An Integration Strategy to Develop Dual-State Luminophores with Tunable Spectra, Large Stokes Shift, and Activatable Fluorescence for High-Contrast Imaging

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE6 Jun 2022An Integration Strategy to Develop Dual-State Luminophores with Tunable Spectra, Large Stokes Shift, and Activatable Fluorescence for High-Contrast Imaging Yongchao Liu, Lili Teng, Chengyan Xu, Tian-Bing Ren, Shuai Xu, Xiaofeng Lou, Lin Yuan and Xiao-Bing Zhang Yongchao Liu State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082 Google Scholar More articles by this author , Lili Teng State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082 Google Scholar More articles by this author , Chengyan Xu State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082 Google Scholar More articles by this author , Tian-Bing Ren State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082 Google Scholar More articles by this author , Shuai Xu State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082 Google Scholar More articles by this author , Xiaofeng Lou State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082 Google Scholar More articles by this author , Lin Yuan State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082 Google Scholar More articles by this author and Xiao-Bing Zhang *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202100935 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Developing dual-state luminophores (DSLs) with strong fluorescence in both the monomer and aggregate states is urgently needed but remains a huge challenge because most current luminophores are either aggregation-induced emission or aggregation-caused quenching molecules. Moreover, limited by the structural conservation of the few existing DSLs, there are not enough response sites that can be used to customize various activatable fluorescent probes for specific molecular imaging. Herein, we engineered a general integration strategy for the fabrication of such DSLs with excellent photophysical properties. The DSLs, with their tunable spectra, a large Stokes shift (>170 nm), and achievable near-infrared (NIR) emission, show great potential for high-contrast imaging. Importantly, DSLs can be used as a universal platform for probe customization due to their activatable fluorescence through protection–deprotection of the phenolic hydroxyl group. Based on this, an NIR fluorescent probe DSL-Gal was developed for sensing of β-galactosidase in solutions, senescent cells, and liver metastases with high contrast, further confirming the superiority and universal feasibility of DSLs in probe design. The integration strategy may provide a novel approach for the generation of other DSLs and have great potential applications in bioimaging. Download figure Download PowerPoint Introduction Fluorescence imaging is a useful tool to illuminate biological information in complex systems and visualize the physiological process and other targets at the molecular level due to its high sensitivity and high spatial resolution.1–3 In recent decades, organic luminophores with excellent biocompatibility and easy modification appear to be a more promising choice for sensing and imaging of molecular targets in living systems.4–7 Such luminophores, however, easily suffer from the aggregation-caused quenching (ACQ) effect, of which the luminophores show strong fluorescence only in the monomer state but remain non- or less fluorescent in the aggregate state (Figure 1a).8 This is because these molecules in the aggregate state may experience strong π–π stacking interactions that lead to ACQ, which may lead to unreliable fluorescence sensing signals due to the generation of nonemissive aggregates.9 To eliminate the ACQ effect in the aggregate state, aggregation-induced emission (AIE) phenomenon, as a noncanonical emission mechanism, was first reported by Tang and co-workers,10–13 in which its applications were greatly extended. The AIE luminophores can be highly emissive in the aggregate state, but their monomeric emission in dilute aqueous solution is often weak (Figure 1b).14 This may lead to compromised fluorescence signals in the monomer state due to the generation of nonemissive monomer and place higher requirements on their application scenarios. Due to the complexity and diversity of the detection microenvironment, luminophores based on ACQ or AIE are still not adequate to meet the growing needs in the field