Automated FRET Analysis for Enhanced Characterization of Protein–Protein Interactions

ABSTRACT Förster Resonance Energy Transfer (FRET) analysis is a powerful technique for studying protein–protein interactions; however, manual methods often introduce variability and user dependency. We present the SONLab FRET Analysis Tool, an open‐source and automated software that integrates Cellpose for cell segmentation with standardized pipelines for bleed‐through correction and FRET efficiency calculation. By minimizing human intervention, the tool improves reproducibility and comparability between experiments. The results demonstrate that the tool achieves FRET efficiencies comparable to those of manual methods but with reduced bias, enabling robust and high‐throughput analysis of protein interactions.