Reversible Chemogenetic Fluorescence Labeling with pFAST in C. elegans

The promiscuous fluorescence-activating and absorption-shifting tag (pFAST) enables reversible chemogenetic labeling with multiple fluorogens. We generated a single-copy tandem pFAST (td-pFAST) transgenic Caenorhabditis elegans strain expressed in pharyngeal muscle. In dissected worms, lime fluorogen produced rapid fluorescence that was efficiently quenched by the competing ligand darth, demonstrating reversibility. Amber and coral fluorogens also produced reversible signals with distinct emission spectra, supporting multicolor labeling. However, fluorogen delivery by soaking intact worms failed, indicating cuticle permeability remains a barrier. These findings establish td-pFAST as a functional probe for reversible, multicolor labeling in dissected C. elegans tissues.