Oxygen-independent imaging of anaerobes with FAST

Why GFP goes dark without oxygen

Green fluorescent protein and every reporter derived from it (EGFP, mCherry, mNeonGreen and the rest) do not arrive fluorescent. After the chain folds, it has to mature: an internal, self-catalysed reaction forms the chromophore, and the final oxidation step consumes molecular oxygen. Take the oxygen away and the chromophore never forms. In an anaerobic culture or the hypoxic core of a biofilm or spheroid, a fluorescent-protein fusion is expressed but stays invisible, and even at the low-oxygen margin, maturation is so slow that the signal lags far behind the biology you are trying to watch.

How FAST stays bright, and lights up in seconds

FAST does not build a chromophore. It binds a small fluorogen present in the medium and switches it on the instant it is bound: no oxygen, no maturation, no waiting. The fluorogen is itself a small, membrane-permeant molecule, so it crosses the cell envelope and reaches the cytoplasm within seconds; in practice, labelling speed is limited only by how fast the fluorogen diffuses in (Acc. Chem. Res. 2022). Because the interaction is non-covalent and reversible, the signal tracks the biology in real time, can be tuned by the amount of fluorogen you add, and can be switched off again by removing it. FAST is also small, a purely monomeric protein of just 14 kDa (a 375 bp gene), so it perturbs your system less than bulkier tags. In practice: put your cells in an anaerobic chamber, add the fluorogen, and image straight away on the microscope, cytometer or plate reader you already use.

How FAST compares with the other oxygen-independent reporters

GFP is not the only reporter people try in anaerobes, and it helps to know where FAST sits among the alternatives.

• Flavin-based reporters (iLOV, phiLOV, CreiLOV, evoglow and other FbFPs). They bind the flavin FMN instead of forming an oxygen-dependent chromophore, so they fluoresce without oxygen and were the first reporters to work in Clostridium. Their limits are brightness and colour: dim, and essentially green-only (Appl. Environ. Microbiol. 2019).
• UnaG. A bright green protein that works without oxygen, but only once it captures its cofactor bilirubin; where that cofactor is scarce or poorly delivered, the signal suffers (Front. Microbiol. 2023).
• Bilin-based near-infrared reporters (IFP, iRFP). Oxygen-independent and useful for deep or turbid samples, but they depend on the pigment biliverdin, whose supply and membrane permeability are often limiting in bacteria (Front. Microbiol. 2023).
• Self-labelling tags (HaloTag®, SNAP-tag®). These also work in strict anaerobes, and with fluorogenic ligands (for example the Janelia Fluor® HaloTag dyes) they light up on binding with no washing steps. What sets FAST apart is speed, reversibility and size. Speed and delivery: FAST switches its fluorogen on the instant it binds, with no covalent bond to form, and the fluorogen is a small molecule (roughly 250 Da, against around 700 Da for the dye-and-linker ligands HaloTag and SNAP-tag need) that crosses the wall and membrane readily; the small size feeds the speed, and together they make FAST far quicker to light up. In a side-by-side test in Clostridium, FAST was instantaneous, while HaloTag took about 15 minutes and SNAP-tag about 30 minutes to form their covalent bond (Appl. Environ. Microbiol. 2020). Bulkier fluorogens may even be kept out of the bacterial cytosol in all but actively growing cells (bioRxiv 2023). Reversibility: FAST binds non-covalently, so labelling is reversible and can be tuned or exchanged in real time, which is what lets you follow transient phenomena and switch colours for multiplexed imaging, whereas HaloTag and SNAP-tag bind permanently. Size: at 14 kDa the FAST tag is smaller than SNAP-tag (about 20 kDa) and HaloTag (about 33 kDa), so it is less likely to disturb the folding, localisation or function of the protein you fuse it to. The three tags are orthogonal, and this same Clostridium group combined FAST, HaloTag and SNAP-tag to follow several subpopulations at once. To be fair to the alternatives, HaloTag and SNAP-tag have a long, well-documented track record of proven constructs, and paired with Janelia Fluor dyes they are very bright.

