English

• Polarity, as one of the key properties of the microenvironment, plays a crucial role in numerous physiological processes, including protein formation, immune system regulation, cell migration, and molecular transmembrane transport [1–3]. Abnormal polarity variations are closely linked to dysfunction of organelles, which can impair the normal physiological activities of cells and induce cancer, diabetes, cirrhosis and a series of senescence-related diseases, etc. [4–7]. While electrochemical analysis, spectroscopic analysis, and chromatographic analysis have made significant advances in detecting polarity [8,9], they still present certain limitations, such as time-consuming and requiring complex sample preparation and operation. Therefore, developing rapid and accurate methods for monitoring polarity changes at the cellular level and in living organisms is essential for elucidating the pathological mechanisms of related diseases.

  Fluorescence imaging technology has garnered significant attention and become powerful tools for studying biological physiological processes due to their nondestructive features, high sensitivity, real-time detection, and superior spatiotemporal resolution [10–16]. As a result, numerous small-molecule fluorogenic probes for rapidly monitoring changes in biological polarity have been developed [17–22]. However, the emission wavelengths of the most existing polarity-sensitive fluorogenic probes remain within the visible light range, leading to limitations in biological applications, such as photobleaching, shallow tissue penetration and high background interference [23–26]. With the expansion of application scenarios, the need for near-infrared (NIR) fluorogenic dyes with longer emission wavelength (650–900 nm) has become increasingly urgent. Extending the conjugated system of fluorescent molecules to achieve longer emission wavelength, however, typically increases in molecular weight. Furthermore, due to the presence of biofilm barriers, these probes often require longer incubation time to monitor and image the cellular polarity. Most of polarity-sensitive fluorogenic probes possess large dipole moments or carry charges, which hinder transmembrane movement and reduce the cellular uptake rate, thereby greatly limiting the biological applications of dyes. Thus, developing novel near-infrared fluorogenic probes with rapid transmembrane capability and high cellular uptake rates is of critical importance for polarity detection.

  In this study, we biomimetically synthesized two dual-emissive fluorogenic dyes, CBA and CBS, which exhibit high cellular uptake rates. The coumarin-benzopyranium fluorophore (CB) was selected as scaffold, while 1,3-dithio-2-heteroarsenic cyclopentane and 1,2-diselenocyclopentane were separately introduced as the promoting cellular uptake group. The control compound, CSF, lacking the promoting cellular uptake group, was also synthesized. Subsequently, we developed a polarity-sensitive dual-emissive fluorogenic probe, CSFNS, with high cellular uptake rate and a control compound, CBNS, which also lacks the promoting cellular uptake group, by modifying the spironolactone structure of CBA into spirolactam. This modification allowed the probe molecule to effectively adjust the dynamic equilibrium between zwitterionic and spirocyclic form, pushing the equilibrium towards the spirocyclic state to enhance the sensitivity of CSFNS to polarity. Using CSFNS and CBNS in living cell confocal imaging, we reconfirmed that the 1,3-dithio-2-heteroarsenic cyclopentane effectively improved the transmembrane efficiency of probe. Furthermore, we applied CSFNS to healthy cells, drug-induced senescent cells and cancer cells to monitor polarity changes, confirming that the polarity of healthy cells is greater than that of cancer cells, and the polarity of healthy cells increases with the deepening of aging in term of overall cellular environment (Scheme 1).

  Scheme 1

  

  Scheme 1.  (a) Proposed mechanism of high cellular uptake NIR fluorogenic dyes and probes in response to polarity. (b) Cellular uptake schematic diagram of probe CSFNS. (c) Schematic representation of cellular polarity in various types of living cells monitored by CSFNS.

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  The probe CSFNS was prepared according to synthesis routes shown in Scheme S1 (Supporting information). The key intermediates (6, 8, 13, 16, CB and CSF) were obtained following methods reported in the literatures [27–30]. 10 was obtained by condensing 8 with N-Boc-ethylenediamine and then deprotection. CBA and CBS were achieved by reacting CSF with either 6 or 10. CSFNS was obtained through four steps involving esterification and dehydration of CSF. The subsequent reaction of CB with N,N-dimethylsulfamide yielded CBNS. All synthesized compounds were systematically characterized using ¹H nuclear magnetic resonance (NMR), ¹³C NMR and high-resolution mass spectrometry (HRMS) techniques (Figs. S15–S63 in Supporting information).

