ArticleMethods in molecular biology (Clifton, N.J.)2025
Measuring Molecular Interactions with Subcellular Resolution: Single-Cell FRET Using the Quantitative Three-Filterset (Intensity-Based) Approach.
Article in Methods in molecular biology (Clifton, N.J.), 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
The actual interaction between signaling species in cellular processes is often more important than their expression levels. Förster resonance energy transfer (FRET) is a popular tool for studying molecular interactions, since it is highly sensitive to proximity in the range of 2-10 nm. Spectral spillover-corrected quantitative (three-filterset) FRET is a cost-effective and versatile approach, which can be applied in flow cytometry and various modalities of fluorescence microscopy, where it provides pixel-by-pixel quantitative FRET efficiency values as well as FRET-corrected fluorescent intensities corresponding to the expression level of the two labeled interacting molecular species. Here, we provide a protocol for implementing such measurements, starting from the preparation of appropriate samples including controls, through a step-by step guide for image processing and derivation of microscopic FRET maps, to various aspects of interpreting the results. The image processing described is aided by a freely available ImageJ/Fiji plugin RiFRET which has been developed with the biology-focused users in mind. The interface guides the user through the evaluation process in a friendly manner and provides the option for automatic processing of large image libraries once the various calibration factors have been correctly set.
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