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Detection efficiency in total internal reflection fluorescence microscopy.

Marcel Leutenegger1, Theo Lasser

  • 1Laboratoire d'Optique Biomédicale, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.

Optics Express
|June 12, 2008
PubMed
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We developed a fast and accurate method to calculate fluorescence detection efficiency near interfaces. This framework is useful for quantitative fluorescence measurements, especially in total internal reflection fluorescence microscopy.

Area of Science:

  • Optics and Photonics
  • Biophysics
  • Materials Science

Background:

  • Accurate calculation of fluorescence detection efficiency is crucial for quantitative microscopy.
  • Interactions with interfaces significantly alter fluorescence emission patterns.
  • Existing methods may lack flexibility or accuracy in complex environments.

Purpose of the Study:

  • To present a rapid and flexible framework for calculating fluorescence detection efficiency.
  • To account for dipole characteristics and total radiated power for absolute efficiency.
  • To validate the framework for quantitative measurements at interfaces.

Main Methods:

  • Developed a computational framework for fluorescence detection efficiency.
  • Incorporated fluorophore dipole characteristics and radiated power.

Related Experiment Videos

  • Applied the framework to analyze fluorescence at a glass-water interface.

    • Main Results:

    • The framework accurately calculates detection efficiency in isotropic media and near interfaces.
    • Demonstrated utility for quantitative fluorescence detection in total internal reflection fluorescence microscopy.
    • Showcased applicability in both epi- and trans-illumination configurations.

    Conclusions:

    • The presented framework offers a robust tool for accurate fluorescence detection efficiency calculations.
    • Enables precise quantitative measurements in diverse optical setups and environments.
    • Facilitates advanced applications in microscopy and interfacial science.