Ultraphotostable Phosphorescent Nanosensors for Sensing the Lysosomal pH at the Single-Cell Level over Long Durations.

Ultraphotostable phosphorescent nanosensors have been designed for continuously sensing the lysosome pH over a long duration. The nanosensors exhibited excellent photostability, high accuracy, and capability to measure pH values during cell proliferation for up to 7 days. By arranging a metal-ligand complex of long phosphorescence lifetime and pH indicator in silica nanoparticles, we discover efficient Förster resonance energy transfer, which converts the pH-responsive UV-vis absorption signal of the pH indicator into a phosphorescent signal. Both the phosphorescent intensity and lifetime change at different pH values, and intracellular pH values can be accurately measured by our custom-built rapid phosphorescent lifetime imaging microscopy. The excellent photostability, high accuracy, and good biocompatibility prove that these nanosensors are a useful tool for tracing the fluctuation of pH values during endocytosis. The methodology can be easily adapted to design new nanosensors with different pKa or for different kinds of intracellular ions, as there are hundreds of pH and ion indicators readily available.

DNA-mediated regioselective encoding of colloids for programmable self-assembly.

How far we can push chemical self-assembly is one of the most important scientific questions of the century. Colloidal self-assembly is a bottom-up technique for the rational design of functional materials with desirable collective properties. Due to the programmability of DNA base pairing, surface modification of colloidal particles with DNA has become fundamental for programmable material self-assembly. However, there remains an ever-lasting demand for surface regioselective encoding to realize assemblies that require specific, directional, and orthogonal interactions. Recent advances in surface chemistry have enabled regioselective control over the formation of DNA bonds on the particle surface. In particular, the structural DNA nanotechnology provides a simple yet powerful design strategy with unique regioselective addressability, bringing the complexity of colloidal self-assembly to an unprecedented level. In this review, we summarize the state-of-art advances in DNA-mediated regioselective surface encoding of colloids, with a focus on how the regioselective encoding is introduced and how the regioselective DNA recognition plays a crucial role in the self-assembly of colloidal structures. This review highlights the advantages of DNA-based regioselective modification in improving the complexity of colloidal assembly, and outlines the challenges and opportunities for the construction of more complex architectures with tailored functionalities.

Single-Stranded DNA-Encoded Gold Nanoparticle Clusters as Programmable Enzyme Equivalents.

Nanozymes have emerged as a class of novel catalytic nanomaterials that show great potential to substitute natural enzymes in various applications. Nevertheless, spatial organization of multiple subunits in a nanozyme to rationally engineer its catalytic properties remains to be a grand challenge. Here, we report a DNA-based approach to encode the organization of gold nanoparticle clusters (GNCs) for the construction of programmable enzyme equivalents (PEEs). We find that single-stranded (ss-) DNA scaffolds can self-fold into nanostructures with prescribed poly-adenine (polyA) loops and double-stranded stems and that the polyA loops serve as specific sites for seed-free nucleation and growth of GNCs with well-defined particle numbers and interparticle spaces. A spectrum of GNCs, ranging from oligomers with discrete particle numbers (2-4) to polymer-like chains, are in situ synthesized in this manner. The polymeric GNCs with multiple spatially organized nanoparticles as subunits show programmable peroxidase-like catalytic activity that can be tuned by the scaffold size and the inter-polyA spacer length. This study thus opens new routes to the rational design of nanozymes for various biological and biomedical applications.

TVH-19, a synthetic peptide, induces mineralization of dental pulp cells in vitro and formation of tertiary dentin in vivo.

Functional peptides derived from the active domains of odontogenesis-related proteins have been reported to promote dental hard tissue regeneration. The purpose of this study was to evaluate the effects of an artificially synthesized peptide, TVH-19, on odontoblast differentiation and tertiary dentin formation in indirect pulp capping (IPC) using in vitro and in vivo experiments. TVH-19 did not exhibit any effect on the proliferation of human dental pulp cells (hDPCs) but significantly promoted cell migration, compared with the control (p < 0.05). TVH-19-treated hDPCs showed significantly higher alkaline phosphatase (ALP) activity and stronger alizarin red staining (ARS) reactivity than the control group (p < 0.05). TVH-19 also upregulated the mRNA and protein expression levels of odontogenic genes. After generating IPC in rats, the samples of teeth were studied using micro-computed tomography (Micro-CT), hematoxylin & eosin (HE) staining, and immunohistochemical staining to investigate the functions of TVH-19. The in vivo results showed that TVH-19 induced the formation of tertiary dentin, and reduced inflammation and apoptosis, as evident from the downregulated expression of interleukin 6 (IL-6) and cleaved-Caspase-3 (CL-CASP3). Overall, the results of our study suggest that TVH-19 induces differentiation of hDPCs, promotes tertiary dentin formation, relieves inflammation, and reduces apoptosis, indicating the potential applications of TVH-19 in IPC.

