Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Related Concept Videos

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.

Super-resolution Fluorescence Microscopy

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...

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

Pump-probe fluorescence lifetime imaging microscopy.

The Journal of chemical physics·2026

Same author

Ultrafast Spectroscopy Reveals Significant Differences in LH2 Exciton Mobility at Cryogenic and Ambient Temperatures.

The journal of physical chemistry letters·2026

Same author

TRPV2 and TRPC5 are potential targets for astringent phytochemicals.

Current research in food science·2026

Same author

Separating Orders of Response in Transient Absorption and Coherent Multidimensional Spectroscopy by Intensity Variation.

The journal of physical chemistry letters·2025

Same author

Fluorescence-Detected Pump-Probe Spectroscopy for Artifact-Free Detection of Stokes Shift Dynamics.

The journal of physical chemistry letters·2025

Same author

Correlation of p53 oligomeric status and its subcellular localization in the presence of the AML-associated NPM mutant.

PloS one·2025

Search research articles

Related Experiment Video

Updated: Jun 13, 2025

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Published on: February 9, 2012

Interferometric excitation fluorescence lifetime imaging microscopy.

Pavel Malý¹, Dita Strachotová², Aleš Holoubek³

¹Faculty of Mathematics and Physics, Institute of Physics, Charles University, Prague, Czech Republic. pavel.maly@mff.cuni.cz.

Nature Communications

|September 13, 2024

Summary

This summary is machine-generated.

Interferometric Excitation Fluorescence Lifetime Imaging Microscopy (ixFLIM) correlates excitation spectra and emission lifetime, overcoming standard FLIM limitations. This novel technique enhances fluorescence imaging by revealing ground-state absorption and excited-state emission correlations.

More Related Videos

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Published on: December 29, 2017

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Published on: November 23, 2021

Related Experiment Videos

Last Updated: Jun 13, 2025

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Published on: February 9, 2012

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Published on: December 29, 2017

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Published on: November 23, 2021

Area of Science:

Spectroscopy
Microscopy
Biophysics

Background:

Fluorescence lifetime imaging microscopy (FLIM) is a powerful imaging technique.
A key limitation of FLIM is its focus solely on the emitting state, neglecting excitation properties.

Purpose of the Study:

To introduce and validate Interferometric Excitation Fluorescence Lifetime Imaging Microscopy (ixFLIM).
To extend FLIM capabilities by correlating excitation spectra with emission lifetimes.
To enable quantitative analysis of molecular interactions, such as resonance energy transfer.

Main Methods:

Development of ixFLIM by integrating interferometric measurement of fluorescence excitation spectra with FLIM.
Application of ixFLIM to increasingly complex biological systems.
Direct comparison of ixFLIM performance against standard FLIM.

Main Results:

ixFLIM successfully measures the correlation between excitation spectra and emission lifetime.
The technique provides insights into the relationship between ground-state absorption and excited-state emission.
Quantitative resonance energy transfer imaging was achieved in a single measurement using ixFLIM.

Conclusions:

ixFLIM significantly expands the utility of fluorescence lifetime imaging microscopy.
The method overcomes previous limitations by incorporating excitation spectral information.
ixFLIM offers a novel approach for advanced molecular imaging and analysis.