of accurate imaging of biological targets because they can only exhibit bright fluorescence in a single monomer or aggregate state.13 To fill in the gap between AIE and ACQ, there is a growing interest in developing dual-state luminophores (DSLs) (Figure 1c), which intensely show fluorescence in both the monomer and aggregate states. Figure 1 | Strategies and characteristic analysis of the emission luminophores. Emission behavior of (a) ACQ luminophore with monomer-dependent emission, (b) AIE luminophore with aggregate-dependent emission, and (c) DSL with dual-state emission. Download figure Download PowerPoint In recent decades, only very few luminophores with dual-state properties have been reported.15,16 For instance, Zhao’s group15 developed several D–A–π–A–D molecules with high emission efficiency both in solution and in the solid state; Qian’s group16 reported a new class of dual-state emitters with apparent solvatochromism by constructing a donor–acceptor pattern and introducing a twisted triphenylamine moiety. Despite their unique emission properties, these DSLs lack appropriate fluorescence regulatory sites, which makes it difficult to customize activatable fluorescent probes to indicate the level of molecular targets via putting out a turn-on or ratiometric fluorescent signal. In addition, these DSLs also showed short emission wavelength (<600 nm), which greatly limits them for low background imaging and in vivo sensing. And it is hard to adjust their emission wavelengths and further derivatize such luminophores. So far, a universal strategy to produce DSLs with desirable photophysical properties is not available. In this study, we reported a general strategy to construct DSLs based on the integrated emission mechanism of excited state intramolecular proton transfer (ESIPT) and intramolecular charge transfer (ICT), wherein the protection–deprotection of the phenolic hydroxyl switch can finely regulate their fluorescence emission. By introducing ESIPT-generating groups into the classic ICT luminophore structure, a series of DSLs were fabricated, which not only retained the aggregate-state emissive characteristics of ESIPT luminophores, but also significantly enhanced the fluorescence of ICT luminophores in the monomer state through hydrogen-bond interaction. Consequently, these DSLs show dual-state emission, that is, they can emit fluorescence both in the monomer and aggregate states, as well as exhibit some excellent photophysical properties, including tunable fluorescence spectra, high fluorescence quantum yield (QY) and a large Stokes shift (>170 nm). Most importantly, by introducing functional groups on the phenolic hydroxyl group, the DSLs can perform OFF–ON fluorescence switching by protection–deprotection of the phenolic hydroxyl, indicating that DSLs could serve as a universal platform for customization of activatable fluorescent probes. As a proof of concept, an near-infrared (NIR) fluorescent probe DSL-Gal was developed for β-galactosidase sensing in solutions, senescent cells, and liver metastases with high contrast that further confirmed the improved superiority and feasibility of DSLs in molecular sensing. This unique emission behavior of DSLs would greatly facilitate the advancement of high-contrast bioimaging. Experimental Methods Reagents and apparatus All chemicals were purchased from commercial suppliers and used without further purification. β-gal was purchased from Sigma-Aldrich (Shanghai, China). The mice were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. (Changsha, Hunan, China). Water was purified and doubly distilled by a Milli-Q system (Millipore, USA). The UV–vis absorption spectra were acquired via a Shimadzu UV-2600 spectrophotometer (Japan). Fluorescence spectra were recorded on a HITACHI F-4600 fluorescence spectrophotometer (Japan) with a 1 cm standard quartz cell. Mass spectra were performed using a Finnigan LCQ Advantage ion trap mass spectrometer (Thermo Fisher Scientific, USA). NMR spectra were recorded on a Bruker DRX400 spectrometer (Switzerland) using tetramethylsilane (TMS) as an internal standard. Thin-layer chromatography (TLC) was conducted using silica gel 60 F254, and column chromatography was carried out over silica gel (200–300 mesh), both of which were obtained from Qingdao Ocean Chemicals (Qingdao, China). Fluorescence images of cells were obtained using an Olympus FV1000-MPE laser scanning confocal-microscope (Japan). Spectral measurements