Luminescent Oxygen-Sensitive Ink to Produce Highly Secured Anticounterfeiting Labels by Inkjet Printing.

A new covert luminescent anticounterfeiting (AC) technology was developed by employing combinatorial chemistry and concentration-dependent stimulus-responsive luminescent patterns. Oxygen-sensitive materials are carefully tailed to be inkjet printable and to form luminescent color inks. The inks are placed in the tanks of a jet printer. The printed luminescent patterns exhibited multilevel and highly secured AC features. Unlike conventional luminescent AC technology that solely relies on luminescent molecules/nanoparticles, the new technique utilizes the following features to fight counterfeiting: (1) the combination of luminescent oxygen-sensitive probes (OSPs) and the oxygen-permeable matrix (OPM), (2) the unique nonlinear oxygen-responsive behavior, (3) the local oxygen concentration, and (4) a luminescence lifetime reading device. The virtually unlimited number of codes is mainly due to the following features: (a) an almost endless number of combinations of OSPs and OPMs and (b) the nonlinearity of the Stern-Volmer plots that describe quenching of luminescence by oxygen. This combinatorial chemistry strategy makes it very difficult for counterfeiters to find the right composition even when the chemical composition of the luminescent molecules/nanoparticles was known. Information encrypted via this new methodology exhibits extremely high security, as counterfeiters need to identify all (not part of them) the following security measures: (1) the right combination of OSPs and OPMs, (2) the right chemical stimulus (here oxygen), (3) the proper oxygen concentration, and (4) the correct luminescence lifetime values.

Antibacterial Effect of Caffeic Acid Phenethyl Ester on Cariogenic Bacteria and Streptococcus mutans Biofilms.

Dental caries is the most common disease in the human mouth. Streptococcus mutans is the primary cariogenic bacterium. Propolis is a nontoxic natural product with a strong inhibitory effect on oral cariogenic bacteria. The polyphenol-rich extract from propolis inhibits S. mutans growth and biofilm formation, as well as the genes involved in virulence and adherence, through the inhibition of glucosyltransferases (GTF). However, because the chemical composition of propolis is highly variable and complex, the mechanism of its antimicrobial action and the active compound are controversial and not completely understood. Caffeic acid phenethyl ester (CAPE) is abundant in the polyphenolic compounds from propolis, and it has many pharmacological effects. In this study, we investigated the antibacterial effects of CAPE on common oral cariogenic bacteria (Streptococcus mutans, Streptococcus sobrinus, Actinomyces viscosus, and Lactobacillus acidophilus) and its effects on the biofilm-forming and cariogenic abilities of S. mutans CAPE shows remarkable antimicrobial activity against cariogenic bacteria. Moreover, CAPE also inhibits the formation of S. mutans biofilms and their metabolic activity in mature biofilms. Furthermore, CAPE can inhibit the key virulence factors of S. mutans associated with cariogenicity, including acid production, acid tolerance, and the bacterium's ability to produce extracellular polysaccharides (EPS), without affecting bacterial viability at subinhibitory levels. In conclusion, CAPE appears to be a new agent with anticariogenic potential, not only via inhibition of the growth of cariogenic bacteria.

Integrating Time-Resolved Imaging Information by Single-Luminophore Dual Thermally Activated Delayed Fluorescence.

The fact that the lifetime of photoluminescence is often difficult to access because of the weakness of the emission signals, seriously limits the possibility to gain local bioimaging information in time-resolved luminescence probing. We aim to provide a solution to this problem by creating a general photophysical strategy based on the use of molecular probes designed for single-luminophore dual thermally activated delayed fluorescence (TADF). The structural and conformational design makes the dual TADF strong in both diluted solution and in an aggregated state, thereby reducing sensitivity to oxygen quenching and enabling a unique dual-channel time-resolved imaging capability. As the two TADF signals show mutual complementarity during probing, a dual-channel means that lifetime mapping is established to reduce the time-resolved imaging distortion by 30-40 %. Consequently, the leading intracellular local imaging information is serialized and integrated, which allows comparison to any single time-resolved signal, and leads to a significant improvement of the probing capacity.

