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Total Internal Reflection Fluorescence Microscopy

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: Jun 28, 2026

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

A programmable light engine for quantitative single molecule TIRF and HILO imaging.

Marcel van 't Hoff¹, Vincent de Sars, Martin Oheim

¹INSERM, Paris, France.

|October 30, 2008

Summary

This summary is machine-generated.

We developed a novel method for total internal reflection fluorescence (TIRF) and highly inclined and laminated optical sheet (HILO) illumination by scanning the laser spot in a circular orbit. This technique enhances fluorescence imaging by reducing scattering and enabling real-time quantitative analysis.

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A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Published on: May 30, 2016

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Last Updated: Jun 28, 2026

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

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Published on: May 30, 2016

Area of Science:

Microscopy
Optical Physics
Biophysics

Background:

Objective-type illumination methods like TIRF and HILO are crucial for advanced fluorescence microscopy.
Traditional methods often face challenges with uniform illumination and quantitative analysis.

Purpose of the Study:

To present a simple yet powerful implementation of objective-type TIRF and HILO illumination.
To improve quantitative fluorescence imaging by achieving uniform illumination and enabling on-line analysis.

Main Methods:

A programmable light engine scans the focused laser spot in a circular orbit at scan rates exceeding 1 MHz.
Excitation light distribution is captured in the objective's back focal plane (BFP) for quantitative analysis.
Crossed acousto-optical deflectors enable arbitrary BFP intensity profiles for variable illumination.

Main Results:

Restoring illumination symmetry via circular spinning significantly reduces scattering.
An evenly lit field-of-view is achieved, facilitating on-line analysis of evanescent-field excited fluorescence without pre-processing.
The system supports variable-angle, multi-color illumination and rapid objective lens exchange.

Conclusions:

This scanning illumination technique offers a robust solution for high-quality, quantitative fluorescence microscopy.
The method simplifies advanced illumination strategies, making them more accessible for various applications.