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Analysis of multi-component fluorescence emission by phase-sensitive detection using one modulation frequency.

S M Keating-Nakamoto1, H Cherek, J R Lakowicz

  • 1Department of Biological Chemistry, University of Maryland, School of Medicine, 660 West Redwood Street, Baltimore, MD 21201, USA.

Biophysical Chemistry
|July 1, 1986
PubMed
Summary
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This study introduces phase-sensitive fluorescence detection for analyzing complex mixtures. It successfully resolves fluorophore lifetimes and intensities from samples with overlapping spectra using a single modulation frequency.

Area of Science:

  • Analytical Chemistry
  • Spectroscopy
  • Photochemistry

Background:

  • Fluorescence spectroscopy is crucial for analyzing molecular properties.
  • Resolving complex mixtures with overlapping spectra and similar lifetimes presents a significant challenge.
  • Existing methods may require multiple measurements or advanced instrumentation.

Purpose of the Study:

  • To develop and validate a method for resolving multi-component fluorescence samples using phase-sensitive detection.
  • To determine fluorophore lifetimes and fractional intensities from mixtures at a single modulation frequency.
  • To assess the impact of spectral overlap and lifetime separation on resolution.

Main Methods:

  • Utilized phase-sensitive detection of fluorescence at a single modulation frequency (30 MHz).

Related Experiment Videos

  • Recorded phase-sensitive spectra at various detector phase angles.
  • Employed nonlinear least-squares fitting to analyze spectral data and extract component parameters.
  • Validated the method with simulations and experimental mixtures of protein-like fluorophores.

    • Main Results:

    • Successfully resolved two- and three-component mixtures of fluorophores with closely spaced lifetimes (1.5-4.5 ns).
    • Demonstrated effective resolution despite extensive spectral overlap among components.
    • Showcased the ability to determine individual fluorophore lifetimes and fractional intensities accurately.

    Conclusions:

    • Phase-sensitive fluorescence detection at a single modulation frequency is a viable technique for resolving complex mixtures.
    • This method offers a simplified approach compared to techniques requiring multiple frequencies or advanced setups.
    • The findings have implications for analyzing complex biological and chemical samples with fluorescence emission.