Transfer Fluorescent Probe Research Articles

A ratiometric fluorescent probe revealing the abnormality of acetylated tau by visualizing polarity in Alzheimer's disease.

Abnormal neuronal polarity leads to early deficits in Alzheimer's disease (AD) by affecting the function of axons. Precise and rapid evaluation of polarity changes is very important for the early prevention and diagnosis of AD. However, due to the limitations of existing detection methods, the mechanism related to how neuronal polarity changes in AD is unclear. Herein, we reported a ratiometric fluorescent probe characterized by neutral molecule to disclose the polarity changes in nerve cells and the brain of APP/PS1 mice. Cy7-K showed a sensitive and selective ratiometric fluorescence response to polarity. Remarkably, unlike conventional intramolecular charge transfer fluorescent probes, the fluorescence quantum yield of Cy7-K in highly polar solvents is higher than that in low polar solvents due to the transition of neutral quinones to aromatic zwitterions. Using the ratiometric fluorescence imaging, we found that beta-amyloid protein (Aβ) inhibits the expression of histone deacetylase 6, thereby increasing the amount of acetylated Tau protein (AC-Tau) and ultimately enhancing cell polarity. There was a high correlation between polarity and AC-Tau. Furthermore, Cy7-K penetrated the blood-brain barrier to image the polarity of different brain regions and confirmed that APP/PS1 mice had higher polarity than Wild-type mice. The probe Cy7-K will be a promising tool for assessing the progression of AD development by monitoring polarity.

Near-infrared Fluorescent Probes with Long-acting Cyclic Monitoring and Effectively Eliminating Peroxynitrite.

Two through-bond energy transfer fluorescent probes with a dihydroxyl naphthyl-pyrenyl conjugated system were synthesized for long-acting cyclic monitoring and eliminating peroxynitrite (ONOO- ). The probes exhibit large Stokes shifts (230 or 280 nm) and the fluorescence at 620 or 652 nm rapidly change in response to continuously variable concentrations of ONOO- under physiological conditions. The probes show good reversibility and can rapidly monitor the concentration changes of ONOO- in real time. In addition, with the additions of the probes, the decomposition of ONOO- is greatly accelerated. Therefore, the probes can effectively eliminate the excess ONOO- as well as sensing it. The biological studies showed that the probes can effectively and reversibly eliminate both exogenous and endogenous ONOO- in-situ as well as sensing its changes in cells, which can help to maintain the normal physiological concentration of ONOO- in organisms. This is the first system that a probe achieves multifunction including real-time detection, long-acting cyclic monitoring and in-situ elimination, thereby maintaining a normal physiological balance for ONOO- .

Protein-activated and FRET enhanced excited-state intermolecular proton transfer fluorescent probes for high-resolution imaging of cilia and tunneling nanotubes in live cells

Excited-state intermolecular proton transfer (inter-ESPT) fluorescent probes responsive to specific bioactive molecules should be greatly promising for protein sensing, DNA mutation simulating and cellular process regulating. However, the inter-ESPT molecules are recessive ESPT fluorophores, which need the assistance of other molecules with both hydrogen-bond accepting and donating abilities to turn on the tautomeric fluorescence. Valid design strategies to create powerful inter-ESPT fluorescent probes are poorly developed, particularly for proteins as targets. We recently reported a unique supramolecular strategy to trigger the inter-ESPT process based on the probe-protein recognition by H-bonding and to image protein-based subcellular structures in live cells. Herein, we found that our inter-ESPT probes (inter-ESPT-01) bearing a 2-amino-3-cyanopyridine scaffold can anchor proteins and light up the “invisible” ESPT state, so as to image the proteins or the protein-based subcellular organelles. More importantly, the inter-ESPT emission of inter-ESPT-01 can be significantly enhanced by the FRET process between amino and imino tautomers, endowing the inter-ESPT-01 probes with super-bright tautomeric fluorescence. The expressed proteins Ecallantide and MarTX were selected as the models to light up the inter-ESPT fluorescence of the probes and revealed that the inter-ESPT process can be triggered by the specific probe-protein recognition events. In the use of the super-bright inter-ESPT fluorescence, not only the proteins, but also the protein-based cilia and tunneling nanotubes (TNTs) can be specifically visualized in living cancer cells. Furthermore, such recognition-driven strategy allows us to construct a unique inter-ESPT probe to track and image specific endogenous proteins in live cells, highlighting the potential of inter-ESPT fluorogens as novel intelligent biomaterials.