In short, FAST keeps the oxygen-independence all of these share and adds the rest: it is the fastest to light up, reversible and tunable, small and monomeric, and it spans green to the near-infrared. Its small fluorogens reach the cell interior in seconds, and non-permeant variants can instead tag the outer envelope of both monoderm and diderm bacteria (Sci. Rep. 2020).

From acetogen engineering to the mammalian microbiome: a short history

FAST reached anaerobic biology in 2019, when Terry Papoutsakis and his team at the University of Delaware built the first strongly fluorescent, oxygen-independent reporter for Clostridium (Appl. Environ. Microbiol. 2019), finally bringing real-time imaging and cell sorting to the acetogenic and solventogenic anaerobes that had long defeated GFP. Until then, gene expression in these organisms was read out mostly with enzymatic assays that meant lysing the cells; FAST let the same measurements be made in live cells, by microscopy and by flow cytometry. A year later the same group turned FAST into part of a multi-colour toolkit, pairing it with orthogonal HaloTag and SNAP-tag reporters so that several strains or subpopulations could be told apart in one mixed culture (Appl. Environ. Microbiol. 2020).

From there it spread across the anaerobic world. Metabolic engineers adopted it to turn one-carbon gases into fuels and chemicals in gas-fermenting acetogens, including the CO2-fixing Eubacterium limosum (Microb. Cell Fact. 2024) and relatives such as Acetobacterium woodii and Clostridium autoethanogenum, and it was extended to strict anaerobic thermophiles that grow hot as well as without air (Front. Bioeng. Biotechnol. 2023). The same freedom from oxygen soon carried FAST beyond bacteria, into methanogenic archaea such as Methanococcus maripaludis and Methanosarcina acetivorans, where it brought live imaging and flow cytometry to organisms that had never been watched this way (J. Bacteriol. 2022); (Appl. Environ. Microbiol. 2023). Because it reports equally under total anoxia and under milder hypoxia, FAST-encoded bacteria have since been used to illuminate the living gut microbiome inside mice (Mater. Today Bio 2022) and to dissect the biology of the human pathogen Clostridioides difficile (PLoS Pathog. 2024). In a handful of years it went from a niche fix for one genus to a general reporter for life without oxygen, from the bioreactor to the gut.

What you can study

• Obligate anaerobes. Visualise protein localisation and characterise gene-expression activity in Clostridium and other strict anaerobes that fluorescent proteins cannot report on.
• Living, multi-species biofilms. Follow community dynamics in bioreactors, where oxygen is depleted within hours and GFP-type signals saturate or vanish.
• Gut microbiome & syntrophic cultures. Track populations for cell sorting, sporulation dynamics and population characterisation in native or synthetic microbiomes.
• Hypoxic tissue and 3D models. Report from the low-oxygen interior of spheroids, organoids and tissue where chromophore maturation stalls.

Recommended fluorogens

^TFLime (green) and ^TFCoral (orange-red) are the validated choices for anaerobic work and were used in the published studies below. For deeper samples where longer wavelengths penetrate better, ^TFCarmine extends the same oxygen-independent readout into the near-infrared. Because binding is oxygen-independent, any ^TFFluorogen works, so the colour choice is yours.

Frequently asked

Does FAST need oxygen at any step?

No. Unlike GFP-family proteins, FAST requires no oxygen to fold, to mature or to fluoresce. Signal appears as soon as the fluorogen is added.

How does FAST compare with iLOV and other flavin (FbFP) reporters?

Both work without oxygen, which is why FbFPs became popular in anaerobes. FAST is brighter, spans many colours including the near-infrared, and its labelling is reversible and tunable, whereas FbFPs are essentially green-only and always on.

Recent papers: FAST & splitFAST for anaerobic labeling