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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: May 7, 2026

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

Published on: October 15, 2015

Rectangle FRAP for measuring diffusion with a laser scanning microscope.

Ranhua Xiong¹, Hendrik Deschout, Jo Demeester

¹Laboratory of General Chemistry and Physical Pharmacy, Ghent University and Centre of Nano- and Biophotonics, Ghent, Belgium.

Methods in Molecular Biology (Clifton, N.J.)

|October 11, 2013

Summary

This summary is machine-generated.

This study details a flexible Fluorescence Recovery After Photobleaching (FRAP) method using rectangular regions to measure molecular mobility and diffusion coefficients. The technique offers precise measurements for various experimental setups.

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Related Experiment Videos

Last Updated: May 7, 2026

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

Published on: October 15, 2015

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

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Published on: October 31, 2015

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

Published on: February 26, 2010

Area of Science:

Microscopy techniques
Biophysics
Molecular dynamics

Background:

Fluorescence Recovery After Photobleaching (FRAP) is crucial for assessing molecular mobility.
Quantifying molecular diffusion coefficients requires precise experimental methods.

Purpose of the Study:

To present a versatile FRAP method utilizing rectangular photobleaching regions.
To provide comprehensive guidelines for FRAP measurements and data analysis.

Main Methods:

Implementing FRAP with rectangular regions of variable size and aspect ratio.
Detailed protocols for experimental setup and image acquisition.
Methods for analyzing FRAP data to determine diffusion coefficients.

Main Results:

Demonstration of a flexible FRAP approach for accurate diffusion coefficient measurements.
Guidelines for optimizing FRAP experiments and data interpretation.
Validation of the rectangle FRAP method using test solutions.

Conclusions:

The described rectangle FRAP method enhances the study of molecular mobility.
This technique provides a robust framework for quantitative diffusion analysis.
Proper application ensures reliable measurements of molecular diffusion coefficients.