pFAST in C. elegans for reversible multicolor labeling. A new microPublication Biology study shows tandem pFAST enables reversible, multicolor chemogenetic fluorescence labeling in dissected C. elegans tissues.
Wang, Langevin, Chen and Bai (Fred Hutchinson Cancer Center) have published the first evaluation of pFAST, the promiscuous Fluorescence-Activating and absorption-Shifting Tag, in Caenorhabditis elegans.
Unlike GFP or mCherry, pFAST only fluoresces when it binds a small fluorogenic ligand. Because that binding is non-covalent and reversible, the signal can be switched on, washed off, or swapped for a different color at will — turning a static reporter into a temporally controllable, spectrally flexible probe. The team built a tandem pFAST (td-pFAST) reporter, codon-optimized for the worm, expressed in pharyngeal muscle under the myo-2 promoter and integrated as a single copy at the ttTi5605 locus by Mos1-mediated insertion.
What pFAST delivered:
pFAST in C. elegans – © 2026 by the authors
- In dissected worms, the tfLime fluorogen (HMBR) lit up the pharynx within 5 minutes, reaching ~50% of maximal intensity by 10 minutes and still rising at 30 minutes.
- Adding the tfDarth ligand (HBIR-3M), a non-fluorescent competitor, quenched more than 80% of the signal within 30 minutes — a direct demonstration of reversibility in living tissue.
- tfAmber (HBR-3,5DM) and tfCoral (HBR-3,5DOM) produced robust signals with distinct emission spectra and comparable quenching, confirming that a single genetic tag can be read out in multiple color channels without re-engineering the strain.
What we learned about pFAST — and its current limits:
- Fluorogen entry and pFAST activation are fast and efficient in dissected tissue, and ligand exchange is clean enough for on/off and color-switching experiments.
- Soaking intact animals failed: no pharyngeal signal at 30 minutes or even 24 hours. The C. elegans cuticle, not fluorogen affinity, is the bottleneck — ligand concentrations used (10 µM) sit well above the submicromolar Kd values reported for pFAST fluorogens.
The takeaway: td-pFAST is now a validated, single-copy, reversible and multicolor labeling tool for dissected C. elegans preparations — and a clear roadmap for extending chemogenetic imaging to intact multicellular organisms.
Read the original open-access article (CC BY 4.0): Wang Z, Langevin D, Chen V, Bai J. “Reversible Chemogenetic Fluorescence Labeling with pFAST in C. elegans.” microPublication Biology, 2026. DOI: 10.17912/micropub.biology.001980
Besides tfCoral, tfAmber and tfDarth used above, The Twinkle Factory offers a wide range of commercial fluorogens, permeant or not, and quenchers, for FAST and derivatives, splitFAST, greenFAST & redFAST, frFAST, nirFAST. On top of those, the range of reagents now includes fluorogenic molecular glues for CATCHFIRE, our reversible and self-reporting protein dimerizer
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