FAST & splitFAST: the fluorogen-activating protein tag
The Science Behind FAST and splitfastFAST: fluorogen-activating protein tags for live-cell imaging
FAST, the Fluorescence-Activating and absorption-Shifting Tag, is a small (14 kDa) genetically encoded protein tag that becomes fluorescent only when it reversibly binds a fluorogenic ligand (a TFfluorogen). First reported by Prof. Arnaud Gautier’s group in PNAS in 2016, FAST has since grown into a whole family of chemogenetic reporters for labelling virtually any protein of interest in living systems.
The principle is simple and powerful: a TFfluorogen is dark in solution and lights up only once bound to FAST. Fusing FAST to a protein of interest (as an N- or C-terminal tag) and adding the fluorogen reveals that protein with high contrast and no washing step, unbound dye stays invisible.
The FAST principle: a TFFluorogen fluoresces only while bound to the FAST-tagged protein of interest (POI), and washing it out reverses the labelling.
Why choose FAST?
- No-wash, high-contrast imaging, only the FAST-bound fluorogen fluoresces, so excess dye never has to be removed.
- Reversible and non-covalent, labelling switches off simply by washing out the fluorogen, enabling pulse-chase and dynamic experiments.
- Oxygen-independent, unlike GFP-type fluorescent proteins, FAST needs no molecular oxygen to fluoresce, so it images anaerobic and hypoxic samples (mature biofilms, gut anaerobes such as Clostridium, tumour cores).
- One tag, many colours, swapping the fluorogen tunes emission from green to far-red without re-engineering the protein.
- Half the size of GFP, 14 kDa minimises steric perturbation of the tagged protein.
- Surface-selective labelling, membrane-impermeant fluorogens (e.g. HBRAA-3E / TFAmber-NP) label only cell-surface proteins, ideal for trafficking studies and flow cytometry.
- Fast binding kinetics, near-instant fluorescence supports real-time recording of rapid events.
How FAST compares with other labelling technologies
FAST belongs to the family of chemical-genetic (chemogenetic) reporters, and its properties often overcome the limits of established methods:
- vs. fluorescent proteins (GFP, RFP): FAST is smaller, matures instantly, needs no oxygen, is spectrally tunable with a single tag, and is reversible, where FPs are bulkier, oxygen-dependent and fixed in colour.
- vs. self-labelling enzyme tags (HaloTag, SNAP-tag, CLIP-tag): these form covalent, irreversible bonds and require washout of excess substrate; FAST labels non-covalently and reversibly, with no wash, at roughly half the size.
- vs. other fluorogen-activating systems (FAPs, aptamer–dye pairs): FAST combines small, cell-permeant HBR-derived fluorogens with a compact single-domain tag, for a well-defined, protein-specific live-cell signal.
For protein–protein interactions, the split version of FAST offers a comparable leap, see below.
A growing FAST family, a short history
Each variant since 2016 has opened a new imaging modality:
2016, FAST, the original tag
The founding PNAS paper introduced the 14 kDa tag and its reversible, oxygen-independent fluorogen chemistry.
2019, splitFAST for protein–protein interactions
Tebo & Gautier (Nature Communications) split FAST into two fragments that reconstitute a fluorescent complex only when their fusion partners interact. Complementation is rapid and fully reversible, so, unlike BiFC (irreversible) and more simply than FRET, splitFAST visualises both the formation and the dissociation of a complex in real time.
2020, frFAST for far-red imaging
frFAST, paired with the fluorogen HPAR-3OM (TFPoppy), pushed the palette into the far-red (Angew. Chem. Int. Ed.) for deeper-tissue imaging with less autofluorescence and scattering, demonstrated in mammalian cells, zebrafish and chicken embryos.
2021, orthogonal and multicolour FAST
greenFAST & redFAST (Nature Chemical Biology) are orthogonal tags, each recognising a specific fluorogen (HMBR / TFLime and HBR-3,5DOM / TFCoral), enabling crosstalk-free two-colour confocal, FLIM, two-photon and super-resolution imaging, plus split versions for multiplexed interaction studies. pFAST added a broadened, tunable fluorogen palette.
2021–2025, nanoFAST, nirFAST and beyond
Structure-guided miniaturisation (nanoFAST) and near-infrared reporters (nirFAST) continue to extend the technology toward smaller tags and longer wavelengths for in vivo work.
Explore the full range of FAST fluorogens in our catalogue, or discover CATCHFIRE, our fluorogenic molecular-glue system for inducible, self-reporting protein proximity.
