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Related Experiment Video

Updated: Jun 17, 2026

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
09:45

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Published on: February 9, 2012

phi2FLIM: a technique for alias-free frequency domain fluorescence lifetime imaging.

Alan D Elder1, Clemens F Kaminski, Jonathan H Frank

  • 1Department of Chemical Engineering, University of Cambridge, Cambridge, CB2 3RA, UK.

Optics Express
|January 7, 2010
PubMed
Summary

A novel alias-free wide-field fluorescence lifetime imaging technique, phi-squared fluorescence lifetime imaging (phi(2)FLIM), uses a supercontinuum source and phase-modulated intensifier. This method enables faster, more versatile frequency-domain fluorescence lifetime imaging (fd-FLIM) for dynamic and complex samples.

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Area of Science:

  • Optics and Photonics
  • Biophotonics
  • Spectroscopy

Background:

  • Frequency-domain fluorescence lifetime imaging (fd-FLIM) is crucial for analyzing fluorophores.
  • Conventional fd-FLIM methods can suffer from aliasing and limited excitation source options.
  • Improving modulation depth and expanding excitation sources are key challenges in fd-FLIM.

Purpose of the Study:

  • To demonstrate a new alias-free wide-field frequency-domain fluorescence lifetime imaging technique.
  • To introduce phi-squared fluorescence lifetime imaging (phi(2)FLIM) for enhanced fd-FLIM measurements.
  • To showcase the advantages of phi(2)FLIM for dynamic and complex biological samples.

Main Methods:

  • Utilized a supercontinuum source for fluorescence excitation.

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  • Employed a phase-modulated image intensifier with square-wave gating for detection.
  • Implemented phi(2)FLIM to suppress higher harmonics and eliminate aliasing.
  • Analyzed results using AB-plots for fluorescence decay characterization.

    • Main Results:

    • Achieved alias-free wide-field fluorescence lifetime imaging.
    • Demonstrated improved modulation depth compared to conventional homodyne fd-FLIM.
    • Successfully imaged dynamic sample mixing at 5.5 Hz.
    • Enabled excitation wavelength-resolved FLIM measurements for multi-fluorophore analysis.

    Conclusions:

    • Phi(2)FLIM offers a robust and versatile approach to frequency-domain fluorescence lifetime imaging.
    • The technique overcomes aliasing limitations and expands excitation source compatibility.
    • Phi(2)FLIM is suitable for rapid measurements in dynamic systems and complex samples.