Dual-Phase Thermally Activated Delayed Fluorescence Luminogens: A Material for Time-Resolved Imaging Independent of Probe Pretreatment and Probe Concentration.

Developing luminescent probes with long lifetime and high emission efficiency is essential for time-resolved imaging. However, the practical applications usually suffer from emission quenching of traditional luminogens in aggregated states, or from weak emission of aggregation-induced emission type luminogens in monomeric states. Herein, we overcome this dilemma by a rigid-and-flexible alternation design in donor-acceptor-donor skeletons, to achieve a thermally activated delayed fluorescence luminogen with high emission efficiency both in the monomeric state (quantum yield up to 35.3 %) and in the aggregated state (quantum yield up to 30.8 %). Such a dual-phase strong and long-lived emission allows a time-resolved luminescence imaging, with an efficiency independent of probe pretreatment and probe concentration. The findings open opportunities for developing luminescent probes with a usage in larger temporal and spatial scales.

Luminescent Silica Nanosensors for Lifetime Based Imaging of Intracellular Oxygen with Millisecond Time Resolution.

Intracellular oxygen concentration was quantitatively imaged and rapidly traced with millisecond time resolution. We have demonstrated a new kind of oxygen nanosensors based on a ruthenium complex doped solid silica nanoparticles, which showed high oxygen sensing performance (I0/I100 = 3.29, t95 < 3 s) and ease of surface functionalization. Their sensing performance can be tuned by changing types of oxygen-sensitive probes and particle morphology. The nanosensors showed excellent control in both sensor size (from 30 to 200 nm), monodispersity, morphology, surface chemistry, and batch to batch consistency. Their uniform size distribution and good biocompatibility made them suitable for intracellular studies. Because the sensor surface can be easily functionalized with arbitrary units (such as transmembrane motifs, drugs, organelle-targeting groups, imaging reagent, and multiple sensor probes), these nanosensors provide a general platform to build easy-to-use tools for intracellular applications. The ease of surface functionalization was demonstrated by modifying the sensors outer surface with morpholinopropylamine and (3-carboxypropyl) triphenyl phosphonium, to actively target intracellular lysosomes and mitochondria of the tested cell lines (HeLa, MCF-7, and MCF-10A). Applying the mitochondria-targeting oxygen nanosensor together with our custom-built rapid phosphorescent lifetime imaging system, variations of intracellular oxygen have been quantitatively imaged and traced (in millisecond intervals) in real time and in situ.

Fully Reversible Optical Sensor for Hydrogen Peroxide with Fast Response.

A fully reversible optical sensor for hydrogen peroxide with fast response is presented. The sensor was fabricated by in situ growing ultrasmall platinum nanoparticles (PtNPs) inside the pores of fibrous silica particles (KCC-1). The nanocomposite was then embedded into a hydrogel matrix and form a sensor layer, the immobilized PtNPs can catalytically convert hydrogen peroxide into molecular oxygen, which is measured via luminescent quenching based oxygen sensor underneath. Owing to the high porosity and permeability of KCC-1 and high local concentration of PtNPs, the sensor exhibits fast response (less than 1 min) and full reversibility. The measurement range of the sensor covers 1.0 µM to 10.0 mM, and a very small amount of sample is required during measurement (200 µL). Because of its high stability, excellent reversibility and selectivity, and extremely fast response, the sensor could fulfill all industry requirements for real-time measurement and fill market vacancy.

Comparative salivary proteomics analysis of children with and without dental caries using the iTRAQ/MRM approach.

