Rational design, synthesis, and spectroscopic and photophysical properties of a visible-light-excitable, ratiometric, fluorescent near-neutral pH indicator based on BODIPY

A visible-light-excitable, ratiometric, brightly fluorescent pH indicator for measurements in the pH range 5-7 has been designed and synthesized by conjugatively linking the BODIPY fluorophore at the 3-position to the pH-sensitive ligand imidazole through an ethenyl bridge. The probe is available as cell membrane permeable methyl ester 8-(4-carbomethoxyphenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (I) and corresponding water-soluble sodium carboxylate, sodium 8-(4-carboxylatophenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (II). The fluorescence quantum yield Φ(f) of ester I is very high (0.8-1.0) in the organic solvents tested. The fluorescence lifetime (ca. 4 ns) of I in organic solvents with varying polarity/polarizability (from cyclohexane to acetonitrile) is independent of the solvent with a fluorescence rate constant k(f) of 2.4×10(8) s(-1). Probe I is readily loaded in the cytosol of live cells, where its high fluorescence intensity remains nearly constant over an extended time period. Water-soluble indicator II exhibits two acid-base equilibria in aqueous solution, characterized by pK(a) values of 6.0 and 12.6. The Φ(f) value of II in aqueous solution is high: 0.6 for the cationic and anionic forms of the imidazole ligand, and 0.8 for neutral imidazole. On protonation-deprotonation in the near-neutral pH range, UV/Vis absorption and fluorescence spectral shifts along with isosbestic and pseudo-isoemissive points are observed. This dual-excitation and dual-emission pH indicator emits intense green-yellow fluorescence at lower pH and intense orange fluorescence at higher pH. The influence of ionic strength and buffer concentration on the absorbance and steady-state fluorescence of II has also been investigated. The apparent pK(a) of the near-neutral acid-base equilibrium determined by spectrophotometric and fluorometric titration is nearly independent of the added buffer and salt concentration. In aqueous solution in the absence of buffer and in the pH range 5.20-7.45, dual exponential fluorescence decays are obtained with decay time τ(1)=4.3 ns for the cationic and τ(2)=3.3 ns for the neutral form of II. The excited-state proton exchange of II at near-neutral pH becomes reversible on addition of phosphate (H(2)PO(4)(-)/HPO(4)(2-)) buffer, and a pH-dependent change of the fluorescence decay times is induced. Global compartmental analysis of fluorescence decay traces collected as a function of pH and phosphate buffer concentration was used to recover values of the deactivation rate constants of the excited cationic (k(01)=2.4×10(8) s(-1)) and neutral (k(02)=3.0×10(8) s(-1)) forms of II.     

2',7'-Difluorofluorescein excited-state proton reactions: correlation between time-resolved emission and steady-state fluorescence intensity

The presence of excited-state buffer-mediated proton exchange reactions influences the steady-state fluorescence signals from dyes in solution. Since biomolecules in general have some chemical groups that can act as proton acceptors/donors and are usually dissolved in buffer solutions which can also behave as appropriate proton acceptors/donors, the excited-state proton exchange reactions may result in distorted steady-state fluorescence signals. In a previous paper (J. Phys. Chem. A 2005, 109, 734-747), we evaluated kinetic and other pertinent parameters for the excited-state proton reactions of the prototropic forms of 2',7'-difluorofluorescein (Oregon Green 488, OG488), recording a fluorescence decay surface at different pH values and acetate buffer concentrations, analyzed by means of global compartmental analysis. In this article we use the rate constants and the corrected pre-exponential factors from the previously recorded fluorescence decay traces to simulate the decay times and associated pre-exponentials at different acetate buffer concentrations and constant pH and compare these theoretically calculated values with new experimental data. We also calculate the steady-state fluorescence intensity vs pH and vs acetate buffer concentration (at constant pH) and compare these calculated emission values with the experimental data previously published. The agreement between the experimental and simulated data is excellent.     

