Quenching Of Triplet States Research Articles

Commentary: Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure.

Commentary: Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure.

Development of photostabilized asymmetrical cyanine dyes for in vivo photoacoustic imaging of tumors.

Photoacoustic imaging (PAI) contributes to tumor diagnosis through the use of PAI probes that effectively accumulate in tumors. Previously, we developed a symmetrical cyanine dye, IC7-1-Bu, which showed high potential as a PAI probe because of its high tumor targeting ability and sufficient in vivo PA signal. However, IC7-1-Bu lacks photostability for multiple laser irradiations, so we developed stabilized PAI probes using IC7-1-Bu as a lead compound. We focused on the effect of singlet oxygen (1O2) generated by excited PAI probes on probe degeneration. We introduced a triplet-state quencher (TSQ) moiety into IC7-1-Bu to quench 1O2 generation and designed three IC-n-T derivatives with different linker lengths (n indicates linker length). The IC-n-T derivatives emitted in vitro PA signals that were comparable to IC7-1-Bu and significantly reduced 1O2 generation while showing improved photostability against multiple irradiations. Of the three derivatives evaluated, IC-5-T accumulated in tumors effectively to allow clear PAI of tumors in vivo. Furthermore, the photostability of IC-5-T was 1.5-fold higher than that of IC7-1-Bu in in vivo sequential PAI. These results suggest that IC-5-T is a potential PAI probe for in vivo sequential tumor imaging.

Pre-denaturing transitions in human serum albumin probed using time-resolved phosphorescence

The investigation of protein dynamics has long been of interest, since protein interactions and functions can be determined by their structure and changes in conformation. Although fluorescence, occurring on the nanosecond timescale, from intrinsic fluorescent amino acids has been extensively used, in order to fully access conformational changes longer timescales are required. Phosphorescence enables processes on the microsecond to second timescale to be accessed. However, at room temperature this emission can be weak and non trivial to measure. It requires the removal of oxygen – a common triplet state quencher and appropriate instrumentation. In this work we make use of a chemical deoxygenator to study room temperature phosphorescence from tryptophan in human serum albumin excited using a pulsed UV light emitting diode. This is extended to monitor the phosphorescence emission upon increasing temperature, allowing pre-denaturing transitions to be observed. Time-resolved data are analysed, both as the sum of exponential decays and using a distribution analysis based on non extensive decay kinetics. These results are compared to a fluorescence study and both the average lifetime and contribution of the different emitting components were found to give more dramatic changes on the phosphorescence timescale.

Modular Design of a Tandem Dye Tunes the Photophysical Properties of a Biosensor

Fluorogen Activating Peptides (FAPs) are protein reporters which activate the fluorescence of a non-covalently bound dark molecule (fluorogen). Light-harvesting dendrimers consisting of multiple chromophores have been built to improve the brightness of a biosensor. The multiple donor dyes transfer energy to the lone acceptor, thus increasing the brightness of the biosensor. Although bright, these light harvesting structures (dyedrons) displayed reduced association constants with the cognate protein, and reduced photostability of the fluorogen-FAP complex. To understand and potentially overcome these limitations, we have synthesized Cy3-Malachite Green based donor-acceptor structures with linker lengths from 10 to 30 atoms and found that longer linker lengths correspond to reduced quantum yield, but not reduced energy transfer efficiency, and that this arises from protein-dye changes that are slow relative to protein-dye association. Ensemble and single molecule level investigation of triplet state quenchers (TSQs) covalently linked to the fluorogen and the tandem dyes provide deeper insights into the development of bright, photostable probes for imaging applications.