BACKGROUND: Dental caries is a major worldwide oral disease afflicting a large proportion of children. As an important host factor of caries susceptibility, saliva plays a significant role in the occurrence and development of caries. The aim of the present study was to characterize the healthy and cariogenic salivary proteome and determine the changes in salivary protein expression of children with varying degrees of active caries, also to establish salivary proteome profiles with a potential therapeutic use against dental caries. METHODS: In this study, unstimulated saliva samples were collected from 30 children (age 10-12 years) with no dental caries (NDC, n = 10), low dental caries (LDC, n = 10), and high dental caries (HDC, n = 10). Salivary proteins were extracted, reduced, alkylated, trypsin digested and labeled with isobaric tags for relative and absolute quantitation, and then they were analyzed with GO annotation, biological pathway analysis, hierarchical clustering analysis, and protein-protein interaction analysis. Targeted verifications were then performed using multiple reaction monitoring mass spectrometry. RESULTS: A total of 244 differentially expressed proteins annotated with GO annotation in biological processes, cellular component and molecular function were identified in comparisons among children with varying degrees of active caries. A number of caries-related proteins as well as pathways were identified in this study. As compared with caries-free children, the most significantly enriched pathways involved by the up-regulated proteins in LDC and HDC were the ubiquitin mediated proteolysis pathway and African trypanosomiasis pathway, respectively. Subsequently, we selected 53 target proteins with differential expression in different comparisons, including mucin 7, mucin 5B, histatin 1, cystatin S and cystatin SN, basic salivary proline rich protein 2, for further verification using MRM assays. Protein-protein interaction analysis of these proteins revealed complex protein interaction networks, indicating synergistic action of salivary proteins in caries resistance or cariogenicity. CONCLUSIONS: Overall, our results afford new insight into the salivary proteome of children with dental caries. These findings might have bright prospect in future in developing novel biomimetic peptides with preventive and therapeutic benefits for childhood caries.

A background-subtraction strategy leads to ratiometric sensing of oxygen without recalibration.

Luminescence-quenching based optical oxygen sensors have wide applications in many fields, which have already replaced almost 40% of the commercial market share dominated previously by the Clark oxygen electrode. The majority of optical oxygen sensors are based on lifetime measurement, which are precise, but are relatively expensive, and require high-speed electronics and detecting circuits. Alternatively, oxygen concentration can be measured via a luminescence intensity change, which is a referenced approach according to the Stern-Volmer equation. However, luminescence intensity based measurement tends to be highly influenced by background light. At a given sensor composition, different instrumentation setups, sensor surface roughnesses and thicknesses, and environmental light will result in significantly different calibration curves and sensitivities. This makes luminescence-intensity based optical sensors almost impossible to use practically, because each sensor needs to be recalibrated before use, and the calibration curve each time is quite different. We have solved this problem by introducing a new background-subtraction strategy. After background subtraction, oxygen sensors with different probe concentrations, instrumentation setups, surface roughnesses, supporting matrixes, and at different temperatures present identical calibration curves. This could greatly reduce the calibration task during practical use. Combined with the advantages of low price and a simple optical configuration, the new method will significantly promote wider applications of optical oxygen sensors.

A lysosome-targeting nanosensor for simultaneous fluorometric imaging of intracellular pH values and temperature.

Lysosomal pH and temperature are two crucial physiological parameters that are involved in regulating intracellular homeostasis, and their precise measurements are extremely important in understanding this process and diseases diagnosis. A lysosome-targeting nanosensor has been designed for simultaneous imaging of pH values and temperature in HeLa cells. Three dyes were covalently immobilized either inside or on silica nanoparticles. The nanosensors have an average diameter of 95 nm. The large surface area of these nanomaterials provides abundant sites for multi-functionality. The surface of nanosensors has been modified with positively-charged amino groups in order to facilitate endocytosis and targeting lysosome. Fluorescein is used as the indicator probe for pH measurement, rhodamine B is the probe for temperature, and a europium complex acts as the reference dye. The dual nanosensor responds to pH values in the range from 3.0 to 9.0, and to temperature in the range from 20 to 60 °C. Owing to its good biocompatibility and good sensitivity, the dual nanosensor has been used to monitor changes in local pH values and temperature in the lysosome of HeLa cells. Graphical abstract A dual nanosensor for simultaneously imaging of pH values and temperature inside the lysosome of HeLa cells was constructed by labelling three luminophores in/on silica nanoparticles. It shows high sensitivity and selectivity, good photostability, and good biocompatibility.

New insights into the electrochemical detection application of p-p junction foam: the effects of the interfacial potential barrier.