Photophysics of a xanthenic derivative dye useful as an "on/off" fluorescence probe

The photophysical behavior of a new fluorescein derivative has been explored by using absorption and steady-state and time-resolved fluorescence measurements. The influence of ionic strength, as well as total buffer concentration, on both the absorbance and fluorescence has been investigated. The apparent acidity constant of the dye determined by absorbance is almost independent of the added buffer and salt concentrations. A semiempirical model is proposed to rationalize the variations in the apparent pKa values. The excited-state proton-exchange reaction around the physiological pH becomes reversible upon addition of phosphate buffer, inducing a pH-dependent change of the steady-state fluorescence and decay times. Fluorescence decay traces, collected as a function of total buffer concentration and pH, were analyzed by global compartmental analysis, yielding the following values of the rate constants describing excited-state dynamics: k01 = 1.29 x 10(10) s(-1), k02 = 4.21 x 10(8) s(-1), k21 approximately 3 x 10(6) M(-1) s(-1), k12B= 6.40 x 10(8) M(-1) s(-1), and k21B = 2.61 x 10(7) M(-1) s(-1). The decay rate constant values of k01, k21, k21B, along with the low molar absorption coefficient of the neutral form, mean that coupled decays are practically monoexponential at buffer concentrations higher than 0.02 M and any pH. Thus, the pH and buffer concentration can modulate the main lifetime of the dye.     

Photophysics of the fluorescent pH indicator BCECF

The photophysical behavior of BCECF [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein]--currently the most widely used fluorescent pH indicator for near-neutral intracellular pH measurements--has been explored by using absorption and steady-state and time-resolved fluorescence measurements. The influence of ionic strength as well as total buffer concentration on the absorbance and steady-state fluorescence has been investigated. The apparent acidity constant of the pH indicator determined by absorbance and fluorescence titration is dependent on the added buffer and salt concentrations. A semiempirical model is proposed to rationalize the variations in the apparent pKa values. The excited-state proton exchange of BCECF at physiological pH becomes reversible upon addition of phosphate buffer, inducing a pH-dependent change of the fluorescence decay times. Fluorescence decay traces collected as a function of total buffer concentration and pH were analyzed by global compartmental analysis yielding the following values of the rate constants describing excited-state dynamics of BCECF: k01 = 3.4 x 10(8) s(-1), k02 = 2.6 x 10(8) s(-1), k21 approximately 1 x 10(6) M(-1) s(-1), k12(B) = 1.4 x 10(8) M(-1) s(-1), and k21(B) = 4.3 x 10(7) M(-1) s(-1).     

Time-resolved fluorescence reveals two binding sites of 1,8-ANS in intact human oxyhemoglobin

Time-resolved fluorescence of 1,8-anilinonaphthalene sulfonate (1,8-ANS) fluorescent probe bound to intact human oxyhemoglobin (HbO2) is investigated. Fluorescence emission spectra of 1,8-ANS in a potassium buffer solution (pH 7.4) of HbO2 undergo a substantial blue shift during first 6 ns after pulsed optical excitation at 337.1 nm. Nonexponential fluorescence kinetics of 1,8-ANS in the HbO2 solution are studied by the decay time distribution and conventional multiexponential analyses for a set of emission wavelength range of lambdaem = 455-600 nm. These fluorescence decays contain components with mean decay times of <0.5 ns, 3.1-5.5 ns, and 12.4-15.1 ns with spectrally-dependent relative contributions. The shortest decay component is assigned to free 1,8-ANS molecules in the bulk buffer environment, whereas the two longer decay components are assigned to two types of binding sites of 1,8-ANS in the HbO2 molecule presumably differing by polarity and accessibility to water molecules. The results represent the first experimental evidence of heterogeneous binding of 1,8-ANS to intact human oxyhemoglobin.     

Fluorescence decay of α-chymotrypsin studied by the picosecond-resolved single photon-counting technique

The fluorescence decay of the multi-tryptophan-containing enzyme α-chymotrypsin in Tris buffer (pH 7.8) at room temperature was studied using a frequency-doubled, synchronously-pumped picosecond rhodamine-6G laser excitation source with time-correlated single photon-counting detection. The fluorescence decay parameters were computed with a non-linear least-squares iterative reconvolution program. The goodness-of-fit was tested with well-known graphical methods such as residuals plots and the autocorrealtion function. Numerical tests (reduced chi-square, ordinary runs test and the Durbin—Watson statistic) were included to improve the reliability of the residuals analysis. Normal distribution of the weighted residuals was checked with the normal probability plot, and with computation of the mean and standard deviation of the weighted residuals. α-Chymotrypsin exhibited triple-exponential fluorescence decay kinetics with decay times of 615 ± 76 ps, 1.7 ± 0.2 ns, and 4.3 ± 0.3 ns. The fractional fluorescence contributions depended on the emission wavelength. The fluorescence spectra of the components contributing to the total fluorescence were calculated from the steady-state fluorescence spectrum and fluorescence decays at different emission wavelengths, and from convoluted time-resolved emission spectra and a fluorescence decay measurement.     

