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Related Experiment Video

Updated: May 5, 2026

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
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Robust calibration and quantification of FRET signals using multiplexed biosensor barcoding.

Jhen-Wei Wu1, Ming Yang1, Chao-Cheng Chen1

  • 1Department of Pathology, Department of Cell Biology, and Center for Cell Dynamics, Johns Hopkins University, Baltimore, MD 21205, USA.

Iscience
|November 10, 2025
PubMed
Summary
This summary is machine-generated.

We developed a calibration method using fluorescent protein barcodes to normalize Förster resonance energy transfer (FRET) biosensor signals. This strategy ensures accurate FRET efficiency measurements independent of imaging conditions, enabling robust live-cell studies.

Keywords:
Biophysical ChemistryBiotechnologyMethodology in biological sciencesSensor

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Area of Science:

  • Live-cell imaging
  • Biophysics
  • Molecular biology

Background:

  • Förster resonance energy transfer (FRET) is crucial for genetically encoded fluorescent biosensors.
  • The FRET ratio is sensitive to imaging parameters, hindering accurate data interpretation.
  • Normalization is needed for reliable FRET biosensor measurements.

Purpose of the Study:

  • To develop a robust calibration strategy for FRET biosensor imaging.
  • To ensure FRET ratio independence from imaging conditions.
  • To enable multiplexed FRET efficiency determination.

Main Methods:

  • Utilized fluorescent protein (FP)-based barcodes as calibration standards within cells.
  • Engineered "FRET-ON" and "FRET-OFF" standards for high- and low-FRET calibration.
  • Incorporated donor-only and acceptor-only controls for simultaneous FRET efficiency measurement.

Main Results:

  • Calibrated FRET ratios were independent of imaging parameters like excitation intensity.
  • The calibration strategy restored reciprocal donor and acceptor signal changes.
  • Enabled simultaneous determination of FRET efficiency for multiple biosensors.

Conclusions:

  • Introduced a simple and robust strategy for multiplexed FRET biosensor imaging.
  • Facilitates accurate cross-experimental and long-term FRET studies.
  • Enhances the reliability and interpretability of FRET biosensor data.