Spectral Properties Echoing the Tautomerism of Milrinone and Its Application to Fe3+ Ion Sensing and Protein Staining.

Knowledge on the spectral properties of the tautomers of milrinone (MLR) in solvents and solid-state, as well as under light conditions is of critical importance from both theoretical and practical points of view. Herein, we investigated the spectral properties of MLR in different conditions using UV-Vis and fluorescence spectroscopies. The experimental results demonstrated that MLR can undergo the tautomerization reaction induced by solvent polarity, light and pH, eliciting four tautomeric structures (enol, keto, anion, and cation forms). The interesting multi-functional groups in MLR enable it to coordinate with metal ions or to recognize gust molecules by H-bonding. In the use of MLR as an excited-state intermolecular proton transfer (inter-ESPT) fluorescent probe, a highly sensitive and selective analysis of Fe3+ was developed, which offered a sensitive detection of Fe3+ with the detection limit of 3.5 nM. More importantly, MLR exhibited the ability of anchoring proteins and led to the recognition-driven turn-on inter-ESPT process, highlighting the potential for the probe to image proteins in electrophoresis gels. The spectral experimental results revealed the possible degradation mechanism, so that we can better understand the side effects of oral preparations. The use of the available drug as an inter-ESPT fluorescent probe is simple and accurate, providing a good method for Fe3+ ion sensing and protein staining.

MtDNA-Specific Ultrasensitive Near-Infrared Fluorescent Probe Enables the Differentiation of Healthy and Apoptotic Cells.

Mitochondrial DNA (mtDNA) as a class of important genetic material is easily damaged, which can result in a series of metabolic diseases, hereditary disease, and so on. mtDNA is an ultrasensitive indicator for the health of living cells due to the extremely short physiological response time of mtDNA toward damage (ca. 5.0 min). Therefore, the development of specific ultrasensitive fluorescent probes that can in real-time monitor mtDNA in vivo are of great value. With this research, we developed a near-infrared twisted intramolecular charge transfer (TICT) fluorescent probe YON. YON is a thread-like molecule with an A-π-D-π-A structure, based on the dicyanoisophorone fluorophore. The molecular design of YON enabled the specific binding with dsDNA (binding constant (K) = 8.5 × 105 M-1) within 1.3 min. And the appropriate water-oil amphiphilicity makes YON significantly accumulate in the mitochondria, enabling the specific binding to mtDNA. The fluorescence intensity at 640 nm of YON enhanced linearly with increasing concentrations of mtDNA. Dicyanoisophorone as the strong electron-withdrawing group that was introduced into both ends of the molecule resulted in YON being a classic quadrupole, so it could ultrasensitively detect trace mtDNA. The minimum detection limit was 71 ng/mL. Moreover, the large Stokes shift (λex = 435 nm, λem = 640 nm) makes YON suitable for "interference-free" imaging of mtDNA. Therefore, YON was used to monitor trace changes of mtDNA in living cells; more importantly, it could be used to evaluate the health of cells by monitoring microchanges of mtDNA, enabling the ultrasensitive evaluation of apoptosis.

An ESIPT fluorescent probe based on Staudinger reaction for detection of triphenylphosphine and tetrakis(triphenyl-phosphine)palladium

Triphenylphosphine (PPh3) and tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) are harmful at extremely low concentration, thus it is essential to explore selective and sensitive method to detect them. Based on Staudinger reaction mechanism, here we present a simple excited-state intramolecular proton transfer (ESIPT) fluorescent probe (HBTN) for detection of PPh3 and Pd(PPh3)4 over other phosphine derivatives with differential channel fluorescence (enol and keto emission). The reaction-typed fluorescence probe with novel Staudinger reaction mechanism offers very low autofluorescence and a large signal-to-noise response signal, which is beneficial for the sensitive detection of PPh3 and Pd(PPh3)4. Moreover, HBTN can be used for efficiently detecting Pd(PPh3)4 residues in highly purified products and imaging PPh3 in living cells.

A Solvent-Mediated Excited-State Intermolecular Proton Transfer Fluorescent Probe for Fe3+ Sensing and Cell Imaging.