Choosing a protein-labeling tag? See our detailed, point-by-point guide: FAST vs GFP, HaloTag & SNAP-tag →
Explore the peer-reviewed evidence
FAST, splitFAST and their variants now feature in dozens of published studies, from multicolour and far-red imaging to anaerobic and in vivo work. Search the database below, filter by reporter, year or type, and open any paper directly.
Reversible Chemogenetic Fluorescence Labeling with pFAST in C. elegans
Made a tandem pFAST (td-pFAST) C. elegans strain for reversible multicolor chemogenetic labeling in worms.
Anaerobic Fluorescent Reporters for Studying Pseudomonas aeruginosa-Specific Responses in a CF Polymicrobial Assay
A PhD thesis developing anaerobic FAST reporters to study Pseudomonas aeruginosa responses.
The fluorescence‐activating and absorption‐shifting tag (FAST), a versatile protein marker for live plant cell imaging
Established FAST, greenFAST/redFAST and splitFAST for versatile multicolor imaging in plant cells.
Chemogenetic Modulation of Luciferase Emission Color for Imaging and Sensing
Introduced LumiFAST, a chemogenetic way to tune luciferase emission color, for bioluminescence imaging and sensing.
Imaging and quantifying organelle contact sites using a reversible splitFAST complementation assay
A methods chapter on imaging and quantifying organelle contact sites with a reversible splitFAST complementation assay.
Random Mutagenesis-Based Optimization of the Fluorogen-Activating Protein picoFAST
Optimized picoFAST, an ultra-small fluorogen-activating protein, by random mutagenesis.
Monitoring intracellular antibiotic concentrations in real-time using allosteric biosensors
Used a FAST-based allosteric biosensor to monitor intracellular antibiotic concentrations in real time.
Bathochromic Shift via C=O to C=S Substitution: A Far-Red Fluorogen for Multiplexed FLIM with FAST Fluorogen-Activating Protein
Extended the FAST palette into the far-red with HBTR-3,5-DOM, a fluorogen made by C=O-to-C=S substitution.
Chemogenetic timestamping for the precise tracing of cell history into protein assemblies
A preprint on chemogenetic 'timestamping' to trace cell history into protein assemblies.
Establishing the fluorescence-activating and absorption-shifting tag as a fluorescent reporter
A preprint establishing FAST as a fluorescent reporter in a new host system.
Kinetic and Mechanistic Characterization of RSpFAST, a Non-Covalent Versatile Chemogenetic Platform for Fluorescence Imaging
Introduces RSpFAST (a pFAST tag paired with a reversibly photoisomerizable fluorogen) and characterizes its thermokinetics, photochemistry and structure. Reveals a dual regime: a stable wash-free label becomes a negative photoswitcher at low fluorogen concentration and high light (photoejection-driven), enabling dynamic-contrast and super-resolution imaging in live and fixed cells.
A color-tailored fluorogenic sensor for hydrogen peroxide
Built HyPerFLEX, a FAST-based color-tunable fluorogenic sensor for high-precision H2O2 detection in living cells.
Characterization of Fluorescent Reporters for Flow Cytometry-Based Single-Cell Studies in Saccharomyces cerevisiae
Benchmarked green/red/blue fluorescent reporters, including frFAST, for flow-cytometry single-cell studies in yeast.
Super-Resolution Live-Cell Mapping of Protein-Protein Interactions Using Chemogenetic splitFAST
Mapped protein-protein interactions at super-resolution in live cells using chemogenetic splitFAST.
Hi-TARGET: A fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui
Built a CRISPR type I-B genome-editing toolkit for the thermophilic acetogen Thermoanaerobacter kivui, with pFAST as a fluorescent reporter.
Toward Far-red Emitting Chemogenetic Labelling for Live Cell Super-Resolution Microscopy using Fluorescence-Activating and Absorption-Shifting Tag
Reported far-red chemogenetic FAST labeling approaches for background-free super-resolution imaging of intracellular targets.
Fluorescence-activating and absorption-shifting tag
The encyclopedia reference entry defining FAST, the 14-kDa tag engineered from photoactive yellow protein (the Wikipedia article itself).
Early HIV-1 maturation drives Env clustering and fusion competence
Studied how early HIV-1 maturation drives Env clustering and fusion competence, using FAST-family imaging.
Deletion of atypical type II restriction genes in Clostridium cellulovorans using a Cas9-based gene editing system
Improved genetic accessibility of the biofuel-relevant anaerobe Clostridium cellulovorans, using FAST as an anaerobic reporter.
