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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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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...

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Related Experiment Video

Updated: Apr 23, 2026

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Published on: November 2, 2009

Real-time absorption reduced surface fluorescence imaging.

Bin Yang, James W Tunnell

Journal of Biomedical Optics

|September 25, 2014

Summary

This summary is machine-generated.

This study presents a new fluorescence imaging technique that reduces light absorption for clearer images at video rates. The method enhances visualization for applications like cancer detection and tumor margin analysis.

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Area of Science:

Biomedical Optics
Medical Imaging Technology
Fluorescence Imaging

Background:

Absorption effects in fluorescence imaging limit image quality and processing speed.
Existing methods often require complex post-processing, hindering real-time applications.
There is a need for efficient techniques to minimize absorption in biological tissues.

Discussion:

The proposed technique utilizes spatial frequency domain imaging with three-phase demodulation.
A single high spatial frequency (f = 0.5 mm−1) effectively minimizes absorption.
This method bypasses the need for extensive image processing, enabling high-speed imaging.

Key Insights:

Demonstrated significant absorption reduction in both in-vitro phantoms and ex-vivo tissue.
Achieved real-time video rate imaging at 19 frames per second.
The technique is robust and applicable to biological samples.

Outlook:

Potential for improved cancer visualization and precise tumor margin detection.
Facilitates real-time intraoperative guidance.
Opens avenues for advanced diagnostic tools in oncology.