Phase-modulation Fluorometry Research Articles

Time-resolved fluorescence anisotropies of diphenylhexatriene and perylene in solvents and lipid bilayers obtained from multifrequency phase-modulation fluorometry

Time-resolved decays of fluorescence anisotropy were obtained from frequency-domain measurements of the phase angle difference between the parallel and perpendicular components of the polarized emission and the ratio of the modulated amplitudes. These data were measured at modulation frequencies ranging from 1 to 200 MHz. To demonstrate the general applicability of this method, we describe the resolution of both simple and complex decays of anisotropy. In particular, we resolved single, double, and triple exponential decays of anisotropy and the hindered rotational motions of fluorophores within lipid bilayers. The ease and rapidity with which these results were obtained indicate that frequency-domain measurements are both practical and reliable for the determination of complex decays of anisotropy.

Application de la fluorimétrie de phase à l'étude des interactions soluté-solvant de quatre amino-9 acridines substituées

The apparent lifetimes of 9-aminoacridine, of atebrin and of two of its derivatives (9-amino-2-methoxy-6-chloroacridine and 9- n-butylamino-2-methoxy-6-chloroacridine) have been measured by phase modulation fluorometry in a glycerol-water mixture (high viscosity) and an ethanol-water mixture (low viscosity). Results are easily interpreted in terms of a relaxation of the first singlet excited state involving a rearrangement of solvent molecules before excitation. In the glycerol-water mixture, the stationary fluorescence spectra of atebrin and its two derivatives can be resolved into fluorescence spectra emitted by the relaxed state and fluorescence spectra emitted by the unrelaxed state. For each compound the lifetimes of these two states have been calculated. In the same solvent mixture the origin of 9-aminoacridine fluorescence is the relaxed state alone. However, the relaxation process is slow enough to be detected by phase modulation fluorometry. The lifetimes of the relaxed and unrelaxed states are still accessible. In the ethanol-water mixture this is no longer true. For each compound under study the origin of the fluorescence is still the relaxed state but the relaxation process is so rapid, compared with the deactivation processes, that it can no longer be detected by phase modulation techniques. In this case, only the lifetime characteristic of the relaxed state is accessible.

Recent Developments in Frequency-Domain Fluorometry

I. SUMMARY Phase-modulation fluorometry is the frequency-domain analogue of time-resolved fluorescence spectroscopy. During the past three years we witnessed the development of variable-frequency phase-modulation fluorometers with modulation frequencies from 1 to 220 MHz. These instruments provide impressive resolution of multi or non-exponential fluorescence decays. To introduce these instruments we describe their design and operational principles. To illustrate the obtainable resolution we present results for the resolution of two and three-component mixtures of fluorophores, the resolution of complex anisotropy decays from non-spherical molecules and the determination of time-resolved emission spectra in the presence of time-dependent spectral relaxation. At present, the resolution obtainable with the frequency-domain fluorometers appears to be at least equivalent to that obtained with pulsed mode-locked laser sources with single photon counting. These mode-locked sources can also be used for phase flu...

Frequency-domain phase-modulation fluorometry: Resolution of complex decays of fluorescence intensity and anisotropy

We used frequency-domain fluorometry to resolve multi-exponential decays of fluorescence intensity and anisotropy. The samples are excited with intensity modulated light, at modulation frequencies ranging from 1 to 200 MHz, and we measure the phase-shifts and demodulation factors. The decay times of a three-component mixture were resolved, 1.2, 4.1 and 7.7 nsec, as well as the two anisotropy decay times of the anisotropic rotator perylene.

Time-resolved fluorescence emission spectra of labeled phospholipid vesicles, as observed using multi-frequency phase-modulation fluorometry

Multi-frequency phase-modulation fluorometry was used to determine the time-resolved spectral parameters of two different fluorescent probes, 6-palmitoyl-2-[(2-trimethylammonium)ethyl[methylamino]naphthalene chloride (Patman) and 2- p-toluidinyl-6-naphthalenesulfonic acid (TNS) in lipid vesicles. The frequency-domain measurements permitted calculation of time-resolved emission spectra, the time-resolved decays of the emission center of gravity and of the emission spectral width. Nanosecond spectral relaxation was found using both probes, and these rates increased with temperature. The detailed time-domain information available using our method indicated that spectral relaxation of TNS is mainly a continuous process, whereas relaxation of Patman shows the characteristic features of a stepwise relaxation. Our results indicate that complex excited-state processes can be quantified using frequency-domain fluorometry.

Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data

Recently it has become possible to measure fluorescence phase-shift and modulation data over a wide range of modulation frequencies. In this paper we describe the analysis of these data by the method of nonlinear least squares to determine the values of the lifetimes and fractional intensities for a mixture of exponentially decaying fluorophores. Analyzing simulated data allowed us to determine those experimental factors that are most critical for successfully resolving the emissions from mixtures of fluorophores. The most critical factors are the accuracy of the experimental data, the relative difference of the individual decay times, and the inclusion of data measured at multiple emission wavelengths. After measuring at eight widely spaced modulation frequencies, additional measurements yielded only a modest increase in resolution. In particular, the uncertainty in the parameters decreased approximately as the reciprocal of the square root of the number of modulation frequencies. Our simulations showed that with presently available precision and data for one emission bandpass, two decay times could be accurately determined if their ratio were greater than or equal to 1.4. Three exponential decays could also be resolved, but only if the range of the lifetimes were fivefold or greater. To reliably determine closely-spaced decay times, the data were measured at multiple emission wavelengths so that the fractional intensities of the components could be varied. Also, independent knowledge of any of the parameters substantially increased the accuracy with which the remaining parameters could be determined. In the subsequent paper we present experimental results that broadly confirm the predicted resolving potential of variable-frequency phase-modulation fluorometry.

Resolution of mixtures of fluorophores using variable-frequency phase and modulation data

We measured fluorescence phase shift and modulation data for one-, two- and, three-component mixtures of fluorophores at modulation frequencies ranging from 1 to 140 MHz. These data were analyzed using the least-squares procedure described in the preceding paper (Lakowicz, J. R., G. Laczko, M. Cherek, E. Gratton, and M. Limkeman, 1984, Biophys. J., 46:463-477). Using data obtained at a single emission bandpass, the lifetimes and preexponential factors of two-component mixtures could be easily resolved if the lifetimes differed by a factor of 2. With currently available instrumental stability, three-component mixtures could be resolved when the overall range of decay times was 10-fold, (e.g., 1.3, 4.4, and 12 ns). Measurement of phase and modulation data at several emission wavelengths, where the ratio of the preexponential factors varied, enhanced our ability to resolve closely spaced two and three-component decays. Two-component mixtures could then be resolved if the lifetimes differed by 30% (4.4 and 6.2 ns). Also, the multiple-wavelength data allowed the lifetimes and emission spectra of the three-components of a mixture to be resolved. These results demonstrated that resolution of multiexponential decay laws was possible using frequency-domain phase-modulation fluorometry.

Determination of time-resolved fluorescence emission spectra and anisotropies of a fluorophore-protein complex using frequency-domain phase-modulation fluorometry.

We report the first time-resolved fluorescence emission spectra and time-resolved fluorescence anisotropies obtained using frequency-domain fluorescence spectroscopy. We examined the fluorophore p-2-toluidinyl-6-naphthalenesulfonic acid (TNS) in viscous solvents and bound to the heme site of apomyoglobin using multifrequency phase fluorometers. Fluorescence phase shift and modulation data were obtained at modulation frequencies ranging from 1 to 200 MHz. For time-resolved emission spectra, the impulse response for the decay of intensity at each emission wavelength was obtained from the frequency response of the sample at the same emission wavelength. The decays have negative pre-exponential factors, consistent with a time-dependent spectral shift to longer wavelengths. These multiexponential decays were used to construct the time-resolved emission spectra, which were found to be in good agreement with earlier spectra obtained from time-domain measurements. Additionally, time-resolved anisotropies were obtained from the frequency-dependent phase angle differences between the parallel and perpendicularly polarized components of the emission. The rotational correlation times of TNS bound to apomyoglobin are consistent with those expected for this probe rigidly bound to the protein. TNS in propylene glycol also displayed a single exponential decay of anisotropy. These results, in conjunction with the previous successful resolution of multiexponential decays of fluorescence intensity (Lakowicz, J. R., Gratton, E., Laczko, G., Cherek, H., and Limkeman, M. (1984) Biophys. J., in press; Gratton, E., Lakowicz, J. R., Maliwal, B. P., Cherek, H., Laczko, G., and Limkeman, M. (1984) Biophys. J., in press) demonstrate that frequency-domain measurements provide information which is, at a minimum, equivalent to that obtainable from time-domain measurements.

Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry

Procedures are described for the treatment of phase and modulation lifetime data in fluorescent systems having multiexponential decay. All computer procedures (called FIT programs) arise from the lifetime resolution theory for phase-modulation measurements (Weber, G. (1981) J. Phys. Chem. 85, 949-953). The programs most successful in resolving heterogeneous lifetimes use a Monte Carlo approach in which phase and modulation lifetime data at three modulation frequencies are simultaneously utilized. These programs are shown to have more utility than the final closed form procedure presented by Weber (1981). The FIT routines are simple and require little computer time while yielding excellent results. To illustrate the applicability of these programs, defined binary (carbazole and pyrene) and ternary systems (carbazole, pyrene and POPOP) were examined. In most cases, the resolved lifetimes were within 5% of the independently measured value and the fractional fluorescence contributions were within 10% of that expected. These results demonstrate that phase-modulation measurements analyzed by appropriate computer programs are capable of solving for lifetimes in both binary and, in selected cases, ternary systems. An example is given from the recent literature (Dalbey, R., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696-4706) in which the above programs allowed the resolution of both binary and ternary lifetimes of a dansyl label on myosin, where Förster energy transfer was occurring. These lifetimes were used to quantify changes in distances between two activity-related thiols on myosin upon the addition of Mg-ATP or its analogs.