A rapid genome-proteome approach to identify rate-limiting steps in the butyrate production pathway in probiotic <i>Clostridium butyricum</i>, CBM588
A genome-proteome approach to find rate-limiting steps in butyrate production by Clostridium butyricum, with FAST reporting.
Design and evaluation of a tripartite chemogenetic fluorescent reporter for visualizing ternary protein complexes
Engineered a tripartite (three-fragment) splitFAST that lights up only when three proteins assemble, visualizing ternary complexes in live cells.
A guide to characterizing the dynamic mitochondria-endoplasmic reticulum contact sites
A review guide to characterizing mitochondria-ER contact sites, referencing reversible splitFAST-type reporters.
Lipid droplet dysmetabolism affects cell homeostasis in an in vitro model of Alzheimer’s disease
Applied splitFAST to link lipid-droplet dysmetabolism to cell-homeostasis defects in an in-vitro Alzheimer's model.
Nano-frFAST: Design of a New Genetically-Encoded Far-Red Fluorescent Label
Designed nano-frFAST, a small far-red fluorogen-activating protein, with a matching series of extended-pi fluorogens.
Thermophilic Chassis-Enabled High-Throughput Selection of a Thermostable Fluorogenic Reporter
Selected thermostable FAST variants (hsFAST/tsFAST) via a thermophilic-chassis high-throughput screen.
Mitophagy and release of mitochondrial derived vesicles in cellular models of inducible unfolded protein accumulation in mitochondrial matrix and intermembrane space.
Studied mitophagy and mitochondrial-derived vesicles in inducible cell models, using FAST-family reporters.
Fluorescence Lifetime-Based Separation of FAST-Labeled Cellular Compartment
A step-by-step protocol for FAST-based fluorescence-lifetime imaging (FLIM) to separate labels sharing a color channel.
A tunable and versatile chemogenetic near-infrared fluorescent reporter
Introduced nirFAST, a bright 14-kDa near-infrared reporter for deep, multiplexed imaging that also doubles as a CATCHFIRE proximity tool.
Synthetically programmed antioxidant delivery by a domesticated skin commensal
Engineered a skin commensal bacterium for programmed antioxidant delivery, using FAST to fluorescently track the strains.
Expression and Subcellular Localization of Lanthipeptides in Human Cells
Expressed and localized lanthipeptides in human cells, using FAST-based fluorescent reporting.
Human MX1 orchestrates the cytoplasmic sequestration of neo-synthesized influenza A virus vRNPs
Applied splitFAST to dissect how human MX1 sequesters newly made influenza vRNPs in the cytoplasm.
FAM122A ensures cell cycle interphase progression and checkpoint control by inhibiting B55α/PP2A through helical motifs
Used splitFAST complementation to characterize how FAM122A binds and inhibits the B55-alpha/PP2A phosphatase to control the cell cycle.
A multi-colour fluorogenic tag and its application in Candida albicans
Applied a multi-colour fluorogenic FAST tag in the fungal pathogen Candida albicans.
A fluorogenic complementation tool kit for interrogating lipid droplet–organelle interaction
A fluorogenic splitFAST toolkit for detecting lipid-droplet-organelle contact sites at nanometer scale in living cells.
Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants
Baranov-group FAST FLIM multiplexing: FAST mutants sharing a fluorogen but differing in fluorescence lifetime, telling several labels apart by lifetime.
A guide to genetically-encoded redox biosensors: state of the art and opportunities
A review of genetically encoded redox biosensors that covers FAST-based fluorogenic designs.
The genetic architecture of protein interaction affinity and specificity
Used splitFAST to quantify how every mutation in the transcription factor JUN reshapes binding across a protein family, mapping interaction affinity and specificity.
Improving Split Reporters of Protein–Protein Interactions through Orthology-Based Protein Engineering
Improved splitFAST's brightness and performance for detecting protein-protein interactions using orthology-based protein engineering.
Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering
Reclassified and metabolically engineered acetogenic Eubacterium strains, using FAST for fluorescent visualization.
Multiplexed In Vivo Imaging with Fluorescence Lifetime-Modulating Tags
Introduced FAST variants tuned to distinct fluorescence lifetimes, enabling highly multiplexed in-vivo imaging by lifetime rather than color.
A Combination of Library Screening and Rational Mutagenesis Expands the Available Color Palette of the Smallest Fluorogen-Activating Protein Tag nanoFAST
Expanded the color palette of nanoFAST, the smallest (98-aa) fluorogen-activating protein, by mutagenesis and screening.