Mechanism of Intramolecular Photostabilization in Self‐Healing Cyanine Fluorophores

Organic fluorophores, which are popular labels for microscopy applications, intrinsically suffer from transient and irreversible excursions to dark-states. An alternative to adding photostabilizers at high concentrations to the imaging buffer relies on the direct linkage to the fluorophore. However, the working principles of this approach are not yet fully understood. In this contribution, we investigate the mechanism of intramolecular photostabilization in self-healing cyanines, in which photodamage is automatically repaired. Experimental evidence is provided to demonstrate that a single photostabilizer, that is, the vitamin E derivative Trolox, efficiently heals the cyanine fluorophore Cy5 in the absence of any photostabilizers in solution. A plausible mechanism is that Trolox interacts with the fluorophore through intramolecular quenching of triplet-related dark-states, which is a mechanism that appears to be common for both triplet-state quenchers (cyclooctatetraene) and redox-active compounds (Trolox, ascorbic acid, methylviologen). Additionally, the influence of solution-additives, such as cysteamine and procatechuic acid, on the self-healing process are studied. The results suggest the potential applicability of self-healing fluorophores in stochastic optical reconstruction microscopy (STORM) with optical super-resolution. The presented data contributes to an improved understanding of the mechanism involved in intramolecular photostabilization and has high relevance for the future development of self-healing fluorophores, including their applications in various research fields.

Photochemistry of excited-state species in natural waters: A role for particulate organic matter

Laser flash photolysis (LFP) was used to characterize a triplet excited state species isolated from Black River and San Joaquin wetlands particulate organic matter (POM). The solubilized organic matter, isolated from POM by pH-independent diffusion in distilled water, was named PdOM. UV–visible absorption spectroscopy, excitation–emission matrix spectroscopy (EEMs), and 1H NMR were used to characterize the PdOM. While LFP of dissolved organic matter (DOM) is known to generate the solvated electron, LFP of the PdOM transient in argon-, air-, and nitrous oxide-saturated solutions indicated that this was a triplet excited state species (3PdOM*). The lifetime and the reactivity of 3PdOM* with sorbic acid, a triplet state quencher, were compared with that of the triplet excited state of benzophenone, a DOM proxy. A second excited state species (designated DOM*), with a longer lifetime, was reported in a number of previous studies but not characterized. The lifetime of DOM*, measured for seventeen organic matter isolates, lignin, tannic acid, and three wetlands plant extracts, was shown to differentiate allochthonous from autochthonous DOM. 3POM* and DOM* were also observed in lake water and a constructed wetlands' water. Aqueous extracts of fresh and aged plant material from the same wetland were shown to be one source of these excited state species. This study provides evidence of a role for POM in the photochemistry of natural and constructed wetland waters.

Photophysical characterization of the plant growth regulator 2-(1-naphthyl) acetamide

Abstract The photophysical properties of the widely used plant growth regulator 2-(1-naphthyl) acetamide (NAD) were studied in water and representative organic solvents (ethanol, ethylene glycol, acetonitrile, chloroform, 1,4-dioxane) employing steady-state and time-resolved spectroscopy. Quantum yields and lifetimes of fluorescence, phosphorescence and triplet formation and triplet–triplet absorption spectra were obtained. From these, all radiative and radiationless rate constants have been determined, together with singlet and triplet excited state energies (4.00 and 2.69 eV, respectively). The fluorescence quantum yield and lifetime increased on going from water ( Φ F 0.066, τ F 35.0 ns) to non-hydrogen bonding solvents ( Φ F 0.357, τ F 51.0 ns in 1,4-dioxane), probably due to decreased internal conversion. Fluorescence was quenched by several anions through an electron transfer process. A limit on the reduction potential of 1 NAD* of E 0 2.1 ± 0.2 V was estimated. The attribution of the transient absorption seen in nanosecond laser flash photolysis to 3 NAD* was confirmed by energy transfer and oxygen quenching. Quenching of triplet states leads to singlet oxygen formation, with quantum yields varying from 0.097 in water to 0.396 in chloroform. However, these are lower than the triplet state quantum yields, particularly in water ( Φ T 0.424), indicating competing quenching pathways, probably involving electron transfer. The relevance of these results to the photoreactivity of NAD under environmental conditions is discussed.