Generalized two-dimensional heterocorrelation analysis of spectrally resolved and temporally resolved fluorescence of the 8-anilino-1-naphthalenesulfonate-apomyoglobin complex with pH perturbation

We demonstrate two-dimensional heterocorrelation analysis between spectrally resolved and temporally resolved fluorescence to investigate the decay dynamics of the 8-anilino-1-naphthalenesulfonate- (ANS-) apomyoglobin complex. The dynamic changes of the lifetime components are disclosed across the emission spectrum with an external pH-perturbation. Two different fluorescence lifetime schemes of the ANS-apomyoglobin complex are revealed. From pH 8.5 to 4.5, the transition of protein conformation from the native state to the folding intermediate, a short lifetime component is found to correlate with a short-wavelength emission whose population diminishes with decreasing pH. The lifetime components reflect the excited-state populations of the nascent and the charge-transfer species. From pH 4.2 to 1.0, the transition from the folding intermediate to the acid-unfolded state, the short lifetime is responsible for a long-wavelength emission and the fraction of this component increases when the solution becomes more acidic. In this pH range, the decay components reflect the ground-state populations of microenvironments. The relative decay dynamics across the emission spectrum are revealed without collecting decays at each wavelength. More importantly, these conclusions are reached without the necessity of statistical fitting of the decay data with an a priori decay model.     

Solvent and pH dependent fluorescent properties of a dimethylaminostyryl borondipyrromethene dye in solution

Steady-state and time-resolved fluorescence techniques have been used to study the photophysical properties of the fluorescent BODIPY-derived dye 3-{2-[4-(dimethylamino)phenyl]ethenyl}-4,4-difluoro-8-(4-methoxyphenyl)-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene. This compound has been synthesized via a microwave-assisted condensation of p-N,N-dimethylaminobenzaldehyde with the appropriate 1,3,5,7-tetramethyl substituted borondipyrromethene unit. The fluorescence properties of the dye are strongly solvent dependent: increasing the solvent polarity leads to lower fluorescence quantum yields and lifetimes, and the wavelength of maximum fluorescence emission shifts to the red. The Catalán solvent scales are found to be the most suitable for describing the solvatochromic shifts of the fluorescence emission. These are dominated by polarity/polarizability effects, as confirmed by quantum-chemical calculations performed in the dielectric continuum approximation. Fluorescence decay profiles of the dye can be described by a single-exponential fit in most solvents investigated, while two decay times are found in alcohols. The dye undergoes a reversible protonation-deprotonation reaction in the acidic pH range with a pK(a) of 2.25 in acetonitrile solution. Fluorimetric titrations as a function of pH produce fluorescence emission enhancements at lower pH. The fluorescence excitation spectra show a hypsochromic shift from 600 nm for the neutral amine to 553 nm for the ammonium form, so that ratiometric measurements can be used to determine pK(a).     

Absorption and emission study of 2',7'-difluorofluorescein and its excited-state buffer-mediated proton exchange reactions

2',7'-Difluorofluorescein (Oregon Green 488) is a new fluorescein-based dye, which has found many applications, above all in biochemistry and neurosciences, and its use has become very popular in the last years. In recent years, we have been investigating the excited-state proton exchange reactions of fluorescein and the effect of suitable proton acceptors and donors which promote these reactions. The excited-state proton transfer reactions may appreciably influence the fluorescence results when using these dyes. We present steady-state emission evidence that acetate buffer species promote an excited-state proton transfer between neutral, monoanionic, and dianionic forms of 2',7'-difluorofluorescein. The time course of the excited species in this reaction was characterized through time-resolved fluorescence measurements, and the kinetics of the reaction was solved by using the global compartmental analysis. A previous identifiability study on the compartmental system set the conditions to design the fluorescence decay surface. This is the first experimental system, studied within this kinetic model, solved under identifiability conditions through global compartmental analysis. The recovered rate constant values for deactivation were 2.94 x 10(8) s(-1) for the monoanion and 2.47 x 10(8) s(-1) for the dianion, whereas the rate constant values of the buffer-mediated excited-state reaction were 9.70 x 10(8) and 1.79 x 10(8) M(-1) s(-1) for the deprotonation and protonation, respectively. With these values, a pK(a) = 4.02 was obtained. In this work, we additionally provide an absorption study, including acid-base equilibria, determination of ground-state pK(a) values (1.02, 3.61, and 4.69), and recovery of molar absorption coefficients of every prototropic species, including absorption and NMR evidence for the existence of three tautomers in neutral species. Steady-state emission spectra of 2',7'-difluorofluorescein in aqueous solution are also described, where the strong photoacid behavior of the cation is noteworthy.     

