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Evanescent interference patterns for fluorescence microscopy.

J R Abney1, B A Scalettar, N L Thompson

  • 1Department of Cell Biology & Anatomy, University of North Carolina, Chapel Hill 27599.

Biophysical Journal
|February 1, 1992
PubMed
Summary
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This study analyzes spatial intensity profiles from interfering evanescent waves, crucial for total internal reflection/fluorescence photobleaching recovery experiments. The findings detail how wave properties influence interference patterns for advanced microscopy applications.

Area of Science:

  • Optics
  • Biophysics
  • Physical Chemistry

Background:

  • Total internal reflection/fluorescence photobleaching recovery (TIRF-FRAP) is increasingly used experimentally.
  • Evanescent waves, generated during total internal reflection, exhibit unique properties compared to plane waves.
  • Understanding evanescent wave interference is key to optimizing TIRF-FRAP techniques.

Purpose of the Study:

  • To theoretically investigate the spatial intensity profiles of two interfering evanescent waves.
  • To analyze how parameters like polarization, incidence angle, and refractive indices affect these interference patterns.
  • To develop expressions for fluorescence photobleaching recovery curves using evanescent interference patterns.

Main Methods:

  • Theoretical calculations of spatial intensity profiles for interfering evanescent waves.

Related Experiment Videos

  • Analysis of periodicity and contrast under varying experimental conditions.
  • Derivation of expressions for fluorescence photobleaching recovery (FRAP) curves.

    • Main Results:

    • Evanescent interference patterns differ significantly from plane wave patterns due to non-transverse nature and angle-dependent propagation.
    • Pattern periodicity and contrast are influenced by beam intensities, polarizations, incidence angles, and refractive indices.
    • Derived intensity profiles enable modeling of FRAP curves for evanescent excitation/bleaching.

    Conclusions:

    • The theoretical framework provides insights into evanescent wave interference crucial for TIRF-FRAP.
    • Calculations offer a basis for predicting and controlling interference patterns in advanced microscopy.
    • Experimental validation of predicted evanescent field polarizations through colliding beam experiments is suggested.