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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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MultiFRET: A Detailed Protocol for High-Throughput Multiplexed Ratiometric FRET.
Masoud Ramuz1, Ivan Diakonov1, Chris Dunsby2,3
1Imperial College London, National Heart and Lung Institute, London, UK.
Methods in Molecular Biology (Clifton, N.J.)
|March 14, 2022
Summary
The new MultiFRET software enables real-time, near high-throughput measurements for multiplexed fluorescent biosensors. This protocol details its setup and use for fluorescence experiments, including advanced features and data analysis.
Area of Science:
- Biophysics
- Biotechnology
- Software Development
Background:
- Fluorescence measurements are crucial for biological research.
- Real-time feedback and high-throughput analysis are needed for complex experiments.
- Existing software may lack flexibility for custom analyses and advanced features.
Purpose of the Study:
- To introduce and detail the setup and use of the MultiFRET software plugin.
- To demonstrate the capabilities of MultiFRET for real-time multiplexed fluorescent biosensor measurements.
- To highlight advanced features for enhanced experimental control and data processing.
Main Methods:
- Development and implementation of the MultiFRET software plugin.
- Protocol description for software setup and experimental integration.
- Application of MultiFRET for real-time cAMP measurements in cardiomyocytes.
Main Results:
- MultiFRET facilitates near high-throughput, real-time measurements of multiplexed fluorescent biosensors.
- The software includes features for beam splitter correction, custom calculations, and customizable Excel output.
- Offline bulk analysis of image stacks is supported.
- Successful demonstration of cAMP measurement in rat neonatal cardiomyocytes.
Conclusions:
- MultiFRET provides a versatile platform for real-time fluorescence measurements.
- The software enhances experimental efficiency and data analysis capabilities.
- It is applicable to various research areas requiring instant feedback during fluorescence experiments.