Effect of extra liganding on spectral and kinetic characteristics and on quenching of excited states of multiporphyrin complexes by molecular oxygen

Based on studies of spectral and kinetic parameters of dimers of Zn porphyrins and of multiporphyrin self-assembling complexes formed on their basis, we find that extra liganding of dimers by pyridine inappreciably lowers the energy of the triplet level E(T 1). We show that, in this case, the nonradiative deactivation T 1 ↝ S 0 of the electronic excitation energy of dimers Zn porphyrins increases not only due to increasing Franck-Condon factor. We discuss mechanisms of the quenching action of an extra ligand related to an accepting role played by high-frequency overtones of vibrations of extra ligand molecules, to an enhancement of the spin-orbit interaction due to energy lowering of σπ* states, and to out-of-plane distortion of dimers caused by the displacement of the Zn2+ ion out of the plane of the tetrapyrrole macrocycle. Quenching of triplet states of extra liganded dimers of Zn porphyrins by molecular oxygen in liquid solutions at 295 K depends on the character of donor-acceptor interactions with pyridine and rigidity of a linking molecular fragment. We find that the rate constants of oxygen quenching of the excited electronic states S 1 and T 1 of multiporphyrin complexes depend on their structure and composition, as well as steric hindrances, created by dimers (screening effects) for contact interactions of a π-conjugated system of the free base (extra ligand) with molecular oxygen. Screening effects of extra ligands by dimer molecules of Zn porphyrins, which reduce the oxygen quenching rate constants k S and k T , are found to barely affect the singlet-oxygen generation quantum yield γΔ.

2H1448 Cysを用いたTrp三重項状態消光によるStaphylococcal nuclease変性状態の研究(蛋白質_物性3,第49回日本生物物理学会年会)

2H1448 Cysを用いたTrp三重項状態消光によるStaphylococcal nuclease変性状態の研究(蛋白質_物性3,第49回日本生物物理学会年会)

Mitigating Unwanted Photophysical Processes for Improved Single-Molecule Fluorescence Imaging

Organic fluorophores common to fluorescence-based investigations suffer from unwanted photophysical properties, including blinking and photobleaching, which limit their overall experimental performance. Methods to control such processes are particularly important for single-molecule fluorescence and fluorescence resonance energy transfer imaging where uninterrupted, stable fluorescence is paramount. Fluorescence and FRET-based assays have been carried out on dye-labeled DNA and RNA-based systems to quantify the effect of including small-molecule solution additives on the fluorescence and FRET behaviors of both cyanine and Alexa fluorophores. A detailed dwell time analysis of the fluorescence and FRET trajectories of more than 200,000 individual molecules showed that two compounds identified previously as triplet state quenchers, cyclooctatetraene, and Trolox, as well as 4-nitrobenzyl alcohol, act to favorably attenuate blinking, photobleaching, and influence the rate of photoresurrection in a concentration-dependent and context-dependent manner. In both biochemical systems examined, a unique cocktail of compounds was shown to be optimal for imaging performance. By simultaneously providing the most rapid and direct access to multiple photophysical kinetic parameters, smFRET imaging provides a powerful avenue for future investigations aimed at discovering new compounds, and effective combinations thereof. These efforts may ultimately facilitate tuning organic dye molecule performance according to each specific experimental demand.

Lanthanide Luminescent Bioprobes (LLBs)

Abstract Lanthanide luminescent bioprobes (LLBs) are amongst the most sensitive luminescent probes because their excited states are long-lived, which allows time-resolved detection (TRD) of their luminescence, boosting the signal-to-noise ratio and the sensitivity; in addition they are fairly insensitive to photobleaching since lanthanide ions are good quenchers of triplet states. Applications of LLBs to immunoassays, DNA analysis, ligand binding assays, analyte sensing, and cellular imaging are reviewed. Future developments in immunohistochemistry, targeting of specific organelles, lab-on-a-chip technology, and instrumental design are evoked.

Nucleosome Dynamics as Studied by Single-pair Fluorescence Resonance Energy Transfer: A Reevaluation

Accessibility of nucleosomal DNA to protein factor binding is ensured by at least three mechanisms: post-synthetic modifications to the histones, chromatin remodeling, and spontaneous unwrapping of the DNA from the histone core. We have previously used single-pair fluorescence resonance energy transfer (spFRET) experiments to investigate long-range conformational fluctuations in nucleosomal DNA (Tomschik M, Zheng H, van Holde K, Zlatanova J, Leuba SH in Proc Natl Acad Sci USA 102(9):3278-3283, 2005). Recent work has drawn attention to a major artifact in such studies due to photoblinking of the acceptor fluorophore. We have now used formaldehyde-crosslinked nucleosomes and imaging in the presence of Trolox, an efficient triplet-state quencher that suppresses photoblinking, to reevaluate our previous conclusions. Careful analysis of the data indicates that most of the events previously characterized as nucleosome 'opening' must have corresponded to photoblinking. There is, nevertheless, evidence for the existence of infrequent, rapid opening events.

