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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.
Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.

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Related Experiment Video

Updated: Jun 18, 2026

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

Published on: October 2, 2012

High-resolution total-internal-reflection fluorescence microscopy using periodically nanostructured glass slides.

Anne Sentenac1, Kamal Belkebir, Hugues Giovannini

  • 1Institut Fresnel (UMR 6133), CNRS, Aix-Marseille Université, Campus de Saint Jérôme, 13013 Marseille, France.

Journal of the Optical Society of America. A, Optics, Image Science, and Vision
|December 4, 2009
PubMed
Summary

Using an optimized grating with interfering light beams significantly enhances the lateral resolution of total-internal-reflection fluorescence microscopy, achieving up to a fourfold improvement for better imaging.

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Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

Published on: January 21, 2019

Area of Science:

  • Microscopy
  • Optics
  • Biophysics

Background:

  • Total-internal-reflection fluorescence microscopy (TIR-FM) is a powerful technique for imaging at the cell surface.
  • Improving the lateral resolution of TIR-FM is crucial for detailed visualization of cellular structures.
  • Substrate and illumination conditions can significantly impact microscope performance.

Purpose of the Study:

  • To investigate the effect of different substrates and illumination strategies on TIR-FM performance.
  • To determine methods for enhancing the lateral resolution of TIR-FM.
  • To optimize TIR-FM for improved nanoscale imaging.

Main Methods:

  • Samples were deposited on homogeneous glass slides and optimized gratings.
  • Microscopy was performed using one or two interfering beams at various incident angles.
  • Fluorophore density was reconstructed from images using a conjugate gradient with positivity a priori information.

Main Results:

  • An optimized grating substrate significantly improved lateral resolution compared to a homogeneous slide.
  • Illumination with two interfering beams further enhanced resolution when using the grating.
  • Up to a fourfold improvement in lateral resolution was achieved under optimal conditions.

Conclusions:

  • Optimized gratings and interfering illumination are effective strategies for enhancing TIR-FM lateral resolution.
  • This approach offers a significant improvement for high-resolution imaging in biological and nanoscale applications.
  • The findings provide a pathway for developing next-generation super-resolution microscopy techniques.