  To investigate the relationship between CBA, CBS, CSF and microenvironmental influencing factors, we initially evaluated the changes in their optical properties by adjusting the proportion of water to 1,4-dioxane to simulate polarity environment in vitro. As shown in Figs. 1a–c and Fig. S1 (Supporting information), the ultraviolet (UV)-absorption spectra of the three compounds did not change significantly with variations in the polarity of mixed solutions. However, their fluorescence intensity at 470 nm increased as the solvent polarity decreased. These phenomena suggest that their polarity sensitivity is due to the coumarin portion of the D-π-A structure. When CBA, CBS and CSF are in a highly polar solvent, the molecules interact with the solvent through dipole-dipole forces, leading to considerable charge separation and energy dissipation in the excited states. This promotes nonradiative transitions, weakening fluorescence. In contrast, in solvents with low polarity, the solvent's impact is minimized, resulting in less charge transfer. This, in turn, increases fluorescence intensity. Simultaneously, the core skeleton CB of these spiropyran dyes underwent reversible ring isomerization (with the ring-opening isomers being amphoteric internal salts) in a low-polarity environment. This process extended the π-conjugated system of these dyes, leading to near-infrared emission at 690 nm. Additionally, we examined their spectral changes in different viscosity environments by varying the ratio of water to glycerol to simulate viscosity. As seen in Fig. S2 (Supporting information), the absorption and fluorescence intensity of the three compounds increased as the viscosity of the mixed solution increased. The results show that although the fluorescence intensity of them gradually increased as solvent viscosity increased, the sensitivity of the compounds to polarity changes was greater than their sensitivity to viscosity.

  Figure 1

  

  Figure 1.  (a–c) Fluorescence spectra of CBA, CBS and CSF (10 µmol/L) in water/1,4-dioxane (1,4-dioxane volume fraction from 0 to 90%) systems (f_d). λ_ex = 400 nm, λ_em1 = 470 nm, λ_em2 = 690 nm; slit widths: 5.0 nm/5.0 nm. (d) HepG2 cells were incubated with CBA, CBS and CSF (5 µmol/L) for different times (1, 10, 20, 30 min) and then were imaged by confocal microscopy. (e) Average fluorescence intensity in the blue channel from parallel images. (f) Average fluorescence intensity in the NIR channel from parallel images. Error bars represent the standard deviation (±S.D.) with n = 3. Blue channel: λ_ex/λ_em = 405/425–475 nm; NIR channel: λ_ex/λ_em = 405/663–738 nm. Scale bar: 20 µm.

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  Subsequently, we studied the effects of different pH on the optical properties for three compounds. As shown in Fig. S3 (Supporting information), the absorption spectra of three compounds only exhibited changes in absorption intensity across various pH values. Additionally, their fluorescence intensity remained stable across both channels within the pH range of 4–9. These results indicate that the optical properties of three compounds are stable, making them suitable for experimental research under physiological pH conditions.

  Given the complexity of the physiological environment, we evaluated the fluorescence spectra of three compounds in the presence of various potential interference species including glutathione (GSH), cysteine (Cys), methionine (Met), glycine (Gly), Al³⁺, Cu²⁺, Mg²⁺, S²⁻, β-galactosidase (β-gal), human serum albumin (HSA, purchased from Nanjing Duly Biotech Co., Ltd.), bovine serum albumin (BSA), rBSA and rHSA. As shown in Fig. S4 (Supporting information), the introduction of these interfering substances did not result in significant changes in fluorescence intensity. Notably, CBA, which contains a well-known specific recognition site for vicinal dithiols proteins (1,3-dithio-2-heteroarsenic cyclopentane) [31], showed no response to rBSA and rHSA. This lack of response may be attributed to steric hindrance, which likely limits interaction between 1,3-dithio-2-heteroarsenic cyclopentane and vicinal dithiols proteins, preventing specifical recognition. Therefore, we suggest that CBA has the potential to be applied in complex biological systems without interference from other potential species.