The fluorescence measurement experiments of DSL-Gal (10 μM) were performed in phosphate-buffered saline (PBS) (10 mM) with dimethyl sulfoxide (DMSO) as cosolvent solution (PBS/DMSO = 9∶1, v/v, 10 mM, pH 7.4). The reaction solution was transferred into a quartz cell to measure the absorbance or fluorescence spectra, with both excitation and emission slits set at 5 nm. The fluorescence spectra were measured with the excitation wavelength at 450 nm. The solutions of various testing species were prepared from (1) Blank, (2) Na+ (10 mM), (3) K+ (10 mM), (4) Ca2+ (10 mM), (5) Fe2+ (1 mM), (6) H2O2 (250 μM), (7) HClO (50 μM), (8) ONOO− (50 μM), (9) O2− (100 μM), (10) t-BuOOH (100 μM), (11) ꞏ OH (100 μM), (12) Cys (500 μM), (13) reduced glutathione (GSH; 1 mM), (14) H2S (100 μM), (15) SO32− (50 μM), (16) β-gal (50 U/L) in twice-distilled water. Calculation of fluorescence QY Fluorescence QY was determined using optically matching solutions of rhodamine 6G (Φf = 0.95 in EtOH solution) and cresol purple as reference (φ = 0.58 in EtOH solution) as the standarde QY was calculated using the following equation: Φ s = Φ r ( A r F s / A s F r ) ( n s 2 / n r 2 ) where, s and r denote sample and reference, respectively. A is the absorbance, F is the relative integrated fluorescence intensity, and n is the refractive index of the solvent. The QYs of DSL2 and DSL4 were determined in tetrahydrofuran (THF)/H2O = 1/1 (v/v) solution by using rhodamine 6G as the reference. The QYs of DSL1 and DSL3 were determined in THF/H2O = 1/1 (v/v) solution by using cresol purple as reference. Cell culture HeLa or OVCAR3 cells were maintained in RPMI-1640 medium with 10% fetal bovine serum (FBS; GIBCO, USA) and 1% penicillin–streptomycin at 37 °C in a humidified atmosphere containing 20% O2 and 5% CO2 as the normoxic condition. Cells were seeded in a 20 mm glass-bottom dish plated and grown to around 80% confluency for 24 h before the experiment. Confocal imaging and in vivo imaging In fluorescence cell imaging, 10 μM of DSLs and ICTs were incubated with the cells for 30 min before conducting the confocal experiments. The fluorescence signal of cells incubated with DSL-Gal (10 μM) was collected in the channel (600–700 nm) by using a semiconductor laser at 488 nm as the excitation source. For (E)-4-(4-hydroxystyryl)-5,5-dimethyl-2-oxo-2,5-dihydrofuran-3-carbonitrile (ICT1), DSL1,3, λex = 488 nm, λem = 600–700 nm, for 2-(3,5,5-trimethylcyclohex-2-en-1-ylidene)-malononitrile (ICT2), DSL2,4, TPE1 and TPE2, λex = 405 nm, λem = 500–600 nm. The fluorescent images of mice were obtained via an IVIS Lumina XR Imaging System (Caliper Life Sciences, USA) equipped with a cooled charge-coupled device (CCD) camera with the collected channel (λex = 430 nm, λem = 600–700 nm). Circular Region of interests (ROIs) were drawn over the areas and quantified by Lumina XR Living Image software (USA), version 4.3. Tumor models of peritoneal metastases and subcutaneous tumor All animal procedures were performed in accordance with the Guidelines for Care and Use of Lboratory Animals of Hunan University, and experiments were approved by the Animal Ethics Committee of the College of Biology (Hunan University). To develop the peritoneal metastases model, 1 × 106 OVCAR3 cells suspended in 300 μL of PBS (pH 7.4) were intraperitoneally injected into female nude mice (BALB/c, 7–8 weeks old) for 36 days. To develop the sc tumor model, 1 × 106 OVCAR3 cells suspended in 25 μL of PBS were subcutaneously injected into the underarm of each female nude mouse (BALB/c, 7–8 weeks old). Tumors with diameters of around 10 mm were formed after 16 days. Fluorescence imaging of DSL1 and ICT1 in liver tissue Liver tissue slices were prepared from freezing microtome. Then these tissues were incubated with DSL1 (50 μM) and ICT1 (50 μM) at 37 °C for 30 min, followed by washing them three times with PBS solution. Under the confocal fluorescence microscope with a 60× objective lens, the probe was excited at 488 nm, and fluorescence emissions in the 600–700 nm channel were gathered, respectively. In tissue depth imaging, the three-dimensional (3D) images were constructed along the z-axis direction. Visualization of β-gal activity in peritoneal metastases tissue and in tumor After 32 days of intraperitoneal injection of OVCAR3 cells, the mice were sacrificed and dissected, and then the peritoneal metastases were collected. Tumor tissue slices were prepared from freezing microtome. Next, these