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Total Internal Reflection Fluorescence Microscopy

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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: Jul 2, 2026

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

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

Total internal reflection fluorescence (TIRF) microscopy.

Andreea Trache¹, Gerald A Meininger

¹Department of Systems Biology and Translational Medicine, College of Medicine, Texas A&M Health Science Center, College Station, Texas, USA.

Current Protocols in Microbiology

|August 30, 2008

Summary

Total internal reflection fluorescence (TIRF) microscopy uses an evanescent field to excite fluorophores near cell membranes. This method offers high-contrast imaging with low background, ideal for studying cell-surface interactions.

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A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

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Last Updated: Jul 2, 2026

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A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Published on: March 22, 2012

Area of Science:

Biophysics
Cell Biology
Microscopy

Background:

Total internal reflection fluorescence (TIRF) microscopy is a specialized imaging technique.
It visualizes fluorophores in the near-membrane region of cells.
The technique relies on the phenomenon of total internal reflection.

Purpose of the Study:

To explain the principle of operation of TIRF microscopy.
To describe the diversity of TIRF instruments.
To highlight applications of TIRF microscopy in biological studies.

Main Methods:

Utilizes total internal reflection of light at a glass-water interface.
Generates an evanescent field that penetrates a limited depth into the lower refractive index medium.
Excites fluorophores specifically at the cell-substrate interface.

Main Results:

Achieves high-contrast fluorescence images.
Minimizes background fluorescence and out-of-focus light.
Enables visualization and spectroscopy of single molecules near surfaces.

Conclusions:

TIRF microscopy provides superior imaging of cell-surface events.
The technique is versatile for various biological sample studies.
It offers a powerful tool for high-resolution live-cell imaging.