Imaging and proteomics toolkits for studying organelle contact sites
A review of imaging and proteomics toolkits for organelle contact sites, including splitFAST-based approaches.
Lactate-mediated mixotrophic co-cultivation of Clostridium drakei and recombinant Acetobacterium woodii for autotrophic production of volatile fatty acids
Engineered a Clostridium/Acetobacterium co-culture for autotrophic fatty-acid production, with FAST as a fluorescent strain marker.
Inactive ryanodine receptors sustain lysosomal availability for autophagy by promoting ER-lysosomal contact site formation
Used splitFAST to study how inactive ryanodine receptors sustain lysosomal autophagy in Alzheimer's models.
Multipartite Fluorogenic Sensors for Monitoring Tyrosine Phosphatase Activity
First steps toward genetically encoded FAST-based sensors for protein tyrosine phosphatase activity.
Simultaneous detection of membrane contact dynamics and associated Ca2+ signals by reversible chemogenetic reporters
Built reversible splitFAST reporters that simultaneously visualize membrane contact-site dynamics and the Ca2+ signals they carry.
Transforming chemigenetic bimolecular fluorescence complementation systems into chemical dimerizers
A preprint turning chemigenetic bimolecular fluorescence complementation systems into chemical dimerizers.
Anaerobic fluorescent reporters for live imaging of Pseudomonas aeruginosa
Developed anaerobic FAST reporters for live imaging of Pseudomonas aeruginosa in biofilms and low-oxygen conditions.
Microbiome imaging goes à la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform
Added click chemistry to FAST imaging to label anaerobic microbiome bacteria in biofilm communities.
Application of the Fluorescence-Activating and Absorption-Shifting Tag (FAST) for Flow Cytometry in Methanogenic Archaea
Applied FAST to flow-cytometry analysis and imaging of methanogenic archaea (M. maripaludis, M. acetivorans).
A fluorescent reporter system for anaerobic thermophiles
Demonstrated a FAST-based reporter that works in anaerobic thermophiles, where oxygen-dependent GFP fails.
Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum
Used endogenous CRISPR/Cas systems to engineer acetogens, with FAST for fluorescent readout.
Engineering of Tunable Allosteric-like Fluorogenic Protein Sensors
Engineered tunable allosteric-like FAST biosensors that switch fluorescence in response to a target biomolecule.
Effeсts of the 70th Amino Acid Residue on the Photostability of FAST Complexes
A FAST T70V mutant showing the 70th residue barely affects brightness or photostability.
Autotrophic lactate production from H2 + CO2 using recombinant and fluorescent FAST-tagged Acetobacterium woodii strains
Engineered fluorescent FAST-tagged Acetobacterium woodii for autotrophic lactate production from H2 + CO2.
Recent Advancements in Tracking Bacterial Effector Protein Translocation
A review of methods for tracking bacterial effector-protein translocation, featuring FAST among the reporters.
Encoding with a fluorescence-activating and absorption-shifting tag generates living bacterial probes for mammalian microbiota imaging
Encoded gut-microbiota bacteria with FAST to create living probes for imaging the mammalian microbiota.
Establishment of Green- and Red-Fluorescent Reporter Proteins Based on the Fluorescence-Activating and Absorption-Shifting Tag for Use in Acetogenic and Solventogenic Anaerobes
Established greenFAST and redFAST reporters for use in acetogenic and solventogenic anaerobes.
Production of propionate using metabolically engineered strains of Clostridium saccharoperbutylacetonicum
Engineered Clostridium saccharoperbutylacetonicum for propionate production, tracked with FAST.
The Fluorescence-Activating and Absorption-Shifting Tag (FAST) Enables Live-Cell Fluorescence Imaging of Methanococcus maripaludis
Showed FAST enables live-cell fluorescence imaging of the methanogenic archaeon Methanococcus maripaludis under anoxic conditions.
Fluorescence-Activating and Absorption-Shifting Tags for Advanced Imaging and Biosensing
An Accounts review by the FAST inventor summarizing how FAST-family tags and biosensors work and their imaging and biosensing applications.
Orthogonal fluorescent chemogenetic reporters for multicolor imaging
Introduced greenFAST and redFAST, spectrally orthogonal FAST variants, for simultaneous two-color imaging in live cells and zebrafish.
Anaerobic fluorescent reporters for cell identification, microbial cell biology and high-throughput screening of microbiota and genomic libraries
A review of real-time fluorescent reporters (FAST prominent among them) for anaerobes in gene expression, screening and cell biology.