Strategies to Improve Photostabilities in Ultrasensitive Fluorescence Spectroscopy

Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes. Apart from reflecting a major pathway for photobleaching, our results also suggest the exploitation of the photoinduced ionization and the subsequent reduction by antioxidants for biomolecular monitoring purposes and as a possible switching mechanism with applications in high-resolution microscopy.

Riboflavin Triplet Quenchers Inhibit Lightstruck Flavor Formation in Beer

We report that the primary photophysical event that leads to the formation of lightstruck flavor in beer exposed to visible light is the excitation of riboflavin to its triplet state followed by electron transfer from iso-α-acids. This conclusion is based on laser flash photolysis measurements that quantitatively characterize the action of various triplet state quenchers combined with sensory analysis. Our findings rationalize the effect of triplet quenching on the lightstruck reaction and provide commercially acceptable methods of control. Beer has an inherent ability to quench the riboflavin triplet, and hence, provide some protection against lightstruck character formation. Now we show how this information could be used to stabilize beer. The concentrations of the naturally occurring quenchers, catechin and tryptophan, required to quench the riboflavin triplet have been determined. The effect of ascorbic acid, a known riboflavin triplet quencher and powerful antioxidant, was also examined. Sensory analysis showed that when dosed at high enough concentrations, riboflavin triplet state quenchers are able to inhibit the formation of lightstruck character.

Zinc-porphyrin Solvation in Folded and Unfolded States of Zn-cytochromec

After a brief review of the use of photochemical triggers and heme metal substitution to probe the folding dynamics of cytochrome c, we present new results on the photophysics and photochemistry of folded and unfolded states of the zinc-substituted protein (Zn-cyt c). Our measurements of Zn-cyt c triplet state decay kinetics reveal a systematic isotope effect on lifetimes: the decay in the folded protein (tau(H)2(O) approximately 10 ms) is only modestly affected by isotopically substituted buffers (k(H)2(O)/k(D)2(O) = 1.2), whereas a reduced triplet lifetime (approximately 1.3 ms) and greater isotope effect (1.4) were found for the chemically denatured, fully unfolded protein. The shortest lifetime (0.1-0.4 ms) and greatest isotope effect (1.5) were found for a fully exposed model compound, zinc-substituted N-acetyl-microperoxidase-8 (ZnAcMP8), implying that the unfolded protein provides some protection to the Zn-porphyrin group even under fully denaturing conditions. Further evidence for partial structure in unfolded Zn-cyt c comes from bimolecular quenching experiments using Ru(NH(3))(6)(3+) as an external Zn-porphyrin triplet state quencher. In the presence of quencher, partially unfolded protein at midpoint guanidinium chloride (GdmCl) and urea concentrations exhibits biphasic triplet decay kinetics, a fast component corresponding to an extended, solvent-exposed state (6.6 x 10(8) M(-1) s(-1) in GdmCl, 6.3 x 10(8) M(-1) s(-1) in urea) and a slow component attributable to a compact, relatively solvent-inaccessible, state (5.9 x 10(7) M(-1) s(-1) in GdmCl, 8.6 x 10(6) M(-1) s(-1) in urea). The variation in Zn-porphyrin solvation for the compact states in the two denaturants reveals that the cofactor in the partially unfolded protein is better protected in urea solutions.

