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Photoluminescence offers a wide range of applications due to its inherent sensitivity and selectivity. This technique allows for both direct and indirect analyses of the analyte. Direct quantitative analysis is possible when the analyte exhibits a favorable quantum yield for fluorescence or phosphorescence. However, an indirect analysis may be feasible if the analyte is not fluorescent or phosphorescent, or if the quantum yield is unfavorable. Indirect methods include reacting the analyte with...
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Related Experiment Video

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Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy
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Expanded applications of bioluminescence microscopy with phasor analysis.

Lila P Halbers1, Caroline K Brennan2, Lorenzo Scipioni3

  • 1Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA.

Cell Reports Methods
|March 31, 2026
PubMed
Summary

Bioluminescent phasor analysis now distinguishes spectrally similar probes, enabling multiplexed imaging of biological processes. This advanced technique resolves subtle spectral changes in complex biological samples and biosensors.

Keywords:
CP: imagingbioluminescencebiosensorimagingluciferaseluciferinspectral phasor

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Area of Science:

  • Biophotonics
  • Molecular Imaging
  • Biotechnology

Background:

  • Bioluminescence is crucial for in vitro and in vivo biological process monitoring.
  • Existing bioluminescent reporters have spectral overlap, hindering simultaneous detection.
  • Bioluminescent phasor analysis was previously developed to resolve spectrally similar probes.

Purpose of the Study:

  • Characterize phasor outputs of common luciferase reporters.
  • Expand bioluminescent phasor analysis for distinguishing complex mixtures in tissues.
  • Apply the method for imaging biosensors in multidimensional models.

Main Methods:

  • Characterization of luciferase reporter phasor outputs.
  • Application of bioluminescent phasor analysis to tissue samples.
  • Imaging of biosensors using multidimensional models.

Main Results:

  • Phasor outputs of common luciferase reporters were characterized.
  • The method successfully distinguished complex mixtures in tissues.
  • Subtle changes in bioluminescent outputs and analyte-dependent spectra were resolved.
  • Bioluminescent phasor imaging was applied to multidimensional biosensor models.

Conclusions:

  • Bioluminescent phasor analysis is expanded for complex biological samples.
  • The technique enables detection of previously indistinguishable spectral changes.
  • This work enhances the utility of bioluminescent phasor imaging for multiplexed applications.