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Related Experiment Videos

Multiple frequency fluorescence lifetime imaging microscopy.

A Squire1, P J Verveer, P I Bastiaens

  • 1Cell Biophysics Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX, U.K.

Journal of Microscopy
|January 29, 2000
PubMed
Summary
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This study introduces multiple frequency fluorescence lifetime imaging microscopy (mfFLIM) for analyzing complex fluorescence decays. The new method accurately resolves fluorescence lifetimes and populations in mixtures and live cells.

Area of Science:

  • * Biophysics and advanced microscopy techniques.
  • * Optical engineering and signal processing.

Background:

  • * Fluorescence lifetime imaging microscopy (FLIM) is crucial for analyzing molecular environments.
  • * Traditional FLIM methods can struggle with complex samples containing multiple fluorophores.
  • * Simultaneous multi-frequency detection offers enhanced resolution for fluorescence decay analysis.

Purpose of the Study:

  • * To describe the experimental setup and computational algorithms for multiple frequency fluorescence lifetime imaging microscopy (mfFLIM).
  • * To demonstrate the capability of mfFLIM in resolving fluorescence lifetimes and fractional populations of complex mixtures.
  • * To validate the mfFLIM technique in biological samples, including live cells.

Main Methods:

  • * Simultaneous homodyne detection of fluorescence emission modulated at harmonic frequencies using a microchannel plate (MCP) image intensifier.

Related Experiment Videos

  • * Excitation source modulated at harmonic frequencies via two standing wave acousto-optic modulators.
  • * Fourier analysis of sampled signals to estimate phase and modulation parameters at each frequency.
  • * Non-linear fitting of dispersion relationships to resolve lifetimes and fractional populations pixel-by-pixel.

    • Main Results:

    • * Successful disentanglement of two populations with distinct fluorescence lifetimes (1 ns and 4 ns) using simulated data.
    • * Significant accuracy improvement by exploiting spatial invariance of lifetimes.
    • * Resolution of fluorescence lifetimes and intensity contributions in a rhodamine dye mixture.
    • * Application to live cells expressing green fluorescent protein variants.

    Conclusions:

    • * The developed mfFLIM system enables simultaneous multi-frequency detection for advanced fluorescence analysis.
    • * The computational approach accurately resolves complex fluorescence decays, lifetimes, and populations.
    • * mfFLIM is a powerful tool for quantitative analysis in both chemical and biological systems, including live-cell imaging.