Related Experiment Video
Updated: May 8, 2026
12:23
Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer
Published on: August 20, 2012
Robust red FRET sensors using self-associating fluorescent domains
Laurens H Lindenburg1, Anne M Hessels, Eduard H T M Ebberink
1Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology , P.O. Box 513, 5600MB Eindhoven, The Netherlands.
ACS Chemical Biology
|August 22, 2013
Summary
Researchers developed new red Förster Resonance Energy Transfer (FRET) pairs to improve imaging of cellular signaling. These enhanced red FRET sensors offer greater sensitivity and dynamic range for studying biological processes.
Area of Science:
- Cellular Biology
- Biophysics
- Molecular Imaging
Background:
- Multiparameter imaging of subcellular signaling networks is limited by the availability of sensitive Förster Resonance Energy Transfer (FRET) pairs compatible with existing spectral channels.
- Traditional red FRET sensors often exhibit poor sensitivity, hindering their utility in complex biological studies.
Purpose of the Study:
- To develop a generic strategy for enhancing the sensitivity and dynamic range of red FRET sensors.
- To create novel red FRET pairs spectrally compatible with cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) systems.
- To enable simultaneous imaging of multiple signaling events using spectrally distinct FRET sensors.
Main Methods:
- Engineered self-associating variants of mOrange and mCherry red fluorescent proteins.
- Introduced a single mutation into the mFruit domain to create well-defined on- and off states for FRET sensors.
- Developed red FRET variants of protease sensors and Zinc (Zn2+) sensors.
- Utilized the enhanced red FRET pair for simultaneous imaging of Zn2+ dynamics.
Main Results:
- The developed red FRET pair demonstrated a significant improvement in dynamic range, up to 10-fold for protease sensors.
- Successfully generated functional red variants of CFP-YFP-based Zn2+ sensors.
- The large dynamic range enabled simultaneous imaging of Zn2+ over a broad concentration range within the same cellular compartment.
Conclusions:
- A novel strategy using self-associating red fluorescent proteins significantly enhances FRET sensor performance.
- The new red FRET pairs are crucial for advancing multiparameter imaging of subcellular signaling.
- This advancement allows for more comprehensive and simultaneous analysis of biological processes in live cells.
