, H. Jia, L. Zhao, D. Cho, J. Fang, Chin. Chem. Lett. 30 (2019) 1704–1716. doi: 10.1016/j.cclet.2019.06.039

  9. [9]

     X. Zhang, C. Wang, L. Jin, Z. Han, Y. Xiao, ACS Appl. Mater. Interfaces 6 (2014) 12372–12379. doi: 10.1021/am503849c

  10. [10]

      S. Pascal, S. David, C. Andraud, O. Maury, Chem. Soc. Rev. 50 (2021) 6613–6658. doi: 10.1039/d0cs01221a

  11. [11]

      L. Wu, J. Liu, P. Li, B. Tang, T.D. James, Chem. Soc. Rev. 50 (2021) 702–734. doi: 10.1039/d0cs00861c

  12. [12]

      L. Yuan, W. Lin, H. Chen, S. Zhu, L. He, Angew. Chem. Int. Ed. 52 (2013) 10018–10022. doi: 10.1002/anie.201303179

  13. [13]

      X. Zhang, T. Ren, Q. Zhang, et al., Cell Rep. Phys. Sci. 2 (2021) 100471. doi: 10.1016/j.xcrp.2021.100471

  14. [14]

      T.B. Ren, W. Xu, Q.L. Zhang, et al., Angew. Chem. Int. Ed. 57 (2018) 7473–7477. doi: 10.1002/anie.201800293

  15. [15]

      Y. Zheng, X.X. Zhang, L. Shi, et al., Chem. Asian J. 17 (2022) e202101197.

  16. [16]

      X. Zhang, T. Ren, L. Yuan, et al., Chin. Chem. Lett. 32 (2021) 3890–3894. doi: 10.1016/j.cclet.2021.06.038

  17. [17]

      H.M. Kim, C. Jung, B.R. Kim, et al., Angew. Chem. Int. Ed. 46 (2007) 3460–3463. doi: 10.1002/anie.200700169

  18. [18]

      C. Zhang, P. Wang, X. Yin, et al., Anal. Chem. 91 (2019) 6371–6377. doi: 10.1021/acs.analchem.9b01455

  19. [19]

      J. Ning, Z. Tian, J. Wang, et al., Angew. Chem. Int. Ed. 61 (2022) e202113191.

  20. [20]

      H. Fang, R. Shi, D. Chen, et al., Chem. Commun. 57 (2021) 11260–11263. doi: 10.1039/d1cc04744b

  21. [21]

      R. Zipfel Warren, M. Williams Rebecca, R. Christie, et al., Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 7075–7080. doi: 10.1073/pnas.0832308100

  22. [22]

      S. Huang, A.A. Heikal, W.W. Webb, Biophys. J. 82 (2002) 2811–2825. doi: 10.1016/S0006-3495(02)75621-X

  23. [23]

      A.J. Radosevich, M.B. Bouchard, S.A. Burgess, B.R. Chen, E.M.C. Hillman, Opt. Lett. 33 (2008) 2164–2166. doi: 10.1364/OL.33.002164

  24. [24]

      C.H. Heo, K.H. Kim, H.J. Kim, et al., Chem. Commun. 49 (2013) 1303–1305. doi: 10.1039/c2cc38570h

  25. [25]

      G. Niu, X. Zheng, Z. Zhao, et al., J. Am. Chem. Soc. 141 (2019) 15111–15120. doi: 10.1021/jacs.9b06196

  26. [26]

      X. Zhang, Kevin D. Belfield, Y. Xiao, J. Qi, J. Qu, et al., J. Org. Chem. 78(2013) 9153–9160. doi: 10.1021/jo401379g

  27. [27]

      D. Kim, H. Moon, S.H. Baik, et al., J. Am. Chem. Soc. 137 (2015) 6781–6789. doi: 10.1021/jacs.5b03548

  28. [28]

      M. Li, X. Wu, Y. Wang, et al., Chem. Commun. 50 (2014) 1751–1753. doi: 10.1039/c3cc48128j

  29. [29]

      S. Singha, Y.W. Jun, S. Sarkar, K.H. Ahn, Acc. Chem. Res. 52 (2019) 2571–2581. doi: 10.1021/acs.accounts.9b00314

  30. [30]

      K. Gu, Y. Xu, H. Li, et al., J. Am. Chem. Soc. 138 (2016) 5334–5340. doi: 10.1021/jacs.6b01705

  31. [31]

      T. Liu, M. Zhang, L. Feng, et al., Sens. Actuators B: Chem. 337 (2021) 129764. doi: 10.1016/j.snb.2021.129764

  32. [32]

      L. He, H. Xiong, B. Wang, et al., Anal. Chem. 92 (2020) 11029–11034. doi: 10.1021/acs.analchem.0c00030

  33. [33]