tissues were incubated with DSL-Gal (50 μM) at 37 °C for 1 h, followed by washing them three times with PBS solution. Under the confocal fluorescence microscope with a 60× objective lens, the probe was excited at 488 nm, and fluorescence emission at the 600–700 nm channel was gathered. In tissue depth imaging, the 3D images were constructed along the z-axis direction. For the sc tumor model, DSL-Gal (50 μM) was intratumorally injected at different times. Subsequently, the mice were immediately imaged via an IVIS Lumina XR Imaging System. Circular ROIs were drawn over each well, and fluorescent intensity was quantified by Living Image software. Results and Discussion Revealing the design and luminous mechanism of DSLs Our group has been engaged in a long-term research project investigating the use of ESIPT-based solid-state luminophores, such as 2-(2’-hydroxyphenyl)-4(3H)-quinazolinone (HPQ) and 6-Chloro-2-(2-hydroxy-5-(1,2,2-triphenylvinyl)phenyl)quinazolin-4(3H)-one (HTPQ), aiming to expand the utility of such luminophores for in situ bioimaging.17–20 However, they have shown short excitation and emission wavelengths (<600 nm), and only emitted fluorescence in the solid state. Recently, we hoped to redshift the excitation and emission wavelengths of such solid-state luminophores by extending their conjugate structure (Figure 2a). We found a convenient approach to integrate HPQ together with the classic ICT dyes to build long-wavelength solid-state luminophores, including ICT1 and ICT2, which show weak solution fluorescence due to the presence of a phenolic hydroxyl group that is not easily ionized ( Supporting Information Figure S1).21,22 Interestingly, a luminophore termed as DSL1, which is fabricated by HPQ and ICT1, immediately grabbed our attention when we discovered its strong fluorescence in both the monomer and aggregate states. Upon excitation at 450 nm, DSL1 displayed similar fluorescence emission at 629 and 642 nm in the monomer and aggregate states, respectively (Figure 2b). In addition, similar fluorescence intensity of polymer-encapsulated DSL-NP and DSL1 in solution was observed ( Supporting Information Figure S2), suggesting that DSL1 can effectively avoid fluorescence quenching caused by molecular aggregation. Figure 2 | Revealing the design and luminous mechanism of DSL1. (a) Chemical structure of HPQ and DSL1. (b) Fluorescence spectra of DSL1 in monomer and aggregate states. (c) Optimized structure of DSL1 and ICT1. (d) DFT molecular orbital plots [lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO)] of DSL1-O−N+, DSL1-OH, ICT1-O−, and ICT1-OH. Oscillator strength and excitation wavelength of (e) DSL1-O−N+ and DSL1-OH, (f) ICT1-O− and ICT1-OH through DFT calculation. Exchange functional: B3LYP. Basis sets: 6-31G*. (g) Probable emissive mechanism of DSL1 and its responsive mechanism for customization activatable probes. Download figure Download PowerPoint To give a theoretical explanation of the emissive mechanism of DSL1, we carried out density functional theory (DFT) calculations (Figure 2c). We used DSL1-O−, DSL1-O−N+, and DSL1-OH to represent the ionized configuration, proton transferred configuration and unionized configuration of DSL1, respectively. Similarly, ICT1-O−, and ICT1-OH were used to represent the ionized configuration and unionized configuration of ICT1, respectively. Compared with DSL1-OH, DSL1-O−N+ showed an obviously increased electron distribution of orbital plots (Figure 2d), as well as increased oscillator strength ( Supporting Information Table S1). These results indicated that the occurrence of the proton transfer process will increase the electron donating ability of the donor of DSL1, which was consistent with the enhanced fluorescence intensity of DSL1 and redshifted wavelength in THF/H2O solutions ( Supporting Information Figure S3). Importantly, DSL1-O− (the ionized configuration of DSL1) and DSL1-O−N+ showed similar electron distribution of orbital plots ( Supporting Information Figure S4), suggesting that the proton transfer process can indeed increase the ICT effect of DSL1, which is near to the ICT effect in the DSL1-O− configuration. These results demonstrate that the quinazolinone moiety could effectively assist the ionization of phenolic hydroxyl through intramolecular hydrogen-bond interaction without the help of any external conditions. For ICT1, the reduced ICT effect was observed in the ICT-OH form from its electron distribution of