Analysis of excited-state processes by phase-modulation fluorescence spectroscopy

Abstract

Fluorescence phase shift and demodulation methods were used in the analysis of excited-state reactions and to investigate solvent relaxation around fluorophores in viscous solvents. The chosen samples illustrate the results expected for fluorophores bound to biological macromolecules. These moderately simple samples served to test the theoretical predictions described in the preceding paper (J.R. Lakowicz and A.B. Balter, Biophys. Chem. 16 (1982) 99.) and to illustrate the characteristic features of phase-modulation data expected from samples which display time-dependent spectral shifts. The excited-state protonation of acridine and exciplex formation between anthracene and diethylaniline provided examples of one-step reactions in which the lifetimes of the initially excited and the reacted species were independent of emission wavelength. Using these samples we demonstrated the following: (1) Wavelength-dependent phase shift and demodulation values can be used to prove the occurrence of an excited-state process. Proof is obtained by observation of phase angles (ø) larger than 90 degrees or by measurement of ratios of m/cos ø greater than 1, where m is the modulation of the emission relative to that of the excitation. (2) For a two-state process the individual emission spectra of each state can be calculated from the wavelength-dependent phase and modulation data. (3) The phase difference or demodulation factor between the initially excited and the reacted states reveals directly the fluorescence lifetime of the product of the reaction. (4) Phase-sensitive detection of fluorescence can be used to prove that the lifetimes of both the initially excited and the reacted states are independent of emission wavelength. (5) If steady-state spectra of the individual species are known, then phase-sensitive emission spectra can be used to measure the lifetimes of the individual components irrespective of the extent of spectral overlap. (6) Spectral regions of constant lifetime can be identified by the ratios of phase-sensitive emission spectra. In addition, we examined 6-propionyl-2-dimethylaminonaphthalene (PRODAN) and N-acetyl-L-tryptophanamide (NATA) in viscous solvents where the solvent relaxation times were comparable to the fluorescence lifetimes. Using PRODAN in n-butanol we used m/cos ø measurements, relative to the blue edge of the emission, to demonstrate that solvent relaxation requires more than a single step. For NATA in propylene glycol we used phase-sensitive detection of fluorescence to directly record the emission spectra of the initially excited and the solvent relaxed states. These measurements can be easily extended to fluorophores which are bound to proteins and membranes and are likely to be useful in studies of the dynamic properties of biopolymers.

Fig. 1.

Fluorescence emission spectra of acridine. Spectra are shown of the neutral and protonated forms of acridine (top) and of acridine with increasing concentrations of ammonium ion (bottom). Excitation was at 340 nm using a 10 nm band pass interference filter. The emission band pass was 8 nm. The temperature was 20°C. Solutions were not purged with inert gas. The acridine concentration was 2 × 10⁻⁵ M.

Fig. 2.

Apparent phase lifetimes of acridine. The inserted axis indicates the phase angles relative to the exciting light. The phase angle difference between the red (ϕ_R) and blue (ϕ_F) regions of the emission reveals the lifetime of the acridinium cation. We note that measured phase angles. relative to the exciting light, do not yield true lifetimes For the acridinium cation (eq. 3 and ref. 1). Additional experimental details are given in the legend to fig. 1.

Fig. 3.

Fluorescences lifetime of acridinium as observed by the phase angle difference between the short- and long-wavelength regions of the emission. The wavelengths chosen for measurement of the F and R states were 400 and 560 nm. respectively. The phase difference between the F and R states reveals the intrinsic lifetime of the directly excited R state (eq. 5).

Fig. 4.

Apparent phase and modulation lifetimes of acridine. Also shown is the wavelength dependence of m/cos ϕ.

Fig. 5.

Measurement and model calculations of m/cos ϕ. Measurements of m/cos ϕ were performed relative 10 the exciting light and relative to the emission at 400 nm. Also shown are model calculations using parameters comparable to the acridine-acridinium system. The emission of the F and R states was assumed to be centered at 430 and 478 nm, respectively, and the standard deviations of the assumed Gaussian distributions were both assumed equal to 1.6 kK (1 kK= 1000 cm⁻¹).

Fig. 6.

Phase-sensitive fluorescence spectra of acridine in 0.2 M ammonium nitrate. These phase-sensitive spectra are identical to the steady-state spectra of acridine in 0.05 N NaOH (left) and in 0.1 N H₂SO₄ (right).

Fig. 7.

Phase-sensitive fluorescence spectra of acridine in 0.2 M ammonium nitrate.

Fig. 8.

Wavelength dependence of the phase angles and apparent fluorescence lifetime as revealed by the phase-sensitive fluorescence spectra The values in square brackets are the apparent lifetimes calculated from the zero crossing points.

Fig. 9.

Wavelength dependence of the ratio of phase-sensitive fluorescence intensities at various detector phase angles. The spectrum obtained at ϕ_D = 81° was chosen as the reference spectrum.

Fig. 10.

Phase angles and demodulation factors for anthracene and its exciplex with diethylaniline. Anthracene was dissolved in toluene and the concentration of diethylaniline was 0.2 M. The excitation was at 357 nm. and the excitation and emission band passes were both 8 nm. The solution was not purged with inert gas. The temperature was 20°C.

Fig. 11.

Resolution of the monomer and exciplex emission of anthracene. The spectra were calculated using eqs. 7–10 and the data shown in fig. 10.

Fig. 12.

Resolution of the initially excited and relaxed states of PRODAN from the wavelength-dependent phase and modulation lifetimes. The top panel shows the apparent phase and modulation Iifetimes at −55°C. Also shown are the steady-state emission spectra at −55 (–––) and 25°C (– – –). Exitation was through a 340 nm interference falter with a band pass of 10 nm. Emission was observed with monochromator and a bandwidth of 8 nm. Polarizers were placed in the excitation and emission beam to eliminate the effects of Brownian rotation [23]. The concentration of PRODAN was 2 × 10⁻⁵ M. The lower panel shows the spectra calculated using eqs. 7–10. We assumed the phase and modulation values measured at 400 and 550 nm were those of the F and R states, respectively.

Fig. 13.

Measurement of m/cos ϕ for PRODAN and TNS in viscous solvents. Excitation of PRODAN and TNS was at 340 nm. The concentrations were 2 × 10⁻⁵ and 5 × 10⁻⁵ M. respectively. The emission band pass was 8 nm.

Fig. 14.

Resolution of the initially excired and relaxed states of N-acetyl-l-tryptophanamide by phase-sensitive detection of fluorescence. Excitation was through a 280 nm interference filter with 10 nm band pass. Emission was observed through a monochromator with a band pass of 8 nm. The concentration of NATA was 5 × 10⁻⁵ M. For the spectra shown polarizers were nor used. Measurements with polarizers set at the magic angle [22] gave equivalent results with a decreased signal-to-noise raatic.