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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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Related Experiment Video

Updated: May 16, 2026

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)
09:14

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Published on: October 2, 2012

Measuring incidence angle for through-the-objective total internal reflection fluorescence microscopy.

Thomas P Burghardt1

  • 1Mayo Clinic Rochester, Department of Biochemistry and Molecular Biology, Rochester, Minnesota 55905, USA. burghardt@mayo.edu

Journal of Biomedical Optics
|December 5, 2012
PubMed
Summary
This summary is machine-generated.

Measuring the incidence angle in through-the-objective total internal reflection fluorescence (TIRF) microscopy is crucial. This study presents a method using objective axial scanning to accurately determine the incidence angle for enhanced TIRF imaging.

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Related Experiment Videos

Last Updated: May 16, 2026

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)
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Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis
10:43

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Published on: November 6, 2019

Area of Science:

  • Optical microscopy
  • Biophysics
  • Surface science

Background:

  • Total internal reflection fluorescence (TIRF) microscopy utilizes an evanescent field generated at a glass/aqueous interface.
  • Accurate measurement of the incidence angle is critical for controlling the evanescent field penetration depth in TIRF.
  • Through-the-objective TIRF configurations complicate direct incidence angle measurement.

Purpose of the Study:

  • To develop and calibrate a method for accurately measuring the incidence angle in through-the-objective TIRF microscopy.
  • To provide a reliable calibration applicable to various interfaces and samples.

Main Methods:

  • Utilizing objective axial scanning to translate the beam-interface intersection point.
  • Tracking the fluorescence center of mass from fluorescent spheres attached to the interface.
  • Calculating the tangent of the incidence angle from axial and center of mass translations.
  • Calibrating the incidence angle by measuring at different radial positions of the focused beam in the back focal plane (BFP).

Main Results:

  • A quantitative relationship between objective translation and center of mass movement was established.
  • The incidence angle was accurately determined by sampling over the objective's trajectory.
  • The calibration method was demonstrated to be dependent on specific microscope and objective parameters.

Conclusions:

  • Objective axial scanning provides a robust method for incidence angle measurement in through-the-objective TIRF.
  • This calibration technique enhances the precision and reliability of TIRF experiments.
  • The developed method is adaptable for diverse experimental setups and sample types.