NanoFAST: structure-based design of a small fluorogen-activating protein with only 98 amino acids
Introduced NanoFAST, a structure-based minimal fluorogen-activating protein of just 98 amino acids.
Protein-fragment complementation assays for large-scale analysis of protein-protein interactions
A review of protein-fragment complementation assays for large-scale PPI analysis, including splitFAST.
Color Tuning of Fluorogens for FAST Fluorogen-Activating Protein
Created a panel of color-shifted benzylidene-imidazolone fluorogens for FAST without losing binding efficiency.
Production of the biocommodities butanol and acetone from methanol with fluorescent FAST-tagged proteins using metabolically engineered strains of Eubacterium limosum
Produced butanol and acetone from methanol in engineered Eubacterium limosum, tracked with fluorescent FAST tags.
Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging
Introduced pFAST, a promiscuous FAST that binds a whole palette of fluorogens, letting one tag be imaged in tunable colors across the visible spectrum.
Developing Bright Green Fluorescent Protein (GFP)-like Fluorogens for Live-Cell Imaging with Nonpolar Protein−Chromophore Interactions
Developed brighter GFP-like fluorogens for FAST using nonpolar protein-chromophore interactions.
Styrene Derivatives of Indole and Pyranone as Fluorogenic Substrates for FAST Protein
Tested styrene derivatives of indole and pyranone as new fluorogens for FAST.
Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins
Developed strong anaerobic FAST reporters for Clostridium acetobutylicum and C. ljungdahlii.
Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells
Showed FAST can tag secreted bacterial effectors, revealing Listeria actively proliferating inside epithelial-cell vacuoles in real time.
Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST
Used FAST to visualize the dynamics of proteins exported to the bacterial cell surface.
A Far-Red Emitting Fluorescent Chemogenetic Reporter for In Vivo Molecular Imaging
Introduced frFAST, a far-red FAST variant, for spectral multiplexing and deep-tissue in-vivo imaging in zebrafish and mammalian cells.
Interspecies Microbial Fusion and Large-Scale Exchange of Cytoplasmic Proteins and RNA in a Syntrophic Clostridium Coculture
Revealed interspecies cytoplasmic exchange in a Clostridium coculture, using FAST-based fluorescent tracking.
A split fluorescent reporter with rapid and reversible complementation
Introduced splitFAST, FAST split into two fragments that fluoresce only when reunited, giving a rapid, reversible readout of transient protein-protein interactions in live cells.
Red-Shifted Substrates for FAST Fluorogen-Activating Protein Based on the GFP-Like Chromophores
Reported a red-fluorescent GFP-like fluorogen for FAST for live-cell microscopy.
Single-Molecule Localization Microscopy with the Fluorescence-Activating and Absorption-Shifting Tag (FAST) System
Demonstrated FAST for single-molecule localization super-resolution microscopy, exploiting its fast fluorogen on/off binding to pinpoint molecules.
A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)
First strongly fluorescent, oxygen-independent FAST reporter and protein-tagging system for anaerobic Clostridium.
Live cell super resolution imaging by radial fluctuations using fluorogen binding tags
Applied FAST to super-resolution radial-fluctuation (SRRF) imaging, exploiting its stochastic fluorogen binding.
The inducible chemical-genetic fluorescent marker FAST outperforms classical fluorescent proteins in the quantitative reporting of bacterial biofilm dynamics
Showed the inducible FAST marker outperforms classical GFP-family reporters for quantifying bacterial biofilm dynamics.
Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy
Introduced improved FAST variants (iFAST and tandem tdFAST) as brighter chemical-genetic markers for live-cell microscopy.
Fluorogenic Probing of Membrane Protein Trafficking
Used FAST with a non-permeant fluorogen to selectively image cell-surface versus intracellular proteins, tracking membrane-protein trafficking.
Fluorogenic Protein-Based Strategies for Detection, Actuation, and Sensing
A BioEssays review by the FAST inventors on fluorogenic protein-based strategies for detection, actuation and sensing; a strong secondary source for Wikipedia.
Dynamic multicolor protein labeling in living cells
Showed FAST can be imaged in several colors by swapping fluorogens, enabling dynamic multicolor labeling of proteins in living cells.
High-throughput, image-based screening of pooled genetic-variant libraries
A high-throughput image-based screening method for pooled genetic-variant libraries, with FAST-relevant readout.
Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo
The founding FAST paper: a 14-kDa tag, half the size of GFP, that fluoresces only while reversibly bound to its fluorogen, giving wash-free, tunable, reversible protein labeling.
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