      A. Gandioso, S. Contreras, I. Melnyk, et al., J. Org. Chem. 82 (2017) 5398–5408. doi: 10.1021/acs.joc.7b00788

  34. [34]

      S.S. Matikonda, J. Ivanic, M. Gomez, G. Hammersley, M.J. Schnermann, Chem. Sci. 11 (2020) 7302–7307. doi: 10.1039/d0sc02566f

  35. [35]

      Brown, J., Wilson, W., Nat. Rev. Cancer 4 (2004) 437–447. doi: 10.1038/nrc1367

  36. [36]

      E. Seki, D.A. Brenner, J. Hepato-Bil-Pan Sci. 22 (2015) 512–518. doi: 10.1002/jhbp.245

  37. [37]

      M. Fernandez, D. Semela, J. Hepatol. 50 (2009) 604–620. doi: 10.1016/j.jhep.2008.12.011

  38. [38]

      T. Kietzmann, E.Y. Dimova, D. Flügel, J.G. Scharf, Z. Gastroenterol. 44 (2006) 67–76. doi: 10.1055/s-2005-858987

  39. [39]

      R. Kumari, D. Sunil, R.S. Ningthoujam, Bioorg. Chem. 88 (2019) 102979. doi: 10.1016/j.bioorg.2019.102979

  40. [40]

      D.J. Manalo, A. Rowan, T. Lavoie, et al., Blood 105 (2005) 659–669. doi: 10.1182/blood-2004-07-2958

  41. [41]

      E. Novo, S. Cannito, E. Zamara, et al., Am. J. Pathol. 170 (2007) 1942–1953. doi: 10.2353/ajpath.2007.060887

  42. [42]

      J. Cai, M. Hu, Z. Chen, Z. Ling, J. Transl. Med. 19 (2021) 186. doi: 10.1186/s12967-021-02854-x

• 

  Scheme 1  (A) The reported red-emitting TP fluorophores. (B) Our design of TP fluorophores with longer emission wavelength.

  下载: 全尺寸图片 幻灯片
  

  Figure 1  Normalized absorption (A) and fluorescence emission (B) spectra of TPQL1, TPCN1 and TPCO2 in ethanol. (C) Optimized frontier molecular orbitals and orbital energies gap of TPQL1, TPCN1 and TPCO2 in the excited state.

  下载: 全尺寸图片 幻灯片
  

  Figure 2  Confocal TP fluorescence images of liver tissues. (A) Liver tissues cultured with TPCO2, Acedan-NH₂ and TPCN1. TPCO2: λ_ex = 810 nm, λ_em = 663–738 nm; Acedan-NH₂: λ_ex = 750 nm, λ_em = 425–475 nm; TPCN1: λ_ex = 810 nm, λ_em = 500–550 nm; Scale bar = 50 µm; Laser power is 3 mW. (B) mean fluorescence intensity and signal-to-background ratio of A. (C-E) Tissue depth of Acedan-NH₂, TPCN1 and TPCO2.

  下载: 全尺寸图片 幻灯片
  

  Figure 3  (A) Structure of TPCO—N₃ and TPCO—NO₂ and its response mechanism to H₂S and NTR. (B) Fluorescence spectra of TPCO—N₃ in the presence of H₂S for 10 min. (C) Fluorescence responses of TPCO—N₃ to various species in PBS (pH 7.4) buffer solution at 37 ℃ for 10 min. (D) Fluorescence spectra of TPCO—NO₂ in the presence of NTR with 500 µmol/L NADH in PBS (pH 7.4) buffer solution at 37 ℃ for 60 min. (E) Fluorescence responses of TPCO—NO₂ to various species in PBS (pH 7.4) buffer solution at 37 ℃ for 60 min. λ_ex = 525 nm, λ_em = 570–800 nm.

  下载: 全尺寸图片 幻灯片
  

  Figure 4  (A) Fluorescence imaging of HeLa cells with different pretreatments before incubation with TPCO—NO₂ (10 µmol/L) by two-photon confocal imaging. Scale bar: 10 µm. (B) Relative fluorescence intensity in HeLa cells from A. (C) Two-photon microscopy imaging of TPCO—NO₂ (10 µmol/L) in liver fibrosis mice liver tissues. (D) Depth fluorescence images of TPCO—NO₂ in 4-week CCl₄ induced mice liver tissue. λ_ex = 810 nm, λ_em = 663–738 nm. (E) Quantification of the relative mean fluorescence levels of cells from the images of A. (F) Quantification of the concentrations of HIF-1α in the normal liver and fibrosis liver tissues by ELISA Kit.

  下载: 全尺寸图片 幻灯片
•