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

J R Lakowicz, G Laczko, H Cherek

    Biophysical Journal
    |October 1, 1984
    PubMed
    Summary
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    This study introduces a nonlinear least squares method for analyzing fluorescence phase-shift and modulation data to determine lifetimes and fractional intensities of fluorophore mixtures. Accurate data, distinct decay times, and multiple wavelengths are critical for resolving complex emissions.

    Area of Science:

    • Analytical Chemistry
    • Biophysics
    • Spectroscopy

    Background:

    • Fluorescence spectroscopy is vital for analyzing molecular properties.
    • Resolving complex mixtures of decaying fluorophores presents analytical challenges.
    • Advancements enable wide-range modulation frequency measurements for fluorescence data.

    Purpose of the Study:

    • To develop and validate a method for analyzing fluorescence phase-shift and modulation data.
    • To determine lifetimes and fractional intensities of mixtures of exponentially decaying fluorophores.
    • To identify critical experimental factors for resolving complex fluorescence emissions.

    Main Methods:

    • Nonlinear least squares analysis of simulated fluorescence phase-shift and modulation data.
    • Evaluation of experimental factors influencing the resolution of fluorophore mixtures.

    Related Experiment Videos

  • Analysis of data across a wide range of modulation frequencies and multiple emission wavelengths.

    • Main Results:

    • Accuracy of experimental data, decay time differences, and multiple emission wavelengths are critical for resolution.
    • Resolution improvement diminishes with additional modulation frequencies.
    • Two decay times resolved if ratio >= 1.4; three decays resolved if lifetime range is fivefold.
    • Multiple wavelengths enhance resolution of closely-spaced decay times by varying fractional intensities.

    Conclusions:

    • Variable-frequency phase-modulation fluorometry, analyzed via nonlinear least squares, effectively resolves complex fluorophore mixtures.
    • Experimental precision and strategic data acquisition (multiple wavelengths) are key to accurate parameter determination.
    • Independent parameter knowledge significantly improves the accuracy of remaining parameter determination.