Constructing excited-state intermolecular proton transfer (ESIPT-e) fluorophores represents significant challenges due to the harsh requirement of bearing a proton donor-acceptor (D-A) system and their matching proton donating-accepting ability in the same molecule. Herein, we synthesized a new-type ESIPT-e fluorophor (2-APC) using the “four-component one-pot” reaction. By the installing of a cyano-group on pyridine scaffold, the proton donating ability of -NH2 was greatly enhanced, enabling 2-APC to undergo ESIPT-e process. Surprisingly, 2-APC exhibited dual-emissions in protic solvents ethanol and normal fluorescence in aprotic solvents, which is vastly different from that of conventional ESIPT-a dyes. The ESIPT emission can be obviously suppressed by Fe3+ due to the coordination reaction of Fe3+ with the A-D system in 2-APC. From this basis, a highly sensitive and selective method was established using 2-APC as a fluorescent probe, which offers the sensitive detection of Fe3+ ranging from 0 to 13 μM with the detection limit of 7.5 nM. The recovery study of spiked Fe3+ measured by the probe showed satisfactory results (97.2103.4%) with the reasonable RSD ranging from 3.1 to 3.8%. Moreover, 2-APC can also exhibit aggregation-induced effect in poor solvent or solid-state, eliciting strong red fluorescence. 2-APC was also applied to cell-imaging, exhibiting good cell-permeability, biocompatibility and color rendering. This multi-mode emission of 2-APC is significant departure from that of conventional extended p-conjugated systems and ESIPT dyes based on a flat and rigid molecular design. The “one-pot synthesis” strategy for the construction of ESIPT molecules pioneered a new route to achieve tricolor-emissive fluorophores.

Aminoindole and naphthalimide based charge transfer fluorescent probes for pH sensing and live cell imaging.

Fluorescent probes are essential for imaging of cancer cells and for tracking organelles inside cells. We have synthesized three molecular rotors AIN, AINP and F-AINP based on 1-aminoindole (AI) as an electron donor and naphthalimide as an electron acceptor. All compounds showed charge transfer (CT) character, aggregation induced emission (AIE) and emission responsiveness towards temperature variation and solvent viscosity. AINP was most sensitive towards viscosity among all molecules with a viscosity sensitivity of ∼0.37. AIN, AINP and F-AINP showed negative temperature coefficients in chloroform with internal sensitivities of -0.04% °C-1, -0.08% °C-1 and -0.1% °C-1, respectively. Furthermore, all the rotors were sensitive towards the pH of the solvent environment as revealed by acid titration and base back-titration and served as colorimetric pH sensors with intriguing photophysical characteristics. Additionally, AINP and F-AINP were used to image the live cancer cell line A549 and the fibroblast cell line L929, and the imaging studies revealed the incorporation of dyes in the cytoplasmic space of the cells except for the nuclei.

Research Advances in Excited State Intramolecular Proton Transfer Fluorescent Probes Based on Combined Fluorescence Mechanism

Fluorescent probes can achieve rapid detection of analytes, and have become a research hotspot in recent years due to their excellent selectivity, high sensitivity and easy operation. Excited state intramolecular proton transfer (ESIPT) is one of the most basic strategies for designing fluorescence probes. Fluorescent probes based on ESIPT possess large Stokes shift, strong luminescence and high fluorescence quantum yield. However, these are still some disadvantages, such as sensitivity to the environment and short emission wavelength. In recent years, the ESIPT combined with other fluorescence mechanisms, can obtain new fluorescent probes with dual mechanism of fluorescence response. This strategy can not only solve the shortage of ESIPT fluorophore, but also realize multi-fluorescence signal channel, and significantly improve the selectivity and sensitivity of the new probe. In this review, we mainly summarized the detection mechanism and imaging application of fluorescence probes of PET-ESIPT, ICT-ESIPT, FRET-ESIPT, AIE-ESIPT for ions, small molecules, biological macromolecules in recent years.

Detection Sensitivity Enhancement of Naphthalimide PET Fluorescent Probes by 4-Methoxy-Substitution.