Features of the Quenching of the Triplet States of Water-Soluble Porphyrins by Molecular Oxygen

Using the methods of time-resolved absorption spectroscopy, we have investigated the features of quenching, by molecular oxygen, of the excited triplet states of water-soluble 5,10,15,20-tetrakis-(4-N-methylpyridyl)-porphyrin (H2TMPyP) and 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrin (H2TSPP) in water–ethanol solutions. It has been revealed that for both compounds the rate constant of quenching of the triplet states increases with increasing viscosity of the medium. Quenching of the excited triplet states of the dissociated (in water) and undissociated (in ethanol) forms of water-soluble porphyrins occurs with a different efficiency, and the rate constant of quenching the triplet states by molecular oxygen kT thereby is higher for the dissociated form. It has been shown by means of mathematical modeling that the experimental results obtained can be described in terms of the change in the rate constants of intracomplex transitions in the porphyrin–oxygen collisional complex at varied solution viscosity and their difference for the dissociated and undissociated forms of water-soluble porphyrin.

Mechanism of Photosensitized Generation of Singlet Oxygen during Oxygen Quenching of Triplet States and the General Dependence of the Rate Constants and Efficiencies of O2(1Σg+), O2(1Δg), and O2(3Σg-) Formation on Sensitizer Triplet State Energy and Oxidation Potential

Rate constants of photosensitized generation of O2(1Σg+), O2(1Δg), and O2(3Σg-) have been determined for a series of ππ* triplet sensitizers with strongly varying oxidation potential (Eox), triplet energy (ET), and molecular structure, in CCl4. We demonstrate that one common dependence on Eox and ET successfully describes these rate constants for the molecules studied here and also for all previously investigated ππ* sensitizers, independently of molecular structure or any other parameter. Photosensitized singlet oxygen generation during O2 quenching of ππ* triplet states can be generally described by a mechanism involving the successive formation of excited noncharge transfer (nCT) encounter complexes and partial charge transfer (pCT) exciplexes of singlet and triplet multiplicity 1,3(T13Σ), following interaction of O2(3Σg-) with the triplet excited sensitizer. Both 1,3(T13Σ) nCT and pCT complexes decay by internal conversion (ic) to yield O2(1Σg+), O2(1Δg), and O2(3Σg-) and the sensitizer ground state. ic is the rate-limiting step in the nCT channel, whereas exciplex formation is rate determining in the pCT channel. Rotation of the O2 molecule within the solvent cage of 1,3(T13Σ) nCT complexes is fast enough to allow for a completely established intersystem crossing (isc) equilibrium, whereas significant noncovalent binding interactions slow rotation and inhibit isc between 1(T13Σ) and 3(T13Σ) pCT complexes. Upon the basis of this mechanism, we propose a semiempirical relationship that can be generally used to estimate rate constants and efficiencies of photosensitized singlet oxygen generation during O2 quenching of ππ* triplet states in CCl4. The data set includes 127 rate constants for derivatives of naphthalene, biphenyl, fluorene, several ketones, fullerenes, porphyrins and metalloporphyrins, and other homocyclic and heterocyclic aromatics of variable molecular structure and size. It is suggested that the general relationship presented here can be used for the optimization of the singlet oxygen photosensitization ability of many molecules, including those used in biological and medical applications, such as the photodynamic therapy of cancer.

Quenching of excited complexes by Fe-octaethylporphin in triplet-triplet annihilation of Mg-phthalocyanine in liquid solutions

The effect of triplet-state quenchers on the kinetics of triplet-triplet annihilation (TTA) of Mg-phthalocyanine (Mg-Phc) is studied. It is found that the rate constant of triplet-state quenching caused by TTA increases with increasing concentration [Q] of quenchers. The maximum values of the relaxation parameter of triplet states are proportional to [Q]2. The experimental data correspond to TTA with the formation of TT complexes from molecules in triplet states. The proportionality of the decay rate of TT complexes into molecules in the ground state to [Q]2 suggests that two quenching molecules are required for quenching one TT complex. It seems that the complex contains two locally excited triplet states of individual molecules. The spin correlation time in the triplet state seems to be longer than the average lifetime of complexes (≤10−4 s). The quenching probability of triplet states in complexes (caused, in particular, by the energy of charge transfer) is lower than the probability of intermolecular triplet energy transfer to the quencher levels.