  Before conducting cell imaging, the cytotoxicity of three compounds was evaluated using standard MTT method. HepG2 cells were co-incubated with varying concentrations of compounds (0, 5, 10, 15, 20 µmol/L) for 12 h. As shown in Fig. S13 (Supporting information), the survival rate of cells slightly decreased with the increasing compounds concentration. However, the survival rate remained above 80% even at the highest concentration (20 µmol/L), indicating that all compounds exhibit low cytotoxicity and are suitable for imaging in living cells.

  We then incubated CBA, CBS and CSF with HepG2 cells for various durations and performed cell imaging. As shown in Figs. 1d–f, the fluorescence intensities of both channels for CBA and CBS were significantly higher than those for CSF with increasing incubation time. This suggests that the introduction of promoting cellular uptake group enhances cellular uptake efficiency. Notably, the fluorescence intensities for CBA in both channels were greater than those of CBS at the same incubation time, indicating that 1,3-disulfine-2-heteroarsenic cyclopentane exhibits more efficient transmembrane capability compared to the previously reported 1,2-selenocyclopentane, a promoting cellular uptake group [32]. The cellular uptake mechanism of CBA and CBS are shown in Figs. S5 and S6 (Supporting information).

  Previous experiments have demonstrated that CBA serves as a near-infrared fluorogenic dye with high cellular uptake rate due to the incorporation of 1,3-dithio-2-heteroarsenic cyclopentane. Consequently, we further modified the spironolactone structure of CBA into spirolactam using N,N-dimethylaminosulfonamide, pushing the modified probe CSFNS into the spirocyclic-locked state. Additionally, we synthesized the control compound CBNS, which lacks the promoting cellular uptake group. Subsequently, the sensitivity of CSFNS and CBNS response to polarity was systematically investigated, where the polarity level was estimated by the Lippert Mataga polarity parameter Δf (Table S1 in Supporting information). As the polarity of the mixed solvent decreased, the absorption intensity of CSFNS at 410 nm gradually increased, while the intensity at 650 nm decreased. Similarly, the fluorescence intensity at 470 nm for CSFNS increased approximately sixty-fold, significantly higher than the fifteen-fold increase observed for CBA. CSFNS also exhibited a strong linear relationship with solvent parameter Δf in both channels (Figs. 2a and b, Figs. S7 and S8 in Supporting information). These findings indicated that CSFNS demonstrates excellent sensitivity to polarity changes.

  Figure 2

  

  Figure 2.  (a, b) Fluorescence spectra of CSFNS and CBNS (10 µmol/L) in water/1,4-dioxane (1,4-dioxane volume fraction from 0 to 90%) systems (f_d). (c, d) Fluorescence spectra of CSFNS and CBNS (10 µmol/L) in THF and MeOH. (e, f) Fluorescence spectra of CSFNS and CBNS (10 µmol/L) in EG and DMSO; λ_ex = 400 nm, λ_em1 = 490 nm, λ_em2 = 690 nm; slit widths: 5.0 nm/5.0 nm.

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  As shown in Fig. S9 (Supporting information), although both compounds exhibited some sensitivity to viscosity, the fluorescence intensity in response to polarity was generally higher than that in response to viscosity at equivalent proportion. This suggests that polarity has a much greater influence on both compounds than viscosity. To further confirm this conclusion, we compared the emission spectra of both compounds in tetrahydrofuran (THF) and methanol (MeOH), where the viscosity of both solvents was the same, but polarity was different. The results showed that both channels exhibited higher fluorescence intensity for both compounds in THF than in MeOH. Additionally, we analyzed the emission spectrum of both compounds in ethylene glycol (EG) and dimethyl sulfoxide (DMSO), where the polarity of both solvents was the same, but their viscosity differed. The results showed that the fluorescence intensities of both compounds were nearly identical in both solvents. Therefore, we conclude that the influence of viscosity environments on fluorescence emission is negligible when detecting polarity (Figs. 2c–f). Furthermore, interference experiments demonstrated that the fluorescence signal of both compounds displayed slight changes in the presence of different pH buffers or other interference substances (Figs. S10–S12 in Supporting information). This means that both compounds possessed excellent selectivity and anti-interference ability in response to polarity.