Naphthalimide photoinduced electron transfer (PET) fluorescent probes are widely used in fluorescence imaging. Thereinto, detection sensitivity is the vital parameter of PET probes. However, the modulation of detection sensitivity is yet to be reported for naphthalimide PET probes. Herein, the detection sensitivity enhancement of naphthalimide PET fluorescent probes through 4-methoxy-substitution is proposed in this work. Taking Zn2+ detection an example, 4-methoxy-naphthalimide PET probe 2-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-6-methoxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (BPNM) and control PET probe 2-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (BPN) are separately synthesized. The addition of 4-methoxy group with ability of strong electron donating to naphthalimide facilitates the construction of electronic push-pull system in the fluorophore resulting in the bathochromic shift of absorption and fluorescence emission spectra of BPNM and is further conducive to the enhancement of molar extinction coefficient ε and fluorescence quantum yield Φf of BPNM. Compared with BPN, BPNM shows lower Zn2+ detection limit in titration assays. Meanwhile, the fluorescence signal change (off-on) before and after Zn2+ addition of intracellular BPNM is more obvious and easier to control in confocal laser scanning imaging. Therefore, 4-methoxy-substitution improves the detection sensitivity of naphthalimide PET probe, which is favorable for the precise sensing of analyte, and further lays a good foundation for the synthesis of PET probe with high sensitivity.

Development of a Pre-assembled Through-Bond Energy Transfer (TBET) Fluorescent Probe for Ratiometric Sensing of Anticancer Platinum(ll) Complexes.

Fluorescence microscopy has emerged as an attractive technique to probe the intracellular processing of Pt-based anticancer compounds. Herein, we reported the first through-bond energy transfer (TBET) fluorescent probe NPR1 designed for sensitive detection and quantitation of PtII complexes. The novel TBET probe was successfully applied for ratiometric fluorescence imaging of anticancer PtII complexes such as cisplatin and JM118 in cells. Capitalizing on the ability of the probe to discriminate between PtII complexes and their PtIV derivatives, the probe was further applied to study the activation of PtIV prodrug complexes that are known to release active PtII species after intracellular reduction.

Imidazole-coumarin containing D – A type fluorescent probe: Synthesis photophysical properties and sensing behavior for F− and CN− anion

A new class of D–A type intramolecular charge transfer (ICT) fluorescent probe, ICU has been designed and synthesized. The solvatochromic behavior, anion interaction and morphological features of the probe have been studied in detail. The molecular probe upon interaction with different class of anions showed good sensitivity for F− and CN− anions in organic medium with high detection limit and rapid response time. The time dependent fluorescence studies suggested about the dynamic and static fluorescence quenching with F− and CN− anions. The scanning electron microscopy (SEM) and diffraction light scattering (DLS) experiments have been performed to understand the morphological behavior and particle size of the probe to distinguish both anions. Upon interaction with F− and CN− the morphology of probe reveals a clear shape reconstruction from the initial aggregated spherical ball shape to fine leaf like and dumbbell like structures, respectively by disaggregation to form minimum energy structures. The photophysical and theoretical (DFT) studies all together suggested about the charge transfer process from imidazole (donor, D) to coumarin (acceptor, A) unit. The probe has been also utilized to detect the anions on test paper strips. The mechanism of interaction between probe and both the anions has been confirmed by 1HNMR and HRMS spectral data analysis.

Insights into intramolecular charge transfer fluorescent probes for recognizing human serum albumin

Development of powerful intramolecular charge transfer (ICT) fluorescent probes that can recognize, detect, and visualize human serum albumin (HSA) is important in the fields of biology and biomedicine. Three ICT probes, namely, butyl gallate (BG), isobutyl gallate (IBG), and isoamyl gallate (IAG), which have simple structures, were selected and used to detect HSA. The probes selectively recognized HSA in aqueous solutions by forming stable complexes with the protein. All of the probes exhibited different changes in fluorescence, and HSA underwent significant changes in fluorescence intensity and decay time. We hypothesized that the probes formed electromagnetic coupling with the whole HSA. Not all biomolecules can offer favorable environment for electromagnetic coupling due to their complex structures. The probes shared the same main binding site (located in Sudlow site II) in HSA. The probes and warfarin fluorescence intensity evidently increased in different hydrophobic cavities of HSA. This finding indicated the lack of limit to the location of the probes. A more suitable electromagnetic environment in protein made the ICT probes all had better response ability in HSA than in BSA. Y-type alkyl chains in IBG and IAG were beneficial for their responsiveness. The binding and response abilities of a specific hydrophobic cavity of a protein should be comprehensively considered when designing a novel fluorescent probe.