Charge Transfer Effects on the Efficiency of Singlet Oxygen Production Following Oxygen Quenching of Excited Singlet and Triplet States of Aromatic Hydrocarbons in Acetonitrile

Rate constants for quenching by molecular oxygen of excited singlet and triplet states, and , respectively, are reported for 12 aromatic hydrocarbons in acetonitrile. Measured values of , except in the case of fluoranthene for which = 6.6 × 109 dm3 mol-1 s-1, are in the range (2.3−4.3) × 1010 dm3 mol-1 s-1, i.e., close to 4.5 × 1010 dm3 mol-1 s-1, the average value obtained for kd, the rate constant for diffusion-controlled reactions of oxygen with aromatic hydrocarbons in acetonitrile. Values of vary from 0.24 to 5.6 × 109 dm3 mol-1 s-1. Thus, /kd was found to be less than one-ninth for 11 compounds. The efficiencies of singlet oxygen production during oxygen quenching of the excited singlet and triplet states, and , respectively, were also measured, as were the oxidation potentials of the hydrocarbons in acetonitrile. Values of were shown to be zero within experimental error for eight compounds and in the range of 0.27 ± 0.05 for the other four compounds. Three different methods, which gave good agreement, were used to measure values of which were found to vary from 0.41 in the case of acenaphthene to 0.85 for anthracene. The fraction of excited singlet states quenched by oxygen which result in triplet states was also measured for all compounds and found to vary from 0.49 to 1.0. Combination of the total quenching rate constants with the fractional efficiencies allows separate net quenching rate constants to be obtained for the various oxygen quenching pathways in acetonitrile. The reasons for variations in these net quenching rate constants and thus in the fractional efficiencies for quenching by the various quenching pathways are discussed. Quenching of both excited singlet and triplet states by energy transfer and by charge-transfer assisted pathways are established. The logarithm of the net rate constants for quenching of the triplet states without energy transfer to oxygen for 11 of the aromatic hydrocarbons shows a linear dependence on the free energy for full charge transfer from the triplet state, with a slope which indicates that the transition states for this quenching pathway only have about 13.5% charge-transfer character.

Mechanism and Cellular Applications of a Green Fluorescent Protein-based Halide Sensor

We report the application of a targetable green fluorescent protein-based cellular halide indicator. Fluorescence titrations of the purified recombinant yellow fluorescent protein YFP-H148Q indicated a pK(a) of 7.14 in the absence of Cl(-), which increased to 7.86 at 150 mM Cl(-). At pH 7.5, YFP-H148Q fluorescence decreased maximally by approximately 2-fold with a K(D) of 100 mM Cl(-). YFP-H148Q had a fluorescence lifetime of 3.1 ns that was independent of pH and [Cl(-)]. Circular dichroism and absorption spectroscopy revealed distinct Cl(-)-dependent spectral changes indicating Cl(-)/YFP binding. Stopped-flow kinetic analysis showed a biexponential time course of YFP-H148Q fluorescence (time constants <100 ms) in response to changes in pH or [Cl(-)], establishing a 1:1 YFP-H148Q/Cl(-) binding mechanism. Photobleaching analysis revealed a millisecond triplet state relaxation process that was insensitive to anions and aqueous-phase quenchers. The anion selectivity sequence for YFP-H148Q quenching (ClO(4)(-) approximately I(-) > SCN(-) > NO(3)(-) > Cl(-) > Br(-) > formate > acetate) indicated strong binding of weakly hydrated chaotropic ions. The biophysical data suggest that YFP-H148Q anion sensitivity involves ground state anion binding to a site close to the tri-amino acid chromophore. YFP-H148Q transfected mammalian cells were brightly fluorescent with cytoplasmic/nuclear staining. Ionophore calibrations indicated similar YFP-H148Q pH and anion sensitivities in cells and aqueous solutions. Cyclic AMP-regulated Cl(-) transport through plasma membrane cystic fibrosis transmembrane conductance regulator Cl(-) channels was assayed with excellent sensitivity from the time course of YFP-H148Q fluorescence in response to extracellular Cl(-)/I(-) exchange. The green fluorescent protein-based halide sensor described here should have numerous applications, such as anion channel cloning by screening of mammalian expression libraries and discovery of compounds that correct the cystic fibrosis phenotype by screening of combinatorial libraries.