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Construction and performance of a variable-frequency phase-modulation fluorometer.

J R Lakowicz, B P Maliwal

    Biophysical Chemistry
    |January 1, 1985
    PubMed
    Summary
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    This study introduces a new variable-frequency phase-modulation fluorometer for advanced fluorescence measurements. The instrument offers high accuracy in resolving complex decay laws for both intensity and anisotropy, proving valuable in chemical and biochemical research.

    Area of Science:

    • Analytical Chemistry
    • Spectroscopy
    • Biophysics

    Background:

    • Frequency-domain fluorometry enables the analysis of fluorescence lifetimes and rotational dynamics.
    • Accurate resolution of complex decay laws is crucial for understanding molecular processes.
    • Existing instruments may have limitations in frequency range or accuracy for challenging samples.

    Purpose of the Study:

    • To describe the construction and performance of a novel variable-frequency phase-modulation fluorometer.
    • To demonstrate the instrument's capability in resolving complex fluorescence intensity and anisotropy decays.
    • To highlight the utility of this technique for chemical and biochemical research applications.

    Main Methods:

    • Construction of a variable-frequency phase-modulation fluorometer using commercially available components.

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  • Operation with a continuous-wave helium-cadmium laser and electro-optic modulators.
  • Measurement of phase angles and demodulation factors using cross-correlation with direct frequency synthesizers.

    • Main Results:

    • The fluorometer operates from 1 to 200 MHz with phase angle accuracy of 0.2 degrees and demodulation factor accuracy of 0.002.
    • Successfully resolved complex multiexponential decays of fluorescence intensity in mixtures of anthracene derivatives and NADH.
    • Determined short rotational correlation times (down to 47 ps) and resolved multiple anisotropy decay times for complex molecules.

    Conclusions:

    • The developed variable-frequency phase-modulation fluorometer provides high accuracy and resolution for complex fluorescence decay analysis.
    • The instrument's performance is comparable to state-of-the-art time-resolved instruments.
    • Frequency-domain fluorometry offers a valuable, accurate, and relatively simple method for chemical and biochemical research.