3D NiO/Co3O4 p-p junction foam was fabricated and applied for electrochemical detection of biomarkers. The theoretical model of employing the interfacial potential barrier as an electrochemical tuning factor was explored in depth. The signals of different targets with similar redox properties could be controllably distinguished by depressing or strengthening the potential barrier. The absorbed positively charged molecules would induce negative charges, inciting a decrease of the potential barrier height Φ and resistance, which is an enhanced tuning factor of the electrochemical signal. However, the effects of the absorbed negatively charged molecules went completely in the inverse direction; the resistance increased following by the increased Φ, which is a weakened tuning factor. Furthermore, the optimum adjustive effects of the p-p junction were validated as both the p-type regions are fully exposed. It is a general strategy to solve the difficulty in selective electrochemical detection of an analyte with similar redox properties. The results build a bridge to connect the potential barrier and electrochemical detection.

Unveiling the mechanism of an amelogenin-derived peptide in promoting enamel biomimetic remineralization.

Amelogenin and its derived peptides have exhibited excellent efficacy in promoting enamel biomimetic remineralization. However, little is known about their specific action mechanisms. Herein, by combining experiments and computer simulation, the mechanism of an amelogenin-derived peptide QP5 in regulating enamel biomimetic remineralization is unveiled for the first time. In experiments, peptide QP5 was separated into (QPX)5 and C-tail domains, the interactions of peptide-minerals in nucleation solution and the regulation of peptide on enamel biomimetic remineralization were explored. QP5 exhibited an unordered conformation when mineral ions existed, and it could adsorb on minerals through its two domains, thereby inhibiting spontaneous nucleation. The remineralized enamel regulated by C-tail showed better mechanical properties and formed more biomimetic crystals than that of (QPX)5, indicating the C-tail domain of QP5 played an important role in forming enamel-like crystals. The simulation results showed that the conformation of QP5 changed greatly, mainly exhibiting ß-bend, ß-turn, and coil structures, and it eventually adsorbed on enamel through negatively charged residues of the C-tail domain, then captured Ca2+ from solution to promote enamel remineralization. This study improved the evaluation methods of the mechanism of biomimetic peptides, and laid a theoretical basis for the amelioration and clinical transformation of peptide QP5.

Mineralization promotion and protection effect of carboxymethyl chitosan biomodification in biomimetic mineralization.

Biomimetic mineralization emphasizes reversing the process of dental caries through bio-inspired strategies, in which mineralization promotion and collagen protection are equally important. In this study, carboxymethyl chitosan (CMC) was deemed as an analog of glycosaminoglycan for biomimetic modification of collagen, both of the mineralization facilitation and collagen protection effect were evaluated. Experiments were carried out simultaneously on two-dimensional monolayer reconstituted collagen model, three-dimensional reconstituted collagen model and demineralized dentin model. In three models, CMC was successfully cross-linked onto collagen utilizing biocompatible 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy sulfosuccinimide sodium salt to achieve biomodification. Results showed that CMC biomodification increased collagen's hydrophilicity, calcium absorption capacity and thermal degradation resistance. In demineralized dentin model, the activity of endogenous matrix metalloproteinases was significantly inhibited by CMC biomodification. Furthermore, CMC biomodification significantly improved cross-linking and intrafibrillar mineralization of collagen, especially in the two-dimensional monolayer reconstituted collagen model. This study provided a biomimetic mineralization strategy with comprehensive consideration of collagen protection, and enriched the application of chitosan-based materials in dentistry.

Promoting effect of a calcium-responsive self-assembly ß-sheet peptide on collagen intrafibrillar mineralization.

Recently, a de novo synthetic calcium-responsive self-assembly ß-sheet peptide ID8 (Ile-Asp-Ile-Asp-Ile-Asp-Ile-Asp) has been developed to serve as the template inducing hydroxyapatite nucleation. The aim of this study was to evaluate the effect of ID8 on intrafibrillar mineralization of collagen making full use of its self-assembly ability. The mineralization experiments were carried out in vitro on both bare Type I collagen and fully demineralized dentin samples. The calcium-responsive self-assembly of ID8 was revealed by circular dichroism spectrum, 8-anilino-1-naphthalenesulfonic acid ammonium salt hydrate assay, attenuated total reflection Fourier transform infrared spectrum (ATR-FTIR) and transmission electron microscope (TEM). Polyacrylic acid (450 kDa) with a concentration of 100 µg ml-1 was selected as the nucleation inhibitor based on the determination of turbidimetry and TEM with selected area electron diffraction (TEM-SAED). The results showed that collagen intrafibrillar mineralization was significantly promoted with the pretreatment of self-assembly ID8 detected by TEM-SAED, SEM, X-ray diffraction and ATR-FTIR. The pretreatment of collagen utilizing self-assembly ID8 not only enhanced intermolecular hydrogen bonding but also contributed to calcium retention inside collagen and significantly increased the hydrophilicity of collagen. These results indicated that peptides with self-assembly properties like ID8 are expected to be potential tools for biomimetic mineralization of collagen.

