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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.
Confocal Fluorescence Microscopy01:16

Confocal Fluorescence Microscopy

Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
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.
Attenuated Total Reflectance (ATR) Infrared Spectroscopy: Overview01:13

Attenuated Total Reflectance (ATR) Infrared Spectroscopy: Overview

Attenuated total reflectance (ATR) infrared spectroscopy is a powerful analytical technique used to study the composition of materials. It is widely employed in chemistry, materials science, forensic science, and other fields where sample characterization is required. ATR has several advantages over traditional transmission IR spectroscopy, including the requirement of little to no sample preparation and the ability to analyze a wide range of samples.
The ATR process begins by directing a beam...

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Related Experiment Video

Updated: Jun 26, 2026

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins
06:43

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Published on: May 3, 2022

Chapter 7: Total internal reflection fluorescence microscopy.

Daniel Axelrod1

  • 1Departments of Physics and Biophysics, University of Michigan, Ann Arbor, Michigan 48109, USA.

Methods in Cell Biology
|January 3, 2009
PubMed
Summary

Total internal reflection fluorescence microscopy (TIRFM) enables high-resolution imaging of single molecules and cellular dynamics near surfaces. This technique uses an evanescent wave to selectively illuminate fluorophores within a thin optical section, reducing background noise.

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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
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Single Molecule Fluorescence Microscopy on Planar Supported Bilayers
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Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

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Related Experiment Videos

Last Updated: Jun 26, 2026

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins
06:43

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Published on: May 3, 2022

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

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers
20:00

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

Published on: October 31, 2015

Area of Science:

  • Biophysics
  • Cell Biology
  • Optical Microscopy

Background:

  • Total internal reflection fluorescence microscopy (TIRFM) is crucial for studying molecular interactions at interfaces.
  • It allows visualization of single molecules and cellular components near substrates.
  • Existing methods face limitations in background reduction and optical sectioning.

Purpose of the Study:

  • To provide a comprehensive review of Total Internal Reflection Fluorescence Microscopy (TIRFM).
  • To cover its history, optical principles, and diverse applications.
  • To discuss various setup configurations and integration with other techniques.

Main Methods:

  • Selective illumination of fluorophores using an evanescent wave.
  • Confocal optical sectioning to a depth of less than 100 nm.
  • Review of established and custom-built TIRFM systems.

Main Results:

  • TIRFM significantly enhances signal-to-noise ratio by minimizing out-of-focus excitation.
  • It provides sub-100 nm optical sectioning for high-resolution imaging.
  • Demonstrates broad applicability in cell biology and biochemistry.

Conclusions:

  • TIRFM is a powerful technique for investigating molecular behavior at interfaces.
  • Its ability to selectively illuminate a thin layer is key to its effectiveness.
  • The review highlights TIRFM's versatility and potential for further advancements.