Detection of the reversibility of an excited-state reaction by phase-modulation fluorometry

We used phase and modulation fluorometry to investigate the excited-state proton transfer from 2-naphthol. Phase-sensitive detection of fluorescence allows determination of the phase angles of the two-excited species, naphthol and naphtholate. If the steady-state spectra of the individual species are known and are not identical, then this general procedure yields the phase angles irrespective of the extent of the spectral overlap. The phase difference (Δφ) between the naphthol and naphtholate emission is given by tan Δφ = ω/(Γ R + k 2 where ω is the circular modulation frequency, Γ R the decay rate of naphtholate fluorescence and k 2 the rate of the reverse reaction. Hence, Δφ reflects both the decay rate of the reaction product and the rate of the reverse action. This back reaction was also detected by comparison of phase shift (φ) and demodulation ( m) values for the initially excited state. Specifically, the reverse reaction results in a double exponential decay of naphthol fluorescence, which is revealed by m/cosφ < 1. The concepts described herein are generally applicable to determination of the reversibility of excited-state reactions.

Theory of phase-modulation fluorescence spectroscopy for excited-state processes.

Theory is presented for the analysis of excited-state reactions by fluorescence phase shift and demodulation methods. Initially, a two-state model with spectral overlap is considered to illustrate most simply the effects of excited-state reactions on the expected phase and modulation values. Secondly, a multistate model is described to illustrate the probable effects of a fluorophore interacting with several solvent molecules. We note the following unique features of phase-modulation data expected from a fluorophore whose emission spectrum shifts during the lifetime of the excited state: (1) The modulation frequency dependence of the apparent phase (tau p) and modulation (tau m lifetimes of the reacted species is opposite to that of a heterogeneous population of fluorophores. (2) For the reacted species tau p greater than tau m. For a heterogeneous sample tau p less than tau m. (3) The phase angle of the reacted species can exceed 90 degrees. For a heterogeneous sample phase angles are always less than 90 degrees. Thus, phase and modulation measurements can distinguish between time-dependent processes and spectral heterogeneity by observation of any feature described above. Additionally: (4) The lifetime of the product species can be measured directly. (5) Reverse relaxation can be identified, and the reverse relaxation rates calculated. (6) The wavelength-dependent phase and modulation data can be used to resolve the individual spectra from a two-state reaction. (7) And finally, under favorable conditions, a two-state excited-state process can be distinguished from a continuous multiple-state process. In each instance, model calculations are presented to illustrate the unique potentials of phase-modulation fluorometry for investigations of excited-state processes.

On the relation between phase-modulation and pulse fluorometry: Analysis of multiexponential fluorescence decays

The paper demonstrates that the phase-modulation fluorometry provides a quick and easy method to distinguish between the fluorescence emission of a mixture of fluorophores and the emission originating from molecules created as a result of some transformation in the excited state of initially excited molecules.

Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry

The measurement of fluorescence lifetimes is known to be hindered by the wavelenght-dependent and photocathode area-dependent time response of photomultiplier tubes. A simple and direct method is described to minimize the effects in photomultiplier tubes for phase-modulation fluorometry. Reference fluorophores of known lifetime were used in place of the usual scattering reference. The emission wavelenghts of the reference and sample were matched by either filters or a monochromator, and the use of a fluorophore rather than a scatter decreases the differences in spatial distribution of light emanating from the reference and sample. Thus photomultiplier tube artifacts are minimized. Five reference fluorophores were selected on the basis of availability, ease of solution preparation, and constancy of lifetime with temperature and emission wavelenght. These compounds are p-terphenyl, PPO, PPD, POPOP and dimethyl POPOP. These compounds are dissolved in ethanol to give standard solutions that can be used over the temperature range from −55 to +55°C. Purging with inert gas is not necessary. The measured phase and modulation of the reference solution is used, in conjunction with the known reference, lifetime, to calculate the actual phase and modulation of the exictation beam. The use of standard fluorophores does not require separate experiments to quantify photomultiplier effects, and does not increase the time required for the measurement of fluorescence lifetimes. Examples are presented which demonstrate the elimination of artifactual photomultiplier effects in measurements of the lifetimes of DADH (0.4 ns) and indole solutions quenched by iodide. In addition, the use of these reference solutions increases the accuracy of fluorescence lifetime measurements ranging ranging to 30 ns. We judge this method to provide more reliable lifetime measurements by the phase and modulation method. The test solutions and procedures we describe may be used by other laboratories to evaluate the performance of their phase fluorometers.