The Amelogenin-Derived Peptide TVH-19 Promotes Dentinal Tubule Occlusion and Mineralization.

In this study, the amelogenin-derived peptide, TVH-19, which has been confirmed to promote mineralization, was evaluated to derive its potential to induce dentinal tubule occlusion. The binding capability of fluorescein isothiocyanate (FITC)-labeled TVH-19 to the demineralized dentin surface was analyzed by confocal laser scanning microscopy (CLSM). Additionally, the sealing function of the peptide was studied through the remineralization of demineralized dentin in vitro. The adsorption results showed that TVH-19 could bind to the hydroxyapatite and demineralized dentin surfaces, especially to periodontal dentin. Scanning electron microscopy analysis further revealed that TVH-19 created mineral precipitates. The plugging rate in the TVH-19 group was higher than that in the PBS group. Moreover, energy-dispersive X-ray spectroscopy (EDX) results indicated that the calcium/phosphorus (Ca/P) ratio of the new minerals induced by TVH-19 was close to that of the hydroxyapatite. Attenuated total internal reflection-Fourier transform infrared (ATR-FTIR) spectrometry and X-ray diffraction (XRD) results indicated that the hydroxyapatite crystals formed via remineralization elongated the axial growth and closely resembled the natural dentin components. These findings indicate that TVH-19 can effectively promote dentin sealing by binding to the periodontal dentin, promoting mineral deposition, and reducing the space between the dentin tubules.

Evaluating the potential of an amelogenin-derived peptide in tertiary dentin formation.

Several novel biomaterials have been developed for dental pulp capping by inducing tertiary dentin formation. The aim of this study was to evaluate the effect of QP5, an amelogenin-based peptide, on the mineralization of dental pulp cells (DPCs) in vitro and in vivo. The cell viability of human DPCs (hDPCs) after treatment with QP5 was determined using the Cell Counting Kit-8 (CCK-8). Migration of hDPCs was assessed using scratch assays, and the pro-mineralization effect was determined using alkaline phosphatase (ALP) staining, alizarin red staining and the expression of mineralization-related genes and proteins. The results showed that QP5 had little effect on the cell viability, and significantly enhanced the migration capability of hDPCs. QP5 promoted the formation of mineralized nodules, and upregulated the activity of ALP, the expression of mRNA and proteins of mineralization-related genes. A pulp capping model in rats was generated to investigate the biological effect of QP5. The results of micro-computed tomography and haematoxylin and eosin staining indicated that the formation of tertiary dentin in QP5-capping groups was more prominent than that in the negative control group. These results indicated the potential of QP5 as a pulp therapy agent.

Long-Term Quantitatively Imaging Intracellular Chloride Concentration Using a Core-/Shell-Structured Nanosensor and Time-Domain Dual-Lifetime Referencing Method.

Luminescence lifetime-based nanosensors for chloride ions were designed by incorporating a luminescent ruthenium dye [Ru(1,10-phenanthroline)3] inside silica nanoparticles and chemically labelling their outer surface with chloride ion-sensitive fluorescent dyes (N,N'-bis(carboxypropyl)-9,9'-biacridine). The nanosensor surface was further functionalized with positively charged amino groups to facilitate intracellular uptake via endocytosis and target lysosomes. The nanosensors have an average diameter of 52 nm and are monodispersed in aqueous solutions. Because of the long lifetime of the reference ruthenium dye, the sensor response can be analyzed using the time-domain dual-lifetime referencing (td-DLR) approach. The use of pulsed excitation in td-DLR rather than intense continuous illumination in ratiometric measurements greatly prevents the dye from photobleaching which significantly improves its measurement stability and reproducibility for long-term monitoring. At optimum conditions, the sensor can measure chloride concentration in the range of 0-200 mM with a large ratiometric signal change from 140.9 to 40.2. Combined with our custom-built microscopic td-DLR system, variations of intracellular chloride concentration in lysosomes were imaged quantitatively with a high spatial resolution and accuracy.