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Experimental method to correct fluorescence intensities for the inner filter effect

N K Subbarao1, R C MacDonald

  • 1Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208.

The Analyst
|July 1, 1993
PubMed
Summary
This summary is machine-generated.

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This study introduces a reliable method to correct fluorescence measurements for the inner filter effect by sequestering fluorophores. This technique enhances accuracy in fluorescence spectroscopy, enabling new research possibilities.

Area of Science:

  • Analytical Chemistry
  • Spectroscopy
  • Biophysics

Background:

  • Inner filter effect (IFE) is a common issue in fluorescence spectroscopy, distorting intensity measurements.
  • Existing correction methods are often unreliable due to potential interactions between fluorophores and chromophores.
  • Accurate fluorescence intensity is crucial for quantitative analysis and understanding molecular interactions.

Purpose of the Study:

  • To develop and validate a robust empirical procedure for correcting fluorescence intensity for the inner filter effect.
  • To enable accurate fluorescence measurements in samples with high absorbance.
  • To facilitate studies of phenomena like protein fluorescence quenching.

Main Methods:

  • Sequestering fluorophores within liposomes or polymer beads to isolate them from the external solution.

Related Experiment Videos

  • Measuring fluorescence intensity of sequestered fluorophores before and after adding a chromophore to induce IFE.
  • Generating a correction curve based on the measured fluorescence reduction.
  • Testing the procedure with calcein-loaded liposomes and fluorophore-embedded polymer beads.
  • Investigating the influence of wavelength, pathlength, slit-width, and turbidity on IFE correction.

    • Main Results:

    • The developed empirical procedure provides reliable IFE correction by preventing direct interaction between fluorophores and chromophores.
    • Correction curves were successfully generated using both liposomes and polymer beads.
    • The magnitude of IFE is instrument-dependent and requires experimental determination for each setup.
    • Wavelength range and pathlength significantly impact IFE, while slit-width and turbidity have minimal effect on the tested instrument.

    Conclusions:

    • This empirical method offers a more accurate way to correct fluorescence intensity for the inner filter effect.
    • The procedure is applicable to various fluorophore systems, including those previously unsuitable for study.
    • Accurate IFE correction is essential and must be determined empirically for specific experimental conditions and instrumentation.