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bioRxiv · 10.1101/2025.09.20.674865

Automated FRET Analysis for Enhanced Characterization of Protein-Protein Interactions

NURSOY, A. Z.·CEVHEROGLU, O.·SON, C. D.
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Abstract

Forster 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. Research HighlightsO_LIWe developed an open-source pipeline using Cellpose for accurate, unbiased FRET analysis. C_LIO_LIIt outperforms manual methods, reduces variability, supports high-throughput studies, and enables reproducible, user-friendly image analysis across labs. C_LI Graphical AbstractA visual summary of the SONLab FRET Tool, displaying the entire workflow--from imaging to the plotting of quantified data. The results provided in this article were obtained using this automated pipeline. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/674865v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@e189cforg.highwire.dtl.DTLVardef@11e933corg.highwire.dtl.DTLVardef@545f48org.highwire.dtl.DTLVardef@13b89c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

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NURSOY, A. Z., CEVHEROGLU, O., SON, C. D.. 2025-09-21. Automated FRET Analysis for Enhanced Characterization of Protein-Protein Interactions. https://doi.org/10.1101/2025.09.20.674865

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