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Phase-resolved fluorescence lifetime measurements for flow cytometry

B G Pinsky1, J J Ladasky, J R Lakowicz

  • 1Becton Dickinson Immunocytometry Systems, San Jose, California 95131.

Cytometry
|January 1, 1993
PubMed
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A novel flow cytometer measures fluorescence lifetimes with picosecond resolution. This intensity-independent method accurately quantifies lifetimes for various biological samples, improving cellular analysis.

Area of Science:

  • Biophysics
  • Analytical Chemistry
  • Cell Biology

Background:

  • Flow cytometry is a crucial technique for cellular analysis.
  • Measuring fluorescence lifetimes offers a unique biophysical parameter for sample characterization.
  • Existing methods may face limitations in resolution or intensity dependence.

Purpose of the Study:

  • To develop and evaluate a flow cytometer for fluorescence lifetime measurements using the phase shift method.
  • To achieve high resolution and intensity independence in fluorescence lifetime measurements.
  • To demonstrate the utility of fluorescence lifetime measurements in analyzing biological samples.

Main Methods:

  • Construction and evaluation of a custom-built flow cytometer.
  • Utilizing the phase shift method for fluorescence lifetime determination.

Related Experiment Videos

  • Employing limiting amplifiers and electronic filtering for pulse intensity normalization.
  • Measuring fluorescence lifetimes of various standards, including dyes, beads, and stained cells.

    • Main Results:

    • Achieved fluorescence lifetime measurement resolution under 200 picoseconds under optimal conditions.
    • Demonstrated intensity-independent lifetime measurements over a 50-fold fluorescence intensity range.
    • Obtained fluorescence lifetimes for diverse samples (dyes, beads, stained cells) that correlate well with standard fluorimeter measurements.
    • Showed that fluorescence lifetime can resolve overlapping spectra, such as phycoerythrin from propidium iodide.

    Conclusions:

    • The developed flow cytometer reliably measures fluorescence lifetimes with high resolution and intensity independence.
    • Fluorescence lifetime measurements provide valuable biophysical information for cellular analysis.
    • This technique enhances the capabilities of flow cytometry for distinguishing complex biological samples and fluorophores.