orbital plots (Figure 2d), as well as decreased oscillator strength ( Supporting Information Table S1), which was consistent with the extremely low fluorescence intensity and the QY of ICT1 in the phenolic hydroxyl state in solution ( Supporting Information Table S2). The results showed that ICT1 can emit strong fluorescence only after the proton on the phenolic hydroxyl group is removed, which requires specific external conditions, such as alkalinity. In addition, compared with ICT1, DSL1 showed a reduced excitation-energy difference before and after the ionization of phenolic hydroxyl (from 81 to 44 nm) (Figures 2e and 2f and Supporting Information Table S1), which also suggests its improved ionization capacity of phenolic hydroxyl. Therefore, the introduction of quinazolinone moiety at the ortho position of the phenolic hydroxyl group of ICT1 can indeed enhance its luminous efficiency in solution by the intramolecular hydrogen bond. Through dynamic light scattering, transmission electron microscopy, and Tyndall effects results, we found that DSL1 was dissolved in 10% H2O/THF mixed solution, but showed in aggregate state in 98% and 60% H2O/THF mixed solution ( Supporting Information Figure S5), indicating their freely switchable molecular states. Moreover, the emission spectra of DSL1 in THF/H2O solution with different water fractions showed its monomer and aggregated state emissions, further confirming its dual-state emissive properties ( Supporting Information Figure S3). These results have also confirmed that the emissive mechanism of DSL1 is ESIPT and ICT (Figure 2g),7,23,24 which allows for customization of activatable probes by the protection–deprotection of the phenolic hydroxyl group. Engineering dual-state emissive luminophores Starting from this initial DSL1 molecule, a series of luminophores were developed based on simple structural modifications using the following general guidelines: (1) start with the quinazolinone and benzothiazole backbone as an ESIPT-generating unit; and (2) introduce an additional electron withdrawing unit at the para position of the phenolic hydroxyl group to form an ICT system (Figure 3a). To our delight, the DSLs all showed fluorescence both in the monomer and aggregate states (Figures 3b and 3c). The synthetic route for DSLs is outlined in Supporting Information Scheme S1, and all the new compound structures are confirmed by NMR ( Supporting Information). The photophysical properties of DSL1-4 were initially investigated in different solvents with a range of polarities ( Supporting Information Figure S6–S9). Relatively high absorbance and low fluorescence of ICT1 and ICT2 were observed, resulting in the extremely low fluorescence QY (<1%). On the contrary, DSL1-4 all showed improved fluorescence and QY in these solvents with different polarities ( Supporting Information Table S2), suggesting that the quinazolinone and benzothiazole groups might be helpful for fluorescence increase. Compared with ICT2, ICT1 had a larger conjugate system and stronger push–pull electron ability in the molecular skeleton. Hence the integrated DSL1,3 showed longer excitation and emission wavelengths than DSL2,4. The absorption and fluorescence spectra of DSL1-4 in the monomer and aggregate states are shown in Figures 3d and 3e, in which DSL2,4 exhibit yellow fluorescence and DSL1,3 exhibit deep-red fluorescence, corresponding to the colors obtained by color coordinates ( Supporting Information Figure S10). Moreover, the photophysical properties of DSLs exhibit less dependence on the solvent polarity ( Supporting Information Figure S11). The statistical results on the photophysical properties of DSL1-4 are summarized in Table 1, giving similar color of fluorescence in both solid and solution states, a large Stokes shift (>170 nm), and relatively high fluorescence QY. Figure 3 | A generalizable molecular engineering strategy for developing DSLs. (a) Illustration of the “integration” strategy for the design of novel DSLs. Fluorescent photographs of DSL1-4 in (b) THF solution and (c) solid state under 365 nm excitation. Normalized fluorescent spectra of DSL1-4 in (d) THF solution and (e) solid state. Download figure Download PowerPoint Table 1 | Photophysical Properties of DSLs Luminophores λabs (nm)a λem (nm)a Stokes Shifta QY (%)b λem (nm)c QY (%)c DSL1 413 629 216 18.6 642 11.8 DSL2 382 556 174 21.2 577 30.7 DSL3 416 649 233 15.3 644 3.5 DSL4 378 572 194 22.4 578 32.5 aThe data were