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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.
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...
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.
Fluorescence and Phosphorescence: Instrumentation01:25

Fluorescence and Phosphorescence: Instrumentation

Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they select excitation and emission wavelengths and the type of light sources they utilize. Fluorometers use absorption interference filters to choose excitation and emission wavelengths. The excitation source in a fluorometer is typically a low-pressure mercury vapor lamp that emits intense lines distributed throughout the ultraviolet and visible regions.

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Related Experiment Video

Updated: May 9, 2026

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

A 'pocket guide' to total internal reflection fluorescence.

M L Martin-Fernandez1, C J Tynan, S E D Webb

  • 1Central Laser Facility, Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Oxford, Didcot, UK.

Journal of Microscopy
|July 30, 2013
PubMed
Summary
This summary is machine-generated.

Total internal reflection fluorescence (TIRF) microscopy uses evanescent fields for optical sectioning near substrates. This technique is crucial for single-molecule experiments, revealing cellular molecular interaction mechanisms.

Keywords:
Evanescent fieldTIRF microscopypolarizationsingle-molecule fluorescence imagingtotal internal reflection

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Last Updated: May 9, 2026

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

Published on: May 3, 2022

Area of Science:

  • Biophysics
  • Optical Microscopy
  • Cell Biology

Background:

  • Total internal reflection fluorescence (TIRF) microscopy, developed over 30 years ago, utilizes evanescent electromagnetic fields.
  • This optical sectioning technique probes sample regions near a substrate where the evanescent field is induced.
  • Early applications focused on investigating phenomena at the cell's basolateral plasma membrane.

Purpose of the Study:

  • To explain the electromagnetic theory behind evanescent fields in TIRF microscopy.
  • To guide users in building or selecting TIRF systems.
  • To explore how TIRF applications leverage evanescent field properties.

Main Methods:

  • Explanation of electromagnetic theory governing evanescent fields.
  • Principles for constructing or selecting TIRF microscopy systems.
  • Analysis of popular TIRF applications and their reliance on evanescent fields.

Main Results:

  • Provides a comprehensive understanding of TIRF microscopy principles.
  • Offers practical guidance for TIRF system implementation.
  • Highlights the utility of TIRF in single-molecule studies for elucidating molecular mechanisms.

Conclusions:

  • TIRF microscopy is a powerful tool for high-resolution imaging near surfaces.
  • Understanding evanescent fields is key to optimizing TIRF applications.
  • TIRF enables critical insights into cellular molecular dynamics and interactions.