The excited-state chemistry of protochlorophyllide a: a time-resolved fluorescence study.

The excited-state processes of protochlorophyllide a, the precursor of chlorophyll a in chlorophyll biosynthesis, are studied using picosecond time-resolved fluorescence spectroscopy. Following excitation into the Soret band, two distinct fluorescence components, with emission maxima at 640 and 647 nm, are observed. The 640 nm emitting component appears within the time resolution of the experiment and then decays with a time constant of 27 ps. In contrast, the 647 nm emitting component is built up with a 3.5 ps rise time and undergoes a subsequent decay with a time constant of 3.5 ns. The 3.5 ps rise kinetics are attributed to relaxations in the electronically excited state preceding the nanosecond fluorescence, which is ascribed to emission out of the thermally equilibrated S(1) state. The 27 ps fluorescence, which appears within the experimental response of the streak camera, is suggested to originate from a second minimum on the excited-state potential-energy surface. The population of the secondary excited state is suggested to reflect a very fast motion out of the Franck-Condon region along a reaction coordinate different from the one connecting the Franck-Condon region with the S(1) potential-energy minimum. The 27 ps-component is an emissive intermediate on the reactive excited-state pathway, as its decay yields the intermediate photoproduct, which has been identified previously (J. Phys. Chem. B 2006, 110, 4399-4406). No emission of the photoproduct is observed. The results of the time-resolved fluorescence study allow a detailed spectral characterization of the emission of the excited states in protochlorophyllide a, and the refinement of the kinetic model deduced from ultrafast absorption measurements.     

The dependence of singlet exciton relaxation on excitation density and temperature in polycrystalline tetracene thin films: kinetic evidence for a dark intermediate state and implications for singlet fission

The excited state dynamics of polycrystalline tetracene films are studied using femtosecond transient absorption in combination with picosecond fluorescence, continuing work reported in an earlier paper [J. J. Burdett, A. M. Muller, D. Gosztola, and C. J. Bardeen, J. Chem. Phys. 133, 144506 (2010)]. A study of the intensity dependence of the singlet state decay is conducted to understand the origins of the discrepancy between the broadband transient absorption and fluorescence experiments seen previously. High-sensitivity single channel transient absorption experiments allow us to compare the transient absorption dynamics to the fluorescence dynamics measured at identical laser fluences. At high excitation densities, an exciton-exciton annihilation rate constant of ~1 × 10(-8) cm(3) s(-1) leads to rapid singlet decays, but at excitation densities of 2 × 10(17) cm(-3) or less the kinetics of the transient absorption match those of the fluorescence. At these lower excitation densities, both measurements confirm that the initially excited singlet state relaxes with a decay time of 80 ± 3 ps, not 9.2 ps as claimed in the earlier paper. In order to investigate the origin of the singlet decay, the wavelength-resolved fluorescence dynamics were measured at 298 K, 77 K, and 4 K. A high-energy J-type emitting species undergo a rapid (~100 ps) decay at all temperatures, while at 77 K and 4 K additional species with H-type and J-type emission lineshapes have much longer lifetimes. A global analysis of the wavelength-dependent decays shows that the initial ~100 ps decay occurs to a dark state and not via energy transfer to lower energy bright states. Varying the excitation wavelength from 400 nm to 510 nm had no effect on the fast decay, suggesting that there is no energy threshold for the initial singlet relaxation. The presence of different emitting species at different temperatures means that earlier interpretations of the fluorescence behavior in terms of one singlet state that is short-lived due to singlet fission at high temperatures but long-lived at lower temperatures are probably too simplistic. The presence of a rapid singlet decay at all temperatures indicates that the initially created J-type singlet exciton decays to an intermediate that only produces free triplets (and delayed fluorescence) at high temperatures.     

Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.     