determined in monomer state. bThe QYs of DSL2 and DSL4 were determined by using rhodamine 6G as reference (φ = 0.98 in EtOH solution). The QYs of DSL1 and DSL3 were determined by using cresol purple as reference (φ = 0.58 in EtOH solution). cThe data were determined in the aggregate state. Improved fluorescence and imaging effects of DSL4 Compared with the low fluorescence of ICT1 and ICT2, DSL1-4 all showed stronger fluorescence in both organic solvent and aqueous solutions (Figure 4a), indicating the improved fluorescence of DSLs after integration. Having demonstrated the huge potential of DSL1 as a probe precursor, we then attempted to explore its advantages in biological applications. First, we tested the cytotoxicity of DSLs (Figure 4b) and observed the negligible cell viability changes after treating cells with different concentrations of DSLs, indicating the good biocompatibility of DSLs. Since DSLs emitted stronger fluorescence than ICTs in both organic solvent and aqueous solutions, strong fluorescence in cell imaging was observed in DSL-incubated cells while weak fluorescence was observed in ICT-incubated cells (Figures 4c and 4d). Relative fluorescence intensity statistics show that DSL1 and DSL4 are 19.5 and 10.2 times stronger than ICT1 and ICT2, respectively (Figures 4e and 4f). Besides, in liver tissue imaging, DSL1 showed deeper tissue penetration under the same concentration and brighter fluorescence under the same penetration depth in imaging of liver tissue than ICT1 (Figure 4g). These results have confirmed the improved fluorescence of DSLs over ICTs in high-contrast cell imaging as a result of our integration strategy. Figure 4 | Improved fluorescence and imaging effects of DSLs over ICTs. (a) Normalized fluorescence intensity of ICT1, ICT2, and DSLs in different solvents under the maximum absorbance. (b) Cell viability of HeLa cells treated with different concentrations of DSLs. (c) Confocal fluorescent images of ICT1 and DSL1,3 in HeLa cells. (d) Confocal fluorescent images of ICT2 and DSL2,4 in living cells. For ICT1, DSL1,3, λex = 488 nm, λem = 600–700 nm, for ICT2, DSL2,4, λex = 405 nm, λem = 500–600 nm. Scale bar = 20 μm. (e and f) Normalized fluorescence intensity in (c) and (d), respectively. (g) Depth fluorescence images of DSL1 and ICT1 in liver tissues. λex = 488 nm, λem = 600–700 nm. Scale bar: 100 μm. Download figure Download PowerPoint Based on the excellent imaging properties and photostability of DSL1 ( Supporting Information Figure S12), we chose DSL1 as a representative luminophore to further clarify the advantages of DSLs in bioimaging, by comparing them with classic monomer-dependent emission luminophores (Rh 6G) and aggregate-dependent emission luminophores (TPE1 and TPE2).25–28 As shown in Supporting Information Figure S13, DSL1-loaded cells showed stronger fluorescence than TPE1 and TPE2-loaded cells under the same concentrations, suggesting its better imaging effects. In addition, with the increased incubation concentrations of these luminophores, the fluorescence in Rh 6G-incubated cells (or tissues) showed a tendency of first increasing and subsequently decreasing, and sharply increasing only in high concentrations of TPE1-incubated cells or tissues ( Supporting Information Figure S14). This phenomenon may be attributed to the fact that the compromised fluorescence of Rh 6G in the aggregate state and TPE1 in the monomer state, respectively. Surprisingly, the fluorescence of DSL1-incubated cells and tissues showed concentration-dependent enhancement, which mainly because the emission that DSL1 showed was not affected by the molecular states. All these results demonstrate that DSLs have great potential in high-contrast bioimaging. Customization of an activatable fluorescent probe DPL-Gal for sensing and imaging of β-gal Next, to further verify the feasibility of DSL1 for probe customization, we applied it to design and synthesize an activatable fluorescent probe for high-reliable imaging of β-gal ( Supporting Information Scheme S2), which has been demonstrated as an important biomarker for cell senescence and primary ovarian cancers.29–34 And to extend the emission wavelength of DSL1, we introduced a chlorine atom into the structure of DSL1, whose photophysical properties are shown in Supporting Information Figure S15 and Table S3, and then synthesized the probe DSL-Gal (Figure 5a). First, we confirmed the response ability of DSL-Gal to β-gal in buffer solutio