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

Area of Science:

• Biophysics
• Analytical Chemistry
• Spectroscopy

Background:

• Fluorescence spectroscopy is crucial for analyzing molecular properties.
• Resolving complex mixtures of fluorescent molecules (fluorophores) presents analytical challenges.
• Frequency-domain fluorometry offers a method for analyzing fluorescence decay dynamics.

Purpose of the Study:

• To investigate the capability of frequency-domain phase-modulation fluorometry for resolving multi-component fluorescence decay.
• To determine the influence of emission bandpass and wavelength selection on the resolution of complex fluorophore mixtures.
• To assess the accuracy in determining lifetimes and preexponential factors for two- and three-component systems.

Main Methods:

• Measurement of fluorescence phase shift and modulation data across a range of modulation frequencies (1-140 MHz).
• Analysis of data using a least-squares procedure.
• Acquisition of data at single and multiple emission wavelengths.
• Application to one-, two-, and three-component mixtures of fluorophores.

Main Results:

• Two-component mixtures with lifetimes differing by a factor of 2 were resolved using single emission bandpass data.
• Three-component mixtures were resolved when decay times spanned a 10-fold range (e.g., 1.3, 4.4, 12 ns).
• Utilizing multiple emission wavelengths significantly enhanced the resolution of closely spaced two- and three-component decays (e.g., resolving 4.4 and 6.2 ns lifetimes).
• Multiple-wavelength data enabled the resolution of lifetimes and emission spectra for three-component mixtures.

Conclusions:

• Frequency-domain phase-modulation fluorometry is a powerful technique for resolving multiexponential fluorescence decay laws.
• The ability to resolve complex mixtures is significantly improved by acquiring data at multiple emission wavelengths.
• This method provides accurate determination of lifetimes and spectral characteristics for individual components within mixtures.