| Summary: | A substantial number of genetically encoded fluorescent sensors rely on the changes in FRET efficiency between fluorescent cores, measured in ratiometric mode, with acceptor photobleaching or by changes in fluorescence lifetime. We report on a modulated FRET acceptor allowing for simplified one‐channel FRET measurement based on a previously reported fluorogen‐activating protein, DiB1. Upon the addition of the cell‐permeable chromophore, the fluorescence of the donorfluorescent protein mNeonGreen decreases, allowing for a simplified one‐channel FRET measurement. The reported chemically modulated FRET acceptor is compatible with live‐cell experiments and allows for prolonged time‐lapse experiments with dynamic energy transfer evaluation. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
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