  In the light of the excellent response to polarity observed in vitro, cell imaging experiments were subsequently conducted. Prior to cellular imaging, the cytotoxicity of both compounds towards HepG2 cells and HL-7702 cells were evaluated. The results demonstrated their negligible cytotoxic effects and excellent biocompatibility (Fig. S14 in Supporting information). We then investigated the effects of CSFNS and CBNS on HepG2 cell uptake by adding these compounds to the cells for co-incubation, followed by cell imaging at different incubation times (1, 10, 20, 30 min) (Fig. 3a). As shown in Figs. 3b–d, the fluorescence intensity of CSFNS in both channels at the same incubation time was significantly higher than that of CBNS. These results indicate that the uptake efficiency of the probe CSFNS was greater than that of CBNS, further confirming that the introduction of the pro-cellular uptake group, 1,3-dithio-2-heteroarsenic cyclopentane, effectively improves the uptake efficiency of the probe molecules.

  Figure 3

  

  Figure 3.  (a) The processes of cell culture and imaging. (b) HepG2 cells were incubated with CSFNS/CBNS (5 µmol/L) for different times (1, 10, 20, 30 min) and then were imaged by confocal fluorescence microscopy. (c, d) Average fluorescence intensity in the blue/NIR channel from parallel images for both probes. (e) Representative confocal imaging for HepG2, HL-7702 cells and DOX-induced senescent HL-7702 cells incubated with CSFNS (5 µmol/L) for 10 min. Scale bar: 20 µm. (f) X-gal staining images of normal HL-7702 cells and DOX-induced senescent HL-7702 cells. Scale bar: 100 µm. (g) Average fluorescence intensity in the blue/NIR channel from parallel images. Error bars represent ±S.D. with n = 3. ns, not significant. **P < 0.01 vs. HepG2 cells are analyzed with two-sided Student's t-test. Blue channel: λ_ex/λ_em = 405/425–475 nm; NIR channel: λ_ex/λ_em =405/663–738 nm.

  DownLoad: Full-Size Img PowerPoint

  In recent years, numerous studies have demonstrated distinct differences in polarity between healthy cells and cancer cells [33,34]. Consequently, the polarity of healthy cells and cancer cells was monitored by CSFNS. We selected HepG2 (cancer cells) and HL-7702 (healthy cells) for imaging experiments. As shown in Fig. 3e, the fluorescence intensities of both channels in HepG2 cells were stronger than these in HL-7702 cells, indicating that the environmental polarity level in cancer cells was lower than that in healthy cells. This finding is consistent with existing literature reports [35–38]. We also utilized CSFNS to visualize polarity changes during cellular senescence. To establish a drug-induced senescent cell model, we treated HL-7702 cells with doxorubicin (DOX, 0.1 µmol/L) for 24 h, as confirmed by the X-gal staining assay. HL-7702 cells stimulated by DOX exhibited abnormal cell morphology and enlarged nuclei, which was consistent with the characteristics of senescent cells (Fig. 3f). As shown in Figs. 3e and g, the fluorescence intensity in senescent cells is lower than that in healthy cells, indicating that the polarity of cells increased with the degree of senescence. These results suggest that the polarity imaging ability of CSFNS can effectively distinguish healthy cells from cancer cells and can be employed to visualize polarity changes during cellular senescence.

