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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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Fluorescence resonance energy transfer microscopy (FRET)
Katarzyna M Kedziora1, Kees Jalink
1Cell Biophysics Group, Department of Cell Biology B5, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands.
Methods in Molecular Biology (Clifton, N.J.)
|November 14, 2014
Summary
Förster Resonance Energy Transfer (FRET) microscopy enables studying protein interactions and cellular processes in living cells. This guide details intensity-based FRET methods, including sensitized emission and ratio imaging, for enhanced biological research.
Area of Science:
- Cell Biology
- Biophysics
- Microscopy
Background:
- Förster Resonance Energy Transfer (FRET) microscopy overcomes the diffraction limit of light, enabling visualization of molecular interactions and conformational changes within living cells.
- Intensity-based FRET detection, including sensitized emission and ratio imaging, are established techniques in cell biology for studying protein dynamics.
- Accurate quantification of FRET signals requires specific mathematical corrections to account for various optical and environmental factors.
Purpose of the Study:
- To provide detailed protocols for implementing intensity-based Förster Resonance Energy Transfer (FRET) microscopy techniques.
- To guide researchers in performing necessary mathematical corrections for accurate FRET signal quantification.
- To demonstrate the application of FRET microscopy in studying protein-protein interactions and cellular signaling pathways.
Main Methods:
- Detailed protocols for sensitized emission FRET measurements, including mathematical corrections for accurate image analysis.
- Step-by-step instructions for ratio imaging FRET using specifically designed sensors, exemplified by monitoring cAMP levels.
- Application examples illustrating FRET for studying the interaction of Sorting Nexin 1 (SNX1) proteins and changes in intracellular cAMP concentration.
Main Results:
- Successful implementation of sensitized emission FRET protocols with necessary mathematical corrections for quantitative analysis.
- Demonstration of ratio imaging FRET for real-time monitoring of cellular cAMP dynamics using an EPAC-based sensor.
- Validation of FRET techniques for investigating specific molecular interactions and cellular signaling events.
Conclusions:
- Intensity-based FRET microscopy, with proper protocol execution and data correction, is a powerful tool for dissecting molecular mechanisms in living cells.
- The provided protocols facilitate the study of protein interactions, such as SNX1, and dynamic cellular processes like cAMP signaling.
- FRET microscopy continues to be an indispensable technique for advancing our understanding of cell biology at the molecular level.
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