Dynamics and energetics of the self-assembly of a hydrophobically modified polyelectrolyte: Naphthalene-labeled poly(acrylic acid)

Steady-state and time-resolved fluorescence studies were performed on aqueous solutions of poly(acrylic acid) hydrophobically modified with two very different levels of naphthalene (Np). It is demonstrated that unique information on association phenomena involving hydrophobe-modifed polymers can be obtained from an extended fluorescence study by using data for a less-modified polymer as a reference. For the more highly modified polymer, the presence of excited-state (as well as ground-state) dimers in addition to monomer emission due to locally excited naphthalene gives evidence for hydrophobic association between naphthalene groups. This association becomes, as expected, much less important at higher pH due to the electrostatic repulsion between different chain segments. However, it is noted that even at high pH there is a significant self-association. The coexistence of static and dynamic quenching phenomena of the Np monomer label was also revealed in the time-resolved fluorescence data. The data are compatible with the existence of two types of monomers and one excimer and suggest that the essential contribution to the monomer emission comes from isolated chromophores, whereas excimer formation arises from both a dynamic route (excited Np chromophores able to produce a dynamic excimer) and a static route (excitation of ground-state Np dimers). At room temperature, the dissociative reaction, excimer-to-monomer, can be neglected, and thus the rate constant for excimer formation and decay could be obtained with and without considering the influence of preformed dimers. Temperature has shown to induce different behavior in the polymer photophysics. In the case of the less-labeled polymer, the decays were found to be single-exponential with the fluorescence lifetime decreasing with increasing temperature. From the temperature dependence of the steady-state fluorescence data, the activation energy for excimer formation and the binding energy of the excimer were evaluated at different pH values, through the Stevens-Ban-type plots of the excimer-to-monomer intensity ratio. With the time-resolved data, measured in the temperature range of 5-60 degrees C, it was possible to extract the intrinsic activation energies for excimer formation. The thermodynamic driving force for the intrapolymeric association was found to be dependent on a balance between hydrophobic and electrostatic interactions, which are dependent on the pH, temperature, and hydrophobic content of the polymer.     

Fluorescence properties of recombinant tropomyosin containing tryptophan, 5-hydroxytryptophan and 7-azatryptophan

Tropomyosin mutants containing either tryptophan (122W), 5-hydroxytryptophan (5OH122W) or 7-azatryptophan (7N122W) have been expressed in Escherichia coli and their fluorescence properties studied. The fluorescent amino acids were located at position 122 of the tropomyosin primary sequence, corresponding to a solvent-exposed position c of the coiled-coil heptapeptide repeat. The emission spectrum of the probe in each mutant is blue-shifted slightly with respect to that of the probe in water. The fluorescence anisotropy decays are single exponential, with a time constant of 2-3 ns while the fluorescence lifetimes of the probes incorporated into the proteins, in water, are nonexponential. Because tryptophan in water has an intrinsic nonexponential fluorescence decay, it is not surprising that the fluorescence decay of 122W is well described by a triple exponential. The fluorescence decays in water of the nonnatural amino acids 5-hydroxytryptophan and 7-azatryptophan (when emission is collected from the entire band) are single exponential. Incorporation into tropomyosin induces triple-exponential fluorescence decay in 5-hydroxytryptophan and double-exponential fluorescence decay in 7-azatryptophan. The range of lifetimes observed for 5-hydroxyindole and 5-hydroxytryptophan at high pH and in the nonaqueous solvents were used as a base with which to interpret the lifetimes observed for the 5OH122W and indicate that the chromophore exists in several solvent environments in both its protonated and unprotonated forms in 5OH122W.     

Excited-state intramolecular charge transfer in 9-aminoacridine derivative

A new fluorochromic dye was obtained from the reaction of 9-aminoacridine with ethyl-2-cyano-3-ethoxyacrylate. It displays complex fluorescence that is ascribed to normal emission from the acridine chromophore in addition to excited-state intramolecular charge transfer (ESICT) formed upon light excitation. The analysis of the fluorescence decays in different solvents reveals two short-lived components in the range of 80-450 ps and 0.7-3.2 ns, ascribed to the formation and decay of the intramolecular charge transfer (ICT) state, in addition to a third component of about 9.0 ns, which is related to the normal emission from the acridine singlet excited state, probably in an enol-imine tautomeric form. The ICT emission is readily quenched by water addition to polar solvents, and this effect is ascribed to changes in the keto-amine/enol-imine equilibrium of this fluorochromic dye.     

