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FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
Published on: August 20, 2012
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SensorFRET: A Standardless Approach to Measuring Pixel-based Spectral Bleed-through and FRET Efficiency using
Paul T Arsenovic1, Carl R Mayer1, Daniel E Conway2
1Virginia Commonwealth University, Department of Biomedical Engineering, Richmond, VA, 23284, USA.
Scientific Reports
|November 17, 2017
Summary
This study introduces sensorFRET, a novel fluorescence microscopy technique for precise FRET efficiency measurement in living cells. It overcomes limitations of existing methods and quantifies measurement uncertainty, enabling accurate nanoscale interaction studies.
Area of Science:
- Cell biology
- Biophysics
- Microscopy
Background:
- Förster Resonance Energy Transfer (FRET) biosensors are crucial for studying nanoscale interactions in living cells.
- Existing FRET measurement techniques have limitations, including the need for single fluorophore standards and intensity dependence.
Purpose of the Study:
- To introduce sensorFRET, a novel spectrally resolved fluorescence microscopy approach for quantitative FRET efficiency measurement.
- To improve upon existing FRET measurement methods by eliminating the need for single fluorophore standards and achieving intensity independence.
Main Methods:
- Developed sensorFRET, a spectrally resolved fluorescence microscopy technique.
- Validated sensorFRET using simulated spectra, zero FRET controls (Fluorescein/TAMRA), and positive FRET controls (Cerulean-Venus).
- Quantified measurement uncertainty by relating spectral fit residuals to FRET efficiency standard deviation.
Main Results:
- SensorFRET enables quantitative FRET efficiency measurement without single fluorophore standards and is intensity independent.
- Identified specific fluorophore-excitation wavelength combinations where spectral methods fail, predictable with sensorFRET.
- Applied sensorFRET to Teal-Venus force-sensitive biosensors in E-cadherin to measure piconewton forces in cell junctions.
Conclusions:
- SensorFRET offers a robust and accurate method for FRET efficiency measurement in live-cell imaging.
- The method provides a priori identification of potential inaccuracies in spectral FRET measurements.
- SensorFRET successfully resolved nanoscale forces in cellular structures, demonstrating its utility in biological research.
