Resolution of multicomponent fluorescence emission by phase sensitive detection at multiple modulation frequencies

Abstract

Phase sensitive emission spectra recorded at multiple frequencies were used to determine the lifetimes and steady-state spectra of the fluorophores In two-component mixtures. This analysis does not require any previous knowledge of the lifetimes, fractional Intensities, or the emission spectra and is thus an Improvement over single frequency phase sensitive detection which requires known emission spectra or known lifetimes. Phase sensitive emission spectra are recorded at several frequencies and arbitrarily chosen detector phase angles. The data are analyzed by use of nonlinear least squares to recover the lifetimes and wavelength-dependent fractional Intensities. The latter values determine the emission spectrum and relative intensity of each component in the mixture. Using this technique, we resolved two-component mixtures of fluorescein and 9-amlnoacrldlne, 2-(p-toluidinyl)-naphthalene-6-sulfonlc acid and 6-proplonyl-2-(dlmethylamlno)naphthalene, and N-acetyl-l-tyrosinamide and N-acetyl-l-tryptophanamide. In these mixtures the lifetimes differ by about 2-fold. Analysis of simulated data is presented to Illustrate the requirements for a satisfactory resolution. For simulated two-component mixtures, the components can be resolved If the lifetimes differ by 2-fold or greater, even with extensive overlap of the emission spectra.

Figure 1.

Determination of the lifetimes and emission spectra of a two-component mixture. The separately measured spectra of FLS (—), 9-AA (—), and the mixture (—) are shown. The calculated spectra are also shown for the 4.51 ns (○) and 9.30 ns (□) components. All solutions were in 100% ethanol. The lifetimes and emission spectra were variables In the analysis.

Figure 2.

Resolution of TNS and PRODAN mixture. The separately measured emission spectrum of the mixture is shown as a bold line (—), and the spectra of TNS and PRODAN as thinner lines (—). The calculated spectra are shown for the 8.08 ns (□) and 3.83 ns (○) components. The solutions were In 100% ethanol.

Figure 3.

Phase sensitive spectra of TNS and PRODAN mixture. Phase sensitive spectra of the TNS and PRODAN mixture measured at 18 MHz (left) and 33 MHz (right). The spectra were measured at 0, 80, 110, 130, and 180° relative to POPOP. The true phase angles relative to the Incident light are 8.7, 88.7, 119, 139, and 189° at 18 MHz and 15.6, 95.6, 126, 146, and 196° at 33 MHz.

Figure 4.

Resolution of a mixture of NATyrA and NATA: steady-state spectra of the mixture (—) and NATyrA and NATA (—). Calculated spectra were as follows: 1.71 ns (○); 3.48 ns (□). The buffer was 25 mM Tris-HCI, pH 7.5.

Figure 5.

Analysis of simulated phase sensitive spectra: simulated emission spectra (—). Calculated emission spectra were as follows: τ₁ = 5 ns and τ₂ = 10 ns (○), τ₁ = 5 ns and τ₂ = 8.5 ns (□).