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Related Experiment Video

Updated: Dec 21, 2025

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
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Live-cell FLIM-FRET using a commercially available system.

Colleen M Castellani1, Ana P Torres-Ocampo2, Jens Breffke3

  • 1Biology Department, University of Massachusetts, Amherst, MA, United States.

Methods in Cell Biology
|May 20, 2020
PubMed
Summary

Förster resonance energy transfer (FRET) sensors visualize cellular processes, but intensity-based measurements have limitations. Fluorescence lifetime imaging microscopy (FLIM)-FRET overcomes these issues by measuring donor fluorophore lifetime for accurate FRET efficiency.

Keywords:
CamuiCdk1CyclinB1FLIMFRETTCSPC

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Area of Science:

  • Cell Biology
  • Biophysics
  • Microscopy

Background:

  • Förster resonance energy transfer (FRET)-based sensors are vital tools in cell biology for visualizing cellular dynamics.
  • Conventional FRET measurements face limitations due to probe concentration, photobleaching, and fluorophore bleed-through.

Purpose of the Study:

  • To introduce the fundamental principles of Fluorescence Lifetime Imaging Microscopy (FLIM)-FRET.
  • To demonstrate how FLIM-FRET overcomes the limitations of intensity-based FRET measurements.
  • To provide practical guidance on using a commercial FLIM system for FLIM-FRET applications.

Main Methods:

  • Utilizing Fluorescence Lifetime Imaging Microscopy (FLIM) to measure the time between photon absorption and emission.
  • Applying FLIM to FRET sensors (FLIM-FRET) to analyze donor fluorophore lifetime.
  • Employing two established FRET reporters as proof-of-concept examples.

Main Results:

  • FLIM-FRET measurements provide accurate FRET efficiency and the percentage of reporters involved in FRET.
  • FLIM-FRET circumvents issues associated with intensity-based FRET, such as probe concentration variations and photobleaching.

Conclusions:

  • FLIM-FRET is a powerful technique that enhances the capabilities of FRET sensors in live-cell imaging.
  • This approach offers a more robust and reliable method for quantifying FRET-based biological processes.