Boronic Acid Appended Naphthyl-Pyridinium Receptors as Chemosensors for Sugars

There remains a need in clinics and research to have simple and sensitive detection systems that allow the detection and quantification of sugar markers of biomedical relevance such as sugars lactulose and mannitol for noninvasive gut permeability assessment. We have prepared a new class of boronic acid-appended naphthyl-pyridinium receptor compounds as chemosensors. These were studied for their ability to act as modular internal charge transfer (ICT) fluorescent probes or donor/acceptor pair ensembles where the receptor compound can act as a quencher for an anionic dye. As an ICT sensor, fluorescence intensity increased upon diol recognition, which stems from the neutralization of the pyridinium nitrogen that is perturbing the chromophoric properties. We found these ICT probes provide good sensitivity for disaccharide lactulose with low micromolar detection and quantification limits. In addition, their ability to form a non-fluorescent ground state complex with anionic reporter dyes, such as HPTS or TSPP, was examined as probes for various sugars. We have identified three receptor/quencher compounds with high quenching efficiency for anionic dyes. Subsequently, a range of sugars and sugar derivatives were tested for chemosenstivity of our probes. This study illustrates an approach for designing boronic acid-based chemoreceptors for the recognition and quantification of sugars and sugar derivatives.

A far-red FRET fluorescent probe for ratiometric detection of l-cysteine based on carbon dots and N-acetyl-l-cysteine-capped gold nanoparticles.

A novel far-red fluorescence resonance energy transfer (FRET) fluorescent probe for ratiometric detection of l-cysteine (l-Cys) has been designed. The system was established a FRET assembly by positively charged carbon dots (CDs) and negatively charged N-acetyl-l-cysteine capped gold nanoparticles (NAC-AuNPs). The fluorescence of CDs at 539 nm could be effectively quenched in the presence of NAC-AuNPs owing to FRET process, while the emission of NAC-AuNPs at 630 nm was appeared. Subsequently, the interactions between l-Cys and NAC-AuNPs resulted in the decreased emission intensity of NAC-AuNPs, but the emission intensity of CDs kept almost constant due to the continuous FRET efficiency. The ratio of emission intensities at 539 and 630 nm (I539/I630) exhibited a linear correlation to the l-Cys concentration in the range of 1.0-110 μM with the detection limit of 0.16 μM. Moreover, this far-red ratiometric sensor also revealed excellent selectivity toward l-Cys over other amino acids, which showed very high potential in the practical application for diagnosing of cysteine-related disease.

Recent Advances in Reaction-Based Excited State Intramolecular Proton Transfer (ESIPT) Fluorescence Probe

Recent Advances in Reaction-Based Excited State Intramolecular Proton Transfer (ESIPT) Fluorescence Probe

Pyrenoimidazolyl-Benzaldehyde Fluorophores: Synthesis, Properties, and Sensing Function for Fluoride Anions.

Two structural isomers of (9H-pyreno[4,5-d]imidazol-10-yl)-benzaldehyde, with para and meta substitution patterns, were synthesized by condensation of 4,5-pyrenedione with terephthalaldehyde and isophthalaldehyde, respectively. These new pyrenoimidazole derivatives were characterized by single-crystal X-ray crystallography, UV–vis absorption spectroscopy, fluorescence spectroscopy, and cyclic voltammetry to elucidate their structural, solid-state packing, and electronic properties. Interactions of these compounds with fluoride anions in polar organic solvents (acetone and dimethyl sulfoxide) were investigated by NMR, UV–vis, and fluorescence techniques in conjunction with density functional theory calculations. UV–vis analysis showed that the binding of the two pyrenoimidazolyl benzaldehydes with fluoride anions resulted in significant colorimetric responses, while fluorescence studies showed that the para-pyrenoimidazolyl benzaldehyde behaved as an intramolecular charge transfer fluorescent probe, exhibiting ratiometric sensing performance to efficiently detect and quantify fluoride anions at the sub-millimolar level.

