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Related Concept Videos

Total Internal Reflection Fluorescence Microscopy01:05

Total Internal Reflection Fluorescence Microscopy

Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.

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Updated: Jun 5, 2026

Polarization-Sensitive Two-Photon Microscopy for a Label-Free Amyloid Structural Characterization
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Changepoint analysis for single-molecule polarized total internal reflection fluorescence microscopy experiments.

John F Beausang1, Yale E Goldman, Philip C Nelson

  • 1Department of Physics and Astronomy, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Methods in Enzymology
|December 29, 2010
PubMed
Summary
This summary is machine-generated.

New microscopy methods reveal molecular motor dynamics. By tracking individual photons, researchers precisely observed myosin V

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Last Updated: Jun 5, 2026

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Microscopy

Background:

  • Studying individual macromolecules reveals their mechanochemical functions.
  • Motor enzymes like myosin are key targets due to their processive motion.
  • Single-molecule studies face limitations like photobleaching and complex dynamics of fluorescent dyes.

Purpose of the Study:

  • To develop methods for extracting maximum information from limited photon detection.
  • To enhance the understanding of molecular motor structural dynamics and temporal resolution.

Main Methods:

  • Extended multiple polarization illumination in total internal reflection fluorescence microscopy (polTIRF) to record photon arrival time and polarization.
  • Applied an optimized analysis technique to determine changes in photon emission rates.
  • Combined polTIRF with advanced analysis for high-resolution molecular motor studies.

Main Results:

  • Successfully recorded individual photon arrival times and polarization states.
  • Optimized analysis accurately identified changes in photon emission rates.
  • Achieved unprecedented detail and temporal resolution in observing myosin V structural dynamics.

Conclusions:

  • The enhanced polTIRF technique and analysis provide superior insights into molecular motor function.
  • This method overcomes limitations of traditional single-molecule fluorescence microscopy.
  • Offers a powerful tool for detailed investigation of biomolecular mechanisms.