  In summary, we initially synthesized two novel dual emissive fluorescence dyes, CBA and CBS, based on the natural biological mechanism of thiol-disulfide dynamic exchange. This was achieved by introducing 1,3-disulf-2-azaarsenic cyclopentane and 1,2-diselenicyclopentane onto the near-infrared fluorescent skeleton (CB) to enhance their cellular uptake rate. Subsequently, we developed the polarity-sensitive dual emissive fluorescent probe CSFNS featured by high cellular uptake via modifying the spiral-ring structure of CBA. CSFNS not only exhibited an excellent optical signal in response to polarity in vitro, but also successfully achieved polarity visualization in healthy cells, cancer cells and senescent cells. This study provides a novel strategy for constructing near-infrared fluorogenic probes with high cellular uptake rate, and we anticipate that the probes designed using this strategy could be widely applied in detecting various diseases in organisms.

  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

  Xu Qu: Writing – original draft, Visualization, Validation, Formal analysis, Data curation. Baohua Ji: Validation, Formal analysis. Haocheng Gong: Validation, Formal analysis. Guangwei Wang: Validation, Formal analysis. Liang-Liang Gao: Validation. Jing Zhang: Writing – review & editing, Formal analysis. Jianjian Zhang: Writing – review & editing, Funding acquisition, Formal analysis. Yuan Guo: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

  Acknowledgments

  We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 22477101, 22277098 and 22037002), the Natural Science Basic Research Plan for Distinguished Young Scholars in Shaanxi Province of China (No. 2020JC-38) and the Shaanxi Fundamental Science Research Project for Chemistry and Biology (No. 22JHQ070).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110766.

  
  
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• 

  Scheme 1  (a) Proposed mechanism of high cellular uptake NIR fluorogenic dyes and probes in response to polarity. (b) Cellular uptake schematic diagram of probe CSFNS. (c) Schematic representation of cellular polarity in various types of living cells monitored by CSFNS.

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  Figure 1  (a–c) Fluorescence spectra of CBA, CBS and CSF (10 µmol/L) in water/1,4-dioxane (1,4-dioxane volume fraction from 0 to 90%) systems (f_d). λ_ex = 400 nm, λ_em1 = 470 nm, λ_em2 = 690 nm; slit widths: 5.0 nm/5.0 nm. (d) HepG2 cells were incubated with CBA, CBS and CSF (5 µmol/L) for different times (1, 10, 20, 30 min) and then were imaged by confocal microscopy. (e) Average fluorescence intensity in the blue channel from parallel images. (f) Average fluorescence intensity in the NIR channel from parallel images. Error bars represent the standard deviation (±S.D.) with n = 3. Blue channel: λ_ex/λ_em = 405/425–475 nm; NIR channel: λ_ex/λ_em = 405/663–738 nm. Scale bar: 20 µm.

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  Figure 2  (a, b) Fluorescence spectra of CSFNS and CBNS (10 µmol/L) in water/1,4-dioxane (1,4-dioxane volume fraction from 0 to 90%) systems (f_d). (c, d) Fluorescence spectra of CSFNS and CBNS (10 µmol/L) in THF and MeOH. (e, f) Fluorescence spectra of CSFNS and CBNS (10 µmol/L) in EG and DMSO; λ_ex = 400 nm, λ_em1 = 490 nm, λ_em2 = 690 nm; slit widths: 5.0 nm/5.0 nm.

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  Figure 3  (a) The processes of cell culture and imaging. (b) HepG2 cells were incubated with CSFNS/CBNS (5 µmol/L) for different times (1, 10, 20, 30 min) and then were imaged by confocal fluorescence microscopy. (c, d) Average fluorescence intensity in the blue/NIR channel from parallel images for both probes. (e) Representative confocal imaging for HepG2, HL-7702 cells and DOX-induced senescent HL-7702 cells incubated with CSFNS (5 µmol/L) for 10 min. Scale bar: 20 µm. (f) X-gal staining images of normal HL-7702 cells and DOX-induced senescent HL-7702 cells. Scale bar: 100 µm. (g) Average fluorescence intensity in the blue/NIR channel from parallel images. Error bars represent ±S.D. with n = 3. ns, not significant. **P < 0.01 vs. HepG2 cells are analyzed with two-sided Student's t-test. Blue channel: λ_ex/λ_em = 405/425–475 nm; NIR channel: λ_ex/λ_em =405/663–738 nm.

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