Location of fluorescent probes (2′-hydroxy derivatives of 2,5-diaryl-1,3-oxazole) in lipid membrane studied by fluorescence spectroscopy and molecular dynamics simulation

2′-Hydroxy derivatives of 2,5-diaryl-1,3-oxazole are known as environment-sensitive ratiometric excited-state intramolecular proton transfer (ESIPT) fluorescent probes, which are used to monitor physicochemical properties of lipid membranes. However, because of their heterogeneous membrane distribution, accurate experimental determination of the probe position is difficult. To estimate the location of the ESIPT probes in lipid membranes we have performed fluorescence measurements and molecular dynamics (MD) simulations. In the series composed of 2-(2′-hydroxy-phenyl)-5-phenyl-1,3-oxazole (1), 2-(2′-hydroxy-phenyl)-5-(4′-biphenyl)-1,3-oxazole (2), and 2-(2′-hydroxy-phenyl)-phenanthro[9,10-d]-1,3-oxazole (3), the structure of the ESIPT-moiety of 2-(2′-hydroxy-phenyl)-oxazole was varied by either aromatic ring substitution or annealing, leading to the systematical increase in the hydrophobic character of the probes. The comparison of the fluorescence behavior of probes 1–3 in a wide variety of solvents with those in phospholipid vesicles revealed that all three probes prefer to reside inside a membrane. Our MD results demonstrate that the probes locate from the glycerol residues and the polar carbonyl groups of phospholipids up to hydrophobic acyl chain units. It has been found that the probe location correlates well with the size of the aromatic moiety, being gradually shifted from 11.1 Å to 7.6 Å from the bilayer center for probes 1 to 3, respectively. Our results may be useful for the design of novel fluorescent probes for fluorescence sensing of specific regions within a lipid membrane.

Synthesis of novel β-cyclodextrin functionalized S, N codoped carbon dots for selective detection of testosterone

A novel functionalized carbon dot has been synthesized by covalently linking β-cyclodextrin to the surface of N, S codoped carbon dots (β-CD-CDs). The characterization was confirmed by transmission electron microscopy, X-ray photoelectron spectroscopy, infrared spectra, ultraviolet-visible, and fluorescence emission spectra. On the basis of this carbon dot and (ferrocenylmethyl) trimethylammonium iodide (Fc+), a photo-induced electron transfer (PET) fluorescent probe system was developed to determine the concentration of testosterone in water and identify testosterone in cell by fluorescence imaging as a visible biomarker. Under the optimum condition, the fluorescent intensity of the probe system linearly responded to the concentration of testosterone from 0μM to 280μM and the limit of detection was 0.51μM. This probe system also performed well at determining testosterone in groundwater with average recoveries of testosterone ranging from 96% to 107% at spiking levels of 0.5–100μM, and the relative standard deviation remained below 13%, which provided a reliable, rapid and easy method to determine testosterone in environmental water. Furthermore, the low cytotoxicity, high anti-interference ability, and excellent biocompatibility of β-CD-CDs made this probe system successfully used in cell fluorescence imaging to monitor levels of testosterone in the cytoplasm of cells with a promising application value in medical research.

Dual Site-Controlled and Lysosome-Targeted Intramolecular Charge Transfer-Photoinduced Electron Transfer-Fluorescence Resonance Energy Transfer Fluorescent Probe for Monitoring pH Changes in Living Cells.

Acidic pH is a critical physiological factor for controlling the activities and functions of lysosome. Herein, we report a novel dual site-controlled and lysosome-targeted intramolecular charge transfer-photoinduced electron transfer-Fluorescence resonance energy transfer (ICT-PET-FRET) fluorescent probe (CN-pH), which was essentially the combination of a turn-on pH probe (CN-1) and a turn-off pH probe (CN-2) by a nonconjugated linker. Coumarin and naphthalimide fluorophores were selected as donor and acceptor to construct the FRET platform. Hydroxyl group and morpholine were simultaneously employed as the two pH sensing sites and controlled the fluorescence of coumarin and naphthalimide units by ICT and PET, respectively. The sensing mechanism of CN-pH to pH was essentially an integration of ICT, PET, and FRET processes. Meanwhile, the morpholine also can serve as a lysosome-targeted group. By combining the two data analysis approaches of the ratios of the two emission intensities (R) and the reverse ratio R' (R' = 1/R), the fluorescent ratio of CN-pH can show proportional relationship to pH values in a very broad range from pH 4.0 to 8.0 with high sensitivity. The probe has been successfully applied for the fluorescence imaging of the lysosomal pH values, as well as ratiometrically visualizing chloroquine-stimulated changes of intracellular pH in living cells. These features demonstrate that the probe can afford practical application in biological systems.