Control of bond-cleaving reactions of free protonated tryptophan ion by femtosecond laser pulses

The excited-state dynamics of protonated tryptophan ions is investigated by photoinduced fragmentation in the gas phase. In contrast to the neutral molecule that decays on the nanosecond time scale, the protonated species exhibits an ultrafast decay with two time constants of about 400 fs and 15 ps. In addition, after UV excitation by a pump photon at 266 nm, specific photofragments, and in particular the NH3-loss channel, can be enhanced by the absorption of a probe photon at 800 nm. The bond-cleaving reactions can thus be controlled by a variation of the pump/probe delay.     

Absorption and emission spectroscopic characterization of lumichrome in aqueous solutions

The spectroscopic behavior of lumichrome (7,8-dimethyl-alloxazine, LC) in aqueous solutions in a pH range from -1.08 to 14.6 is studied. Absorption spectra, fluorescence quantum distributions, quantum yields, and lifetimes are determined. The ionization stage of ground-state LC changes with rising pH from the cationic form (LCH(2)(+)) to the neutral form (LCH) with a mid-point pH of pK(c) ≈ -0.53, and to the anionic form (LC(-)) with a mid-point pH of pK(a) ≈ 12.5. Above pH 7 a partial ground-state tautomerization of LCH to 7,8-dimethyl-isoalloxazine (IAH) occurs by N1-N10 intra-molecular proton transfer. For pH > pK(a) ≈ 12.5 LCH and IAH change to the anionic forms LC(-) and IA(-), and above pH 14 LC(-) tautomerizes completely to IA(-). In the excited state some neutral lumichrome (LCH*) converts to cationic lumichrome (LCH(2)(+)) at low pH by proton transfer from H(3)O(+) to LCH*. No photoinduced excited-state tautomerization of lumichrome was observed. LCH for pH > 3 and IAH are reasonably fluorescent. The fluorescence efficiencies of LC(-) and IA(-) are lower than those of LCH and IAH. The fluorescence of LCH(2)(+) is strongly quenched likely by intra-molecular diabatic charge transfer and excited-state relaxation by potential surface touching with the ground state.     

Anomalous "unquenching" of the fluorescence decay times of beta-lactoglobulin induced by the known quencher acrylamide

Picosecond time-resolved fluorescence, together with the addition of quenching agents, was employed to discriminate the fluorescence contributions of the two tryptophans of beta-lactoglobulin (Trp19 and Trp61) to the fluorescence decays of the protein. The fluorescence decays of beta-lactoglobulin at pH 3, 5 and 8 are best fitted using sums of three exponentials and show a dominant contribution (98%) of the components associated with the buried Trp19, which decays according to a double exponential function. The addition of acrylamide (0.05 M) causes an increase of the decay times associated with Trp19. This effect is observed at all pH values studied, but the effect is stronger at pH 3 and pH 5, than at pH 8. The unexpected increase of the decay times of Trp19 and the variation of the respective amplitudes were rationalized in terms of alterations of Trp19 mobility. The hindrance of Trp19 upon acrylamide binding was also monitored and supported by fluorescence anisotropy measurements.     

CALCIUM-DEPENDENT FLUORESCENCE LIFETIMES OF INDO-1 FOR ONE- AND TWO-PHOTON EXCITATION OF FLUORESCENCE

Abstract— We characterized the fluorescence intensity decays of Indo-1, which is commonly used as an emission wavelength-ratiometric calcium probe. The apparent lifetime of the long-wavelength side of the emission of Indo-1 is dependent on Ca²⁺. This long-wavelength emission displays the characteristics of an excited-state reaction, that is, a negative preexponential component in thc multiexponential analysis. The emission spectra and lifetime of Indo-1 appear to be identical for one-photon and two-photon excitation at 351 and 702 mn, respectively, suggesting that the relative one- and two-photon cross sections are similar for the calcium-free and calcium-bound forms of Indo-1. Also, the two-photon cross section of Indo-1 is relatively high, about 4 × 10⁻⁴⁹ cm⁴ s/photon molecule at 690 nm for both the calcium-free and calcium-bound forms. Hence, Indo-1 can be used for calcium imaging based on one- or two-photon excitation, using either emission wavelength ratios or lifetime imaging methods.