A Software Tool for High-Throughput Real-Time Measurement of Intensity-Based Ratio-Metric FRET

Area of Science:

• Cell biology
• Biophysics
• Software development

Background:

• Förster resonance energy transfer (FRET) is a key technique for measuring molecular interactions in live cells.
• Current methods can be limited in throughput and real-time analysis capabilities.

Purpose of the Study:

• To develop and validate a freely available software tool, MultiFRET, for enhanced live cell FRET imaging.
• To enable simultaneous, real-time analysis of FRET signals from multiple cells within a single experiment.

Main Methods:

• Development of MultiFRET, a Java plugin for Micro-Manager software.
• Integration with a motorized microscope stage for multi-cell acquisition.
• Real-time, ratio-metric signal calculation and data export to Excel.
• Validation using various cAMP FRET sensors in different cardiomyocyte models (adult, neonatal, hiPSC-CM).

Main Results:

• MultiFRET enables simultaneous recording and real-time ratio-metric calculations from multiple cells.
• The software is flexible, supporting multiple spectral acquisition channels and custom live calculations.
• Validation showed no significant difference compared to established FRET measurement methods.
• Successful application in diverse cell models including cardiomyocytes expressing various FRET sensors.

Conclusions:

• MultiFRET is a validated, open-source software solution for efficient live cell FRET imaging.
• It significantly improves experimental throughput by allowing simultaneous multi-cell analysis.
• The tool is broadly applicable to live cell imaging requiring real-time ratio-metric calculations.